Hua Xi Yi Ke Da Xue Xue Bao, 1993 Jun, 24(2), 143 - 6 {The effects of salvia miltiorrhiza, polysaccharide sulphate, dextran 40 and mannitol on the viscoelasticity properties of red blood cell suspension}; Yang Y et al.; In vitro, the effects of Dextran 40(DX40), mannitol, salvia miltiorrhiza and polysaccharide Sulphate (PSS) on the viscoelasticity properties of red cell suspensions were studied . The results demonstrated that when the concentration of the drugs increased, mannitol increased eta 5 x 96, eta 51 x 2, eta', eta" and G', and DX 40 increased the values of eta' eta" and G', but it had no obvious effect on eta 5 x 96 and eta 51 x 2; salvia miltiorrhiza and PSS had no obvious effect on eta 5 x 96, eta 51 x 2, eta', eta'' and G' . However, the average values of eta', eta'' and G' of Salvia miltiorrhiza and PSS groups were lower than those of DX 40 and Mannitol groups . Clinically, these four drugs in treatment doses might improve viscoelasticity properties of whole blood . For treating the ischemic cerebral vascular diseases and hyperviscosity syndromes, Salvia miltiorrhiza and PSS could be infused faster, but DX40 and mannitol should be infused slowly. J Immunol, 1993 May 15, 150(10), 4236 - 43 Phosphatidylserine enhances the ability of epidermal Langerhans cells to induce contact hypersensitivity; Girolomoni G et al.; Phosphatidylserine (PS) modulates several immune functions in vitro, including T cell activation, antibody and cytokine production, and macrophage growth . In the present work we studied the effects of PS on the induction of contact hypersensitivity (CH) in mice . BALB/c mice painted with PS (9.4-75 mg/kg) and with a sensitizing dose of DNFB or oxazolone on the same skin site exhibited a dose-dependent augmentation of CH reactions to either DNFB (> 60%) or oxazolone (> 35%), respectively . Bovine brain PS-enriched phospholipid mixture, lyso-PS, and dipalmitoyl-PS also induced similar enhanced CH responses, whereas phosphatidylglycerols had no effect . Increased CH was observed only when PS was applied from 2 days before to 12 h after DNFB . Immunization of naive syngeneic mice with skin grafts that were treated with PS and DNFB also led to enhanced (> 50%) CH responses . In addition, immunization by iv injection of epidermal cell suspensions enriched for Langerhans cells (LC) or of purified LC that were treated with PS (1-100 microM, 30 min, 37 degrees C), and then modified in vitro with DNBS (1 mg/ml, 30 min, 37 degrees C) led to increased (> 30-75%) CH responses in recipient syngeneic animals . Finally, adoptive transfer of DNFB-immune lymph node cells obtained from mice that were treated with PS induced augmented CH responses in recipient animals . The results suggest that PS is capable of up-regulating the induction of CH in mice by stimulating the APC function of epidermal LC. J Biol Chem, 1993 May 5, 268(13), 9762 - 70 A transgenic mouse model for studying the lineage relationships and differentiation program of type II pneumocytes at various stages of lung development; Hansbrough JR et al.; A pedigree of transgenic mice has been characterized that contains a H2-Kb/LacZ fusion gene that exhibits integration site-dependent expression from the earliest stages of lung development through adulthood . Histochemical and immunocytochemical studies indicate that the LacZ reporter appears throughout the pulmonary endoderm by embryonic day 11 (E11) . A proximal-to-distal wave of extinction of transgene expression occurs during E13-14 that parallels the wave of cytodifferentiation of the pulmonary endoderm . By E16, the LacZ reporter is restricted to the distal portion of epithelial tubules and by birth to scattered cells located in alveoli . Crude epithelial cell suspensions were prepared from lungs harvested from E16 and 14 day postnatal transgenic mice, labeled with the fluorescent LacZ substrate fluorescein di-(beta-galactopyranoside), and the LacZ expressing population isolated by fluorescence-activated cell sorting . Electron microscopic, immunocytochemical and histochemical studies of this purified cell population establish that type II pneumocytes are the only cell lineage that support H2-Kb/LacZ expression in the mature postnatal lung . Fluorescence-activated cell sorting of E16 lung suspensions yielded a homogeneous population of cells that produced surfactant protein A, that could be maintained in cell culture, and that are likely precursors of adult type II pneumocytes . Together these studies indicate that (i) expression of the transgene in this pedigree of mice provides a marker for describing early differentiation of the pulmonary epithelium; (ii) the transgene may be useful as an enhancer trap to isolate cis-acting sequences that regulate gene transcription within this lineage; (iii) the LacZ reporter expression can be used to purify specific embryonic pulmonary epithelial cell populations; and (iv) primary cultures of these embryonic populations represent a potentially useful model system for analyzing the cellular components and signaling pathways necessary to support and complete passage through the type II pneumocyte differentiation program. Muscle Nerve, 1993 May, 16(5), 498 - 505 Purification of human muscle satellite cells by flow cytometry; Baroffio A et al.; To purify satellite cells directly from human muscle biopsies, we have developed a method based on size separation of dissociated cells by flow cytometry . Immediately after tryptic dissociation of human muscle biopsies and elimination of erythrocytes, microscopic observation and flow cytometry analysis of cell suspensions revealed two populations of cells differing in size and nucleocytoplasmic ratio . Clonal cultures of these two cell types with a manual procedure demonstrated that only the small cells were myogenic satellite cells . Flow cytometry-sorting and analysis of the small cell population showed that (1) all sorted cells contained desmin immediately after dissociation and plating; (2) more than 98% of the cells expressed the 5.1.H11 epitope after 2 weeks of proliferation in culture; and (3) 90% of the sorted cells were able to form myotubes when cultivated at low density or in clonal cultures . Thus, human muscle satellite cells can be directly purified from human muscle samples using flow cytometry. Plant Mol Biol, 1993 May, 22(2), 239 - 53 Stress responses in alfalfa (Medicago sativa L.) . 15 . Characterization and expression patterns of members of a subset of the chalcone synthase multigene family; Junghans H et al.; We have identified five different full length chalcone synthase (CHS) cDNA clones from a cDNA library produced from transcripts isolated from an elicitor-treated alfalfa cell suspension culture . Nucleotide sequence similarity between the clones varied from 88-93% . Oligonucleotides based on divergent sequences in the 5'-untranslated regions of the clones could distinguish individual genes, or groups of genes, and their corresponding transcripts . Developmentally regulated expression of the CHS transcripts was predominantly in roots and root nodules; other unidentified members of the CHS gene family are expressed in stems, leaves and nodules . One of the CHS transcripts was strongly expressed in floral tissue . All the CHS transcripts studied were induced in elicitor-treated cell suspension cultures . Transcripts were also induced in roots in response to wounding or spraying with various elicitors, and in leaves infected with Phoma medicaginis (but not in wounded leaves) . The induction kinetics of CHS2 transcripts were more rapid and/or transient than those of other members of the CHS family in CuCl2-treated roots and Phoma-infected leaves . The results are discussed in terms of the evolution and functions of the CHS gene family in legumes. Anat Rec, 1993 May, 236(1), 41 - 52 Cell biology of pulmonary neuroepithelial bodies--validation of an in vitro model . I . Effects of hypoxia and Ca2+ ionophore on serotonin content and exocytosis of dense core vesicles; Cutz E et al.; Pulmonary neuroendocrine (NE) cells including the innervated clusters of NE cells--neuroepithelial bodies (NEB)--are difficult to study because of their small numbers and diffuse distribution within the airway mucosa of the lung . We have previously reported a method for isolation and culture of NE cells from rabbit fetal using a combination of mechanical and enzymatic dissociation followed by gradient centrifugation . This method provides single cell suspension of mixed lung cells enriched in NE cells, particularly those originating from NEB . This study further validates our in vitro model by detailed morphologic characterization of cultured NEB cells using high resolution light microscopy, transmission and scanning electron microscopy, HPLC for detection of serotonin (5-HT), and molecular (Northern blot) analysis of mRNA encoding for 5-HT synthesizing enzymes, tryptophane hydroxylase, and aromatic L-amino acid decarboxylase . In addition the effects of hypoxia on NEB cells in vitro were investigated to define the role of these cells as possible airway chemoreceptors . Exposure of NEB cultures to hypoxia resulted in decreased intracellular content of 5-HT accompanied by increased exocytosis of dense core vesicles (DCV) . The amount of 5-HT release correlated with the degree of hypoxia, suggesting modulation by ambient pO2 levels . The role of Ca2+ ions in exocytosis of DCV and 5-HT release from NEB cells was tested in experiments with Ca2+ ionophore (A23187) . Exposure of cultures to 5 micrograms/ml of ionophore resulted in up to 40% reduction in 5-HT content of NEB cultures as well as increased exocytosis of DCV . Our overall findings are consistent with a view that NEB cells are chemosensory in nature and that Ca2+ signaling pathway is involved in stimulus-secretion coupling . Further refinements in cell separation and culture methodology are required before more detailed investigation of NEB cell membrane properties, signal transduction mechanisms, and intracellular signaling pathways can be carried out. Am Rev Respir Dis, 1993 May, 147(5), 1259 - 63 Peripolesis in alveolar sarcoidosis; Van Maarsseveen TC et al.; We observed that in bronchoalveolar lavages (BAL) of patients with active sarcoidosis (SARC) a mononuclear cell infiltrate is present that often contains clusters consisting of lymphocytes adhering to a macrophage . In order to investigate what kind of cellular interactions are involved in such a process, cell suspensions obtained from BAL of patients with SARC or extrinsic allergic alveolitis (EAA) were cultured for 1 to 2 days, during which time lapse cinematography was applied . We were able to show that such clusters consist of lymphocytes gathered around a macrophage . This is known as peripolesis . Peripolesis, as observed in our BAL, could last for some minutes or for some hours during which time a number of lymphocytes were moving around a single alveolar macrophage, without losing contact with the macrophage . Short interactions were mostly observed in EAA, whereas SARC was characterized by long periods of lymphocyte-macrophage cooperation . We also found a correlation between the time-dependent peripolesis t > 30 min/t < 30 min and the CD4/CD8 ratio . Although the precise mechanisms of peripolesis are not well understood, some interactions between lymphocytes and macrophages have now become more comprehensive. Eur J Immunol, 1993 May, 23(5), 1112 - 7 Activation of uterine intraepithelial gamma delta T cell receptor-positive lymphocytes during pregnancy; Meeusen E et al.; Intraepithelial lymphocytes (IEL) of the uterus of non-pregnant sheep were analyzed by single- and two-color flow cytometry . Very few lymphocytes carrying classical B and T cell markers (CD5, surface immunoglobulin) were detected in the uterine epithelial cell suspensions and all IEL expressed the CD8 surface marker although with varying intensities . Three distinct subpopulations were identified including a major (46-56%) population of CD8+CD45R- gamma delta T cell receptor (TcR)-negative cells and approximately equal numbers of CD8+CD45R+ gamma delta TcR- and CD8+CD45R+ gamma delta TcR+ lymphocytes . The same three subpopulations were also present in the interplacentomal areas of the uterus of ewes at a late stage of pregnancy but there was a dramatic increase (60-70%) in the gamma delta TcR+ subpopulation . In addition, a pronounced increase in both size and granularity was observed in the IEL population of pregnant uteri and this was attributed to the gamma delta TcR+ cells . Light and electron microscopic examination of these gamma delta TcR+ IEL revealed an increase in metabolic activity and the formation of exceptionally large cytoplasmic granules and confirmed their restricted localization within the uterine epithelium close to the trophoblast . These results represent for the first time, a clear example of the activation of gamma delta TcR+ cells which is not associated with an ongoing disease process or infection . gamma delta TcR+ cells have recently been observed in the epithelium of the murine reproductive tract and were characterized by their unique homogeneous receptor structure . The present results indicate that these cells may play an important physiological role during pregnancy. Am J Vet Res, 1993 May, 54(5), 808 - 12 Effects of lead on glucose metabolism, ion flux, and collagen synthesis in cerebral capillaries of calves; Ahrens FA; Brain capillary function was assessed in 4- to 6-week-old calves given lead acetate (15 mg/kg of body weight) orally for 7 to 8 days . Neurologic signs of lead poisoning included CNS depression, blindness, and hyperesthesia . Brain capillaries were isolated from cerebral cortex of control and lead-treated calves and evaluated for metabolic indicators, ion transport, and prolyl hydroxylase activity . In lead-treated calves, the rate of glucose metabolism was less than half that in controls . Ion efflux of 45Ca or 36Cl from endothelial cell suspensions was not affected by lead treatment . Prolyl hydroxylase activity in endothelium and proline-to-hydroxyproline ratio in endothelial basement membranes were similar