Cell suspensions

Anticancer Res, 1996 Nov-Dec, 16(6B), 3709 - 14
Boron substituted deoxyribonucleosides as cytotoxic agents; Hall IH et al.; Base substituted boronated nucleosides and phosphate modified nucleotides were examined for their cytotoxic activity in both murine and human tissue cultured cancer cells . These derivatives demonstrated better activity against the growth of single cell suspensions than solid cell tumor cell growth . A detailed mode of action study showed that 2'deoxyriboadenosine-N7-cyanoborane 6 suppressed Tmolt3 DNA synthesis preferentially with the major target of the agent being the purine de novo pathway . The activities of one of the regulatory enzymes of the pathway were reduced by the agents, i.e . PRPP-amido transferase . Other sites in the cell which were moderately affected by the agent were nucleoside kinase activities . DNA polymerase alpha and dihydrofolate reductase activities . The DNA molecule itself did not appear to be a target of the compound.

J Androl, 1996 Nov-Dec, 17(6), 708 - 17
Isolation of highly purified type A spermatogonia from prepubertal rat testis; Morena AR et al.; We have developed a new method that allows isolation of highly purified type A spermatogonia from prepubertal rats . The procedure is based on the maximal release of spermatogonia from the seminiferous epithelium obtained by the complete enzymatic digestion of the tubular basal lamina, followed by removal of contaminating somatic cells through adhesion to plastic dishes coated with the lectin Datura stramonium agglutinin and fractionation on a discontinuous Percoll gradient . The cell suspension obtained contains up to 85% type A spermatogonia . Besides morphological criteria, the identification of germ cells and somatic cells has been performed by means of immunocytochemical markers, such as c-kit receptor, which is present only in germ cells, and vimentin, which is present only in somatic cells . All type A spermatogonia isolated were c-kit positive, thus suggesting that c-kit receptor is present in both undifferentiated and differentiating type A spermatogonia . Preliminary culture experiments demonstrate that spermatogonia survival in vitro was significantly improved by the addition of 10% fetal calf serum or horse serum to the culture medium; however, optimal culture conditions remain to be established . In vitro studies on isolated spermatogonia may provide a significant contribution toward elucidation of the mechanisms regulating spermatogonial proliferation and differentiation.

J Androl, 1996 Nov-Dec, 17(6), 603 - 14
Ultrastructural observations of spermatogenesis in mice resulting from transplantation of mouse spermatogonia; Russell LD et al.; The objective of the present study was to provide a morphological characterization of spermatogenesis following germ cell transplantation into the seminiferous tubular lumen of another mouse . The recipient mice (W-locus) were sterile because of a defect in spermatogenesis resulting from the failure of virtually all germ cell precursors to migrate to the genital ridge during embryonic development . Recipient mice containing intratubular injections of testis cell suspensions from C57 mice were allowed to develop for over 1 year, whereupon animals were sacrificed and testis tissue examined by light and electron microscopy . Donor mouse cells formed normal cell associations (stages) as viewed in cross-sectioned tubules . Spermatogonia were found exclusively in the basal compartment, indicating that they were translocated from the tubule lumen through the Sertoli cell junctions, eventually to reside on the basal lamina . Some tubules looked entirely normal from both a quantitative and qualitative standpoint . Others showed qualitative and quantitative impairment . In some tubules a generation of cells was missing from a cell association . A variety of degenerating cells and structural abnormalities were responsible for this impairment, however, the most common abnormalities were seen during the elongation phase of spermatogenesis . Elongation abnormalities and the subsequent degeneration of these cells led to the presence of fewer-than-expected elongate spermatids . There were regions of the testis where no spermatogenesis was noted and only Sertoli cells were present . These regions were generally typical of the testis histology seen in animals not exposed to injected germ cells . However, Sertoli cells in these regions phagocytosed sperm produced in spermatogenically active regions of the tubules . Because transplantation of germ cells, either from fresh or from frozen cells, had wide-ranging implications in biology and medicine, characterization of spermatogonial transplants is an important step in improving this procedure.

J Orthop Res, 1996 Nov, 14(6), 907 - 13
Osteogenic potential of allogeneic rat marrow cells in porous hydroxyapatite ceramics: a histological study; Sempuku T et al.; Hydroxyapatite ceramics facilitate osteogenesis but cannot induce bone formation by themselves . We studied the feasibility of bone formation supported by allogeneic bone marrow cells in porous hydroxyapatite ceramics . Coralline hydroxyapatite discs were soaked in a marrow cell suspension harvested from either ACI (RT1a), Lewis (RT1(1)), or Fischer 344 (RT1(1v)) male rats, and these discs were implanted subcutaneously into 56 male Fischer 344 rats . FK-506 (tacrolimus hydrate), an immunosuppressant, or saline was injected intramuscularly into the recipients every day for 2 weeks after surgery, and additional injections were given to 19 of the rats every 2 days for 2 more weeks . Neither of the mismatched major (ACI rat) or minor (Lewis rat) marrow cell transplants showed any bone formation without administration of FK-506 . However, in rats treated with FK-506, bone formed in the pores of all the three types of ceramics implanted, which each contained the marrow cells from one of the three kinds of rats used . There were no differences among the three groups of donors with regard to the bone formation ratio . We previously reported that subcutaneous implantation of porous hydroxyapatite combined with isogeneic marrow cells resulted in consistent bone formation, even at ectopic sites . Since it would be difficult to harvest a large number of autologous marrow cells in clinical cases, we attempted to use allogeneic marrow cells and have shown the allogeneic murine marrow cells to have osteogenic potential.

Plant Mol Biol, 1996 Nov, 32(3), 415 - 26
Coordinated activation of as-1-type elements and a tobacco glutathione S-transferase gene by auxins, salicylic acid, methyl-jasmonate and hydrogen peroxide; Xiang C et al.; The molecular mechanism of signal transduction pathways which mediate the action of phytohormones are poorly understood . Recently, we and others have shown that the as -1 type cis-acting elements can respond to auxin and salicylic acid, two well-characterized signaling molecules in plants . In the present work, we have examined a comprehensive set of physiological and abiotic agents and found that auxin, salicylic acid and methyl-jasmonate are three effective inducers of the as-1-type elements in transgenic tobacco . Using a cell suspension culture containing a synthetic promoter-GUS fusion, we demonstrated rapid and sensitive induction of the as-1-type element by these phytohormones . Furthermore, a tobacco glutathione S-transferase gene, GNT35, that contains an as-1-type binding site in its promoter is also inducible by auxin, salicylic acid and methyl-jasmonate with similar kinetics . As Ulmasov et al . have recently reported, we found that the as-1-type elements can also respond to weak/inactive analogues of auxin and salicylic acid . In addition, we show that hydrogen peroxide can also effectively activate the expression of GNT35 as well as the as-1-type element in a cell suspension culture, but not with whole seedlings . These results are discussed with respect to the possible mechanism(s) through which a single cis element may respond to a diverse array of molecules.

Biol Chem, 1996 Nov, 377(11), 689 - 93
Cytolysis of B-16 melanoma tumor cells mediated by the myeloperoxidase and lactoperoxidase systems; Odajima T et al.; Halide-dependent cytolysis of B-16 melanoma cells mediated by myeloperoxidase and lactoperoxidase systems was observed by turbidimetry . A significant decrease in turbidity, which is indicative of cytolysis, was found when a system consisting of myeloperoxidase, a source of hydrogen peroxide (glucose+glucose oxidase), and chloride or bromide were added to a B-16 melanoma cell suspension in the pH 4.7-6.0 region . The myeloperoxidase could be replaced by lactoperoxidase in the system containing bromide, but not that containing chloride . B-16 melanoma cells exposed to myeloperoxidase or lactoperoxidase systems at pH 5.5 or 7.0 were implanted by subcutaneous inoculation into C57BL/6CrSlc mice . After 14 days, a significant suppression of the growth of black tumors was detected in the groups of mice inoculated with melanoma cells exposed to the systems containing myeloperoxidase, glucose, glucose oxidase and chloride or bromide, or the system containing lactoperoxidase, glucose, glucose oxidase and bromide, at pH 5.5, but no significant suppression was observed at pH 7.0 . From these findings, we concluded that the exposure of B-16 melanoma cells to a system consisting of myeloperoxidase, hydrogen peroxide (generated by the glucose+glucose oxidase system) and chloride or bromide, or of lactoperoxidase, the hydrogen peroxide and bromide, at moderately acidic pH, causes cytolysis is accompanied by cell death.

Acad Radiol, 1996 Nov, 3(11), 929 - 35
Radio-frequency tissue ablation of VX2 tumor nodules in the rabbit lung; Goldberg SN et al.; RATIONALE AND OBJECTIVES: The authors investigated whether small pulmonary malignancies could be treated with computed tomography (CT)-guided, percutaneously placed radio-frequency (RF) electrodes . METHODS: Pulmonary tumors were created in 11 New Zealand white rabbits by using CT-guided injection of a VX2 sarcoma cell suspension into the lower portion of the right lung . Tumors were allowed to grow 14-21 days to achieve a diameter of 6-12 mm . Electrodes were placed coaxially into the tumors via insulated 19-gauge Turner needles . Seven tumors were treated with RF for 6 minutes at 90 degrees C . Four tumors served as controls and were not treated . Follow-up CT and histopathologic analysis were performed on days 0-28 . Specimens from treated rabbits were examined histopathologically on days 0 and 3 (n = 2 each), and days 1, 5, and 28 (n = 1 each) . RESULTS: Immediately following treatment, CT images showed rounded opacities enveloping the tumor . This corresponded histologically to coagulation necrosis of tumor and surrounding alveoli . In all cases, at least 95% of treated tumor nodules were necrotic at histopathologic analysis . Peripheral residual nests of histologically viable tumor were seen in three rabbits (43%) . Control rabbits showed growing tumor nodules without necrosis at autopsy (mean survival, 23 days after inoculation) . Two RF-treated rabbits (29%) and one control rabbit (25%) had pneumothoraces . CONCLUSION: Percutaneous RF tissue ablation can be used to successfully treat small parenchymal tumor nodules within the lung in an animal model.

J Endocrinol, 1996 Nov, 151(2), 301 - 7
The steroidogenic effects of beta-endorphin and joining peptide: a potential role in the modulation of adrenal androgen production; Clarke D et al.; This study examines the androgen-stimulating properties of pro-opiomelanocortin-derived peptides, ACTH, beta-endorphin (beta-End) and joining peptide (JP) . Ten different cell suspensions were prepared from ten human adrenal glands . ACTH and JP stimulated cortisol, androstenedione (delta 4) and dehydroepiandrosterone (DHEA) production (P < 0.05); beta-End stimulated only delta 4 and DHEA production . beta-End brought about significant increases in the delta 4 or DHEA to cortisol ratios . The addition of beta-End (10(-10) M) suppressed ACTH-stimulated cortisol production from 7573 +/- 2960 to 5994 +/- 2654 pmol/10(6) cells (means +/- S.E.M.; P < 0.05) . The addition of beta-End did not affect ACTH-stimulated delta 4 production (210 +/- 88 and 236 +/- 105 pmol/10(6) cells) . JP (10(-10) M) inhibited ACTH-stimulated cortisol production so that the mean values fell to 5186 +/- 2588 and also inhibited DHEA production, from 240 +/- 48 to 180 +/- 33 pmol/10(6) cells . These results suggest that the relative production of androgen to cortisol is greater in response to beta-End and JP than in response to ACTH . If blood levels of these peptides rise to herald adrenarche as reported for beta-End, suppression of cortisol production may result in an increase in ACTH to correct cortisol levels resulting in an increase in delta 4 and DHEA levels . This may explain the occurrence of increasing androgen levels at adrenarche.

Cell Transplant, 1996 Nov-Dec, 5(6), 599 - 611
A comparative study of preparation techniques for improving the viability of striatal grafts using vital stains, in vitro cultures, and in vivo grafts; Fricker RA et al.; Cell suspension grafts from embryonic striatal primordia placed into the adult rat striatum survive well and are able to alleviate a number of behavioral deficits caused by excitotoxic lesions to this structure . However, neither the anatomical connectivity between the graft and host nor the functional recovery elicited by the grafts is completely restored . One way in which the survival and function of embryonic striatal grafts may be enhanced is by the improvement of techniques for the preparation of the cell suspension prior to implantation, an issue that has been addressed only to a limited extent . We have evaluated a number of parameters during the preparation procedure, looking at the effects on cell survival over the first 24 h from preparation using vital dyes and the numbers of surviving neurons in vitro, after 4 days in culture, in addition to graft survival and function in vivo . Factors influencing cell survival include the type of trypsinization procedure and the age of donor tissues used for suspension preparation . The presence of DNase has no effect on cell viability but aids the dissociation of the tissue to form single cells . These results have important implications for the use of embryonic striatal grafts in animal models of Huntington's disease, and in any future clinical application of this research.

Plant Physiol, 1996 Nov, 112(3), 997 - 1004
Lipoxygenase gene expression in the tobacco-Phytophthora parasitica nicotianae interaction; Veronesi C et al.; A recently isolated cDNA clone of tobacco (Nicotiana tabacum L.) lipoxygenase (LOX) was used to study LOX gene expression in tobacco cell-suspension cultures and intact plants in response to infection with Phytophthora parasitica nicotianae (Ppn) . Southern blot analysis of tobacco DNA indicated that only a small number of LOX genes hybridize to this probe . These genes were not constitutively expressed to a detectable level in control cells and healthy plants . In contrast, a rapid and transient accumulation of transcripts occurred in cells and plants after treatment with elicitor and inoculation with zoospores of Ppn, respectively . In cell cultures LOX gene expression could also be induced by linolenic acid, a LOX substrate, and by methyl jasmonate, one of the products derived from the action of LOX on linolenic acid . In the infection assays, LOX gene expression and enzyme activity were observed earlier when the plants carried a resistance gene against the race of Ppn used for inoculation . The differential expression of LOX during the race-cultivar-specific interaction between tobacco and Ppn, as well as its regulation by elicitors and jasmonate, suggest a role of LOX in plant resistance and establishment of the defense status against this pathogen.

Transfusion, 1996 Nov-Dec, 36(11-12), 960 - 5
Time-dependent, spontaneous release of white cell- and platelet-derived bioactive substances from stored human blood; Nielsen HJ et al.; BACKGROUND: The mechanisms of the detrimental effects of perioperative allogeneic blood transfusion are still unclear . Previous studies have suggested a higher incidence of adverse effects after the use of blood stored for prolonged time . Therefore, a possible time-dependent release of various white cell- and platelet-derived bioactive substances in stored human red cell suspensions was studied . STUDY DESIGN AND METHODS: Whole blood (6 units), plasma-reduced whole blood (6 units), and saline-adenine-glucose-mannitol blood (6 units) from 18 unpaid, normal blood donors were stored under standard blood bank conditions at 4 degrees C for 35 days . After refrigeration, samples were collected from all blood bags on Days 0, 2, 5, 9, 14, 21, 28, and 35 of storage . Extracellular concentrations of eosinophil cationic protein, eosinophil protein X, plasminogen activator inhibitor 1, myeloperoxidase, and interleukin 6 were analyzed by enzyme-linked immunosorbent assay and radioimmunoassay . The total intracellular and donor plasma levels of these substances also were analyzed at the time of blood donation . RESULTS: Eosinophil cationic protein, eosinophil protein X, and myeloperoxidase increased 10- to 25-fold (p < 0.05) in a time-dependent manner in whole blood, plasma-reduced whole blood, and saline-adenine-glucose-mannitol blood during storage for 35 days . Plasminogen activator inhibitor 1 increased threefold to sixfold (p < 0.05) in whole blood and plasma-reduced whole blood, but not in saline-adenine-glucose-mannitol blood . Interleukin 6 was not detected in either plasma or samples obtained from the blood bags . CONCLUSION: Stored whole blood, plasma-reduced whole blood, and saline-adenine-glucose-mannitol blood may release white cell- and platelet-derived bioactive substances in a time-dependent manner, which may be related to the detrimental effects of perioperative blood transfusions . Therefore, prestorage white cell reduction should be considered for further improvement of red cell suspensions.

No Shinkei Geka, 1996 Nov, 24(11), 987 - 93
Unilateral fetal mesencephalic grafting in two patients with Parkinson's disease: short-term result after transplantation; Chung CK et al.; Fundamental pathological and neurochemical changes in Parkinson's disease are loss of midbrain dopamine neurons that innervate the caudate and putamen . In an effort to replenish the striatal dopaminergic innervation, fetal mesencephalic tissue containing dopamine cells was implanted into the unilateral putamen in two patients with severe Parkinson's disease . The tissue was obtained from three fetuses with gestational ages of 7 to 9 weeks . The cell suspension was stereotactically injected into the unilateral putamen using 5 needle trajectories . Postoperative immune suppression was not performed . Clinical improvement appeared after 2 months . Both patients showed improvement according to the Activities of Daily Living Scale during the off and practically-defined off state 9 and 14 months after surgery . The motor scores of the Unified Parkinson's Disease Rating Scale improved during the off and practically-defined off state 9 and 14 months after surgery . Dyskinesia and off state were shorter and less severe than before the transplantation . Although the long-term effects need to be ascertained, our short-term observation in these two patients with unilateral transplantation is encouraging and justifies further research trials in selected patients.

J Histochem Cytochem, 1996 Nov, 44(11), 1337 - 43
Reconstructed three-dimensional images of flow-sorted nuclei hybridized with chromosome-specific probes; Matsuta M et al.; We demonstrated that the three-dimensional (3-D) locational and morphological differences of chromosome 17 are dependent on each cell cycle phase in the clinical materials . Cell suspensions prepared from hypertrophied tonsil were hybridized with chromosome 17 whole painting probe or its centromeric probe and the probes were detected with fluorescein isothiocyanate . Then the cells were sorted from G(0+1), S-, and G(2+M)-phase fractions by flow cytometry and observed by confocal laser scanning microscopy to obtain the serial optical sections . The 3-D images were obtained by assembling these sections using a computerized image analysis device . The distribution of centromeric copies was analyzed statistically, and the data values were not a population of random distribution within a sphere . The copies were observed in the periphery of the nuclei in G(0+1)- and S-phase . The 3-D images revealed that chromosome 17 was oval in shape in the G(0+1)-phase nucleus, and was changing into a flame shape in the S-phase, with arms stretching out along the nuclear membrane, and looked bush shaped in G2-phase . The eccentric distribution of chromosome 17 in G(0+1)- and S-phase nuclei may reflect the optimal efficiency of incorporating and/or releasing essential materials and products.

Int Arch Allergy Immunol, 1996 Nov, 111(3), 230 - 7
Characterization of the spontaneous apoptosis of rat thymocytes in vitro; Rinner I et al.; Unlike splenic or blood lymphocytes, rat thymocytes spontaneously undergo continuously increasing apoptosis during culture . In this study we characterized apoptotic thymus cells of rats according to cell size, nuclear dye binding and surface marker expression . Furthermore, the effects of cell density in culture, the age of the donor animals, glucocorticoids, and inhibition of protein synthesis were studied . It was found that: (1) apoptotic rat thymocytes are recognized in flow cytometry as small, acridine orange low, CD4low, CD8high cells; (2) the rate of apoptosis is dependent on the cell density in the culture in a biphasic manner; (3) thymic apoptosis increases with age of the donor animal in fresh, as well as in 24-hour cultivated cell suspensions; (4) neither adrenalectomy nor in vivo or in vitro treatment with the glucocorticoid antagonist RU 38486 influenced spontaneous apoptosis of thymocytes, and (5) inhibition of protein synthesis, which decreases apoptosis induced by corticosterone, had no effect on spontaneous apoptosis of thymocytes.

Ann Surg Oncol, 1996 Nov, 3(6), 580 - 7
Improved in vivo efficacy of tumor-infiltrating lymphocytes after restimulation with irradiated tumor cells in vitro; Burger UL et al.; BACKGROUND: We investigated different culture conditions for tumor-infiltrating lymphocytes (TILs) with regard to proliferation, phenotypic changes, in vitro cytotoxicity, and in vivo therapeutic efficacy . METHODS: After enzymatic digestion of the murine fibrosarcoma, MCA-105, TIL cultures were initiated as pure lymphocyte (groups 1 and 2) or mixed lymphocyte/tumor suspensions (groups 3 and 4) . Group I TILs were grown in culture medium containing 100 IU/ml recombinant interleukin-2 (rIL-2) . Group 2 TILs were stimulated with solid-phase anti-CD3 monoclonal antibody (mAb) for 48 h and cultured in rIL-2 (100 IU/ml)-containing medium . Group 3, which consisted initially of a surplus of tumor cells, received the same treatment as group 2 . Group 4 was also activated with anti-CD3 mAb and rIL-2 but was additionally restimulated weekly with irradiated tumor cells (TILs to tumor, 20:1) . RESULTS: Groups 1 and 2 showed up to twofold higher increases in TIL numbers compared with groups 3 and 4 by the end of culture week 5 . Although the original lymphocyte/tumor cell suspension consisted of 12.0 +/- 3.8% CD4+ T cells and 5.3 +/- 3.3% CD8+ T cells, all four TIL cultures showed approximately 80% CD8+ TILs and no CD4+ TILs by the end of culture week 4 . In vitro cytotoxicity did not correlate with in vivo efficacy of the examined TIL cultures . By using the MCA-105 pulmonary metastases model in C57BL/6 mice, only suboptimal doses of TILs (2 x 10(6)) from group 4, which had been restimulated weekly with irradiated tumor, showed significant tumor eradication compared with all other treatment groups (p < 0.01) . CONCLUSIONS: We conclude that in vitro tumor restimulation of TILs improves in vivo efficacy, most likely through the education of tumor-reactive T cells.

Exp Neurol, 1996 Nov, 142(1), 36 - 46
Contributions of donor and host blood vessels in CNS allografts; Baker-Cairns BJ et al.; The contributions of blood vessels in various transplantation paradigms of solid CNS tissue or cell suspension allografts placed into adult host brains were investigated immunohistochemically using the PVG-RT1C and PVG-RT1U inbred rat strains and a panel of highly specific monoclonal antibodies . The monoclonal antibodies included OX-27 and U9F4 against major histocompatibility complex (MHC) class I antigens of the PVG-RT1C and PVG-RT1U rats, respectively; OX-26 against the rat transferrin receptor located on blood-brain barrier (BBB) endothelia; and OX-7 against rat neuronal Thy 1.1 for evaluating graft survival . Our study is the first to address the immunogenicity of blood vessels in surviving CNS allografts . Solid fetal or neonatal PVG-RT1C cortex was grafted into the third or lateral cerebral ventricle or caudate/putamen of PVG-RT1U adult hosts for 30 days to 7 months . All allografts expressed demonstrable Thy 1.1 immunoreactivity with OX-7 antibody and appeared well-vascularized with blood vessels that immunostained with the OX-26 antibody against the transferrin receptor . For the most part, the allografts were supplied sparsely with donor (PVG-RT1C) MHC class I-positive (OX-27) blood vessels clustered in pockets . Donor MHC class I-positive vessels entered the host brain only from allografts in the third ventricle; these vessels were restricted to the host median eminence and no longer immunostained with OX-26 for the transferrin receptor (normally the median eminence is supplied with non-BBB vessels that do not possess the transferrin receptor and do not stain with OX-26) . In host brains harboring a third ventricle allograft, host MHC class I-positive vessels immunostained with the U9F4 antibody were evident throughout the host CNS, including the median eminence, and throughout the allografts excluding sites inhabited by donor PVG-RT1C vessels . Cell suspension neural allografts (donor PVG-RT1C) placed within the brain parenchyma of PVG-RT1U hosts revealed no significant differences in vascular contributions between donor and host when compared to results obtained from solid CNS allografts . A unique immunohistochemical approach of introducing ascites fluid OX-27 as the primary antibody intravenously to the PVG-RT1U host demonstrated that in donor PVG-RT1C posterior pituitary allografts, donor and not host vessels predominate and are restricted to the graft . Finally, blood vessels isolated from adult PVG-RT1C brains were mixed with solid fetal PVG-RT1U cortical tissue and grafted into the brain parenchyma of adult PVG-RT1U hosts . Immunostaining with OX-27 antibody against MHC class I of the PVG-RT1C rat strain disclosed that the PVG-RT1C blood vessels survived and were confined to the PVG-RT1U syngeneic graft . The results suggest that blood vessels supplying CNS allografts placed within the host brain are predominantly of host origin; surviving donor vessels are restricted to the allograft with rare exceptions, which may be dictated by the type of neural allograft and the host CNS site receiving the allograft . The survival of isolated allogeneic CNS blood vessels grafted into the host brain suggests that such blood vessels can present an endothelial genotype and phenotype different from those of host vessels indigenous to the CNS site receiving the allogeneic vessel graft . This finding may have implications in the circumvention of the blood-brain fluid barriers for the CNS delivery of blood-borne therapeutics.

Brain Res Dev Brain Res, 1996 Oct 23, 96(1-2), 11 - 27
Lineage specification of olfactory neural precursor cells depends on continuous cell interactions; Magrassi L et al.; We transplanted, as a single cell suspension, cells dissociated from the mature and immature olfactory epithelium of rats or TgR(ROSA26)26Sor mice expressing constitutively the LacZ gene into the developing brain (cerebellum, striatum, inferior colliculus, lateral ventricles) of E15 rat fetuses . Grafted cells or their descendants were still present in the central nervous system more than a month after transplantation . Transplanted cells either integrated as isolated cells or, during the first day after transplantation, reaggregated into clusters . Scattered cells, despite their placodal origin, differentiated into neuron or glial cells with a central phenotype . This was demonstrated by anatomical methods and selective amplification of cDNA encoding for neuronal specific transcripts (microtubule-associated protein 2 and middle-molecular-mass neurofilament protein) expressed by the engrafted cells . Cells in large clusters generated an epithelium containing mature olfactory neurons . Some of them were immunoreactive for the olfactory marker protein . Our findings show that cells dissociated from the developing and adult olfactory organs when transplanted into the rat fetal brain can either completely change their fate and differentiate according to their final position or generate an olfactory epithelium if they reaggregate into large clusters.

Ugeskr Laeger, 1996 Oct 21, 158(43), 6098 - 102
{10 years' experiences with flow cytometric immunological phenotyping in malignant hematologic diseases}; Kristensen JS et al.; Immunological phenotyping of mononuclear cell suspensions from bone marrow and/or peripheral blood from 2341 patients (6140 examinations) suspected of malignant haematological diseases was performed during a 10-year period . The cells were labelled with a panel of monoclonal antibodies and subjected to flowcytometry . The results showed a clear distinction between acute lymphoblastic leukaemia and acute myeloid leukaemia, and a new group of acute hybrid leukaemia (3.3% of all acute leukaemia cases) was established . During the 10-year period immunological phenotyping has changed from being a research method to a widely employed method for cell characterisation early in the course of malignant haematological diseases.

J Immunol Methods, 1996 Oct 16, 197(1-2), 85 - 95
The ABL-MYC retrovirus generates antigen-specific plasmacytomas by in vitro infection of activated B lymphocytes from spleen and other murine lymphoid organs; Largaespada DA et al.; ABL-MYC is a recombinant retrovirus that constitutively expresses the v-abl and c-myc oncogenes . When used to infect immunized mice this virus rapidly and efficiently induces plasmacytomas of which an unusually high percentage secrete antigen (Ag)-specific monoclonal antibodies . These findings suggested that ABL-MYC targets Ag-stimulated B cells for transformation and that infection of lymphoid cells in vitro might be a useful, alternative method for generating monoclonal, Ag-specific plasmacytomas (ASPCTs) . Therefore, we used helper virus-free ABL-MYC to infect suspensions of cells from spleens and other lymphoid organs from mice that had been immunized with a variety of Ags and transplanted them into naive mice . The results show that ABL-MYC preferentially transforms splenocytes that are Ag-reactive . They also demonstrate that ASPCTs can be produced by in vitro infection of cell suspensions from the spleen, lymph nodes and Peyer's patches of mice that had been immunized intraperitoneally with sheep red blood cells, Escherichia coli core RNA polymerase or Epstein-Barr virus gp340 protein or immunized orally with live Giardia lamblia parasites . The ASPCTs usually consisted of one to three colnes, secreted antibodies that were quantitatively and qualitatively similar to those obtained from hybridomas, and could continue to secrete Ag-reactive antibody over eight transplant generations.

J Immunol Methods, 1996 Oct 16, 197(1-2), 51 - 67
Quantitation of the density of cell surface carbohydrate antigens on cancer cells with a sensitive cell-suspension ELISA; Ravindranath MH et al.; The density of carbohydrate epitopes on the surface of tumor cells is a governing factor for immune recognition and antibody-mediated targeting of tumor-associated carbohydrate antigens in cancer immunotherapy . A sensitive cell-suspension ELISA (cs-ELISA) is developed for quantitation of the functionally exposed carbohydrate epitopes on the cell surface . The factors affecting the measurement of tumor-cell surface glycoconjugates are evaluated using three human melanoma cell lines before and after exposure to various cell preservation treatments . The results of cs-ELISA are compared with the quantitative profile obtained by biochemical and flow cytometry assays . Cs-ELISA measures the density of the functionally exposed specific sugar epitopes on the surface of tumor cells, even in the presence of other similar carbohydrate antigens, provided that the monoclonal antibodies to carbohydrate epitopes are monospecific and sensitive, and that the cells are viable and present in optimal density . Of the three melanoma cell lines, M10-v and M101 expressed disialolactosyl residues of GD3 at concentrations of 5-6 pmol/10(6) cells and 2-3 pmol/10(6) cells, respectively . In both cell lines, the cell-surface GD2 was less than 1.0 pmol/10(6) cells . M24 melanoma cells expressed trace quantities (< 0.1 pmol/10(6) cells) of GD3 and GD2 . Trypsinization of M10-v and M101 cells significantly reduced the cell-surface expression of GD3, suggesting GD3 loss, but increased the expression of GD2, suggesting crypticity of membrane-bound GD2 . Cs-ELISA results showed that cryopreservation with 10% DMSO and irradiation at 15 krad decreased melanoma cell viability and ganglioside expression for M10-v but not M101 and M24 . Formalinization did not affect cs-ELISA measurement of cell-surface carbohydrates . Cs-ELISA was used to monitor the quantity of incorporation of exogenous GD3 onto the surface of GD3-deficient M24 cells . Cs-ELISA for assessment of density of cell surface carbohydrate epitopes may be useful to characterize different types of tumors, to develop carbohydrate-based whole cell vaccines from tumor biopsies, to monitor the effects of cell preservation treatments commonly used in a whole cell vaccine preparation, and to evaluate the incorporation of a particular glycolipid (antigen or adjuvant) into glycolipid-deficient cells that are useful for carbohydrate-based active specific immunotherapy.

J Biochem Biophys Methods, 1996 Oct 15, 33(1), 9 - 23
Determination of relative amounts of ribosome and subunits in Escherichia coli using asymmetrical flow field-flow fractionation; Nilsson M et al.; The 30S and 50S subunits and the 70S ribosome of Escherichia coli were separated in 6 minutes by using asymmetrical flow field-flow fractionation (FFF) . The total analysis time for determination of the relative amounts of ribosomes and free subunits in a preparation from a cell suspension was 8 min . The method can detect a change in the mass fraction of ribosomes if it exceeds approx, 10% . The separation is based on differences in diffusion coefficients, i.e., hydrodynamic diameters, and these can be determined from observed retention times . The hydrodynamic diameters were in good agreement with literature values obtained from electron microscopy . The mass fraction of ribosomes changed as a function of the magnesium ion concentration which confirms previous knowledge and shows the accuracy of the method . The method appears as an alternative to ultracentrifugation analysis and avoids some of its drawbacks and artefacts . An obvious application can be the optimisation of cell design in metabolic engineering in order to maximise translation and protein production.

Virology, 1996 Oct 15, 224(2), 564 - 7
Efficient translation of distal cistrons of a polycistronic mRNA of a plant pararetrovirus requires a compatible interaction between the mRNA 3' end and the proteinaceous trans-activator; Edskes HK et al.; Caulimoviruses, a type of plant pararetrovirus, employ a highly unusual mechanism to express the multiple cistrons of their pregenomic RNA . It involves translation of a polycistronic mRNA utilizing cis-acting viral RNA sequences and a transacting virus-encoded protein (P6) . In addition to its role in polycistronic translation, the translational trans-activator protein P6 also activates its own expression from a monocistronic subgenomic RNA . Using Nicotiana Edwardsonii cell suspension protoplasts, we analyzed the ability of P6 proteins from three different caulimoviruses to activate viral RNA-based reporter constructs . Cis-acting elements present in figwort mosaic caulimovirus (FMV) are functional not only in the presence of the cognate P6 activator protein, but also in the presence of the heterologous activators from cauliflower mosaic caulimovirus (CaMV) and peanut chlorotic streak caulimovirus (PCISV) . However, when 3' cis-acting elements essential for efficient polycistronic expression of FMV are replaced by their counterparts from PCISV, reporter gene expression is only observed in the presence of PCISV P6 . Derepression of monocistronic reporter constructs tailed with FMV or CaMV 3' proximal sequences is less efficient in the presence of PCISV P6 than with either FMV or CaMV P6, but more efficient when the constructs contain a cognate PCISV 3' cis-element . Efficient expression of polycistronic and monocistronic caulimovirus mRNAs in plant cells thus requires compatible interactions between P6, a translational trans-activator, and its cognate cis-element at the 3' end of the mRNA.

Biochem Biophys Res Commun, 1996 Oct 14, 227(2), 589 - 93
4-hydroxynonenal specifically inhibits c-myb but does not affect c-fos expressions in HL-60 cells; Barrera G et al.; 4-Hydroxynonenal, an aldehyde produced from lipid peroxidation of cellular membranes, inhibits growth and induces differentiation of HL-60 human leukemic cell line . Since it is highly unstable in the culture medium, its effectiveness is increased when added repeatedly to the cell suspension . We have previously demonstrated that HNE inhibits c-myc but not N-ras expression in HL-60 cells . Here we investigate its effect on the expression of c-myb and c-fos, two early genes involved in the induction of myeloid and monocytic differentiation . Moreover, since c-fos is directly correlated with the intracellular level of cAMP, we also analysed the cAMP concentration after aldehyde treatment . HNE significantly inhibits c-myb expression during and after repeated treatments . A single administration of 1 microM HNE decreases c-myb mRNA at 1 hour whereas 10 microM HNE inhibits c-myb expression from 3 to 6 hours after treatment, and then the expression returns to the control level . By contrast, c-fos expression and intracellular cAMP concentration do not show any significant change after HNE treatments.

Exp Cell Res, 1996 Oct 10, 228(1), 50 - 7
Establishment of the permanent microvascular endothelial cell line PBMEC/C1-2 from porcine brains; Teifel M et al.; Porcine brain microvascular endothelial cells (PBMEC) were isolated from fresh brains by several enzymatic digestion steps followed by a gradient centrifugation . Cells of the primary culture were transfected with pRNS-1, encoding for the small and large T-antigens of SV 40, by means of lipofection . After selection with G-418, clones were isolated and one clone, PBMEC/C1-2, was further characterized by microscopic examination of morphology, immunofluorescence, and lectin binding . PBMEC/C1-2 was cultivated for nearly 1 year with 250 cumulative population doublings and showed the typical morphology of capillary endothelial cells in vitro . Postconfluent cultures showed the formation of a tubular network as a second layer, which is characteristic of capillary endothelial cells . SV 40 T-antigens were present in the nuclei and the cells showed the typical granular staining of von Willebrand factor (vWF) and were positive for Bandeiraea simplicifolia isolectin B4 . Furthermore, PBMEC/C1-2 exhibited the uptake of acetylated LDL, expression of nonmuscular- and smooth-muscle-type myosins, and the presence of the blood-brain barrier-associated markers gamma-glutamyltranspeptidase (gamma-GT), the glucose transporter Glut-1, and apolipoprotein A-1 . In addition, enzymatic activity of gamma-GT and alkaline phosphatase could be detected in cell suspensions . In summary, PBMEC/C1-2 represents an immortalized PBMEC line exhibiting endothelial characteristics as well as typical markers of the blood-brain barrier.

J Obstet Gynaecol Res, 1996 Oct, 22(5), 481 - 8
A method of measuring dipalmitoylphosphatidylcholine (DPPC) by high-performance liquid chromatography (HPLC): effect of dexamethasone on DPPC secretion in fetal rat alveolar type II cell culture; Suzuki R et al.; OBJECTIVE: To establish a method by which to measure dipalmitoylphosphatidylcholine (DPPC), which is the most prevalent phospholipid in lung surfactant, by high-performance liquid chromatography (HPLC), and to measure DPPC secretion in type-II alveolar cell culture to determine whether glucocorticoid has a direct accerelating effect . METHOD: Type-II alveolar cell suspensions were made with fetal rat lungs and then cultured . Dexamethasone, (10(-9), 10(-8) M) was added to some of the culture dishes . DPPC was extracted from the culture medium, purified, and measured by high-performance liquid chromatography (HPLC) . RESULTS: In the measurements of DPPC, the specificity of isolation and measurement parity were excellent {coefficient of variation: intraassay; 5.66% (0.005 mg/ml), 25.37% (0.05 mg/ml): interassay; 8.35% (0.005 mg/ml), 0.08% (0.05 mg/ml)} . The DPPC concentration in the culture dishes of the dexamethasone was not significantly different than that of the control dishes (Student's t-test) . CONCLUSION: The above results prove that dexamethasone does not directly stimulate lung alveolar type-II cells.

Exp Dermatol, 1996 Oct, 5(5), 272 - 8
Expression, but lack of calcium mobilization by high-affinity IgE Fc epsilon receptor I on human epidermal and dermal Langerhans cells; Shibaki A et al.; In atopic dermatitis (AD) patients, IgE molecules are demonstrated on the surface of Langerhans cells (LC) . Fc epsilon RI molecules, which are present on the surface of LC in AD patients as well as normal individuals, are responsible for this binding . In this study, we have investigated phenotypic and functional characteristics of Fc epsilon RI on epidermal and dermal cell populations . Epidermal and dermal cell suspensions were prepared enzymatically with dispase followed by either trypsin or collagenase treatment, respectively . Peripheral blood basophils were negatively selected by excluding other leukocytes with surface marker staining . Consistent with previous reports, both peripheral blood basophils and epidermal LC were positively stained with anti Fc epsilon RI monoclonal antibody . In addition, an Fc epsilon RI positive population was demonstrated among dermal HLA-DR positive cells . These cells express significant amounts of HLA-DR molecules (DRHi) and co-express CD 1 a molecules, which identifies them as LC-like dendritic APC of the dermis . No other Fc epsilon RI positive population was found in the other dermal DRMid or DR- populations, except for a minor DRLo population, presumably mast cells . To analyze whether these Fc epsilon RI molecules are signal transducing for LC, intracellular calcium mobilization after crosslinking of Fc epsilon RI was measured with flow cytometry . Following crosslinking, peripheral blood basophils clearly increased intracellular calcium . On the other hand, neither normal epidermal LC nor dermal DRHiCD1a + cells changed their intracellular calcium level after Fc epsilon RI crosslinking . These data indicate that normal epidermal and dermal LC, but not basophils, are resistant to calcium flux following Fc epsilon RI engagement.

J Pathol, 1996 Oct, 180(2), 214 - 22
Three-dimensional confocal laser scanning DNA ploidy cytometry in thick histological sections; Tekola P et al.; DNA ploidy measurement by flow (FCM) or image cytometry (ICM) of single cell suspensions of solid tumour has prognostic value, but it would be a definite advantage if the assessment could be done on histological sections . However, this is usually not possible by means of standard ICM, due to the capping of nuclei in thin sections, or overlap in thick sections . Three-dimensional (3D) microscopy by means of confocal laser scanning microscopy (CLSM) could solve this problem in theory but the results published so far are not very satisfactory . A new method has been developed in which the DNA content of haploid (human testis spermatozoa), diploid, tetraploid, octaploid (human and rat liver and human spermatogonia), and near-triploid (human breast cancer) nuclei stained with YOYO-1 iodide has been measured by a newly developed 3D image cytometry method (3DICM) in 20 microns thick histological sections . YOYO-1 iodide is a new highly sensitive, specific, stoichiometric, and stable fluorescent dye for DNA . DNA ploidy of a breast cancer which was near-triploid with FCM and ICM was also assessed with 3DICM in a tissue section adjacent to the section used for FCM and ICM and the results were compared . The integrated 3DICM fluorescence intensity showed good linearity (r = 0.99) with the real DNA content of all nuclei analysed . In human tissue, the coefficient of variation of 3DICM for haploid (n = 12), diploid (n = 63), triploid (n = 13), tetraploid (n = 12), and octaploid (n = 3) ploidy distributions was 5.1, 6.6, 4.2, 4.0, and 0.6 per cent, respectively (n = the number of nuclei) . For the rat liver, the CV of the diploid (n = 21), tetraploid (n = 31), and octaploid (n = 3) peaks was 6.7, 4.8, and 1.6 per cent, respectively . Repeated "blind' measurements of nuclei with different DNA indices showed excellent reproducibility between different observers (r = 0.98) . It is concluded that the 3DICM method used is accurate, reproducible, and clinically feasible in thick histological sections . This is especially important in small lesions, or if the results of DNA ploidy measurement of single cell suspensions (by FCM) or imprints (by ICM) are inadequate.

Crit Rev Clin Lab Sci, 1996 Oct, 33(5), 423 - 55
Lung lymphocytes: origin, biological functions, and laboratory techniques for their study in immune-mediated pulmonary disorders; Semenzato G et al.; Different types of immunocompetent cells, including T lymphocytes and alveolar macrophages, account for pulmonary host defense . Taking advantage of the availability of the monoclonal antibody technique, cell culture facilities, pure recombinant cytokines, and molecular probes for their genes, in the last few years it has been possible to keenly study the different steps that lead to the compartmentalization of immune response in human lung . Furthermore, the immunological analysis of cells retrieved from bronchoalveolar lavage (BAL) allowed recognition of the importance of immune mechanisms in the evolution of immune-mediated pulmonary disorders . The purpose of this review is to summarize recent advances on the immunologic characterization of lung lymphocytes in health and disease . Following a brief description of the pathways through which the pulmonary lymphoid system contributes to removing potentially harmful inhaled antigenic materials, available laboratory techniques to evaluate the lymphoid component of the pulmonary immune system and their byproducts are discussed . These techniques cover methods for preparing lymphocytes from the BAL fluid and for characterizing lung lymphocytes both in cell suspensions and pulmonary tissue biopsies . Other sections of this review describe the techniques for measuring the immunologic effector functions of lung lymphocytes . We also provide the reader with a flavor of the molecular biology methods used to characterize lymphocytes in the pulmonary microenvironment . The final sections of the review article highlight the pathogenetic role envisaged for lymphoid cells in pulmonary disease states and emphasize the importance of the BAL analysis in the clinical management of the most relevant immune-mediated lung disease.

Glia, 1996 Oct, 18(2), 79 - 91
Immunocytochemical and biochemical characterization of glial phenotypes in normal and immortalized cultures derived from 3-day-old chick embryo encephalon; Kentroti S et al.; We examined properties of glia derived from very early neurogenesis in the chick embryo, as well as their behavior in response to extended cell passages and at various periods in culture . Primary cultures derived from the telencephalic region of 3-day-old chick embryo (stage 19) exhibited intense staining for vimentin (Vim; indicative of immature glial phenotypes) throughout the 17-day culture period, transitioning to Vim-positive/glial fibrillary acidic protein (GFAP)-positive astroblasts after a single cell passage at 13 days in vitro (DIV) . With subsequent passage (P; through P6), cell continued to coexpress Vim/GFAP with the occasional appearance of fibronectin (Fib) . By P11, Vim staining was faint, whereas GFAP staining gained in intensity, indicating the presence of mature astrocytes . These cultures also featured significant activity of glutamine synthetase (GS), which increased slightly with both cell passage and days in culture, correlating well with immunocytochemical findings . The activity of 2'3'-cyclic nucleotide 3'-phosphohydrolase (CNP) remained low, indicating a low percentage of mature oligodendrocytes . Thus, embryonic day (E)3H glia cultures mature into astrocytes given sufficient time in culture . To obtain a stable population of glia at various stages of astrocytic differentiation, we immortalized and subcloned homogenous colonies of E3H glia precursors at specific stages of development . A cell suspension was electroporetically transfected with the pSV40neo gene for large T antigen (E3H.SV5) . After 5 passages, the parent colonies consisted of a heterogeneous population of cells, most of which were vim and GFAP positive . Following subcloning, one line (colony 7) displayed the staining pattern of mature astrocytes . However, with advancing age in culture, staining for both vim and GFAP became increasingly faint . Compared with control (normal) cultures, transfected cells exhibited a significantly lower activity of GS that did not fluctuate with days in culture but decreased with advancing cell passage . Furthermore, CNP activity in E3H.SV5 colony 7 was approximately double that observed in normal cultures at the same cell passage . This observation suggests that the phenotype of transfected glia was less stable than that observed in normal glial cultures.

J Nat Prod, 1996 Oct, 59(10), 944 - 51
Cloning and heterologous expression of a second (+)-delta-cadinene synthase from Gossypium arboreum; Chen XY et al.; Screening of a Gossypium arboreum L . cv . Nanking cDNA library resulted in the identification and cloning of a second (+)-delta-cadinene synthase . A probe for the screens was prepared by PCR using primers based on conserved sequences in farnesyl diphosphate cyclases and genomic DNA as a template . This second cDNA clone encodes a protein that is 80% identical to the recently described (+)-delta-cadinene synthases CAD1-C1 and C14 from G . arboreum and maintains a significant degree of homology to the other known mono-, sesqui-, and diterpene synthases . As in the case of CAD1-C1 (+)-delta-cadinene synthase from cultured cotton cells, the synthesis of the second CAD1-A mRNA was induced by treatment of cotton cell suspension cultures with a partially purified elicitor preparation from the phytopathogenic fungus Verticillium dahliae . Expression of CAD1-A mRNA was quantitated with reverse transcription PCR and showed that CAD1-A mRNA was maximally increased 8-fold, 6 h after addition of elicitor . Heterologous expression of this second cDNA produced a 64 kD protein that catalyzed the cyclization of farnesyl diphosphate to (+)-delta-cadinene, the identical product produced by CAD1-C1 . The steady-state kinetic parameters of CAD1-A were similar to CAD1-C, showing a Km of 7 mM farnesyl diphosphate and kcat of 0.039 s-1 at 30 degrees C . However, the optimal pH and Mg2+ concentration for CAD1-A activity were significantly higher than those observed for CAD1-C.

Cytometry, 1996 Oct 1, 25(2), 200 - 4
Coaxial flow mixer for real-time monitoring of cellular responses in flow injection cytometry; Blankenstein G et al.; Improved time resolution of kinetic cellular events in flow cytometry is demonstrated by using a coaxial flow-mixing device integrated within a flow-injection (FI) system . The instrument is used in combination with a Becton Dickinson FACS Analyzer for on-line reagent addition, rapid sample mixing, and temperature control of cell suspensions . The coaxial flow device can instantaneously (< 60 ms) mix reagent and sample streams, allowing cytometric analysis of subsecond events to be performed . Kinetic measurements can be performed on the FACS analyzer in a variable time range of from 100 ms to 3 min . The system also allows the collection of unlimited cellular events at a specific incubation time point . Because the system operates continuously and no boost in core flow is required, disturbances of flow conditions are avoided . The capabilities of the flow injection cytometer have been demonstrated by the determination of internal {Ca2+}i mobilization in Jurkat T lymphocytes perfused internally with INDO-1 and stimulated by ionomycin.

