Gastrointest Endosc, 1994 Mar-Apr, 40(2 Pt 1), 146 - 9 An in vitro, randomized, prospective study to maximize cellular yield during bile duct brush cytology; Baron TH et al.; Endoscopic retrograde brush cytology is useful for the evaluation of biliary strictures . Access across a stricture can be maintained by removing the cytology brush while leaving the sheath in the bile duct . We examined the potential for loss of diagnostic cellular material in this setting using the canine biliary system . Twenty consecutive samples were randomly collected by (1) pushing the brush from the end of the sheath or (2) pulling the brush through the length of the sheath . Slides prepared from cell suspensions were scored by a cytopathologist blinded to the collection method . Pulling the brush resulted in a significant loss of cellular material (p < 0.001) . In a second phase, 23 consecutive samples were randomly obtained in the same fashion . Combining salvage cytology of material from the sheath with cytology of the pulled-brush specimens produced cellular yields similar to those of specimens obtained by pushing the brush from the sheath . If the brush is pulled from the sheath during bile duct brush cytology, we suggest that salvage cytology be performed in order to maximize the diagnostic sensitivity. Br J Biomed Sci, 1994 Mar, 51(1), 35 - 43 Monocyte isolation by flow cytometer-monitored centrifugal elutriation: a preparative tool for antibody-dependent cell-mediated cytotoxicity (ADCC) assays; Garner SF et al.; The value of isolating monocytes by flow cytometer-monitored centrifugal elutriation (CE) after initial density gradient centrifugation (DGC) was investigated . A pooled cell suspension prepared by DGC+CE had a monocyte purity of 90% while a pool prepared by DGC alone had a monocyte purity of only 30% . Monocyte suspensions prepared from 15 separate blood donations demonstrated that, despite wide variation in initial monocyte purity following DGC (i.e . 13-32%), CE consistently increased the purity to 85-92% . Antibody-dependent cell-mediated cytotoxicity (ADCC) studies showed that the most sensitive and reproducible assays were those performed with the DGC+CE pool . At a monocyte concentration of 2 x 10(6)/ml the DGC+CE pool was almost twice as active as the DGC pool (60.2% lysis vs 31.2% lysis, respectively) . Considerable variation in ADCC activity was seen when using monocytes from individual donors . We conclude that centrifugal elutriation is a valuable tool for preparing effector cells for use in monocyte driven ADCC assays. Eur J Immunol, 1994 Mar, 24(3), 585 - 92 MRC OX19 recognizes the rat CD5 surface glycoprotein, but does not provide evidence for a population of CD5bright B cells; Vermeer LA et al.; To clone the rat CD5 gene we first produced two rat CD5 probes . The probes were obtained by polymerase chain reaction (PCR) on rat genomic DNA using primers designed on conserved regions between mouse and human CD5 . The screening of a rat cDNA library at high stringency using these probes resulted in a 1.5-kb positive clone . The DNA sequence of this clone confirmed its CD5 nature, but the clone appeared to lack part of the 5' and part of the 3' end . These missing 5' and 3' ends were obtained by PCR on rat thymus RNA . By ligating these PCR products to the original 1.5-kb CDM8 clone, a full-length rat CD5 gene was constructed . The full-length clone showed high identity with mouse and human CD5; however, at the 5' site of the gene a region of 36 nucleotides is present which is not seen in either mouse or human CD5 . We have evidence that this sequence is a normal constituent of the rat CD5 gene: first, it is in frame with the rest of the CD5 coding sequence; second, it does not contain a stop codon; and third, it is also present in the CD5 gene of other rat strains . We transfected the full-length CD5 construct in COS cells and demonstrated that indeed the CD5 protein is recognized by MRC OX19 . Although we showed that CD5 mRNA is present in rat B cells, extensive flow cytometry analysis using MRC OX19 as antibody failed to detect B cells expressing significant levels of CD5 on their cell surface compared to other B cells in any tissue or cell suspension tested from a variety of rat strains . This is in contrast with the mouse where a distinct population of B cells (B-1a cells) can be found expressing more CD5 than the other B cells . Either B-1 cells are not present in rats or CD5 is not the right phenotypic marker for rat B-1 cells . It still remains to be investigated whether a population of B cells with functions similar to those of murine B-1 cells is present in rats. Br J Cancer, 1994 Mar, 69(3), 546 - 9 Flow cytometric analysis of S-phase fraction in breast carcinomas using gating on cells containing cytokeratin . South East Sweden Breast Cancer Group; Wingren S et al.; We investigated distant recurrence and S-phase fraction (SPF), estimated by flow cytometry with and without selection of the epithelial cell population, in 201 stage II breast carcinomas . The tumour tissue was disintegrated mechanically by scissors and one part of the cell suspension was treated with a detergent-trypsin method for single-parameter analysis, and the other part, for immunological selection of epithelial cells, was incubated with a monoclonal antibody (CAM 5.2) recognising cytokeratins 8 and 18 and a secondary fluorescein isothiocyanate-labelled antibody . DNA was stained with propidium iodide . In order to compare the methods, statistical analysis was performed on the 152 tumours with S-phase fraction estimated by both methods . Sixty-five tumours were diploid, 81 were aneuploid and six tumours had different ploidy determined by the two methods . Using univariate regression analysis, SPF of the epithelial cell population predicted recurrence more effectively than SPF from single-parameter analysis . In multivariate regression analysis, SPF of the cytokeratin-containing population added significant prognostic information to the SPF of the non-selected cells . We concluded that the use of flow cytometric selection of epithelial breast carcinoma cells enhances the predictability value of SPF. Transplantation, 1994 Feb 27, 57(4), 563 - 8 Reduction of the incidence of rejection by adjunct immunosuppression with photochemotherapy after heart transplantation; Meiser BM et al.; In this study, photochemotherapy (PCT) was used for adjunct immunosuppression in the first six months after heart transplantation (HTx) . Fifteen patients after orthotopic HTx were included in the study; all received standard triple-drug immunosuppression including cyclosporine, azathioprine and glucocorticoids, but no adjunct therapy with mono- or polyclonal antibodies . The patients were divided into three groups: group I served as control with no additional treatment; group II received adjunct treatment with 10 courses of PCT (single-day treatments); and group III received 20 PCT courses since it was given each time on two consecutive days . PCT was started in both groups on day one after HTx; it was applied with a higher frequency in the early postoperative period and thereafter continued at four-week intervals for a total of 6 months . The photopheresis method for PCT included extracorporeal UVA irradiation of mononuclear cells that were treated with the photosensitive drug 8-methoxypsoralen (8-MOP) and subsequently retransfused to the patient . A new liquid form of 8-MOP was added directly to the buffy coat, resulting in reliable and sufficient drug levels in the cell suspension during the irradiation period; problems caused by oral application due to unpredictable variations in gastrointestinal absorption were thus prevented . Analysis of the total numbers of acute rejection episodes (AREs) within the first four weeks after HTx revealed a more impressive decrease by double PCT (group III, 3 AREs) than by single PCT (group II, 5 AREs) in comparison with the control (group I, 6 AREs) . Over the total observation time (mean: 9.6 months), however, both PCT schedules reduced the total number of AREs observed in the control group (20 AREs) equally by more than 50% (9 AREs each in groups II and III) (P = 0.007) . Furthermore, PCT treated patients had significantly fewer infections (6 infections in each group) than control group patients (15 infections) (P = 0.026); this, however, may be accounted for by the higher number of acute rejections in the control group and consequent increase in unspecific immunosuppression treatment . Our results suggest that PCT is a safe and effective method of adjunct immunosuppression that can be applied early in the postoperative period; it reduces the number of rejection episodes and does not increase the risk of infections. J Immunol, 1994 Feb 15, 152(4), 1888 - 97 Acute inflammatory activity of the S100 protein CP-10 . Activation of neutrophils in vivo and in vitro; Devery JM et al.; The murine S100 chemotactic protein of m.w . 