in control and lead-poisoned calves . Results indicate that lead may inhibit energy metabolism, but not ion transport or collagen biosynthesis in brain capillaries of calves and, compared with suckling rats, damage to the blood-brain barrier is less important . In calves, neuronal tissue may be the primary target for the CNS effects of lead. Anat Rec, 1993 May, 236(1), 35 - 40 An overview of culture and isolation methods suitable for in vitro studies on pulmonary neuroendocrine cells; Speirs V et al.; Successful isolation and culture of pulmonary neuroendocrine cells (PNEC) is essential for the investigation of cellular and membrane properties of these cells . Such studies are important to define the functional role for PNEC but are hampered by their scant numbers and widespread distribution within the pulmonary epithelium . Several in vitro methods for the isolation and culture of these cells have been described over the past decade, including organ culture, isolation of single cell suspensions enriched for PNEC, and immunomagnetic cell separation techniques . This paper reviews the various methods and discusses their advantages and pitfalls. Dev Biol, 1993 May, 157(1), 277 - 80 Proliferation of mouse primordial germ cells in vitro: a key role for cAMP; De Felici M et al.; Two agents known to enhance the level of intracellular cAMP (dibutyryl cAMP and forskolin) markedly increase the number of 8.5, 10.5, and 11.5 days postcoitum (dpc) mouse primordial germ cells (PGCs) cultured on TM4 cell feeder layers . Forskolin (FRSK) caused a significant increase of PGC number also in monodispersed cell suspensions obtained from PGC-containing tissues of the three embryonic ages studied and in purified 11.5 dpc PGCs cultured without feeder layers . The addition to the culture medium of adenosine-3',5'-cyclic monophosphorothioate RP isomer (Rp-cAMPS, a competitive antagonist for cAMP-dependent protein kinases), significantly reduced the effects of FRSK . Last, FRSK stimulated PGC proliferation, as assessed by 5-bromo-2'-deoxyuridine incorporation . We conclude that cAMP-dependent mechanisms play a crucial role in the control of mitotic proliferation of mouse PGCs in culture. Mod Pathol, 1993 May, 6(3), 270 - 5 Image analysis versus flow cytometry for DNA ploidy quantitation of solid tumors: a comparison of six methods of sample preparation; Danque PO et al.; With the availability of user-friendly interactive image analysis instruments for DNA analysis, there is a growing need for comparison with the established methodology of flow cytometry . We have compared the results of DNA ploidy quantitation in 12 solid tumors prepared by six different techniques of sample preparation: flow cytometry of fresh cell suspensions and of nuclei isolated from formalin-fixed, paraffin-embedded tissue; and image analysis of touch preparations, of disaggregated cells from paraffin-embedded tissue as well as of 3- and 7-microns-thick tissue sections . Complete agreement in DNA ploidy results obtained by the six methods was found in six out of 12 solid tumors . Image analysis of touch preparations detected most tetraploid and multiple aneuploid peaks . Sections of 7-microns-thick tissue gave better histogram quality than 3-microns-thick sections, however tetraploid peaks were not resolved in one case . Image analysis of disaggregated paraffin-embedded tumor showed comparable ploidy to fresh touch preparations in seven out of 12 cases, the discrepancies being due to loss of tetraploid or multiple aneuploid peaks . Flow cytometry gave good histograms, but tetraploid and multiple aneuploid peaks were occasionally not detected . Each method presents advantages and disadvantages . Flow cytometry and image analysis are complementary methods for DNA quantitation, and more than one method may be necessary to confirm the DNA content of solid tumors. J Appl Toxicol, 1993 May-Jun, 13(3), 161 - 8 Early cytotoxic effects induced by bis-chloroethyl sulphide (sulphur mustard): {Ca2+}i rise and time-dependent inhibition of B77 fibroblast serum response; Hua A et al.; Early cytotoxic events were studied on B77 fibroblasts . Cells were treated with sulphur mustard (SM) in short-term experiments in which cell viability was unchanged, as evaluated by the neutral red cytotoxicity test . This treatment was correlated to two early signs of cytotoxicity . The intracellular Ca2+ concentration {Ca2+}i level in SM-treated Fura-2-loaded fibroblasts showed a significant dose-dependent increase . This observed rise was sustained, in contrast to the Ca2+ signal induced by serum, and was already visible 5-10 min after the addition of SM to cell suspensions in vitro . Modification of the extracellular Ca2+ concentration in the medium had no effect on the cytosolic calcium rise caused by SM, suggesting release from intracellular Ca2+ pools . Furthermore, a time-dependent inhibition of the {Ca2+}i transient increase induced by growth-factors (as evaluated by the fetal calf serum (FCS) response) was observed within the first hour of exposure . These latter results suggest that early alterations of calcium distribution induced by SM could be one of the earliest markers of SM intoxication. Zhonghua Zhong Liu Za Zhi, 1993 May, 15(3), 195 - 7 {Establishment of a model of human gastric cancer with liver metastasis in nude mice}; Yang SM; An animal model of liver metastasis was established by injection of suspending tumor cells into subcapsule of spleen in nude mice . With additional mechanic disaggregation, single-tumor cell suspension was prepared by enzymatic disaggregation, including trypsin-collagenase disaggregate medium treatment, from the subcutaneous MGC 80-3 xeno-transplanted tumor and filtered through filter holders with different pore size . To investigate the effect of liver damage by carbon tetrachloride (CCL4) on the metastatic potential of MGC 80-3 cells injected intrasplenically, the nude mice were divided into 2 groups: Group 1 received intrasplenic injection of 1.25 x 10(6) MGC 80-3 cells without CCL4 treatment; Group 2 received intrasplenic injection of equal cells with CCL4 treatment . The incidence of liver metastasis was 60% in Group 1, but 100% in Group 2 . The mean numbers of metastatic nodules per liver were 1.80 in Group 1, but 4.85 in Group 2 . It is obvious that the incidence and nodules of liver metastasis were increased in the mice treated with CCL4 . This is a useful model to study antimetastatic agents and metastatic behaviors. Thymus, 1993 May, 21(3), 141 - 57 Involvement of IL-7 in the development of gamma delta T cells in the thymus; Tomana M et al.; The effect of IL-7 on the growth of thymic T lymphocytes was investigated by adding recombinant IL-7 into cell suspension cultures and submersion organ cultures (SOC) of murine fetal thymuses (FT) and newborn thymuses (NBT) . FT and NBT were obtained from C57BL/6 mice at day 15 of gestational age and at day 3 after birth, respectively . In both cell suspension cultures and SOC, addition of IL-7 highly improved the cell recovery . In cell suspension cultures, addition of IL-7 resulted in the growth of gamma delta T-cells from FT-cells, whereas the same cytokine promoted the growth of both alpha beta and gamma delta T-cells from NBT-cells . These results may indicate that this cytokine is able to support the proliferation of T-cells of both alpha beta and gamma delta lineages . In marked contrast, in SOC, addition of IL-7 resulted in the growth of gamma delta T-cells not only in FT but also in NBT, despite the fact that the SOC of NBT without exogenous cytokine exclusively promoted the growth of alpha beta T-cells . A similar effect was also seen when IL-2 was added to NBT-SOC, though the skewing to gamma delta lineage was not so strong as in the case of IL-7 . In addition, we found that IL-7 mRNA is expressed in the day 15 FT at a much higher level than in the adult thymus . These results strongly suggested that the production of a large amount of IL-7 synthesized in the FT is one of the major factors leading to the generation of gamma delta T-cells in FT. Chem Res Toxicol, 1993 May-Jun, 6(3), 252 - 60 Cytotoxicity of polycyclic aromatic hydrocarbon o-quinones in rat and human hepatoma cells; Flowers-Geary L et al.; A novel pathway of polycyclic aromatic hydrocarbon (PAH) metabolism involves the oxidation of non-K-region trans-dihydrodiols by dihydrodiol dehydrogenase (DD) to yield PAH o-quinones whose cytotoxicity and genotoxicity are unknown . The cytotoxicity of several PAH o-quinones derived from this reaction {naphthalene-1,2-dione (NPQ), benzo{a}pyrene-7,8-dione (BPQ), and 7,12-dimethylbenz{a}anthracene-3,4-dione (DMBAQ)} was examined in rat (H-4IIe) and human (Hep-G2) hepatoma cells which are known to express DD . 2-Methylnaphthalene-1,4-dione (menadione), a known cytotoxic p-quinone, was used as a positive control . Hepatoma cells (1 x 10(6) cells/mL) were exposed to PAH o-quinones (1-100 microM) for 0-4 h, and cell viability and survival were measured and related to O2.- production and changes in redox potential {GSSG/GSH and NAD(P)+/NAD(P)H} . Three different modes of cytotoxicity were observed: (1) NPQ (no bay region) and DMBAQ (methylated bay region) were as cytotoxic as menadione in reducing cell survival but had less effect on cell viability . These o-quinones adversely affected GSH levels and the redox state of the cell and caused an increase in the production of O2.- in cell suspensions . This cytotoxicity was not enhanced by dicoumarol (10 microM), a DT-diaphorase inhibitor, implying that this enzyme is unable to prevent these PAH o-quinones from entering one-electron redox-cycles . (2) BPQ (bay region only) was the least cytotoxic of the PAH o-quinones studied . BPQ decreased cell viability (< 40% at 20 microM) but did not adversely affect cell survival or the redox state of the cell.(ABSTRACT TRUNCATED AT 250 WORDS) In Vitro Cell Dev Biol Anim, 1993 May, 29A(5), 379 - 87 Serial culturing of human bronchial epithelial cells derived from biopsies; de Jong PM et al.; In the present study we describe the establishment of serial cultures of human bronchial epithelial cells derived from biopsies obtained by fiberoptic bronchoscopy . The cell cultures were initiated from small amounts of material (2 mm forceps biopsies) using either explants or epithelial cell suspensions in combination with a feeder-layer technique . The rate of cell proliferation and the number of passages (up to 8 passages) achieved were similar, irrespective of whether the explants or dissociated cells were used . To modulate the extent of differentiation, the bronchial epithelial cells were cultured either under submerged, low calcium (0.06 mM) (proliferating), normal calcium (1.6 mM) (differentiation enhancing) conditions, or at the air-liquid interface . Characterization of the bronchial epithelial cell cultures was assessed on the basis of cell morphology, cytokeratin expression, and ciliary activity . The cells cultured under submerged conditions formed a multilayer consisting of maximally three layers of polygonal-shaped, small cuboidal cells, an appearance resembling the basal cells in vivo . In the air-exposed cultures, the formed multilayer consisted of three to six layers exhibiting squamous metaplasia . The cytokeratin profile in cultured bronchial epithelial cells was similar in submerged and air-exposed cultures and comparable with the profile found in vivo . In addition to cytokeratins, vimentin was co-expressed in a fraction of the subcultured cells . The ciliary activity was observed in primary culture, irrespective of whether the culture had been established from explants or from dissociated cells . This activity was lost upon subculturing and it was not regained by prolongation of the culture period . In contrast to submerged cultures and despite the squamous metaplasia appearance, the cells showed a reappearance of cilia when cultured at the air-liquid interface . Human bronchial epithelial cell cultures can be a representative model for controlling the mechanisms of regulation of bronchial epithelial cell function. Cancer, 1993 May 1, 71(9), 2732 - 8 Chronic lymphocytic leukemia with low lymphocyte count; Batata A et al.; BACKGROUND . A peripheral blood absolute lymphocyte count (ALC) of greater than 5 x 10(9)/l is considered the required minimum for diagnosis of chronic lymphocytic leukemia (CLL) . Cases with low ALC (CLL-LLC), less than 5 x 10(9)/l, have not been included in the current staging systems, and would not be suspected of having CLL, or investigated for the disease, especially in the absence of clinical manifestations . On the other hand, the diagnostic value of the differential lymphocyte counts have not been emphasized . METHODS . Cell suspensions from peripheral blood of previously untreated cases of CLL-LLC (n = 12) and typical CLL (n = 189) were analyzed for immunologic evaluation of surface immunoglobulin (SIg), mouse erythrocyte rosettes, CD5, CD19, CD20, CD22, and CD2, as well as cytochemical evaluation of tartrate-resistant acid phosphatase (TRAP) . The results in CLL-LLC were compared statistically with typical CLL . RESULTS . The ages of the 12 patients with CLL-LLC ranged from 47 to 84 years . The absolute lymphocyte counts ranged from 1.5 x 10(9)/l to 4.9 x 10(9)/l, and the percentage of lymphocytes in the differential leukocyte counts ranged from 52% to 93% . None of the patients had signs and symptoms of CLL or other conditions that may cause reactive lymphocytosis . CLL-LLC demonstrated similar characteristics to typical CLL, i.e., weak expression of monoclonal SIg, mouse rosette formation, positive CD5, high CD19 and CD20, negative CD22 and TRAP . No statistical differences existed between the immunologic markers or between SIg isotype distribution in the two groups . CONCLUSIONS . Cases of CLL-LLC constituted 6% of B-CLL and would have been missed if immunologic investigation was not carried out because of the absence of absolute lymphocytosis and clinical manifestations of CLL . Persistent relative lymphocytosis of > or = 50% of the differential leukocyte count in older individuals (older than 50 years