Plant Physiol, 1996 Oct, 112(2), 705 - 15
Novel molecular markers for late phases of the growth cycle of Arabidopsis thaliana cell-suspension cultures are expressed during organ senescence; Callard D et al.; In an Arabidopsis thaliana T87-C3 cell-suspension culture, entry into the growth-arrest phase is rapidly followed by a loss of cell viability . Three cDNA clones, SRG1, SRG2, and SRG3, corresponding to genes with transcripts that accumulate during these late phases, were isolated by the mRNA differential display method . Amino acid sequence analysis shows that the putative SRG1 protein is a new member of the Fe(II)/ascorbate oxidase superfamily, and that SRG2 codes for a protein with significant homology to beta-glucosidases . Significantly, all three SRG genes are expressed in senescing organs of Arbidopsis plants . Two previously characterized genes, SAG2 and SAG4, induced during natural senescence in Arabidopsis, were also found to be expressed in cell-suspension cultures and have expression kinetics similar to those observed for the SRG1 gene . Taken together these finding suggest that certain molecular events are common to both plant senescence and growth arrest in arabidopsis cell suspensions . Both internucleosomal cleavage of nDNA and an apparent compaction of chromatin, two characteristic features of programmed cell death in animal cells, have been observed in Arabidopsis cell cultures at a stage corresponding to loss of cell viability.

Scand J Immunol, 1996 Oct, 44(4), 394 - 400
A study of natural cytotoxic (NC) activity in the metrial glands of rats and mice; Stewart IJ et al.; Cell suspensions prepared from the metrial glands of rats killed on days 10, 13 and 20 of pregnancy and mice killed on day 12 of pregnancy were assessed for their ability to kill natural cytotoxic cell target Wehi 164 cells in a 51chromium-release cytotoxicity assay . High cytotoxicity indices were found with metrial gland cell suspensions from rats killed on days 10 and 13 of pregnancy and from mice killed on day 12 of pregnancy . Cell suspensions from rats killed on day 20 of pregnancy, when there are relatively few of the granulated metrial gland cells which characterize the metrial gland, had little natural cytotoxic cell cytotoxic activity . Direct observation of mouse granulated metrial gland cells killing co-cultured Wehi 164 cells were made using time-lapse video recordings . The results indicate that granulated metrial gland cells are a type of natural cytotoxic cell.

Dis Colon Rectum, 1996 Oct, 39(10 Suppl), S7 - 13
Trocar site recurrence is unlikely to result from aerosolization of tumor cells; Whelan RL et al.; PURPOSE: This study was undertaken to investigate the ability of a high-pressure CO2 environment to aerosolize tumor cells in both in vitro and in vivo models . (An aerosol is defined as a stable gaseous suspension of insoluble particles) . Also, this study was designed to determine if rapid desufflation is capable of transporting fluid laden with tumor cells . METHODS: The four in vitro aerosol experiments were performed in an 18.9-1 plastic vessel fitted with two 7-mm ports and a compliant latex balloon affixed to the top . After CO2 insufflation, the vessel was desufflated through a sterile soluset containing 25 ml of culture media that was subsequently emptied into a culture dish, incubated for two weeks, and periodically assessed for growth . At the bottom of the vessel, one of the following was placed: Study 1 and 2, a suspension of B16 melanoma or colon 26 tumor cells in liquid culture media; Study 3, colon 26 cells in saline solution; Study 4, several pieces of solid colon 26 tumor . In Studies 1 to 3, cell preparations were subjected to the following high-pressure CO2 conditions (pneumo): 1) static pneumo of 15 and 30 mmHg (10 minute dwell); 2) a continuous flow (CF) of CO2 (1O l) while maintaining a pressure of 15 or 30 mmHg in the vessel . In Study 4, only the 30 mmHg static and CF conditions were tested . Between 6 and 12 determinations were performed for each condition and cell preparation . In vivo aerosol experiments consisted of Spraque Dawley rats that received intraperitoneal injections of 10-5 B16 cells in 0.1 ml of liquid media . Two laparoscopic ports were placed in the abdomen, one each for insufflation and desufflation . Study groups were: 1, static CO2 pneumo of 15 mmHg; 2 and 3, continuous CO2 flow (10 l) at a stable pneumo pressure of 5 and 10 mmHg . Desufflation was performed via the same collecting device and handled in an identical manner to the in vitro experiments described above . The in vitro balloon experiment was designed to investigate the ability of desufflation to transport fluid-containing tumor cells; latex balloon model was used . To prevent complete loss of volume on desufflation, a wire coil was placed inside the balloon . Twenty ml of media containing 20 x 10(-6) B16 cells was placed in the bottom of the balloon . The balloon was insufflated with 1 to 2 l of gas . There were three study groups that differed in the degree to which the cell suspension was agitated before desufflation . Study conditions were as follows: 1) no agitation; 2) moderate agitation to coat the lower walls and coil; 3) maximum agitation to coat the entire balloon . To verify the viability of tumor cells, at the end of each in vitro and in vivo study, a sample of tumor cells or peritoneal washing was incubated in sterile media . These samples served as positive controls . RESULTS: In vitro aerosol studies consisted of the following . At the end of two weeks of incubation, no tumor growth was noted in any of the 124 test dishes . The 14 control samples all demonstrated tumor growth . In vivo aerosol studies consisted of the following . Zero of 18 experimental dishes grew tumor . All three peritoneal washing samples demonstrated growth . In vitro balloon studies consisted of the following . Zero of 12 test dishes in Groups 1 and 2 demonstrated growth, whereas five of six dishes did so in Group 3 (maximally agitated before desufflation) . Again, positive controls all grew tumor cells . SUMMARY: We were unable to demonstrate aerosol formation in any of the in vitro and in vivo studies performed . In the balloon experiment, desufflation-related transport of tumor cells was demonstrated but only when the entire balloon surface was coated with the tumor cell suspension before desufflation . CONCLUSION: Aerosols of tumor cells are not likely to form . Free intraperitoneal tumor cells are most likely found in liquid suspension . Desufflation is a potential means of transport of cell-laden fluid.

Calcif Tissue Int, 1996 Oct, 59(4), 265 - 70
Species differences in growth requirements for bone marrow stromal fibroblast colony formation In vitro; Kuznetsov S et al.; The marrow stromal fibroblast (MSF) population has been shown to include precursor cells for at least five types of connective tissue: bone, cartilage, adipose tissue, fibrous tissue, and hematopoiesis-supporting reticular stroma . In this study, growth requirements for MSF colony formation were studied in vitro . In order to exclude the influence of nonadherent cells, after a period of initial adhesion of bone marrow cells in serum-containing medium nonadherent cells were removed . Further cultivation was carried out in either serum-containing or serum-free conditions, with or without feeder cells (irradiated bone marrow cells) . This approach revealed differences between animal species in initial MSF growth requirements . In serum-containing conditions, mouse MSF precursor cells (colony-forming units-fibroblast, CFU-Fs) were shown to be feeder cell dependent: MSF colonies were formed only in the presence of feeder cells . Guinea pig CFU-Fs were partially feeder cell dependent, whereas human CFU-Fs were feeder cell independent . In serum-free conditions, CFU-Fs of all three species were feeder cell dependent . The difference between the growth requirements for mouse and human MSFs was not caused by serum origin or concentration, feeder cell origin, or differences in the preparation of marrow cell suspensions.

Transplantation, 1996 Sep 27, 62(6), 715 - 21
Graft-infiltrating cells in rats receiving orthotopic semiallogeneic small intestine transplantation with portal or systemic venous drainage; Sullivan B et al.; The effect of alterations in venous drainage, from either ivc to portal vein (pv), along with peritransplant systemic (ivc) or portal (pv) venous alloimmunization with irradiated semiallogenic cells, on cell subset recovery in lymphoid organs of Lewis rats receiving orthoptic small bowel allografts (from LewisXBrown Norway) F1, LBNF1) was examined . Combined portal, venous drainage and alloimmunization has been reported to increase graft/recipient survival in this model . FACS analysis using monoclonal antibodies specific for different lymphocyte subsets was performed on cell suspensions of peripheral (P) and mesenteric (M) lymph node (LN), small bowel intraepithelial lymphocytes (SBIEL), and Peyer's patch (PP) lymphocytes on days 2 and 8 posttransplantation . Donor cell contributions to these cellular analyses were estimated by comparison of FACS staining with polyclonal anti-Lewis or Lewis anti-LBNF1 antibodies . Control animals received syngeneic grafts . In both syngeneic and semi-allogenic transplants with pv or ivc drainage there was no consistent difference in cell subsets from in PLN compared with those of control nongrafted rats . Approximately 50% to 60% of these cells were alphabetaTcR+ with a CD4+/CD8+ ratio of 3-4:1 and a (CD4++CD8+)/alphabetaTcR+ ratio of 1:1 . Some 5% to 12% ED3+ cells were also present . In IEL, MLN, and PP by contrast, there were significant differences in cells recovered from rats with ivc vs . pv drainage of grafts . The most striking changes reflected a decreased CD4+/CD8+ and alphabetaTcR+gammadeltaTcR+ cells in these tissues in rats predestined to show prolongation of allograft survival (ivc vs . pv injected IEL CD4/CD8+ ratios and alphabetaTcR+gammadeltaTcR+ ratios 1.0, 0.7 and 5.0, 1.0, respectively . These data are consistent with a proposed role for such gammadeltaTcR+ cells in the local regulation of graft rejection.

J Neurosci Res, 1996 Sep 15, 45(6), 723 - 34
Phenotype and function of hematopoietic-derived cells in the CNS of SCID mouse-Lewis rat bone marrow chimeras; Jones RE et al.; Severe combined immunodeficient (SCID) mice previously transplanted with Lewis rat hematopoietic cells (SCID mouse-Lewis rat chimeras) developed experimental autoimmune encephalomyelitis (EAE) following injection with myelin basic protein (BP)-specific Lewis rat T lymphocytes . Rat T cells did not cause EAE in non-chimeric SCID mice . Thus, in addition to BP-specific rat T cells, transplanted rat hematopoietic cells were involved in the development of EAE in SCID mice . In order to examine the role of hematopoietic rat cells in the development of EAE, chimeras were constructed in SCID mice by transplanting 40 x 10(6) T cell-depleted adult Lewis rat bone marrow cells . Single cell suspensions of brain, blood and spleen from chimeric mice were phenotyped by monoclonal antibody staining specific for mouse or rat cellular differentiation markers at 2 week intervals . Brain cells from chimeric mice were also evaluated for the presence of rat antigen-presenting cells (APC) . Four and six weeks after hematopoietic cell transfer, mouse brain contained rat cells expressing the phenotypic markers (CD45+, CD11b/c+) of CNS antigen-presenting cells (APC) . Six weeks after hematopoietic cell transfer, rat cells populating the CNS of chimeras were shown to function as APC, stimulating BP-specific Lewis rat T lymphocytes in vitro.

Radiats Biol Radioecol, 1996 Sep-Oct, 36(5), 671 - 5
{Characteristics of microwave and thermal heating of samples of cell suspensions and solutions of several compounds}; Morozov II et al.; The influence of microwaves and heat on dynamics of heating of samples of intact and inactivated bacteria Escherichia coli and solutions of some basic molecular components of cell was investigated . It was shown that microwaves induce different dynamics of heating of all samples . On the contrary, thermal action induces identical dynamics of heating of all this samples except vegetable oil which was heated more intensively.

Br J Dermatol, 1996 Sep, 135(3), 379 - 84
Epidermal cell DNA content and intermediate filaments keratin 10 and vimentin after treatment of psoriasis with calcipotriol cream once daily, twice daily and in combination with clobetasone 17-butyrate cream or betamethasone 17-valerate cream: a comparative flow cytometric study; Glade CP et al.; Calcipotriol and corticosteroids, two therapy modalities frequently prescribed in the treatment of psoriasis, are often used in combination . The aim of the present study was to determine whether the cell biological response pattern of concurrent use of calcipotriol and corticosteroids is different from calcipotriol monotherapy . Forty patients with chronic plaque psoriasis were divided at random in four parallel groups and treated for 8 weeks with: (1) calcipotriol cream (50 micrograms/g once daily); (2) calcipotriol cream twice daily; (3) calcipotriol and clobetasone 17-butyrate (0.5 mg/g) creams; and (4) calcipotriol and betamethasone 17-valerate (1 mg/g) creams . Before and after treatment keratotome biopsies were taken and single cell suspensions prepared for flow cytometric analysis . Flow cytometric multiparameter quantification of markers for proliferation (TO-PRO-3), differentiation (antikeratin 10) and inflammation (antivimentin) was used to evaluate all four therapy modalities . A statistically significant decrease of the percentage of basal cells in S- and G2M-phase (proliferation) was obtained with all therapy modalities, except for calcipotriol monotherapy applied once daily . A significant reduction of the number of vimentin-positive cells (non-keratinocytes) was observed following combined treatment with calcipotriol and clobetasone butyrate . In contrast, monotherapy with calcipotriol had virtually no effect on the number of vimentin-positive cells . It can be concluded that: (i) calcipotriol monotherapy, applied once daily was less antiproliferative compared with twice daily applications of calcipotriol or the combined treatment with corticosteroids and that (ii) the combination of calcipotriol and corticosteroids proved to have a marked effect on the percentage of non-keratinocytes, in contrast to the modest effect of calcipotriol.

Anticancer Res, 1996 Sep-Oct, 16(5A), 2533 - 6
A rapid and simplified technique for analysis of archival formalin-fixed, paraffin-embedded tissue by fluorescence in situ hybridization (FISH); Kopf I et al.; We here present a simplification of the entire procedure for preparing formalin-fixed, paraffin-embedded tissue to be used for FISH-analysis . The steps for deparaffinisation and disintegration of the tissue to produce intact cell nuclei in a monodispersed suspension are detailed as well as the hybridisation steps . The procedure results in a clear cell suspension with intact cell nuclei and without clumped debris particles . The enzymatic digestion is restricted to the trypsinisation step of the deparaffinisation procedure . No further pretreatments prior to hybridization are performed . This modified technique has been successfully applied to different formalin-fixed, paraffin-embedded materials.

Plant Mol Biol, 1996 Sep, 31(6), 1129 - 39
Isolation and expression in transgenic tobacco and rice plants, of the cassava vein mosaic virus (CVMV) promoter; Verdaguer B et al.; The cassava vein mosaic virus (CVMV) is a double stranded DNA virus which infects cassava plants (Manihot esculenta Crantz) and has been characterized as a plant pararetrovirus belonging to the caulimovirus subgroup . Two DNA fragments, CVP1 of 388 nucleotides from position -368 to +20 and CVP2 of 511 nucleotides from position -443 to +72, were isolated from the viral genome and fused to the uidA reporter gene to test promoter expression . The transcription start site of the viral promoter was determined using RNA isolated from transgenic plants containing the CVMV promoter:uidA fusion gene . Both promoter fragments were able to cause high levels of gene expression in protoplasts isolated from cassava and tobacco cell suspensions . The expression pattern of the CVMV promoters was analyzed in transgenic tobacco and rice plants, and revealed that the GUS staining pattern was similar for each construct and in both plants . The two promoter fragments were active in all plant organs tested and in a variety of cell types, suggesting a near constitutive pattern of expression . In both tobacco and rice plants, GUS activity was highest in vascular elements, in leaf mesophyll cells, and in root tips.

Immunology, 1996 Sep, 89(1), 126 - 34
Expression and modulation of C5a receptor (CD88) on skin dendritic cells . Chemotactic effect of C5a on skin migratory dendritic cells; Morelli A et al.; Although it is known that dendritic cells (DC) migrate in response to inflammatory stimuli . There is little information about the expression of receptors for chemotactic factors on DC . The present study has demonstrated by double immunostaining and flow cytometry of Langerhan's cell (LC)-enriched epidermal cell suspensions that a small subpopulation (5-6%) of epidermal resident DC (rLC) expresses receptors for C5a (C5aR) . Epidermal rLC positive for C5aR show a round-shape morphology, were located next to the basement membrane and express HLA-DR molecules higher than C5aR negative rLC . These observations suggest that rLC would express C5aR as part of their process of maturation during tissue trafficking . To investigate whether epidermal LC up-regulate C5aR along their differentiation pathway . LC were differentiated in vitro after culture in epidermal cell suspensions supplemented with granulocyte macrophage colony-stimulating factor (GM-CSF) . As a result, in vitro differentiated LC increased the expression of C5aR up to 69% of the DC population . In accordance with this observation, interdigitating DC of secondary lymphoid organs (lymph node and tonsil) also expressed (5aR . Migratory CD1a positive DC that spontaneously migrated out of dermal or split-skin organ explants were also positive for C5aR and were used for chemotaxis and chemokinesis assays in response to human recombinant C5a (rC5a) . Optimum migration to rC5a was observed at 10(-8)M with a sigmoidal dose response curve . Checkboard analysis demonstrated that locomotion in response to rC5a was chemotaxis and not chemokinesis.

In Vivo, 1996 Sep-Oct, 10(5), 463 - 9
Efficiency of orthotopic xenograft models for human colon cancers; Pocard M et al.; It is feasible to graft human colon cancer tissue into immuno-deficient nude mice, in order to maintain these tumors in vivo . Analysis of the properties tumors under such conditions might increase our knowledge of their biological characteristics . Xenografts are usually implanted into subcutaneous tissue, a site easily accessible for both graft procedure and observation of tumor growth . However these subcutaneous tumors are usually non-invasive and fail to metastasize . To override these limitations we, as others, have tried to improve the tumor xenograft model by transplanting tumors into their original location, designated as the orthotopic site . Transplanted into the cecum of nude mice, human colon cancers were frequently locally invasive and developed liver metastases . These transplantations may be done by injection of colon cancer cell suspensions into the cecal wall . Alternatively, orthotopic implantations of histologically intact tissue were performed and the thus-obtained tumors were more metastatic than tumors obtained after tumor cell injections . The chemosensibility of tumors xenografted into their orthotopic site was different from that of their subcutaneously implanted counterpart . This, added to the enhancement of invasive and metastatic properties, leads us to conclude that colon cancer xenografted into the caecum might be more representative of the clinical situation . We have reviewed the literature and report on the possibilities and limitations different models of colon cancer in vivo.

Int J Radiat Oncol Biol Phys, 1996 Sep 1, 36(2), 377 - 83
Radiosensitization of hypoxic tumor cells in vitro by nitric oxide; Griffin RJ et al.; PURPOSE: The effects of nitric oxide (NO) on the radiosensitivity of SCK tumor cells in oxic and hypoxic environments in vitro were studied . METHODS AND MATERIALS: NO was delivered to cell suspensions using the NO donors 2,2-diethyl-1-nitroso-oxyhydrazine sodium salt (DEA/NO), and a spermine/nitric oxide complex (SPER/NO), which release NO at half-lives of 2.1 min and 39 min at pH 7.4, respectively . The cells were suspended in media containing DEA/NO or SPER/NO for varying lengths of time under oxic or hypoxic conditions, irradiated, and the clonogenicity determined . RESULTS: Both compounds markedly radiosensitized the hypoxic cells . The drug enhancement ratios (DER) for 0.1, 1.0, and 2.0 mM DEA/NO were 2.0, 2.3 and 3.0, respectively, and those for 0.1, 1.0, and 2.0 mM SPER/NO were 1.6, 2.3, and 2.8, respectively . Aerobic cells were not radiosensitized by DEA/NO or SPER/NO . When DEA/ NO and SPER/NO were incubated in solution overnight to allow release of NO, they were found to have no radiosensitizing effect under hypoxic or oxic conditions indicating the sensitization by the NO donors was due to the NO molecule released from these drugs . At the higher concentrations, SPER/NO was found to be cytotoxic in aerobic conditions but not in hypoxic conditions . DEA/NO was only slightly toxic to the cells in both aerobic and hypoxic conditions . CONCLUSIONS: NO released from NO donors DEA/NO and SPER/NO is as effective as oxygen to radiosensitize hypoxic cells in vitro . Its application to the radiosensitization of hypoxic cells in solid tumors remains to be investigated.

Inflamm Res, 1996 Sep, 45(9), 442 - 7
Characteristics of the histamine release from hamster cheek pouch mast cells stimulated by lectins from Brazilian beans and concanavalin A; Ferreira RR et al.; We have characterized the histamine releasing effects of lectins extracted from Brazilian beans, in comparison to concanavalin A, in hamster cheek pouch cell suspensions containing mast cells . The lectins from Dioclea virgata, Canavalia brasiliensis, and Dioclea rostrata induce histamine release in a similar manner to concanavalin A, but appear to differ in potency and efficacy . The effects depended on the temperature, pH, and metabolic energy, demonstrating the non-cytotoxic nature of the histamine release . It is suggested that the lectins studied act by the same mechanism as concanavalin A (interacting with sugars in the antibodies bound to the mast cells), since high concentrations of glucose inhibit the histamine release . The lectins at high concentrations quench the histamine release . This suppression is reversed by increasing calcium concentration, suggesting that the lectins bind to the calcium that is essential for the secretion, thereby confirming and extending our previous data using the lectin from Dioclea virgata in rat peritoneal mast cells.

Exp Brain Res, 1996 Sep, 111(2), 187 - 207
A comparison of behavioural effects and morphological features of grafts rich in cholinergic neurons placed in two sites of the denervated rat hippocampus; Hofferer E et al.; This study compared the morphological characteristics and the behavioural effects of intrahippocampal septal cell suspension grafts injected either just above the pyramidal cell layer of the hippocampal region CA1 or within the dorsal leaf of the dentate gyrus (DG) in rats subjected to electrolytic fimbria-fornix lesions . The behavioural tests determined home-cage and open-field activity, as well as radial-maze performance . Cresyl-violet staining, acetylcholinesterase (AChE) histochemistry, and parvalbumin, glial fibrillary acidic protein and glutamic acid decarboxylase immunocytochemistry were used for morphological assessments . The cross-sectional area of the grafts was measured between 0.8 mm and 5.3 mm posterior to Bregma and used as an index of their development . Whether injected into CA1 or DG, the grafts provided the partially denervated hippocampus with a dense AChE-positive reinnervation . Both types of grafts were devoid of reactive astrocytes (although reactive astrocytes were found close to the graft-host interface), contained almost no parvalbumin-positive neurons and showed a high density of GAD-positive terminals . One of the main differences between the two groups of grafted rats was that the suspension injected into the DG yielded grafts that, in the vicinity of the injection sites (between 2.3 mm and 4.3 mm posterior to Bregma), had a cross-sectional area exceeding that of the grafts placed into CA1 by about 63-110% (average 79%), the latter being more dispersed than the former in the coronal plane . In addition, rats with grafts in the DG exhibited granule cell degeneration in the vicinity of the injection sites, whereas rats with grafts in region CA1 showed no damage near the injection sites . Concerning the behavioural data, we found that fimbria-fornix lesions induced hyperactivity in both the home cage and the open field and impaired radial-maze performance . Compared with the lesion-only rats, the grafted rats in both groups had further increased open-field and home-cage activity . While the grafts placed into region CA1 slightly, but significantly, accentuated the lesion-induced deficit in radial-maze performance, those placed into the DG had no effect . These results suggest that intrahippocampal grafts may, in some (still unspecified) conditions, produce adverse behavioural effects or no behavioural effects, despite an acceptable graft-induced cholinergic reinnervation of the hippocampus . They do not allow a clear answer to the question of whether intra-DG and intra-CA1 septal suspension grafts exhibiting almost comparable morphological features (except in their size and their dispersion in the vicinity of the injection sites) induce behavioural effects that would depend on intrahippocampal location of the grafts . They suggest, however, that the granule cell degeneration caused by the implantation procedure, in conjunction with the intragyral development of the graft, probably does not account for some of the reported adverse behavioural effects of intrahippocampal basal forebrain grafts . Finally, the finding that septal cell suspensions placed into the DG yielded larger grafts than when an equivalent number of cells was injected into CA1 might be explained by a larger lesion-induced neurotrophic activity in DG than in region CA1, although both regions had undergone a similar degree of cholinergic denervation.

Cell Transplant, 1996 Sep-Oct, 5(5 Suppl 1), S49 - 50
A simple method for cryopreservation of purified adult pig pancreatic cells; Sato S et al.; Cryopreservation of islet cells may benefit many aspects of islet transplantation including storage, transport of islets, immunomodulation, reduction of the exocrine contaminants . Isolation and purification of islets from large mammalian pancreases have been encountered by many problems . We reported an autodigestion method for preparation of adult pig pancreatic cells . In this paper, we describe a newly invented simple method for cryopreservation of purified adult pig pancreatic cells . The viability and function of islet cells before (precryo.) and after (postcryo.) the cryopreservation procedure were compared . Islet cell harvested by the autodigestion method were suspended in cryoprotectant "Cell banker." A biofreezing vessel "Bicell" containing a cryogenic vial with cell suspension was frozen at -80 degrees C . After 4 wk, the cryogenic vial was rapidly thawed at 37 degrees C water, the islet cells were washed and cultured for overnight . Number of dithizone-stained precryo . and postcryo . cells were 1.71 +/- 0.5 x 10(5) and 1.75 +/- 0.98 x 10(5) cells/pancreas respectively; therefore, the viability was 99% . Insulin release following high and low concentrations of glucose in precryo . (12.02 +/- 3.63 ng/ml: low-glucose, 9.81 +/- 3.59 ng/ml: high glucose) and precryo . (5.57 +/- 4.03 ng/ml: low-glucose, 10.24 +/- 8.88 ng/ml: high glucose) cells showed a marked improvement in response in postcryo . cells . The insulin content of precryo . and postcryo . cells were 914.4 +/- 277.4 and 93.6 +/- 15.4 ng/ml, respectively . This simple and efficient cryopreservation protocol may be applied for a large-scale storage of adult pig islet cells to establish an islet bank for transplantation.

Scand J Plast Reconstr Surg Hand Surg, 1996 Sep, 30(3), 169 - 75
Contact hypersensitivity is suppressed after sensitisation by dinitrofluorobenzene of early stage iso-skin grafts; Yasuda H et al.; Isologous free skin grafts were applied to the backs of BALB/c mice and contact hypersensitivity studied by epicutaneous application of DNFB (2,4-dinitro-1-fluorobenzene) to the grafted skin . In the first experiment, the grafted skin was sensitised and the elicitation reaction assessed by measuring the ear swelling after five days . Sensitisation was not successful until two weeks after grafting . In such non-responding animals, re-sensitisation with DNFB on the ungrafted skin area was also unsuccessful, indicating the establishment of immunological tolerance . This tolerance was considered antigen-specific, as re-sensitisation with oxazolone was successful . In the second experiment, spleen cell suspension, both untreated and treated in vitro with antiThy 1.2, antiLyt 1.2, and antiLyt 2.2 antibody, from non-DNFB-responding mice was transferred into normal mice . Subsequently these mice were sensitised with DNFB . Sensitisation was not successful in the untreated group or in those treated with antiLyt 1.2 antibody . On the other hand, the groups treated with antiThy 1.2 and antiLyt 2.2 antibody did become sensitised . These results indicate that the unsuccessful delayed hypersensitivity of the grafted skin was caused by suppressor T cells . In addition, the density of epidermal Langerhans cells was reduced in the early stages of skin grafting and morphological abnormalities were present . From these results, we conclude that contact sensitisation during the early stage of grafting skin not only produces suppression of ear swelling but also induces antigen-specific tolerance . These results also suggest that the suppression depends on the antigen-specific suppressor cells and that acquirement of tolerance is associated with a reduction in the number of epidermal Langerhans cells in the grafted skin and abnormalities in their structure.

J Dermatol Sci, 1996 Sep, 12(3), 263 - 74
Regulation of Langerhans cell function via blood borne factor(s); Xie Y et al.; Langerhans cells (LC) are epidermal dendritic cells that are functionally labile . Freshly obtained LC (fLC) readily activate allogeneic T cells, but they are incapable of activating autologous T cells; and even when pulsed with antigen, they fail to activate naive, antigen-specific T cells . When fLC are cultured for 2-3 days in the presence of keratinocytes, LC swiftly up-regulate surface expression of class I and II MHC molecules, and express de novo the co-stimulatory molecules B7 and ICAM-1 . In addition to displaying enhanced ability to activate allogeneic T cells, cultured LC acquire the novel functional property of activating autologous T cells . It is believed that keratinocyte-derived GM-CSF is the primary driving force responsible for the conversion of fresh to cultured LC in vitro . However, in vivo administration of GM-CSF, either intracutaneously or systematically, fails to induce LC to undergo functional transformation in situ . Moreover, despite a high level of GM-CSF in the circulation, fLC from mice bearing GM-CSF-producing tumors display no ability to activate syngeneic T cells . These observations suggest that a homeostatic factor that antagonizes the effect of GM-CSF may be present in vivo . To test this possibility, we have examined the functional properties of LC prepared from mouse skin that had been explanted in vitro for 3 days . We found that the functional and phenotypic features of these cells closely resembled those of LC cultured in single cell suspension: the cells strongly expressed B7-1 and B7-2, and displayed enhanced expression of class II MHC molecules; they readily activated naive autologous T cells . Strikingly, when explants or suspensions of fresh epidermal cells were cultured in the presence of 10% mouse serum they failed to acquire syngeneic T cell activating properties; and surface up-regulation of Ia and de novo expression of B7 was inhibited . However, the cultured cells still expressed surface Ia and readily activated allogeneic naive T cells . If mouse serum was added only during the last 24 h of culture, the LC displayed full functional transformation . Human, rabbit and bovine serum showed no inhibitory effect on mouse LC . Our data suggest that mouse serum contains a factor (or factors) that inhibits, in a species-specific manner, epidermal LC from undergoing functional transformation in vitro, and this factor may maintain epidermal LC in the 'fresh' functional program in vivo.

Int J Obes Relat Metab Disord, 1996 Sep, 20(9), 874 - 81
Microcalorimetric and biochemical investigations of thermogenesis and metabolic pathways in human white adipocytes; Bottcher H et al.; OBJECTIVE: Validation of a novel culture technique for human white adipocytes, facilitating direct microcalorimetry and simultaneously performed biochemistry for 3 days . DESIGN: Subcutaneous adipocytes were cultured in a 3-dimensional matrix of agarose gel . Biochemical measures were obtained every 24 h, while thermogenesis was continuously monitored for 72 h . DNA content of the cultures served as reference . SUBJECTS: 73 men and women undergoing uncomplicated surgery . MEASUREMENTS: Cell viability (LDH release), total cellular thermogenesis, oxygen consumption, glycolysis, lipolysis (basal, catecholamin-stimulated) triglyceride/free fatty acid substrate cycle, adenine nucleotides, insulin-induced thermogenesis . RESULTS: LDH release was 0.4% of total LDH per hour . Cellular ATP, ADP and AMP (3.77, 0.39 and 0.06 nmol/microgramDNA, resp.) were constant . The rates of glucose consumption, lactate and pyruvate production and basal glycerol release (56.4, 43.1, 2.7 and 28.1 nmol/microgramDNA.h, resp.) were stable during 72 h . Isoprenaline (1 microM) enhanced lipolytic rate by the same extent at any time of the study (glycerol release 67.8 nmol/microgramDNA.h) . FFA release (initially 26.0 nmol/ microgramDNA.h) declined during the experiment, due to an increase of reesterfication rate . Unstimulated heat production was 6.5 microW/microgramDNA, 68% of which were of oxidative origin . Insulin (0.1 microM) induced thermogenesis was 8.8 microW/ microgramDNA . CONCLUSION: The results were in accordance with data from human white adipocytes in suspensions . However, in contrast to fat cell suspensions, gel cultured adipocytes were viable for 3 days without metabolic alterations.

Mod Pathol, 1996 Sep, 9(9), 925 - 9
Use of CD23 (BU38) on paraffin sections in the diagnosis of small lymphocytic lymphoma and mantle cell lymphoma; Kumar S et al.; The CD23 antigen is a low-affinity immunoglubulin E receptor that is expressed during B-cell activation . Recently, it has been shown to be of diagnostic utility in distinguishing between small lymphocytic lymphoma (SLL) and mantle cell lymphoma (MCL), two entities that can have similar morphologic and immunophenotypic features . Such studies, however, generally required viable cells in cell suspension or cryostat sections for detection of CD23 . We evaluated staining for the CD23 antigen in paraffin sections, using BU38, an antibody that detects a fixation-resistant epitope of the antigen . We analyzed 44 SLLs, 3 lymphoplasmacytoid lymphomas, and 39 MCLs . Staining was performed on formalin- or B5-fixed paraffin-embedded tissue sections using L26 (CD20), CD3, Leu22 (CD43), and BU38 (CD23) antibodies . All of the cases were of B-cell phenotype (CD20+), and 42/44 SLLs, 3/3 lymphoplasmacytoid lymphomas, and 33/39 MCLs coexpressed the CD43 antigen . CD23 was positive in 41 (93%) of 44 SLLs . The majority of neoplastic cells (75% or more) stained positively, with a membranous pattern of staining . The staining was moderate in intensity and easily interpreted . Only 1/39 MCLs and 1/3 lymphoplasmacytoid lymphomas were CD23 positive . CD23-positive follicular dendritic cells were, however, present in all of the MCLs, either in residual follicles or in large, disordered meshworks . These results demonstrate that the BU38 antibody can detect CD23 on the cells of SLLs in paraffin sections and that this antibody can have diagnostic utility in routine diagnosis.

Magn Reson Med, 1996 Sep, 36(3), 359 - 65
A computational strategy for the deconvolution of NMR spectra with multiplet structures and constraints: analysis of overlapping 13C-2H multiplets of 13C enriched metabolites from cell suspensions incubated in deuterated media; Laatikainen R et al.; A computational strategy for the deconvolution of complex spectra involving scalar multiplet patterns is presented . This approach fits spectra that can be composed of single resonances as well as scalar coupling multiplets for which resonance frequencies, intensities, and lineshape parameters can be optimized . For multiplets, the coupling constant also is optimized . Any external information about the optimizable parameters can be taken into account as external constraints . A lineshape described by absorptive and dispersive Lorentzian and Gaussian contributions and the baseline with up to 40 Fourier and polynomial terms can likewise be optimized . The effectiveness of the procedure is assessed on the basis of computer simulated deconvolutions of a composite of 1J(13C-2H) multiplets arising from a mixture of all possible 13C-2H isotopomers of deuterated L-{3-13C}lactate generated from cell preparations incubated with D-{1-13C}glucose in D2O, which was analyzed previously with a manual deconvolution procedure (R . Willem, M . Biesemans, F . Kayser, W . J . Malaisse, Magn, Reson . Med . 31, 259-267 (1994)) . The use of constraints is shown to lead to an improvement in the results . The fitting strategies and the importance of the baseline as an origin of bias are discussed.

Cytometry, 1996 Sep 1, 25(1), 104 - 8
Comparison of Vindelov et al . and bromodeoxyuridine/DNA double-staining flow cytometry methods for analysis of cell cycle distribution in rat thymocytes; Orfao A et al.; This study compares the cell cycle distribution in rat thymocytes obtained by means of bromodeoxyuridine (BrdUrd) labeling of S-phase cells and the analysis of the S-phase fraction obtained according to the technique of Vindelov et al . (Cytometry 3:332-338, 1983) . The proportion of BrdUrd-labeled cells was analyzed in single cell suspensions of adult rat thymocytes after in vivo injection of BrdUrd and the results then compared with those obtained after measuring the cell DNA contents according to the Vindelov et al . method . The percentage of BrdUrd-positive cells was greater than the S-phase fraction obtained using the Vindelov et al . technique . By contrast, no major differences were observed between the percentage of BrdUrd-positive cells and the S-phase fraction obtained after analyzing the DNA histograms of the same data files with the RFIT mathematical model . The elimination of trypsin treatment used in the Vindelov et al . method did not alter the results, whereas the use of DNA denaturation with 2N HCl was shown to increase the percentage of S-phase rat thymocytes (calculated from DNA histograms) independently of whether trypsin treatment was used or not . However, the value of the S-phase fraction was not as great as that obtained after BrdUrd labeling . Thus when comparing BrdUrd-labeling and the Vindelov et al . technique, important differences in the percentage of S-phase adult rat thymocytes were observed . Selective G0/G1 cell loss during washing and centrifugation steps performed after the DNA denaturation used for BrdUrd detection was the main reason for these differences.

AIDS, 1996 Sep, 10(10), F35 - 8
Reduced CD4 cell counts in blood do not reflect CD4 cell depletion in tonsillar tissue in asymptomatic HIV-1 infection; Rosok BI et al.; OBJECTIVE: To investigate whether the loss of CD4 cells seen in peripheral circulation of HIV-1-positive individuals reflects a similar depletion of CD4 cells from lymphoid tissue . DESIGN: CD4 and CD8 cells in tonsillar mononuclear cell suspensions were quantified relative to tonsillar B cells, as these were thought to remain numerically unchanged in the course of HIV infection . Results were related to the CD4 cell counts in blood and to the clinical status of the patients . METHODS: Blood samples and tonsillar tissue were obtained from 13 HIV-1-seropositive individuals and six seronegative controls . B cells and T-cell subsets in mononuclear cells were quantified using a three-colour flow cytometry protocol . Histological sections were morphologically classified and B-cell areas were quantified by morphometry . RESULTS: The B-cell fraction was confirmed to be relatively unchanged in asymptomatic HIV-1-seropositive individuals compared with controls . The tonsillar CD4 : B-cell ratios in asymptomatic individuals was similar to those seen in controls, whereas the CD4 : B-cell ratios in symptomatic HIV-1-infected individuals were greatly reduced . The tonsillar CD4 : CD8 cell ratios in HIV-1-infected individuals were much lower than those seen in controls, in the asymptomatic group due to a considerable expansion of the tonsillar CD8 cell subset, and in the symptomatic group also due to a loss of CD4 cells . CONCLUSIONS: We found no evidence of CD4 cell depletion in tonsillar tissue in asymptomatic HIV-1-infected individuals despite low CD4 cell counts in blood . Loss of CD4 cells from this lymphoid tissue seems to occur as a late-stage phenomenon correlated with the onset of clinical symptoms.

Biophys J, 1996 Sep, 71(3), 1616 - 20
Infrared nonlinear optical measurements of membrane potential in photoreceptor cells; Ben-Oren I et al.; In the past it has not been possible to measure optically the membrane potential of cells and collections of cells that are either naturally photosensitive or that can be activated by photolyzable caged transmitter molecules . This paper reports on a unique application of nonlinear optics that can monitor the potential of cellular membranes with a near-infrared source . Among many other singular advantages, this nonlinear optical approach to measuring membrane potential does not activate light sensitive cells or cell suspensions and cellular networks surrounded with photolyzable molecules . To demonstrate this capability we show that the technique can be applied to living photoreceptor cells that are very sensitive to visible light . These cells are ideal for characterizing such a new technique, not only because of their unmatched sensitivity to light, but also because their electrical responses have been extensively characterized (Minks and Selinger, 1992).

Mov Disord, 1996 Sep, 11(5), 522 - 32
Reversal of behavioural abnormalities by fetal allografts in a novel rat model of striatonigral degeneration; Wenning GK et al.; We have developed a rodent model of striatonigral degeneration, one of the core pathologies underlying the disease multiple system atrophy (MSA) . 6-Hydroxydopamine (6-OHDA) was administered into the left medial forebrain bundle of male Wistar rats, followed 3-4 weeks later by intrastriatal injection of quinolinic acid into the ipsilateral striatum . The 6-OHDA lesion resulted in ipsilateral rotation to (+)-amphetamine and contralateral rotation to apomorphine . Following the subsequent striatal lesion, amphetamine-induced ipsilateral rotation persisted, but apomorphine-induced contralateral rotation was reduced or abolished . Subsequently, the lesioned striatum was implanted with fetal CNS allografts consisting of cell suspensions derived from striatal primordium alone or combined with cografts of ventral mesencephalon . Cografted rats showed a reduction or reversal of amphetamine-induced rotation . This was not observed in animals receiving striatal grafts alone . Apomorphine-induced contralateral rotation was restored after striatal grafts alone, but only partially in animals receiving sham or cografts . Tyrosine hydroxylase (TH) and dopamine- and cyclic adenosine 3':5'-monophosphate-regulated phosphoprotein (DARPP 32) immunocytochemistry showed mesencephalic and striatal graft survival in most animals . However, dopaminergic outgrowth was restricted to the graft deposit . The latter was surrounded by a markedly gliotic glial fibrillary acidic protein-positive capsule continuous with corpus callosum . Dopaminergic reinnervation of denervated and lesioned adult striatum itself was absent, suggesting that rotational recovery was due to diffuse dopamine release . The study shows that combined unilateral lesioning of rodent medial forebrain bundle and striatum results in a characteristic drug-induced rotational response that can be partly restored by mesencephalic/striatal cografts.

Neuroscience, 1996 Sep, 74(2), 553 - 65
Change in the molecular phenotype of Schwann cells upon transplantation into the central nervous system: down-regulation of c-jun; Vaudano E et al.; Activated Schwann cells such as those in the distal stump of a cut peripheral nerve, or those cultured in vitro, develop a molecular phenotype very different from that of quiescent Schwann cells, and express high levels of the transcription factor c-jun . We studied the expression of c-jun messenger RNA, by in situ hybridization, and Jun-like immunoreactivity of Schwann cells in segments of peripheral nerve, or in cell suspensions grafted into the adult rat brain . Schwann cells rapidly lost their Jun immuno-positivity, and down-regulated expression of c-jun messenger RNA once implanted into the brain, and only the Schwann cells contained in the portion of peripheral nerve which remained outside the brain maintained Jun-like immunopositivity . c-jun messenger RNA was also down-regulated in the grafts, but more slowly than the protein; however, a proximodistal gradient in the level of expression of c-jun messenger RNA along the graft, comparable to that found for Jun immunoreactivity, was not detected . Schwann cells transplanted into the lesioned central nervous system promote regeneration of some injured central nervous system axons, but this regenerative response is always much more limited than peripheral nervous system regeneration . We suggest a correlation between the limited regeneration of central nervous system axons into peripheral nerve grafts and the loss of c-jun expression in Schwann cells following exposure to the central nervous system environment.

J Clin Microbiol, 1996 Sep, 34(9), 2312 - 5
Evaluation of an infectivity standard for real-time quality control of human immunodeficiency virus type 1 quantitative micrococulture assays . Participating Laboratories of The AIDS Clinical Trials Group; Scott WA et al.; Quantitative microculture assays of cryopreserved human immunodeficiency virus type 1-infected cell suspensions and culture supernatants were compared among seven assays sites . There was no significant change in titer during 1 year of storage . The overall standard deviation for infected cell suspensions was approximately 0.8 log10 virus titer . A method for detecting deviant assay results was developed and was used to identify two donor cell preparations (n = 54) that gave consistently low titers.

Plant Cell, 1996 Sep, 8(9), 1555 - 67
Distinct UV-B and UV-A/blue light signal transduction pathways induce chalcone synthase gene expression in Arabidopsis cells; Christie JM et al.; UV and blue light control the expression of flavonoid biosynthesis genes in a range of higher plants . To investigate the signal transduction processes involved in the induction of chalcone synthase (CHS) gene expression by UV-B and UV-A/blue light, we examined the effects of specific agonists and inhibitors of known signaling components in mammalian systems in a photomixotrophic Arabidopsis cell suspension culture . CHS expression is induced specifically by these wavelengths in the cell culture, in a manner similar to that in mature Arabidopsis leaf tissue . Both the UV-B and UV-A/blue phototransduction processes involve calcium, although the elevation of cytosolic calcium is insufficient on its own to stimulate CHS expression . The UV-A/blue light induction of CHS expression does not appear to involve calmodulin, whereas the UV-B response does; this difference indicates that the signal transduction pathways are, at least in part, distinct . We provide evidence that both pathways involve reversible protein phosphorylation and require protein synthesis . The UV-B and UV-A/blue light signaling pathways are therefore different from the phytochrome signal transduction pathway regulating CHS expression in other species.

J Cell Physiol, 1996 Sep, 168(3), 695 - 704
Role of integrins in regulation of collagen phagocytosis by human fibroblasts; Lee W et al.; Phagocytosis of collagen fibrils by fibroblasts is an important pathway for degradation of extracellular matrix in mature connective tissues . To study regulatory mechanisms in phagocytosis, 2-microns fluorescent beads coated with either collagen (COL) or bovine serum albumin (BSA) were incubated with human gingival fibroblasts in vitro . For these studies single cell suspensions were prepared by trypsinization, and bead internalization and collagen receptor expression were assessed by flow cytometry . After 3-h incubations, up to 8-fold more cells internalized COL beads than BSA-coated beads . Increased collagen coating concentration was associated with elevated proportions of cells that internalized COL beads, and was observed also in the presence of competing fibronectin-coated beads . The number of beads per cell and the percent of phagocytic cells increased proportionally with higher bead loadings . At > 4 beads per cell a maximum of approximately 80% of cells were phagocytic . Cells reacted with mAbs against the alpha 1, alpha 2, and alpha 3 integrin subunits were, respectively, 5%, 98% and 93% positively stained above background controls . All cells that internalized COL beads exhibited alpha 2 staining but there were large proportions of phagocytic cells that were not stained for alpha 1 . In unfixed cells, bead internalization caused an immediate reduction of surface staining of membrane-bound alpha 2 by approximately 55% which returned to control levels within 3 h, indicating that cell-surface alpha 2 was internalized by phagocytosis . Preincubation of cells with up to 8 COL beads per cell reduced the proportion of phagocytic cells and the number of internalized beads after a second COL bead incubation 4 h later . To assess the relationship between the percent of phagocytic cells and alpha 2 integrin levels, serum starvation and cycloheximide experiments were conducted . Compared to controls, serum starvation for 24 h induced a 3.2-fold increase of cells internalizing COL beads but did not alter alpha 2 staining levels . In contrast, 3 h cycloheximide treatment reduced alpha 2 staining to 60% of control levels and this treatment also inhibited COL bead internalization . GRGDTP peptide as well as mAbs against the alpha 1 and alpha 2 subunits significantly reduced internalization of COL beads by 1.8 to 2.6-fold, whereas GRGESP peptide and alpha 3 mAb exerted no effect . Internalization of BSA beads was not affected by any of these treatments . Collectively, these data indicate that the alpha 2 integrin, along with other, as yet unidentified components, is likely involved in COL bead internalization . The alpha 2 integrin subunit is rapidly recycled or synthesized following a phagocytic load . In contrast, the alpha 1 integrin is not directly required for phagocytosis but may regulate the internalization step.

Eur J Immunol, 1996 Sep, 26(9), 2035 - 42
Subepithelial B cells in the human palatine tonsil . I . Morphologic, cytochemical and phenotypic characterization; Dono M et al.; This study describes the purification of a subset of tonsillar B cells which share phenotypic, morphologic and cytochemical features with subepithelial (SE) B cells . These cells, which represented the 5-10% of the total tonsillar B cells, were found in the Percoll gradient fraction of highest density, together with resting follicular mantle (FM) B cells . The latter B cells, however, expressed surface CD5 and could be removed by an immune rosetting procedure . The remaining small CD5- B cells had a surface phenotype (IgM+, IgD+, CD23-, CD38+/-, CD10-, CD44+) that was different from that of FM (IgM+, IgD+, CD23+, CD39+, CD38-, CD10-, CD44+2) and of germinal center (GC) (CD23-, CD39-, CD38+, CD10+, CD44+/-, IgG+) B cells isolated from the same cell suspensions . Furthermore, the absence of surface activation markers (CD71 and CD69) and of surface IgG allowed us to distinguish small CD5- B cells from activated and memory cells migrating within Percoll fractions of lower density . In situ immunohistochemical studies revealed that B cells with an identical phenotype as that of small CD5- B cells could be detected predominantly in the SE region (lamina propria) of the tonsil, and also within the epithelium lining the cryptae . This area was also comprised of a relatively minor proportion of activated B cells, not found in the small CD5- B cell fraction owing to the separation procedure used . Consistent with the notion that the SE area could be a site of B cell activation was also the presence of activated macrophages and of plasma cells . Thirty to forty percent of small CD5- B cells isolated in suspension were positive for the endogeneous alkaline phosphatase (ALP) activity . In contrast, only a few FM B cells were ALP+, while GC cells were consistently ALP- . In situ studies also demonstrated a prevalent expression of ALP activity by the B cells in the SE area . At the ultrastructural level, small CD5- B cells were clearly different from both FM and GC B cells . They displayed a cytoplasm more extended than that of FM B cells with abundant endosomes and plasma membrane projections, and a speckled pattern of nuclear heterochromatin distribution . When fixed tissue sections were examined, cells with identical ultrastructural features could be demonstrated in the tonsillar lamina propria . Collectively, the above data demonstrate an identity of features between the small CD5- B cells isolated in suspension and SE B cells analyzed in situ . Since tonsillar SE B cells are generally thought to represent the homolog of the extrafollicular B cells (including those of the splenic marginal zone), these studies may provide new opportunities for functional studies on this so far incompletely characterized B cell subset.