10,000 termed (CP-10), has potent chemotactic activity for murine and human myeloid cells . We examined the ability of a synthetic CP-10 hinge region peptide CP-10(42-55) and rCP-10 to act as chemotactic agents and induce expression of the adhesion molecule Mac-1 (CD 11b/CD 18) in vivo . Maximal neutrophil (PMN) accumulation occurred between 2 to 8 h after mouse footpad injection of rCP-10 (10(-7) M) or CP-10 peptide (10(-6) M) . The infiltrating PMN expressed high levels of Mac-1, and low levels of the murine L-selectin Mel-14 . Injection of CP-10 peptide i.p . also induced infiltration of PMNs that expressed high levels of Mac-1 . Cell suspensions obtained after i.p . injection of CP-10 peptide could be significantly inhibited from adhering to fibrinogen-coated plates when incubated with anti-Mac-1 antibody . The chemotactic activity of CP-10 peptide toward murine inflammatory PMN in vitro was also inhibited by anti-Mac-1 antibody . Neither CP-10 analogue stimulated or primed murine inflammatory or human blood neutrophils for superoxide production or granular enzyme release . The localization of CP-10 in vivo was examined using murine footpads injected with LPS and was found to be concentrated around the endothelial cells of the small blood vessels . This distribution suggests that the accumulated CP-10 the may contribute to the generation of a chemotactic gradient. Thromb Res, 1994 Feb 15, 73(3-4), 205 - 14 Platelet aggregation inhibition by mononuclear leukocytes; Schattner MA et al.; In this study we have investigated the effect of human mononuclear leukocytes (ML) on platelet aggregation . The results obtained demonstrated that coincubation of platelets with nonstimulated ML decreased platelet aggregation induced by collagen or thrombin in a concentration-dependent manner . The inhibitory effect increased with the incubation period of the cells, reaching a plateau at 5 minutes . T and non-T enriched ML suspensions exerted an inhibitory effect similar to the total population of ML . Supernatants from ML or mixed cell suspensions also diminished platelet aggregation . 6-keto PGF1 alpha concentration in the supernatants was less than 10 pg/ml . Hemoglobin, L-arginine and cytochrome C did not modify the antiaggregating activity of ML, whereas superoxide dismutase potentiated the inhibition of aggregation mediated by ML . The inhibitory effect was not modified by monoclonal antibody (MoAb) against the lymphocyte function-associated antigen 1, alpha subunit (LFA-1 alpha) or by a MoAb directed against P-selectin . Our results demonstrated that ML inhibited platelet aggregation, at least partially, by the release of a soluble factor(s) distinct of prostacyclin or nitric oxide . Surface adhesion molecules seem also not to be involved. Exp Neurol, 1994 Feb, 125(2), 268 - 77 Myelination by cryopreserved xenografts and allografts in the myelin-deficient rat; Archer DR et al.; This study examined the ability of freshly prepared and cryopreserved canine oligodendrocytes to myelinate axons following transplantation into the myelin deficient (md) rat . The effects of immunosuppression, and the age of the donor tissue, were also examined . Canine glial cells, dissociated from the spinal cords at E50, P2, P20, P28, and P50, were transplanted into the spinal cords of myelin-deficient rats as single cell suspensions . Both cryopreserved (E50 and P28) and freshly dissociated tissue (P2, P20, and P50) were able to form myelin within 13 days of transplantation . Cells from younger donors (< P20) myelinated more md axons than those from older donors . In those rats which received xenografts and which were treated with cyclosporin A there was markedly less cellular infiltration than in untreated animals . For comparison with these xenografts, fresh and cryopreserved adult rat glia were also transplanted . Eight days after transplantation, myelination by allografts of cryopreserved rat glia was qualitatively similar to that produced by freshly prepared cells . These results show that oligodendrocytes transplanted as xenografts are capable of myelinating rat axons, and that cryopreserved glia retain their capacity to myelinate in vivo. Anesthesiology, 1994 Feb, 80(2), 372 - 82 Volatile anesthetic effects on sarcoplasmic reticulum Ca content and sarcolemmal Ca flux in isolated rat cardiac cell suspensions; Wheeler DM et al.; BACKGROUND: Cardiac cellular Ca metabolism is central to the control of the inotropic state of the heart and is altered in various ways by the volatile anesthetics halothane, enflurane and isoflurane . Specifically, differences among the agents regarding their effect on the uptake and release of Ca from the sarcoplasmic reticulum (SR) have been found, but the nature of such differences is not yet certain . At the sarcolemma, the effects of the anesthetics on the peak Ca current generally are believed to be similar among the three agents, but their impact on other aspects of sarcolemmal Ca transport is less understood . The authors sought to measure the direct action of these agents on SR Ca content and, in the same preparation, to provide a measure of Ca transfer across the sarcolemma during sustained depolarizations . METHODS: In stirred suspensions of quiescent rat cardiac cells, the effects were measured of halothane, enflurane, and isoflurane on changes in quin2Ca fluorescence produced by the addition of caffeine (10 mM) and by depolarization with increased extracellular K+ . The peak of the fluorescence response to caffeine, which is due to a sudden release of Ca from the SR into the cytoplasm, was used as an index of SR Ca content . Analysis of the fluorescence increase that occurred after increasing extracellular K+ from 5 mM to 30 mM in the presence of caffeine provided a measure of net Ca influx across the sarcolemma during sustained depolarizations . RESULTS: The Ca channel blocker nitrendipine maximally inhibited 77% of the initial net Ca influx during 30 mM K+ depolarization, indicating that most of this influx involves L-type Ca channels . Of the volatile anesthetics, isoflurane (2.6 vol% or 0.57 mM) and enflurane (4.3 vol% or 1.25 mM) inhibited initial net Ca influx during K depolarization significantly more than halothane (1.7 vol% or 0.50 mM), which had no apparent effect . Isoflurane caused no transient change in cytoplasmic Ca concentration and had no effect on the SR Ca content of these quiescent cells . Enflurane (4.3 vol%) caused a significant reduction in SR Ca content . CONCLUSIONS: As previously reported, halothane depleted the SR of Ca in quiescent rat cardiac cells, and the present results indicate that enflurane had a similar effect . However, isoflurane did not produce any SR Ca depletion and thus must not significantly alter the balance between SR Ca efflux and uptake in these quiescent cells . The different effects of the three volatile anesthetics on a Ca influx largely carried by L-type Ca channels stand in contrast to the reported findings of similar inhibition of peak L-channel current among the three agents . This result may indicate a differential action (at least in the case of halothane) on peak and