of age), is an indication for further investigation of CLL by immunophenotyping of peripheral blood lymphocytes and examination of bone marrow. Thromb Haemost, 1993 Apr 1, 69(4), 335 - 8 Different expression of procoagulant activity in human cancer cells cultured "in vitro" or in cells isolated from human tumor tissues; Zucchella M et al.; We studied in a homologous system the procoagulant activity of human tumor cells cultured "in vitro" (1402 primary melanoma, Me 7110/2 metastatic melanoma, Hep G2 hepatoma and GLC1 small cell lung carcinoma) or of cells freshly isolated from different human tumor tissues . Tumor cells cultured "in vitro" possessed and released a factor VII dependent procoagulant activity, which was inhibited by concanavalin A and unaffected by iodoacetamide or HgCl2 . The activity released by the cells of metastatic melanoma was higher than that released by the cells of the primary tumor . On the contrary, cancer cells isolated from tumor tissues possessed and released a factor VII independent activity which was inhibited by iodoacetamide of HgCl2 and was not modified by concanavalin A . Therefore, different methods for the preparation of tumor cell suspensions have to be used for the study of tumor procoagulants, since their expression depends very largely on the source of tumor cells . Furthermore, cultured human tumor cells are not an appropriate model for the "in vivo" procoagulant effect of tumor cells. Immunology, 1993 Apr, 78(4), 526 - 33 Induction and suppression of cytokine release (tumour necrosis factor-alpha; interleukin-6, interleukin-1 beta) by Escherichia coli pathogenicity factors (adhesions, alpha-haemolysin); Konig B et al.; Escherichia coli bacteria expressing mannose-resistant fimbriae/haemagglutination induced the production of substantial amounts of tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) from a peripheral human lymphocyte, monocyte, basophil (LMB) cell suspension . In this regard, E . coli bacteria with S-mannose-resistant fimbriae/haemagglutination were the most potent inducers of IL-6 and TNF-alpha secretion, followed by the E . coli strain with P-mannose-resistant fimbriae/haemagglutination . The E . coli alpha-haemolysin did not stimulate cytokine release from human LMB . In fact, this toxin, at non-toxic concentrations, depressed the spontaneous as well as the E . coli-induced production of TNF-alpha, IL-6, IL-1 beta . Our results indicate that two mechanisms may contribute to the severity of E . coli infection: (a) stimulation of cytokine release by type-specific fimbriae/haemagglutination properties and (b) depression of immune response by the E . coli alpha-haemolysin. Appl Environ Microbiol, 1993 Apr, 59(4), 1231 - 5 Use of autoradiography to assess viability of Helicobacter pylori in water; Shahamat M et al.; Autoradiographic methods have been developed to detect metabolic activity of viable but nonculturable cells of Helicobacter pylori in water . Four strains of H . pylori were studied by using microcosms containing suspensions of 72-h cultures in water . The suspensions of aged, nonculturable cells of H . pylori were incubated with {3H}thymidine for 24 to 72 h, after which the cell suspensions were exposed to Kodak NTB2 emulsion for 3 to 28 days . Each sample was processed with three separate controls to rule out false-positive reactions . The organism remains viable and culturable under these conditions for up to 48 h and, in some cases, 20 to 30 days, depending on physical conditions of the environment . We found that temperature was a significant (P < or equal to 0.01) environmental factor associated with the viability of H . pylori cells in water . Autoradiographs of tritium-labeled cells of H . pylori revealed aggregations of silver grains associated with uptake by H . pylori of radiolabelled substrate . Findings based on the autoradiographic approach give strong evidence supporting the hypothesis that there is a waterborne route of infection for H . pylori . The possibility that H . pylori may persist in water in a metabolically active stage but not actively growing and dividing is intriguing and relevant to public health concerns. Phys Med Biol, 1993 Apr, 38(4), 511 - 20 Time-domain dielectric spectroscopy applied to cell suspensions; Bone S et al.; A precision difference time-domain reflectometry (TDR) technique is described for the investigation of the dielectric properties of cell suspensions . The dielectric spectra obtained for erythrocytes using TDR are comparable with those reported in previous dielectric studies employing frequency-domain technique. Burns, 1993 Apr, 19(2), 99 - 104 Improvement of human keratinocyte isolation and culture using thermolysin; Germain L et al.; We propose a modification of the conventional keratinocyte isolation method which has shown a significant improvement in the purity, colony forming efficiency (c.f.e.) and growth capacity of the isolated epidermal cell population . This method utilized thermolysin since it selectively digests the dermo-epidermal junction . Following separation from the dermis, the epidermis was digested with trypsin to obtain a single cell suspension . Compared with the conventional procedure, this isolation method was shorter and resulted in (i) cells displaying a higher colony forming efficiency, (ii) cells reaching confluence 1-3 days earlier, (iii) cells not contaminated by fibroblasts, (iv) a cell population containing all the basal layer keratinocytes . These cells were suitable for the establishment of primary cultures and could be subcultured . Such cell populations should be advantageous in studies of epithelial-mesenchymal interactions in which keratinocyte populations, free of fibroblasts, are desirable . In the treatment of extensively burned patients using cultured epidermal sheets, the main problem remains the time required for their production . Thus, the absence of fibroblast overgrowth of the keratinocyte cultures and the significantly reduced time to obtain confluent cultures and epidermal sheets with our method have very important implications for the treatment of large burn wounds. J Cell Physiol, 1993 Apr, 155(1), 185 - 96 Heparin-like glycosaminoglycans participate in binding of a human trophoblastic cell line (JAR) to a human uterine epithelial cell line (RL95); Rohde LH et al.; In vitro studies in our laboratory have indicated that heparan sulfate proteoglycans (HSPGs) play an important role in murine embryo implantation . In order to investigate the potential function of HSPGs in human implantation, two human cell lines (RL95 and JAR) were used to model uterine epithelium and embryonal trophectoderm, respectively . A heterologous cell-cell adhesion assay was developed to determine if binding of JAR cells to RL95 cells was heparan sulfate-dependent . Labeled, single cell suspensions of JAR cells attached to confluent monolayers of RL95 cells in a dose- and time-dependent manner . Heparin-like glycosaminoglycans and JAR cell proteoglycans competitively inhibited JAR cell adhesion to RL95 cells by 50% or more . A panel of chemically modified heparins were used to demonstrate that O-sulfation and amino group substitution were critical for inhibition of cell-cell adhesion . Treatment with chlorate, an inhibitor of ATP-sulfurylase, resulted in a 56% reduction in cell-cell binding compared to untreated controls . Heparinase and chondroitinase ABC markedly inhibited JAR-RL95 binding, while chondroitinase AC had no significant effect . These observations indicated that HSPGs as well as dermatan sulfate-containing proteoglycans participated in cell-cell binding . Collectively, these results indicate that initial binding interactions between JAR and RL95 cells is mediated by cell surface glycosaminoglycans (GAGs) with heparin-like properties (i.e., heparan sulfate and dermatan sulfate) . These observations are consistent with an important role for HS and heparin-like GAGs as well as their corresponding binding sites in early stages of human trophoblast-uterine epithelial cell binding. Magn Reson Med, 1993 Apr, 29(4), 571 - 4 Diffusion in red blood cell suspensions: separation of the intracellular and extracellular NMR sodium signal; van der Veen JW et al.; It is shown that the signal of intracellular and extracellular sodium of red blood cells can be separated by a difference in diffusion . Comparison with proton diffusion experiments conducted in parallel showed that this difference was caused by restriction to the cell volume . The measured proton and sodium root mean square displacements agreed well with the cell dimensions . However, this experiment is limited to use in vitro by the required gradient strength. Magn Reson Med, 1993 Apr, 29(4), 563 - 6 A perifusion loop-gap resonator NMR probe for aerobic cell suspensions; Anderson ME et al.; NMR studies of aerobic cell metabolism require that spectral data of high sensitivity and resolution be acquired from samples maintained under suitable conditions . The loop-gap resonator 31P NMR probe described here was designed for investigations of highly aerobic-dependent renal proximal tubules in an environment similar to those of conventional incubation flasks . The inherently higher sensitivity of the loop-gap resonator design and associated circuitry made it possible to obtain adequate spectra in short periods of time with small amounts of sample . Cellular physiological properties of perifused aerobic renal proximal tubule cell suspensions in the loop-gap resonator probe were found to be similar to equivalent tissue samples incubated in conventional flasks . Furthermore, the loop-gap perifusion probe was found to provide useful 31P NMR spectra of the kidney tubule system after acquisition times as short as 3 min. Membr Biochem, 1993 Apr-Jun, 10(2), 107 - 18 Duramycin increases intracellular calcium in airway epithelium; Cloutier MM et al.; Duramycin increases short-circuit current (Isc) and net Cl- secretion in tracheal epithelium . We measured the intracellular free calcium ({Ca2+}i) response to duramycin using Indo-1 and bovine and canine tracheal cell suspensions, and the effect of an intracellular calcium chelator, BAPTA, and the protein kinase C inhibitor, staurosporine, on the Isc and {Ca2+}i response to duramycin . {Ca2+}i increased in a dose-dependent manner from basal levels of 34 +/- 5 to 949 +/- 136 nM at 5 x 10(-6) M duramycin . Both BAPTA (50 microM) and staurosporine (5-50 nM) pretreatment blunted the increase in Isc and net Cl- secretion produced by duramycin . BAPTA also blunted the rise in {Ca2+}i produced by duramycin (5 x 10(-6) M) in the presence of extracellular calcium (499 +/- 122 nM) . In the absence of extracellular calcium, the duramycin-induced (5 x 10(-6) M) rise in {Ca2+}i was blunted from 949 +/- 136 nM (stimulation in the presence of Ca2+) to 621 +/- 122 nM, and was further decreased in the presence of BAPTA to 197 +/- 42 nM . In contrast, staurosporine (50 nM) pretreatment had no effect on the rise in {Ca2+}i produced by duramycin (basal 90 +/- 27 to 861 +/- 110 nM at 5 x 10(-6) M) . Duramycin had no effect on {Ca2+}i in human neutrophils . These data demonstrate that duramycin releases calcium from intracellular stores and stimulates the influx of calcium in airway epithelial cells . These data also demonstrate that, in the presence of protein kinase C pathway blockade, an increase in intracellular free calcium is not sufficient for chloride secretion; thus, duramycin-stimulated chloride secretion may depend upon protein kinase C. Sheng Li Xue Bao, 1993 Apr, 45(2), 103 - 10 {A study of transpial migration of implanted serotonergic neurons in rat spinal cord}; Chen ZF et al.; Transpial migration of implanted 5-HT neurons from the subarachnoid space into the spinal cord was studied in adult Wistar rats . Embryonic raphe tissue or cell suspension containing 5-HT cells was used as grafts . The implanted 5-HT cells were monitored by 5-HT immunohistochemical method . The results are as follows: (1) 10 d after cutting the spinal cord at lower thoracic level, 5-HT fibers disappeared in the transected spinal cord . (2) Raphe tissue was implanted into the subarachnoid space of the thoracic lumbar segment after the spinal cord was cut . One month later, 5-HT positive cells could be found in the transected spinal cord with fibers extending into both the grey and the white matters . (3) If the raphe cell suspension instead was implanted, a number of 5-HT positive cells appeared in the grey matter near the implanted region and the distribution of these cells in the grey matter was quite consistent with the implanted range of the cell suspension in the subarachnoid space . The 5-HT positive cells which had entered into the spinal cord sent out fibers and reestablished a new fiber network in the grey matter . (4) After implantation, the number of the 5-HT positive fibers in the transected grey matter became more and more sparsely distributed with increasing distance from the cell bodies and the 5-HT positive fibers reappeared in the white matter were much less than that in the grey matter . Present results show that the implanted 5-HT neurons are able to migrate transpially from the subarachnoid space into the spinal cord. Pharmacol Res, 1993 Apr, 27(3), 241 - 52 Accumulation of salicylic acid and indomethacin in isolated proximal tubular cells of the rat kidney; Cox PG et al.; The handling and accumulation of salicylic acid (SA) and indomethacin was examined in freshly isolated proximal tubular (PT) cells of the rat kidney in order to determine whether these cells provide a useful tool for studying accumulation of nonsteroidal anti-inflammatory drugs . A PT cell suspension was prepared by collagenase digestion, followed by filtration and isopycnic centrifugation . SA uptake was concentration-dependent and could be inhibited by probenecid . SA accumulated in the PT cells, and therefore, uptake is probably an active process . In the presence of probenecid, no SA accumulation was observed . Indomethacin uptake increased with time up to 2 min . Thereafter, a sharp decrease occurred, probably caused by inhibition of the oxidative