Photochem Photobiol, 1996 Sep, 64(3), 537 - 41
Role of enterobactin and intracellular iron in cell lethality during near-UV irradiation in Escherichia coli; Hoerter J et al.; In Escherichia coli, fur mutants that constitutively express their native iron chelating agent, enterobactin, are significantly more sensitive to near-UV radiation (NUV) than wild type, An entA mutant, which is incapable of synthesizing enterobactin, is equal to wild type in resistance to NUV irradiation . However, the addition of Fe+3 enterobactin but not AI+3 enterobactin to entA cell suspensions just prior to irradiation results in an increased sensitivity to NUV irradiation . A fes mutant, which is unable to reduce and release iron from enterobactin, is significantly more sensitive to NUV irradiation than wild type . The addition of nontoxic levels of H2O2 (5 microM) just prior to irradiation significantly increases sensitivity of both fur and fes mutants . These results suggest that one mechanism by which NUV irradiation leads to cell lethality is by creating a transient iron overload, producing very favorable conditions for the production of highly deleterious free radicals through a variety of mechanisms that lead to oxidative stress and DNA damage including lethal and mutagenic lesions . These results are consistent with the hypothesis that enterobactin is an endogenous chromophore for NUV and contributes to cell lethality via the destruction of its ligand, releasing Fe+2 into the cytoplasm to catalyze the production of highly reactive hydroxyl radicals and other toxic oxygen species via the Haber-Weiss reaction.

Exp Neurol, 1996 Sep, 141(1), 79 - 93
The time course of loss of dopaminergic neurons and the gliotic reaction surrounding grafts of embryonic mesencephalon to the striatum; Barker RA et al.; Grafts of embryonic ventral mesencephalic tissue placed in the striatum of 6-hydroxydopamine-lesioned rats survive, and make and receive connections to and from the host brain . The dopaminergic neurons of the graft can grow processes into the host brain, and thereby alleviate many of the behavioral deficits of this form of experimental Parkinson's disease . However, when examined some weeks after implantation, grafted substantia nigra only contains about 5% of the expected complement of dopaminergic neurons . We have examined the time course of loss of grafted neurons . We find that the majority die during the first 7 days after transplantation . However, we have shown previously that three-dimensional cultures with the same dimensions as a graft, made of identical cell suspensions, have much better dopaminergic neuronal survival . There must, therefore, be features in the environment surrounding a graft that are toxic to dopaminergic neurons . A limiting factor in the efficacy of dopaminergic grafts is the small distance over which the neurons are able to grow neurites and form connections in the host brain . We find that the growth of neurites from dopaminergic neurons into the host striatum occurs in two phases . Neurites reach their maximum length within 7 days of transplantation, and this is followed by a much slower process of branch and terminal formation . Since axon growth in the adult brain may be inhibited by a number of factors associated with reactive gliosis, we have immunostained various ages of graft for vimentin, tenascin, chondroitin sulfate proteoglycan (CS-PG) using the CS56 antibody, the DSD-1 proteoglycan, and microglia using the OX-42 antibody . We have compared this staining with that surrounding a simple stab wound . Vimentin staining was initially seen in the graft and in astrocytes immediately surrounding it . By 7 weeks staining was restricted to a ring of astrocytes surrounding the graft . Tenascin, DSD-1, and CS-PG were initially seen in and around the grafts . By 7 weeks they had disappeared from grafts, but CS-PG and tenascin persisted in small amounts around stab wounds . In general, immunostaining of these molecules persisted longer around a stab lesion than around a graft . There was also an intense local microglial reaction surrounding both grafts and stab wounds which had largely resolved by 7 weeks.

Anal Chem, 1996 Sep 1, 68(17), 2932 - 8
Human norepinephrine transporter kinetics using rotating disk electrode voltammetry; Burnette WB et al.; Rotating disk electrode (RDE) voltammetry is applied to the measurement of the transport of the catecholamine neurotransmitters norepinephrine (4-(2-amino-1-hydroxyethyl)-1,2-benzenediol, NE) and dopamine (3,4-dihydroxyphenethylamine, DA) in suspensions of LLC-NET cells, a line of porcine kidney cells expressing the human norepinephrine transporter (hNET) . Initial rate of transport was assessed by following the initial decrease in neurotransmitter after its addition to the cell suspension, as measured by the decrease in oxidation current at +0.45 V vs Ag/AgCl . The initial rate of norepinephrine uptake was saturable, with Vmax and KM of 197 +/- 17 amol min-1 cell-1 and 1.64 +/- 0.46 microM, respectively . The RDE method also allows observation of outward transport (efflux) of the DA or NE previously taken up by the cells . Outward transport was induced by the addition of either d-amphetamine (d-AMPH) or p-tyramine (4-hydroxyphenethylamine, p-TYR), which are also substrates for the NE transporter . The technique was also used to monitor accelerated NE uptake by cells preloaded with p-TYR, a phenomenon distinguishing carriers from channels . Together, these findings document the utility of RDE for the nonisotopic measurement of neurotransmitter influx and efflux from transfected mammalian cells.

Diabetes, 1996 Sep, 45(9), 1223 - 8
Regulation of proliferation and differentiation of human fetal pancreatic islet cells by extracellular matrix, hepatocyte growth factor, and cell-cell contact; Beattie GM et al.; Ex vivo expansion of human fetal pancreatic endocrine cells is important for biological studies and as a potential tissue source for transplantation in insulin-deficient states . In tissue culture experiments involving the use of hepatocyte growth factor/scatter factor and selected extracellular matrices, we obtained a 30-fold increase in cell number of human fetal pancreatic epithelial cells . This proliferation in monolayer culture was associated with marked downregulation of insulin and glucagon gene expression . However, gene expression increased when the cells were combined into three-dimensional aggregates, suggesting that cell-cell contact mediated mechanisms regulate the transcription of islet-specific genes, a process enhanced by nicotinamide (NIC) . After transplantation into nude mice, either as cell suspensions or aggregates, only the cell aggregates treated with NIC developed into mature functional islet-like structures . These are the first experiments to describe the interactions of specific matrices and growth factors in the ex vivo expansion of human fetal pancreatic cells, and they also show the importance of cell aggregates in the context of cellular and molecular events that might positively influence islet cell transplantation.

Pflugers Arch, 1996 Sep, 432(5), 753 - 9
Effect of temperature on the resistance of individual red blood cells to flow through capillary-sized apertures; Lecklin T et al.; Low temperature can be expected to increase the resistance to deformation of red blood cells, but the effect of such changes on microcirculatory perfusion are unknown . We therefore analysed resistance to flow through capillary-sized apertures for individual human red blood cells, by micropipette aspiration (approximately 3 microm aperture) and pore transit analysis (approximately 5 microm), as well as average resistance to flow of red cell suspension through multipore filters (5-microm pores) . Over a range decreasing from 37 to 0 degrees C, rates of flow of single cells through the 3- and 5-microm apertures decreased monotonically by 2.5- to 3-fold . The changes were similar in magnitude to that expected for the viscosity of aqueous fluid (2.5-fold increase) . Average flow resistance measured by bulk filtration also increased in line with viscosity of water, while tendency to block pores was not increased . Micropipette aspiration of small membrane tongues showed that membrane rigidity increased as temperature was lowered, but by a factor rather less than the viscosity . Cell volume also responded rapidly to change in temperature, with lower temperature being associated with swelling, although this effect was much reduced in plasma compared with saline buffer . We conclude that, although increased resistance to deformation of red cells may impair microcirculation at low temperature, there is no structural change likely to induce more dramatic occlusion of flow . Moreover, the effect is comparable in magnitude to the increase predicted for changes in plasma and blood viscosity.

Int Arch Allergy Immunol, 1996 Sep, 111(1), 64 - 70
An approach to predictive testing of contact sensitizers in vitro by monitoring their influence on endocytotic mechanisms; Lempertz U et al.; Endocytotic activation of epidermal Langerhans cells (LC) by immunogenic haptens is an early event during development of allergic contact dermatitis . In this work a fast and objective flow-cytometric assay for predictive in vitro testing of contact sensitizers by monitoring their influence on endocytotic mechanisms in murine LC was developed . Epidermal cell suspensions were labelled with a monoclonal antibody directed to MHC class II molecules and pH-sensitive fluorochrome-coupled second-step reagents . For untreated LC a significant quenching of fluorescence intensity by internalization of the MHC-antibody complexes into acidic compartments was noticed . Similar results were obtained in the presence of irritants, the lectin concanavalin A and the phorbol ester phorbol 12-myristate 13-acetate . In contrast stimulation with several well-defined sensitizing compounds resulted in partial conservation of the fluorescence intensity due to the internalization of the labelled complexes into less acidic compartments . Monitoring this modulation of endocytosis is an effective in vitro method to test for properties typical for moderate and strong contact sensitizers . It will help to assess the risk of unknown chemicals to act as haptens and should be useful for restriction of animal experimentation in this field.

Int Arch Allergy Immunol, 1996 Sep, 111(1), 30 - 5
Eosinophil peroxidase deficiency in New Zealand white mice; Ohmori J et al.; Eosinophil peroxidase (EPO) is one of the granule enzymes in the eosinophil-specific granules and is distinct from myeloperoxidase . Here we report that peroxidase activity was absent in eosinophils of New Zealand White (NZW) mice . When NZW, New Zealand Black and their F1 mice were treated with cyclophosphamide followed by Toxocara canis infection, the kinetic changes in the number of eosinophils in peripheral blood, determined by counting in Hinkelman's diluting fluid, were almost comparable among the three strains . However, when their blood films were stained for peroxidase reaction, eosinophils of NZW mice, but not of the other strains, lacked EPO activity, though their specific granules were stained by eosin Y . Sudan black staining for phospholipid was also negative in eosinophils of NZW mice . EPO deficiency in NZW eosinophils was further confirmed by electron-microscopic observations and by measuring EPO activity in the extracts of eosinophil-rich cell suspensions . These results indicate that NZW eosinophils share most of the features with human EPO-deficient eosinophils, suggesting that the NZW mouse is a murine counterpart of human EPO deficiency.

Cancer Res, 1996 Sep 1, 56(17), 3986 - 92
Intrathecal chemotherapy with 1,3-bis(2-chloroethyl)-1-nitrosourea encapsulated into hybrid liposomes for meningeal gliomatosis: an experimental study; Kitamura I et al.; 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), one of the chloroethyl nitrosoureas, is effective against malignant glioma . To develop its use in intrathecal chemotherapy, we encapsulated BCNU in hybrid liposomes composed of dimyristoylphosphatidylcholine and micellar surfactants (Tween 20) and dissolved it in artificial cerebrospinal fluid (lipo-BCNU) . We then studied the toxicity of hybrid liposomes and cellular proliferation inhibition of lipo-BCNU in vitro . We found that 3 mM hybrid liposomes did not affect the viability of human endothelial cells and that lipo-BCNU inhibited the proliferation of human glioma cell lines U-105MG, U-251MG, and U-373MG, and rat glioma cell lines C6 and 9L in a concentration-dependent fashion . Wistar rats that were administered lipo-BCNU intracisternally showed no weight loss, neurological symptoms, or histological changes of the brain and spinal cord . A Wistar rat model of meningeal gliomatosis was established by intracisternal inoculation of 0.1 ml cell suspension containing 1 x 10(6) or 5 x 10(6) viable C6 glioma cells . Two days after inoculation, lipo-BCNU (BCNU, 2.5 mg/kg) was administered intracisternally . When 1 x 10(6) glioma cells were inoculated (experiments 1 and 2), the median survival times were 24.5 and 26 days in the control groups and 32 and 45 days in the lipo-BCNU-treated groups . respectively . When 5 x 10(6) glioma cells were inoculated (experiments 3-6), the median survival times were 17-29.5 days in the control groups and 23-44 days in the treated groups, respectively . Significantly prolonged survival was obtained in three of six experimental groups . After the administration of 1 ml lipo-BCNU (BCNU, 4.67 mM) or 1 ml BCNU solubilized with 5% dextrose/water (BCNU, 4.67 mM) into the cisterna magna of dogs, the cisterna magna cerebrospinal fluid was sampled, and the BCNU concentrations were assayed by high-performance liquid chromatography . The half-life of the lipo-BCNU was longer than that of BCNU solubilized with 5% dextrose/water . These results suggest that the intrathecal administration of lipo-BCNU may be possible for the treatment of meningeal gliomatosis.

J Invest Dermatol, 1996 Sep, 107(3), 354 - 9
Epidermal protein kinase C-beta 2 is highly sensitive to downregulation and is exclusively expressed in Langerhans cells: downregulation is associated with attenuated contact hypersensitivity; Goodell AL et al.; Treatment of mice with multiple topical applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) or diacylglycerol resulted in a preferential decrease in epidermal protein kinase C-beta 2 (PKC-beta 2) compared with PKC-alpha as determined by western analysis . When PKC-alpha was decreased by 40%, PKC-beta 2 could no longer be detected, suggesting that PKC-beta 2 is more sensitive to downregulation, and/or specific epidermal cell types that contain PKC-beta 2 are more sensitive to TPA/diacylglycerol . To address this issue, we isolated Langerhans cells (LCs) from epidermal cell suspensions with immunomagnetic beads and an antibody to the class II major histocompatibility complex . Northern blot analysis revealed a PKC-beta 2 signal in isolated LCs that was 40-fold greater than that observed in unfractionated epidermal cells, and no PKC-beta 2 signal was detected in epidermal cells depleted of LCs, indicating that PKC-beta 2 is expressed exclusively in LCs within the epidermis . Western blot analysis confirmed the presence of PKC-beta 2 in LCs . PKC-beta 2 was highly sensitive to downregulation, because a single application of TPA resulted in a 90% loss of PKC-beta 2 within 6 h without a decrease in the number of LCs . To determine whether the decreased level of PKC-beta 2 within LCs was associated with an alteration in contact hypersensitivity, we treated mice with only a single application of TPA, and 6 hours later mice were sensitized with 2,4-dinitrofluorobenzene on the same dorsal area . Subsequent challenge revealed a 60% decrease in contact hypersensitivity in TPA-treated mice . These data indicate that (i) within the epidermis, PKC-beta 2 is highly sensitive to downregulation and is exclusively expressed in LCs, and (ii) the downregulation of PKC-beta 2 is associated with impaired LC function with respect to contact hypersensitivity.

Biochem Biophys Res Commun, 1996 Aug 23, 225(3), 924 - 31
Promotion of heat-induced apoptosis in FM3A cells by protease inhibitors; Zhu WG et al.; Although it has been shown that proteases may play a positive role in in causing apoptosis of some cells, we report here that, on the contrary, protease inhibitors can promote heat-induced apoptosis in FM3A cells . Cysteine protease inhibitor, trans-Epoxy-succinyl-L-leucylamido-(4-guanidino)butane (E-64, 100 micrograms/ml) and aspartate protease inhibitor, pepstatin-A (100 micrograms/ml) were used to test hyperthermic effect on FM3A cells and showed remarkable cytotoxicity when they were present in cell suspension during heating at 44 degrees C . The cytotoxicity was due to promotion of heat-induced apoptosis as judged by DNA agarose electrophoresis . Furthermore, using flow cytometric analysis, we observed a decrease in the G0/G1 phase cell and an increase in the S phase cell as well as increased apoptosis after heat shock . E-64 and pepstatin-A exhibited a promotive effect on the changes of cell cycle induced by heat . The data presented suggest that the enhancement of hyperthermic cell killing by protease inhibitors may be related to promotion of heat-induced apoptosis and changes of cell cycle.

J Clin Invest, 1996 Aug 15, 98(4), 906 - 13
Syntaxin-4 is localized to the apical plasma membrane of rat renal collecting duct cells: possible role in aquaporin-2 trafficking; Mandon B et al.; To evaluate the possible role of a putative vesicle-targeting protein, syntaxin-4, in vasopressin-regulated trafficking of aquaporin-2 water channel vesicles to the apical plasma membrane of renal collecting duct cells, we have carried out immunoblotting, immunocytochemistry, and reverse transcription (RT)-PCR experiments in rat kidney . Immunochemical studies used an affinity-purified, peptide-directed polyclonal antibody to rat syntaxin-4 . Immunoblots using membrane fractions from inner medullary collecting duct (IMCD) cell suspensions revealed a solitary protein of 36 kD, the expected molecular mass of syntaxin-4 . This protein was enriched in a plasma membrane-enriched membrane fraction from IMCD cells . Immunoperoxidase immunocytochemistry in 0.85-microm cryosections from rat inner medulla revealed discrete labeling of the apical plasma membrane of IMCD cells . RT-PCR demonstrated the presence of syntaxin-4 mRNA in microdissected IMCD segments, confirmed by direct sequencing of the PCR product . In addition, RT-PCR experiments demonstrated syntaxin-4 mRNA in glomeruli, vasa recta, connecting tubules, and thin descending limbs of Henle's loops . The demonstrated localization of syntaxin-4 in the apical plasma membrane of collecting duct principal cells, coupled with previous demonstration of syntaxin-4's putative cognate receptor VAMP2 in aquaporin-2-containing vesicles, supports the view that these proteins could play a role of aquaporin-2 vesicle targeting to the apical plasma membrane.

Cancer, 1996 Aug 15, 78(4), 819 - 26
Flow cytometric quantitation of the proliferation-associated nuclear antigen p105 and DNA content in patients with renal cell carcinoma; Yokogi H; BACKGROUND . Although tumor stage and grade are important prognostic parameters for patients with renal cell carcinoma, postnephrectomy survival is often difficult to predict . Therefore identifying patients at high risk for disease progression is critical . Using two-color flow cytometry, DNA and proliferation-associated nuclear antigen p105 contents were measured simultaneously in 75 patients with renal cell carcinomas and the ability of these results to predict the survival was assessed . METHODS . Flow cytometric study of the proliferation-associated nuclear antigen p105 was done on cancer cell suspensions from 75 patients with renal cell carcinomas . By setting the cutoff line at the level between the brighter and the dimmer subpopulations in the diploid G0G1 region, the p105-labeling rate was calculated by the p-105DNA dual fluorescence analysis . RESULTS . The mean p105-labeling rate was 66.8% (median: 67.6%; range: 33.9-93%) . The 5-year survival rate of patients with high p105-labeling tumors was significantly lower than that of patients with low labeling tumors (P < 0.05) . The following factors were examined univariantly as prognostic factors: Robson's stage, DNA ploidy pattern, grade, and p105-labeling rate . All of these factors except for DNA ploidy pattern were prognostic . The Cox multivariate regression analysis was performed with the three statistically significant variables . Accordingly, these three factors were significantly associated with survival rate and were found to be independent prognostic factors . CONCLUSIONS . The measurement of p105 may provide useful information for predicting prognosis of patients with renal cell carcinoma.

FEBS Lett, 1996 Aug 5, 391(1-2), 175 - 80
Levels of endogenous cytokinins, indole-3-acetic acid and abscisic acid during the cell cycle of synchronized tobacco BY-2 cells; Redig P et al.; Correlation between cell cycle progression and endogenous levels of plant hormones was studied in synchronized tobacco BY-2 cell suspension cultures . Sixteen different cytokinins, indole-3-acetic acid (IAA) and abscisic acid (ABA) were extracted using solid-phase anion exchange chromatography in combination with immunoaffinity purification, and quantified by mass spectrometry . No significant correlation could be identified for IAA and ABA . In contrast, there were sharp peaks in the levels of specific cytokinins (zeatin- and dihydrozeatin-type) at the end of the S phase and during mitosis . The levels of other cytokinins analyzed, including zeatins N- and O-glucosides, remained low, suggesting that the increased amounts of their corresponding non-glucosylated form resulted from de novo synthesis . These findings suggest that zeatin- and dihydrozeatin-type cytokinins might play a specific regulatory role in the progression of the plant cell cycle . One hypothesis to explain cytokinin action is based on a specific interaction with kinases that regulate cell cycle progression, as has been recently shown for the cytokinin analogue olomoucine.

J Reprod Immunol, 1996 Aug, 31(1-2), 81 - 95
A progesterone-dependent immunomodulatory protein alters the Th1/Th2 balance; Szekeres-Bartho J et al.; In the presence of progesterone, lymphocytes from pregnant females produce an immunomodulatory protein known as progesterone induced blocking factor (PIBF) . We tested the effect of this protein on cytokine production by mitogen-activated lymphocytes . Spleen cells from Balb/c mice were incubated with Con A in the presence or absence of PIBF . Supernatants from the activated cells were collected and the concentrations of IL-3, IL-4, IL-10 and IFN gamma were determined by ELISA . In supernatants from spleen cells activated in the presence of PIBF the concentration of IFN gamma was not substantially different from controls however, the same spleen cells produced significantly more IL-10, IL-3 and IL-4 than those cultured without the progesterone-induced protein . When CD4+ and CD8+ enriched cell suspensions were used as producers of the cytokines it was found that both populations reacted with an equally increased production of IL-3, IL-4 and IL-10 in the presence of PIBF . Although cytokine-producing Th cells can be identified within the CD4+ population, the present findings suggest that involvement of CD8+ cells in altered cytokine production cannot be excluded . These data indicate that the PIBF affects the Th1/Th2 balance, and via altered cytokine ratios it contributes to decreased cell-mediated responses during pregnancy.

J Hematother, 1996 Aug, 5(4), 339 - 49
Concomitant mobilization of plasma cells and hematopoietic progenitors into peripheral blood of patients with multiple myeloma; Lemoli RM et al.; In this article, we review neoplastic contamination in the peripheral blood (PB) of patients with multiple myeloma (MM) upon stem cell mobilization . We first evaluated PB samples from pretreated MM patients following administration of high-dose cyclophosphamide (Cy, 7 g/m2 or 4 g/m2) and granulocyte colony-stimulating factor (G-CSF) for the presence of myeloma cells as well as hematopoietic progenitors . Plasma cells containing intracytoplasmic immunoglobulin (cIg) were counted by immunofluorescence microscopy after incubation with appropriate antisera against light and heavy chain Ig . Flow cytometry studies were performed to determine the presence of malignant B lineage elements, using monoclonal antibodies against the CD19 antigen and the monotypic light chain . Prior to PBSC mobilization, circulating plasma cells were detected in all MM patients at 0.1%-1.8% of the mononuclear cell (MNC) fraction (mean value 0.7 +/- 0.4% SD) . In these patients, a higher absolute number of PB neoplastic cells was detected after administration of chemotherapy and G-CSF . Kinetic analysis showed a pattern of tumor cell mobilization similar to that of normal hematopoietic progenitors, with the peak coinciding with the optimal period for the collection of PBSC . The absolute number of plasma cells showed a 10-50-fold increase over the baseline value . Apheresis products contained 0.7 +/- 0.2% SD myeloma cells (range 0.2%-2.7%), which demonstrated the capacity of plasma cells to proliferate, differentiate, and mature in response to c-kit ligand (SCF), IL-3, IL-6, and a combination of IL-3 and IL-6 . Subsequently, in an attempt to reduce tumor cell contamination prior to autologous transplantation, circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, cryopreserved, and used to reconstitute bone marrow (BM) function after myeloablative therapy in 13 patients . The median purity of the enriched CD34+ cell population was 89.5% (range 51%-94%), with a 75-fold enrichment compared with the pretreatment samples . The median overall recovery of CD34+ cells and CFU-GM was 58% (range 33%-95%) and 45% (range 7%-100%), respectively . Positive selection of CD34+ cells resulted in 2.5-3 log depletion of plasma cells and CD 19+ B lineage cells as determined by immunofluorescence studies, although DNA analysis of the CDR III region of the IgH gene demonstrated the persistence of minimal residual disease (MRD) in 5 of 6 patient samples studied . Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (TBI, 1000 cGy) and high-dose melphalan (140 mg/m2) or melphalan (200 mg/m2) alone . They received a median of 5 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis . The median time to 0.5 x 10(9) neutrophils, 20 x 10(9) and 50 x 10(9) platelets/L of PB was 10, 11, and 12 days, respectively . These results, as well as other clinically significant parameters, did not significantly differ from those of patients (n = 13) receiving unmanipulated PBSC following the same pretransplant conditioning regimen . Our data demonstrate the concomitant mobilization of tumor cells and hematopoietic progenitors in the PB of MM patients . Positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3 log and provides a cell suspension capable of restoring normal hematopoiesis following a TBI-containing conditioning regimen.

Neurol Res, 1996 Aug, 18(4), 297 - 304
CNS immunological modulation of neural graft rejection and survival; Borlongan CV et al.; Neural transplantation therapy as a possible alternative treatment for neurological movement disorders, such as in Parkinson's disease (PD), has accentuated research interest on the immune status of the central nervous system (CNS) . Most animal studies concerned with neural transplantation for the treatment of PD have utilized dopamine (DA) neurons from tissues of the embryonic ventral mesencephalon . Rat embryonic DA neurons, grafted either as solid blocks or dissociated into a cell suspension and stereotaxically injected intraparenchymally into a rat lesion model of PD, have been shown to survive and form connections with the host brain, and ameliorate the behavioral deficits of PD . Similarly, studies on nonhuman primate models of PD provide considerable support for neural transplantation of DA neurons as an experimental clinical procedure for the treatment of PD . To this end, experimental clinical trials have been centered upon transplantation of the embryonic ventral mesencephalic cells for PD patients . Although not conclusive, the findings from clinical studies have provided some evidence that most patients with marked increases in fluorodopa uptake (indicating graft survival) have been immunosuppressed . Furthermore, immune reactions have been observed in rats xenografted with human embryonic tissue . Of note, embryonic ventral mesencephalic tissues compared to adult tissues produce better morphological and long-lasting behavioral amelioration of the neurobehavioral deficits of PD, thus advocating the use of grafts from young donors (embryo) to circumvent the CNS immune rejection . The possible graft rejection due to CNS immune reactions, coupled with the social and ethical problems surrounding the use of embryonic neural tissue, and the logistical problems concerning tissue availability have prompted the development of alternative sources of DA-secreting cells . To circumvent these obstacles, several methods have been suggested including the use of immunosuppressants such as Cyclosporine-A, transplantation of autografts, polymer-encapsulated DA-secreting cells, co-culturing and co-transplantation of DA-secreting cells with microcarrier beads, with Sertoli cells, or with fragments of a monoclonal antibody that can mask the MHC class I antigens, and genetically modifying cells that can withstand CNS immune reactions . Some of these techniques allow transplantation of allograft (same species transplantation), or even xenograft (cross species transplantation) without immunosuppression of the recipient . We discuss recent CNS immunosuppression techniques that pose some promise for enhanced survival of neural grafts . When possible, advantages and disadvantages of each method are presented . Hopefully, such critical analysis of different immunosuppression techniques will produce innovated ideas that will lead to a better understanding of CNS immune response and its modulatory function on graft rejection and survival.

Graefes Arch Clin Exp Ophthalmol, 1996 Aug, 234 Suppl 1, S96 - 100
Nerve growth factor delays retinal degeneration in C3H mice; Lambiase A et al.; BACKGROUND: The aim of the present study was to investigate the biological role of nerve growth factor (NGF) on retinal degeneration in the C3H mouse strain . This strain is characterized by a single gene mutation (rd) which leads to photoreceptor degeneration resembling human retinitis pigmentosa . METHODS: Neural retinas from 1- to 25 day-old C3H mice were dissected from outer ocular tissues, dissociated in cell suspension, stained with a vital dye and counted in a hemocytometer . For in vivo study, NGF was injected into the intraocular or retro-ocular area, and at the end of the treatment the mice were killed . The eyes were enucleated, fixed and cut by cryostat into 14-microns serial sections . The serial sections were stained with hematoxylin-eosin and the outer nuclear layer (ONL) was measured using a computerized image analysis system . RESULTS: An intraocular injection of NGF, or repeated retro-ocular injections, induced a significant increase in ONL thickness compared to controls . CONCLUSION: Our data show that NGF inhibits retinal degeneration in C3H mice . The mechanism(s) underlying the protective action of NGF against retinal cell death remains to be established.

Plant Mol Biol, 1996 Aug, 31(5), 983 - 92
Characterization of Vitis vinifera L . glutamine synthetase and molecular cloning of cDNAs for the cytosolic enzyme; Loulakakis KA et al.; Grapevine (Vitis vinifera L.) glutamine synthetase (GS) was analysed into two distinct classes of isoforms; one of them was present in both leaf and root tissues while the other one showed leaf specificity . Western blot analysis revealed that grapevine GS consists of three types of polypeptides of distinct size and differential tissue specificity . Two structurally distinct cDNA clones, pGS1;1 and pGS1;2, encoding grapevine GS were isolated from a cell suspension library and characterized . Both clones contained open reading frames encoding for polypeptides of 356 amino acids with a predicted molecular mass of about 39 kDa . Although the coding sequences of pGS1;1 and pGS1;2 were 84% similar, their 5'- and 3'-untranslated sequences showed only 40% similarity . The coding sequences of the two clones and the derived amino acid sequences showed higher homology to cytosolic than to chloroplastic GSs of other higher plants indicating that the cDNAs isolated encode for cytosolic isoforms of grapevine GS . Southern blot analysis suggested the existence of more than two GS genes in the grapevine genome . In northern blots both clones were hybridized to mRNAs of about 1.4 kb that are differentially expressed in the various tissues . Supply of nitrate or ammonium in the cell suspension culture medium, as a sole nitrogen source, resulted in differential response of the pGS1;1- and pGS1;2-related genes.

Biol Reprod, 1996 Aug, 55(2), 439 - 44
Isolation of the synchronized A spermatogonia from adult vitamin A-deficient rat testes; van Pelt AM et al.; A method for isolating A spermatogonia from the adult vitamin A-deficient (VAD) rat testis is described . After removal, the testes were decapsulated and tubules were dissected . An enzymatic digestion with collagenase, hyaluronidase, and trypsin was performed first to eliminate most of the interstitial cells . A second digestion with collagenase and hyaluronidase was performed to obtain a cell suspension with a high number of A spermatogonia . The cell suspension was further enriched with A spermatogonia by preplating on peanut agglutinin and separating on a discontinuous Percoll gradient . By this procedure, purification of the suspension to 70-90% A spermatogonia was obtained . In the seminiferous tubules of the VAD rats, only Sertoli cells, A spermatogonia, and some preleptotene spermatocytes are present . In our rats, the A spermatogonia are almost all arrested in the G1 phase of the cell cycle before the S phase of A1 spermatogonia, and presumably before their differentiation into A1 spermatogonia . After administration of vitamin A, spermatogenesis starts synchronously from these A spermatogonia . The isolation of these synchronized A spermatogonia opens ways to investigate the regulation of differentiation and proliferation of A spermatogonia and the biochemical characteristics of the subsequent types of A spermatogonia.

Int J Radiat Oncol Biol Phys, 1996 Aug 1, 36(1), 125 - 34
Feasibility of measuring radiation-induced DNA double strand breaks and their repair by pulsed field gel electrophoresis in freshly isolated cells from the mouse RIF-1 tumor; van Waarde MA et al.; PURPOSE: To examine the technical feasibility of pulsed field gel electrophoresis (PFGE) as a predictive assay for the radioresponsiveness of tumors . Induction and repair of DNA double strand breaks (DSBs) in a freshly prepared cell suspension from a RIF-1 tumor (irradiated ex vivo) was compared with DSB induction and repair in exponentially growing RIF-1 cells in culture (irradiated in vitro) . METHODS AND MATERIALS: A murine RIF-1 tumor grown in vivo was digested, and cells were exposed to x-rays (ex vivo) at doses of 1 to 75 Gy . DNA damage was measured using CHEF (clamped homogeneous electric fields) electrophoresis . Repair kinetics were studied at 37 degrees C for 4 h after irradiation . Radiosensitivity was determined by clonogenic assay, and cell cycle distributions by flow cytometry . For comparison, a trypsinized suspension of exponentially growing RIF-1 cells in vitro was run parallel with each ex vivo experiment . RESULTS: Induction of DSBs, expressed as % DNA extracted from the plug, was similar in the in vitro and ex vivo irradiated cells . Compared to repair rates in vitro cultured RIF-1 cells, repair kinetics in a freshly prepared cell suspension from the tumor were decreased, unrelated to differences in radiosensitivity . Differences in repair could not be explained by endogenous DNA degradation, nor by influences of enzymes used for digestion of the tumor . A lower plating efficiency and differences in ploidy (as revealed by flow cytometry) were the only reproducible differences between in vivo and in vitro grown cells that may explain the differences in repair kinetics . CONCLUSIONS: The current results do not support the idea that PFGE is a technique robust enough to be a predictive assay for the radiosensitivity of tumor cells.

J Exp Med, 1996 Aug 1, 184(2), 387 - 96
Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation; Asano M et al.; Neonatal thymectomy (NTx), especially around day 3 after birth, causes various organ-specific autoimmune diseases in mice . This report shows that: (a) T cells expressing the interleukin 2 receptor alpha chains (CD25) ontogenically begin to appear in the normal periphery immediately after day 3, rapidly increasing within 2 wk to nearly adult levels (approximately 10% of CD3+ cells, especially of CD4+ cells); (b) NTx on day 3 eliminates CD25+ T cells from the periphery for several days; inoculation immediately after NTx of CD25+ splenic T cells from syngeneic non-Tx adult mice prevents autoimmune development, whereas inoculation of CD25- T cells even at a larger dose does not; and furthermore, (c) similar autoimmune diseases can be produced in adult athymic nu/nu mice by inoculating either spleen cell suspensions from 3-d-old euthymic nu/+ mice or CD25+ cell-depleted spleen cell suspensions from older, even 1-yr-old, nu/+ mice . The CD25- populations from neonates or adults are also similar in the profile of cytokine formation . These results, taken together, indicate that one aspect of peripheral self-tolerance is maintained by CD25+ T cells that sustain potentially pathogenic self-reactive T cells in a CD25- dormant state; the thymic production of the former is developmentally programmed to begin on day 3 after birth in mice . Thus, NTx on day 3 can, at least transiently, eliminate/reduce the autoimmune-preventive CD25+ T cells, thereby leading to activation of the self-reactive T cells that have been produced before NTx.

J Exp Med, 1996 Aug 1, 184(2), 325 - 36
Differential effects of interleukin-15 and interleukin-2 on differentiation of bipotential T/natural killer progenitor cells; Leclercq G et al.; Bipotential T/natural killer (NK) progenitor cells are destined to differentiate mainly into T cell receptor (TCR) alpha beta and TCR gamma delta cells in a thymic microenvironment, whereas extrathymically they selectively develop into NK cells . The exact environmental conditions that are required for differentiation into these three leukocyte populations are largely unknown . In this report, we have investigated and compared the effect of interleukin (IL)-15 and IL-2 in this process . The IL-15 receptor is composed of the gamma and beta chains of the IL-2 receptor (IL-2R gamma and IL-2R beta) and of a specific alpha chain (IL-15R alpha) . Here, it is shown that IL-15 mRNA is mainly expressed in thymic epithelial stromal cells, whereas IL-2 mRNA is exclusively expressed in thymocytes . IL-2R beta-expressing cells were present in the fetal thymus with a CD25-CD44+Fc gamma R+HSA-/low TCR- phenotype, which is characteristic of progenitor cells . These cells also expressed IL-15R alpha messenger RNA . Sorted IL-2R beta + TCR- cells differentiated into TCR alpha beta and TCR gamma delta cells after transfer to alymphoid thymic lobes, whereas culture of the same sorted cells in cell suspension in the presence of IL-15 resulted in the generation of functional NK cells . This shows that IL-2R beta +TCR- cells of the fetal thymus contain bipotential T/NK progenitors . Addition of low concentrations of IL-15 to fetal thymic organ culture (FTOC) resulted in an increase of all T cell subpopulations . The largest expansion occurred in the TCR gamma delta compartment . In contrast, low concentrations of IL-2 did not result in a higher total cell number and did not induce outgrowth of TCR gamma delta cells . High concentrations of IL-15 blocked TCR alpha beta development and shifted differentiation towards NK cells . Differentiation towards TCR gamma delta cells still proceeded . High concentrations of IL-2 similarly induced development into NK cells, but the cell number was fourfold lower than in IL-15 cultures . Importantly, blocking of IL-2R alpha in IL-2-treated FTOC resulted in a drastic increase in cell number, indicating that IL-2R alpha negatively regulates cell expansion . Collectively, these experiments provide direct evidence that IL-15 and IL-2 differentially affect the differentiation of bipotential T/NK progenitors.

J Appl Bacteriol, 1996 Aug, 81(2), 133 - 8
Development of a method based on alkaline gel electrophoresis for estimation of oxidative damage to DNA in Escherichia coli; Zirkle RE et al.; A method for estimating DNA strand breakage and subsequent repair based on alkaline gel electrophoresis was developed and tested with isogenic strains of Escherichia coli deficient in DNA repair enzymes . Samples from a cell suspension were removed at 2 min intervals following a 15 min exposure to 20 mmol l-1 H2O2 . Catalase was added and the cells were embedded in blocks of low-melting point agarose and lysed . After alkaline gel electrophoresis, photographs of the gels were taken and the relative lengths of the distributions of DNA fragments were measured with a scanner and computer . The lengths were correlated with survival of the cells exposed to H2O2 and with the importance of particular DNA repair enzymes . Alkaline gel electrophoresis appears to be a relatively simple method for analysing the level of H2O2-caused DNA damage and repair in E . coli.

Blood, 1996 Aug 1, 88(3), 1005 - 12
Antisense oligodeoxynucleotide combination therapy of primary chronic myelogenous leukemia blast crisis in SCID mice; Skorski T et al.; The proliferation of chronic myelogenous leukemia (CML) cells and the transformation of normal hematopoietic cells by BCR-ABL appear to require the expression of a functional MYC protein, suggesting an approach to treatment of Philadelphia leukemias based on simultaneous targeting of BCR-ABL and c-MYC . To test this hypothesis, CML-blast crisis (CML-BC) primary cells were treated in vitro with bcr-abl and c-myc antisense phosphorothioate oligodeoxynucleotides ({S}ODNs), individually or in combination . Compared with antisense ODNs targeting of individual oncogenes, downregulation of both BCR-ABL and c-MYC by specific antisense {S}ODNs resulted in a synergistic antiproliferative effect . Colony formation of normal bone marrow cells was not affected by either treatment . To assess the therapeutic potential of multiple oncogene downregulation, SCID mice injected with CML-BC primary cells were treated systematically with equal doses of bcr-abl or c-myc antisense {S}ODNs or with a combination of both antisense {S}ODNs . Compared with mice treated with individual compounds, the disease process was significantly retarded in the group treated with both {S}ODNs as revealed by flow cytometry, clonogenic assay, and RT-PCR analysis to detect leukemic cells in mouse tissue cell suspensions . These effects correlated with a markedly increased survival of leukemic mice treated with both antisense {S}ODNs . Leukemic cells harvested from antisense {S}ODN-treated mice were sensitive to the effects of antisense {S}ODNs in vitro, suggesting that the treatment can be successfully repeated . These data demonstrate the therapeutic potential of targeting multiple cooperating oncogenes.

J Biol Chem, 1996 Jul 26, 271(30), 17944 - 8
Release of leukotriene A4 versus leukotriene B4 from human polymorphonuclear leukocytes; Sala A et al.; The reactive intermediate formed by 5-lipoxygenase metabolism of arachidonic acid, leukotriene A4, is known to be released from cells and subsequently taken up by other cells for biochemical processing . The objective of this study was to determine the relative amount of leukotriene A4 synthesized by human polymorphonuclear leukocytes (PMNL) that is available for transcellular biosynthetic processes . This was accomplished by diluting cell suspensions and measuring the relative amounts of enzymatic versus nonenzymatic leukotriene A4-derived metabolites after challenge with the Ca2+ ionophore A23187 . Nonenzymatic leukotriene A4-derived metabolites were used as a quantitative index of the amount of leukotriene A4 released into the extracellular milieu . The results obtained demonstrated that in human PMNL, the relative amounts of nonenzymatic versus enzymatic leukotriene A4-derived metabolites increased with decreasing cell concentrations . After a 20-fold dilution of PMNL in cell preparations, a doubling in the amount of nonenzymatic leukotriene A4-derived metabolites was observed following challenge (from 53.9 +/- 1.3 to 110.4 +/- 8.9 pmol/10(6) PMNL, p < 0.01) . Reduction of possible cell-cell interactions by dilution suggested that over 50% of leukotriene A4 synthesized is released from the PMNL . These data provide evidence that, in human PMNL preparations, transfer of leukotriene A4 to neighboring PMNL is taking place, resulting in additional formation of leukotriene B4 and its omega-oxidized metabolites 20-hydroxy- and 20-carboxy-leukotriene B4 . Neutrophil reuptake of extracellular leukotriene A4 leads to an underestimation of the fraction of leukotriene A4 that is in fact available for transcellular metabolism when tight cell-cell interactions occur, such as during PMNL adhesion to the microvascular endothelium and diapedesis.

Blood, 1996 Jul 15, 88(2), 505 - 10
The supportive effects of erythropoietin and mast cell growth factor on CD34+/CD36- sorted bone marrow cells of myelodysplasia patients; Brada S et al.; In the present study, we analyzed the capacity of CD34+/CD36- sorted bone marrow cells of myelodysplasia patients (n = 4) to differentiate along the erythroid lineage in the presence of erythropoietin (Epo) and mast cell growth factor (MGF) . Two subgroups could be identified . In 6 patients, a normal number of burst-forming units-erythroid (BFU-Es) were cultured from CD34+/CD36- sorted cells . Cells from these patients did have the capacity to differentiate to colony-forming units-erythroid (CFU-Es) progenitors in cell suspension cultures with Epo plus MGF followed by Epo in the culture assay . Moreover, the cells became CD34-/CD36+/gly-cophorin A (GpA)+ after 7 days of culture with Epo plus MGF, a pattern comparable to that of normal progenitors . In contrast, in 8 patients, a different pattern was observed . No BFU-Es or a low number of BFU-Es were cultured from the CD34+/CD36- sorted cell fraction that was, in most of the cases, incapable of differentiating to CFU-E progenitors . Flow cytometry of the sorted population showed that, after 7 days of culture with Epo plus MGF, a high proportion of CD34+/CD36- cells persisted, whereas a low proportion of cells became CD34-/CD36+/GpA+ . The unresponsiveness is not caused by the used growth factor combination, because the addition of interleukin-3 did not correct the defect . Evi-1 expression was studied in 9 cases to show whether an aberrant Evi-1 expression correlates with a disturbed erythroid development . Evi-1 expression was shown in 4 of 9 cases, whereas 3 of 9 cases did have a disturbed erythroid differentiation . In summary, the results show that the defects in the erythroid development in a subpopulation of patients with myelodysplasia is localized at an early stage of the erythroid differentiation and is associated with the persistent expression of the CD34 antigen and, in some cases, with the expression of Evi-1.

Neuroimmunomodulation, 1996 Jul-Aug, 3(4), 247 - 53
Effect of cholinergic stimulation on free intracellular Ca2+ concentration in human lymphocytes; Laskowska-Bozek H et al.; Binding of ligand to receptor, in various types of cells, results in changes in calcium concentration, which is an important factor in cellular signal transduction . Lymphocytes receive signals from the parasympathetic nervous system through the cholinergic receptors . Cholinergic receptors mediate response to stimuli through changes of the IP3, or cAMP level and Ca2+ mobilization in various types of cells . The aim of this work was to measure changes in calcium concentration in cytosol of lymphocytes stimulated with acetylcholine agonists - carbachol (analogue of acetylcholine) or nicotine . Human peripheral blood mononuclear cells (PMBC) and lymphocytes from the leukemic cell lines Jurkat and Raji were used . Immune cells were preactivated with mitogen phytohemagglutinin (PHA) for PBMC and Jurkat cells or lipopolysaccharide for Raji cells . Two methods of measurement of calcium concentration were used . With the first, calcium concentration was measured in the suspension of cells loaded with the fluorescent dye Fura-2AM . With the other method, calcium concentration was assessed in single cells loaded with the fluorescent dye Indo-1AM . Using the method of single cell investigation, we observed an increase in the level of calcium concentration induced by carbachol and nicotine . The method of measuring the Ca2+ concentration in a cell suspension was found to be not sensitive enough for this purpose . The increase in calcium concentration resulted both from the stimulation of Ca2+ influx and from the release of Ca2+ from intracellular stores, most likely due to the increase in the concentration of IP3 . In conclusion, we suggest that the lymphocytes activated with PHA respond to cholinergic stimulation with an increase in their free cytoplasmic Ca2+ concentration.

Indian J Exp Biol, 1996 Jul, 34(7), 710 - 1
A simple technique for improving chromosome spreads from clumped metaphases; Rao NM et al.; A method has been described by which clumped metaphases either due to inadequate hypotonic KCl treatment or prolonged storage at 4 degrees C can be rescued . The cell pellet obtained from cell suspension following centrifugation was resuspended in freshly prepared Carnoy's fixative (1:3, acetic acid: methanol) at room temperature by vortexing . Twenty microliters of Triton X-100 at a concentration of 0.5% was added drop by drop while vortexing . Three changes with fixative containing 0.5% Triton X-100 were optimal for obtaining good metaphase spreads with complete removal of the cytoplasmic background . The advantage of this technique is that important patients' samples having clumped metaphases otherwise not useful for G-banding can be rescued and karyotyped by this method.

Biofizika, 1996 Jul-Aug, 41(4), 876 - 86
{Mechanism of human blood neutrophil activation by electric field pulses}; Malinin VS et al.; High voltage electric field pulses, was shown previously, induced activation of blood neutrophil respiratory burst, registered as increase in luminol-dependent chemiluminescence of cell suspension . The quantitative analysis of this phenomenon by fluorescent probes and radioisotope methods have shown that electric pulse induced neutrophil chemiluminescence is a result of Ca2+ ions entering the cells through reversible pores in plasma membrane . Electric pulse of amplitude 5 kV/cm generates two tens of reversible pores with average diameter nearly 2 nm and lifetime 1 minute . Total amount of calcium penetrating at this conditions from the medium (1 mM Ca2+) into the cells was as high as 1.2 fmole/cell that is nearly 3.6 mM concentration per cell volume . Whereas the increase in the cytoplasmic concentration of free calcium did not exceed 1.3 microM, that is more then 3 order less . Data presented is discussed with relation to possible biological role of electroporation by natural electric fields presented in living systems.

J Endod, 1996 Jul, 22(7), 346 - 51
Production of interleukin-1 by polymorphonuclear leukocytes resident in periradicular tissue; Miller GA et al.; Twenty-one patients undergoing endodontic surgery were identified . Periradicular tissue samples were recovered, and those showing significant numbers of polymorphonuclear leukocyte (PMN) infiltration were prepared for immunoperoxidase identification of interleukin (IL)-1 alpha and IL-1 beta-producing cells using specific polyclonal antibodies . In selected tissue specimens, 90% or more of the PMN's were found to stain positively for IL-1 alpha and IL-1 beta . In addition, significant numbers of plasma cells and tissue histiocytes stained positively for these IL's . Cell suspensions from selected periapical granuloma specimens, as well as from purified peripheral blood PMN's and peripheral blood mononuclear cells, were also subjected to IL-1 quantitation using a commercial ELISA procedure . Such cell suspensions were found to produce significant levels of IL and could be stimulated to produce increased levels after coculture with lipopolysaccharide . These results suggest that PMN's in inflammatory periradicular tissues may be a significant source of IL-1, and their possible roles in the establishment and resolution of periradicular lesions need to be re-evaluated.