steady-state Ca currents. J Neurosci, 1994 Feb, 14(2), 697 - 711 Cholinergic basal forebrain transplants restore diminished metabolic activity in the somatosensory cortex of rats with acetylcholine depletion; Jacobs SE et al.; It has been known for several years that stimulus-evoked metabolic activity is reduced in the somatosensory cortex of animals with basal forebrain lesions that deplete the neocortex of acetylcholine (ACh) . During 2-deoxyglucose (2-DG) experiments, animals with unilateral basal forebrain lesions demonstrate a decreased response to somatic stimulation, while background metabolic activity in the surrounding cortical regions remains normal . In an attempt to ameliorate these deficits, we examined the ability of embryonic cholinergic basal forebrain transplants inserted into neocortex to innervate surrounding cortical regions and restore functional 2-DG activity in adult host rats previously depleted of ACh by basal forebrain lesions . To accomplish this goal, a series of experiments were conducted in which we (1) depleted the cerebral cortex of ACh by injecting an excitotoxin into the rat basal forebrain, (2) transplanted embryonic basal forebrain or embryonic neocortical (control) tissue into the ACh-depleted cortex and, (3) 6-12 months later, used the 2-DG metabolic mapping technique to examine effects of the transplants on metabolic activity evoked by whisker stimulation in rat somatosensory (barrel) cortex . Histochemical analysis revealed that acetylcholinesterase (AChE) staining within 2 mm of the basal forebrain transplants was similar in density to the contralateral normal hemisphere . AChE staining farther than 2 mm from the basal forebrain transplants and throughout hemispheres containing neocortical (control) transplants was greatly reduced, with few AChE-positive fibers present, a finding typical of cerebral cortex in basal forebrain-lesioned animals . Stimulus-evoked 2-DG uptake in barrels adjacent to the basal forebrain transplants, and therefore within AChE-rich territory, was similar to that in corresponding barrels identically activated in the contralateral hemisphere . 2-DG activity was reduced, however, in stimulated barrels outside the region of dense AChE-positive staining, as well as in all activated barrels in hemispheres containing control transplants of embryonic neocortex . These results indicate that transplantation of cell suspensions containing embryonic cholinergic basal forebrain, but not neocortex, can ameliorate basal forebrain lesion-induced deficits in functional activity, and that the restoration of activity is influenced by proximity to the transplant. J Vet Med Sci, 1994 Feb, 56(1), 161 - 3 Circadian oscillation of 64-kDa polypeptide in the rat suprachiasmatic nucleus; Nishi R et al.; In cell suspensions of suprachiasmatic nucleus harvested every 3 hr from rats kept under 12 hr: 12 hr light-dark cycle and constant darkness, we have detected a M(r) 64-kDa protein whose synthesis exhibits two distinct daily peaks in SDS-PAGE . Analysis of densitometer tracings revealed that the synthesis of other proteins was independent of the time of day or not reproducible . Maximum synthesis of the 64-kDa polypeptide occurred at around CT6 and CT21, which are almost coincident with the phase advance regions of circadian activity rhythm induced by anisomycin and light pulses {15}, respectively . These results suggest that the 64-kDa protein in SDS-PAGE may be a part of the circadian clock mechanism. Anal Cell Pathol, 1994 Feb, 6(2), 137 - 47 Computer assisted image analysis of bladder tumour nuclei for morphonuclear and ploidy assessment; Colombel MC et al.; Morphonuclear analysis using a quantitative image analysis system has been demonstrated to be a potentially useful technique in the prognostic evaluation of bladder carcinoma . The integrated optical density parameter permits DNA content evaluation in addition to 15 other morphonuclear parameters . We assessed the reliability of morphonuclear analysis by image analysis and flow cytometry for 46 bladder carcinomas and 14 normal bladder specimens . Frozen sample material was obtained from endoscopic resection, radical cystectomy and from cadaveric donors . The grade and staging of the tumours according to the World Health Organization was as follows: 8 G1, 18 G2, 20 G3 and 28 T1, 7 T2, 7 T3, 4 T4 . Quantitative image analysis was made on imprint smears stained by the Feulgen method . Simultaneously cell suspensions were obtained by mechanical dissociation and stained with propidium iodide for Flow cytometry analysis . There was a good agreement between quantitative image analysis and flow cytometry for DNA content measurement indicating that image analysis is a reliable method for the quantitation of DNA content (P = 0.001) . Moreover we found a good correlation between five of the morphonuclear parameters: surface (P < 0.001), chromatin clumps distribution (P < 0.001), frequency of small (P < 0.001) to large chromatin clumps (P < 0.001) and the grade of bladder tumours . These results indicate that morphonuclear analysis may be a valuable method of quantitating DNA and morphonuclear parameters in a single analysis to provide information which may have some prognostic significance for patients with bladder carcinoma. Vet Immunol Immunopathol, 1994 Feb, 40(2), 105 - 17 Distribution of lymphocyte subpopulations in thymus, spleen, and peripheral blood of specific pathogen free pigs from 1 to 40 weeks of age; Joling P et al.; Using flow-microfluorometry analysis and cluster determinant (CD) markers, we studied how lymphocyte subpopulations in lymphoid organs of specific-pathogen-free pigs developed in pigs from birth to young adulthood . Cell suspensions of the thymus and spleen were prepared and peripheral blood cells were collected at 1, 4, 10, and 40 weeks of age . Tissue sections of the thymus and spleen were stained with monoclonal antibodies directed against CD2 and immunoglobulin to localize the CD2-Ig- lymphocyte subpopulation . In the thymus, only limited changes were observed in the lymphocyte subpopulations with time . Most thymocytes expressed CD4 or CD8 or both . Most CD2-Ig- cells or, 'null cells', (5-13%) were observed in the medulla of the thymus and probably represented a recirculating cell type . In the spleen and blood the percentage of CD2+ and Ig+ cells increased significantly with time, the former increasing from about 30-60% owing to an increase of CD8+ cells . Therefore, the selective increase of the CD8+ population also caused the CD4/CD8 ratio to change . Although CD2+ cells in the spleen and blood are positive for CD4 or CD8, but not for both, quantities of CD4+ CD8+ cells were also observed . Half of the lymphocytes in the spleen and blood were typed as null cells at 1 week of age and decreased in proportion to the increase of the CD8+ and Ig+ cells . Nevertheless, quantities of null cells were still present in the spleen blood at 40 weeks of age . Almost all these were located in the red pulp of the spleen . This study indicates an effect of age and housing conditions on the distribution of the lymphocyte subpopulations, and especially on the CD8+ subset . Quantities of CD4+CD8+ cells as well as CD4-CD8- were observed in blood, but also in spleen of pigs . The function of high numbers of null cells directly after birth are discussed. Cryobiology, 1994 Feb, 31(1), 31 - 8 Localization of freezing injury in articular cartilage; Muldrew K et al.; In order to improve techniques for cryopreservation of articular cartilage, a study has been carried out to assess localization of cryoinjury in intact articular cartilage . Osteochondral dowels taken from the femoral condyles of sheep were subjected to graded freezing in the presence and absence of a cryoprotectant (10% DMSO) . The graded freezing technique involves slow cooling (1 degree C/min) to various subzero temperatures before either rapid warming or rapid cooling by plunging in liquid nitrogen . This protocol allows assessment of the separate effects of rapid and