phosphorylation . In the presence of probenecid, uptake was significantly reduced and no longer time-dependent . Indomethacin accumulated in the PT cells by a factor of more than 25 . In the presence of probenecid, accumulation was decreased but was still considerable (approximately 10), probably as a result of binding to cellular protein . We conclude that as a result of carrier-mediated transport which is probenecid-sensitive, SA and indomethacin accumulate in the PT cells . The observed accumulation values are in accordance with previously observed values in the isolated perfused rat kidney . Therefore, freshly isolated PT cells appear to be a simple and useful model for studying the accumulation process of drugs like SA and indomethacin. Plant Physiol, 1993 Apr, 101(4), 1275 - 82 cDNA sequence, expression, and transcript stability of a cold acclimation-specific gene, cas18, of alfalfa (Medicago falcata) cells; Wolfraim LA et al.; The nucleotide sequence of a full-length cDNA, the deduced amino acid sequence, and the regulation of expression of a cold acclimation-specific gene, cas18, in cell suspension cultures of a freezing-tolerant cultivar of alfalfa (Medicago falcata cv Anik) have been determined . The deduced polypeptide, CAS18, is relatively small (17.6 kD), is highly hydrophilic, is rich in glycine and threonine, and contains two distinctive repeat elements . It exhibits homology with members of the LEA/RAB/dehydrin family of proteins, which accumulate in response to abscisic acid (ABA) or water stress . It is intriguing that cas18 is induced by neither ABA nor water stress . The cas18 cDNA hybridizes to three transcripts of 1.6, 1.4, and 1.0 kb, and the cDNA characterized here corresponds to the 1.0-kb transcript . The expression of this gene is about 30-fold greater in cold-acclimated cells than in nonacclimated cells . Although the accumulation of transcripts during cold acclimation is relatively slow, their disappearance during deacclimation is dramatically rapid, becoming undetectable in less than 5 h . Studies of nuclear run-on transcription show that cold acclimation enhances the transcription of this gene nearly 9-fold . The stability of cas18-detectable transcripts during deacclimation is considerably increased if transcription is inhibited with cordycepin . It therefore appears that low temperature regulates the expression of cas18 at both the transcriptional and posttranscriptional levels. Eur J Neurosci, 1993 Apr 1, 5(4), 299 - 310 Localization of janusin mRNA in the central nervous system of the developing and adult mouse; Wintergerst ES et al.; Janusin (formerly termed J1-160/180) is an oligodendrocyte-derived extracellular matrix molecule which is restricted to the central nervous system and which is expressed late during development (Pesheva et al., J . Cell Biol., 1765-1778, 1989) . To gain insights into the molecule's morphogenetic functions and to identify its cellular source in vivo, we have studied the localization of janusin messenger RNA in the optic nerve, retina and spinal cord and the expression of janusin protein in the spinal cord of developing and adult mice . Moreover, we have analysed optic nerve cell cultures and retinal cell suspensions in double-labelling experiments using a janusin-specific anti-sense complementary RNA probe and cell type-specific antibodies to identify the cell types containing janusin transcripts . In developing animals, oligodendrocytes were strongly labelled with the janusin anti-sense cRNA probe during the period of myelination . The number of labelled cells and intensity of the hybridization signal decreased significantly with increasing age . Interestingly, expression of janusin was not confined to oligodendrocytes . Some neuronal cell types and type-2 astrocytes present in optic nerve cell cultures also contained janusin transcripts . In contrast to oligodendrocytes, the number and labelling intensity of neurons containing janusin transcripts remained constant during postnatal development and into adulthood . Expression of janusin protein in the spinal cord was developmentally regulated, with a peak of expression in 2- or 3-week-old animals . The molecule was visible in the white and grey matter . In myelinated regions, it was associated with myelinated fibres and accumulated at nodes of Ranvier . These observations suggest that janusin may be of functional relevance for myelination. Chest, 1993 Apr, 103(4), 1051 - 8 Antibodies to collagen in patients with idiopathic pulmonary fibrosis; Nakos G et al.; STUDY OBJECTIVE: The pathogenesis of idiopathic pulmonary fibrosis (IPF) is uncertain . This investigation was undertaken to determine if antibodies to human native collagens and their chains are present in the serum of the patients with IPF and to examine their relationship with clinical factors . MATERIALS: Serum specimens were obtained from 45 subjects . The subjects were separated into three distinct groups: group 1 consisted of 16 patients with IPF; group 2a, 9 patients with pulmonary fibrotic scars from previous tuberculosis were examined as a control group; group 2b, 20 normal individuals matched by age and sex . The collagen antigens used in this study consisted of four genetically distinct types, I, II, III, IV and their corresponding chains alpha 1(I) + alpha 2(I), alpha 1(II), and alpha 1(III) . METHODS: a passive microhemagglutination assay was used to test the antibody activity in the sera of both patients and controls . Titration was performed in microtiter plastic plates, using 0.5 percent cell suspension . The specificity of anticollagen antibodies was then tested using an absorption serum technique with native collagens and their chains . Hemagglutination enhancement and inhibition tests, as well as chromatography, were used to determine the type of antibodies . RESULTS: Thirteen of 16 (81 percent) patients with IPF (group 1) had antibodies against at least one type of native collagen or one type of collagen chain in titers up to 1:512 . Twelve patients exhibited anticollagen antibodies with titers above 1:16 . By contrast, only 2 of 9 (22 percent) subjects with fibrotic scars (group 2a) and 3 of 20 (15 percent) normal subjects (group 2b) had antibody activity in titers up to 1:8 . The differences between group 1 and group 2a and 2b were statistically significant, (p < 0.05 and p < 0.001, respectively) . There was an inverse, statistically significant correlation between duration of the disease (IPF) and antibody activity . The correlation coefficient between duration of the disease and titers of antibodies to type III collagen (native and/or chain) was higher than the correlation coefficient between duration of the disease and titers to collagen I . The enhancement and inhibition of agglutination tests, as well as the chromatography, showed that the agglutination factors were antibodies of IgG and IgM classes . The antibody absorption test revealed that the anticollagen antibodies were specific for each collagen and its chain and there was no cross-reaction . CONCLUSION: This study suggests that the anticollagen antibodies could be a marker of IPF activity and may perpetuate the lung tissue inflammation . It is still unclear if the autoimmunity of the collagen participates in the pathogenesis of IPF or the presence of the anticollagen antibodies is simply an epiphenomenon. J Neural Transplant Plast, 1993 Apr-Jun, 4(2), 147 - 55 Reconstruction of GABAergic transmission and behavior by striatal cell grafts in rats with ischemic infarcts in the middle cerebral artery; Nishino H et al.; Fetal striatal cell suspensions were grafted stereotaxically into the infarcted striatum of rats, and reconstruction of striatopallidal GABA transmission and behavior were investigated . Occlusion of the middle cerebral artery (MCA) for one hour induced ischemic infarcts mainly in the lateral striatum, as detected by magnetic resonance imaging (MRI) and histology . Ischemic rats had deficits in the performance of a passive avoidance task, both acquisition and retention, but no changes in general circadian actograms . In these animals pallidal GABA, detected by microdialysis, decreased to about half of control levels . There were suggestions of an improvement in passive avoidance performance in the grafted animals . Pallidal GABA concentrations recovered almost to control levels, and were increased by infusions of the GABA uptake blocker nipecotic acid . These data indicate that neural transplantation is a promising approach to improve the deficits in chemical transmission and behavior following ischemic infarcts in rat striatum. Mem Inst Oswaldo Cruz, 1993 Apr-Jun, 88(2), 235 - 41 Improved enzyme-linked immunoadsorbent assay (ELISA) for the study of Trypanosoma cruzi-host cell interaction in vitro; Luz MR et al.; We herein present an improved assay for detecting the presence of Trypanosoma cruzi in infected cultures . Using chagasic human sera (CHS), we were able to detect T . cruzi infection in primary cultures of both peritoneal macrophages and heart muscle cells (MHC) . To avoid elevated background levels--hitherto observed in all experiments especially in those using HMC--CHS were preincubated with uninfected cells in monolayers or suspensions prior to being used for detection of T . cruzi in infected monolayers . Preincubation with cell suspensions gave better results than with monolayers, reducing background by up to three times and increasing sensitivity by to twenty times . In addition, the continuous fibroblastic cell line L929 was shown to be suitable for preadsorption of CHS . These results indicate that the high background levels observed in previous reports may be due to the presence of human autoantibodies that recognize surface and/or extracellular matrix components in cell monolayers . We therefore propose a modified procedure that increases the performance of the ELISA method, making it an useful tool even in cultures that would otherwise be expected to present low levels of infection or high levels of background. Jpn J Cancer Res, 1993 Apr, 84(4), 445 - 50 Enhancement of cytosine arabinoside cytotoxicity by granulocyte/macrophage colony-stimulating factor and granulocyte colony-stimulating factor in a human myeloblastic leukemia cell line; Takauji R et al.; Enhancement of the cytotoxicity of cytosine arabinoside (ara-C) by granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), and the mechanisms involved, were studied in the AML-193 human leukemia cell line . AML-193 cells require GM-CSF and G-CSF(CSFs) for optimum growth, and 24 h deprivation of CSFs decreased DNA synthesis measured in terms of 3H-thymidine incorporation . The DNA synthesis gradually recovered upon addition of CSFs . To examine the sensitivity to ara-C under different growth conditions, two groups of cell suspensions, one pretreated with CSFs after 24 h deprivation (CSFs(+) cells), and the other held continuously under CSFs-free conditions (CSFs(-) cells), were exposed to 1.0 microgram/ml of ara-C for 16 h . In clonogenic assays, CSFs(+) cells showed higher sensitivity to ara-C than CSFs(-) cells . These cell groups showed no significant difference in ara-C triphosphate accumulation or retention, though the amount of ara-C incorporated into the acid-insoluble fraction was two times greater in CSFs(+) cells than CSFs(-) cells, and that difference became even clearer in the retention pools . These data suggest that the enhancement of cytotoxicity by CSFs was due to the promotion of ara-C incorporation into DNA as a result of an increase of the cell fraction in the S phase. Exp Mol Pathol, 1993 Apr, 58(2), 105 - 13 Application of the disc angiogenesis system to tumor-induced neovascularization; Nelson MJ et al.; Solid tumors elaborate soluble substances that (directly or indirectly) induce angiogenesis by a step-wise process which ultimately results in a microvascular network that nourishes the growing tumor . To study angiogenesis induced by brain tumors we have used a rat glioma model . Modifying the disc angiogenesis system (DAS) we evaluated quantitatively the angiogenic response to cultured, live RT-2 rat glioma cells placed in the center of the discs . DAS were implanted in the subcutaneous tissue of rats and evaluated for vessel proliferation 2 weeks later . Recognition of vessels was greatly facilitated by the staining of their basement membrane using a polyclonal anti-collagen IV antibody . Experimental discs containing 10(3) or 10(5) glioma cells as well as positive control discs containing the agonist prostaglandin E1 consistently demonstrated greater vessel growth than negative controls (discs containing a balanced salt solution) . The disc angiogenesis system is a useful tool for the measurement of angiogenic response to living tumor cell suspensions. Biochem Biophys Res Commun, 1993 Mar 31, 191(3), 1224 - 9 Dual action of the neuropeptide galanin on the cytoplasmic free calcium concentration in RIN m5F cells; Fridolf T et al.; The neuropeptide, galanin, potently inhibits insulin secretion and is thought to be an adrenergic neurotransmitter in the pancreas . In this study, the effects of galanin and the galanin receptor antagonist, galantide, on the cytoplasmic free Ca2+ {Ca2+}i, were investigated in FURA 2-AM-loaded cells of the rat insulinoma cell line, RIN m5F, in cell suspensions in a cuvette . It was found that galanin (100 nmol/l) after a prior addition of D-glyceraldehyde (15 mmol/l), both at 3.3 and 8.3 mmol/l glucose, induced a dual, biphasic, action on the {Ca2+}i: a rapid and transient peak was followed by a reduction below the prestimulatory levels . The rapid peak was similar to that induced by the cholinergic agonist, carbachol (100 mumol/l) . A prior addition of the galanin receptor antagonist, galantide (500 nmol/l), abolished the changes in {Ca2+}i after galanin . However, galantide by itself induced the same biphasic changes in {Ca2+}i as those induced by galanin . Hence, the study demonstrates a) that galanin induces a dual response in {Ca2+}i in insulin-producing RIN m5F cells with a rapid peak preceeding a reduction below prestimulatory levels, and b) that the galanin receptor antagonist, galantide, is a partial galanin agonist . It is proposed that the changes in {Ca2+}i induced by galanin are much more complex than previously thought and, therefore, that galanin does not inhibit insulin secretion by simply reducing the {Ca2+}i. Biochem J, 1993 Mar 15, 290 ( Pt 3), 671 - 7 Mechanisms of elevation of adenosine levels in anoxic hepatocytes; Bontemps F et al.; Previous work has shown that normoxic isolated rat hepatocytes continuously produce adenosine from AMP and that the nucleoside is not catabolized further but immediately rephosphorylated by adenosine kinase {Bontemps, Van den Berghe and Hers (1983) Proc . Natl . Acad . Sci . U.S.A . 