Gut, 1996 Jul, 39(1), 77 - 81
Methionine derivatives diminish sulphide damage to colonocytes--implications for ulcerative colitis; Roediger WE et al.; BACKGROUND: Bacterial production of anionic sulphide is increased in the colon of ulcerative colitis and sulphides can cause metabolic damage to colonocytes . AIMS: To assess the reversal of the damaging effect of sulphide to isolated colonocytes by methionine and methionine derivatives . METHODS AND SUBJECTS: Isolated colonocytes were prepared from rat colons and 12 human colectomy specimens . In cell suspensions 14CO2/acetoacetate generation was measured from {1-14C}-butyrate (5.0 mmol/l) in the presence of 0-2.0 mmol/l sodium hydrogen sulphide . The effect of 5.0 mmol/l L-methionine, S-adenosylmethionine 1,4 butane disulphonate and DL-methionine-S-methylsulphonium chloride on sulphide inhibited oxidation was observed . RESULTS: In rat colonocytes sodium hydrogen sulphide dose dependently reduced oxidative metabolite formation from n-butyrate, an action reversed in order of efficacy by S-adenosylmethionine 1,4 butane disulphonate > DLmethionine-S-methyl-sulphonium chloride > L-methionine . In human colonocytes S-adenosylmethionine 1,4 butane disulphonate most significantly improved 14CO2 production (p = < 0.005) suppressed by sodium hydrogen sulphide . CONCLUSION: Sulphide toxicity in colonocytes is reversible by methyl donors . The efficiency of sulphide detoxification may be an important factor in the pathogenesis and treatment of ulcerative colitis for which S-adenosylmethionine 1,4 butane disulphonate may be of therapeutic value.

Acta Derm Venereol, 1996 Jul, 76(4), 282 - 6
Expression of gastrin-releasing peptide receptor in human skin; Staniek V et al.; Bombesin-related peptides are expressed in the skin of batrachians and mammals . As gastrin-releasing peptide belongs to this family, we searched for the presence and distribution of gastrin-releasing peptide receptors (GRPr) in the skin of healthy human adults by immunohistochemistry, flow cytometry and electron microscopy . The results indicated that GRPr are expressed on nerves and vessels in the dermis, on eccrine sweat glands, sebaceous glands and erector pili muscle . Within epidermis, staining was localized only on basal and suprabasal layer cells, or in the whole epidermis, according to the samples studied . Interestingly, suprabasal epidermal dendritic cells occasionally showed a strong labelling . Some of these epidermal dendritic cells were identified as Langerhans' cells by immunoelectron microscopy studies . Flow cytometry analysis of crude epidermal cell suspensions resulted in the expression of GRPr on about 43% of the cells . Therefore, we investigated whether human GRPr could modulate Langerhans' cells antigen-presenting functions . For this purpose, we added increasing concentrations of GRP (10(-12) to 10(-5) M) to mixed epidermal cell lymphocyte reactions . Allogeneic T-cell proliferation was not significantly modified when added to GRP-pretreated epidermal cells . In conclusion, we demonstrated the presence of GRPr in human skin, suggesting that GRP may modulate epidermal cell functions but does not modify antigenic presentation.

Neurosci Res, 1996 Jul, 25(3), 203 - 8
Trypsin promotes C6 glioma cell proliferation in serum- and growth factor-free medium; Amano H et al.; C6 glioma cells could be successively subcultured and maintained in serum- and growth factor-free medium (SF/GFF medium) . C6 cell proliferation in SF/GFF medium was positively correlated with the initial cell density at plating . This correlation disappeared when the medium had been renewed early after cell adhesion (3 h after plating), suggesting that C6 cell growth depends on some diffusible factor in the medium before renewal, and that this factor is not secreted from C6 cells in the assay culture but is transferred from the cell suspension . The supernatant of trypsinized C6 cell suspension (SCS), trypsin-EDTA solution for routine cell harvesting use, and modified trypsin of protein sequencing grade all promoted C6 cell proliferation at, appropriate dilutions or concentrations under SF/GFF conditions . The growth promoting effects of SCS and trypsin-EDTA solution were completely inhibited by soybean trypsin inhibitor . These results demonstrate that the serine protease trypsin has a proliferative effect on C6 cells continuously subcultured in SF/GFF medium . In addition, it is suggested that trypsin used for cell dispersion is transferred from cell suspension into the culture, where it promotes C6 cell growth after passage in our SF/GFF subculture system.

Cancer Immunol Immunother, 1996 Jul, 42(6), 351 - 6
Mucin-1-related T cell infiltration in colorectal carcinoma; Mulder WM et al.; Mucins (MUC) are highly glycosylated molecules widely expressed on epithelia of different origins, including colonic mucosa . Altered glycosylation processes in tumour cells result in the exposure of normally cryptic peptide epitopes, which may then be recognized as tumour-specific antigens . Recently, MUC1-specific antibodies were detected in the serum of a broad range of cancer patients, and from different tumours tumour-specific cytotoxic T lymphocytes (CTL) were isolated that recognized MUC1 . Absence of HLA restriction in the recognition has been ascribed to the highly repetitive sequence of the polypeptide core, allowing simultaneous recognition of multiple identical epitopes and cross-linking and aggregation of T cell receptor on mucin-specific T cells . We investigated the expression of MUC1 epitopes in 56 cell suspensions from Dukes' B to D colorectal carcinomas using antibodies that recognize distinct peptide sequences on the glycosylated or deglycosylated MUC1 protein backbone . No relation was observed between MUC1 expression, or the extent of its glycosylation, and Dukes' stage, tumour location and tumour differentiation, but a positive correlation was detected between the percentages of tumour cells expressing mucin-1 and the numbers of CD3+ infiltrating cells . These tumour-infiltrating lymphocytes contained, however, only a few MUC1-specific T lymphocytes, as CTL showing preferential killing of MUC1-expressing target cells were only obtained from one tumour . Since, in addition, the majority of colorectal carcinomas were found to express the fully glycosylated MUC1 glycoprotein, its potential role as a target antigen for T-lymphocyte-mediated immunotherapy in this tumour type is probably limited.

Brain Res Mol Brain Res, 1996 Jul, 39(1-2), 127 - 36
Expression of dopamine transporter mRNA and its binding site in fetal nigral cells transplanted into the striatum of 6-OHDA lesioned rat; Fujita M et al.; Neurological disorders in rat model of hemi-Parkinson's disease can be compensated by the transplantation of fetal nigral cells . However, the role of the dopamine transporter (DAT) in this recovery has not been clarified . To clarify this mechanism, we examined the expression of DAT in the caudate putamen (CPu) by in situ hybridization histochemistry (mRNA) and autoradiography (using the ligand {125I} beta-CIT, which labels DAT) and compared them with the recovery of motor disturbance revealed with methamphetamine-induced rotation . Models were made with the stereotaxic injection of 6-hydroxydopamine into the left side of the substantia nigra pars compacta . Cell suspensions from rat fetus (embryonic day 14-15) were transplanted into the lesioned side of CPu . Methamphetamine-induced rotation, expression of DAT mRNA, and {125I} beta-CIT binding were evaluated 2, 4 and 12 weeks after the transplantation . Methamphetamine-induced rotation recovered partly in the 2nd week and significantly in the 4th week . {125I} beta-CIT binding increased with time and the dense binding was detected 4 and 12 weeks after the transplantation . In all transplanted rats, cells expressing DAT mRNA were found in CPu . These results indicated that transplanted fetal dopaminergic cells maturated in CPu of host animals and extended nerve terminals where high density of DAT binding sites were found.

Endocrinology, 1996 Jul, 137(7), 2791 - 8
Signaling mechanisms underlying the insulinotropic effect of pituitary adenylate cyclase-activating polypeptide in HIT-T15 cells; Klinteberg KA et al.; The signaling mechanisms underlying the insulinotropic effect of the pancreatic neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) were studied in insulin-producing HIT-T15 cells . It was found that unlabeled PACAP38 dose-dependently displaced {125I}PACAP27 from HIT-T15 cell membranes, showing binding of the peptide to these cells . Furthermore, at levels above 0.1 nM, PACAP38 stimulated insulin secretion in a Ca2+ -dependent manner, requiring 0.5 mM glucose . The peptide also markedly increased the cellular cAMP content and slightly stimulated the formation of inositol phosphates . Moreover, PACAP38 elevated the cytoplasmic Ca2+ concentration ({Ca2+}cyt) in fura-2-AM-loaded cell suspensions . The PACAP38-induced increase in cellular cAMP content was also seen at zero glucose, whereas the increases in insulin secretion and {Ca2+}cyt were abolished by removal of glucose or extracellular Ca2+ . We conclude that PACAP38 binds to HIT-T15 cell membranes and acts in a glucose-dependent manner on adenylate cyclase to form cAMP, which both directly and indirectly, through increased {Ca2+}cyt, stimulates exocytosis.

Br J Cancer Suppl, 1996 Jul, 27, S61 - 4
Tirapazamine: hypoxic cytotoxicity and interaction with radiation as assessed by the micronucleus assay; Shibata T et al.; We investigated the cytotoxicity and the interaction with low-dose radiation (1-4Gy) of tirapazamine by the in vitro cytokinesis-block micronucleus (MN) assay . Murine SCCVII and human melanoma (G-361) cells were treated with tirapazamine under aerobic or hypoxic conditions for 1 h and the MN frequency was determined using cytochalasin-B . The cells were also treated with or without tirapazamine or KU-2285 (hypoxic cell sensitiser) under hypoxic conditions and irradiated with or without reaeration of the cell suspensions . A dose-dependent increase of MN frequency was observed by tirapazamine treatment and the hypoxic toxicity ratio was about 130 for SCCVII and 37 for G-361 . The radiation dose-response curves of MN frequency suggested that the interaction of tirapazamine with irradiation appeared to be essentially additive in both cell lines . In contrast, the dose-response curve became steeper by KU-2285 treatment . Combined effects of tirapazamine and irradiation on the hypoxic cells were much higher than the radiation effect on aerobic cells at low doses, while the effects of KU-2285 did not exceed that of aerobic irradiation . In conclusion, tirapazamine appeared to be superior to hypoxic radiosensitisers at clinically relevant doses, not because of aerobic radiosensitisation but because of its potent hypoxic cytotoxicity additive to radiation effect.

Am J Physiol, 1996 Jul, 271(1 Pt 1), G104 - 12
Insulin potentiates mitogenic effect of epidermal growth factor on cultured guinea pig gastric mucous cells; Ogihara S et al.; Epidermal growth factor (EGF) stimulated proliferation of gastric mucous epithelial cells from guinea pigs in serum-free culture conditions . Western blot analysis with antiphosphotyrosine antibody showed that EGF initiated tyrosine phosphorylation of a 170-kDa protein, and this protein was identical to the EGF receptor . Insulin was not mitogenic, but it potentiated the mitogenic effect of EGF . Tyrosine phosphorylation of additional proteins was not induced by the combined actions of insulin and EGF . Stimulation by EGF and/or insulin did not cause a calcium response . However, when insulin was added to cells pretreated with EGF for > 6 h, it elicited a rapid intracellular calcium concentration rise that was reproducible in both cell suspension and single cell analyses . This calcium response coincided with the translocation of protein kinase C (PKC) from the cytosolic to the particulate fraction . Phorbol 12-myristate 13-acetate also caused the translocation and stimulated proliferation of the cells . These results suggest that the calcium-dependent activation of PKC may participate in the potentiation of the mitogenic effect of EGF by insulin.

Am J Physiol, 1996 Jul, 271(1 Pt 1), C235 - 41
Measuring volume perturbation of proximal tubular cells in primary culture with three different techniques; Raat NJ et al.; Osmotic cell volume perturbations of rabbit proximal tubule (PT) in primary culture were measured using three independent techniques . Automatic cell thickness monitoring of PT monolayers revealed that cell volume rapidly increased by 39 +/- 2% in hypotonic medium (150 mosM), which was followed by partial regulatory volume decrease (RVD) . Subsequent incubation in hypertonic medium (500 mosM) rapidly decreased cell volume by 54 +/- 2% not followed by regulatory volume increase (RVI) . When cell volume in PT monolayers was derived from concentration changes in the trapped fluorescent dyes, fura 2 or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, osmotically induced cell volume changes appeared much smaller (17 +/- 1 and 22 +/- 2% for similar hypo- and hypertonicity, respectively) . However, changes in fluorescence intensity were most often not in agreement with anticipated cell volume changes . With the Coulter counter, a much larger shift in cell volume was observed in PT cell suspensions . In this situation, cell swelling in hypotonic medium amounted to 74 +/- 2% but was still followed by partial RVD . Hypertonicity resulted in a decrease in cell volume of 42 +/- 3% not followed by RVI . In conclusion, our study indicates that automatic cell thickness monitoring of an epithelial cell layer cultured on a permeable support provides more reliable data than monitoring changes in fluorescence intensity of trapped dyes.

Exp Neurol, 1996 Jul, 140(1), 21 - 36
Reconstruction of transected postcommissural fornix in adult rat by Schwann cell suspension grafts; Stichel CC et al.; Schwann cell (SC) transplantation has emerged as a powerful tool to promote regeneration in the lesioned central nervous system (CNS) . Most studies focused on the use of guidance channels to introduce the SCs into CNS neuropil; a technique that itself causes extensive damage to host tissue and is only applicable in superficial brain areas . The present study examines the efficacy of microinjected SC suspensions to promote structural reconstruction of the transected postcommissural fornix in the adult rat . Stereotactic, unilateral fornix transection was performed using a tungsten wire knife . Immediately after transection lesion cavities received either a Dulbecco's modified Eagle's medium injection or a pure SC suspension graft derived from a highly purified SC culture that was prepared from syngeneic rat (P1) sciatic nerves . After 4 days to 8 months, the implant characteristics as well as the structural reconstruction of the tract were analyzed using immunocytochemical methods and anterograde tracing techniques . Numerous SCs of the graft could be identified for up to 8 months . They rapidly dispersed from the injection site and migrated freely for considerable distances into the host tissue . The SCs exhibited a low proliferation activity that ceased within 2 weeks after transplantation . They did not prevent retrograde axonal degeneration of the fornix tract for a short distance (600 micron) but promoted structural reconstruction of the transected fornix tract . Regenerating fibers traversed the lesion site and extended along their former pathway up to the mammillary body, their proper target . Moreover, the applied transplantation technique allowed remyelination of the regenerating fibers by host oligodendrocytes . In conclusion, microtransplantation of SC suspensions represents a promising strategy for promoting structural reconstruction of lesioned CNS projections.

Eur J Biochem, 1996 Jun 15, 238(3), 737 - 43
Electropermeabilization of intact maize cells induces an oxidative stress; Sabri N et al.; By applying electric field pulses through cell suspensions, cell membranes can be permeabilized transiently, giving free access to the cytosol . Electropulsation is now routinely used in cell biology when introducing various molecules such as proteins and nucleic acids into the cell . But the molecular and cellular bases of cell electropermeabilization are still unclear . In the present study, we observed that electropermeabilization of intact black Mexican sweet (BMS) maize cells induces a generation of oxygen species (oxidative jump) . Using the chemiluminescent probe lucigenin, we have shown that the electro-induced chemiluminescent response depends on the level of the stress factor as shown by its dependence on the electric parameters (electric field intensity, duration, and number of pulses) . While the electroinduced cell permeabilization has a short life, the oxidative jump that is triggered by this electropermeabilization is a much longer-lived response . The electroinduced loss in viability is linearly correlated to permeabilization . However, there is no correlation between the oxidative jump and the loss in viability . The modulation of oxygen species electroinduction by antioxidant products (dimethylsulfoxide, sodium L-ascorbate, and glutathione) does not lead to an increase in cell viability . Such results are different to those observed with mammalian cells and indicate that even if the same phenomenon is observed with mammalian cells and indicate that even if the same phenomenon is observed when pulsing mammalian or intact plant cells, the associated metabolic response is not the same.

Transplantation, 1996 Jun 15, 61(11), 1618 - 24
Quantitating immunosuppression . Estimating the 50% inhibitory concentration for in vivo cyclosporine in mice; Batiuk TD et al.; Cyclosporine (CsA) blocks T cell responses in vitro by inhibiting the phosphatase activity of calcineurin (CN) and thus preventing the activation of cytokine transcription . In the study presented here, we measured the extent of inhibition of these functions in the tissues of CsA-fed mice . Mice fed increasing doses of CsA were assessed for CsA blood and tissue levels, spleen cell CN activity, ex vivo spleen cell cytokine induction by A23187, and in vivo interferon-gamma induction during an allogeneic response . The CN activity of spleen homogenates and cell suspensions and the ex vivo cytokine responses of spleen cells from CsA-treated mice were inhibited with a 50% inhibitory concentration (IC50) greater than 300 microg/L . The in vivo interferon-gamma response to an allogeneic ascites tumor was also inhibited by CsA treatment, with IC50s between 517 and 886 microg/L . The true IC50 for CsA in vivo may be even higher, as CsA levels in spleen and kidney were 4-fold higher than concomitant blood levels . We conclude that inhibition of CN activity by systemically administered CsA leads to a parallel reduction in cytokine gene induction in response to an allogeneic stimulus . In light of our previous clinical findings that therapeutic levels of CsA in renal transplant patients were associated with only partial inhibition of CN activity, these current results support the concept that partial CN inhibition can account for both the immunosuppression and the immunocompetence of CsA-treated patients.

Eur J Pharmacol, 1996 Jun 13, 306(1-3), 325 - 33
Cytosolic Ca2+ evaluation in rabbit parietal cells: a novel method to screen gastrin receptor antagonists; Letari O et al.; We have evaluated the application of the fura-2 method to detect cytosolic Ca2+ increase in gastric cells expressing CCKB/gastrin receptors, in order to screen gastrin receptor antagonists, as an alternative to functional studies . We have characterized the receptors on parietal cell suspension from rabbit gastric mucosa and validated the method using both the CCKB and CCKA receptor agonists and antagonists . Human gastrin I (gastrin) (0.1 nM-4 microM) and sulfated cholecystokinin 26-33 (CCK-8) (0.01 nM-2 microM) dose-dependently augmented cytosolic Ca2+ . The efficacies of the two agonists were similar, but the potency of CCK-8 (EC50 1.03 nM) was about 10-fold greater than that of gastrin (11 nM) . Response to a submaximal dose of gastrin (50 nM) was dose-dependently blocked by the CCKB-receptor antagonists CAM-1028 (4-{{2-{{3-(1 H-indol-3-yl)-2-methyl-1-oxo-2-{{{1,7,7-trimethylbicyclo{2, 2,1}hept-2-yl)oxy}carbonyl}amino}propyl}amino}-1-phenylethyl}amino-4-oxo -{1 S-1 alpha, 2 beta {S'(S')4 alpha}}-butanoate-N-methyl-D-glucamine) (IC50 1.9 nM), L-365,260 (3 R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1 H-1, 4-benzodiazepin-3-yl)-N'-(3-methylphenyl)urea) (IC50 10 nM) and spiroglumide ((R)-4-(3,5-dichlorobenzamido)-5-(8-azaspiro{4.5}decan -8-yl)-5-oxopentanoic acid) (IC50 2 microM) . The results were in agreement with those obtained from binding studies in guinea-pig cortical membranes . The model was employed to optimize the synthesis of a new class of spiroglumide analogues which led to a new molecule, (S)-4- inverted question mark(R)-4'-(3,5-dichlorobenzoylamino)-5'-(8-azaspiro{4.5} decan-8-yl)-5'-oxo)-pentanoylamino-5-(1-naphthylamino)-5-oxo pentanoic acid (CR 2622), whose potency was about 100-fold greater than that of spiroglumide . CR 2622, as well as the other CCKB receptor antagonists tested, exhibited no effect on basal {Ca2+}i . The simplicity and the reproducibility of this method suggest that it is a useful model to screen gastrin and antigastrin activity in parallel or as an alternative to binding studies.

Biochim Biophys Acta, 1996 Jun 13, 1274(3), 143 - 8
Dielectric properties of yeast cells as simulated by the two-shell model; Raicu V et al.; The paper reports a re-evaluation of the previous studies on yeast by considering the influence of vacuole upon the dielectric properties of the cell . In this respect, relative permittivity and conductivity of yeast cells dispersed in KCI solutions of various concentrations were measured in the frequency range from 0.1 to 100 MHz . The analysis of data revealed that the beta-dielectric dispersion of yeast cell suspensions is a composite of three (or probably four) distinct sub-dispersions . Since the dielectric response of the cell wall was experimentally avoided (according to Asami et al . (1976) J . Membr . Biol . 28, 169-180), the two-shell model, related to the plasma membrane and the vacuolar membrane, respectively, appeared to be the best approximation for yeast cells . The most relevant parameters obtained with the aid of the two-shell model were as follows . Specific capacitance of the plasma membrane and the vacuolar membrane were 0.703 +/- 0.011 microF/cm2 and 0.483 +/- 0.029 microF/cm2, respectively; electrical conductivity of the cytoplasm and the vacuole interior were 0.515 +/- 0.028 S/m and 3.22 +/- 0.48 S/m; finally, the permittivity of the cytoplasm was 50.6 +/- 2.

Neurosci Lett, 1996 Jun 7, 210(3), 185 - 8
Foetal ventral mesencephalic cell suspension grafts to the 6-hydroxydopamine-lesioned rat reduce the rate of dopamine uptake in the contralateral striatum; Earl CD et al.; The present study employed fast cyclic voltammetry, at carbon-fibre microelectrodes, to monitor and compare the rate of dopamine uptake in the rat striatum contralateral to (a) the 6-hydroxydopamine (6-OHDA)-lesioned/grafted striatum and (b) the 6-OHDA-lesioned/ sham grafted striatum . Cell suspensions of foetal rat ventral mesencephalic tissue were grafted into the dopamine-depleted striatum of unilaterally 6-OHDA-lesioned rats . Six weeks after grafting, animals with functional, mature grafts were monitored for dopamine elimination in the contralateral striatum following electrical stimulation of the median forebrain bundle, before and after treatment with the dopamine uptake inhibitor GBR 12909 . Compared to animals with sham grafts, amphetamine-amplified rotational behaviour was significantly reduced in animals with grafts of foetal ventral mesencephalic tissue . Fast cyclic voltammetric measurements followed by evaluation with the aid of a kinetic model revealed that in grafted animals, the rate of dopamine uptake via the high affinity uptake mechanism, following treatment with GBR 12909, was significantly reduced when compared to sham grafted animals.

Biochemistry, 1996 Jun 4, 35(22), 7069 - 76
Sterol biosynthesis: strong inhibition of maize delta 5,7-sterol delta 7-reductase by novel 6-aza-B-homosteroids and other analogs of a presumptive carbocationic intermediate of the reduction reaction; Rahier A et al.; A series of mono- and diazasteroids have been synthesized as analogs of a predicted carbocationic intermediate of delta 5,7-sterol delta 7-reductase (delta 7-SR) . 6-Aza-B-homo-5 alpha-cholest-7-en-3 beta-ol (4), a novel compound whose synthesis is described for the first time, and 6,7-diaza-5 alpha-cholest-8(14)-en-3 beta-ol (6) were shown to be very powerful inhibitors of delta 7-SR in a preparation isolated from maize (Zea mays) (K(i),app = 50-70 nM, Ki,app/Km,app = 1.0 x 10(-4) to 1.3 x 10(-4) . The data are consistent with a carbonium ion mechanism for the reduction; compounds 4 and 6 probably act as reaction intermediate analogs . Compound 4, in contrast to compound 6, displayed in the same microsomal preparation more than 50-fold selectivity for inhibition of the delta 7-SR versus delta 8-delta 7-sterol isomerase, cycloeucalenol isomerase, and delta 8,14-sterol delta 14-reductase, the mechanism of these four enzymes involving presumptive cationic intermediates centered respectively at C7, C8, C9, and C14 . These observations highlight the paramount importance of the location of the positively charged nitrogen atom(s) in the B-ring structure for selectivity among these enzymes involving structurally close cationic reaction intermediates . Efficient in vivo inhibition of sterol biosynthesis in bramble cell suspension cultures by a low concentration of compound 4 was demonstrated and confirmed the in vitro properties of this derivative.)

Gastroenterology, 1996 Jun, 110(6), 1835 - 46
Selective ligand-induced intracellular calcium changes in a population of rat isolated gastric endocrine cells; Zeng N et al.; BACKGROUND & AIMS: Peripheral regulation of acid secretion depends mainly on stimulation or inhibition of the three major gastric endocrine cells (enterochromaffin-like, gastrin, and somatostatin) . The aim of this paper was to define physiological responses of enterochromaffin-like, gastrin, and somatostatin cells in a mixed endocrine cell population by measuring ligand-selective changes of intracellular calcium ({Ca2+}i) in individual cells . METHODS: Endocrine cells were enriched from a rat gastric cell suspension by elutriation, a density-gradient fractionation, and a 48-hour short-term culture . {Ca2+}i responses of individual cells to various ligands such as gastrin/carboxy-terminal cholecystokinin octapeptide and selective cholecystokinin antagonists, carbachol, and gastrin-releasing peptide were monitored using video imaging in a perfusion chamber . Characteristic {Ca2+}i changes distinguished the three cell types, confirmed by immunostaining . RESULTS: All enterochromaffin-like cells respond to cholecystokinin-B receptor stimulation, but only a few respond to carbachol . Gastrin cells respond to both gastrin-releasing peptide and carbachol but not to cholecystokinin-receptor agonists . Somatostatin cells have both stimulatory cholecystokinin-A and cholecystokinin-B receptors and inhibitory muscarinic receptors . All cells have inhibitory somatostatin receptors . CONCLUSIONS: Calcium-signaling responses of gastric endocrine cells are distinctive . This allows individual cell types in a mixed population to be characterized and permits an analysis of the hormones and transmitters that act directly on a specific cell type.

J Endocrinol Invest, 1996 Jun, 19(6), 342 - 7
Differences in PTH (1-84) release in response to ambient calcium concentrations of parathyroid adenoma fragments and dispersed parathyroid adenoma cells in culture; Yu M et al.; In a previous study we observed that during perfusion of normal human parathyroid tissue, the release of PTH (1-84) was modulated by ambient extracellular calcium (Ca++) and lithium (Li+) concentrations in the media and preliminary studies indicated that this stimulus-response coupling was absent in human parathyroid adenoma fragments . The present study compares the responsiveness of parathyroid adenoma fragments and isolated parathyroid adenoma cells from the same adenoma and their response to Ca++ changes and Li+ presence in culture media . The data indicate that parathyroid adenoma tissue fragments fail to respond to ambient changes in Ca++ and Li+ . In contrast, dispersed parathyroid cells preparations responded with a significant increase of PTH (1-84) release (50%) under the influence of low ambient calcium concentrations . Six of the dispersed cell preparations also responded with a 45% decrease in PTH release under the influence of a high Ca concentration in the medium . Isolated parathyroid cells obtained from the same adenoma's did not respond to the presence of Li++ in the medium . These data suggest tat human parathyroid adenoma tissue functions autonomously and is not sensitive to calcium regulation in the tissue configuration as opposed to the isolated cell suspensions . The nature of this difference remains elusive.

Anal Cell Pathol, 1996 Jun, 11(1), 55 - 70
Options of flow cytometric three-colour DNA measurements to quantitate EGFR in subpopulations of human bladder cancer; Brockhoff G et al.; Flow cytometric multi-parameter analysis has proven to be a powerful tool to characterize subpopulations of cell suspensions, and is applied routinely in hematology . However, in studies of cancer where there is interest in defining phenotypic markers in conjunction with DNA content, this method has hardly been applied {6} . Our objective was to develop a methodology that extends previous investigations on relative and absolute quantitation of the epidermal growth factor receptor (EGFR) in dual parameter analysis in vitro on human bladder cancer cell lines {3} . In order to quantitate EGFR content in tumours and to relate it to DNA content, tumour selection, DNA-content, and EGFR-content measurements should be carried out simultaneously . Different fluorescent dyes were used to optimize DNA assessment and antibody staining, using a single laser instrument as a practical approach for clinical routine . In vitro cultures were used to validate the quality of tumour cell selection and antigen quantitation . Therefore, two urothelial tumour cell lines-lowly and highly differentiated-were incubated under different conditions: monolayer (ML), three-dimensional multi-cellular spheroids (MCS) and cocultures (COCU) with the fibroblast cell line NI were investigated and EGFR quantitation was related to S-phase fraction (SPF) . Accurate determination of instrument settings allows simultaneous three-colour analysis with DNA assessment . Tumor cell selection based on staining with phycoerythrine (R-PE) against a highly expressed urothelial glycoprotein, detected with the antibody Uro5 or against cytokeratin appeared to be possible in FL2, using the fluorochrome combination fluorescein-isothiocyanate (FITC), R-PE and propidiumiodide (PI) . Using this staining protocol, relative and absolute EGFR quantitation (quantum simply cellular beads) is shown to be accurate, when FITC is used for EGFR staining and measured in the green fluorescence channel (FL1) . Using this colour combination EGFR content and SPF of tumour cells were compared in different growth states, and could be monitored reliably . In spite of a higher emission spectrum of 7-aminoactinomycin-D (7-AAD), this DNA stain provided no advantage over PI . Broad coefficients of variation (CV) were found when intact cells were stained, thus hindering accurate assessment of ploidy and S-phase fraction . Similarly, Syto-13, a DNA dye detected in FL1, could not be optimized for multi-parameter measurements . Although the emission maximum is at 520 nm, the spectrum is too wide to compensate fluorescence overlap in FL2 or FL3 . Quantum Red (QR), used as a streptavidin conjugate in FL3, could not be combined with two other colours for DNA staining, since sufficient compensation was not obtainable when an argon ion laser is used . The coculture model allows verification of tumour cell selection and discrimination . The high differentiated tumour cell line RT4 shows an unambiguous correlation between EGFR content and S-phase fraction . The low differentiated tumour cell line J82 presents a similar pattern of post-transcriptional EGFR regulation with respect to culture condition, however, the S-phase fraction is basically unaffected.

Anal Cell Pathol, 1996 Jun, 11(1), 43 - 54
Cell cycle kinetics in normal human skin by in vivo administration of iododeoxyuridine and application of a differentiation marker--implications for cell cycle kinetics in psoriatic skin; van Erp PE et al.; Renewal of epidermal cells is a highly coordinated process in which terminal differentiation balances the proliferative rate in the germinative compartment . Exact quantitative data on cell cycle parameters of normal human epidermis are fragmentary, and do not allow firm conclusions on issues such as cell cycle time, duration of the various cell cycle phases, and the pool sizes of the different cellular populations . As part of a study on bone marrow cell cycle kinetics, 14 lymphoma patients were infused with the thymidine analogue iododeoxyuridine (IdUrd) . This provided us with the unique opportunity to study the cell cycle kinetics in normal epidermis obtained from these patients . Single epidermal cell suspensions were prepared from skin, stained with propidium iodide (PI) for relative DNA content, and simultaneously labeled with an anti-IdUrd antibody to detect DNA-synthesizing cells . In parallel samples suprabasal cells were analyzed by labeling with an anti-cytokeratin 10 antibody . Analysis was performed using bivariate flow cytometry . The results showed that 3.5% of the total epidermal cell population was in S-phase . An S-phase duration of 9.7 +/- 0.6 h and a cytokeratin 10-positive pool size of 59.6 +/- 4.6% were obtained . The duration of the G1-phase and the G2M-phase were calculated to be 7.6 +/- 2.0 h and 11.1 +/- 2.0 h, respectively . From these data a total cell cycle time can be calculated of 28.4 h . Combining this data with previous findings we were able to determine a similar cell cycle time of 27.8 h, and pool sizes of the epidermal cells: 30% quiescent (resting, G0) and 10% cycling cells . The implications of these findings for the interpretation of deviations in growth control as found in hyperproliferative skin diseases (e.g . psoriasis) are discussed.

Genes Chromosomes Cancer, 1996 Jun, 16(2), 106 - 12
Clonal origin of trisomy for chromosome 7 in the epithelial compartment of colon neoplasia; Herbergs J et al.; In this study, we demonstrated the clonal origin of trisomy for chromosome 7 in epithelial cells of colon neoplasia . By using the double-target fluorescence in situ hybridization (FISH) technique in frozen tissue sections that were also immunostained for keratin and vimentin, ratio analysis of FISH signals for chromosomes 7 and 17 could be performed in epithelial (cytokeratin-positive) or stromal (vimentin-positive) areas . The data demonstrated that trisomy for chromosome 7 is found exclusively in the epithelial compartments and not in the stroma of colon adenocarcinoma . We then demonstrated the occurrence of trisomy for chromosome 7 in the different types of epithelial neoplastic cells, i.e., columnar and goblet cells, which were isolated from frozen tissue sections by mechanical disaggregation of colon tissue and mild lysis of the cells while protease activity was inhibited . In these cell suspensions, the columnar cells were detected with an antibody to villin, and the goblet cells were stained for mucin, whereas all cells were subsequently subjected to FISH for chromosome 7 . For analysis of neuroendocrine cells, which are present in a very low frequency in colon neoplasia, frozen tissue sections that were immunostained for Chromogranin A could be used . Individual neuroendocrine cells could be distinguished in these thin frozen tissue sections . The presence of trisomy for chromosome 7 in all three different epithelial cell types strengthens our suggestion that this chromosomal aberration is found in the epithelial stem cell compartment of colon neoplasia.

AIDS, 1996 Jun, 10(7), 717 - 27
Thymocyte and thymic microenvironment alterations during a systemic HIV infection in a severe combined immunodeficient mouse model; Autran B et al.; OBJECTIVE: A new model for systemic and multifocal HIV-1 infection was developed in severe combined immunodeficient (SCID) mice to study the alterations of thymocytes and of the thymic microenvironment that occur during a disseminated HIV infection . DESIGN AND METHODS: We grafted SCID mice with the classical human fetal thymus/liver co-implants together with fragments of autologous lungs (SCID-huLLT) . These organs achieved normal differentiation and were productively infected after an intraperitoneal inoculation of two HIV-1 primary isolates . At time of sacrifice, thymic biopsies and thymic cell suspensions were analysed by immunohistochemistry, flow cytometry and lymphocyte function assays . RESULTS: At weeks 2-4 post-inoculation we observed the following thymocyte abnormalities: a minor to severe depletion of the immature CD1+CD4+CD8+ T cells (range, 0-73% thymocytes), compared with the persistence of mature CD4+ cells (11-50%) and amplification of CD8+ T cells (6-92%) . The immature subset depletion was inversely related to the thymic HIV-1 viral load, suggesting the preferential infection of this subset . The residual mature thymocytes were functional as assessed by their sustained proliferative responses to CD3-triggering which contrasted with the lack of HIV-specific cytotoxic activity . A quantitative analysis of immunostained thymic sections revealed a disorganization and a densification of the thymic epithelial cells (TEC) network which occurred in all HIV-infected SCID-hu mice independently of the thymic CD1+CD4+CD8+ T-cell depletion . CONCLUSION: These results suggest that a systemic HIV infection induces in human thymuses from SCID-huLLT mice a preferential depletion of the immature thymocytes in the absence of mature CD4+ T-cell depletion, HIV-specific cytotoxic T-lymphocyte activity or thymic epithelial cell death, but is associated with dysplasia of the thymic microenvironment, and is therefore opening new perspectives for studying immune cell reconstitution strategies in HIV infection.

J Virol Methods, 1996 Jun, 60(1), 59 - 64
A simplified technique for determining the sensitivity of cytomegalovirus strains to ganciclovir; Calico I et al.; A technique for determining the susceptibility to ganciclovir of cytomegalovirus (CMV) strains isolated in clinical samples is described . The inoculum was composed of a partially infected suspension of cells from a young positive culture (< 10 days), usually the first passage of the primary culture . The appropriate dilution of the cell suspension to provide a suitable inoculum was based on a previous study of five strains grown in different dilutions which provided a countable number of plaques and avoided titration of each of the isolated strains . Fifty-three strains were studied at three different dilutions . Five from patients on maintenance ganciclovir therapy with poor clinical response had a 50% inhibitory dose (ID50) between 21.46 and 13.35 microM and the remainder an ID50 between 2.31 and 10.5 microM, comparable to results obtained by other authors using susceptibility techniques with a sonicated inoculum . Three of these strains were studied by both methods using sonicated inoculum and cell suspension inoculum . The mean time which elapsed between seeding the specimen and obtaining sensitivity was 39.00 and 27.66 days, respectively . The technique reduces significantly the time involved since relatively young cultures can be studied and previous titration is not required.

Vet Immunol Immunopathol, 1996 Jun 1, 51(3-4), 353 - 63
Identification of duck T lymphocytes using an anti-human T cell (CD3) antiserum; Bertram EM et al.; Duck lymphocytes have not been classified into cells resembling B or T cells of mammals . Reagents used in the past to identify lymphocyte populations in other species have not been useful for this purpose and antibodies raised to duck immunoglobulin bind in high proportions to blood and organ lymphocytes of ducks as well as to their red blood cells . Here we report that a polyclonal rabbit antiserum reacting to the CD3 marker on human T cells has been used to identify duck T lymphocytes . These antibodies react with the intracytoplasmic portion of the human CD3 epsilon chain (amino acids 156-168), an epitope highly conserved between mammals . Immunohistochemical staining with this antiserum of sections of duck lymphoid organs and FACScan analysis of duck lymphoid cell suspensions identified a population of duck lymphocytes with a staining pattern similar to that seen for mammalian T cells . This anti-human CD3 immunoprecipitated a 23 kDa protein from a duck lymphoblast lysate: a size similar to the human CD3 epsilon chain . This is the first direct identification of duck T lymphocytes.

Glia, 1996 Jun, 17(2), 103 - 20
Cellular immune reactions in brain transplantation: effects of graft pooling and immunosuppression in the 6-hydroxydopamine rat model of Parkinson's disease; Schwarz SC et al.; We used high immunogenic mouse and low immunogenic rat brain transplants to investigate the effect of pooling of tissue with immunogenetic disparity on cellular immune reactions . Foetal xenogenic mouse striatum and allogenic rat substantia nigra were implanted into i) the 6-hydroxydopamine lesioned striatum of outbred female Sprague-Dawley rats as a pooled cell suspension, or into ii) the unlesioned and lesioned striata as non-pooled separate deposits, with or without immunosuppressive treatment with cyclosporin A (Cy A) . In control animals, iii) mouse striatum was replaced by rat striatum, and iv) sham grafts with and without immunosuppression . Six weeks post grafting, brains were semiquantitatively processed using immunocytochemical markers for microglia, astrocytes, T-helper cells, and macrophages, major histocompatibility class (MHC) I and II expression . The total amount of immunoreactivity (PA) for microglial cells and astrocytes was pronounced and the PA for T-helper cells and macrophages was doubled in recipients of pooled rat and mouse cografts compared to non-pooled deposits, indicating ongoing immune reactions with participation of glial cells . MHC I expression was significantly increased in pooled xeno- and allogenic cografts with and without immunosuppression compared to allogenic controls . Expression of MHC II was significantly increased in pooled cografts without immunosuppression . In recipients of separate, non-pooled heteroimmunogenic cotransplants, MHC I and II expression was significantly increased in xenogenic deposits with and without immunosuppression . MHC II was as well significantly increased in allogenic deposits without immunosuppression . Immunosuppressed animals with non-pooled allogenic mouse cografts showed low levels of cellular immune parameters . In conclusion non-pooled heteroimmunogenic grafts lead to less pronounced immune reactions compared to pooled grafts and immunosuppressive treatment with Cy A has a beneficial effect on acute transplant-associated immune parameters.

Phytochemistry, 1996 Jun, 42(3), 667 - 9
Biotransformation of digitoxigenin by cultured ginseng cells; Kawaguchi K et al.; Nine compounds, including a new compound (digitoxigenin beta-D-glucoside malonyl ester), were isolated as biotransformation products of digitoxigenin by cell suspension cultures of Panax ginseng (Pg-3 cell line) . At the same time, two known products were identified by TLC and HPLC.

Parasite, 1996 Jun, 3(2), 119 - 23
{Cryopreservation of isolates of Toxoplasma gondii in cell culture}; Cotty F et al.; For a better preservation and identification of Toxoplasma gondii isolates, we propose a new method of freezing of toxoplasma growth in THP-1 cell culture . A cystogenic strain isolated from foetal blood has been grown in these cells and frozen in liquid nitrogen . After thawing, toxoplasma recover the same growth rate and morphology in vitro and the same capacity to form brain cysts into mice compared to the initial strains . The freezing of the cell suspension provides a simple and appropriate method for preservation of Toxoplasma gondii within "bank" isolates.

Neuroscience, 1996 Jun, 72(4), 959 - 88
Contrasting effects of fetal CA1 and CA3 hippocampal grafts on deficits in spatial learning and working memory induced by global cerebral ischaemia in rats; Hodges H et al.; Functional effects of fetal hippocampal field grafts were assessed in rats with spatial learning and memory impairments following global cerebral ischaemia . Experiment 1 examined effects of grafts dissected from fields CA1 and CA3 at embryonic day 19 and from the dentate gyrus at postnatal day 1 . Cell suspensions (15,000 cells/site) were implanted bilaterally at two points above the dorsal CA1 area two weeks after four-vessel occlusion (electrocoagulation of the vertebral arteries followed the 24 h later by occlusion of the carotid arteries for 15 min) . Histological examination showed that CA1 neuronal loss (60-70%) was equivalent in all ischaemic groups and that 80% of CA1 and 60% of CA3 grafts survived and were sited appropriately in the alveus or corpus callosum above the area of ischaemic CA1 damage in the host, but there was no survival of dentate grafts . Results from rats with poor pyramidal cell graft survival were excluded, but those from rats with non-surviving dentate grafts were retained as an additional control group . Acquisition in the water maze was examined nine and 25 weeks after transplantation, and spatial working memory was assessed in three-door runway and water maze matching-to-position tasks 19 and 28 weeks after grafting, respectively . For water maze acquisition rats were trained with two trails/day and a 10 min inter-trial interval for 10-12 days to locate a submerged platform . Ischaemic rats with CA1 grafts learned the platform position as rapidly as non-ischaemic controls, searched appropriately in the training quadrant and were accurate in heading towards the platform, but were initially impaired on recall of the precise platform position on probe trials with the platform removed . Performance of ischaemic controls and groups with CA3 and non-surviving dentate graft groups was significantly impaired relative to controls and to the CA1 grafted group . The CA1 grafted group was also as successful as controls in matching-to-position in the water maze and substantially superior to the other ischaemic groups, assessed using three trials/day, with a 30-s inter-trial interval and a different platform position on each day . In a more complex matching-to-position task in the three-door runway, the performance of the CA1 grafted group was significantly impaired relative to controls, although superior to that of the other ischaemic control and graft groups . Functional recovery with CA1, but not CA3, grafts in ischaemic rats was replicated in a second experiment which assessed water maze acquisition and working memory at 10 and 14 weeks after transplantation, in rats with 90% graft survival . These results indicate that long-lasting, task-dependent improvements can be seen in ischaemic rats with CA1 fetal grafts in both aversively and appetitively motivated spatial learning tasks . The findings suggest that functional recovery requires homotypic replacement of CA1 cells damaged by ischaemia, rather than provision of structurally similar glutamate-releasing CA3 pyramidal cells.

J Immunol, 1996 Jun 1, 156(11), 4298 - 308
Insight into the mechanism(s) through which TNF promotes the generation of T cell-mediated antitumor cytotoxicity by tumor bearer splenic cells; Gorelik L et al.; We have shown previously that addition of TNF to stimulation cultures of MOPC-315 tumor bearer splenic cell suspensions containing metastatic tumor cells capable of secreting TGF-beta greatly enhances the generation of anti-MOPC-315 lytic activity by their CD8+ T cells . The current studies were undertaken to gain some insight into the mechanism(s) through which TNF potentiates the in vitro generation of anti-MOPC-315 cytotoxicity by such tumor bearer splenic cells . Here we show that TNF is capable of 1) preventing completely the inhibitory activity of TGF-beta for CTL generation when both cytokines are added at the time of initiation of a 5-day stimulation culture and 2) reversing at least part of the inhibitory activity of TGF-beta when TNF is added as late as 3 days after TGF-beta addition . The costimulatory molecule B7-2 is shown here to be important for the realization of the potentiating activity of TNF for CTL generation by tumor bearer splenic cells . However, despite the importance of the B7-2 molecule, TNF does not mediate its immunopotentiating activity for CTL generation through up-regulation in IL-2 production . In addition, we show here that GM-CSF, but not IL-12, is important for the potentiating effect of TNF for CTL generation by tumor bearer splenic cells . Taken together, these studies identify several factors that are important for the realization of the potentiating effect of TNF for the in vitro generation of antitumor cytotoxicity by tumor-infiltrated splenic cells . It is not known at present, however, whether these factors utilize distinct and/or overlapping mechanisms in realizing the TNF effect.

J Cell Physiol, 1996 Jun, 167(3), 394 - 405
Transforming growth factor beta (TGF-beta) expression in isolated and cultured rat hepatocytes; Gao C et al.; It is still a subject of debate whether hepatocytes have the ability to express TGF-beta . Therefore, we investigated in freshly isolated and in monolayer cultures of rat hepatocytes the expression of TGF-beta isoform s at the RNA and protein level applying RT-PCR, immunocytochemistry, immunoblotting, and functional assays of TGF-beta . TFG-beta 1, -beta 2, and -beta 3 transcripts were detected in cultured cells, and the level of m RNA increased up to 48/72 h, but TGF-beta 1 transcripts were absent in freshly isolated cells . Using APAAP stainings the proteins of all three TGF-beta isoforms were observed in hepatocyte cultures from 5-96 h, but in hepatocytes in the liver in situ and in freshly isolated cell suspensions TGF-beta staining was negative . SDS-PAGE under reducing conditions followed by Western blotting detected in cell lysates the subunit of mature TGF-beta at about 13 kd . Analysis of TGF-beta bioactivity with the mink cell (Mv1Lu) proliferation inhibition assay and competitive radioligand assay confirmed in activated (i.e., acidified and subsequently neutralized) hepatocyte-conditioned media the presence of TGF-beta, which, however, is almost entirely in the latent form . It is concluded that TGF-beta can be expressed in cultured hepatocytes and that the level of expression is quickly upregulated under abnormal, not yet known, microenvironmental conditions.

Arch Biochem Biophys, 1996 Jun 1, 330(1), 33 - 47
Biosynthesis of cyclic diterpene hydrocarbons in rice cell suspensions: conversion of 9,10-syn-labda-8(17),13-dienyl diphosphate to 9beta-pimara-7,15-diene and stemar-13-ene; Mohan RS et al.; The biosynthesis of diterpene hydrocarbons with enzyme extracts from rice cell suspension cultures was investigated to verify proposed pathways and intermediates in the production of the momilactone and oryzalexin phytoalexins . Diterpene synthase activity in cells treated with chitin to elicit the phytoalexin response was compared with the activity in untreated cells using the acyclic substrates {1-3H}(E,E,E)- and {1-3H} (E,Z,E)-geranylgeranyl diphosphates (GGPPs 4-OPP and 11-OPP) as well as the bicyclic substrates {15-3H}ent-copalyl and {15-3H} syn-copalyl diphosphates (CPPs, 5-OPP, and 6-OPP) . ent-kaurene (7), ent-sanda, racopimaradiene (8), 9 beta H-pimara-7,15-diene (9), and stemar-13-ene (10) were identified as major products by comparisons with authentic standards . Marked increases in diterpene synthase activities were observed with enzyme from chitin-treated cells: (E,E,E)-GGPP (approximately 100 fold), ent-CPP (approximately 3 fold), and syn-CPP (approximately 60 fold) . The very low conversions of (E,Z,E)-GGPP to hydrocarbon products excludes its role in the biosynthesis of 9,10-syn-diterpenes in rice cells . ent-Kaurene was the major diterpene formed from ent-CPP with enzyme from unelicited cells . In contrast the enzyme from chitin-treated cells converted ent-CPP to a mixture of ent-kaurene, ent-sandaracopimaradiene, and a third unidentified diterpene . With syn-CPP as substrate the induced syntheses afforded a mixture of 9 beta-pimaradiene, stemarene, and a third, unidentified syn-diterpene . Overall the results are consistent with the hypothesis that rice cells respond to treatment with chitin fragments by producing new diterpene synthases not present in the untreated cells . These induced cyclases initiate phytoalexin biosynthesis by diverting (E,E,E)-GGPP into new cyclization modes that produce ent-sandaracopimaradiene, stemarene, and 9 beta-pimaradiene, the presumed precursors to oryzalexins A-F, oryzalexin S, and momilactones A-C, respectively . The intermediate role of 9,10-syn-CPP in syn diterpene biosynthesis is verified.