slow freezing which damage cells in different ways, and the effects of cryoprotectants on the different types of damage . To assay damage, thin slices of cartilage were cut with a vibratome, which allows viable cells within the matrix to be observed microscopically . Injury was assessed by staining with fluorescent dyes to indicate damage to the plasma membrane . In general, tissue response was similar to that of cell suspensions, showing at least two mechanisms of injury acting on the cells: one at slow cooling rates and another at rapid cooling rates . The primary effect of DMSO was to reduce injury due to slow cooling . When the location of injury within the tissue was examined, it was found that chondrocytes of the intermediate layer were injured more extensively than those of either the deep or superficial layers. J Immunother Emphasis Tumor Immunol, 1994 Feb, 15(2), 105 - 12 The use of interleukin-6 to generate tumor-infiltrating lymphocytes with enhanced in vivo antitumor activity; Marcus SG et al.; Tumor-infiltrating lymphocytes (TIL) are cytotoxic T cells isolated from solid tumors and expanded in vitro in recombinant interleukin-2 (rIL-2) . TIL have antitumor effects in murine models and in some patients with melanoma . In an effort to generate murine TIL with enhanced in vivo therapeutic efficacy, viable tumor cells were coinjected with a collagen matrix plus recombinant human IL-6 (rIL-6) subcutaneously into syngeneic mice to achieve sustained local concentrations of rIL-6 at the tumor site from which TIL were derived . In five separate experiments, single cell suspensions of tumors were admixed with either (a) Hanks' balanced salt solution (HBSS), (b) 2% (20 mg/ml) collagen matrix only, (c) 250 micrograms rIL-6 only, or (d) 250 micrograms rIL-6 in a 2% collagen matrix (prolonged release) before subcutaneous inoculation . These tumors were subsequently resected and TIL were isolated and expanded in vitro . TIL generated from tumors admixed with matrix plus rIL-6 were significantly more effective than TIL expanded from tumors admixed with HBSS (four of five experiments), TIL from tumors admixed with matrix only (five of five experiments), and TIL from tumors admixed with rIL-6 only (three of four experiments) in an established tumor treatment model . In no experiment was any other TIL culture superior to TIL grown from tumors augmented with collagen matrix plus rIL-6 . These results suggest that strategies designed to increase the local concentrations of cytokines at tumor sites may lead to the generation of more potent TIL for clinical administration. Radiat Res, 1994 Feb, 137(2), 196 - 201 Visible-light and X irradiations of Chinese hamster lung cells treated with hematoporphyrin derivative; Kavarnos G et al.; Single cell suspensions of Chinese hamster lung cells were treated with hematoporphyrin derivative (HPD) and were exposed under aerobic conditions to visible light alone, X rays alone or light and X rays concurrently . Cytotoxicity was assayed using the colony formation ability of cells as the end point . The drug toxicity, phototoxicity, radiosensitization and photoradiosensitization of HPD were examined for a drug concentration of 1 microgram/ml and incubation time of 24 h . In these experiments, the X-ray dose was 3 Gy and the energy fluence of visible light was 0.35 kJ/m2, and both irradiations lasted for 10 min . A significant enhancement in cytotoxicity was observed when the HPD-treated cells were irradiated concurrently with light and X rays . However, no significant enhancement was observed when visible-light and X irradiations were performed sequentially with a 15-min waiting time between the two irradiations. Plant Mol Biol, 1994 Feb, 24(3), 505 - 14 Selection for kanamycin resistance in transformed petunia cells leads to the co-amplification of a linked gene; Jones JD et al.; A cell suspension culture was established from a transgenic petunia (Petunia hybrida L.) plant which carried genes encoding neomycin phosphotransferase II (nptII) and beta-glucuronidase (uidA, GUS) . Two selection experiments were performed to obtain cell lines with increased resistance to kanamycin . In the first, two independently selected cell lines grown in the presence of 350 micrograms/ml kanamycin were eight to ten-fold more resistant to kanamycin than unselected cells . Increased resistance was correlated with amplification of the nptII gene and an increase in nptII mRNA levels . Selection for kanamycin resistance also produced amplification of the linked GUS gene, resulting in increased GUS mRNA levels and enzyme activity . Selected cells grown in the absence of kanamycin for twelve growth cycles maintained increased copy numbers of both genes, and GUS enzyme activity was also stably overexpressed . In a second selection experiment, a cell line grown continuously in medium containing 100 micrograms/ml kanamycin exhibited higher nptII and GUS gene copy numbers and an increase in GUS enzyme activity after eleven growth cycles . In this cell line, amplification of the two genes was accompanied by DNA rearrangement. Anal Quant Cytol Histol, 1994 Feb, 16(1), 25 - 8 Correlation of flow cytometric and microdensitometric DNA content analysis in lung cancer; Kuo SH et al.; The DNA histograms of 29 histologically verified primary lung cancers were analyzed with flow cytometry and microdensitometry . Fresh tumor tissues were imprinted on the slide and air dried for modified Feulgen staining . Then the smears were scanned with a scanning microdensitometer at a wavelength of 550 nm . The S-phase fraction value was estimated from the DNA histogram with a cumulated probability scale . The remaining parts of the tumors were minced and prepared for dispersal as a single cell suspension for propidium iodide staining . The tumor cells were then analyzed with a flow cytometer at 488 nm . The correlation between the analyses measured with these two methods showed r = .75 for the DNA index, r = .71 for the S-phase fraction and r = .61 for the G0G1 population; P < .001 . The DNA indices, S-phase fraction values and G0G1 populations obtained with flow cytometry and microdensitometry correlated well. Eur J Cell Biol, 1994 Feb, 63(1), 68 - 76 Intracellular free calcium concentrations in cell suspensions during hyperthermia; Wierenga PK et al.; The aim of this study was to explore the possibility that heat-induced alterations in calcium homeostasis are the cause of hyperthermic cell killing . Therefore, the intracellular free calcium concentration ({Ca2+}i) was determined spectrofluorometrically, using the fluorescent calcium probe fura-2/acetoxy methylester (AM), at both physiological and hyperthermic temperatures in cell suspensions from six different tumor cell lines . For all cell lines fura-2 leakage appears to contribute to a change in the fluorescence signal and hence leads to a false indication of an increase in {Ca2+}i, especially at the hyperthermic temperature . Two methods were introduced that circumvent this problem and results in true values of {Ca2+}i . Also, measurements of {Ca2+}i in single cells using a fluorescent microscopical technique (not affected by dye leakage) were used for comparison . All three approaches show that a hyperthermic treatment that kills > 90% of the cells does not lead to changes in the {Ca2+}i in most cell lines . Therefore, heat-induced alterations of calcium homeostasis cannot be considered the general cause for hyperthermic cell killing. Bull Tokyo Dent Coll, 1994 Feb, 35(1), 33 - 9 Effects of low molecular chitosan on pH changes in human dental plaque; Shibasaki K et al.; Effects of six kinds of low molecular chitosan (LMCS) on pH changes in dental plaque were compared in vitro and in vivo in order to clarify the relationship between their characteristics and effectiveness . Six LMCS with different molecular weights (MW) (500-3,000) and degrees of deacetylation (DAC) (50-95%) were prepared for this study . Evaluations using S . mutans cell suspensions in vitro showed that all samples had almost