80, 2829-2833} . We now report the effect of anoxia on adenosine production and on the AMP/adenosine substrate cycle . In cell suspensions incubated in O2/CO2, the adenosine concentration was about 0.4 microM . It increased 30-fold in cells incubated in N2/CO2 or with 5 mM KCN, and 20-fold in cells incubated with 2 mM amytal . Adenosine production, measured in hepatocytes in which adenosine kinase and adenosine deaminase were inhibited by 5-iodotubercidin and deoxycoformycin respectively, was about 18 nmol/min per g of cells in normoxia; it increased about 2-fold in anoxia, although AMP increased 8-16-fold in this condition . From studies with inhibitors of membrane 5'-nucleotidase and of S-adenosylhomocysteine hydrolase, it was deduced that adenosine is produced by the latter enzyme and by cytosolic 5'-nucleotidase in normoxia, and by cytosolic and membrane 5'-nucleotidases in anoxia . Unlike in normoxic hepatocytes, inhibition of adenosine kinase by 5-iodotubercidin neither elevated the adenosine concentration nor enhanced total purine release from adenine nucleotides in cells treated with N2/CO2 or KCN; it had only a slight effect in cells treated with amytal . This indicates that recycling of adenosine is suppressed or profoundly inhibited in anoxia . The rate of accumulation of adenosine in anoxia was several-fold lower than the rate of its rephosphorylation upon reoxygenation . It is concluded that the elevation of adenosine in anoxic hepatocytes is much more dependent on decreased recycling of adenosine by adenosine kinase than on increased production by dephosphorylation of AMP. Biochim Biophys Acta, 1993 Mar 10, 1176(1-2), 77 - 82 Uptake of tyramine by rat hepatocytes; Zhong ZD et al.; Observations on the uptake of tyramine by hepatocytes indicate that the amine is taken up by simple diffusion and a transporter mediated system, with a Km of 39 microM and a Vmax of 270 pmol/min/10(5) cells . The carrier-mediated process is pH- and temperature-dependent and requires an activation energy of 12.9 kcal/mol . An overshoot uptake is achieved a few minutes after adding this amine to the cell suspension, suggesting that active transport is involved . This is supported by the finding that partial inhibition of the uptake can be induced by oligomycin, azide, cyanide and dinitrophenol . NO3-, SCN- and SO4(2-), which change the membrane potential significantly, and depress the transporter mediated uptake further, suggesting that the membrane potential is the driving force for the entry of this amine across hepatic membrane . Cysteine is essential for the normal carrier function; whereas, histidine, tryptophan, arginine and lysine do not directly deal with the activity of the carrier . Many substances, but not amino acids, H, M, and N receptor agonists, can inhibit the uptake of tyramine . It is possible that other amines can enter hepatocytes by using this transporter. Plant Mol Biol, 1993 Mar, 21(6), 1085 - 95 Molecular cloning and expression of a Eucalyptus gunnii cDNA clone encoding cinnamyl alcohol dehydrogenase; Grima-Pettenati J et al.; Cinnamyl alcohol dehydrogenase (CAD) catalyses the reduction of hydroxycinnamyl aldehydes (sinapyl, paracoumaryl, coniferyl aldehydes) to the corresponding alcohols which are the direct monomeric precursors of lignins . Recently, we have purified from Eucalyptus gunnii two isoforms of CAD (CAD1 and CAD2), distinct in their biochemical and functional properties . In this paper, we report the cloning of a CAD cDNA (pEuCAD2) isolated by screening a lambda gt11 library generated from cell suspension culture of Eucalyptus gunnii, using a tobacco CAD cDNA as a probe . This full-length clone (1392 bp) encodes a protein of 356 amino acids which corresponds to the subunit molecular weight of the CAD2 isoform . Sequence analysis revealed that CAD2 is very well conserved among species (78% homology with CAD from tobacco, a herbaceous angiosperm, and 81% with the partial sequence from a gymnosperm, loblolly pine) . The identity of this clone was unambiguously demonstrated (1) by comparison with peptide sequence data from purified CAD2 and (2) by functional expression of the recombinant enzyme in Escherichia coli . Recombinant CAD showed the same properties as the natural isoform CAD2, in terms of electrophoretic mobility, polypeptide structure, substrate specificity and antigenicity . The CAD2 transcript is equally abundant in stems and leaves and at the limit of detection in roots . At the tissue level the CAD2 gene is highly expressed in xylem and virtually undetectable in phloem. Biophys J, 1993 Mar, 64(3), 709 - 15 Cholesterol is required for the fusion of single unilamellar vesicles with Mycoplasma capricolum; Tarshis M et al.; Small unilamellar vesicles (SUV) were prepared from the total lipid extract of Mycoplasma capricolum . The SUV were labeled with the fluorescent probe octadecylrhodamine B chloride (R18) to a level at which the R18 fluorescence was self-quenched . At pH 7.4 and 37 degrees C, and in the presence of 5% polyethylene glycol, an increase in the R18 fluorescence with time was observed when the R18-labeled SUV were introduced to a native M . capricolum cell suspension . The fluorescence dequenching resulting from dilution of the R18 into the unlabeled membranes of M . capricolum, was interpreted as a result of lipid mixing during fusion between the SUV and the mycoplasma cells . The presence of cholesterol in the SUV was found to be obligatory to allow SUV-mycoplasma fusion to occur . Adaptation of M . capricolum cells to grow in a medium containing low cholesterol concentration provided cells in which the unesterified cholesterol content was as low as 17 micrograms/mg cell protein . The fusion activity of the adapted cells was very low or nonexistent . Nonetheless, when an early exponential phase culture of the adapted cells was transferred to a cholesterol-rich medium, the cells accumulated cholesterol and regained their fusogenic activity . The cholesterol requirement for fusion in the target mycoplasma membrane was met by a variety of planar sterols having a free beta-hydroxyl group, but differing in the aliphatic side chain, e.g., beta-sitosterol or ergosterol, even though these sterols, having a bulky side chain, are preferentially localized in the outer leaflet of the lipid bilayer.(ABSTRACT TRUNCATED AT 250 WORDS) Cell Prolif, 1993 Mar, 26(2), 147 - 59 Hydroxyurea exposure alters mouse testicular kinetics and sperm chromatin structure; Evenson DP et al.; The effects of hydroxyurea (HU) on testicular cell kinetics and sperm chromatin differentiation were investigated in mice . Whole testis, minced testicular cell suspensions and caudal epididymal sperm cells were obtained at 8 and 29 days after i.p . injections containing 0, 25, 50, 100, 200, 400 and 500 mg/kg HU x 5 days . Testis weights were unaffected by 25 mg/kg HU while 500 mg/kg caused up to a 50% loss of testicular weight by 29 days . Flow cytometrically measured acridine-orange (AO) stained testicular cells revealed altered population ratios at the highest dosages at 8 days and for all dosages except 25 mg/kg HU at 29 days . At 8 days, 400-500 mg/kg HU caused a near depletion of tetraploid cells . Flow cytometry of AO stained sperm, previously treated with acid to potentially induce DNA denaturation, was used to follow the shift from normal chromatin structure to an abnormal form with increased sensitivity to DNA denaturation in situ . The extent of DNA denaturation was quantitated for each cell by the computer-derived value alpha t, alpha t = {red/(red+green) fluorescence} . The flow cytometry measures, standard deviation of alpha t (SD alpha t), mean of alpha t (X alpha t) and cells outside the main peak of alpha t (COMP alpha t), gave similar dose response curves to the sperm head morphology assay . SD alpha t was more sensitive than the X alpha t as a measure of HU-induced alteration of chromatin structure . The major conclusions reached are that HU inhibits DNA synthesis, probably by inhibiting ribonucleotide reductase, causing maturation depletion of pachytene spermatocytes and, subsequently, depletion of meiotic daughter cells and differentiated cell types leading to mature sperm . This inhibition of DNA synthesis is related to an alteration of sperm chromatin structure and abnormal sperm head morphology. Cell Prolif, 1993 Mar, 26(2), 115 - 24 In vitro BrdUrd incorporation of colorectal tumour tissue; Williamson K et al.; This study describes a novel in vitro method for the incorporation of the thymidine analogue, bromodeoxyuridine (BrdUrd), in fresh colorectal tumour tissue . Disaggregation by pronase, collagenase and DNAse resulted in high cell yields of viable single cell suspensions, representative of the original tumour, which could be infiltrated with BrdUrd . A modified ELISA identified optimal incubation times and BrdUrd concentrations . This technique has been used in preliminary studies to investigate two important areas intrinsic in the analysis of BrdUrd colorectal cell proliferation data: 1) to determine the effects of the individual constituents of the cell culture media, in particular glutamine, on BrdUrd incorporation in suspensions of colorectal cells and 2) to examine the denaturation step . This method will have wide applicability in investigations of cell proliferation status in both normal and diseased tissue. Plant Mol Biol, 1993 Mar, 21(5), 923 - 7 Molecular characterization of salt-stress-associated protein in citrus: protein and cDNA sequence homology to mammalian glutathione peroxidases; Holland D et al.; A gene encoding for a citrus salt-stress-associated protein (Cit-SAP) was cloned from Citrus sinensis salt-treated cell suspension . The gene, designated csa, was isolated from a cDNA expression library . The partial amino acid sequence of the protein, as well as that deduced from the nucleotide sequence of csa, revealed a considerable homology to mammalian glutathione peroxidase (GP), and to clone 6P229 from tobacco protoplasts . The increased expression of Cit-SAP in NaCl-treated cultured citrus cells and in citrus plants irrigated with saline water, and its similarity to GP, raise the possibility that one of the effects of salt stress in plants may be the increase of the level of free radicals. Arch Dis Child, 1993 Mar, 68(3), 393 - 8 Rapid diagnosis of malignancy using flow cytometry; Williams DM et al.; The rapid and accurate diagnosis of childhood malignancy is important both in the planning of appropriate treatment and in relieving the inevitable family anxiety . The use of flow cytometry to analyse monoclonal antibody coated single cell suspensions is widely accepted as having increased the speed and accuracy of diagnosis in leukaemias, though its use in solid tumour diagnosis is not widely reported . Ten cases of childhood malignancy in whom the diagnosis was initially made by flow cytometry and subsequently confirmed histologically are described . The technique has a number of advantages . Only a small sample is required as the analysis is carried out on a single cell suspension, the method is rapid, a diagnosis being reached within three hours of receipt of the sample, and information is obtained on cell lineage and stage of differentiation . Diagnostic accuracy is good when compared with histological results. Stem Cells, 1993 Mar, 11(2), 105 - 11 To do tissue culture in two or three dimensions? That is the question; Hoffman RM; Alexis Carrel introduced the in vitro culture of tissues in the beginning of the century utilizing a culture system that allowed the three-dimensional growth of tissues . Leighton improved upon this system by developing a substrate of sponge matrices . Other methods of three-dimensional culture include collagen gels and what are known as organ culture systems on filters or meshes . In addition, cell suspensions can be converted into multicellular spheroids, another form of three-dimensional culture . Comparison of the three-dimensional culture methods with two-dimensional culture methods has shown critical differences in the behavior of biological systems in culture . For example, in vivo-like drug responses are observed in three-dimensional but frequently not in two-dimensional cultures, indicating that drug response may be a function of tissue architecture . The in vivo mechanism of drug resistance may involve alterations in cell-cell interaction which may occur in three-dimensional culture as opposed to monolayer culture . Practical applications of three-dimensional culture include the development of a drug-response assay that correlates not only with drug resistance but also with drug sensitivity and survival of cancer patients . It has been shown that gene expression may be more in vivo-like in three-dimensional cultures than in two-dimensional monolayer cultures . For example, tumor antigens may be expressed in three-dimensional culture and not in monolayer culture . Thus, future studies utilizing three-dimensional cultures may significantly enhance our understanding of gene expression and resistance to drugs and enhance the efficacy of cancer chemotherapy by correctly predicting active drug regimens for individual patients. J Invest Dermatol, 1993 Mar, 100(3), 282 - 7 Epidermal Langerhans cells are resistant to the permeabilizing effects of extracellular ATP: in vitro evidence supporting a protective role of membrane ATPase; Girolomoni G et al.; Extracellular adenosine 