Proc Soc Exp Biol Med, 1996 Jun, 212(2), 160 - 4
Transplantation of immortalized, nontumorigenic parotid acinar cells into the allogeneic rat parotid gland and oral submucosa; Krause GE et al.; The recent establishment of an immortalized clonal cell line of rat parotid acinar cells (2RSG) by transfecting isoproterenol-stimulated parotid cells with a plasmid vector, pSV3neo which carries the large T-antigen gene from SV40 virus, afforded the opportunity to develop a model for parotid acinar cell transplantation . Single cell suspensions of 2RSG cells labeled with a fluorescent tracer, DiI, were injected into the parotid gland or oral submucosa of allogeneic adult rats . The grafted cells survived and were functionally viable for at least 30 days . Histological sections revealed no evidence of infiltration of leukocytes or lymphocytes . Grafted cells did not form tumors . Results suggest that allogeneic parotid acinar cell transplantation is a feasible technique in the animal model.

J Mol Biol, 1996 May 24, 258(5), 778 - 88
Characterization of an Arabidopsis thaliana gene that defines a new class of putative plant receptor kinases with an extracellular lectin-like domain; Herve C et al.; We have characterized an Arabidopsis receptor-like serine/threonine kinase gene, Ath.lecRK1 (Arabidopsis thaliana lectin-receptor kinase), defining a new and putatively important class of plant receptor kinases . Structural features of the predicted polypeptide include an amino-terminal membrane-targeting signal sequence, a legume lectin-like extracellular domain, a single membrane-spanning domain, and a characteristic serine/threonine protein kinase domain . A recombinant protein containing the kinase domain can be autophosphorylated on a serine residue . Ath.lecRK1 is a member of a gene family of at least two closely related genes . Northern blot analysis indicates that the Ath.lecRK1 gene is weakly expressed in a variety of organs and is regulated in Arabidopsis cell suspension cultures according to the growth phase of cells . The role this new class of plant receptor kinase could play is discussed with regard to the transduction of oligosaccharide and plant hormone signals.

Biochim Biophys Acta, 1996 May 21, 1290(1), 46 - 52
Hydrogen peroxide increases Na+/K(+)-ATPase function in alveolar type II cells; Gonzalez-Flecha B et al.; We have studied the regulation of Na+/K(+)-ATPase function in alveolar type II cells submitted to oxidative stress . Alveolar type II cells were isolated from Sprague Dawley rats and suspended in Dulbecco's modified Eagle's medium . 500 muM xanthine plus 0.5 or 5 mU/ml xanthine oxidase (group 1 and 2, respectively) were added to the cell suspensions . Following various exposure times the reaction was stopped by adding allopurinol and cells were processed to assay H2O2 steady state concentrations, enzymatic activity of catalase and Na+/K(+)-ATPase function . Hydrogen peroxide production by the xanthine-xanthine oxidase system reached maximal values at 30 min of incubation in both groups . H2O2 steady state concentration increased 2- and 10-fold, respectively . Catalase activity was not changed after slight oxidative stress (group 1) but decreased in severe oxidative stress (group 2) . Decreases in the Na+/K(+)-ATPase activity (10 and 60% for groups 1 and 2) were found during the first hour of exposure coinciding with the peak in H2O2 steady state concentration . This early inactivation was followed by progressive increases in the activity up to 70% over the control value in group 1, and to the control value in group 2 . {3H}Ouabain binding studies showed that the increase in Na+/K(+)-ATPase activity after oxidative stress was due to an increase in the number of phosphorylated pump molecules in the plasma membrane of alveolar type II cells.

J Biol Chem, 1996 May 17, 271(20), 11824 - 30
Cell surface expression of HIP, a novel heparin/heparan sulfate binding protein, of human uterine epithelial cells and cell lines; Rohde LH et al.; Previous studies established that uterine epithelial cells and cell lines express cell surface heparin/heparan sulfate (HP/HS)-binding proteins (Wilson, O., Jacobs, A . L., Stewart, S., and Carson, D . D . (1990) J . Cell . Physiol . 143, 60-67; Raboudi, N., Julian, J., Rohde, L . H., and Carson, D . D . (1992) J . Biol . Chem . 267, 11930-11939) . The accompanying paper (Liu, S., Smith, S . E., Julian, J., Rohde, L . H., Karin, N . J., and Carson, D . D . (1996) J . Biol . Chem . 271, 11817-11823) describes the cloning of a full-length cDNA corresponding to a candidate cell surface HP/HS interacting protein, HIP, expressed by a variety of human epithelia . A synthetic peptide was synthesized corresponding to an amino acid sequence predicted from the cDNA sequence and used to prepare a rabbit polyclonal antibody . This antibody reacted with a protein with an apparent Mr of 24,000 by SDS-polyacrylamide gel electrophoresis that was highly enriched in the 100,000 x g particulate fraction of RL95 cells . This molecular weight is similar to that of the protein expressed by 3T3 cells transfected with HIP cDNA . HIP was solubilized from this particulate fraction with NaCl concentrations > or = 0.8 M demonstrating a peripheral association consistent with the lack of a membrane spanning domain in the predicted cDNA sequence . HIP was not released by heparinase digestion suggesting that the association is not via membrane-bound HS proteoglycans . NaCl-solubilized HIP bound to heparin-agarose in physiological saline and eluted with NaCl concentrations of 0.75 M and above . Furthermore, incubation of 125I-HP with transblots of the NaCl-solubilized HIP preparations separated by two-dimensional gel electrophoresis demonstrated direct binding of HP to HIP . Indirect immunofluorescence studies demonstrated that HIP is expressed on the surfaces of intact RL95 cells . Binding of HIP antibodies to RL95 cell surfaces at 4 degrees C was saturable and blocked by preincubation with the peptide antigen . Single cell suspensions of RL95 cells formed large aggregates when incubated with antibodies directed against HIP but not irrelevant antibodies . Finally, indirect immunofluorescence studies demonstrate that HIP is expressed in both lumenal and glandular epithelium of normal human endometrium throughout the menstrual cycle . In addition, HIP expression increases in the predecidual cells of post-ovulatory day 13-15 stroma . Collectively, these data indicate that HIP is a membrane-associated HP-binding protein expressed on the surface of normal human uterine epithelia and uterine epithelial cell lines.

J Neurosci, 1996 May 15, 16(10), 3199 - 208
Restoration of normal conduction properties in demyelinated spinal cord axons in the adult rat by transplantation of exogenous Schwann cells; Honmou O et al.; Although remyelination of demyelinated CNS axons is known to occur after transplantation of exogenous glial cells, previous studies have not determined whether cell transplantation can restore the conduction properties of demyelinated axons in the adult CNS . To examine this issue, the dorsal columns of the adult rat spinal cord were demyelinated by x-irradiation and intraspinal injections of ethidium bromide . Cell suspensions of cultured astrocytes and Schwann cells derived from neonatal rats transfected with the (beta-galactosidase) reporter gene were injected into the glial-free lesion site . After 3-4 weeks nearly all of the demyelinated axons were remyelinated by the transplanted Schwann cells . The dorsal columns were removed and maintained in an in vitro recording chamber; conduction properties were studied using field potential and intra-axonal recording techniques . The demyelinated axons exhibited conduction slowing and block, and a reduction in their ability to follow high-frequency stimulation . Axons remyelinated by transplantation of cultured Schwann cells exhibited restoration of conduction through the lesion, with reestablishment of normal conduction velocity . The axons remyelinated after transplantation showed enhanced impulse recovery to paired-pulse stimulation and greater frequency-following capability as compared with both demyelinated and control axons . These results demonstrate the functional repair of demyelinated axons in the adult CNS by transplantation of cultured myelin-forming cells from the peripheral nervous system in combination with astrocytes.

Eur J Cancer, 1996 May, 32A(5), 883 - 7
Cytotoxic activity of calcein acetoxymethyl ester (Calcein/AM) on primary cultures of human haematological and solid tumours; Jonsson B et al.; The aim of this study was to determine the in vitro cytotoxicity of calcein acetoxymethyl ester (Calcein/AM) on primary cultures derived from solid and haematological human tumours . Calcein/AM is a fluorescent dye that localises intracellularly after esterase-dependent cellular trapping and which has shown cytotoxic activity against various established human tumour cell lines at relatively low concentrations . The semi-automated fluorometric microculture cytotoxicity assay, based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate to fluorescein, in microtitre plates was used for the evaluation of Calcein/AM activity in tumour cell suspensions from patients . The cytotoxicity was measured as a survival index (SI), defined as the fluorescence as a percentage of control cultures . A total of 163 evaluable samples from various tumours were tested with continuous drug exposure . The activity of Calcein/AM was compared with representatives of six major classes of standard chemotherapeutic drugs . Calcein/AM was found to induce concentration-dependent decreases in the SI of both haematological and solid tumour cells . The ratio of solid over haematological tumour activity increased at a rate that was concentration dependent . Although it was relatively less active than cisplatin against solid tumours, Calcein/AM showed higher solid tumour activity compared to leukaemic specific agents (cytarabine and amsacrine), vincristine and doxorubicin (Dox) . Among the solid tumours tested, childhood tumours, non-small cell lung cancer and sarcomas were the most sensitive to Calcein/AM . The best correlation between SI values was seen between Calcein/AM and Dox, with weaker correlations to representatives of antimetabolites, platinum compounds, topoisomerase II inhibitors, tubulin interactive agents and alkylators . Non-cytotoxic concentrations of cyclosporin A significantly potentiated calcein-induced cytotoxicity . The results show that Calcein/AM is differentially active against haematological tumours, but with substantial activity against solid tumours . The drug may represent a new class of anticancer compound with a unique means of drug delivery.

Biorheology, 1996 May-Jun, 33(3), 267 - 83
Effects of sedimentation of small red blood cell aggregates on blood flow in narrow horizontal tubes; Murata T; The flow properties of aggregating red cell suspensions flowing at low flow rates through horizontal tubes are analyzed using a theoretical model . The effects of sedimentation of small aggregates, which will be formed at comparatively high flow rates, on the relative apparent viscosity are considered . In the case in which a large number of small aggregates are formed in a suspension flowing through a horizontal tube, it seems that red cells are transported as a concentrated suspension through the bottom part of the tube because of sedimentation of aggregates . A two-layer flow model is used for the distribution of red cells . It consists of plasma in the upper part and a concentrated red cell suspension in the bottom part of the tube divided by a smooth and horizontal interface . It is assumed that the suspension is a Newtonian fluid whose viscosity increases exponentially with hematocrit . The velocity distribution, the relative apparent viscosity and the flux of red cells are calculated as functions of width of plasma layer for a different discharge hematocrit . The theoretical results are compared with the results obtained from experimental data . The relative apparent viscosity increases rapidly with an increasing degree of sedimentation over a wide range of plasma layer widths.

J Periodontal Res, 1996 May, 31(4), 285 - 93
Isolation of alkaline phosphatase-positive gingival fibroblasts from patients with chronic inflammatory periodontal disease; Abe T et al.; We have reported recently that increased expression of membrane alkaline phosphatase (ALP) activity is a phenotypical characteristic of gingival fibroblasts located in chronic inflammatory periodontal lesions . To understand the cellular properties of these cells, we isolated ALP-positive gingival fibroblasts from patients with adult periodontitis and evaluated their proliferative potential . Using an enzymatic digestion procedure, we prepared gingival cell suspensions containing ALP-positive fibroblasts without affecting their ALP activities . These cell suspensions were then subjected to 1 g sedimentation, followed by allowing cells to adhere to substrata . Using this procedure, 71.9% of isolated cells were ALP-positive . Dissociation of ALP-positive fibroblasts and contamination by non-fibroblastic cells were examined by cytochemical and immunocytochemical analyses . The proliferative capacity of ALP-positive fibroblasts in culture was assessed by monitoring the proportion of ALP-positive cells after repeated subculture passages and by labelling DNA-synthesizing cells with bromodeoxyuridine (BrdU) . The proportion of ALP-positive fibroblasts decreased during cell culture passages without an apparent change in the ALP-positive phenotype . The percentage of BrdU-positive cells was significantly lower among ALP-positive than among ALP-negative fibroblasts . These results indicate that ALP-positive fibroblasts in chronic inflammatory periodontal lesions have low growth potential . We suggest that their reduced capacity to grow in vitro reflects a more differentiated state induced under inflammatory conditions in vivo.

In Vivo, 1996 May-Jun, 10(3), 329 - 33
The effect of high dose vitamin A on the morphology and proliferative activity of xenograft lung and head and neck cancer; Mourad WA et al.; In vitro studies have suggested that vitamin A lowers invasive potential of squamous cell carcinoma . Epidemiological data have also indicated that high dose vitamin A may improve survival in patients with previously resected lung carcinoma . To our knowledge, no studies have attempted to test the in vivo effect of vitamin A on the morphology and growth rate of lung and head and neck cancer . Freshly resected tumor cell suspensions were obtained by ex vivo fine needle aspiration and injected subcutaneously in duplicate in athymic male nude mice . Two to six weeks post-engraftment tests and controls were separated for each xenograft . Mice with test xenografts were given water soluble vitamin A (Aquasol ATM, Astra pharmaceutical, Westborough, MA, U.S.A) at a dose of 10,000 U/Kg/day intraperitoneally for 6 to 10 weeks (median 8 weeks) . One to two hours prior to sacrifice bromodexouridine (BrdU) was injected intraperitoneally to assess the S-phase fraction in both test and control xenografts . Blood vitamin A levels in test and control animals were measured after sacrifice using high performance liquid chromatography (HPLC) . Sections of test and control xenografts were routinely stained to assess morphologic differentiation and mitotic counts . Unstained sections of xenografts were immunostained by the antibody to BrdU to test for BrdU labeling index (BLI) reflecting S-phase fraction (SPF) and also by the MIB-1 antibody to assess proliferative activity . Eighteen tumors were studied . These included 9 squamous cell carcinomas of the lung, 5 squamous cell carcinomas of the head and neck, and 4 adenocarcinomas of the lung . Blood levels of vitamin A in test animals were 7 to 23 times those of the control animals (median 13 times) . Neovascularization of the xenografts was seen in all cases . The morphology and mitotic activity of the test and control xenografts showed no significant difference . SPF and proliferative activity measured by BrdU and MIB-1 immunolabelling respectively showed no significant difference between test and control xenografts . Our study suggests that there is no significant in vivo effect of high dose vitamin A on the morphology and growth rate of xenografted non small cell carcinoma of the lung or squamous cell carcinoma of the head and neck.

Anticancer Drugs, 1996 May, 7(3), 275 - 80
Antiproliferative effects of interleukin-12 treatment on human tumor colony-forming units taken directly from patients; Izquierdo MA et al.; Interleukin-12 (IL-12) has important immunomodulatory effects on T and natural killer (NK) cells that might be exploited in anticancer treatment . Murine IL-12 models have shown antimetastatic and antitumor effects against murine tumors in vivo . Data on the effects of human IL-12 on human tumors are confined to 51Cr-release assay studies showing that IL-12 increases NK activity against cancer cells . We used a human tumor cloning assay (HTCA) to investigate the effects of human IL-12 on solid tumors taken directly from patients . The HTCA is suitable to test direct, as well as immune-mediated, antitumor effects of cytokines on heterogeneous cell preparations derived from fresh tumors . Single cell suspensions prepared from 193 tumors were continuously exposed (14 days) to 10, 100 and 1000 ng/ml of human IL-12 in a capillary HTCA . Seventy-four (38%) specimens were evaluable . Inhibition of tumor growth was observed in 35 specimens (47%; concentration-related in 33 cases), including cancers of the ovary, lung, prostate, breast, colon and kidney, as well as melanoma . Antitumor effect was observed in 10 (14%), 18 (24%) and 32 (43%) tumors, at 10, 100 and 1000 ng/ml of IL-12, respectively . One specimen (1%), a melanoma, showed stimulation of tumor proliferation only at 100 mg/ml of IL-12 . Our results show that IL-12 has substantial in vitro activity against a variety of solid tumors taken directly from patients . Clinical trials of IL-12 in patients with solid tumors are warranted.

J Burn Care Rehabil, 1996 May-Jun, 17(3), 231 - 6
Differences in IgM synthesis to gut bacterial peptidoglycan polysaccharide after burn injury and gut ischemia; Tabata T et al.; Both burn injury and intestinal ischemia have been proven to induce bacterial translocation from the gut . It is still unknown, however, whether the bacteria induces immune response in these different models . To assess this, we measured in vitro IgM synthesis to peptidoglycan polysaccharide (PGPS), a ubiquitous gut bacterial antigen, after burn injury or gut ischemia-reperfusion in a mouse model . Eighty-five BALB/c mice were divided into four groups . Gut ischemia was produced by placing a vessel loop around the superior mesenteric artery at celiotomy (group Isc; n = 31) . After 45 minutes, the abdomen was reopened, and the vessel loop removed . All animals had visible gut ischemia . Control mice (group Isc-C; n = 15) underwent two sham operations . Burn injury was 25% body surface area full-thickness to the dorsum (group B; n = 27) . Another control group (B-C; n = 12) was also used . Animals were euthanized 24 hours after recirculation or 5 days after the burn injury . All spleens were removed, and cell suspensions prepared . Cells were cultured in 2.5 micrograms/ml lipopolysaccharide for 5 days, and anti-PGPS IgM level in the supernatant was measured by an enzyme-linked immunosorbent assay . Intestinal ischemia produced a significant rise in in vitro anti-PGPS IgM synthesis per 10(5) lymphocytes, which is the principal immunoglobulin response to infection . However, anti-PGPS IgM in mice after burn injury was significantly decreased . This decreased IgM synthesis after burn injury compared to gut ischemia may represent continued immune impairment from the burn wound, and may account for organ dysfunction related to bacterial translocation after burn injury.

Cell Growth Differ, 1996 May, 7(5), 563 - 72
Nonconstitutive expression of the gastrin-releasing peptide autocrine growth system in human small cell lung carcinoma NCI-H345 cells; Aguayo SM et al.; Constitutive, unregulated autocrine growth is thought to be an important mechanism whereby cancer cells gain a proliferative advantage over nonmalignant cells . The question addressed here was whether the autocrine growth system for gastrin-releasing peptide (GRP) in human small cell lung carcinoma cells is, in fact, always expressed in a constitutive, unregulated fashion . Lag, rapid, and plateau growth states were defined for small cell lung carcinoma NCI-H345 cells based on periods during which they expressed different growth rates after plating as single cell suspensions . Immunoreactive GRP in the conditioned medium and in NCI-H345 cells harvested during each of these growth states, as well as cell DNA content, GRP mRNA expression, specific 125I-GRP uptake, specific 125I-GRP binding to solubilized membranes, and GRP and neuromedin B receptor mRNA expression by reverse transcription-PCR were analyzed . Maximal levels of GRP expression were observed during the lag growth state, with the highest concentration of immunoreactive GRP in the conditioned medium during the rapid growth state . Specific 125I-GRP uptake and binding were also highest during the lag growth state; however, GRP receptor mRNA did not significantly change . In contrast to prevailing concepts, these studies support the conclusion that the expression of the GRP autocrine growth system in NCI-H345 cells is indeed regulated . Furthermore, the components are maximally expressed before rapid growth begins, suggesting that other mechanisms are activated to support the actual proliferation.

Ann Surg Oncol, 1996 May, 3(3), 317 - 24
B7-1 gene transfer into human cancer cells by infection with an adenovirus-B7 (Ad-B7) expression vector; Dessureault S et al.; BACKGROUND: Transfection of the costimulatory molecule B7-1 into some murine tumors can increase antitumor immunity and eradicate tumor growth . The purpose of this work was to construct an adenovirus-B7 (Ad-B7) expression vector and study B7-1 gene transfer into human cancer cells . METHODS: The human B7-1 cDNA was ligated into an expression cassette containing the human cytomegalovirus immediate early gene promoter and then inserted into the E1 region of the Ad5 genome by homologous recombination . The resulting Ad-B7 vector was used to infect established cancer cell lines and freshly resected cancers . Resected tumors were disaggregated into single cell suspensions by mechanical mincing and enzymatic digestion . Surface expression of B7-1 after infection was verified by flow cytometry . RESULTS: Expression kinetics in three cell lines showed that infected cells began to express B7-1 within 24 h . The proportion of B7-1+ cells continued to increase during the next 48 h, after which expression remained relatively constant during the next 5 days (up to 98% B7-1+ cells) . Fresh tumor cells from various cancers displayed similar kinetics, but with greater variability in the proportion of cells expressing B7-1 (13% to 95% B7-1+ cells) . Cancers which were successfully infected included 3 colorectal adenocarcinomas, 2 leiomyosarcomas, 2 lung squamous cell carcinomas, and 1 renal cell carcinoma . CONCLUSIONS: The Ad-B7 vector is a rapid and efficient means of gene transfer which does not require host cell proliferation . The ultimate objective is to engineer autologous tumors to express B7-1 and vaccinate cancer patients in an adjuvant or palliative setting.

Biotech Histochem, 1996 May, 71(3), 115 - 7
An improved method for preparing the chromosomes of pines and other gymnosperms; Murray BG et al.; A simple technic is described to produce well spread gymnosperm chromosomes . Root tip meristems are digested with a pectinase:cellulase mixture to produce a cell suspension which then is squashed to yield flat, well spread chromosome complements that can be stained or used for in situ hybridization.

Immunology, 1996 May, 88(1), 147 - 52
Antigen presentation in the murine oral epithelium; Eriksson K et al.; We have previously reported that the buccal mucosa can support delayed type hypersensitivity (DTH) reactions to contact sensitizers . In the present study, we show that cells isolated from the buccal epithelium are able to present soluble exogenous antigens to specific T cells . Single cell suspensions obtained by enzymatic dispersion of buccal epithelial sheets could present the native protein antigen hen-egg lysozyme (HEL) to the I-Ak-restricted CD4+ T-cell hybridoma specific for a.a 46-61 on HEL . T-cell activation resulted in interleukin-2 (IL-2) production which could be inhibited by anti-major histocompatibility complex (MHC) class-II antibodies of pertinent specificity . Immunohistochemical staining of whole buccal epithelial sheets revealed that all MHC II positive cells had a dendritic morphology and expressed ATPase activity, indicating that these cells represent a major antigen-presenting cell (APC) population in this tissue . Furthermore, single cell suspensions isolated from buccal epithelium (BEC) after local in vivo administration of either a native soluble protein, a synthetic dodecapeptide, or a contact sensitizer were able to activate antigen-specific T cells ex vivo . Kinetic analyses indicated that maximal APC activity in the oral epithelium occurred within 1 hr after local antigen administration, and had essentially vanished after 24 hr . Conversely, APC activity was undetectable in draining cervico-mandibular lymph node cell suspensions recovered 1 hr after local antigen injection but became manifest after 3-24 hr . These observations suggest that dendritic cells can acquire antigens in the buccal epithelium and migrate to draining lymph nodes where they present processed antigen to MHC class II-restricted T cells . This APC population may thus be a critical element in the initiation of Th1-driven DTH responses in the oral mucosa.

Gut, 1996 May, 38(5), 679 - 86
Influence of cell interactions in a novel model of postnatal mucosal regeneration; Patel HR et al.; BACKGROUND AND AIMS--Conventional models of postnatal mucosal regeneration are cumbersome and limited: a novel model is described here . In addition, the influence of cell interactions on mucosal regeneration is examined within the model . METHODS--Postnatal rat small intestinal mucosa was digested by enzymes to yield heterotypic cell aggregates (CA) . CA colony forming ability, growth, and limited cytodifferentiation were assessed in vitro . CA were transplanted subcutaneously and retrieved for histological examination at staggered intervals to assess neomucosal morphogenesis and cytodifferentiation in vivo . Cell interactions in CA were disrupted by enzymes, thus producing cell suspensions (CS) . Regeneration by CA and CS were compared . RESULTS--CA produced proliferative colonies in vitro and showed a temporal sequence of neomucosal morphogenesis and differentiation in vivo . CA colonies were more numerous within 24 hours of primary culture and had greater cellularity by 96 hours than CS colonies . Alkaline phosphatase was expressed only by 258 of 696 CA colonies (37%) . CA subcutaneous grafts (48 of 56 (87%)) regenerated small intestinal neomucosa while CS were unsuccessful . CONCLUSION--These methods provide a model of mucosal regeneration which includes constituent processes of colony formation, growth, neomucosal morphogenesis, and cytodifferentiation . Preservation of cell interactions within CA seems advantageous to regeneration within the model.

Z Naturforsch {C}, 1996 May-Jun, 51(5-6), 432 - 4
Radiation-induced apoptosis in thymocytes: pH sensitization; Ojeda F et al.; Thymocytes were used as a model system to study the effect of microenvironmental pH changes on the radiation-induced apoptosis . We found that the sensitivity of thymocytes toward radiation induced apoptosis is increased by increasing the pH of the incubation medium . The major sensitivity change occurs between pH 7 and 8 . In a given cell suspension the results obtained where similar when the apoptosis evaluation was carried out either by counting the picnotic nuclei, or monitoring the fraction of apoptotic nuclei by flow cytometry; both methods show a radiosensitization when the pH value of incubation media rises from 7 to 8 . These results may be important when "in vitro" experiments are performed with lymphoid cells, since changes in pH of the media may determine important changes in the results.

Eur J Immunol, 1996 May, 26(5), 1083 - 7
Development in vitro of human CD4+ thymocytes into functionally mature Th2 cells . Exogenous interleukin-12 is required for priming thymocytes to produce both Th1 cytokines and interleukin-10; Mingari MC et al.; Fresh postnatal thymocyte cell suspensions were directly cloned under limiting dilution conditions with either phytohemagglutinin or toxic shock syndrome toxin-1 (TSST-1), a bacterial superantigen . Cultures contained allogenic irradiated feeder cells and interleukin (IL)-2, in the absence or presence of exogenous IL-4, interferon (IFN)-gamma or IL-12 . The resulting CD4+ T cell clones generated under these different experimental conditions were then analyzed for their ability to produce IL-2, IL-4, IL-5, IL-10, IFN-gamma and tumor necrosis factor (TNF)-beta in response to stimulation with phorbol 12-myristate 13-acetate (PMA) + anti-CD3 monoclonal antibody or PMA + ionomycin . Different from T cell clones generated from peripheral blood, virtually all CD4+ T cell clones generated from human thymocytes produced high concentrations of IL-2, IL-4 and IL-5, but no IFN-gamma, TNF-beta or IL-10 . Moreover, after activation, these clones expressed on their surface membrane both CD30 and CD40 ligand, but not the product of lymphocyte activation gene (LAG)-3, and provided strong helper activity for IgE synthesis by allogeneic B cells . The Th2 cytokine pattern could not be modified by the addition of IFN-gamma . However, upon addition of exogenous IL-12, the resulting CD4+ thymocyte clones produced TNF-beta, IFN-gamma, and IL-10 in addition to IL-4 and IL-5 . These results suggest that CD4+ human thymocytes have the potential to develop into cells producing the Th2 cytokines IL-4 and IL-5, whereas the ability to produce both Th1 cytokines and IL-10 is acquired only after priming with IL-12.

Oncology, 1996 May-Jun, 53(3), 241 - 6
Effect on leukemia cell numbers of in vivo administration of immunotherapeutic agents is age-dependent; Dussault I et al.; On the basis of our previous findings that erythroleukemia-bearing mice of different ages responded positively to immunotherapy {indomethacin +/- recombinant interleukin (rIL-2)} in vivo {stimulated natural killer (NK) cells and increased life span}, we aimed, in the present study, to determine if changes in these parameters could be correlated to change in erythroleukemia cell numbers . Infant (< 3 weeks old), young adult (5-8 weeks) and aged (> 10 months) DBA/2 mice were injected with erythroleukemia cells . Some mice remained untreated for the 10-day duration of the tumor-bearing period, while others were treated with either indomethacin alone for the 10 days from tumor inoculation, rIL-2 alone for the last 4 days of the 10-day tumor-bearing period, or with both indomethacin and rIL-2 as above . In all cases, both treated and untreated mice were killed at 10 days after tumor inoculation . Cytospot preparations from single cell suspensions of spleen and bone marrow, in all cases, were stained with MacNeal's tetrachrome hematologic stain and the relative and absolute number of erythroleukemia cells in these organs were determined using light microscopy . The results show that indomethacin- and/or rIL-2 treated leukemic infant and young adult mice have significantly lower numbers of erythroleukemia cells in both the spleen and bone marrow relative to untreated, leukemic mice of corresponding age . Leukemic aged mice, however, show no change in erythroleukemia cell numbers in either organ, regardless of treatment, paralleling our observations of a lack of immunotherapeutic value of these agents on NK cell production/function in aged mice.

Br J Haematol, 1996 May, 93(2), 295 - 8
Leucocyte filterability: comparing diluted blood with purified cell suspensions; Evans SA; Current rheological trends in filtration studies involve purification of cell subpopulations in order to study near-homogenous populations of cells, and this means that time-consuming and possibly cell-damaging procedures are used . Filtration of diluted blood offers the possibility of determining the properties of subpopulations of cells with minimal cell manipulation . The filtration properties of granulocytes and lymphocytes result in their being detected as one kinetic population, and therefore a combined granulocyte/lymphocyte pore transit time is calculated . There was no significant change seen in the combined granulocyte/lymphocyte transit time over the normal range of differential count . Filtration of diluted blood is a more reproducible way of measuring granulocyte and lymphocyte filterability, because purification of these cells gives rise to more scatter and larger ranges in the measured pore transit time, and significant day-to-day variation . A combined granulocyte/lymphocyte transit time is therefore an acceptable and reproducible way of assessing leucocyte rheology in diluted blood, when the differential count lies within the normal range . If changes in the granulocyte/lymphocyte transit time are detected, then cell purification may yield additional information on the subpopulations . Therefore, particularly in pathological samples, a combination of these approaches will yield more information than either test alone.

Cell Tissue Res, 1996 May, 284(2), 327 - 30
Thymocyte-directed enhancement of apoptosis via soluble factor(s) derived from a cortical and a medullary thymic epithelial cell line; Rinner I et al.; Apoptosis of murine thymocytes was examined either in intact fetal thymus lobes or in thymus cell suspensions, both cultured alone or in the presence of either a cortical (TEC 1.4) or a medullary (TEC 2.3) thymic epithelial cell line . Both TECs induced a pronounced increase of apoptosis in 24-h cultivated single thymus cell suspensions but not in spleen or bone marrow cell cultures . Co-culture of thymocytes with murine fibroblasts did not enhance apoptosis of the thymus cells . A similar enhancement of thymocyte apoptosis was observed with dialysed culture supernatants derived from both TEC lines, the active component(s) having a molecular weight of > 30 kDa . In contrast, the cortical TEC 1.4 had a pronounced apoptosis inducing effect on intact fetal thymus lobes cultivated for six days, whereas the medullary TEC 2.3 had only a marginal influence . TEC 1.4 also induced a significant alteration in the ratio of CD4+CD8+ to CD4-CD8- cells . It is concluded that both the cortical and medullary epithelial cell lines are able to induce thymocyte apoptosis but that a large proportion of the cells within the intact thymus stroma is refractory to the respective signal(s) of the medullary epithelial cell line.

Cell Tissue Res, 1996 May, 284(2), 303 - 16
A morphometric analysis of exocytosis in KCl-stimulated bovine chromaffin cells; Fox GQ; Transmission electron microscopy has been used to morphometrically evaluate exocytosis in bovine adrenal medulla chromaffin cells as the mechanism of catecholamine release . Purified cell suspensions were stimulated with KCl at varying strengths and durations and then conventionally processed for ultrastructural analysis . Quantitation of exocytotic images of dense cored chromaffin granules was a major objective and such images were found in all preparations, attesting to the efficacy of chemical fixation to preserve this event . However, because hundreds of cell profiles had to be screened to find a single granule in the process of release this low frequency precluded any meaningful correlations with estimates of granular involvement based on catecholamine release . Neither KCl molarity nor duration altered this finding nor did these variables significantly affect other parameters linked to exocytotic activity . For example, cell size and numbers of "empty' granules and vesicles remained constant and attempts to label "any' organelle with 30-nm colloidal gold or lanthanum precipitate proved unsuccessful . In short, if exocytosis is responsible for release, it would appear to function without leaving a morphological trace . An alternative hypothesis, therefore, is outlined which better accommodates existing data.

Br J Cancer, 1996 May, 73(9), 1031 - 6
Apoptosis of human seminoma cells upon disruption of their microenvironment; Olie RA et al.; One of the main obstacles encountered when trying to culture human seminoma (SE) cells in vitro is massive degeneration of the tumour cells . We investigated whether dissociation of tumour tissue, to obtain single-cell suspensions for in vitro culture, results in the onset of apoptosis . Using morphological analysis and in situ end labelling, less than 4% of apoptotic tumour cells were detected in intact tissue from 11 out of 14 SEs . In these 11 tumours, apoptosis-specific DNA ladders, indicative of internucleosomal double-strand DNA cleavage, were not detected on electrophoresis gels . In contrast, three SEs with over 12% of apoptotic tumour cells in the intact tissue and all analysed (pure) SE cell suspensions, obtained after mechanical dissociation of intact tumour tissue, showed DNA ladders . Flow cytometric analysis of end labelled SE suspensions showed DNA breaks in up to 85% of the tumour cells . As indicated by cell morphology and DNA degradation, SE cells appear to rapidly enter the apoptotic pathway upon mechanical disruption of their microenvironment . No expression of p53 and of the apoptosis-inhibitor bcl-2 was detectable in intact SE tissue or cell suspensions . Our data suggest that abrogation of apoptosis might be crucial to succeed in culturing human SE cells in vitro.

J Invest Dermatol, 1996 May, 106(5), 1042 - 6
In vivo retinoic acid modulates expression of the class II major histocompatibility complex and function of antigen-presenting macrophages and keratinocytes in ultraviolet-exposed human skin; Meunier L et al.; Because retinoic acid (RA) can alter photoaging of the skin and repeated ultraviolet (UV)-induced immunologic injury may play a role in chronic photoaging, we asked whether RA alters the acute photoimmunologic effects of UV radiation . Two sites from each volunteer were treated with 0.1% RA or vehicle continuously for 24 h before and 24 h after a 4-minimal erythema dose UVB exposure . RA did not function as a sunscreen, as determined by quantitating the increase in redness after 1 minimal erythema dose to vehicle- and RA-pretreated sites (n = 12) . By flow cytometric analysis of epidermal cell suspensions harvested 3 d after the UV-EC, RA treatment did not protect CD1+ Langerhans cells from being depleted by UV light and did not modify the number of UV-induced infiltrating CD36+CD11b+CD1-DR+ macrophages . RA treatment did, however, result in a 40% downregulation of human leukocyte antigen (HLA)-DR expression on these infiltrating macrophages (p = 0.016) (n = 11), in conjunction with a decrease in alloantigen-presenting cell activity of RA-treated UV-EC as measured by T-cell proliferations . RA also induced a 72% inhibition of the autologous T suppressor-inducer cell proliferation induced by UV-EC (vehicle: 21,813 +/- 7,302 cpm; RA; 5,299 +/-635 cpm) (n = 3) . The downregulation could be due to RA-modulated keratinocytes; RA-treated UV-EC keratinocytes depleted of CD1a+ and DR+ antigen-presenting cells displayed a greater ability, relative to similarly treated vehicle-EC keratinocytes, to inhibit alloantigen presentation . In conclusion: (i) in vivo RA treatment did not protect human Langerhans cells from being depleted by UV and did not block infiltration of macrophages into sunburned skin; and (ii) RA did decrease autologous and allogeneic T-cell reactivity induced by macrophage antigen-presenting cells in UV-exposed epidermis, at least in part by downregulating their HLA-DR expression and by upregulating inhibitory signals from UV-irradiated keratinocytes.

Neurosci Lett, 1996 Apr 19, 208(2), 89 - 92
Regional study of spinal alpha 2-adrenoceptor densities after intraspinal noradrenergic-rich implants on adult rats bearing complete spinal cord transection or selective chemical noradrenergic denervation; Roudet C et al.; One of the challenges of restorative neuronal transplantation in the CNS of mammals is the appropriate integration of grafted cells in the host circuitry . One key parameter is the specific influence of grafted cells upon corresponding receptors . In order to test this issue on the lesioned spinal cord of adult rats, two models of spinal cord denervation were used: the first one consisted of a complete transection 1 week prior to an intraspinal transplantation of embryonic locus coeruleus (LC) primordia cell suspension; the second one was a chemical destruction of the spinal noradrenergic (NA) system 1 month prior to a similar transplantation . Five weeks after transplantation, spinal sections were processed for autoradiographic quantification of alpha 2-adrenoceptor binding sites densities . In most regions, alpha 2-adrenoceptor densities remained comparable or higher than before graft; interestingly, in lumbar dorsal horn, lumbar intermediate zone and sacral distal dorsal horn of transected-grafted rats, they returned to control level . Results are discussed in relation to the parallel study performed concerning alpha 1-adrenoceptors.

J Biol Chem, 1996 Apr 19, 271(16), 9384 - 9
Identification and characterization of an S-adenosyl-L-methionine: delta 24-sterol-C-methyltransferase cDNA from soybean; Shi J et al.; In plants, the dominant sterols are 24-alkyl sterols, which play multiple roles in plant growth and development, i.e . as membrane constituents and as precursors to steroid growth regulators such as brassinosteroids . The initial step in the conversion of the phytosterol intermediate cycloartenol to the 24-alkyl sterols is catalyzed by S-adenosyl-L-methionine: delta 24-sterol-C-methyl-transferase (SMT), a rate-limiting enzyme for phytosterol biosynthesis . A cDNA clone (SMT1) encoding soybean SMT was isolated from an etiolated hypocotyl cDNA library by immunoscreening using an anti-(plasma membrane) serum . The deduced amino acid sequence of the SMT1 cDNA contained three conserved regions found in S-adenosyl-L-methionine-dependent methyltransferases . The overall structure of the polypeptide encoded by the SMT1 cDNA is most similar to the predicted amino acid sequence of the yeast ERG6 gene, the putative SMT structural gene . The polypeptide encoded by the SMT1 cDNA was expressed as a fusion protein in Escherichia coli and shown to possess SMT activity . The growing soybean vegetative tissues had higher levels of SMT transcript than mature vegetative tissues . Young pods and immature seeds had very low levels of the SMT transcript . The SMT transcript was highly expressed in flowers . The expression of SMT transcript was suppressed in soybean cell suspension cultures treated with yeast elicitor . The transcriptional regulation of SMT in phytosterol biosynthesis is discussed.

Biochim Biophys Acta, 1996 Apr 3, 1280(1), 34 - 40
Time domain dielectric spectroscopy study of human cells . I . Erythrocytes and ghosts; Lisin R et al.; A method, allowing correction of electrode polarization effect in case of cell suspensions of small volume fraction is proposed . The dielectric behavior of human erythrocytes and erythrocyte ghosts suspensions was studied by time domain dielectric spectroscopy (TDDS) and the estimation of the dielectric constants of cell's structural parts (membrane, cytoplasm) on the basis of suitable models are presented . It was shown that in the case of small volume fraction of cells in suspension, the electrode polarization effect can be taken into account by the additional measurement of the corresponding supernatant . The optimal volume fraction of cells in suspensions in the TDDS measurements was found to be 3%-10% . The erythrocyte and erythrocyte ghost suspensions were found to demonstrate a single dispersion which can be described by the Debye equation . The membrane dielectric constant of different erythrocytes and ghosts was distributed near 5.

Arch Dermatol Res, 1996 Apr, 288(4), 203 - 10
Multiparameter flow cytometric characterization of epidermal cell suspensions prepared from normal and hyperproliferative human skin using an optimized thermolysin-trypsin protocol; Glade CP et al.; Reliable flow cytometric analysis of normal and diseased skin requires pure epidermal single-cell suspensions . Several methods to separate the dermis from the epidermis are available . The proteolytic enzyme thermolysin separates the epidermis from the dermis at the lamina lucida and therefore permits reliable dermoepidermal separation . In the present study an optimized cell isolation procedure using thermolysin and trypsin is described, which is particularly suitable for punch biopsies . A 16-20-h (overnight) incubation of biopsies taken from normal and hyperproliferative skin with thermolysin (0.5 mg/ml) at 4 degrees C produced a selective separation of the dermis and epidermis . After a 30-min trypsin incubation (0.25 mg/ml) at 37 degrees C a cell suspension was produced which was characterized by minimal cell damage (cellular debris and clumps), a high recovery of basal cells and high quality DNA histograms . Furthermore, dermal contamination was very low . The thermolysin-trypsin separation methodology followed by triple-labelling flow cytometry provided a precise quantification of the percentage of keratin 10-positive cells, vimentin-positive cells and cells in S and G2M phases . Proliferative activity was selectively measured in the basal, the suprabasal and the non-keratinocyte compartment at various time intervals during epidermal regeneration after adhesive tape stripping . In contrast to the non-keratinocytes, the percentage of cells in S and G2M phases in the basal keratinocytes and in the suprabasal compartment increased 44-48 h after stripping . The increased proliferation following tape stripping was paralleled by an increased invasion of vimentin-positive cells into the epidermis and preceded by a decreased number of keratin 10-positive cells . Thermolysin-trypsin separation followed by three-colour flow cytometry permits a highly selective characterization of normal and hyperproliferative epidermis.

Noshuyo Byori, 1996 Apr, 13(1), 17 - 20
MIB-1 immunostaining and DNA flow cytometry in meningiomas; Yasue M et al.; The correlation between S- and G2/M-phase fractions and MIB-1 index in meningiomas was investigated . Flow cytometric analyses were performed on cell suspensions from 81 samples of paraffin-embedded tissue from 29 patients with a total of 28 meningiomas and 1 hemangiopericytoma using the modified Hedley's method . Sections from the paraffin-embedded tissues were stained with anti-MIB-1 monoclonal antibody after microwave oven processing . Five hundreds cells were scored . Correlations between data were estimated using linear regression . DNA aneuploidy was present in 4/29 tumors . Mean S- and G2/M-phase fractions in the 25 nonrecurrent tumors were 1.77 and 4.76%, and in the recurrent tumors were 2.41 and 5.81%, respectively . The proliferative index (PI = S + G2/M fraction) was 6.52% in the nonrecurrent tumors and 8.29% in the recurrent tumors . The mean MIB-1 score in the nonrecurrent tumors was 1.25% and in the recurrent tumors was 2.69% . There was a linear correlation between percentage of S-phase fraction or PI and the MIB-1 score . Both methods are useful to assess the proliferative activity in meningioma.

Cytometry, 1996 Apr 1, 23(4), 279 - 83
Karyotyping of individual cells with flow cytometry; Stepanov SI et al.; As the study of metaphase chromosomes with flow cytometry presupposes mixing all chromosomes from many cells before analysis, important information is lost . To overcome this limitation we have developed a novel cell-oriented flow cytometric method for chromosome analysis . A flow cytometer supplied with a special device for disruption of metaphase chromosomes is the heart of the method . Cells with stained chromosomes are pressed into the disruption device and then converted in batches of chromosomes . Chromosome fluorescence and time intervals between fluorescence pulses are registered . There are large time intervals between neighboring batches because a sparse cell suspension is used and, in turn, there are small time intervals inside batches . Taking account of time intervals, it is possible to identify individual cell flow karyotypes . This highly productive method for individual cell analysis (100-200 cells/min) ensures detecting the changes in cell flow karyotypes, i.e., under- and overrepresentation of chromosomes, aberrations, and amplification of DNA.

Gen Physiol Biophys, 1996 Apr, 15(2), 145 - 63
Low pH induced shape changes and vesiculation of human erythrocytes; Gros M et al.; Shape changes and vesiculation were induced in intact human erythrocytes by gradually decreasing pH in the cell suspension . A sequence of different shapes preceding vesiculation was documented, i.e . discocytes, stomatocytes, and stomatoacantocytes . The final state was characterized by spherical mother cells and vesicles released . Low pH-induced vesiculation was also studied in the presence of stomatocytogenic or echinocytogenic compounds . The action of stomatocytogenic compounds was inhibitory, and echinocytogenic compounds had no effect on low pH-induced vesiculation . Vesiculation induced by low pH was studied also in isotonic solutions of different sucrose/salt composition . It was concluded that (i) low intracellular pH is responsible for cell shape transformations as well as for release of vesicles, (ii) at temperature 37 degrees C the intracellular pH value which induces the release of vesicles is 5.4, and (iii) the sequence of typical shape changes preceding vesiculation does not include echinocytes . The results are discussed on the basis of the layered membrane model of the shape formation and shape transformations of the human erythrocyte, and additionally considering the partial detachment of the membrane skeleton from the bilayer part of the membrane.

Bioseparation, 1996 Apr, 6(2), 81 - 9
Freeze/thawing and sonication of Escherichia coli TB1 cells for cytochrome b5 recovery; Santos JA et al.; The influence of sonication power, suspension volume and cell concentration on the kinectics of cytochrome b5 and intracellular protein release by sonication of Escherichia coli TB1 cells was studied . The influence of freezing and thawing of the cell suspension was also evaluated . Freezing and thawing increased the recovery yield of cytochrome b5 . The sonication efficiency increased with the increase of sonication power and with the decrease of the suspension volume and cell concentration.

Exp Neurol, 1996 Apr, 138(2), 318 - 26
Transplantation of Mesencephalic Cell Suspension in Dopamine-Denervated Striatum of the Rat
Di Loreto S, Florio T, Capozzo A, Napolitano A, Adorno D, Scarnati E.
These studies have examined the extent to which intrastriatal grafts of embryonic mesencephalic neurons induce recovery of normal discharge patterns in striatal neurons of rats after a unilateral 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal dopamine (DA) pathway . Lesioned rats were tested for rotational behavior induced by amphetamine and apomorphine . Animals which responded positively to these tests received two suspensions of mesencephalic embryonic neurons into the dorsal striatum (ST) ipsilateral to the denervated side . Sham-grafted rats received the suspension medium only . The vitality of the graft was assessed by the disappearance or reversion of rotational movements induced by amphetamine . Extracellular recordings of neurons located throughout the ST were carried out 3 months after grafting, when the animals reached the age of 6 months . The 6-OHDA-induced nigral lesion caused a net increase both in the number of striatal neurons spontaneously active and in their discharging rates . The signs of increased neuronal activity were also present in sham-grafted animals . The grafting of embryonal cells strongly reduced the number of active neurons and decreased significantly their discharging rate . The effects of the intrastriatal graft appeared to be present within a radius of 1.5-2 mm from the core of the grafted area . The presence of tyrosine-hydroxylase-immunopositive neurons innervating the host ST confirmed the viability of the grafts at the time of electrophysiological recording . The results show that besides compensating motor asymmetries caused by DA denervation, intrastriatally grafted dopaminergic neurons are able to only partially restore the electrophysiological action of DA in discrete striatal domains.