equal abilities to reduce pH fall in dental plaque . They were found to have no influence on the glycolytic activity of S . mutans . However, clinical evaluations of pH fall in dental plaque using an ion-sensitive field-effect transistor (ISFET) electrode showed unexpected results as follows; The most reduction in pH fall was obtained with the LMCS with a MW of 3,000 and a DAC of 50%, which had been presumed to be the most ineffective sample . The second ranked LMCS in this experiment had a MW of 500 and a DAC of 95%; it had been expected to be the most effective one . The findings indicated that LMCS has a high ability to inhibit pH fall in dental plaque and suggested that such properties as molecular weight and degree of deacetylation can have great influences upon its activity in clinical application. Exp Neurol, 1994 Feb, 125(2), 228 - 46 Behavioral assessment of the effects of embryonic nigral grafts in marmosets with unilateral 6-OHDA lesions of the nigrostriatal pathway; Annett LE et al.; Grafts of embryonic nigral tissue were made into the striatum of marmosets (Callithrix jacchus) which had previously received a unilateral 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal bundle . The grafts comprised injections of cell suspensions prepared from embryonic (74 day) marmoset ventral mesencephalic tissue targeted at multiple striatal sites in the caudate nucleus, the putamen, and the nucleus accumbens on the same side as the initial lesion . A series of behavioral tests was used to assess the monkeys prior to surgery, following the 6-OHDA lesion, and at regular intervals for 6 months after transplantation surgery . Lesioned and grafted (n = 6) or lesion alone (n = 4) monkeys were matched as far as possible with respect to their scores prior to transplantation so that explicit graft-derived recovery could be distinguished from any spontaneous recovery that might occur . Sham-lesioned or unoperated monkeys served as further controls (n = 5) . The grafts were functionally effective as measured by a reduction, and in some cases a reversal, of spontaneous, amphetamine- and apomorphine-induced rotation . The reversal of amphetamine-induced rotation correlated with the number of dopaminergic neurons in the grafts visualized by tyrosine hydroxylase immunohistochemistry . Successful use of the hands was restored by the grafts on tasks in which the monkeys reached into tubes to retrieve food . However, functional recovery was not seen on some other behavioral tests . In particular, grafts did not influence ipsilateral biases induced by the lesion, including the position of the head with respect to the rest of the body, hand preference while reaching for food at a conveyor belt, and neglect of contralateral stimuli either at the conveyor belt or of adhesive labels placed around the feet . Indeed, the graft group was impaired compared with the lesion group in the accuracy of reaches at the conveyor belt . Overall, these results indicate that embryonic nigral grafts can yield a partial recovery from the symptoms induced by unilateral nigrostriatal lesions in a primate model of hemiparkinsonism. Appl Microbiol Biotechnol, 1994 Feb, 40(6), 851 - 6 Expression of recombinant antibody and secreted alkaline phosphatase in mammalian cells . Influence of cell line and culture system upon production kinetics; Racher AJ et al.; The growth and productivity of an Sp2/0 cell line, F3b10, expressing a recombinant antibody (rAb) and BHK21 cells expressing either the same rAb from the same plasmids (BHK.IgG) or secreted alkaline phosphatase (SEAP) (BHK.SEAP) were investigated . The F3b10 line was grown as a single cell suspension . The BHK lines were grown either as suspended natural aggregates or on Cytodex 3 microcarriers . The data for F3b10 showed that the cell-specific rAb production rate (QsrAb) increased in parallel with increases in the specific growth rate (mu) . A similar result was obtained for suspended aggregate cultures of both recombinant BHK cell lines . In contrast, for microcarrier cultures of both BHK cell lines, Qsproduct increased as mu decreased . This report shows that the relationship between cell growth and Qsproduct for the cell lines and products studied is dependent upon the culture process . In systems where recombinant cells are growing as a single cell suspension or within a natural suspension aggregrate, Qsproduct increased with increases in mu . In such systems, the cells have a rounded morphology . When cells were grown on microcarriers . Qsproduct decreased as mu increased . Cells growing attached to a surface are flat and elongated . The observed differences in the relationship of Qsproduct to mu are correlated with changes in cell morphology . The relationship between Qsproduct and mu is also affected by the choice of cell line. Immunology, 1994 Feb, 81(2), 303 - 8 Langerhans' cell expression of the selectin ligand, sialyl Lewis x; Ross EL et al.; Cellular adhesion molecules play a central role in leucocyte migration through peripheral blood and tissues . A crucial stage in these events in selectin-mediated adhesion involving E-selectin expressed on activated endothelium interacting with a range of carbohydrate ligands expressed by specific subpopulations of leucocytes . As such mechanisms may be relevant to bone marrow-derived dendritic epidermal Langerhans' cell (LC) migration, expression of these carbohydrate ligands was assessed immunocytochemically in whole skin biopsies and in epidermal cell suspensions obtained from adult humans . Double-labelling experiments revealed that sialyl Lewis x, recognized by the monoclonal antibody CSLEX1, was expressed on epidermal LC (n = 9) . Furthermore, expression was enhanced at 24 hr following epicutaneous application of antigen and in the inflammatory disorder psoriasis (n = 10) . E-selectin was concomitantly strongly expressed on dermal endothelium in psoriasis and allergic contact dermatitis . Intradermal injection of the T-cell-derived cytokine interferon-gamma (IFN-gamma) led to increased LC expression of sialyl Lewis x . In epidermal cell suspensions, in contrast to keratinocytes, CD1a+ cells expressed sialyl Lewis x, intensity of which was enhanced after 4 days in culture . CSLEX1 staining could be abolished and CD15 (non-sialated Lewis x) expression induced by saponification and treatment with neuraminidase . Expression of other selectin ligands was also examined . While the cutaneous lymphocyte antigen defined by the monoclonal antibody HECA-452 reacted with a small minority of LC, sialyl Lewis a and sulphatide were not expressed under any experimental conditions . These studies indicate that E-selectin-sialyl Lewis x interactions are potentially important in LC migration, both into and out of skin. FEMS Microbiol Lett, 1994 Jan 15, 115(2-3), 335 - 9 Study of the influence of plasmids on the arbitrary primer polymerase chain reaction fingerprint of Escherichia coli strains; Elaichouni A et al.; To study the effect of plasmids on the arbitrary primer-polymerase chain reaction fingerprint of bacterial strains, the Escherichia coli strains DH5, Top10, and W3110 were transformed with plasmids of different sizes: respectively, pUC19, pCEP and two clinically important plasmids carrying resistance to several antibiotics . Total DNA, i.e . both chromosomal and plasmid DNA, was prepared from transformed cells by boiling the cell suspensions and by phenol-chloroform extraction; chromosomal DNA was prepared by the same methods from the non-transformed, plasmid-free strains; plasmid DNA of pUC19 was purchased; plasmid DNA of pCEP was purified from the transformed strains by caesium chloride density gradient centrifugation . Arbitrarily primed polymerase chain reaction was carried out for all of these preparations . Amplification carried out independently with three different primers resulted in similar patterns for the chromosomal preparations whether or not plasmid was present . Amplification of plasmid DNA gave different patterns, characterized by fragments larger than