5'-triphosphate (ATPo) can induce pore formation in cell membranes, leading to cell permeabilization and eventual cell death . In this study, we examined the sensitivity of human epidermal Langerhans cells to ATP-induced permeabilization and tested the possibility that the Mg(++)- or Ca(++)-dependent plasma membrane ectonucleotidase (mATPase) on Langerhans cells provides protection against the cytotoxic effects of ATPo . Membrane permeability was assessed by using the fluorescent tracer propidium iodide, which confers red nuclear fluorescence to permeabilized cells . Langerhans cells were identified within human epidermal cell suspensions with fluorescein isothiocyanate-conjugated MoAb against CD1a or human leukocyte antigen-DR (HLA-DR) antigens . Cultured human keratinocytes and J774 macrophages were both highly sensitive to permeabilization induced by incubation with ATP (0.5 to 20 mM at 37 degrees C), whereas Langerhans cells were relatively resistant . The non-hydrolyzable ATP analog, adenosine 5'-(beta,gamma-imido) triphosphate, but not other nucleotides such as ADP, AMP, GTP, or UTP, was also able to induce permeabilization comparable to that of ATP, thereby suggesting that ATP hydrolysis is not required for this effect . ATP4- is the moiety most likely responsible for permeabilization, because propidium iodide uptake occurred only when the pH of the medium was > or = 7.4 . Permeabilization induced by ATP was augmented by chelation of divalent cations with ethylene-diamine-tetraacetic acid and by the addition of lanthanum or cerium (0.01 to 1 mM) . Finally, incubation with the adenosine analog, 5'-p-fluorosulfonylbenzoyl-adenosine (1 mM), inhibited mATPase staining of Langerhans cells in human epidermal sheets, but markedly augmented ATP-induced permeabilization of Langerhans cells . The results indicate that epidermal LC are resistant to the lytic effects of ATPo and that mATPase is involved in such resistance. Cancer Res, 1993 Mar 1, 53(5), 1204 - 8 Nude mouse metastatic models of human stomach cancer constructed using orthotopic implantation of histologically intact tissue; Furukawa T et al.; Nude mice have been used to develop s.c . growing human stomach tumors, but these rarely metastasize . Recently, I . J . Fidler and others have developed orthotopic implantation metastatic models using cell suspensions which are inoculated into the corresponding organ of nude mice from which the tumor cells were originally derived in the human . However, recent work has indicated that disaggregated cell suspensions may not always express their full metastatic potential . In this light, we have recently developed an orthotopic implant model utilizing intact tissue such as that obtained directly from surgery . This approach has yielded high take rates and frequent metastases in colon cancer, bladder cancer, lung cancer, pancreatic cancer, and prostate cancer . We report here the application of this intact tissue orthotopic implant technique to stomach cancer resulting in the formation of metastases in 100% of the mice with extensive primary growth to the regional lymph nodes, liver, and lung . In contrast, when cell suspensions were used to inject stomach cancer cells at the same site, metastases occurred in only 6.7% of the mice with local tumor formation, emphasizing the importance of using intact tissue to allow full expression of metastatic potential . Injuring the serosa similar to that occurring in intact tissue transplantation did not increase the metastatic rate after orthotopic injection of cell suspensions of stomach tumor cells . This intact tissue orthotopic implantation model should allow development of new treatment modalities and further study of the biology of human stomach cancer. Diabetes, 1993 Mar, 42(3), 496 - 500 Islet cell DNA is a target of inflammatory attack by nitric oxide; Fehsel K et al.; NO has been identified recently as the prime islet-toxic product of inflammatory macrophages . The adverse effects of IL-1 on isolated islets also have been reported to involve NO . We now show that exposure of an islet cell suspension to the NO donor nitroprusside or to activated macrophages leads to DNA strand breaks . Macrophages did not induce DNA damage in the presence of the NO synthase inhibitor NG-methyl-L-arginine . DNA strand breaks were demonstrated at the level of single cells by a modified nick-translation procedure and confirmed by analysis of DNA fragmentation by gel electrophoresis . DNA strand breaks occurred within 1 h and preceded islet cell lysis . DNA damage could not be prevented by inhibitors of endogenous endonucleases . We conclude that islet cell DNA is an early target of NO action. J Physiol, 1993 Mar, 462, 609 - 26 Volume-activated Cl(-)-independent and Cl(-)-dependent K+ pathways in trout red blood cells; Guizouarn H et al.; 1 . Swelling of trout erythrocytes can be induced either by addition of catecholamine to the cell suspension, thus promoting NaCl uptake via beta-adrenergic-stimulated Na(+)-H+ exchange (isotonic swelling) or by suspending red blood cells in a hypotonic medium (hypotonic swelling) . In both cases cells tend to regulate their volume by losing K+, but the characteristics of the volume-activated K+ pathways are different: after hormonally induced swelling the K+ loss is strictly Cl- dependent; after hypotonic swelling the K+ loss is essentially Cl- independent . 2 . In order to determine the nature of these volume regulatory pathways (i.e . whether the net K+ loss was conductive or was by electroneutral K(+)-H+ exchange or KCl co-transport), studies were performed to analyse ion fluxes and associated electrical phenomena . The cell membrane potential and intracellular ionic activities of volume-regulating and volume-static cells were measured by impalement with conventional microelectrodes and double-barrelled ion-sensitive microelectrodes . 3 . The information gained from the electrical and ion flux studies leads to the conclusion that both Cl(-)-independent and Cl(-)-dependent K+ loss proceed via electrically silent pathways . 4 . Experiments were designed to distinguish between electroneutral K(+)-H+ exchange or KCl co-transport . These were based upon the inhibition of Cl(-)-OH- exchange to evaluate the degree of coupling between K+ and Cl- (KCl stoichiometry, pH change) . The experimental observations are consistent with the fact that both Cl(-)-independent and Cl(-)-dependent K+ loss are mediated by coupled K(+)-anion co-transport and not by K(+)-H+ exchange . 5 . On the basis of previous data, we suggest that only one type of K(+)-anion co-transport exists in the cell membrane, for which the selectivity for anions varies according to the change in cellular ionic strength induced by swelling. Microvasc Res, 1993 Mar, 45(2), 149 - 57 A micropipette which allows in situ perfusion of arterioles and capillaries; Damon DN et al.; In order to investigate capillary physiology, a glass micropipette system was developed that allowed in situ perfusion of microvessels as well as rapid changes of perfusion solutions . Theta tube (WPI, Inc.; 1.5-mm o.d . glass stock capillary tubing which is divided into two hemicylindrical sides by a central glass septum) was pulled to a smaller diameter of approx 300-600 microns and inserted into the shank of a sharpened cannulating micropipette tip constructed from large-bore glass stock (1.6 mm i.d.) . The resulting dead volume between the end of the Theta supply tube and the tip of the outer cannulating tip was approximately 90 nl . The perfusate was driven in a circuit from a pressurized feed reservoir down one side of the Theta supply tube pipette and back through the second side into a reservoir maintained at a lower pressure . The pressure gradient between the two reservoirs established a high-volume flow rate and subsequently a short perfusate transit time from the feed to the collection reservoir . The average pressure in the two reservoirs determined the pressure which drove the perfusate from the cannulating tip . At normal pressures and flows, the time required to change perfusion fluid composition at the pipette tip was less than 1 min, and discharge hematocrit of a red blood cell suspension was indistinguishable from the hematocrit measured in the feed reservoir.(ABSTRACT TRUNCATED AT 250 WORDS) Plant Physiol, 1993 Mar, 101(3), 1089 - 96 Molecular cloning of abscisic acid-responsive mRNAs expressed during the induction of freezing tolerance in bromegrass (Bromus inermis Leyss) suspension culture; Lee SP et al.; Abscisic acid (ABA) increases the freezing tolerance of bromegrass (Bromus inermis Leyss) cell-suspension cultures at 23 degrees C and elicits many metabolic changes similar to those observed during cold acclimation . Induction and maintenance of freezing tolerance by ABA is accompanied by the expression of novel polypeptides and translatable RNAs . The objective of this study was to isolate and characterize ABA-responsive cDNAs associated with ABA-induced freezing tolerance in bromegrass cell cultures . Among the 16 ABA-responsive cDNA clones isolated, 9 were expressed only with ABA treatment, 7 showed increased transcript level, and 1 was transiently expressed . Cold responsiveness was determined in three clones with increased transcript levels and in the transiently expressed clone . Deacclimation of ABA-hardened cells was a relatively slow process, because all of the novel transcripts persisted for at least 7 d after cells were cultured in ABA-free medium . Preliminary sequencing of cDNAs has identified several clones that share high sequence homology with genes associated with sugar metabolism, osmotic stress, and protease activity . Clone pBGA61 was fully sequenced and tentatively identified as an NADPH-dependent aldose reductase . The predicted amino acid sequence of the coding region shared 92% similarity with that predicted for barley aldose reductase cDNA . It is proposed that expression of genes related to sugar metabolism and osmotic stress may be required for ABA-induced hardening. Cell Transplant, 1993 Mar-Apr, 2(2), 175 - 82 Metabolic activity and proliferation of CHO cells in hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) microcapsules; Uludag H et al.; To better understand encapsulated cell behaviour, Chinese Hamster Ovary (CHO) fibroblasts were encapsulated in HEMA-MMA microcapsules and short-term (<2 wks) proliferation and changes in metabolic activity were investigated in vitro . CHO cells were observed to undergo rapid proliferation in the first week following encapsulation after which a growth arrest was obtained at approximately 3500 cells/capsule . The cell growth was localized in aggregates in the capsule core, resulting in high local cell density but low cell density in the whole capsule interior (approximately 10(7) cells/mL) . The total metabolic activity, as determined by the MTT (3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyl tetrazolium) assay, within the microcapsules increased in the first week, with no significant change afterwards . A broad variation in metabolic activity among the individual capsules was obtained . Supplementing the cell suspension with 20% Ficoll 400 during the encapsulation process resulted in significantly higher morphological uniformity among the individual capsules (with reduced capsule wall thickness and eccentricity); however, this did not change the extent of heterogeneity in metabolic activity . We conclude that viability and proliferation ability (at least to a limited extent) of CHO cells are maintained in HEMA-MMA microcapsules . The local cell growth and subsequent growth arrest remain issues to be addressed in order to obtain better utilization of the microcapsule core volume . Alternatively, small diameter (< 400 microns as opposed to the present approximately 750 microns diameter) capsules are necessary. Neuroscience, 1993 Mar, 53(2), 403 - 15 Characterization of GABA release from intrastriatal striatal transplants: dependence on host-derived afferents; Campbell K et al.; Extracellular levels of GABA, derived from cell suspension transplants of embryonic day 14-15 rat striatal primordia implanted into the previously excitotoxically lesioned striatum, were measured using intracerebral microdialysis in halothane-anaesthetized rats . GABA overflow was monitored using loop type dialysis probes implanted into grafted, age-matched ibotenic acid-lesioned and intact striata, under baseline conditions and after different pharmacological manipulations . Basal and evoked GABA release, which was reduced by 58 and 96%, respectively, in the excitotoxin-lesioned striatum, was restored by the striatal grafts to levels close to or above those observed in normal striata . The graft-derived release of GABA was most likely of neuronal origin, since the K(+)-evoked (100 mM) GABA overflow was reduced by almost 80% when Ca++ was replaced by 20 mM Mg++ in the perfusion medium, and blockade of GABA uptake by nipecotic acid (0.5 mM), induced a greater than six-fold increase in GABA overflow . However, perfusion of the graft with 1 microM tetrodotoxin in combination with K+ (100 mM) resulted in little if any reduction in the K(+)-evoked overflow . Histological analysis demonstrated a dense tyrosine hydroxylase-positive fibre network in the grafts, which was removed after a 6-hydroxydopamine lesion of the ipsilateral nigrostriatal pathway . The dopamine denervating lesion resulted in an increased K(+)-evoked GABA overflow both in the intact (+76%) and the grafted striata (+181%), suggesting that the tonic dopaminergic inhibitory control of GABA release, seen in the intact striatum, is also present in the grafted striata . The glutamate analogue, kainic acid (1 mM added to the perfusion fluid), evoked a 60-74% increase in GABA overflow both in intact striata (with or without dopaminergic denervation) and in the striatal grafts . This effect seemed to be dependent on an intact corticostriatal projection, since knife-cut transections of the frontal cortex at the level of the forceps minor, abolished the response in both the intact and grafted striata . These results demonstrate that grafts of