Rinsho Shinkeigaku, 1996 Apr, 36(4), 557 - 61
{Fornix transection stimulates locus coeruleus neurons transplanted in the rat hippocampus to increase noradrenaline}; Furui E; Fetal noradrenergic neurons from the brain stem locus coeruleus region were transplanted, as a cell suspension, into the hippocampus of rats . Adult male rats were subjected to removal of the superior cervical ganglia and were used as recipients . In the fornix transection (FT) group, rats were sacrificed before fornix transection, or received fornix transection and were sacrificed 3, 5 or 7 weeks after fornix transection . In the transplantation (TP)-FT group, rats received transplants 3 weeks before fornix transection and were sacrificed 3, 5 or 7 weeks after fornix transection . In the FT-TP group rats received transplants a week after fornix transection and were sacrificed 7 weeks after fornix transection . At sacrifice, the noradrenaline (NA) concentration in the hippocampus was measured . The differences in the NA concentration between the FT group and either the TP-FT group or the FT-TP group were statistically significant 7 weeks after fornix transection, respectively . Results suggested that the fornix transection stimulates transplanted neurons to increase NA . This stimulation depended not on the time after transplantation but on the time after fornix transection.

Exp Brain Res, 1996 Apr, 109(1), 179 - 84
Foetal nigral cell suspension grafts influence dopamine release in the non-grafted side in the 6-hydroxydopamine rat model of Parkinson's disease: in vivo voltammetric data; Earl CD et al.; The present study employed differential-pulse voltammetry to assess the influence of foetal ventral mesencephalic grafts on dopamine overflow in the contralateral caudate putamen of the 6-hydroxydopamine rat model of Parkinson's disease . The experimental design involved measurements of dopamine overflow in the grafted and contralateral striatum . Control measurements of dopamine overflow were performed in 6-hydroxydopamine-lesioned rats only and the caudate putamen of normal control rats . Cell suspensions of foetal rat ventral mesencephalic tissue were grafted into the dopamine-depleted caudate putamen of unilaterally 6-hydroxydopamine-lesioned rats . At 6 weeks, animals with functional, mature grafts (as assessed by amphetamine-amplified behavioural asymmetry), were pretreated with pargyline (75 mg/kg i.p.), and both striatal sides were monitored for dopamine overflow for 90 min following amphetamine sulphate administration (5 mg/kg i.p.) . The time course of dopamine overflow inside the graft was similar to that in the contralateral caudate putamen of the same animal, the normal control animal and the contralateral caudate putamen of 6-hydroxydopamine-lesioned animals . However, in grafted animals the mean dopamine overflow detected in the contralateral caudate putamen was approximately 34% lower than the concentration of dopamine detected in the contralateral caudate putamen of 6-hydroxydopamine-lesioned control animals and approximately 39% lower than the concentration of dopamine detected in the caudate putamen of the normal control animal . There was no statistical difference in the concentration of amphetamine-induced dopamine overflow between the caudate putamen contralateral to the 6-hydroxydopamine lesion and the caudate putamen of the normal control animal . These data suggest that intrastriatal foetal ventral mesencephalic suspension grafts reduce amphetamine-induced dopamine release in the contralateral non-grafted caudate putamen.

J Hepatol, 1996 Apr, 24(4), 468 - 77
Phagocytic function and metabolite production in thioacetamide-induced liver cirrhosis: a comparative study in perfused livers and cultured Kupffer cells; Petermann H et al.; BACKGROUND/AIMS: The aim of the study presented here was to evaluate the basal and stimulated phagocytic activities and the metabolite production of isolated perfused livers, and also the phagocytic capacity of cultured Kupffer cells from rats with macronodular cirrhosis . METHODS: Rats were made cirrhotic by oral administration of thioacetamide . The phagocytic activity was assessed by the rate of removal of colloidal carbon . The Kupffer cells were prepared by a pronase/collagenase digestion method followed by elutriation . RESULTS: The phagocytic activity and production of glucose, lactate and pyruvate were reduced in cirrhotic livers when calculated per g liver . Due to hyperplastic-regenerative processes the mass of the cirrhotic livers was markedly augmented so that the colloidal carbon uptake calculated per cirrhotic liver was not significantly different from the controls . Colloidal carbon-induced glucose release increased more markedly in the controls than in cirrhotic livers . Isoproterenol considerably stimulated phagocytosis and glucose production in controls, whereas the response was clearly reduced in cirrhotic livers when calculated either per g liver or per total liver weight . The cyclic AMP analogue elicited a marked glycogenolytic response in the controls, whereas there was only a slight increase in glucose production in cirrhotic livers . Phagocytosis of cirrhotic livers was only moderately stimulated by opsonized zymosan when compared with the controls . Freshly isolated Kupffer cells exhibited a reduced phagocytic activity . Stimulation by zymosan was observed only in cell suspensions of the controls . In contrast, Kupffer cells from cirrhotic livers did not differ from controls with respect to basal or zymosan-stimulated phagocytic activity after 48-h cultivation . CONCLUSION: The stimulated phagocytic function was disturbed in perfused macronodular-cirrhotic livers as compared to controls . In contrast, 48-h cultured Kupffer cells from cirrhotic livers exhibited the same basal and stimulated phagocytic capacity as controls . The glucose release from perfused livers, initiated by stimulation of Kupffer cells or hepatocytes, was significantly reduced in cirrhotic livers . Therefore, we postulate an impaired intra- and/or intercellular signalling in macronodular-cirrhotic livers.

Eur J Cancer, 1996 Apr, 32A(4), 715 - 21
Characterisation of platelet aggregation induced by PC-3 human prostate adenocarcinoma cells and inhibited by venom peptides, trigramin and rhodostomin; Swaim MW et al.; PC-3 cells, a metastatic human prostate adenocarcinoma line, caused dose-dependent platelet aggregation in heparinised human platelet-rich plasma (PRP) . PC-3 tumour cell-induced platelet aggregation (TCIPA) was completely inhibited by hirudin (5 U/ml) and limited by increasing concentrations of apyrase . This TCIPA was unaffected by cysteine proteinase inhibition with E-64 (10 microM), but was limited by cell pretreatment with phospholipase A2 . PC-3 cell suspension caused marked, dose-dependent decreases in plasma recalcification times using normal, Factor VIII-deficient and Factor IX-deficient, but not Factor VII-deficient, human plasma . This effect was potentiated in cell lysates, but was inhibited in intact cells preincubated with sphingosine . Overall, these data suggest that PC-3 TCIPA arises from PC-3 tissue factor activity expression . Trigramin and rhodostomin, RGD-containing snake venom peptides which antagonise the binding of fibrinogen to platelet membrane glycoprotein IIb-IIIa, prevented PC-3 TCIPA . Similarly, synthetic peptide GRGDS as well as monoclonal antibodies against platelet membrane glycoproteins IIb-IIIa and Ib prevented PC-3 TCIPA, which was unaffected by control peptide GRGDS . On a molar basis, trigramin (IC50, 0.11 microM) and rhodostomin (IC50, 0.03 microM) were approximately 5000 and 18000 times, respectively, more potent than GRGDS (IC50, 0.56 mM).

J Exp Med, 1996 Apr 1, 183(4), 1865 - 78
A life stage of particle-laden rat dendritic cells in vivo: their terminal division, active phagocytosis, and translocation from the liver to the draining lymph; Matsuno K et al.; Initiation of an adoptive immune response against pathogenic organisms, such as bacteria and fungi, may involve phagocytic activity of dendritic cells (DC) or their immature precursors as a prelude to antigen processing and presentation . After intravenous injection of rats with particulate matter, particle-laden cells were detected in the peripheral hepatic lymph . Since it has been known there is a constant efflux of DC from nonlymphoid organs into the draining peripheral lymph, we examined whether these particle-laden cells belonged to the DC or macrophage lineage . The majority of particle-laden cells in lymph showed immature monocyte-like cytology, and the amount of ingested particles was small relative to typical macrophages . We identified these particle-laden cells as DC based on a number of established criteria: (a) they had a phenotype characteristic of rat DC, that is, major histocompatibility complex class Ihigh+ and IIhigh+, intercellular adhesion molecule 1+ and 80% positive with the rat DC-specific mAb OX62; (b) they showed strong stimulating capacity in primary allogeneic mixed leukocyte reaction; (c) in vitro, they had little phagocytic activity; and (d) the kinetics of translocation was similar to that of lymph DC in that they migrated to the thymus-dependent area of the regional nodes . Furthermore, bromodeoxyuridine feeding studies revealed that most of the particle-laden DC were recently produced by the terminal division of precursor cells, at least 45% of them being <5.5 d old . The particle-laden DC, defined as OX62+ latex-laden cells, were first found in the sinusoidal area of the liver, in the liver perfusate, and in spleen cell suspensions, suggesting that the site of particle capture was mainly in the blood marginating pool . It is concluded that the particle-laden cells in the hepatic lymph are recently produced immature DC that manifest a temporary phagocytic activity for intravascular particles during or after the terminal division and that the phagocytic activity is downregulated at a migratory stage when they translocate from the sinusoidal area to the hepatic lymph.

J Magn Reson B, 1996 Apr, 111(1), 9 - 14
Measuring Nitrate in Plant Cells by in Vivo NMR Using Gd3+ as a Shift Reagent
Shachar-Hill Y, Pfeffer PE, Ratcliffe RG.
NMR investigations of nitrate in plant cells and tissues have hitherto been limited by the indistinguishability of the signals from intracellular and extracellular nitrate . Gd3+ is shown to be an effective shift reagent for 14N and 15N nitrate NMR signals, resolving the internal and external nitrate signals in plant tissues, including cell suspensions and root material . However, time-course experiments show that, while the use of Gd3+ allows nitrate levels to be monitored over extended periods, it also has adverse effects on growth and nitrate uptake . Accordingly, a number of chelated forms of gadolinium were investigated, and it is concluded that the NMR contrast agent Gd(DTPA-BMA) is likely to be a suitable shift reagent for physiologically relevant studies of nitrate transport in roots.

Int Arch Allergy Immunol, 1996 Apr, 109(4), 376 - 82
Purification of mast cells with an improved nonsynchronous flow-through coil planet centrifuge; Okada T et al.; A method for cell purification was designed without using high-density media which may impair membrane receptors . Rat and mouse mast cells were separated with an improved nonsynchronous flow-through coil planet centrifuge . Peritoneal cells were suspended at a concentration of 2-3x10(7) cells/ml in conditioned RPMI 1640, supplemented with 50% heat-inactivated FCS and 0.32% sodium citrate . In each separation 3 ml of cell suspension were loaded into the coiled column and elutriated at 4 degrees C . Several conditions, including the centrifugal force, revolution/rotation ratio, density of separation media, flow speed, and designs of both coiled column and flow tubes, were examined and optimized for mast cell purification . Rat mast cells were separated at the purity of 99.2%, with an average yield of 40% under sterile conditions . Nearly 90% pure mouse mast cells were harvested, despite a very low population of mast cells available in murine peritoneal cells . Purified cells were morphologically intact and discharged granules by exocytosis as indicated by electron-microscopic observations . The average histamine release with antigenic specificity was 34 and 61%, in passive sensitization in vitro and in vivo, respectively . Mast cells sensitized with mouse monoclonal IgE antibody released histamine, similar to cells sensitized with homologous antibody . This newly devised method of cell separation will be useful to purify biologically intact mast cells.

Exp Neurol, 1996 Apr, 138(2), 318 - 26
Transplantation of mesencephalic cell suspension in dopamine-denervated striatum of the rat.I . Effects on spontaneous activity of striatal neurons; Di Loreto S et al.; These studies have examined the extent to which intrastriatal grafts of embryonic mesencephalic neurons induce recovery of normal discharge patterns in striatal neurons of rats after a unilateral 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal dopamine (DA) pathway . Lesioned rats were tested for rotational behavior induced by amphetamine and apomorphine . Animals which responded positively to these tests received two suspensions of mesencephalic embryonic neurons into the dorsal striatum (ST) ipsilateral to the denervated side . Sham-grafted rats received the suspension medium only . The vitality of the graft was assessed by the disappearance or reversion of rotational movements induced by amphetamine . Extracellular recordings of neurons located throughout the ST were carried out 3 months after grafting, when the animals reached the age of 6 months . The 6-OHDA-induced nigral lesion caused a net increase both in the number of striatal neurons spontaneously active and in their discharging rates . The signs of increased neuronal activity were also present in sham-grafted animals . The grafting of embryonal cells strongly reduced the number of active neurons and decreased significantly their discharging rate . The effects of the intrastriatal graft appeared to be present within a radius of 1.5-2 mm from the core of the grafted area . The presence of tyrosine-hydroxylase-immunopositive neurons innervating the host ST confirmed the viability of the grafts at the time of electrophysiological recording . The results show that besides compensating motor asymmetries caused by DA denervation, intrastriatally grafted dopaminergic neurons are able to only partially restore the electrophysiological action of DA in discrete striatal domains.

J Nutr, 1996 Apr, 126(4), 849 - 59
The CD4/CD8 ratio in the blood does not reflect the response of this index in secondary lymphoid organs of weanling mice in models of protein-energy malnutrition known to depress thymus-dependent immunity; Lee WH et al.; A low ratio of cellular numbers within CD4+ (helper/inducer) relative to CD8+ (suppressor/cytotoxic) thymic lymphocyte subsets (low CD4/CD8 ratio) is widely accepted as fundamental to the depression in thymus-dependent immunocompetence associated with wasting protein-energy malnutrition (PEM) . The objective of this investigation, therefore was to determine the CD4/CD8 ratio in peripheral lymphoid compartments of diverse murine models of protein-energy malnutrition which produce systemic wasting (loss of approximately 1.8% of initial body weight per day), lymphoid involution and (as shown in many previous studies) depression in thymus-dependent immunocompetence . In the first of two experiments, male and female weanling mice of disparate inbred strains, CBA/J and C57BL/6J, were allocated to a zero-time control group (23- and 19-d-old, respectively), or to groups fed for 14 d as follows: ad libitum intake of a complete purified diet (19% crude protein, 17 kJ/g gross energy), restricted intake of the complete diet, or ad libitum intake of an isocaloric low protein diet (0.6% crude protein) . In a supplementary experiment, (0.6% crude protein) . In a supplementary experiment, male and female C57BL/6J weanling mice were fed the complete diet or the low protein diet for either 6 or 21 d . CD4+ and CD8+ thymic lymphocytes were enumerated by flow cytometry in mononuclear cell suspensions from blood, spleen and mesenteric lymph nodes . A low CD4/CD8 ratio is common in the blood in wasting protein-energy malnutrition, but appears uncharacteristic of the profoundly involuted lymphoid organs which generate acquired immune responses . The CD4/CD8 ratio is irrelevant to the thymus-dependent immunoincompetence previously demonstrated in the rodent models used in this investigation.

J Neurosurg, 1996 Apr, 84(4), 655 - 62
Combined fetal neural transplantation and nerve growth factor infusion: effects on neurological outcome following fluid-percussion brain injury in the rat; Sinson G et al.; This study was designed to evaluate the histological and behavioral impact of fetal neural transplantation with and without neurotrophin infusion in rats subjected to traumatic brain injury using a clinically relevant model of lateral fluid-percussion brain injury . Adult male Sprague-Dawley rats received lateral fluid-percussion brain injury of moderate severity (2.1-2.3 atm) . Twenty-four hours after injury, minced fetal cortical grafts (E16) were stereotactically transplanted into the site of injury cavity formation (in 32 rats) . Ten control animals received injections of saline . A third group of 29 animals that received transplants also underwent placement of a miniosmotic pump (immediately after transplantation) to continuously infuse nerve growth factor (NGF) directly into the region of graft placement for the duration of the experiment . A fourth group of eight animals underwent transplantation of fetal cortical cells that had been dissociated and placed in suspension . Animals were evaluated at 72 hours, 1 week, and 2 weeks after injury for cognitive function (using the Morris water maze), posttraumatic motor dysfunction, and transplant survival and morphology (using Nissl and modified Palmgren's silver staining techniques) . Robust survival of whole-tissue transplants was seen in 65.5% of animals and was not increased in animals receiving NGF infusion . Animals receiving transplants of cell suspension had no surviving grafts . Brain-injured animals receiving transplants showed significant cognitive improvements compared with controls at the 2-week evaluation . Significantly improved memory scores were seen at all evaluation times in animals receiving both NGF and transplants compared with injured controls and compared with animals receiving transplants alone at the 72-hour and 1-week evaluations . Neurological motor function scores were significantly improved in animals receiving transplants alone and those receiving transplants with NGF infusion . Histological evaluation demonstrated differentiation of grafted cells, decreased glial scarring around transplants when compared with control animals, and the presence of neuronal fibers bridging the interface between graft and host . This study demonstrates that fetal cortical cells transplanted into the injured cortex of the adult rat can improve both posttraumatic cognitive and motor function and interact with the injured host brain.

J Surg Res, 1996 Apr, 62(1), 23 - 8
PKH26 and 125I-PKH95: characterization and efficacy as labels for in vitro and in vivo endothelial cell localization and tracking; Ford JW et al.; PKH26, a fluorescent cell label, and PKH95, a 125 I-radioactive cell label, are both potentially valuable endothelial cell labels because they bind irreversibly within cell membranes . These labels would be particularly well suited to tract transplanted endothelial cells in vivo . However, no previous studies documenting lack of transfer of the label to unlabeled endothelial cells, as well as the effect of the label on endothelial cell function, have been undertaken . The purpose of this study was to determine the optimal method of endothelial cell (EC) labeling with PKH26 and PKH95, whether significant to EC-to-EC transfer of the label occurs, the effects of these labels on EC proliferation and membrane function, and the feasibility of using these labels for long-term quantitative EC tracking in vivo . Canine ECs in confluent monolayers or in cell suspension were labeled by exposure to 1.0 or 5.0 microM PKH26 for 1, 3, or 5 min . Cell viability was determined by trypan blue exclusion . The percentage of cells labeled and their fluorescence intensity were determined in a fluorescent-activated cell sorter (FACS) . Effect of the label on cell function was assessed by measuring EC proliferation rates as well as intercellular adhesion molecule (ICAM) expression before and after induction with tumor necrosis factor (TNF) . To determine if transfer of the label occurs, both labeled and nonlabeled ECs were grown in coculture and subjected to FACS analysis . For in vivo cell tracking, doubly labeled ECs were injected into the femoral artery of rat hind-limbs, and whole-body tissue analysis was undertaken to determine labeled-EC distribution at 60 days . Endothelial cells were labeled with equal efficacy as monolayers or in suspension . Labeling had no effect on EC proliferation rates nor on TNF-induced upregulation of ICAM expression . Coculture experiments revealed no significant label transfer to nonlabeled ECs . In vivo cell tracking studies documented that 8% of label remained within the skeletal muscle capillaries at 60 days after injection . PKH26 and PKH95 labels incorporate stably into EC membranes, do not alter endothelial cell function, and provide a precise means for quantitative EC tracking and histologic localization both in vitro and in vivo . These labels should prove to be very useful for studies of endothelial cell biology and transplantation.

Br J Obstet Gynaecol, 1996 Apr, 103(4), 359 - 65
The suitability of DNA cytometry for the prediction of the histological diagnosis in women with abnormal cervical smears; van Leeuwen AM et al.; OBJECTIVE: To analyse the suitability of DNA cytometry for predicting the histological diagnosis in women with cervical dyskaryosis . DESIGN: Survey with the use of diagnostic information to revise disease probability . SETTING: Colposcopy clinic of a university hospital . PARTICIPANTS: One hundred and ten women with two mildly or moderately dyskaryotic cervical smears and 98 women with one severely dyskaryotic smear . INTERVENTIONS: DNA cytometric analysis using cytocentrifuge preparations of single cell suspensions from a cervical scrape . The main DNA cytometric parameter was N5C (i.e . the absolute number of cells with a DNA content of more than 5C on a given surface with a predefined cell density) . MAIN OUTCOME MEASURE: The probability of finding CIN II or worse . On arbitrary grounds, a positive test should point to a probability of 85% or higher . RESULTS: In the patients with cervical neoplasia, the value of N5C increased significantly with an increasing CIN grade (P < 0.001) . In the patients with one severely dyskaryotic smear and in those with two mildly or moderately dyskaryotic smears, the prior probability of finding CIN II or worse was 94% and 53%, respectively . Therefore, DNA cytometric analysis might be particularly useful in women with mild or moderate dyskaryosis; further analysis was restricted to this group . All of the women in whom the N5C value was higher than 52 were diagnosed as having CIN II or worse . Only 16 (14.5%) of the 110 women had an N5C value of 52 or higher . When the N5C value was 27, the probability of finding CIN II or worse was estimated to be 85% . Only 28 (25%) patients had an N5C value of 27 or higher . CONCLUSIONS: DNA cytometry produced significant diagnostic information, as was shown by the relation between N5C and the histological diagnosis . However, the N5C value could not discriminate sufficiently between women with CIN II or worse and CIN I or better . Therefore, the management of individual patients with cytological abnormalities cannot be based on the results of DNA cytometric analysis.

Clin Exp Immunol, 1996 Apr, 104(1), 124 - 32
Distribution of human colonic dendritic cells and macrophages; Pavli P et al.; To define the phenotype of intestinal dendritic cells and macrophages, resected colonic specimens were used to obtain lamina propria cell suspensions by EDTA treatment, then enzymatic digestion . The phenotype of dendritic cell-enriched suspensions was compared with that of macrophage-enriched populations by immunocytochemistry using the avidin-biotin-peroxidase (ABC) system and immunoelectron microscopy . Dendritic cells expressed HLA-DR (L243) and HLA-DQ-associated (RFD) antigens and CD68 in a perinuclear distribution . Staining for S100 was weak or absent . Macrophages also expressed HLA markers (L243 and RFD1) and CD68 . The 25F9 antigen was expressed strongly, whilst CD14 was absent from cells isolated from noninflamed tissues . To determine their anatomic distribution, immunohistochemistry was performed using single- and double-labelling techniques (ABC +or- alkaline phosphatase anti-alkaline phosphatase method) . Mutually exclusive subsets of 25F9+ and S100+ cells were seen: 25F9+ macrophages were concentrated in a band immediately beneath the luminal epithelium; S100+/HLA-DR+ dendritic cells formed a reticular network throughout the lamina propria and beneath the basement membrane of the crypts . This distribution suggests that macrophages may help regulate intestinal responses by acting as the first line of defence against the entry of luminal antigens . A breach of the macrophage 'barrier' by invading antigens may necessitate the recruitment of T cell responses by immunostimulatory dendritic cells.

Am J Respir Cell Mol Biol, 1996 Apr, 14(4), 309 - 15
Isolation and primary culture of murine alveolar type II cells; Corti M et al.; Previous attempts to culture mouse alveolar type II (ATII) cells have been hampered by limited purity and cell recovery . We have now obtained culturable ATII cells from female C57BL/6 mice at a purity of 92% +/- 3 (mean +/- SD; n = 20), with viabilities of 96% +/- 2 and total yields of 5.1 +/- 0.7 X 10(6) cells per mouse . Crude lung cell suspensions were prepared by intratracheal instillation of Dispase and agarose followed by mechanical disaggregation of the lungs . Crude cell suspensions were purified by negative selection using a biotinylated-antibody, streptavidin-coated biomagnetic particle system . Cell purities were determined by Pap staining and confirmed ultrastructurally . Purified ATII cells were cultured on fibronectin-coated chamber slides and maintained for up to 5 days in DMEM with 10% fetal bovine serum . Cultures exhibited minimal contamination by Clara cells, mesenchymal cells, or endothelial cells, and the epithelial nature of the cultures was confirmed by positive cytokeratin staining in at least 97% of the cells through day 5 . Day 3 cultures demonstrated osmium tetroxide/tannic acid-stained granules consistent with lamellar bodies in 76% +/- 3.6 of the cells . The cultures displayed features distinct from those previously described for adult rat ATII cells, including irregularly-shaped cells and the formation of numerous cytoplasmic projections in direct contact with other cells . These studies indicate that excellent yields of highly purified, culturable ATII cells can be obtained from genetically defined mice . These techniques may provide powerful new models for the study of parenchymal lung disease in vitro.

Clin Chim Acta, 1996 Mar 29, 247(1-2), 7 - 21
The activity of a blood type B specific exoglycosidase from Glycine max; Hobbs L et al.; Soluble antigens, enzyme-linked immunosorbent assays (ELISA), and cell suspension assays were used to study the blood group B activity of Glycine max (soybean) alpha-D-galactosidase . The enzyme readily hydrolyzed the terminal alpha-D-galactosyl of the B antigen under a variety of conditions, converting it to H antigen . Conversion of the B antigen to H antigen produces blood type O which is universally transfusable . These preliminary studies are important in determining optimal conditions for enzymatic conversion of blood type B to O erythrocytes if efficient large-scale production of enzymatically converted, universally transfusable red blood cells is to be achieved.

J Immunol Methods, 1996 Mar 28, 190(1), 1 - 10
A novel method of microwave treatment for detection of cytoplasmic and nuclear antigens by flow cytometry; Lan HY et al.; Flow cytometry has recently become a useful technique for the quantitative analysis of cytoplasmic and nuclear antigens . We report here a rapid, simple, reproducible, and sensitive method for the simultaneous detection of cytoplasmic and nuclear antigens by flow cytometry . This technique involves the treatment of cell suspensions with 60 s of microwave oven heating after fixation with 2% paraformaldehyde . Following this treatment a number of cytoplasmic and nuclear antigens were detected on the human myelomonocytic cell line U937 (CD68, PCNA and Ki-67), peripheral blood leukocytes from both normal donors and leukemia patients (CD68, lipocortin-1 and PCNA) and a rat mesangial cell line 1097 (desmin, alpha-smooth muscle actin) using a standard indirect immunofluorescent staining with mouse monoclonal antibodies (mAbs) . There are several advantages of this technique over the routinely used methods currently available . Firstly, microwave treatment is a rapid, simple, and reproducible method, which largely reduces both time and cost expenditure, and makes this technique widely available for flow cytometric analysis in many areas of diagnostic and research purposes . Secondly, microwave treatment produces optimal results for simultaneous detection of both cytoplasmic (CD68, lipocortin-1, desmin, alpha-smooth actin) and nuclear (PCNA, Ki67) antigens . Thirdly, microwave treatment also produces a discrete profile for DNA content analysis . Finally, microwaving retains a clear discrimination between cells and debris as measured by light scatter . This study demonstrates that microwave treatment is a powerful technique which will be particularly applicable to flow cytometric analysis in the detection of many cytoplasmic and nuclear antigens.

J Physiol, 1996 Mar 15, 491 ( Pt 3), 773 - 7
Distribution of chloride permeabilities in normal human red cells; Raftos JE et al.; 1 . The rate of dehydration of K+ permeabilized red cells is influenced by their Cl- permeability (PCl) . In instances of pathological K+ permeabilization, cell-to-cell differences in PCl may determine which red cells dehydrate most . The present study was designed to investigate whether PCl differed significantly among red cells from a single blood sample . 2 . Previously available methods measure only the mean PCl of red cell populations . We describe a 'profile migration' method in which dilute red cell suspensions in low-K+ media were permeabilized to K+ with a high concentration of valinomycin, rendering PCl the main rate-limiting factor for cell dehydration . As the cells dehydrated, samples were processed to obtain full haemolysis curves at precise times . Variations in PCl among cells would have appeared as progressive changes in the profile of their haemolysis curves, as the curves migrated towards lower tonicities . 3 . Red cells from five normal volunteers showed no change in profile of the migrating haemolysis curves, suggesting that their PCl distributions were fairly uniform . Quantitative analysis demonstrated that intercell variation in PCl was less than 7.5% . 4 . Results obtained with this technique were analysed using the Lew-Bookchin red cell model . The calculated PCl was within the normal range described in earlier studies.

Biochim Biophys Acta, 1996 Mar 15, 1289(2), 231 - 7
Plant and human neutrophil oxidative burst complexes contain immunologically related proteins; Dwyer SC et al.; Generation of the microbicidal oxidative burst in human neutrophils requires participation of four proteins, a membrane bound flavocytochrome beta-558, two soluble proteins termed p47-phox and p67-phox, and the Ras-related GTPase Rac . Because plant cells exposed to pathogens produce a similar oxidative burst, we have looked for similarities between the oxidase complexes of the two systems . Antibodies against human neutrophil p47-phox and p67-phox were used to immunoblot cell extracts from several plant cell lines and were found to cross-react with proteins of the same molecular weight . Furthermore, plant cell lines not previously shown to produce an oxidative burst, yet found to express these immunoreactive proteins, rapidly generated hydrogen peroxide in response to elicitation . Finally, diphenylene iodonium (DPI) and alpha-naphthol, known specific inhibitors of the NADPH oxidase in neutrophils, also inhibited the oxidative burst in soybean cell suspensions with similar Ki values (about 15 microM and 30 microM respectively) . These results provide evidence for involvement of proteins related to the neutrophil oxidase complex in the defense-related oxidative burst of plants.

Artif Cells Blood Substit Immobil Biotechnol, 1996 Mar, 24(2), 143 - 51
Simulation of oxygen saturation of hemoglobin solution, RBC suspension and hemosome by a neural network system; Kan P et al.; Hemoglobin-based artificial blood substitutes as oxygen carrier is advantageous over current plasma expander . In this study, oxygen saturation of hemoglobin solution, red blood cell suspension and artificial blood substitute under various conditions were measured by yeast-consuming-oxygen experiments instead of spectrophotometer . The empirical results were assigned into training feedforward back-propagation neural network system in order to simulate the oxygen saturation model modulated by those factors such as pH, {Cl-}, {2,3-DPG}, pO2 and pCO2 . Consequently, this neural network is able to simulate accurately the oxygen saturation of Hb solution . The prediction of hemosome is not agreed well possible because of the resistance of transport of oxygen . However, the results showed neural net can offer a simple and convenient way in comparison with the conventional methods, especially in dealing with complex and ambiguous problem.

Arch Androl, 1996 Mar-Apr, 36(2), 119 - 25
Reactive oxygen species generation in human sperm: luminol and lucigenin chemiluminescence probes; McKinney KA et al.; The objective of this study was to compare measurements of reactive oxygen species (ROS) generation from human spermatozoa in vitro using the luminol and lucigenin chemiluminescent probes . Luminol reacts with a variety of reactive oxygen species (H2O2, O2-, OH) and allows both intra- and extracellular ROS to be measured . Lucigenin, however, yields a chemiluminescence that is more specific for superoxide anions released extracellularly . Therefore, measurements made with both probes on the same samples should allow the intra- and extracellular components of ROS generation to be identified . Sperm samples from 47 men were divided into two equal aliquots, then processed by centrifugation and swim-up . Following further division into aliquots and the addition of the two chemiluminescent probes, Phorbol 12-myristate 13-acetate was added to trigger ROS release . Forty three percent of the sperm samples generated detectable levels of ROS . In the centrifuged preparations luminol produced a significantly higher peak luminescence than lucigenin . However, the sperm prepared by swim-up showed no significant differences in peak luminescence between luminol and lucigenin . The higher level of ROS generation produced by centrifugation may be due to membrane disruption or possibly the use of unfractionated cell suspensions . Extracellular ROS generation is more clinically important because surrounding healthy spermatozoa may be damaged . Therefore the lucigenin probe may be a more useful diagnostic tool than luminol for identifying sperm at risk of peroxidative damage after swim up preparation . The patients identified in this way may benefit from the addition of ROS scavengers to the culture medium in order to protect healthy sperm from collateral damage.

Res Virol, 1996 Mar-Jun, 147(2-3), 89 - 95
In vitro HIV1 infection of CD34+ progenitor-derived dendritic/Langerhans cells at different stages of their differentiation in the presence of GM-CSF/TNF alpha; Charbonnier AS et al.; Langerhans cells (LC) are antigen-presenting cells which are found in areas at risk of inoculation by the human immunodeficiency virus (HIV) . LC were shown to be sensitive to in vitro infection by HIV1 . They could be generated in vitro by culturing CD34+ haematopoietic progenitors with GM-CSF+TNF alpha . In this study, we tested the sensitivity to HIV1 infection of in vitro generated LC throughout their differentiation and we investigated the effect of such an infection on in vitro differentiation . Phenotypic controls were performed using FACS analysis on day 6 for the presence of a CD1a+ cell population, and differentiation was assessed by transmission electron microscopy on day 13 for the presence of Birbeck granules . CD34+ cells were purified from cord blood mononuclear cells by magnetic separation . Cell suspensions were infected with either a T-lymphotropic, syncytium-inducing isolate (HXB2) or a macrophage-tropic, non-syncytium-inducing isolate (Ba-L) . Viral particle release was measured by p24 antigen production in the culture supernatant . A high level of p24 production was noted on day 13 of postinfection only when infection was carried out with Ba-L isolate on cells generated after 6 days in culture with GM/CSF+TNF alpha . No infection of CD34+ progenitor cells was obtained either with Ba-L isolate or HXB2 . The sensitivity of Langerhans cell/dendritic cell (LC/DC) precursors to NSI isolate (Ba-L) seemed to coincide with the early stage of differentiation (CD1a antigen appearance) . The infection did not alter the differentiation of in vitro generated LC, which presented their specific ultrastructural marker of epidermal environment, i.e . Birbeck granules from day 15 of the culture as compared to control culture . These results highlight the HIV infectibility of a differentiated population of LC/DC generated in vitro from CD34+ progenitors.

Immunopharmacology, 1996 Mar, 31(2-3), 171 - 81
Local popliteal lymph node reactions to hexachlorobenzene and pentachlorobenzene: comparison with systemic effects; Schielen P et al.; The effects of the presumed autoimmunogenic chemical hexachlorobenzene (HCB), and the closely related non-autoimmunogenic pentachlorobenzene (PCB) in the local popliteal lymph node assay (PLNA) were investigated . To that end 1-5 mg of HCB, equimolar amounts of PCB or the vehicle only, were injected into the hind footpads of rats or mice, and the reaction in the draining lymph node was evaluated on days 7 and 21 after injection . PLN were isolated, weighed, and cell suspensions were prepared to determine PLN cell numbers, and antibody production of PLN cells with an ELISPOT assay or a line immunoassay . The extent of the lymphoproliferative effect was examined by detection of proliferating cells with the BrdU method, and by measurement of paracortex and follicle areas, by combined immunohistochemistry and morphometry of PLN cryosections . We demonstrate here that HCB elevated PLN weights and cell numbers of the rat PLN, by day 7 after injection, but no elevation of antibody production in the PLN . Moreover, HCB caused an enlargement of both the PLN paracortical and follicular areas, and an elevation of proliferating paracortical T cells . None of the HCB-induced effects were found on day 21 . HCB caused the same effects in the mouse PLNA, but they tended to sustain at least until day 21 . Hardly any of the HCB-induced changes were found when PCB was injected . Previously, we have shown that oral exposure of Wistar rats to HCB elevated the number of splenic T cells and B cells, but also the serum levels of (auto-)antibodies and the production of these antibodies in the spleen, which is thus only partly in accordance with the results of the local reaction to HCB described in this study . This seeming contradiction is discussed.

J Pharmacol Exp Ther, 1996 Mar, 276(3), 1174 - 9
Isolated rat gastric mucosal cells: optimal conditions for cell harvesting, measures of viability and direct cytoprotection; Glavin GB et al.; Controversial data have been obtained with direct cellular protection by prostaglandins and sulfhydryls . In the present studies, we compared the merits and liabilities of currently available cell viability assays, some of which have not been previously employed in studies of the gastric mucosa . We also tested the hypothesis that length of incubation of isolated cells with protective agents might influence the degree of cellular damage . Gastric mucosal cells were isolated from nonfasted rats and digested with various concentrations of pronase and/or EGTA . Cell viability was assessed by trypan blue and fast green exclusion, fluorescein diacetate hydrolysis, chromium release, LDH release, mitochondrial succinate dehydrogenase activity and nuclear fluorescence induced by ethidium bromide . The experiments revealed that both pronase and EGTA are needed to obtain mucosal cells with optimal yield and initial viability and that sequential additions of pronase (30 min) and EGTA (30 min), rather than their combination (60 min), increased viability without decreasing yield . Cell viability and yield were better when pronase was used in the first incubation . For LD50 determinations, a cell suspension was incubated with ethanol (0%-15%) for 5 min . For studying the effects of protective agents, the cells were pretreated with 16,16-dmPGE2 or cysteamine HCl at 37 degrees C for 30 min before a 5-min exposure to 7.5% or 15% ethanol . The LD50 value for ethanol injury was approximately 15% for all assays except LDH, where the LD50 value was 8.5% . Preincubating gastric mucosal cells for 30 min with 16,16-dmPGE2 or cysteamine resulted in no preservation of cell viability . However, when cells were preincubated with one of the protective agents for 60 min and then exposed to 8% or 10% ethanol for 5 min, partial protection was observed when assessed by succinate dehydrogenase activity and, in certain cases, by LDH release . We conclude that all seven cell viability assays yield a measurable LD50 value for ethanol-induced cell injury, but the results may vary by as much as 82% . Low concentrations of both pronase and EGTA are needed to obtain isolated mucosal cells with both high yield and initial viability . Biochemical measures of mitochondrial activity and nuclear damage provide reliable evidence of cell viability and should be used to complement membrane permeability assays . Long preincubation of cells (60 min) with protective agents resulted in only slight protection of mitochondrial function, in contrast to the rapid induction of gastroprotection seen with these compounds in vivo . We therefore surmise that processes that contribute to organ protection occur faster and more efficiently than those that control direct cell injury and protection.

Electrophoresis, 1996 Mar, 17(3), 526 - 8
Sodium chloride in preparative free-flow cell electrophoresis: recent developments; Bauer J et al.; Three buffer systems for free-flow electrophoresis have been designed, which proved useful for performing cell electrophoresis in the presence of 50 mM NaCl . Each system consists of one central cell suspension buffer with 50 mM NaCl, two margin buffers, and two electrode buffers . With the aid of a bromophenol blue/maxilon blue accumulation test the various buffers were adjusted to ensure a laminar flow and remain unchanged on their way through an electrophoresis chamber.

Phytochemistry, 1996 Mar, 41(5), 1309 - 14
O-acetylated xyloglucan in extracellular polysaccharides from cell-suspension cultures of Mentha; Maruyama K et al.; Extracellular polysaccharide produced by suspension-cultured Mentha cells consisted of 50% neutral sugars, 32% uronic acid and 10% of protein . The ammonium oxalate-soluble fraction of this polysaccharide contained 30% hemicellulose and 70% pectic substances . The purified hemicellulose contained xylose, glucose, arabinose, galactose, mannose and fucose residues in a molar ratio of 41.6:31.3:13.1:11.1:1.3:1.6 . It was identified as a xyloglucan from its neutral sugar composition and by methylation analysis and cellulase treatment: the principal neutral sugar was arabinose . The presence of O-acetyl residues was confirmed with 1H NMR, 13C NMR and GC-mass spectrometry . The total acetyl content in the polysaccharide was 4% . The point of attachment of the O-acetyl residue was shown to be at position 6 of the galactosyl residue.

Ecotoxicol Environ Saf, 1996 Mar, 33(2), 186 - 92
Innate immune function as a bioindicator of pollution stress in fish; Rice CD et al.; Immunotoxicological studies, based on processing of samples in the field and laboratory, were conducted on fish collected from a stream receiving point-source contaminants near its headwaters . Previous studies in this stream have revealed that cytochrome P4501A activity, liver somatic indices, macrophage aggregates, and parasitic liver lesions are significantly elevated in sunfish with the degree of impact decreasing with distance from the contaminant source . Fish collected from each sampling site were equally divided, One group was sacrificed in the field and the spleen and anterior kidney tissues were removed and placed in buffer on ice . The other group was kept in MS-222 for 2 hr and transported to the laboratory for processing . The spleen and anterior kidney from each fish were then prepared as a single cell suspension and shipped overnight to Mississippi State University . Cells were then evaluated for PMA-stimulated phagocyte oxidative burst and non-specific cytotoxic cell (NCC) activity against K562 tumor targets . Oxidative burst responses were dramatically suppressed in both groups at sampling sites near the headwaters but returned to reference levels further downstream . There were no differences between processing strategies at each station . NCC activities did not follow gradient-response patterns observed with phagocyte oxidative burst data and there were inconsistent differences between processing strategies at each site . These data indicate that simple immune function assays, such as phagocyte oxidative burst responses, can be used as a ancillary bioindicator in fish health monitoring and that immune function in these fish can be reliably assessed even if samples are not immediately processed.

Graefes Arch Clin Exp Ophthalmol, 1996 Mar, 234(3), 155 - 63
Partial characterization of a putative new growth factor present in pathological human vitreous; Pombo C et al.; BACKGROUND: Several growth factors have been implicated in the development of proliferative eye diseases, and some of those are present in human vitreous (HV) . The effects of HV on cellular responses which modulate proliferative cell processes were studied . This study describes the partial characterization of a vitreous factor activity which does not correspond to any of the previously reported growth factors in pathological HV . METHODS: Vitreous humour was obtained from medical vitrectomies, from patients with PDR and PVR . The biological activity of the vitreous factor was determined by its ability to increase cytosolic calcium concentration ({Ca2+}i), increase production of inositol phosphates, and induce cell proliferation in the cell line EGFR T17 . In some experiments other cell lines, such as NIH 3T3, 3T3-L1, FRTL5, A431, PC12, Y79, and GH3, were also employed . Measurement of {Ca2+}i in cell suspensions was performed using the fluorescent Ca2+ indicator fura-2 . The activity of the factor present in HV was compared with other growth factors by means of: (a) {Ca2+}i mobilization pattern, (b) sequential homologous and heterologous desensitization of receptors, (c) effects of phorbol esters on their action, and (d) inactivation after treatment with different proteolytic enzymes . RESULTS: The HV-induced cell proliferation and increases in {Ca2+}i concentration were characterized by a peculiar time pattern . The different approaches used ruled out its identity with PDGF, bFGF, EGF, TGF-beta, IGFs, TNF-alpha, NGF, and other compounds such as ATP, angiotensin I, and bradykinin . Vitreous factor actions are mediated by specific receptors apparently regulated by PKC . This factor is able to induce {Ca2+}i mobilization in most of the cell lines studied, indicating that its effects are not tissue specific . CONCLUSIONS: These results suggest the presence of a growth factor activity in pathological HV which may be due to the presence of an undescribed growth factor in the eye.

J Periodontol, 1996 Mar, 67(3), 184 - 96
In vitro comparison of aged and young osteogenic and hemopoietic bone marrow stem cells and their derivative colonies; Dodson SA et al.; The purpose of this in vitro study was to determine whether there were differences in the number and size of osteogenic and hemopoietic colonies derived from bone marrow stem cells of aged and young adult male Sprague-Dawley rats . Using a Ficoll-Paque gradient, stem cells were harvested from aged male rats 18 to 22 months old and young adult males 55 days of age . Single cell suspensions from the red marrow of the long bones were cultured 14 days in vitro and subsequent colonies were assessed by light microscopy for number and size . A computerized histomorphometric linear measuring system was utilized to assess colony area in square millimeters . The results clearly show that young animals have a statistically significant increased cellular potential for osteogenic and hemopoietic colony formation . Cultures from aged animals showed an average formation of 0.45 +/- 0.6863 osteogenic colonies while those from younger animals had an average of 3.6 +/- 2.3523 osteogenic colonies per 3 million cells plated . Hemopoietic colonies from aged animal cell cultures numbered 5.25 +/- 2.2449 while those from the young animals averaged 8.23 +/- 3.3601 per 3 million cells plated . The difference in size of the osteogenic and hemopoietic colonies between age groups was not statistically significant . The area of osteogenic colonies derived from aged animals measured 0.1244 +/- 0.0891 mm2, while those derived from the young animals averaged 0.1276 +/- 0.0518 mm2 . Hemopoietic colonies from the aged cells measured 0.0759 +/- 0.0514 mm2, while hemopoietic colonies from the young animal cells measured 0.06010 +/- 0.0180 mm2 . The results of this study may have implications for consideration in the cellular healing aspects of aged versus young individuals.

Int J Urol, 1996 Mar, 3(2), 128 - 33
In vitro parathyroid hormone release in patients with secondary hyperparathyroidism; Yano S et al.; BACKGROUND: The purpose of this study was to determine the precise endocrine characteristics of parathyroid function in secondary hyperparathyroidism (sHPT) . METHODS: We examined the effects of extracellular ionized calcium (Ca2+) varying from 0.5 to 2.0 mM on parathyroid hormone (PTH) release in parathyroid cell suspensions using a mid-regional PTH assay . Cells were obtained from 26 patients with sHPT who were divided into two groups according to the type of hyperplasia they exhibited, either nodular (n = 16) or diffuse (n = 10) . For comparison, we also analyzed data from nine patients with primary hyperparathyroidism (pHPT; adenomas) . RESULTS: Significant in vitro suppression of PTH release by Ca2+ was observed in the majority of subjects, regardless of the histologic abnormality . The pHPT group exhibited no significant relationship between clinical and in vitro data . In contrast, in the sHPT group (taken as a whole), suppression of PTH release by Ca2+ exhibited a plateau at a total serum calcium concentration of 2.5 mmol/L, and a parathyroid gland weight of 2 g . CONCLUSIONS: These findings suggest that there is a curvilinear relationship in sHPT, but not pHPT, between the in vitro calcium sensitivity of parathyroid cells and total serum calcium, as well as gland weight . The in vitro calcium sensitivity in sHPT remains constant when the total serum calcium concentration exceeds 2.5 mmol/L, or when the gland weight exceeds 2 g.

Cell Transplant, 1996 Mar-Apr, 5(2), 269 - 77
Amelioration of the behavioral phenotype in genetically ataxic mice through bilateral intracerebellar grafting of fetal Purkinje cells; Triarhou LC et al.; We have previously applied neural grafting to "Purkinje cell degeneration" mutant mice (gene symbol pcd, mouse chromosome 13), a model of recessively inherited cerebello-olivary atrophy, to create appropriate interactions between wild-type and mutant cells in elucidating gene effects on the involved neuron populations and to address issues of the structural integration of donor Purkinje cells into the disrupted cerebellar loop . Behaviorally, pcd homozygotes manifest ataxic signs beginning at 3-4 wk of age . The functional effects of cerebellar transplants on motor performance have long remained an open question . The aim of the present study was to determine the recovery of motor responses in pcd mutants in a battery of behavioral tasks after bilateral transplantation of cerebellar cell suspensions (prepared from wild-type mice) into the parenchyma of the deep cerebellar nuclei of the hosts, according to a protocol that emphasizes the reconstruction of the missing inhibitory cortico-nuclear projection . With this approach, the denervated deep nuclei of the host receive a new Purkinje axonal innervation; further, most transplanted Purkinje cells end up occupying cortical localities anyway and display a correct dendritic tree orientation toward the pia . Motor coordination and fatigue resistance were assessed in a rotarod treadmill apparatus, a behavioral paradigm useful in studying various brain abiotrophies and treatments, including developmental perturbations of the cerebellar cytoarchitecture . Locomotor activity was quantified by the number of squares mice crossed as they moved about in an open-field matrix . Grafted pcd mice performed significantly better than sham-operated mutants in both of these tasks . Moreover, graft-recipient mice were able to sustain their abdomen above the floor on their limbs during movement, contrasting to the typical lowered, widened stance of sham-operated pcd mutants . These findings clearly demonstrate that bilateral transplants of fetal Purkinje cells have functional effects on motor performance in the pcd model of hereditary cerebellar ataxia.

Undersea Hyperb Med, 1996 Mar, 23(1), 27 - 30
Pressure antagonism of nitrous oxide depression of intracellular calcium in a neuroblastoma cell line; Resendes MC et al.; While the ability of increased pressure to reverse anaesthesia has been well documented, the cellular or molecular mechanism(s) responsible for their mutual antagonism have remained elusive . Previous work in our laboratory, using diverse cell types, has indicated several processes requiring Ca2+ are affected in opposite directions by hydrostatic pressure {as represented by helium (He)}, narcotic gases, and some anesthetics . Here we report on the effects of elevated pressures of He, and of 1 atm abs of the anesthetic gas nitrous oxide (N2O), when present alone and in combination, on calcium mobilization in the human neuroblastoma cell line SK-N-SH . Cytosolic-free Ca2+ ({Ca2+}i) was monitored by fluorescence spectrophotometry in cell suspensions loaded with the intracellular Ca2+ indicator fura-2 . N2O reversibly depressed the carbachol-stimulated increase in {Ca2+}i (P < 0.01) . The application of both 18 and 35 atm abs He attenuated this N2O-induced depression of carbachol-stimulated increase in {Ca2+}i . These findings support the hypothesis that pressure/anesthetic antagonism may be due in part to effects on neuronal {Ca2+}i and its regulation.