those obtained when total or chromosomal DNA were used as the target . These data illustrate that the plasmids studied here do not influence the chromosomal arbitrarily primed PCR fingerprint, although plasmids alone are amplified in the absence of chromosomal DNA . Experiments comparing different relative concentrations of plasmid and chromosomal DNA indicate that under natural conditions the amount of chromosomal DNA per cell is sufficient to inhibit observable amplification of the plasmid(s) present. J Biotechnol, 1994 Jan 15, 32(1), 1 - 10 Transgenic Indica rice (Oryza sativa L.) plants obtained by direct gene transfer to protoplasts; Ghosh Biswas GC et al.; We have established a reproducible procedure for transformation of protoplasts and regeneration of transgenic plants for an improved Indica rice cultivar IR43 . Mature embryo-derived calli were placed in liquid culture medium containing maltose to establish meristematically active, embryogenic cell suspension lines . In order to obtain transgenic plants, a chimeric hygromycin phosphotransferase hph gene under the control of the cauliflower mosaic virus CaMV 35S promoter was introduced into protoplasts from these cell suspension lines using polyethylene glycol . Protoplasts were cultured in maltose-containing medium . Hygromycin B selection was applied to 14-day-old dividing cell colonies . Resistant calli were readily obtained after 3 weeks of selection . Seventy-three plantlets were regenerated from resistant calli from several independent experiments, and a few of the 29 plants grown in the greenhouse reached maturity . Stable integration of the transgene in the genome of these plants was confirmed by Southern blot analysis and the expression of the transgene in plants by hygromycin phosphotransferase assay . The procedure described yielded 5 to 18 resistant colonies and approximately four transgenic plantlets per million treated protoplasts. Biochem Biophys Res Commun, 1994 Jan 14, 198(1), 236 - 42 Occurrence of specific sterols in Pneumocystis carinii; Florin-Christensen M et al.; We have examined the sterol composition and biosynthesis of rat Pneumocystis carinii . We found a number of lipid components among the nonsaponifiable fraction that appear unique to P . carinii . These lipids are present in P . carinii purified preparations and P . carinii-infected whole lungs but absent from control lungs . They show chromatographic properties typical of sterols on GC and HPLC; they are present in free and esterified sterol fractions isolated by thin layer chromatography, they are precipitated by digitonin and they are labeled upon incubation of purified P . carinii cell suspensions with {3H} mevalonate and {3H} squalene . The finding of these sterols opens new opportunities for the identification of chemotherapeutic targets against P . carinii. Brain Res, 1994 Jan 7, 633(1-2), 133 - 43 Improved graft survival and striatal reinnervation by microtransplantation of fetal nigral cell suspensions in the rat Parkinson model; Nikkhah G et al.; A microtransplantation approach has been used in order to achieve more complete reinnervation of the dopamine denervated rat striatum by fetal nigral cell suspensions injected into multiple striatal sites . A total of 450,000 cells, obtained from the ventral mesencephalon of embryonic day 14 rat fetuses, were implanted either in the conventional way as two 1.8-microliters deposits centrally in the head of the caudate-putamen ('Macro grafts'), or as eighteen 0.2-microliter deposits disseminated over six needle penetrations in the same area using a 50-70 microns glass capillary tip ('Micro grafts') . Non-grafted lesioned rats served as controls . Dopamine neuron survival (as assessed by tyrosine hydroxylase immunohistochemistry at 4 months after transplantation) was 2.8-fold greater in the Micro grafts as compared to the Macro grafts . Striatal dopamine tissue levels (determined in a separate group of rats) was increased 2.5-fold in the head of the caudate-putamen (from 12.5% of normal in the Macro graft group to 30% of normal in the Micro graft group) . Consistent with this, the overall graft-derived tyrosine hydroxylase positive fiber outgrowth was more extensive in the Micro graft group and covered larger areas of the previously denervated caudate-putamen . The results show that distribution of the fetal nigral tissue in multiple small deposits provides for increased dopamine neuron survival, probably because of a closer contact between the implanted cells and the surrounding host striatal tissue in the small-sized graft deposits . Less bleeding and necrosis at the implantation site may also have contributed to this effect . The present microtransplantation procedure is an efficient means to increase overall dopamine neuron survival and to achieve more complete reinnervation of the denervated striatum in the rat Parkinson model . It also substantially increased the reproducibility of DA graft survival between animals. Thymus, 1994-95, 24(1), 9 - 28 Isolation, cultivation and phenotypic characterization of rat thymic dendritic cells; Ilic V et al.; Rat thymic dendritic cells (TDC) were isolated from thymic cell suspension by Nycodenz gradients of different densities and osmolarities . After cultivation of these cells for 3 days in the conditioned medium (TE-R 2.5 + HT supernatant) prepared by cocultivation of a medullary thymic epithelial cell line (TE-R 2.5) and hydrocortisone resistant thymocytes, the purity (75-85%) and survival of TDC significantly increased . The supernatant contained moderate activities of IL-1 and IL-6, low levels of TNF-alpha and IL-2 and factor(s) that strongly stimulated the proliferation of a mouse macrophage cell line RAW 264.7 . TDC survival in culture was significantly increased by GM-CSF and decreased by IL-6 and IFN-gamma . The phenotype of TDC was studied by flow cytometry using a panel of monoclonal antibodies (mAb) to rat cell surface markers . It was found that almost all freshly isolated TDC expressed major histocompatibility complex (MHC) class I and class II molecules as well as CD45 . Most TDC were LFA-1 (CD11a)+, CD18+, ICAM-1 (CD54)+ and CD53 (OX- 44)+, but only certain subsets expressed CD11b and thymocyte markers Thy1, CD2, CD4 and CD8 . Upregulation in the expression of almost all the markers was observed after cultivation of TDC . In addition, cultivated, but not freshly isolated TDC expressed CD25 (IL-2R alpha) and CD45RC (OX-22) molecules . Cultivated TDC had strong accessory function in autologous thymocyte proliferation. J Appl Biomater, 1994 Winter, 5(4), 361 - 7 Improved sensitivity and decreased sample size in a cytotoxicity test for biomaterials: a modified colony microassay using a microplate and crystal violet staining; Tsuchiya T et al.; Modified Eagle's minimum essential medium supplemented with 5% fetal calf serum was highly sensitive to cytotoxicity and formed large control colonies in the V79 colony assay . A highly sensitive cytotoxicity test was developed using 96-well microtiter plates . Test chemicals or extracts of polyurethane materials containing the same chemicals were added 24 h after inoculation of cell suspensions . The cells were fixed and stained with crystal violet after additional culture for 6 days (V79 cells) or 10 days (Balb/3T3 cells) . In terms of sensitivity and rapid quantitative measurement, this modified colony microassay, using a low cell density in 96-well microplates, was superior to various cytotoxicity tests such as colony, growth inhibition, cytolethality, and agar diffusion assays. Am J Surg, 1994 Jan, 167(1), 67 - 72 Colonic mucosal replacement by syngeneic small intestinal stem cell transplantation; Tait IS et al.; A novel method of colonic mucosal replacement by transplantation of disaggregated small intestinal epithelium is described . Thirty-one inbred rats had the ascending colon isolated, and surgical mucosectomy was performed on the "free" loop . Epithelial cell aggregates were isolated from postnatal small intestine using collagenase and