fetal striatal tissue implanted into the excitotoxically lesioned striatum restore striatal GABA overflow in a neuron-dependent manner, close to or above that seen in the normal intact striatum . Furthermore, the graft-derived GABA release appears to be under normal regulatory control from the host dopaminergic and glutamatergic systems . Since the GABAergic striatal output system is critical for the expression of striatum-related behaviours, it is proposed that the graft-induced behavioural recovery in the striatal lesion model, at least in part, may depend on the restoration of striatal GABAergic neurotransmission. Am J Obstet Gynecol, 1993 Mar, 168(3 Pt 1), 831 - 6 Gamma delta T cells in human decidua; Ditzian-Kadanoff R et al.; OBJECTIVE: Our purpose was to assess the presence and frequency of gamma delta T cells in the decidua of term and first-trimester pregnancies . STUDY DESIGN: Term and first-trimester placentas were obtained from normal subjects . Frozen sections and cell suspensions were prepared from decidual tissue and stained with monoclonal antibodies to T cell markers . Cell sorter analysis was performed . RESULTS: gamma delta T cells in term decidual cell preparations were enriched 2.4-fold compared with peripheral blood . Immunohistochemical staining of term decidual tissue demonstrated many gamma delta + and CD3+ T cells, fewer CD8+ cells, and rare CD4+ cells . In contrast, first-trimester decidua was found to be devoid of gamma delta + T cells, by both cell sorter analysis and immunohistochemical methods . CONCLUSIONS: Term, but not early, decidua harbors a resident T-cell population that is significantly enriched in gamma delta T cells compared with blood . These lymphocytes may provide an added defense mechanism against infection during the peripartum. Transfus Med, 1993 Mar, 3(1), 43 - 50 Half-strength citrate CPD and new additive solutions for improved blood preservation . I . Studies of six experimental solutions; Hogman CF et al.; Poor stability of plasma factor VIII in whole blood and loss of erythrocyte 2,3-bis-phosphoglycerate (BPG) during red cell storage are limitations with systems for blood component preparation in current use . This study presents attempts to improve post-collection storage conditions in both these respects using half-strength citrate CPD solution (0.5CPD) for blood collection, which has been shown by others to improve the stability of factor VIII, and some compositions of hypotonic additive solutions for red cell storage containing citrate, adenine, mannitol, and phosphate . Guanosine was also included in some of the media . The erythrocyte BPG concentration was maintained at a normal level for 3-4 weeks with the best of the tested compositions . Total adenine nucleotide concentration was maintained at the original level for 49 days and adenosine triphosphate for 28 days . Spontaneous storage haemolysis was low, 0.31% (mean) +/- 0.08-0.10% (SD) after 49 days in the two best compositions . The intracellular pH was 0.2-0.3 pH units higher than the extracellular pH at the beginning of storage, but this difference gradually diminished and disappeared after 4-5 weeks . We suggest two likely explanations of the effects: the maintenance of intracellular pH at a level sufficiently high not to impair BPG synthesis until after several weeks of storage, and a sufficient supply of phosphate needed in the synthesis of organic phosphate compounds . The content of citrate was selected such that the total amount supplied to a patient in a massive transfusion, when using a combination of 0.5CPD plasma and red cell suspension, would be smaller than that provided by a transfusion of CPD whole blood. J Periodontal Res, 1993 Mar, 28(2), 145 - 51 Lectin binding to chronic inflammatory gingival tissue: possible adhesion mechanisms based on lectin-carbohydrate interactions; Krugluger W et al.; In this study, we analyzed the expression of different leukocyte surface antigens, of the adhesion molecules ELAM-1 and GMP-140 and binding of various lectins and neoglycoproteins in inflamed gingival tissue . Cell suspensions from collagenase-digested gingiva were analyzed by flow cytometry in a FACScan . The expression of ELAM-1, GMP-140, carbohydrate structures and lectins in gingival specimens was also studied by immunohistochemistry . Gingival tissue of patients with active periodontal disease contained between 5% and 50% CD45+ mononuclear cells, consisting mainly of CD19+ cells (B lymphocytes) . CD62, resembling GMP-140, and ELAM-1 were strongly expressed on endothelial cells of these patients . Control subjects usually contained almost no CD45+ cells in their gingiva and no CD62+ or ELAM-1-positive endothelial cells could be found in 5 of 6 control persons . Analysis of the glycosylation pattern revealed staining of infiltrating cells by peanut agglutinin (PNA; specificity for galactose), whereas soy bean agglutinin (SBA; specificity for N-acetyl-galactosamine) bound to epithelial cells . An endogenous lactosyl-specific lectin could be detected on endothelial cells by binding of lactosyl-BSA . Ulex europeus I agglutinin (UEA-1, specific for fucose) showed selective staining of endothelial and epithelial cells . Expression of a fucose-binding lectin, demonstrated by binding of fucosylated BSA, could be found on infiltrating cells . The adhesion molecules ELAM-1 and GMP-140 seem to be involved in cell adhesion during chronic inflammation of the gingiva . Interaction of other carbohydrate residues with endogenous lectins might resemble additional adhesion mechanisms in inflamed gingiva. J Surg Res, 1993 Mar, 54(3), 179 - 88 Detection of c-erbB-2 oncoprotein expression in breast tissue by multiparameter flow cytometry; Li BD et al.; A novel technique in multiparameter flow cytometry (FCM) using dual laser excitation of three fluorescent dyes has been developed to differentiate breast epithelial cells from stromal components . This technique has been applied to determine the expression of the oncoprotein c-erbB-2 in breast epithelial tumors . SK-BR-3, a breast cancer cell line with c-erbB-2 overexpression, can be identified by FCM from a mixed cell suspension using a monoclonal anti-human cytokeratin antibody conjugated with fluorescein isothiocyanate . Using the mouse monoclonal anti-c-erbB-2 antibody, TA-1 (4.8 +/- 1.0 x isotype control), the c-erbB-2 oncoprotein overexpression in breast epithelial cells can be detected as an increase in indirect blue fluorescence by aminomethyl coumarin . MCF-7, a breast cancer cell line with normal c-erbB-2 expression, has baseline blue fluorescence (1.0 +/- 0.5 x isotope control) . Twenty-one fresh breast specimens have been examined by FCM . Overexpression of c-erbB-2, defined by blue fluorescence ratio of TA-1/isotype control > or = 1.5 (> 3 SD from baseline), is detected in 0 of 3 patients with Stage I cancer, 5 of 14 patients with Stage II and III cancer, and 3 of 4 patients with proliferative disease . Patients with elevation of oncoprotein detected by FCM have corresponding RNA overexpression detected by Northern blot hybridization and increased gene amplification detected by Southern blot hybridization . FCM allows for the simultaneous identification of breast epithelial cells and the selective examination of these cells for the expression of c-erbB-2 oncoprotein, thus minimizing stromal contamination . This represents a novel application of FCM with potential for wide clinical applicability. J Bone Miner Res, 1993 Mar, 8(3), 331 - 6 Effect of medium pH on osteoclast activity and osteoclast formation in cultures of dispersed rabbit osteoclasts; Shibutani T et al.; We investigated the effect of medium pH on activity of isolated osteoclasts and have also looked at the possibility that medium pH affects osteoclast numbers during culture . Osteoclast-containing cell suspensions prepared from neonatal rabbits were cultured on bovine bone slices at pH 6.5, 7.0, or 7.5 . After 24 or 48 h of culture, the cells and bone slices were fixed and stained for tartrate-resistant acid phosphatase (TRAP) . After counting the osteoclasts, the cells were removed and the resorption lacunae stained by immunostaining using anticollagen type I antibody and then quantitated . We found that the resorptive activity of isolated rabbit osteoclasts was sharply increased at pH 6.5-7 . Osteoclast differentiation and proliferation, on the other hand, were optimal at pH 7.0-7.5 but decreased at pH 6.5 . The results thus imply that pH regulation of the bone surface environment can dramatically alter both the number of osteoclasts and their resorptive activity. Transplantation, 1993 Mar, 55(3), 646 - 50 Inhibition of the multidrug efflux pump in isolated hepatocyte couplets by immunosuppressants FK506 and cyclosporine; Takeguchi N et al.; Fluorescence image analysis of isolated rat hepatocyte couplets, which retain a bile canaliculus between them, has shown the presence of transport systems for the bile acid and non-bile acid organic anion in the canalicular membrane . The cells also transported Fura-2 and BCECF, which are fluorescent indicators of intracellular Ca2+ and H+ concentrations, respectively, into the canaliculus . Both Fura-2 and BCECF transports were inhibited by progesterone, verapamil, vinblastine, and daunomycin, indicating that the transports are due to a multidrug efflux pump (P-glycoprotein) in the canalicular membrane . Interestingly, the transport by the multidrug efflux pump was inhibited by immunosuppressants FK506 (tacrolimus) and cyclosporine, their half-maximal inhibitory concentrations in the cell suspension being 10 microM and 0.6 microM, respectively . In contrast, the reported immunosuppressive potency of FK506 is 10- to 100-fold that of cyclosporine . Inhibition by immunosuppressants of the multidrug efflux pump, which is a transporter of cytotoxic and other drugs and present in normal human tissues--such as kidney, liver, the blood-brain barrier, and colon--may, at least partly, explain side effects caused by cyclosporine in these tissues of transplant recipients . FK506 at its clinical concentrations may not inhibit the multidrug efflux pump. Am J Clin Pathol, 1993 Mar, 99(3), 304 - 10 Combined assay of surface immunoglobulin intensity and mouse rosettes . A practical parameter in the differential diagnosis of small lymphocytic and follicular center cell lymphomas; Batata A et al.; Cell suspensions from the lymph nodes of small lymphocytic lymphoma (n = 94) and nodular and diffuse follicular center cell lymphomas (n = 330) were analyzed to evaluate the diagnostic significance of the surface immunoglobulin (SIg) intensity and mouse rosette assay (MR) . In small lymphocytic lymphoma, SIg was monoclonal in 65 cases (69.15%), with weak fluorescence in 59 (90.77%) . It was not detected in 29 cases (30.85%) . The MR findings were positive in 68 cases (72.34%) and negative in 26 (27.66%) . The combined results of these two assays showed the following: weak SIg/MR+, 35 (37.23%); weak SIg/MR-, 24 (25.53%); strong SIg/MR+, 6 (6.38%); strong SIg/MR-, 0; undetected SIg/MR+, 27 (28.72%); and undetected SIg/MR-, 2 (2.13%) . By performing the assays for these two markers and accepting weak SIg/MR+, weak SIg/MR-, strong SIg/MR+, or undetected SIg/MR+ as sufficient for diagnosis, 92 cases (97.87%) were diagnosed . In diffuse follicular center cell lymphomas, SIg was monoclonal in 287 cases (86.97%), with strong fluorescence in 258 (89.9%) and weak fluorescence in 29 (10.1%) . It was not detected in 43 cases (13.03%) . The MR results were positive in 34 cases (10.3%) and negative in 296 (89.7%) . The combined findings of these two assays showed that strong SIg/MR- was present in 244 cases (73.94%) . The diagnostic value of the combined assay in the differential diagnosis between small lymphocytic lymphoma and diffuse follicular center cell lymphomas was proved using five statistical parameters. Biochem J, 1993 Feb 15, 290 ( Pt 1), 241 - 7 Enkephalin activates the phospholipase C/Ca2+ system through cross-talk between opioid receptors and P2-purinergic or bradykinin receptors in NG 108-15 cells . A permissive role for pertussis toxin-sensitive G-proteins; Okajima F et al.; In an NG 108-15 neuroblastoma x glioma hybrid cell suspension, extracellular ATP (via P2-purinergic receptors) and bradykinin stimulated Ins(1,4,5)P3 formation, which was accompanied by an increase in the cytosolic Ca2+ concentration ({Ca2+}i) . Leucine enkephalin (EK) also slightly increased {Ca2+}i in the absence, but not in the presence, of apyrase, which hydrolyses extracellular ATP and ADP to AMP . When the cells were stimulated by P2-agonists or bradykinin prior to the application of EK, EK induces a remarkable rise in {Ca2+}i . This P2-agonist- or bradykinin-assisted EK action was also observed in single cells on a coverslip . A decrease in the extracellular Ca2+ concentration only slightly lowered the EK-induced rise in {Ca2+}i, but treatment of the cells with thapsigargin, an agent which depletes Ca2+ in the Ins(1,4,5)P3-sensitive pool, almost completely abolished EK action . The observed permissive stimulation by EK of Ins(1,4,5)P3 formation induced by a P2-agonist or bradykinin may be a primary event for the EK-induced {Ca2+}i rise . These actions of EK were antagonized by naloxone and completely reversed by prior treatment of the cells with pertussis toxin, whereas the toxin hardly affected the actions of P2-agonists and bradykinin themselves . Thus EK can induce phospholipase C activation and subsequent Ca2+ mobilization, provided that the cells have been previously or are simultaneously stimulated by endogenous adenine nucleotides or by externally applied P2-agonists or bradykinin . In this cross-talk mechanism between opioid receptors and these Ca(2+)-mobilizing agonist receptors, pertussis toxin-sensitive G-proteins play a permissive role. Brain Res, 1993 Feb 12, 603(1), 143 - 7 Effect of embryonic hippocampal transplantation in amygdaloid kindled rat; Miyamoto O et al.; Embryonic neural tissue was transplanted into previously kindled rats . A thirteen- to fourteen-day embryonic hippocampal cell suspension was