J Invest Dermatol, 1996 Mar, 106(3), 446 - 53
Immunomorphological and ultrastructural characterization of Langerhans cells and a novel, inflammatory dendritic epidermal cell (IDEC) population in lesional skin of atopic eczema; Wollenberg A et al.; We investigated epidermal cell suspensions prepared from lesional and nonlesional atopic eczema skin, other inflammatory skin conditions, and normal human skin for high-affinity IgE receptor (Fc epsilon RI) expression on dendritic CD1a cells by quantitative flow cytometric analysis . A single CD1a bright/CD1b neg/Fc epsilon RI dim/CD23 neg/CD32 dim/HLA-DR bright/CD36 neg population was found in normal skin . In contrast, lesional skin of atopic eczema and other inflammatory skin diseases harbored variable proportions of two distinct CD1a populations . Both populations exhibited typical ultrastructural features of Langerhans cells, but the second one lacked Birbeck granules and was unreactive to the Birbeck granule-specific LAG antibody . Both populations differed phenotypically: classical Langerhans cells were CD1a bright/CD1b neg/Fc epsilon RI dim/CD23 neg/CD32 dim/HLA-DR bright/CD36 dim, while the second population was CD1a dim/CD1b dim/Fc epsilon RI bright/CD23 dim/CD32 dim/HLA-DR bright/CD36 bright . The highest Fc epsilon RI expression was found on the second CD1a population in lesional atopic eczema skin . Furthermore, Fc epsilon RI expression on CD1a cells correlated significantly with the serum IgE level of the patients . Thus, a distinct population of CD1a inflammatory dendritic epidermal cells different from classical Langerhans cells appears in the epidermis of lesional skin and is subjected to specific signals leading to the upregulation of Fc epsilon RI in atopic eczema skin.

Invest Ophthalmol Vis Sci, 1996 Mar, 37(4), 561 - 73
Development and maintenance of outer segments by isolated chick embryo photoreceptor cells in culture; Saga T et al.; PURPOSE: To investigate the capacity of isolated chick embryo photoreceptors to develop and maintain outer-segment processes in dissociated cell cultures, in the absence of pigment epithelial and glial cells . METHODS: Cells were obtained from the retinas of embryonic day (ED) 17 chick embryos, after the onset of outer-segment formation in vivo . After 5 to 12-minute incubation in Ca++ and Mg++-free Hank's balanced salt solution, neural retinas were freed from other optical tissues, including the pigment epithelium . Retinal cell suspensions were prepared by repeated pipetting after mild trypsinization and were grown in serum-containing medium on a polyornithine-coated substratum . Cell differentiation was evaluated using phase-contrast and transmission electron microscopes and by autoradiographic analysis of the uptake of putative amino acid neurotransmitters, lectin cytochemical analysis, and immunocytochemical analysis with rod and cone-specific antibodies . Cells isolated from ED 8 retinas, before the onset of outer-segment formation in vivo, were also studied . RESULTS: At culture onset, ED 17 cells appeared morphologically undifferentiated and devoid of processes; differentiated features could be detected after 24 to 48 hours in vitro . Photoreceptor cells were the most abundant cell type after 6 days in vitro, followed by nonphotoreceptor multipolar neurons and morphologically undifferentiated cells . Autoradiographic analysis showed extensive Na+ -dependent uptake of (2,3,4-(3)H)gamma- aminobutyric acid in nonphotoreceptor neurons, whereas photoreceptors were labeled predominantly with 3H-glutamate . Most of the photoreceptors were labeled with fluorescent peanut lectin and with a sheep polyclonal antibody against bovine rhodopsin . Subsets of photoreceptors, on the other hand, were immunoreactive with cone- or rod-specific monoclonal antibodies COS-1, OS-2, 50-1B11, or Rho-4D2 . Approximately 50% to 65% of the photoreceptors positive with these monoclonal antibodies showed a remarkable polarization of immunoreactive materials, which accumulated predominantly, or even exclusively, in an outer-segment-like apical process . When viewed on the transmission electron microscope, these outer-segment-like processes appeared as distal expansions of the photoreceptor cilium and contained disc-like membranous profiles . Outer-segment-like processes also could be detected using the electron microscope and by immunocytochemical analysis of cultures of ED 8 retinal cells . CONCLUSIONS: After undergoing morphologic dedifferentiation as a result of tissue dissociation, isolated retinal photoreceptors, grown in the absence of contact-mediated cell interactions and of pigment epithelial and glial cells, can regenerate and maintain a highly polarized pattern of structural and molecular organization, including the formation of outer-segment-like processes . The cultures provide an experimental system for the investigation of cellular and molecular mechanisms regulating further development and maturation of these photoreceptor structures.

Biochem J, 1996 Feb 15, 314 ( Pt 1), 91 - 4
Temporal patterns of changes in ATP/ADP ratio, glucose 6-phosphate and cytoplasmic free Ca2+ in glucose-stimulated pancreatic beta-cells; Nilsson T et al.; Closure of ATP-sensitive K+ (K(ATP)) channels is part of the stimulus-secretion coupling mechanism in the pancreatic beta-cell, leading to membrane depolarization and influx of Ca2+ through voltage-sensitive L-type Ca2+ channels . The elevated ATP/ADP ratio seen in the presence of high levels of glucose has been postulated to mediate the glucose-induced closure of the K(ATP) channels and rise in cytoplasmic free Ca2+ concentration ({Ca2+}i), or alternatively to be a consequence of activation of mitochondrial dehydrogenases by the increase in {Ca2+}i . To distinguish between these two possibilities, the time course of the change in the ATP/ADP ratio was determined in comparison with that of {Ca2+}i . We here show that a severalfold rise in the ATP/ADP ratio occurs rapidly on stimulation of suspensions of mouse pancreatic beta-cells with glucose . The change in the ATP/ADP ratio is an early event that begins within 20-40 s and precedes the rise in {Ca2+}i . The temporal relationship indicates that the adenine nucleotide changes cannot be a consequence of the {Ca2+}i changes and may indeed be the connecting link between glucose metabolism and {Ca2+}i changes . When the cells were sequentially treated with high glucose concentration, clonidine and finally high extracellular Ca2+ concentration to induce synchronized oscillations in {Ca2+}i in the cell suspension, corresponding oscillations in the ATP/ADP ratio were observed . Glucose 6-phosphate levels oscillated out of phase with the ATP/ADP ratio . These results support the hypothesis that the Ca2+ oscillations previously observed in glucose-stimulated single islets or beta-cells may reflect oscillations in the ATP/ADP ratio that accompany oscillatory glycolysis.

Transplantation, 1996 Feb 15, 61(3), 492 - 7
Administration of rabbit anti-asialo GM1 antiserum facilitates the development of human Epstein-Barr virus-induced lymphoproliferations in xenografted C.B-17 scid/scid mice; Lacerda JF et al.; Mice with severe combined immune deficiency (C.B-17 scid/scid {SCID mice}) lack functional B and T lymphocytes and are permissive for the growth of human xenografts . However, the development of functional NK cells is not affected by the scid mutation . Mouse NK cells express the surface glycolipid asialo GM1 and are implicated in the rejection of heterotransplanted cells . In the present study, we demonstrate that SCID mice treated with rabbit anti-asialo GM anti-serum (alpha-asialo GM1), for in vivo depletion of endogenous NK cell function, develop lethal Epstein-Barr virus (EBV)-induced lymphoproliferative disorders (EBV-LPD) at lower doses od inoculated EBV-transformed lymphoblastoid B cell lines (EBV-LCL) than untreated animals . Furthermore, at any given dose of EBV-LCL inoculated, EBV-LPD developed earlier and induced lethality sooner in alpha-asialo GM1-treated animals . We also demonstrate that SCID mice treated with alpha-asialo GM1 have reduction in the number of asialo GM1-expressing splenocytes . Moreover, splenic cell suspensions derived from alpha-asialo GM1-treated SCID mice show lower cytotoxicity against the mouse NK-sensitive cell line YAC-1 and human EBV-LCL than splenocytes obtained from untreated SCID mice.

Blood, 1996 Feb 15, 87(4), 1625 - 34
Concomitant mobilization of plasma cells and hematopoietic progenitors into peripheral blood of multiple myeloma patients: positive selection and transplantation of enriched CD34+ cells to remove circulating tumor cells; Lemoli RM et al.; One advantage of the use of peripheral blood stem cells (PBSCs) over autologous bone marrow would be a reduced risk of tumor cell contamination . However, the level of neoplastic cells in the PB of multiple myeloma (MM) patients after mobilization protocols is poorly investigated . In this study, we evaluated PB samples from 27 pretreated MM patients after the administration of high dose cyclophosphamide (7 g/m2 or 4 g/m2) and granulocyte-colony stimulating factor for the detection of myeloma cells as well as hematopoietic progenitors . Plasma cells containing intracytoplasmic lg were counted by microscope immunofluorescence after incubation with appropriate antisera directed against light- and heavy-chain lg . Moreover, flow cytometry studies were performed to determine the presence of malignant B-lineage elements by using monoclonal antibodies against the CD19 antigen and the monotypic light chain . Before initiation of PBSC mobilization, circulating plasma cells were detected in all MM patients in a percentage ranging from 0.1% to 1.8% of the mononuclear cell fraction (mean value, 0.7% +/- 0.4% SD) . In these patients, a higher absolute number of PB neoplastic cells was detected after chemotherapy and granulocyte colony-stimulating factor . Kinetic analysis showed a pattern of tumor cell mobilization similar to that of normal hematopoietic progenitors with a maximum peak falling within the optimal time period for the collection of PBSCs . The absolute number of plasma cells showed a 10 to 50-fold increase as compared with the baseline value . Apheresis products contained 0.7% +/- 0.2% SD of myeloma cells (range, 0.2% to 2.7%) . Twenty-three MM patients were submitted to PBSC collection . In 10 patients, circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, were cryopreserved, and used to reconstitute bone marrow function after myeloablative therapy . The median purity of the enriched CD34+ cell population was 89.5% (range, 51% to 94%), with a 75-fold increase as compared with the pretreatment samples . The median overall recovery of CD34+ cells and colony-forming unit-granulocyte-macrophage was 58% (range, 33% to 95%) and 45% (range, 7% to 100%), respectively . Positive selection of CD34+ cells resulted in 2.5- to 3-log depletion of plasma cells and CD19+ B-lineage cells as determined by immunofluorescence studies, although DNA analysis of CDR III region of IgH gene showed the persistence of minimal residual disease in 5 of 6 patient samples studied . Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (1,000 cGy) and highdose melphalan (140 mg/m2) . They received a median of 4 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis; the median time to 0.5 x 10(9) neutrophils and to 20 and 50 x 10(9) platelets per liter of PB was 10, 11, and 12 days, respectively . These results, as well as other clinically significant parameters, did not significantly differ from those of patients (n = 13) receiving unmanipulated PBSCs after the same pretransplant conditioning regimen . In summary, our data show the concomitant mobilization of tumor cells and hematopoietic progenitors in the PB of MM patients . Positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3-log and provides a cell suspension capable of restoring a normal hematopoiesis after a total body irradiation-containing conditioning regimen.

FEBS Lett, 1996 Feb 12, 380(1-2), 97 - 102
Ionic transport in the plasma membrane of carrot protoplasts from embryogenic cell-suspension cultures; Bregante M et al.; Ionic transport properties of protoplasts obtained from embryogenic carrot suspension cells were studied by the patch-clamp technique . In the whole-cell configuration, carrot protoplasts presented macroscopic time-dependent outward currents, showing kinetics of activation which did not depend appreciably on the amplitude of the stimulus . Time- and voltage-dependent whole-cell inward rectifying currents as well as instantaneous non-selective currents were also observed . Both time-dependent inward and outward currents are carried by potassium ions . In a cell-attached configuration, two types of single-channel signals, displaying conductances of 10 and 17 pS, were observed; the instantaneous 10 pS channel was also present in outside-out excised patches.

Biophys Chem, 1996 Feb 8, 58(3), 273 - 9
Analytical model for effects of shear rate on rouleau size and blood viscosity; Chen J et al.; In this paper, a theoretical model is proposed by using the reversible kinetic equation of colloid coagulation to reveal the effects of erythrocyte aggregation and shear rate on the rouleau size and the blood viscosity . With this model, shear rate gamma dependences of the average size of rouleaux n(e) and the viscosity eta in concentrated red blood cell suspension are derived analytically . In this model, we consider not only the shear rate effect but also the rouleau size effect on the aggregation and degradation mechanism . By comparing with experimental results obtained by Shiga et al., some theoretical parameters have been determined . The variation of rate of rouleau formation with shear rate derived in our model is in fairly good agreement with the experimental results, and the viscosity of the concentrated red cell suspension derived in our model showing shear thinning agrees qualitatively with the experimental results obtained by Chien and Sung.

Am J Med Genet, 1996 Feb 2, 61(4), 387 - 93
Dynamics of chromosome spreading; Spurbeck JL et al.; Consistency of optimum chromosome spreading during harvest of cytogenetic specimens remains a major concern . We have tested the idea that a precise control of the drying rate (the time with which metaphase cells dry), as fixed cell suspension is placed on a slide or an in situ culture in last fixation, may be the answer . Amniocyte and lymphocyte cultures were allowed to dry at defined combinations of relative humidity (RH) and temperature (T) in a modified Thermotron environmental control unit . We were able to demonstrate, based on 2,250 amniocytes and 1,650 lymphocytes, that the metaphase area after drying was a function of RH and T for both in situ and non-in situ culture systems . As the RH and T increase, the metaphase area increases until a threshold is reached . Also, as RH increases, the slide drying time increases . Data obtained using a response surface regression, proportional hazards regression analysis and slide drying time studies are consistent with our model of chromosome spreading . Optimum metaphase areas can be achieved at various combinations of RH and T . We propose that the use of an environmental control unit is a practical way of achieving optimum chromosome spreading routinely and in a highly consistent manner.

Arch Dermatol Res, 1996 Feb, 288(2), 68 - 73
Adhesion molecules and IL-1 costimulate T lymphocytes in the autologous MECLR in psoriasis; Prens E et al.; Membrane molecules such as CD36 (OKM5), intercellular adhesion molecule-1 (ICAM-1, CD54), gamma interferon-induced protein 10 (gamma-IP10) and IL-1 are induced and/or upregulated in psoriatic epidermis . These molecules have important accessory, trafficking or signalling functions in the immune system and also play a role in the pathophysiology of psoriasis . The relevance of adhesion molecules, CD36 and epidermal IL-1 in psoriasis was studied in vitro in the autologous mixed epidermal cell - T lymphocyte reaction (MECLR) . Their level of expression was quantitated in epidermal cell suspensions (ECS) from patients with psoriasis and their function was assessed by blocking with specific mAbs and antisera or by depleting CD36+ cells from the ECS prior to the MECLR . ECS from psoriatic lesions contained increased numbers of CD36+ (23 +/- 12%), ICAM-1(+) (31 +/- 14%) and IL-1(+) (57 +/- 21%) cells . The autologous MECLR was inhibited in samples from all patients by mAb to CD2 (LFA-2), CD11a (LFA-1alpha), CD18 (LFA-1beta), ICAM-1, CD58 (LFA-3) and an antiserum to IL-1beta . Thus, adhesion molecules facilitate inflammation in psoriasis not only via adhesion and recruitment of T lymphocyte in psoriatic lesions, but also via activation of T cells . Furthermore CD36 molecules on psoriatic epidermal cells do not costimulate autologous T lymphocytes in psoriasis . The observed costimulatory function of IL-1beta in the MECLR emphasizes its relevance in psoriasis.

Vaccine, 1996 Feb, 14(3), 199 - 206
Parenteral vaccination of mice and piglets with F4+ Escherichia coli suppresses the enteric anti-F4 response upon oral infection; Bianchi AT et al.; We studied with a mouse model and in piglets the requirements to prime for a secondary, mucosal B-cell response against Escherichia coli F4 fimbriae, an important virulence factor of enterotoxigenic E . coli, the agent associated with postweaning diarrhoea in piglets . The major observation obtained with the mouse model was verified for piglets . Mice and piglets were primed orally or parenterally with purified F4ac antigen or whole bacterial cells carrying the F4ac antigen and were later orally infected with live F4ac+ E . coli bacteria . Cell suspensions of murine spleen or porcine serum were used to study the systemic B-cell response . Cell suspensions were also made of murine and porcine enteric lamina propria and were used to study the mucosal B-cell response . Enzyme-linked immunospot assays and enzyme-linked immunosorbent assays specific to E . coli F4ac antigen were used to quantify either the antibody-secreting cells or antibody titres in serum . Results showed that in mice only primary oral immunization with live bacteria induced an enteric immune response against the E . coli F4ac+ fimbriae . Oral immunization with killed bacteria induced hardly any mucosal immune response . Parenteral immunization induced a state of suppression that was reflected by the lack of an enteric immune response upon a subsequent oral infection with live bacteria . A comparable induction of suppression was observed in piglets using the same protocol . We conclude that parenteral vaccination of piglets with the E . coli F4ac antigen is ineffective to induce protective immunity at the mucosal level against postweaning diarrhoea and is possibly detrimental.

J Endourol, 1996 Feb, 10(1), 71 - 5
High-intensity focused ultrasound ablation of rabbit kidney tumors; Adams JB et al.; High-intensity focused ultrasound (HIFU) is a noninvasive surgical technique in which ultrasound energy is delivered transcutaneously to a discrete area within the body . This energy can result in a well-defined zone of cellular death within the targeted tissue . We used HIFU in an effort to ablate rabbit VX-2 kidney tumors . A tumor cell suspension was injected into a renal segmental artery (Phase 1, nine rabbits) or directly into the lower pole parenchyma (Phase 2, nine rabbits) . After a 2-week incubation period, open direct contact (Phase 1) or transcutaneous ablation (Phase 2) was performed . In Phase 1, after sonablation, there was pathologic evidence of tissue destruction in nine animals, and seven had both gross and histologic evidence of tumor ablation . There was sharp demarcation between viable and ablated tissue . In Phase 2, pathologic evidence of kidney ablation was seen in seven of nine animals . However, only two rabbits showed the well-demarcated effects of ablation in the tumor . High-intensity focused ultrasound can be effective at causing cell death in renal tumors and surrounding renal tissue . However, with the present ultrasound technology, imaging of renal lesions in the rabbit model is not adequate to consistently localize and completely ablate tumor.

Biophys J, 1996 Feb, 70(2), 715 - 22
Detection of Cl- binding to band 3 by double-quantum-filtered 35Cl nuclear magnetic resonance; Liu D et al.; We have applied double-quantum-filtered (DQF) NMR of 35Cl to study binding of Cl- to external sites on intact red blood cells, including the outward-facing anion transport sites of band 3, an integral membrane protein . A DQF 35Cl NMR signal was observed in cell suspensions containing 150 mM KCl, but the DQF signal can be totally eliminated by adding 500 microM 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), an inhibitor that interferes with Cl- binding to the band 3 transport site . Therefore, it seems that only the binding of Cl- to transport sites of band 3 can give rise to a 35Cl DQF signal from red blood cell suspensions . In accordance with this concept, analysis of the single quantum free induction decay (FID) revealed that signals from buffer and DNDS-treated cells were fitted with a single exponential function, whereas the FID signals of untreated control cells were biexponential . The DQF signal remained after the cells were treated with eosin-5-maleimide (EM), a noncompetitive inhibitor of chloride exchange . This result supports previous reports that EM does not block the external chloride binding site . The band 3-dependent DQF signal is shown to be caused at least in part by nonisotropic motions of Cl- in the transport site, resulting in incompletely averaged quadrupolar couplings.

Phys Med Biol, 1996 Feb, 41(2), 255 - 68
Triexponential decomposition of 1H spin-lattice relaxation decay curves of paramagnetically doped red cell suspensions at 7 T; Mulkern RV et al.; The spin-lattice relaxation behaviour of protons in paramagnetically doped red cell suspensions at two haematocrit levels has been studied using high-field (7 T) inversion recovery data . The resulting relaxation decay curves were found to be best characterized by triexponential functions . The two-site exchange model, generally applied to biexponential relaxation data in calculating red cell water exchange parameters, was applied to the two fastest-relaxing fractions of the triexponential fits . The intracellular to extracellular water exchange rates so obtained were in good agreement with literature values . Additional exchange parameters including intracellular and extracellular water volume fractions and extracellular relaxation rates were also calculated directly from the relaxation data and found to be consistent with sample haematocrits and with independent relaxation rate measurements of the resuspension medium . The small-amplitude, slowly relaxing third component recovered from the triexponential fits of the relaxation data is attributed to intracellular haemoglobin protons.

J Neurosci Methods, 1996 Feb, 64(2), 173 - 9
Methylcellulose during cryopreservation of ventral mesencephalic tissue fragments fails to improve survival and function of cell suspension grafts; Sautter J et al.; Cryopreservation may allow long-term storage of fetal ventral mesencephalon (VM) for transplantation in patients suffering from Parkinson's disease (PD) . We investigated whether the polymer methylcellulose protects fetal rat VM during cryopreservation in liquid nitrogen and improves survival and function of this tissue as intrastriatal suspension grafts in the 6-hydroxydopamine (6-OHDA) rat model . VM tissue fragments (E14-E15) were either immediately dissociated and grafted as a cell suspension (FRESH) or cryopreserved under controlled conditions for 7 days in a conventional cryoprotective medium (CRYO) or a medium containing 0.1% methylcellulose (mCRYO) and then dissociated and grafted . Rats from the cryo-groups showed only limited behavioral compensation in contrast to complete compensation observed in rats from the FRESH group . Cryopreservation of fetal rat VM decreased the viability of cell suspensions in vitro to about 70%, survival of grafted tyrosine hydroxylase-immunoreactive (TH-IR) neurons to 11% and 20%, and transplant volume to 8% and 17% (mCRYO and CRYO, respectively, compared to FRESH) . The addition of 0.1% methylcellulose to tissue fragments during freezing did neither improve in vitro viability nor survival of TH-IR neurons nor behavioral compensation when compared to the control CRYO group . These results suggest that methylcellulose failed to improve survival of cryopreserved dopaminergic ventral mesencephalic neurons.

Immunology, 1996 Feb, 87(2), 317 - 25
Flow cytometric analysis of cytokine receptors on human Langerhans' cells . Changes observed after short-term culture; Larregina A et al.; It is well established that granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-1 and tumour necrosis factor-alpha (TNF-alpha) are involved in Langerhans' cell (LC) development and dendritic cell traffic . However, little is known about the pattern of cytokine receptors on human LC and their modulation during different stages of maturation . The expression of cytokine receptors was studied by flow cytometry on both freshly isolated LC (fLC) and 72-hr cultured LC (cLC) . Epidermal cell suspensions enriched in LC were obtained after skin trypsinization and Ficoll-Hypaque gradient . LC were identified by their CD1a positivity . Although the majority of fLC were positive for the alpha chain of GM-CSF receptor (GM-CSFR), the beta chain of GM-CSFR was detected only on 15% of CD1a+ cells . fLC were also positive for IL-1 receptor (IL-1R) type 1, IL-1R type 2, 75,000 molecular weight TNF receptor (TNFR) and interferon-gamma receptor (IFN-gamma R) . IL-6R and its transducing signal gp130 were present in a subset of fLC . Granulocyte colony-stimulating factor receptor (G-CSFR), macrophage colony-stimulating factor receptor (M-CSFR), the alpha and beta chain of IL-2R, IL-4R, IL-7R, IL-8R and 55,000 molecular weight TNFR were not detected on fLC . After culture, LC up-regulated the expression of both the alpha and beta chains of GM-CSFR, IL-1R type 2, alpha and beta chains of IL-2R, IL-6R and gp130 . In contrast, IL-1R type 1 and 75,000 molecular weight TNFR were down-modulated and the expression of IFN-gamma R was not affected by culture . These results suggest that LC undergo changes in the cytokine receptor repertory during in vitro maturation.

Immunology, 1996 Feb, 87(2), 310 - 6
Cultured human Langerhans' cells are superior to fresh cells at presenting native HIV-1 protein antigens to specific CD4+ T-cell lines; Girolomoni G et al.; Cultured Langerhans' cells (CLC) exhibit enhanced antigen-presenting function compared to freshly isolated LC (FLC), but they are commonly believed to be inefficient at processing intact proteins . In this study, FLC and CLC from normal, human immunodeficiency virus (HIV) seronegative volunteers were compared for their ability to present the HIV-1 envelope glycoprotein gp120 or reverse transcriptase (p66) antigens to autologous, specific CD4+ T cell lines . Epidermal cell suspensions enriched for LC were prepared from suction blister roofs . FLC stimulated T cells at lower antigen concentrations compared to unfractionated peripheral blood mononuclear cells (PBMC) . CLC were more potent on a per cell basis than FLC, PBMC or adherent monocytes at presenting native gp120, native p66 or immunogenic peptides . CLC were also more efficient than FLC or PBMC in terms of the amount of antigen required for T-cell activation . Chloroquine and leupeptin inhibited presentation of intact p66, but not of an immunodominant peptide, by FLC or CLC, thus indicating that both cells utilize antigen-processing mechanisms that are based on intracellular acidification and protease activity . Incubation of CLC with monoclonal antibodies against HLA-DR, CD11b, CD18, CD50, CD54, CD58 or CD80, but not anti-major histocompatibility complex class I (MHC-I), inhibited antigen-specific T-cell proliferation to varying degrees . We conclude that human CLC retain the ability to process and present protein antigens potently to CD4+ T cells . Thus, CLC have the capacity to participate actively in the generation and maintenance of T-helper cell immunity to viral antigens during HIV-1 infection.

J Mol Endocrinol, 1996 Feb, 16(1), 57 - 62
Angiotensin II stimulation of the basolateral located Na+/H+ antiporter in eel (Anguilla anguilla) enterocytes; Vilella S et al.; The pH-sensitive fluorescent dye, 2',7'-bis-carboxyethyl-5, 6-carboxyfluorescein acetoxymethyl ester, was used to examine the effects of fish or human angiotensin II (Ang II) on the activity of the basolateral located Na+/H+ antiporter in eel intestinal cell suspensions . Exposure of eel enterocytes to either hormone led to an increased activity of the antiporter . This time- and dose-dependent stimulatory effect was inhibited by the specific antiporter inhibitor dimethylamiloride (DMA) . Preincubation with a monoclonal antibody (6313/ G2), directed against the N-terminal extracellular domain of the mammalian AT1 Ang II receptor, prevented the stimulatory effect of the hormone and inhibited the binding of {3,5-3H} Tyr4-Ile5-Ang II to intestinal cell suspensions, suggesting specific binding of the antibody to the eel Ang II receptor . The results indicate that both fish and human Ang II stimulate the DMA-sensitive Na+/H+ antiporter present in eel intestinal cells by means of a mammalian AT1-like receptor.

Int Immunol, 1996 Feb, 8(2), 203 - 9
Effect of superantigens on human thymocytes: selective proliferation of V beta 2+ cells in response to toxic shock syndrome toxin-1 and their deletion upon secondary stimulation; Mingari MC et al.; The effects of toxic shock syndrome toxin-1 (TSST-1) on human thymocytes and their CD4/CD8-defined subsets have been analyzed . Postnatal thymocyte cell suspensions were cultured with 5 ng/ml TSST-1 for different time intervals . A strong cell proliferation of CD3+/TCR+ cells, characterized by selective expansion of cells expressing TCR V beta 2, occurred . In these cultured thymocytes, V beta 2+ cells were detected in all subsets including CD4-CD8+ cells . CD4-CD8+ thymocyte populations (obtained by depletion of CD4+ cells) were further analyzed for their ability to directly respond to TSST-1 . An efficient cell proliferation occurred; however, it was completely abrogated upon removal of HLA class II+ cells (representing 10% of fresh thymocytes depleted of CD4+ cells) . The HLA class II dependency of TSST-1 mediated functions was further documented at the clonal level . Thus, in the presence of TSST-1, CD4-CD8+ V beta 2+ clones efficiently lysed the HLA class II+ Raji target cells but not the corresponding HLA class II- variant RJ 2.2.5 . Analysis of the effect of TSST-1-induced secondary stimulation on cultured (V beta 2+-enriched) thymocytes resulted in a selective depletion of V beta 2+ cells due to apoptotic cell death.

Geburtshilfe Frauenheilkd, 1996 Feb, 56(2), 70 - 8
-Chemosensitivity testing in gynecologic oncology . Experiences with an ATP bioluminescence assay-; Kurbacher CM et al.; In the present study, our first experiences with the ATP Tumour Chemosensitivity Assay (ATP-TCA), a novel method for pretherapeutic drug testing, are reported . During one year, a total of 111 samples obtained from patients suffering from different gynaecological malignancies (ovary: 53, breast: 41, others: 17) were sent for chemosensitivity testing . The quality of 104 single cell suspensions was sufficient for further processing . Evaluability rates ranged from 71% (breast cancers and non-ovarian tumours) to 83% (ovarian cancers) with an average of 77% . Evaluability of recurrences was slightly better compared to primaries . A total of 18 different antineoplastic agents and 67 drug combinations was used for in vitro testing with an average of 12.4 drugs/combinations tested per each sample (ovary: 14.8, breast: 9.7, others: 10.2) . In 70 of 111 cases, the ATP-TCA was followed by chemotherapy with 44 cases treated in respect of the assay results and 29 cases receiving an antineoplastic regime, which differed from the standard treatment protocol . Compared to untreated patients, recurrences were treated more frequently based on the ATP-TCA, with a majority receiving an alternative protocol as proposed by the assay . In ovarian carcinoma, chemosensitivity testing was of particular importance for the further therapy . 41 of 53 patients were treated by chemotherapy with 29 therapies basing on the results of the assay (alternative protocol: 17) . The good correlation of the ATP-TCA and the individual clinical course of the patients was demonstrated by two case reports of recurrent ovarian carcinomas, one of them responding well to platinum-based combinations and the other presenting in vitro and in vivo platinum-resistance . In conclusion, our first experiences with the ATP-TCA were promising . Our results, however, warrant confirmation by studies with a sufficient number of cases and an extended observation period.

J Pharmacol Toxicol Methods, 1996 Feb, 35(1), 35 - 43
Continuous monitoring of mitochondrial membrane potential in hepatocyte cell suspensions; Palmeira CM et al.; We report a simple fluorometric method for the continuous monitoring of mitochondrial membrane potential and cell viability in suspensions of hepatocytes exposed in vitro to cytotoxic agents . Suspensions of freshly isolated hepatocytes (10(6) cells/mL) preloaded with rhodamine 123 (Rh 123, 100 mumol/L) are transferred to a thermostatically controlled mixed cuvette to which the desired cytotoxic agent is added . Rh 123 is a cationic fluorophore that is actively accumulated by cells in direct proportion to the mitochondrial membrane potential . Cell viability was estimated by monitoring propidium iodide (PI) fluorescence . Exposure of cell suspensions to the mitochondrial uncoupling agent FCCP caused an immediate and titratable increase in Rh 123 fluorescence . Subsequent treatment with digitonin did not change Rh 123 fluorescence, suggeseting that Rh 123 equilibrates rapidly across the intact cell membrane . Likewise, treatment of hepatocyte suspensions with inhibitors of mitochondrial respiration (rotenone, cyanide, or menadione) caused an immediate increase in Rh 123 fluorescence . This was accompanied by a progressive increase in PI fluorescence, suggesting a causal relationship between mitochondrial depolarization and cell injury . In contrast, 1,4-benzoquinone caused a time-dependent and linear increase in PI fluorescence that paralleled changes in Rh 123 fluorescence . Comparing the time courses for changes in PI and Rh 123 fluorescence suggests that for benzoquinone, the depolarization of the mitochondria is a consequence rather than a cause of the cell injury . This modified procedure provides a simple and specific technique for continuously monitoring mitochondrial membrane potential and cell viability in suspensions of freshly isolated hepatocytes . The advantage is that there is no need to separate cells from the incubation medium, making it possible to record real-time changes in mitochondrial membrane potential and cell viability throughout the in vitro exposure period.

Exp Hematol, 1996 Feb, 24(2), 246 - 52
Goralatide (AcSDKP) selectively protects murine hematopoietic progenitors and stem cells against hyperthermic damage; Wierenga PK et al.; Hyperthermic purging procedures may be improved by methods that selectively inhibit the proliferative activity of normal hematopoietic progenitors and stem cells, since active proliferation of these subsets is accompanied by increased heat sensitivity . For this reason, bone marrow cells from CBA/H mice were incubated with Goralatide (tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro), a well-known inhibitor of normal hematopoietic progenitor cells to enter the S phase of the cell cycle . Subsequently, the cell suspensions were heat-treated at 43 degrees C for up to 90 minutes . After an exposure of 8 hours to 10(-9) M Goralatide, the number of CFU-GM cells in S phase decreased from 30 to 10%, resulting in an almost 10-fold increase in survival after 90 minutes at 43 degrees C . No effect on the primitive subsets could be detected because of their quiescent cell cycle state in normal bone marrow . To investigate the potential vulnerability of these subsets for Goralatide, bone marrow cells from 5-fluorouracil (5-FU)-pretreated mice were used . 5-FU induced increase in proliferative activity of the CFU-S-12 (day-12 colony-forming units-spleen), and the stem cell with marrow repopulating ability could be abolished by an incubation period with 10(-9) M Goralatide for 16 and 24 hours, respectively . Hence, this decrease in proliferative activity confers a decrease in hyperthermic sensitivity for the primitive hematopoietic subsets . The cytotoxic effect of the incubation on the absolute number of the hematopoietic progenitors and stem cells was <10% . Goralatide treatment (10(-8), 10(-9), and 10(-10) M) up to 24 hours had no effect on the growth kinetics and cell cycle distribution and consequently on the hyperthermic sensitivity of L1210 cells . Based on these results, it can be concluded that Goralatide will have a positive effect on the survival of hematopoietic progenitors and stem cells after hyperthermia and may lead to a gain in the therapeutic window of this purging modality.

Exp Neurol, 1996 Feb, 137(2), 324 - 32
Early cytopathic features in rat ischemia model and reconstruction by neural graft; Onizuka K et al.; Using a silver impregnation (argyrophil III) and immunohistochemistry, acute cytopathic features after cerebral ischemia were investigated . Additionally, functional recovery and interconnection between the host and graft was also explored after neural graft . Animals were embolized in unilateral middle cerebral artery for 1 h . Argyrophil III method demonstrated "collapsed" dark neurons in the striatum, cortex, reticular thalamus, amygdala, and hypothalamus on ischemic side . These neurons exhibited characteristic shrunken somata with corkscrew-like dendrites, suggesting changes in cytoskeletal protein . In the above mentioned areas, the loss of immunoreactivity for mu-calpain proenzyme and microtubule-associated protein 2 was also detected . Neural graft into the ischemic striatum was made 2 weeks after the ischemia paradign . The grafted striatal cells were prepared from E15 fetuses to make cell suspension marked by rhodamine-labeled latex microspheres . Methamphetamine-evoked rotations were detected after ischemia . These motor alterations were reduced gradually but significantly at 8 weeks after the graft . Interconnecton between the host and grafted cells was then studied in a brain slice preparation after loading fura-2 AM . About 10% of grafted cells tested from rats that showed motor amelioration exhibited {Ca2+}i increase to the electrical stimulation applied to the neighboring host tissue . Data indicate that, in the very early stage after ischemia, cytoskeletal damages, especially on microtubules, started and this would lead to later infarct . The graft survived in the ischemic striatum having connections with the host, and this might be partly involved in the amelioration of motor function.

Exp Neurol, 1996 Feb, 137(2), 309 - 17
Labeling and identification of living donor cells in brain slices of recipient hemiparkinsonian model rats for physiological recordings: methods for physiological assessments of neural transplantation; Fukuda A et al.; Physiological properties of grafted neurons, such as membrane and intr acellular properties, have not been reported . To fill this lack in knowledge, physiological recordings from the identified grafted cells are required . Fluorescent latex microspheres (FLM) are non-toxic and stable, and thus seem suitable for long-term labeling of donor cells . Therefore, we tested the feasibility of labeling with FLM to identify living donor cells in the recipients' brain slices . We also tested if physiological recordings from the identified cells are possible or not . Cell suspensions were prepared from the substantia nigra (SN) of Embryonic Days 15 or 16 rats with enzymatic and mechanical trituration . Cell suspensions were then incubated with 0.5% FLM for 30 min to 2 h . The longer cells were incubated, the more FLM were taken up . The FLM-labeled SN cell suspensions were injected in the striatum of the hemiparkinsonian model rats . Eight to 13 weeks later, 150-mu m thick coronal brain slices including the graft track were prepared from the recipients . Slices were kept in vitro for several hours . Grafted cells could be clearly identified in the slice preparations by the uptake of FLM under a fluorescence microscope . Voltage-dependent currents and intracellular Ca2+ transcients were successfully recorded from the identified grafted neurons . It is suggested that labeling and identification of living donor cells with FLM is feasible and thus can provide a powerful tool to study the mechanisms underlying graft-induced amelioration of neurological deficits in parkinsonism by enabling physiological assessments of grafted cells.

Plant Mol Biol, 1996 Feb, 30(3), 427 - 38
Stress responses in alfalfa (Medicago sativa L.) . XX . Transcriptional activation of phenlpropanoid pathway genes in elicitor-induced cell suspension cultures; Ni W et al.; Nuclear transcript run-on analysis was used to investigate++ the relative transcription rates of genes encoding enzymes of isoflavonoid phytoalexin biosynthesis and related pathways in elicitor-treated alfalfa (Medicago sativa L.) cell suspension cultures . Genes encoding L-phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and chalcone reductase (CHR) were most rapidly activated, with increases in transcription measurable within 10-20 min after elicitation . Cinnamic acid 4-hydroxylase (C4H), chalcone isomerase (CHI), isoflavone reductase (IFR) and caffeic acid 3-0-methyltransferase (COMT) genes were also rapidly activated, but at a slower initial rate . Transcription of chalcone 2'-O-methyltransferase (CHOMT), and 1,3-beta-D-glucanase genes was less rapid, with lag periods of 60 and 30 min post-elicitation, respectively . Treatment of cells with a PAL inhibitor L-alpha-aminooxy-beta-phenylpropionic acid (AOPP) resulted in increased transcription of PAL, CHS and CHR, but reduced transcription of CHOMT, indicating a role for phenylpropanoid products as both negative and positive regulators of gene expression within the phenylpropanoid pathway.

J Invest Dermatol, 1996 Feb, 106(2), 305 - 11
Identification of a human dermal macrophage population responsible for constitutive restraint of primary dermal fibroblast proliferation; Gonzalez-Ramos A et al.; Primary human dermal cell suspensions prepared from the papillary dermis of keratomed skin strips were used to investigate the effect of indigenous dermal macrophages (HLA-DR+, CD11c+ CD11b+ CD1c- phagolysosome+) upon dermal fibroblast proliferation . Rapid dermal fibroblast expansion was induced upon immunomagnetic bead removal of CD11b+ or CD11c+ cells as well as by removal of more inclusive subsets contained within the DR+ population, but the removal of mast cells, endothelial cells, and CD1c+ dermal Langerhans cells from dermal cell suspensions failed to result in proliferation of the remaining cell subsets . Removal of 1B10+ fibroblasts from macrophage depleted (CD11b-) dermal cell suspensions essentially abrogated the unrestrained proliferation of the CD11b- dermal cells . Flow cytometric cell cycle analysis of cultured macrophage-depleted dermal cells confirmed that the unrestrained proliferating cells contain procollagen I+ as well as procollagen I- dermal fibroblasts . Inhibition of primary fibroblast expansion by adding a supernatant from unfractionated dermal cells suggested that a growth-inhibitory soluble activity of >30,000 kDa dominates the cytokine mixture released by unfractionated fresh dermal cells ex vivo . Inhibitory activity counterbalanced positive fibroblast growth- stimulatory cytokines released by dermal cells because neutralizing antibodies to insulin-like growth factor 1 and interleukin-1 beta resulted in decreased CD11b- dermal cell fibroblast proliferation . These data indicated an important role for dermal macrophages of the DR+ CD11b+ CD11c+ DC1c- phenotype in the normal homeostatic restraint of primary human dermal fibroblast proliferation.

Exp Hematol, 1996 Feb, 24(3), 459 - 65
BCL2 oncogene protein expression in human hematopoietic precursors during fetal life; Bonati A et al.; BCL2 proto-oncogene encodes a 25-kD protein that is characteristically localized in the inner mitochondrial membrane of the cell . It has been reported that BCL2 protein has the unique functional role of blocking programmed cells death without affecting proliferation . We have analyzed the expression of the BCL2 protein in fetal hematopoietic tissues from the 10th week of gestational age onward . Fetal thymus, liver, and bone marrow and cord blood were investigated . The experiments were performed by the alkaline-antialkaline phosphatase (APAAP) technique by staining air-dried acetone-fixed cytospins and by dual-color immunofluorescent assay by staining mononuclear cell suspensions with monoclonal antibodies detecting BCL2 protein and antigens expressed by different hematopoietic subsets . Flow cytometric analyses were performed on FACSort's Comsort 32 (Becton Dickinson, San Jose, CA) . The results have shown that the BCL2 protein is expressed in human fetal ontogenesis at the earliest stages examined . The major conceptual aspects of the results are 1) BCL2 is largely expressed in the hematopoietic cells during ontogenesis . BCL2+ cells include both immature and more differentiated subsets . Moreover, the 25-kD protein is expression in cell subsets well known to be high proliferating . This behavior suggests that BCL2 could have more complex functions than those previously described . 2) The expression in the major part of CD34+ cells suggests that BCL2 could play a role in stem cell survival . 3) BCL2 is expressed in not only medullary but also cortical thymocytes, where it could cooperate in positive selection processes . 4) The involvement of BCL2 in the immunosurveillance is indicated not only by its role in B and T cell lineages but also by its expression in particular subsets like that of the cytoplasmic CD3+ fetal liver NK cells . 5) The discrepancy observed between the results of transgenic mice analysis and in vitro inhibition experiments by antisense oligonucleotides performed for understanding BCL2 functions must stress the importance of the direct immunologic analysis of BCL2 in human hematopoietic cells.

Circ Res, 1996 Feb, 78(2), 283 - 8
In vivo survival and function of transplanted rat cardiomyocytes; Li RK et al.; Recent studies have demonstrated the feasibility of transplanting fetal mouse cardiomyocytes into the hearts of adult syngeneic mice . However, the function of the transplanted cardiomyocytes and their capacity to survive in fibrous connective tissue were not assessed . In the present study, we evaluated the viability and contractility of transplanted fetal and neonatal rat cardiomyocytes in the connective tissue of the adult rat hindlimb . Purified fetal or neonatal rat cardiomyocytes were cultured . These cells contained sarcomeres, formed junctions composed of desmosomes and fascia adherens, and contracted regularly and spontaneously . A fetal or neonatal cardiomyocyte suspension was injected into the subcutaneous tissue of adult rat hindlimbs . Cyclosporin A (5 mg/kg) was administered subcutaneously daily for the 3-month duration of the study, at which time the animals were killed . The transplanted cardiomyocytes formed 'tissue' in vivo that increased in size for the first 2 weeks and remained the same size at the third week . The tissue derived from the transplanted fetal cardiomyocytes contracted spontaneously at a rate of 73 +/- 12 bpm, and that from the neonatal cardiomyocytes contracted at a rate of 43 +/- 21 bpm . The electrocardiogram was similar to that seen in myocardium with an idioventricular rhythm . Histologically, the tissue appeared to be cardiac muscle with sarcomeres . Angiogenesis occurred in the cardiomyocyte graft . In summary, a cell suspension of cultured fetal and neonatal rat cardiomyocytes transplanted into the adult rat hindlimb formed contractile cardiac tissue in the subcutaneous connective tissue.

J Bacteriol, 1996 Feb, 178(3), 728 - 34
Low-affinity potassium uptake system in the archaeon Methanobacterium thermoautotrophicum: overproduction of a 31-kilodalton membrane protein during growth on low-potassium medium; Glasemacher J et al.; During growth on low-K+ medium (1 mM K+), Methanobacterium thermoautotrophicum accumulated K+ up to concentration gradients ({K+}intracellular/{K+}extracellular) of 25,000- to 50,000-fold . At these gradients ({K+}extracellular of < 20 microM), growth ceased but could be reinitiated by the addition of K+ or Rb+ . During K+ starvation, the levels of a protein with an apparent molecular weight of 31,000 increased about sixfold . The protein was associated with the membrane and could be extracted by detergents . Cell suspensions of M . thermoautotrophicum obtained after K+-limited growth catalyzed the transport of both K+ and Rb+ with apparent Km and Vmax values of 0.13 mM and 140 nmol/min/mg, respectively, for K+ and 3.4 mM and 140 nmol/min/mg, respectively, for Rb+ . Rb+ competitively inhibited K+ uptake with an inhibitor constant of about 10 mM . Membranes of K+-starved cells did not exhibit K+-stimulated ATPase activity . Immunoblotting with antisera against Escherichia coli Kdp-ATPase did not reveal any specific cross-reactivity against membrane proteins of K+-starved cells . Cells of M . thermoautotrophicum grown at a high potassium concentration (50 mM) catalyzed K+ and Rb+ transport at similar apparent Km values (0.13 mM for K+ and 3.3 mM for Rb+) but at significantly lower apparent Vmax values (about 60 nmol/min/mg for both K+ and Rb+) compared with K+-starved cells . From these data, it is concluded that the archaeon M . thermoautotrophicum contains a low-affinity K+ uptake system which is overproduced during growth on low-K+ medium.

Neuroreport, 1996 Jan 31, 7(2), 449 - 53
Increased food intake and body weight gain after lateral hypothalamic dopaminergic cell implantation; Yang ZJ et al.; To examine our hypothesis that dopamine activity in the lateral hypothalamic area (LHA) may play a role in enhancing the process of eating, a fetal cell suspension of predominantly dopaminergic cells was bilaterally transplanted into the LHA of study rats via direct injection; controls had carrier medium injection . Thereafter, mean daily food intake was 1 g per day greater in dopaminergic cell transplanted rats vs . controls for each day of the 10-week observation period . This resulted in a significantly greater cumulative body weight gain in study rats vs . controls (386 +/- 5.1 g vs . 354 +/- 3.8 g, respectively) . On sacrifice at the end of the study, transplanted cells in the LHA were viable . Our data suggest that bilateral LHA dopaminergic cell transplant which presumably resulted in chronically and persistently enhanced dopaminergic activity in the LHA is associated with overeating and consequently, an excess weight gain.

Biochim Biophys Acta, 1996 Jan 31, 1278(2), 205 - 12
The role of efflux systems and the cell envelope in fluorescence changes of the lipophilic cation 2-(4-dimethylaminostyryl)-1-ethylpyridinium in Escherichia coli; Sedgwick EG et al.; The interaction of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethlypyridinium cation (DMP+) with cells of Escherichia coli AN120 (uncA) and AS-1 (acrA) was studied to elucidate the role of the envelope and of efflux systems in the uptake of lipophilic cations . DMP+ bound to the two strains in a different manner . With AS-1 the bound dye was displaced only to a small extent by addition of Mg2+ or other divalent cations . By contract, 50% of the DMP+ was displaced by micromolar concentrations of Mg2+ from resting cells of AN120 . Energization of the cells by substrate oxidation resulted in the loss in AN120 of 50% of the bound dye and a decrease of the fluorescence in the cell suspension . With AS-1, energization caused more DMP+ to be taken up from the medium . This was associated with an increase in fluorescence in the cell suspension . The extent of the quenching by addition of Mg2+ was not increased . Right-side out vesicles from AN120, like those of AS-1, showed DMP+ fluorescence behaviour which resembled that of intact cells of AS-1 . Transformation of AS-1 with plasmids encoding the E . coli Mvr and EmrAB efflux systems resulted in the DMP+ fluorescence response of this strain becoming like that of AN120 . It is suggested that with strain AN120 the changes in binding of DMP+ and fluorescence intensity were associated with activation of efflux systems on cell energization . With AS-1, it is suggested that the observed fluorescence and binding changes are due to inactivation of the AcrAB efflux system by the acrA mutation . Thus, the net entry of lipophilic cations is facilitated . Energization of dye update and release is driven by an electrochemical gradient of protons . ATP is not directly involved in energizing the movement of the dye.