dispase digestion, then 20 microL of the cell suspension was "seeded" over the denuded colonic muscle of 25 recipient rats . Six control rats had surgical mucosectomy only . All loops were retrieved after 14 days for histologic examination . Stem cell lineage studies were used with selective staining protocols to identify enterocytes, goblet cells, entero-endocrine cells, and Paneth cells . A neomucosa with typical small bowel morphology including crypts and villi and all four stem cell lineages was regenerated by transplanted cells on the colonic muscle in 19 of 25 (76%) recipients . Control loops showed no epithelial regrowth confirming total mucosectomy . With appropriate stromal support, transplanted small intestinal stem cells have the capacity to re-epithelialize denuded colonic muscle with small bowel neomucosa. Am J Physiol, 1994 Jan, 266(1 Pt 1), C58 - 66 Characteristics and regulation of a muscarinically activated K current in HSG-PA cells; Izutsu KT et al.; Whole cell currents were measured in HSG-PA cells (a proposed model for salivary gland duct cells) after muscarinic receptor activation or exposure to known signaling agents . Exposure to carbachol or oxotremorine M produced large and often oscillatory increases in outward current whose reversal potentials indicated a K current . The current was sensitive to extracellular atropine, charybdotoxin, and quinine, but not apamin, and to 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in the pipette . The response was prolonged or increased by guanosine 5'-O-(3-thiotriphosphate) and mimicked by D-myo-inositol 1,4,5-trisphosphate (IP3) or heparin in the pipette and by extracellular Ca ionophores . Tetraethylammonium indirectly inhibited the response via the muscarinic receptor . Fura 2 in cell suspensions showed that muscarinic agonists increased cytosolic Ca ion concentration ({Ca2+}i) five- to sevenfold, and measurements with indo 1 in individual cells showed that the oscillatory changes in outward current were tightly correlated with parallel changes in {Ca2+}i . The results indicate that muscarinic receptor stimulation of HSG-PA cells activates Ca(2+)-activated K channels through a signaling pathway involving a G protein, IP3 production, and increased {Ca2+}i levels . These findings are similar to those in salivary gland acinar cells. Am J Physiol, 1994 Jan, 266(1 Pt 1), C222 - 33 Regulation of intracellular free Mg2+ and contraction in single adult mammalian cardiac myocytes; Silverman HS et al.; Studies in isolated cardiac myocytes have increased our understanding of intracellular Ca2+ regulation . Because less is known about Mg2+ regulation, adult rat ventricular myocytes were loaded with the Mg(2+)-sensitive fluorescent probe mag-indo 1, and changes in intracellular Mg2+ concentration ({Mg2+}i) and cell length were examined under a variety of conditions . The fluorescent signal was calibrated intracellularly and found to differ slightly from that for the probe in solution . Roughly 40% of the signal was intramitochondrial; the remainder was localized in the cytosol . Basal {Mg2+}i averaged 1.02 +/- 0.03 mM (n = 53 cells) . No change in {Mg2+}i was observed during a single electrically stimulated contraction, and only a minor increase was seen during rapid electrical stimulation, which was expected to raise intracellular Ca2+ concentration ({Ca2+}i) to approximately 1 microM . An acid shift in intracellular pH of approximately 1 pH unit was accompanied by a small change in {Mg2+}i (0.34 +/- 0.03 mM, n = 6, P < 0.05) . No change in {Mg2+}i was observed when cells were superfused with 15 mM Mg2+, despite marked changes in contraction . {Mg2+}i more than doubled when cells were depleted of ATP by exposure to hypoxia or metabolic inhibitors . The increase in {Mg2+}i was abrupt and occurred at the time of the failure of contraction, plateauing as rigor contracture developed . Reoxygenation was accompanied by a gradual fall in {Mg2+}i in cells that recovered mechanical function, and in a subset of cells that underwent hypercontracture . Studies in cell suspensions confirmed that rapid cellular energy depletion was accompanied by increases in {Mg2+}i and parallel decreases in ATP . Thus {Mg2+}i was largely insensitive to changes in {Ca2+}i or pHi and extracellular {Mg2+} but was rapidly altered by changes in energy state in a manner that was related to specific changes in cell morphology and contractile function. Am J Obstet Gynecol, 1994 Jan, 170(1 Pt 1), 202 - 6 Transferrin receptor (CD71) expression on circulating mononuclear cells during pregnancy; Bianchi DW et al.; OBJECTIVE: We studied transferrin receptor (CD71) expression in peripheral blood mononuclear cells from healthy pregnant women, to determine if a relationship existed between gestational age and circulating CD71+ mononuclear cells . STUDY DESIGN: Cell suspensions were prepared from venous blood from 139 pregnant women (7 to 26 weeks of gestation), incubated with monoclonal anti-CD71 antibody, and analyzed by flow cytometry . RESULTS: When only the first sample from each woman was analyzed, extensive biologic variation between women was shown . An apparent biphasic increase in the percentage of CD71+ cells with advancing gestation was suggested . A subgroup of 13 women studied on multiple occasions demonstrated linear increases in CD71+ cells as pregnancy progressed . CONCLUSIONS: Pregnant women, when compared with each other, may have differences in the baseline number of circulating CD71+ cells . The increases seen in individuals studied repeatedly are likely to reflect maternal hematopoiesis and current fetomaternal transfusion. Dev Biol, 1994 Jan, 161(1), 59 - 69 Keratinocytes are involved in regulating the developmental changes in the proliferative activity of mouse epidermal melanoblasts in serum-free culture; Hirobe T; When epidermal cell suspensions derived from 0.5-, 2.5-, and 4.5-day-old mice were plated onto uncoated polystyrene dishes and cultured with serum-free medium supplemented with dibutyryl cyclic adenosine 3',5'-monophosphate and basic fibroblast growth factor, melanoblasts proliferated dramatically around keratinocyte colonies and after 12-14 days pure and enriched cultures of melanoblasts (ca . 75%) and melanocytes (ca . 25%) were obtained . In contrast, when epidermal cell suspensions derived from 7.5-, 20.5-, and 60.5-day-old mice were cultured similarly, keratinocytes failed to attach to the dish and melanoblasts did not proliferate at all . However, when epidermal cell suspensions of older mice were plated onto type I collagen-coated dishes and cultured similarly, keratinocytes attached well to the dish and melanoblasts proliferated dramatically around keratinocyte colonies . Moreover, pure melanoblasts and melanocytes derived from primary cultures of young and old mice could be subcultured on collagen-coated dishes with the medium in the presence of secondary keratinocytes that were subcultured from a pure population of primary keratinocytes . No differences were observed in the proliferative activity of secondary melanoblasts between young and old mice . These results suggest that keratinocytes are involved in regulating the proliferation of mouse epidermal melanoblasts and that the developmental changes in the proliferative activity of epidermal melanoblasts in culture are due to the developmental changes in the substrate attachment and proliferation of keratinocytes, rather than to intrinsic changes in melanoblasts. Acta Cytol, 1994 Jan-Feb, 38(1), 33 - 6 Cell yield . ThinPrep vs . cytocentrifuge; Papillo JL et al.; Cell yields on cytologic preparations made in the Cytospin II cytocentrifuge and the ThinPrep Processor were compared . Slides were prepared by each method using calibrated volumes (25 microliters) of cell suspensions from 13 nongynecologic specimens . Cell counts for each slide were calculated by counting cells in predetermined fields using a gridded reticle at 40 x magnification, then extrapolating to the total surface area of the preparation . The cell counts