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