Brain Res, 1996 Jan 29, 707(2), 245 - 55
Transplantation of embryonic noradrenergic neurons in two models of adult rat spinal cord injury: ultrastructural immunocytochemical study; Gimenez y Ribotta MG et al.; The synaptic connections established by grafted noradrenergic (NA) neurons into the lesioned adult rat spinal cord were analysed using immunocytochemistry at the electron microscopic level . An embryonic cell suspension of the locus coeruleus region from E-13 rat embryos was transplanted into the spinal cord following either: (1) spinal cord transection or (2), partial selective denervation by 6-hydroxy dopamine (6-OH DA) . One month after grafting, the NA-neurons established, in the two models, an innervation pattern similar to that found in the intact spinal cord . In both models, the transplanted NA-immunoreactive neurons formed extensive synaptic contacts with dendrites, spines and perikarya . The proportion of axodendritic and axospinous contacts was inverse in the two models . The first model thus reproduced more closely the normal synaptic pattern prefering dendritic targets, which could correspond to a better integration of the graft . In the second model, a partially NA-denervated spinal cord, there existed a competition between residual intrinsic and grafted neuron-derived fibres, which presumably affects synaptogenesis . In conclusion, the present study illustrate the complexity of cell interations conducting to the formation of a specific circuitry . Recognition phenomenon are likely modulated by space constraints, which ultimately shape-up the geometry of synaptic contacts.

J Immunol, 1996 Jan 15, 156(2), 621 - 7
Rhinovirus enters but does not replicate inside monocytes and airway macrophages; Gern JE et al.; Potential interactions between rhinovirus (RV) and both the airway macrophage and its precursor cell, the blood monocyte, were investigated in terms of direct binding, intracellular replication, cell survival, and cytokine production . When HeLa cell suspensions are inoculated with RV as a positive control, virus titer increases by 100-fold in the first 24 h, confirming intracellular replication . In contrast, RV titer in monocyte and macrophage suspensions steadily decreased . Despite a lack of productive RV replication, cell-associated RV RNA was detectable using a biotin-labeled cDNA probe as early as 6 h after inoculation . Direct binding of RV16 to macrophages was confirmed using radiolabeled virus, although preincubation with anti-ICAM-1 mAb did not block this interaction . Synthesis of RV RNA, as indicated by {3H}uridine incorporation in actinomycin D-treated cells, was detected in HeLa cells but not macrophages, suggesting that the viral RNA detected inside macrophages was from input virus and was not newly synthesized . RV inoculation did not adversely affect monocyte or macrophage viability . Finally, RV caused macrophage activation, as indicated by the induction of TNF-alpha secretion . These in vitro findings suggest that macrophages interact with major group RV in vivo, and raise the possibility that there is a second cellular receptor for these viruses . Furthermore, macrophages do not serve as permissive host cells during in vivo RV infection, but instead may be active participants in anti-RV immunity and RV-induced airway inflammation.

Dev Biol, 1996 Jan 10, 173(1), 279 - 91
Skeletal myogenesis: the preferred pathway of chick embryo epiblast cells in vitro; George-Weinstein M et al.; The epiblast layer of the chick embryo gives rise to all embryonic tissues . In vitro analyses were carried out to determine whether epiblast cells could form skeletal muscle prior to entry into the primitive streak . Epiblasts were separated from the mesoderm, hypoblast, and primitive streak, dissociated to produce a single cell suspension, and plated at high density . Myogenesis began on the first day in culture, and by the fifth day most cells had differentiated into skeletal muscle . Some cells differentiated without replicating . MyoD messenger RNA was present in epiblast tissue and translated in practically all cells in culture . Cells from regions of the epiblast which do not form muscle later in the embryo did so in vitro . Epiblasts cultured for 2 days as an intact epithelium, or in the presence of the mesoderm and hypoblast, did not undergo myogenesis . These findings demonstrate that myogenic potential is wide-spread within the primitive streak stage epiblast, and that muscle differentiation, which occurs relatively autonomously in culture, can be prevented by cell and tissue interactions.

Chem Biol Interact, 1996 Jan 5, 99(1-3), 227 - 38
Redox behaviour of nifuroxazide: generation of the one-electron reduction product; Squella JA et al.; The electrochemical properties of nifuroxazide have been investigated in aqueous and aqueous-DMF mixed solvents . In aqueous media, a single, irreversible four-electron reduction occurs to give the hydroxylamine derivative . In mixed media, a reversible one-electron reduction to form a nitro radical anion takes place . Cyclic voltammetric studies show that the anion radical product is stable, although the nitro radical anion intermediate shows a tendency to undergo further chemical reactions . A comparison with the voltammetric behaviour of other nitrofurans such as nifurtimox, nitrofurazone and furazolidone is made . The electrochemically-obtained parameters are correlated with the in vivo studies of oxygen consumption on Trypanosoma cruzi cell suspensions.

Ultrasound Med Biol, 1996, 22(9), 1267 - 75
Biophysical effects of high-energy pulsed ultrasound on human cells; Feigl T et al.; Human benign and malignant cells of different human origin (pancreas, liver, kidney, pharynx, tongue, lip) were exposed to high-energy pulsed ultrasound (HEPUS) in vitro to evaluate the effects of various physical parameters and sonication conditions on cell viability . This included the number of pulses, focal pressure, pulse repetition rate, pulse shape, cell suspension volume, water level of the basin and cell density . Cell viability was found to depend significantly on the number of pulses (exponential), the focal pressure (linear) and the pulse repetition rate (minimum at 1 Hz) . Other parameters showed no marked influence . Furthermore, electron microscopy revealed intracellular damage, and proliferation rates of cells surviving sonication were normal after HEPUS exposure . The experimental piezoelectric ultrasound transducer used in the experiments generated oscillating bipolar pulses with high negative pressure amplitudes . Measurements were made of the pulse shape and ultrasonic field of the experimental device and of a conventional lithotripter for comparison.

Biomed Pharmacother, 1996, 50(10), 505 - 9
Purine metabolism in HIV-1 virus-infected T lymphocyte population; Carlucci F et al.; Purine nucleotide metabolism has been studied in T-lymphocyte population in healthy subjects and in AIDS bearing patients . Nucleotide content was determined by HPLC . The overall rate of purine nucleotide synthesis was measured following the incorporation of 14C-formate into the nucleotides of a T cell suspension . The authors discuss the results, which indicate interesting variations in nucleotide content and a lower nucleotide synthesis, determined by kinetic studies.

Nat Toxins, 1996, 4(6), 254 - 60
Transformation of the mycotoxin ochratoxin A in plants: 1 . Isolation and identification of metabolites formed in cell suspension cultures of wheat and maize; Ruhland M et al.; The metabolism of the mycotoxin ochratoxin A in plant cells was investigated using cell suspension cultures of wheat and maize . A number of metabolites were detected by HPLC-chromatography with fluorescence detection . The main metabolites were ochratoxin alpha, ochratoxin A methyl ester, two isomers of hydroxyochratoxin A, and the glucosides and methyl esters of both hydroxyochratoxin A isomers . The compounds were isolated by TLC and preparative HPLC and identified by mass spectrometry and specific enzymic reactions.

Cytobios, 1996, 86(346), 193 - 200
Neutrophil activation in nickel sensitized subjects; Testa A et al.; Conflicting results were obtained from the evaluation of nickel-mediated effects on immunoresponsiveness . A reduction of B cell polyclonal response, mixed lymphocyte reaction, T lymphocyte proliferation and natural killer cytotoxicity was evident following nickel salt exposure . On the contrary, an enhancement of cytokine release, T cell proliferative capacity and adhesion molecule expression was found in nickel sensitized donors . In addition, under different experimental conditions, respiratory burst induction and myeloperoxidase release by polymorphonuclear cells (PMN) in a group of individuals exhibiting nickel hypersensitivity were investigated . Results provide evidence that nickel allergic individuals displayed a significant increase of superoxide anion (O2-) generation by suspended PMN in comparison with similar cell suspensions from healthy donors, but this was not the case when adherent neutrophils were used as effector cells . PMN from nickel sensitized donors exhibited a significant enhancement of hydrogen peroxide (H2O2) and myeloperoxidase release . The results imply the occurrence of neutrophil activation in nickel hypersensitivity, which in turn may be responsible for the low frequency of life-threatening infections in these subjects.

Mycopathologia, 1996, 134(2), 97 - 102
Transformation of the mycotoxin ochratoxin A in plants . 2 . Time course and rates of degradation and metabolite production in cell-suspension cultures of different crop plants; Ruhland M et al.; Ochratoxin A, one of the most toxic mycotoxins, can be metabolized nearly completely by suspension cultures of various plant cells . The transformation products identified in this study were almost the same in the cell-suspension cultures of maize, carrot, tomato, potato, soybean, wheat and barley, but the quantitative distribution differed strongly depending on incubation time and species of plant-cell culture . The compounds were extracted with ethyl acetate and detected by reversed-phase HPLC with gradient elution . From the result it is supposed that besides ochratoxin A also ochratoxin derivatives may occur in food and feedstuff of plant origin.

Cancer Chemother Pharmacol, 1996, 39(1-2), 67 - 70
Electropermeabilization in bladder cancer chemotherapy; Kubota Y et al.; PURPOSE: Electropermeabilization has been used for the introduction of genes into cells . Using this technique, we introduced the cytotoxic drug bleomycin (BLM) into cells and examined whether the technique might be useful for the treatment of bladder cancer . MATERIALS AND METHODS: For electropermeabilization in vitro, we used YTS-1 cells, a human transitional cell carcinoma line . Aliquots of cell suspension were mixed with a solution of BLM and immediately exposed to electric pulses . A high-power pulse generator was used to supply square-shaped pulses of 1250 V/cm (100 micros, eight pulses) . After a 2-h post-shock incubation, cells were washed and incubated for one further hour . Then the concentration of BLM in the cells was measured using a bioassay . For electropermeabilization of tissue, we used normal male Wistar rats . The bladder was exposed and 10 mg/kg BLM was injected into the caudal vein . A series of eight pulses with a time constant of 100 micros at an electric field intensity of 1000 V/cm was applied . The bladder, liver and lungs were extracted 1 h later and prepared for quantification of the BLM concentration using the bioassay . RESULTS: Electrotreated cells contained significantly higher concentrations of BLM than nonelectrotreated cells . The concentration of BLM 1 h after electrotreatment in bladder tissue was 2.7 times higher than that in nonelectrotreated bladder tissue . CONCLUSION: The electropermeabilization technique has the potential to serve as a new and effective modality for the treatment of bladder cancer.

Biotherapy, 1996, 9(1-3), 55 - 9
Profiles of cytokine production in recipients of transfer factors; Alvarez-Thull L et al.; Transfer factors (TF) are proteins that transfer the ability to express cell-mediated immunity from immune donors to non-immune recipients . The mechanisms of these effects have not been defined . The experiments described in this report were undertaken to test the hypothesis that a mechanism through which the beneficial effects of TF are expressed in clinical situation is through "education" of the immune system to produce certain cytokines in response to antigenic stimulation . BALB/c mice were sensitized to Herpes simplexvirus (HSV) either by sublethal systemic or cutaneous infections by administration of a HSV-specific TF . One week later their spleen cells were collected and single cell suspensions were stimulated in vitro with irradiated HSV or concanavalin . A Culture supernatants were collected and assayed for content of IL-2, IL-4, IL-10 and IFN-g . Spleen cells from infected mice responded to concanavalin A and to HSV by secreting large amounts of IL-2 and IFN-g, modest amounts of IL-10, and no IL-4 . Transfer factor recipients produced similar cytokine profiles in response to concavalin A . These mice, however, responded to HSV by secreting IFN-g, but no IL-2 . Thus, TF treatment selectively affects cytokine production in response to antigenic stimulation.

Exp Clin Endocrinol Diabetes, 1996, 104 Suppl 3, 56 - 9
Model of the athymic nude mouse for the study of benign goiter disease; Gerber H et al.; Since Shimosato et al., in the mid 70s transplanted for the first time thyroid carcinoma tissue onto nude mice, other research groups have made use of the nude mouse model for the investigation of xenotransplanted thyroid tissue . The use of this model for the investigation of benign goiters is briefly discussed in this article . Normal human thyroid tissue has been transplanted either as a control in experiments with benign and malignant goiter tissue, or for the study of thyroid tissue response to stimulators such as TSH or thyroid stimulating antibodies (TSAb) . Thyroid glands from 8- to 10-week old human fetuses obtained at the time of legal abortion were cryopreserved in liquid nitrogen and successfully transplanted into nude mice . Moreover, all the variants of human benign goiter tissue have been xenotransplanted: tissue from nodular and diffuse goiters, hot and cold nodules or goiter areas, rapidly growing nodules, etc . Two examples of animal thyroid tissue xenotransplantation onto nude mice are briefly discussed: Nude mice bearing normal thyroid tissue transplants from 4 different species (man, rat, pig, guinea-pig) have been used for the study of the species specific effect of bovine TSH and TSAb . In studies aiming at elucidating the pathogenesis of hyperthyroidism, toxic goiter tissue from hyperthyroid cats has been transplanted . In methodological terms, these experiments have shown that surgically removed goiter tissue can be shipped by air in cell culture medium at 4 degrees C over long distances and then successfully transplanted.-Finally, cell lines such as the rat cell line FRTL-5 can be transplanted onto nude mice either as cell suspension or embedded in collagen, for example for the study of proliferation and folliculogenesis . Using the xenotransplantation model, function and proliferation, morphogenesis and differentiation, as well as thyroid autonomy and response to stimulators have all been studied in xenotransplanted human and animal thyroid thyroid tissue and cell lines under various experimental conditions . Although new research tools, for example transgenic animals, are now increasingly and successfully used, xenotransplantation still offers the possibility of addressing some specific questions which cannot be answered so easily with other experimental models . For example, studies with human tissue, involving drugs or radioactive tracers which cannot be applied to the intact human being, can relatively easily be performed with xenotransplanted human tissue and application of the drug or tracer to the host mouse . Or embryological development can be followed and studied using fetal thyroid (and other) tissue transplanted onto nude mice; here, of course, difficult ethical issues have to be considered . Finally, it should be mentioned that, although many scientific questions can be studied nowadays by cell culture or other in vitro systems, animal models are still needed . Extrapolation to the human being, however, should always be done with caution and we should always keep in mind that for the understanding of a human disease indeed human experimental models remain the goldstandard.

Beitr Infusionsther Transfusionsmed, 1996, 33, 184 - 90
{Experimental principles and general practice of intraoperative autotransfusion with blood irradiation in tumor operation}; Hansen E et al.; Clinical studies fail to verify or to exclude the lethal risk of tumor spread after intraoperative autotransfusion in tumor surgery . An alternative approach is the development of methods for the elimination of tumor cells in the salvaged blood . The radiosensitivity of tumor cells especially in oxygenated single cell suspensions is well known, while the non-nucleated red blood cells are radioresistant . With 50 Gy a 12 log reduction in proliferating cells is expected . This we have tested experimentally and put into clinical practice . The suppression of colony formation in cell culture by irradiation with 50 Gy was tested with tumor cells from established cell lines or from solid tumors after admixture in high cell number to red blood cells from volunteer blood donations . DNA metabolism was tested by incorporation of bromodesoxyuridine (BrdUrd) and staining with mcab-anti-BrdUrd . Colony formation and DNA metabolism was absent in all samples of cell lines or tumor cells in blood after irradiation with 50 Gy, reflecting a 10 log, or 7 log reduction, respectively . In clinical practice the method of intraoperative blood salvage during tumor surgery with blood irradiation showed its practicability and efficacy in reducing homologous transfusions.

J Hirnforsch, 1996, 37(1), 15 - 24
Specific innervation of the rat thalamus by grafted noradrenergic locus coeruleus neurons; Murata Y et al.; Growth and distribution of noradrenaline (NA) fibres from the implant into the thalamus of host rats were examined at 5-13 months after the implantation by immunohistochemistry using NA or tyrosine hydroxylase antisera . Cell suspension dissociated from the locus coeruleus (LC) region of 14-day-old rat fetuses was implanted into the center of the unilateral thalamus in adult rats from which the noradrenergic afferents to the thalamus had been eliminated with 6-hydroxydopamine treatment . A dense network of varicose NA-immunoreactive (NA-IR) fibres extended laterally into the posterior thalamic nuclear group and the ventral posterolateral thalamic nucleus from the implant in a pattern similar to that the intrinsic noradrenergic fibres form in the normal thalamus, i.e . laterally rich and medially poor NA fibres . Electron microscopic observations revealed that varicosities of NA-IR fibres formed symmetrical as well as asymmetrical axodendritic synapses and axo-axonic synapses with the host neurons as seen in the normal thalamus . labelled dendrite-like fibres of graft origin penetrated deep into the host brain and received afferents from non-labelled axon terminals . Varicosities of NA-IR fibres in the LC implanted animal formed axo-dendritic synapses at the higher ratio than those in the normal animal did . These results show that implanted fetal noradrenergic neurons innervate target regions of the thalamus specifically as the noradrenergic fibres in the normal thalamus do and maintain the innervation for a long time in the noradrenergically denervated rats.

Transpl Int, 1996, 9 Suppl 1, S492 - 6
Comparative analysis of immunological reconstitution induced by vascularized bone marrow versus bone marrow cell transplantation; Lukomska B et al.; We have reported previously that vascularized bone marrow transplantation (VBMT) in an orthotopic hind limb graft brings about complete repopulation of bone marrow cavities in lethally irradiated syngeneic recipients within 10 days . Intravenous infusion of an equivalent volume of bone marrow cell suspension was evidently less effective . The purpose of this study was to investigate the reconstitution of immunocompetent compartments of lethally irradiated syngeneic rats after VBMT . Lewis rat hind limbs were transplanted orthotopically into irradiated recipients . Ten days after irradiation and bone marrow transplantation, bone marrow, mesenteric lymph nodes, and sera from rats were harvested . Mesenteric lymph node lymphocytes were analyzed . The responsiveness for mesenteric lymph node lymphocytes (MLNL) to mitogens and cell proliferation in the presence of sera and bone marrow cell (BMC) culture supernatants were measured . Our studies have shown that vascularized bone marrow transplantation brings about rapid replenishment of lymphoid organs of lethally irradiated syngeneic recipients . The repopulating subsets are fully responsive to mitogens . Sera from reconstituting rats had no effect on the proliferation of mature lymphocytes . Intravenous infusion of a number of BMC in suspension equivalent to that grafted in hind limb transplant was less efficient in reconstitution of lymphoid tissue.

Breast Cancer Res Treat, 1996, 41(2), 147 - 59
Selective growth of freshly isolated human breast epithelial cells cultured at low concentrations in the presence or absence of bone marrow cells; Emerman JT et al.; In this study, we show that conditions previously found to promote the selective growth of human breast epithelial cells (HBEC) in serum-free primary cultures established from normal or malignant tissue can be extended to cultures initiated at low seeding densities (< 5000 cells/cm2) . The epithelial nature of the cells produced was documented by their positive staining with antibodies specific for keratins 8, 14, and 18, and 2 antibodies that recognize epithelial-specific antigens (Ber-EP4 and HB8630) . HBEC growth was not affected, either positively or negatively, by the use of a medium containing a combination of fetal calf and horse serum, which promotes the growth of many types of stromal cells and associated hematopoietic precursors, or by the inclusion in the initial cell suspension of marrow cells at HBEC to marrow cell ratios typical of bone marrow samples from patients with metastatic breast cancer . The presence of fibroblast feeders from a variety of sources enhanced the growth of HBEC to different degrees . In cultures initiated with low numbers of cells obtained from samples of breast carcinoma, HBEC growth was generally reduced by comparison to cultures of normal HBEC . With the detection methods used, it was not possible to determine the extent to which this decreased growth was due to a reduced frequency of malignant HBEC with in vitro precursor activity, or the presence of reduced numbers of residual normal HBEC precursors, or both . However, preliminary data indicate that this approach also allows the detection of some breast carcinoma cells with proliferative ability that are present in the marrow or pleural effusions of some breast cancer patients . These studies demonstrate the feasibility of detecting normal and malignant HBEC with growth potential when these are cultured at low density and/or as rare contaminants of marrow cell suspensions, and provide a starting point for their further characterization.

Free Radic Biol Med, 1996, 20(4), 507 - 14
Different effects of thiol and nonthiol ace inhibitors on copper-induced lipid and protein oxidative modification; Fernandes AC et al.; Differences among angiotensin-converting enzyme inhibitors (ACEI) in scavenging reactive oxygen species were described and mainly attributed to the presence or absence of a thiol group . Plasma constituents and red cells are known targets for oxidative damage . Transition metals, like copper, are well known catalizers of free radical generation . In the present study we compared the abilities of captopril (a thiol ACEI), enalaprilat, and lisinopril (two nonthiol ACEI) for inhibiting copper-induced thiobarbituric acid reactive substances (TBARS) formation and fluorescence generation in whole human plasma and low-density lipoprotein . The effects of those ACEI on copper/hydrogen peroxide-induced fluorescence development and electrophoretic mobility modification in albumin and on copper-induced TBARS formation and hemolysis in human red cells were also compared . Captopril was more effective than the two nonthiol ACEI in inhibiting plasma and LDL lipid peroxidation, but it was ineffective in inhibiting the albumin oxidative modification that was moderately inhibited by enalaprilat and lisinopril . On the contrary, the inhibitory effects of the three ACEI on copper-induced lipid peroxidation and hemolysis in red cell suspensions were more uniform . This as yet unreported red cell protective effect may deserve pharmacological evaluation . Our results show that captopril is a more effective antioxidant than the nonthiol ACEI in some systems . However, the nonthiol ACEI also have the ability to partially protect some targets against oxidative damage . These observations suggest that the presence of a thiol group in the ACEI structure is not the only determinant for the antioxidant properties.

Cancer Treat Res, 1996, 82, 101 - 14
In vitro pharmalogic rationale for intraperitoneal regional chemotherapy; Link KH et al.; We performed basic in vitro studies on cell lines and individual tumor cell suspensions to support the concept of intraperitoneal regional chemotherapy, and to improve the rationale for drug selection in this regional chemotherapeutic method . We defined the concentration-response behavior and the dependence of drug cytotoxicity on time using the two human colorectal carcinoma cell lines HT29 and NMG 64/84 . In addition, the drugs concentration-response behavior and cytotoxic potency for IPRC after a single drug exposure at 10 micrograms/ml (5-FU at 100 micrograms/ml) was preclinically defined with in vitro phase II studies using single cell suspensions of human solid tumor biopsies in the human tumor colony assay (HTCA) . The drugs doxorubicin (ADM), cisplatin (CDDP), epidoxorubicin (EPI), 5-fluorouracy (5-FU), 5-fluorodeoxyuridine (5-FUDR), melphalan (LPAM), mitomycin C (MMC), and mitroxantrone were incubated at increasing concentrations up to 1000 micrograms/ml at 10, 30, 60, 360, and 1440 minutes with the cell lines . These drugs, as well as vindesine (VDS) and mafosfamide (MAF) were also tested in the HTCA at increasing concentrations . The HTCA response rates at 10 micrograms/ml (5-FU and MAF at 100 micrograms/ml) were used for in vitro phase II comparisons of potential drug clinical activities . All test drugs showed a time- and concentration-dependent cytotoxic activity against the cell lines . Based on the cytotoxicity test results with HT29 and NMG 64/84, specific times were recommended for clinical therapy with each drug . In the HTCA, the drugs showed different cytotoxic concentration responses . The concentration-response behavior of each drug varied in individual tumor biopsies of the same histology . Comparing the response rates at 1 microgram/ml (5-FU and MAF 10 micrograms/ml) and 10 micrograms/ml (5-FU and MAF 100 micrograms/ml), an overall increase of in vitro response by a factor of 2.1 +/- 0.7 (1.1-3.7) was noted . We were able to prove this principle qualification of various test drugs in our in vitro studies and to suggest the optimal exposure times for their use in intraperitoneal chemotherapy . Based on these results, NOV was successfully used in an IPRC clinical study.

Brain Res Bull, 1996, 39(1), 23 - 32
Transplantation of human striatal tissue into a rodent model of Huntington's disease: phenotypic expression of transplanted neurons and host-to-graft innervation; Pundt LL et al.; The present study was undertaken to investigate the phenotypic expression and integration of human striatal neurons transplanted into an animal model of Huntington's disease . Sprague-Dawley rats were anesthetized and subjected to quinolinic acid lesions of the left striatum . Three human fetal cadavers were utilized for transplantation in this study (7, 8, and 10 weeks in gestation) . The striatal primordia was dissected from each fetus and subsequently dissociated into cell suspensions . Following the initial lesion surgeries (3-4 months), the rats were reanesthetized and transplanted with human striatal cells (400,000 cells per rat) . The animals were processed for histochemical analysis 9-17 weeks posttransplantation . Histochemistry was performed utilizing thionin (Nissl staining), acetylcholinesterase, NADPH-diaphorase, and antibodies against tyrosine hydroxylase and glial fibrillary acidic protein . Examination of stained brain sections demonstrate that human striatal transplants grow to fill a substantial portion of the remaining striatum, and contain clusters of immature and mature cells . Acetylcholinesterase activity is present in the transplant neuropil, varying in intensity, and distributed in a heterogeneous fashion . In addition, host afferent dopaminergic fibers penetrate into the transplant, and are occasionally found in patches . NADPH-diaphorase histochemistry revealed medium sized aspiny striatal neurons of donor origin in the transplants . The results of this study are similar to those obtained with rodent fetal striatal transplants, and suggest that human striatal tissue is capable of surviving, expressing normal striatal cell phenotypes, and receiving host dopaminergic innervation.

Ann Oncol, 1996, 7 Suppl 4, 35 - 9
Single cell PCR for the analysis of Hodgkin's disease: four years later; Kupper M et al.; BACKGROUND: Single cell-based studies represent a promising alternative to conventional molecular approaches in the study of Hodgkin's disease since the malignant Hodgkin and Reed-Sternberg cells (H & RS) represent only a small minority of the cellular infiltrate in affected nodes . METHODS: Single cell polymerase chain reaction (PCR) assays were developed for the analysis of specific genomic DNA sequences and the detection of gene expression . Single H & RS cells were isolated by micromanipulation from cytospin slides or fresh cell suspensions after staining with an anti-CD 30 MoAB . RESULTS: The status of oncogenes and immune receptor genes was examined by DNA-PCR . So far, no IgH or TCR gamma rearrangements were detected in H & RS cells of T- and B-antigen negative classical Hodgkin's cases but were detected in two cases of nodular paragranuloma . Global cDNA amplification was successfully performed from single H & RS cells, and specific gene transcripts were detected with a novel PCR method . CONCLUSION: Single cell PCR is a novel and promising method that will help to elucidate many of the open questions in the biology of Hodgkin's disease . In the case of contradictory results, collaborations between different groups utilizing similar approaches have to be performed.

Cancer Treat Res, 1996, 81, 31 - 40
Intraperitoneal regional chemotherapy with mitroxantrone; Link KH et al.; Pharmacokinetic considerations and tests with cell lines and individual cell suspensions from metastatic human solid tumor biopsies suggested testing the efficacy of mitoxantrone (NOV) in intraperitoneal regional chemotherapy (IPRC) . Twenty-seven patients with intraperitoneal metastatic disease of various solid tumors received cyclic IPRC with NOV for treatment of malignant ascites (N = 16) or of peritoneal carcinomatosis (N = 11) at a NOV instillate concentration of 10 micrograms/ml . A total of 125 cycles (1-5 per patient) were applied . Response and toxicity were registered according to WHO criteria . The response rate (CR+PR) was 56 percent in malignant ascites, 45 percent in peritoneal carcinomatosis, and 52 percent overall . There were no systemic toxicities . Regional side effects were bacteriemia (4 of 125 cycles), pain (2 of 125 cycles), small bowel stricture (1 of 27 patients), and small bowel perforation (1 of 27 patients) . From these results we can conclude that NOV appears to be effective in IPRC for malignant ascites and peritoneal carcinomatosis at tolerable toxicities.

J Membr Biol, 1996 Jan, 149(2), 141 - 59
Shrinkage-induced activation of the Na+/H+ exchanger in Ehrlich ascites tumor cells: mechanisms involved in the activation and a role for the exchanger in cell volume regulation; Pedersen SF et al.; Amiloride-sensitive, Na(+)-dependent, DIDS-insensitive cytoplasmic alkalinization is observed after hypertonic challenge in Ehrlich ascites tumor cells . This was assessed using the fluorescent pH-sensitive probe 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) . A parallel increase in the amiloride-sensitive unidirectional Na+ influx is also observed . This indicates that hypertonic challenge activates a Na+/H+ exchanger . Activation occurs after several types of hypertonic challenge, is a graded function of the osmotic challenge, and is temperature-dependent . Observations on single cells reveal a considerable variation in the shrinkage-induced changes in cellular pHi, but the overall picture confirms the results from cell suspensions . Shrinkage-induced alkalinization and recovery of cellular pH after an acid load, is strongly reduced in ATP-depleted cells . Furthermore, it is inhibited by chelerythrine and H-7, inhibitors of protein kinase C (PKC) . In contrast, Calyculin A, an inhibitor of protein phosphatases PP1 and PP2A, stimulates shrinkage-induced alkalinization . Osmotic activation of the exchanger is unaffected by removal of calcium from the experimental medium, and by buffering of intracellular free calcium with BAPTA . At 25 mM HCO3(-), but not in nominally HCO3(-)-free medium, Na+/H+ exchange contributes significantly to regulatory volume increase in Ehrlich cells . Under isotonic conditions, the Na+/H+ exchanger is activated by ionomycin, an effect which may be secondary to ionomycin-induced cell shrinkage.

J Basic Microbiol, 1996, 36(1), 3 - 11
The effect of ultrasound on Escherichia coli viability; Allison DG et al.; The effect of continuous-wave ultrasound on the viability of Escherichia coli HB101 was assessed using a 20 kHz ultrasonic processor . A standardised cell suspension of fixed concentration was used to investigate the influence of different physical and environmental conditions on ultrasound susceptibility . Cell viability decreased exponentially with time at different intensities of ultrasound . Increasing intensity caused a decrease in decimal reduction times . Loss of cell viability occurred primarily from the mechanical effects of ultrasound rather than free radical damage . E . coli susceptibility was also shown to vary with growth conditions, whereby cells cultivated either on agar or harvested from the stationary phase of liquid culture were significantly more susceptible to ultrasound than an equivalent population obtained from the exponential phase of liquid growth . The implication of these results is discussed in relation to the use of ultrasound as a novel means of bacterial transformation.

J Cancer Res Clin Oncol, 1996, 122(9), 533 - 40
An attempt to enhance chemosensitivity of quiescent cell populations in solid tumors by combined treatment with nicotinamide and carbogen; Masunaga S et al.; cis-Diamminedichloroplatinum(II) (cisplatin) was intraperitoneally injected into mice bearing SCC VII or EMT6/KU tumors after ten administrations of 5-bromo-2'-deoxyuridine (BrdU) to label all the proliferating tumor cells . The tumors were excised 1 h after the cisplatin injection, minced, and trypsinized . The tumor cell suspensions were then incubated with cytochalasin-B (a cytokinesis blocker) . The micronucleus frequency was determined, using immunofluorescence staining for BrdU . Cells that were not labeled with BrdU were regarded as quiescent . The micronucleus frequency in the total number of tumor cells was determined in tumors that had not been pretreated with BrdU . To modify the sensitivity to cisplatin, nicotinamide was intraperitoneally injected before the administration of cisplatin or mice were placed in a circulating carbogen (95% O2, 5% CO2) chamber for 30 min after cisplatin administration . In both tumor systems, the micronucleus frequency in quiescent cells was lower than that in the total cells . Nicotinamide pretreatment increased the micronucleus frequency in total and in quiescent cells in both tumor systems, and to a higher extent in total cells . The combination of nicotinamide and carbogen increased the micronucleus frequency more markedly than treatment with either nicotinamide or carbogen alone . In total cells of both tumors, the nicotinamide injection increased the uptake of {195mPt}cisplatin . The combined treatment raised the uptake more markedly than did treatment with either agent alone . In total cells of the SCC VII tumor, these increases in micronucleus frequency and the {195mPt}cisplatin uptake following nicotinamide or combined pretreatment were significant . In both tumors, carbogen breathing also elevated the micronucleus frequency to some degree in total and quiescent cells and the {195mPt}cisplatin uptake in total cells . The combined nicotinamide and carbogen treatment was considered to be useful for sensitizing tumor cells to chemotherapy with cisplatin in vivo.

Protein Sci, 1996 Jan, 5(1), 114 - 20
Two mutations in recombinant Hb beta F41(C7)Y, K82 (EF6)D show additive effects in decreasing oxygen affinity; Dumoulin A et al.; Based on the properties of two low oxygen affinity mutated hemoglobins (Hb), we have engineered a double mutant Hb (rHb beta YD) in which the beta F41Y substitution is associated with K82D . Functional studies have shown that the Hb alpha 2 beta 2(C7)F41Y exhibits a decreased oxygen affinity relative to Hb A, without a significantly increased autooxidation rate . The oxygen affinity of the natural mutant beta K82D (Hb Providence-Asp) is decreased due to the replacement of two positive charges by two negative ones at the main DPG-binding site . The functional properties of both single mutants are interesting in the view of obtaining an Hb-based blood substitute, which requires: (1) cooperative oxygen binding with an overall affinity near 30 mm Hg at half saturation, at 37 degrees C, and in the absence of 2,3 diphosphoglycerate (DPG), and (2) a slow rate of autooxidation in order to limit metHb formation . It was expected that the two mutations were at a sufficient distance (20 A) that their respective effects could combine to form low oxygen affinity tetramers . The double mutant does display additive effects resulting in a fourfold decrease in oxygen affinity; it can insure, in the absence of DPG, an oxygen delivery to the tissues similar to that of a red cell suspension in vivo at 37 degrees C . Nevertheless, the rate of autooxidation, 3.5-fold larger than that of Hb A, remains a problem.

Biomed Pharmacother, 1996, 50(2), 85 - 91
Suppressive effect of met-enkephalin on bone marrow cell proliferation in vitro shows circadian pattern and depends on the presence of adherent accessory cells; Krizanac-Bengez LJ et al.; The cellularity of femoral bone marrow and the content of the granulocyte-macrophage colony forming cells (GM-CFC) were followed in mice between 0600 h and 1800 h . The cellularity increased at the beginning of the light period, and the GM-CFC content at the end . Opioid pentapeptide methionine-enkephalin reduced the GM colony forming ability of the bone marrow cell suspensions in proportion to the GM-CFC content . Removal of the accessory cells reversed the enkephalin sensitivity pattern of the GM-CFC . The circadian variations have been ascribed to a neuroendocrine regulatory network involving the opioid peptides and affecting the bone marrow accessory cells . The work draws attention to the circadian activity pattern of hemoregulatory oligopeptides applicable as adjuvants to antineoplastic chemotherapy.

Free Radic Res, 1996 Jan, 24(1), 9 - 18
Semiquinone free radical formation by daunorubicin aglycone incorporated into the cellular membranes of intact Chinese hamster ovary cells; Malisza KL et al.; The production of semiquinone free radicals has been measured by electron paramagnetic resonance spectroscopy (EPR) in Chinese hamster ovary cells in which 7-hydroxy daunorubicin aglycone had been incorporated . The highly lipophilic daunorubicin aglycone was incorporated into the cellular membrane by swirling a cell suspension over a thin layer of daunorubicin aglycone . Thus, the observed semiquinone free radical was likely formed directly in the lipophilic environment of the cellular membrane . The linewidth of the observed EPR signal suggested that a neutral protonated semiquinone species was formed . In the presence of the cell-impermeant paramagnetic line broadening agent chromium(III) oxalate, no detectable signal was observed . This result indicates that even though the semiquinone is embedded in the membrane, it is still partly accessible to the external chromium(III) oxalate . Analysis of chloroform extracts of the cells after EPR experiments indicated that daunorubicin aglycone was extensively metabolized . The results of a growth inhibition assay carried out on cells into which daunorubicin aglycone had been incorporated showed almost no effect on cell growth . This result indicates that in spite of significant daunorubicin aglycone-induced radical formation taking place directly in the cell membrane, little cell damage results.

Free Radic Biol Med, 1996, 20(7), 905 - 14
Inhibition of anthracycline semiquinone formation by ICRF-187 (dexrazoxane) in cells; Malisza KL et al.; The formation of semiquinone free radicals of doxorubicin, epirubicin, daunorubicin, and idarubicin was measured by electron paramagnetic resonance (EPR) spectroscopy in hypoxic suspensions of chinese hamster ovary (CHO) cells . The amount of semiquinone produced was in the order idarubicin >> doxorubicin > daunorubicin > epirubicin . The idarubicin semiquinone signal was both the fastest to be formed and to decay . Idarubicin, which was the most lipophilic of the anthracyclines studied, also displayed the fastest fluorescence-measured cellular uptake of drug . Thus, it was concluded that semiquinone formation was dependent upon the rate of cellular uptake . Lysed cell suspensions were also shown to be capable of producing the doxorubicin semiquinone in the presence of added NADPH . The cardioprotective agent dexrazoxane (ICRF-187) was observed to decrease the amount of doxorubicin semiquinone observed in cell suspensions . Dexrazoxane also decreased the amount of doxorubicin semiquinone observed in the NADPH-lysed cell suspension mixture . Neither bipyridine nor deferoxamine decreased NADPH-dependent doxorubicin semiquinone formation . These results suggest that dexrazoxane does not decrease doxorubicin semiquinone formation through an iron complex formed from hydrolyzed dexrazoxane . Dexrazoxane may be inhibiting an NADPH-dependent enzyme.

J Photochem Photobiol B, 1996 Jan, 32(1-2), 27 - 32
Merocyanine 540 mediated photoirradiation of leukemic cells . In vitro inference on cell survival; Lydaki E et al.; In order to evaluate the selective killing of merocyanine 540 (MC 540) mediated photoirradiation in neoplastic cells, bone narrow cells from children with leukaemia or neuroblastoma and normal children as well as peripheral blood cells and Reh-6 and HL-60 cell lines were studied . Cell suspensions were incubated with MC 540 and exposed to various argon laser 514 nm doses . Cell survival was estimated with trypan blue supravital stain following a 24 h incubation and has been followed in continuous cell cultures of 4 weeks duration . Our results showed that the inhibition of survival of neoplastic haemopoietic cells by laser in the presence of MC 540 is proportional to the MC 540 and photoirradiation doses . A 99.9999% inhibition of Reh-6 and HL-60 was noted at irradiation doses where the corresponding mean survival of normal bone narrow cells was (33.6 +/- 15.5)% and (50.6 +/- 10.7)% respectively . Peripheral blood mononuclear cells were not sensitive to MC 540 mediated photoirradiation . The inhibition of survival of bone marrow metastatic neuroblastoma cells was (69.9 +/- 4.1)% . In conclusion, it seems that MC 540 mediated photoirradiation in neoplastic cells exerts selective cytotoxicity and can be used in ex vivo purging of malignant cells in the bone marrow.

Planta, 1996, 198(3), 397 - 403
Purification and characterization of glycosyltransferases involved in anthocyanin biosynthesis in cell-suspension cultures of Daucus carota L; Rose A et al.; The major anthocyanins accumulated by an Afghan cultivar of Daucus carota L . are cyanidin 3-(xylosylglucosylgalactosides) acylated with sinapic or ferulic acid . The formation of the branched triglycoside present as a common structural element requires an ordered sequence of glycosylation events . Two of these enzymic glycosylation reactions have been detected in protein preparations from carrot cell-suspension cultures . The first step is a galactosyl transfer catalyzed by UDP-galactose: cyanidin galactosyltransferase (CGT) resulting in cyanidin 3-galactoside . The putative second step is the formation of cyanidin 3-(xylosylgalactoside) catalyzed by UDP-xylose: cyanidin 3-galactoside xylosyltransferase (CGXT) . Both enzyme activities were characterized from crude protein preparations . The CGT was purified 526-fold from the cytosolic fraction of UV-irradiated cell cultures by ion-exchange chromatography on diethylaminoethyl (DEAE)-Sephacel, affinity chromatography on Blue Sepharose CL-6B, gel permeation chromatography on Sephadex G-75 and elution from the gel matrix after non-dissociating PAGE . Its molecular mass was estimated by SDS-PAGE and by calibrated gel permeation chromatography on Sephadex G-75 . In both cases a molecular mass of 52 kDa was determined, indicating that the native protein is a monomer of 52 kDa . The galactosyl transfer and the xylosyl transfer are presumed to be catalyzed by separate enzymes.

Leuk Lymphoma, 1996 Jan, 20(3-4), 291 - 5
Memory T cell subsets in B cell non-Hodgkin's lymphomas; Diaz JI et al.; Memory T cells were quantitated by three color flow cytometry in cell suspensions from biopsy specimens of 34 B cell non-Hodgkin's lymphomas (NHL) and 10 benign lymphoid hyperplasias (BLH) . CD3+CD45R0-CD45RA+ (naive), CD3+CD45R0+CDRA- (memory) and total CD3+ T cells were compared in BLH, low grade (LG), intermediate (IG) and high grade (HG) B cell NHL . Mean percentage +/- s.e . of CD45R0 + T cells for BLH, LG, IG and HG NHL were: 14.7 +/- 3.8, 11.2 +/- 3.5, 28.9 +/- 7.5 and 45 +/- 15, respectively . Mean percentage +/- s.e . of CD45RA+ T cells were: 10.2 +/- 2.6, 7.1 +/- 2.3, 5.4 +/- 1.4 and 3.5 +/- 1.2, respectively . Mean percentage +/- s.e . of total CD3+ T cells were: 42.5 +/- 11, 22.4 +/- 7.5, 39.3 +/- 10.1 and 62.7 +/- 22.2, respectively . Memory and total T cells progressively increased from LG toward IG and HG NHL while naive T cells simultaneously decreased . Although the differences in naive T cells were non-significant, the differences in memory T cells were significant between LG and IG and LG and HG NHL (both p < .0100) . We previously reported an increase in activated-cytotoxic T cells in IG -HG B cell NHL . We now report that this subset of lymphocytes has also acquired memory function . Future studies should determine if in vitro expansion and adoptive transfer of this subset of T cells results in tumor cytolysis.

Plant Mol Biol, 1996 Jan, 30(2), 351 - 8
Characterization of two class II chitinase genes from peanut and expression studies in transgenic tobacco plants; Kellmann JW et al.; Two different genes encoding class II chitinases from peanut (Arachis hypogaea L . cv . NC4), A.h.Chi2;1 and A.h.Chi2;2, have been cloned . In peanut cell suspension cultures, mRNA levels of A.h.Chi2;2 increased after ethylene or salicylate treatment and in the presence of conidia from Botrytis cinerea . The second gene, A.h.Chi2;1, was only expressed after treatment with the fungal spores . Transgenic tobacco plants containing the complete peanut A.h.Chi2;1 gene exhibited essentially the same expression pattern in leaves as observed in peanut cell cultures . Expression characteristics of transgenic tobacco carrying a promoter-GUS fusion of A.h.Chi2;1 are described.

Plant Mol Biol, 1996 Jan, 30(2), 307 - 20
Identification of a tobacco cDNA encoding a cytosolic NADP-isocitrate dehydrogenase; Galvez S et al.; A cDNA which encodes a specific member of the NADP-dependent isocitrate dehydrogenase (ICDH) multi-isoenzyme family has been isolated from a tobacco cell suspension library, and the expression pattern of ICDH transcripts examined in various plant tissues . To assign this cDNA to a specific ICDH isoenzyme, the major, cytosolic ICDH isoenzyme of tobacco leaves (ICDH1) was purified to homogeneity and its N-terminus as well as several tryptic peptides, representing 30% of the protein, were sequenced . The comparison of these amino acid sequences with the deduced protein sequence of the cDNA confirmed that this clone encodes for ICDH1 . The total ICDH specific activity and protein content were higher in vascular-enriched tobacco leaf tissue than in deveined (depleted in midrib and first-order veins) leaves . Taking advantage of antibodies raised against either ICDH1 or the chloroplastic ICDH2 isoenzyme from tobacco cell suspensions, an immuno-cytochemical approach indicated that the ICDH1 isoenzyme, located in the cytosolic compartment of tobacco leaf cells, is responsible for this expression pattern . This observation was confirmed by northern blot analyses, using a specific probe obtained from the 3' non-coding region of the ICDH1 cDNA . A comparison of ICDH protein sequences shows a large degree of similarity between eukaryotes (> 60%) but a poor homology is observed when compared to Escherichia coli ICDH (< 20%) . However, it was found that the amino acids implicated in substrate binding, deduced from the 3-dimensional structure of the E . coli NADP-ICDH, appear to be conserved in all the deduced eukaryotic ICDH proteins reported until now.

Acta Cytol, 1996 Jan-Feb, 40(1), 107 - 19
Would monolayers provide more representative samples and improved preparations for cervical screening? Overview and evaluation of systems available; McGoogan E et al.; Conventional cervical smears prepared on site by the smear taker are subject to great variation in technical quality and allow little control of the critical parameters required for optimal microscopic diagnosis . More critical, however, is that a significant proportion of the cells removed from the cervix are discarded along with the collecting device and that the material placed on the slide is not transferred in a representative way and may not fully represent the cells removed from the cervix . If the cells were placed directly into preservative fluid, all the material scraped from the cervix would be sent to the laboratory in a well-preserved state, and fully representative slides could be prepared . Several studies have suggested that this would result in an increased cell harvest with a reduction of inadequate slides and an increase in the detection of abnormal cells . Automated preparation devices are now available . Two such devices, CytoRich and ThinPrep, were recently evaluated at the University of Edinburgh Department of Pathology (UEPD) . The operational characteristics of each device were evaluated and consumables costed according to 1993-1994 prices . The consumables for the CytoRich were calculated to be more expensive, while operator time for the ThinPrep was more expensive . More mechanical problems were encountered with the ThinPrep, which was also considered more tedious to use . Cytotechnologists required considerable retraining before reaching competence in screening monolayers . They could assess them in approximately half the time required for conventional smears but found it more tiring . Almost no monolayer slides were considered unsatisfactory for laboratory interpretation due to the cells' being obscured by blood, pus or thick streaks of other cells . Scanty monolayers proved particularly difficult to read and interpret . Despite the bias introduced by using only the "leftover" material to make the monolayer, the diagnostic results of the monolayers from both devices were broadly similar to those for the matched split sample conventional smears . UEPD concluded that both devices produced monolayers that were adequate for diagnostic purposes . Neither device was ideal for routine laboratory use in cervical cytopathology in the model tested by UEPD, but both companies recently modified their devices in the light of the deficiencies identified by UEPD . The substantial loss of potentially diagnostic cells during the conventional transfer of cervical scrape material to glass slide alone merits consideration of alternative methods of preparation to obtain representative cell samples for optimal conventional microscopic diagnosis . Monolayers prepared by automated devices offer such an improvement for routine cervical cytopathology . The general deployment of such machines (involving substantial changes in the methods of working of laboratory staff responsible for tens of thousands of clinically important decisions each year) must await the completion of extensive and exhaustive laboratory and field trials . It is recommended that such trials be designed so that all the cellular material removed from the cervix is placed in the cell suspension, and the assessments should be carried out in a routine laboratory environment by cytotechnologists working in a routine cervical cytopathology service.

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