grafted in the stratum oriens near the CA2 area of the hippocampus . Almost 80% of the animals had a good recovery and became seizure-free . Injection of neocortical cells or saline did not show any positive effect on the kindling susceptibility . Although 20 day embryonic cell transplantation was also effective, the effect did not last as long as the 13- to 14-day embryonic transplantation . These observations open the possibility that the neural grafts may be used for therapy of medically intractable epilepsies. Neurosci Lett, 1993 Feb 5, 150(1), 89 - 94 Injections of fluid or septal cell suspension grafts into the dentate gyrus of rats induce granule cell degeneration; Cassel JC et al.; This study was originally aimed at investigating the effects of intragyral cell suspension grafts which had been enriched in basic fibroblast growth factor (bFGF) before being implanted into the rat hippocampus denervated by aspiration of the septohippocampal pathways . Whether treated with vehicle alone, vehicle + bFGF, cell suspension with or without bFGF, and irrespective of the surgical treatment (sham-operation, lesions or lesions + grafts), we unexpectedly found approximately 80% of the rats to show morphological alterations in the dentate gyrus (20 weeks post-grafting) . These alterations consisted of loss of a part of the granule cells; this loss was most often located in the dorsal leaf of the dentate gyrus . Also, in the close vicinity of the degeneration area, we found severe shrinkage of the molecular layer and disappearance of the typical laminae pattern of acetylcholinesterase distribution . These observations confirm previous findings which showed that fluid injections into the dentate gyrus, a widely used technique for intracerebral administration of drugs, trophic factors or neural grafts, may induce undesirable granule cell necrosis. J Immunol Methods, 1993 Feb 3, 158(2), 207 - 14 Flow cytometric analysis of surface major histocompatibility complex class II expression on human epithelial cells prepared from small intestinal biopsies; Madrigal L et al.; A technique for preparing viable, single cell suspensions of the epithelial layer of small intestinal tissue obtained endoscopically is described . Constant agitation of four biopsies for 60 min in the presence of chelating and reducing agents gave yields of 1.2-6.7 x 10(6) cells, of which 11-30% were intraepithelial lymphocytes (IEL) . Passage through a nylon wool column removed dead cells . This preparation was suitable for flow cytometric analysis . Using this technique, surface MHC class II molecule expression was studied in 14 patients with normal small intestinal mucosa . Fluorescence labelling of these cells showed strong HLA-DR expression by epithelial cells (EC), DP was expressed less strongly, while little DQ expression could be detected . This technique demonstrates that small intestinal biopsies taken during routine endoscopy can yield adequate numbers of viable epithelial cells to perform flow cytometric analysis. Eur J Clin Microbiol Infect Dis, 1993 Feb, 12(2), 98 - 104 Cell-associated haemolytic activity of Helicobacter pylori; Ansorg R et al.; Helicobacter pylori cells cultured on solid medium were quantitatively tested for haemolytic activity against erythrocytes of man, sheep, the guinea pig and rabbit . Using 4-day and 8-day cultures of two standard strains (ATCC 43504, IMMi 676), human erythrocytes were not lysed by 10% bacterial suspensions . Rabbit erythrocytes were the most sensitive to 8-day cultures . Hot-cold incubation yielded the highest haemolysis titres . The extent of haemolysis strongly correlated with the number of bacterial cells . Supplementation of the test medium (PBS, pH 7.4) with L-cysteine, dithiothreitol, MgCl2, EDTA, cholesterol, lecithin or sphingomyelin did not influence the haemolysis titres . They were significantly reduced in the presence of pronase E, human serum, bovine serum albumin or CaCl2, and by heat treatment of the bacteria . Supplementation of the test medium with cardiolipin strongly increased the haemolysis titres . Comparing the cell-associated haemolytic activity of 18 strains, the titres ranged from < 2 to 64, with a median titre of 16 . No correlation was found between the haemolytic activity and phospholipase C activity of the cell suspensions . It was concluded that the formation of lysophosphatides and non-enzymatic factors rather than a sulphydryl-activated cytolysin or phospholipase C are responsible for the cell-associated haemolytic activity . This property may be involved in the pathogenicity and virulence of Helicobacter pylori. Xenobiotica, 1993 Feb, 23(2), 205 - 13 Effects of diethyl maleate on phenyl-hydroquinone-induced cytotoxicity in isolated rat hepatocytes; Nakagawa Y et al.; 1 . The effects of diethyl maleate (DEM) on the cytotoxicity of phenyl-hydroquinone (PHQ) and other hydroquinones were studied in freshly isolated rat hepatocytes . 2 . Addition of PHQ (0.5 or 0.75 mM) to hepatocytes resulted in dose-dependent cell death accompanied by the abrupt depletion of both GSH and protein thiols and the accumulation of phenyl-benzoquinone (PBQ) . 3 . Pretreatment with DEM (1.25 mM), which causes an abrupt depletion of cellular GSH in hepatocytes, delayed the onset of PHQ-induced cytotoxicity . The delay correlated with inhibition of PBQ formation . 4 . Although the pH of the cell suspension was increased slightly (mean pH 0.18) by incubation under carbogen flow, the addition of DEM to the cell suspension inhibited both the increase in pH and the formation of PBQ from PHQ . 5 . In hepatocyte suspensions without DEM, PHQ cytotoxicity was dependent on pH, and toxicity was associated with oxidation of PHQ and accumulation of PBQ . 6 . Among other hydroquinones (0.5 mM), tert-butyl-hydroquinone-induced cytotoxicity was decreased by DEM (1.25 mM), but DEM did not affect the cytotoxicity of 2,5-di(tert-butyl)-1,4-benzohydroquinone . 7 . PHQ-induced cytotoxicity correlated with the accumulation of PBQ in the cell, and the inhibition of PHQ-induced cytotoxicity by DEM correlated with pH-dependent changes in PBQ formation. Int J Microcirc Clin Exp, 1993 Feb, 12(1), 17 - 32 Erythrocyte-leukocyte interactions in the vascular bed of isolated perfused rat lungs; Wikstrom T et al.; An earlier study of perfused rat lungs showed that leukocytes, given as bolus injections, seemed to become more or less permanently trapped in the pulmonary microvascular bed under erythrocyte-free perfusion . The aim of the present study was, therefore, to investigate the rheologic effects of erythrocytes on the leukocyte microcirculation of perfused rat lungs . Leukocyte and erythrocyte suspensions were given as bolus injections during cell-free, constant pressure perfusion of ventilated rat lungs . Leukocyte numbers were counted in samples of the venous effluent and flow resistance changes were computed from registrations of flow rate, arterial and venous pressures . The preparations showed a continuous efflux of leukocytes which had been trapped in the pulmonary microcirculation before the perfusion was started . A bolus infusion of erythrocytes (3 ml, hematocrit: 30%) caused a transient flow resistance increase during the passage of the erythrocyte bolus through the vascular bed . This initial peak was accompanied by an increased venous efflux of leukocytes and followed by a lower second peak, attributed to a redistribution of trapped leukocytes in the capillary bed . Infusions of mixed cell suspensions (20-30 x 10(6) leukocytes in 30% hematocrit) caused a transient resistance increase similar to that caused by erythrocytes and a sustained resistance increase, less persistent than that caused by leukocytes . The present data suggest that the infusion of erythrocytes caused a re-distribution and an increased efflux of leukocytes, pooled in the pulmonary microcirculation . The rheological effects of erythrocytes could, hypothetically, result from mechanical interactions with the leukocytes in the pulmonary microvessels. Anal Quant Cytol Histol, 1993 Feb, 15(1), 23 - 31 Heterogeneity of DNA ploidy, proliferation index and nuclear size in human colorectal carcinomas; Dangou JM et al.; Measurements of DNA ploidy, proliferation index and nuclear area were performed on 210 samples taken from 15 human colorectal tissues . The tissues were divided into four groups labeled G1, G2, G3 and C . For each of the 15 tissues 9 samples were taken from the so-called unaffected--i.e., marginal--mucosa (G1-G3 groups) and 5 from the tumor (C group) . The 9 samples from the unaffected mucosa of each tumor were obtained at a distance of 10 cm (3 samples/tissue, G1 group), 5 cm (3 samples/tissue, G2 group) and 1 cm (3 samples/tissue, G3 group) from the tumor . Computerized cell image analysis was carried out on Feulgen-stained cell suspensions obtained from paraffin-embedded, formalin-fixed tissues . The results revealed that four to five analyses are necessary to detect minor aneuploid cell nuclei populations in human colorectal tumors . A definite homogeneous diploid pattern was found in the G1-G3 samples . In contrast, proliferative activity varied widely between the normal and tumor samples, with such variations observed at both the sample-to-sample and tissue-to-tissue level . The nuclear area also varied markedly across the samples from a given tissue--i.e., both marginal and tumoral and across the tissues themselves . Finally, we observed that the diploid tumors, the nuclear sizes of which varied as widely as those of the aneuploid tumors, possessed a higher proportion of highly proliferating samples than did the aneuploid. J Nat Prod, 1993 Feb, 56(2), 165 - 74 Production of an anti-allergic triterpene bryonolic acid, by plant cell cultures; Tabata M et al.; Cell suspension cultures of Luffa cylindrica, Citrullus lanatus, and related cucurbitaceous plants accumulate large quantities of bryonolic acid (3 beta-hydroxy-D:C-friedoolean-8-en-29-oic acid) {1}, an acidic, pentacyclic triterpene found exclusively in the roots of the intact plants . This compound could readily be isolated from cultured cells with CHCl3 and purified simply by recrystallization . Pharmacological tests using mice demonstrated that bryonolic acid or its derivative is active against at least three types of allergies and that its activity could be increased significantly by preparing synthetic derivatives, in particular a potassium salt of its succinate ester . The biosynthesis of bryonolic acid from mevalonic acid via isomultiflorenol has been elucidated by tracer and enzymological experiments using cultured cells of watermelon both in vitro and in vivo . Furthermore, cell fractionation and electron microscopic studies on subcellular structures of luffa cells suggested that minute vesicles originating from elongated, rough endoplasmic reticulum probably play an important role in the transport of bryonolic acid which largely accumulates in the cell wall of cultured cells . The results obtained from the present study indicate that plant cell culture would be useful not only as a biological system for elucidating biosynthetic mechanisms but also as a potential source of new pharmacologically active compounds. Acta Med Okayama, 1993 Feb, 47(1), 13 - 9 Distribution of lectin receptors in the human hyperplastic tonsil: histochemical and flow cytometric analyses; Sarker AB et al.; The distribution of lectin receptors in the human tonsil was studied using 16 biotinylated lectins . The avidin-biotin-peroxidase complex (ABC) method was used on frozen and paraffin-embedded tissue sections . Cell suspensions were also analysed by dual flow cytometry using respective fluorescein isothiocyanate-conjugated lectins and phycoerythrin-labeled anti-CD3 and anti-human immunoglobulin . Frozen sections fixed with acetone and paraffin-embedded materials fixed in three solutions were compared for lectin affinity; ethanol-fixed sections gave best results followed by frozen and buffered formalin-fixed ones, then nonbuffered formalin . Con-A, RCA-1, LcH, WGA, MPA, PHA, PSA, PNA, SJA and GSA-1 reacted with all tissue components of the tonsil in immunohistochemical studies, but binding intensity was fixative dependent . Binding of Lotus and BPA to lymphocytes was limited to germinal center lymphocytes . Other tissue components were also reactive but staining intensity was weaker in Lotus compared with BPA . SBA and DBA did not react with lymphocytes, but reacted with macrophages/histiocytes, vascular endothelia, and epithelial cells . LBA and LPA were constantly negative with all tissue components irrespective of fixatives . Flow cytometric analyses showed that all but three (DBA, LBA and LPA) partially or totally stained lymphocyte surfaces . Lotus receptors were expressed exclusively on B-lymphocytes. Lymphokine Cytokine Res, 1993 Feb, 12(1), 1 - 8 A tumor-elaborated supernatant factor chemotactic for IL-2 expanded tumor infiltrating T-lymphocytes; Averbook BJ et al.; Very little is known about factors influencing the migration of highly activated T-lymphocytes . One such lymphocyte population is the IL-2 expanded population of T cells infiltrating tumors . These tumor-infiltrating lymphocytes (TIL) can cause tumor regression in patients with metastatic cancer and in murine tumor models when given in adoptive transfer . In patients with melanoma, these TIL have been shown to migrate to sites of tumor and this may be a critical factor in their antitumor activity . In this study, a 48-well microchemotaxis chamber and a 5 microns pore nitrocellulose filter membrane system was utilized to study the motility of murine TIL . A chemotactic response was observed to supernatants from freshly explanted, autologous, and nonautologous tumor cultured for 24 h . Serially passaged autologous and nonautologous tumors also produced supernatants with chemotactic activity . Supernatants from single cell suspensions of normal tissues prepared and cultured identically did not elicit chemotaxis . Chemotactic activity for TIL was not removed by dialysis (2000 MW exclusion limit), its activity was undiminished by heat treatment at 60 degrees C for up