demonstrated that when processing equal amounts of cell suspension, the ThinPrep method retained three times as many cells as the cytocentrifuge method . The ThinPrep method, with a higher rate of cell recovery, may provide a valuable tool toward more accurate cytologic diagnosis, particularly for cytologic samples with small numbers of cells. Clin Chem, 1994 Jan, 40(1), 43 - 7 Manipulation and flow of biological fluids in straight channels micromachined in silicon; Wilding P et al.; Analysis of minute sample volumes is a major analytical challenge that requires an understanding of fluid flow in microstructures . Accordingly, flow dynamics of biological fluids and cell suspensions in straight glass-capped silicon microchannels (40 to 150 microns wide, 20 and 40 microns deep) were studied . We demonstrated that these microstructures are appropriate components for microfluidic analytical devices . Different fluids were easily manipulated in the microchannels, and measurements of flow rate as a function of pressure for whole human blood, serum, plasma, and cell suspensions revealed non-Newtonian behavior . By means of micromachined filters (5 microns) located in channels, blood cells and microparticles were effectively separated from nanoliter-sized samples, clearly indicating the future role of microstructures for a variety of analytical purposes. Endocrinology, 1994 Jan, 134(1), 377 - 82 Suppression in gonadotropes of gonadotropin-releasing hormone-stimulated follicle-stimulating hormone release by the gonadal- and neurosteroid 3 alpha-hydroxy-4-pregnen-20-one involves cytosolic calcium; Wiebe JP et al.; The gonadal- and neurosteroid 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP) suppresses FSH release in cultures of anterior pituitary cells . In a previous report, we showed that this suppression is achieved at least in part by an interaction at the plasma membrane level . We undertook to examine the possible interaction of 3 alpha HP at the level of intracellular Ca2+ . Anterior pituitary cells from adult randomly cycling female rats were treated for 4 h with 10 nM GnRH and 0.1 nM 3 alpha HP with or without protein kinase C activator (SC10), antagonist (H-7), intracellular Ca2+ chelator (TMB-8), and intracellular Ca2+ mobilizer (glutamate), and with or without EGTA and Ca2+ in the medium . FSH content in media and cells was determined by RIA . The protein kinase C (PKC) activator, SC10, increased basal levels of secreted FSH . 3 alpha HP suppressed (P < 0.05) SC10-stimulated basal FSH release . The PKC inhibitor, H7, decreased GnRH-induced FSH release; FSH was further suppressed (P < 0.05) by 3 alpha HP in the presence of H7 . These results were interpreted to indicate that 3 alpha HP may act in part at the level of PKC and also at another site(s) . The intracellular Ca2+ chelator, TMB-8, suppressed released and cellular GnRH-stimulated FSH to the same extent as 3 alpha HP; FSH was not further decreased by 3 alpha HP in the presence of TMB-8 . 3 alpha HP suppressed glutamate-stimulated FSH release in Ca(2+)-free medium (P < 0.01) . Moreover, GnRH-induced release of FSH was suppressed to the same degree by 10(-10) M 3 alpha HP as by 10(-4) M EGTA . In pituitary cell suspensions, the GnRH-induced {Ca2+}i elevations were significantly (P < 0.05) attenuated by 3 alpha HP . From these and previous results, a model is proposed for the action of 3 alpha HP . The model suggests that 3 alpha HP may interact with gonadotropes at the level of the PKC cell signaling pathway and intracellular Ca2+ mobilization, in addition to the plasma membrane/calcium channel . The interaction effects a decrease in intracellular Ca2+, leading to decreases in FSH release from those pituitary gonadotropes that are responsible for FSH . The consistent decrease in total FSH (released plus cellular content) by 3 alpha HP suggests that this neurosteroid may also suppress FSH synthesis. Virology, 1994 Jan, 198(1), 377 - 80 De novo generation and accumulation of tomato bushy stunt virus defective interfering RNAs without serial host passage; Law MD et al.; Studies were initiated to monitor generation and accumulation of defective interfering (DI) RNAs associated with tomato bushy stunt virus (TBSV) in the absence of serial, high multiplicity of infection passage . Infections were initiated in Nicotiana clevelandii host plants and protoplast cell suspensions by inoculation with in vitro-synthesized infectious TBSV RNA transcripts containing a genomic marker . The infections were then assayed for DI-size RNAs by both Northern blot analysis and reverse transcription coupled with PCR amplification . DI-size RNAs could not be detected by Northern blot analysis in either plants or protoplasts after an evident viral infection . However, RT-PCR amplification permitted the isolation of DI-size cDNAs (600-700 nt) from plant, but not protoplast, infections as early as 8 days postinoculation . Sequence analysis of these DI-size cDNA clones revealed that they contained the four conserved regions found in all previously identified competent DI RNAs . Several DI RNA clones contained the genomic marker which confirmed their de novo generation from the input transcript inoculum . A comparison of the nucleotide sequence of these clones to previously sequenced DI RNAs, isolated from plants after multiple passages, showed that differences existed at the junctions between regions . These results demonstrate that a heterogeneous population of DI RNAs accumulated in plants in the absence of serial host passage . In addition, the similarity of these DI RNAs to previously characterized DI RNAs that accumulate upon passage indicates that evolution can occur very rapidly within the initially inoculated plant. Pol J Pathol, 1994, 45(1), 45 - 53 The effect of deoxycholic acid on calcium ions in the cytoplasm of gastric mucosal cells in rabbits; Dziki AJ; Lack of decrease in the level of calcium ions in the cytoplasm after incubation of cell suspension with deoxycholic acid (DC) may be an early sign of cellular damage . The purpose of the study was to find out whether an increase in free calcium level in the cytoplasm preceded other signs of damage and whether calcium channel blockade may inhibit calcium increase in the cytoplasm . The cell suspension from the gastric mucosa in rabbits was incubated with DC in various concentrations . The calcium level was measured by means of spectrofluorimetry after prior incubation with FURA 2/AM . The viability of cells was determined by measuring oxygen consumption and using trypan blue test . Deoxycholic acid in the concentration of 0.2 mM produced an increase in {Ca2+}i from 177 +/- 5 to 285 +/- 24 nM . Incubation of the cell suspension with 0,5 mM DC also produced an abrupt increase in {Ca2+}i from 177 +/- 5 nM to 639 +/- 49 nM . In addition, the studies showed that 0,2 mM DC despite increasing free calcium level in the cytoplasm did not reduce the cell viability . It was revealed on the basis of oxygen consumption and trypan blue test . The studies showed that an increased intracellular {Ca2+}i may be a very early sign of their damage. Biorheology, 1994 Jan-Feb, 31(1), 103 - 13 Rheologic and hemodynamic characteristics of red cells of mouse, rat and human; Chen D et al.; The present study compares hematologic, rheologic and hemodynamic characteristics of red cells from mouse, rat and human . Red cells in these species are biconcave discs that show significant differences in diameter and mean corpuscular volume (MCV) . However, differences in mean corpuscular hemoglobin concentration (MCHC) are not significant . Viscosity measurement of washed red cell suspensions (in each case the medium osmolarity adjusted to match plasma osmolarity) showed significant interspecies differences at shear rates of 37.5 and 750 sec-1 as follows: Human > rat > mouse . Hemodynamic and microcirculatory behavior of these red cells was investigated in the artificially perfused ex vivo mesocecum vasculature of the rat . Hemodynamic measurements in the whole ex vivo mesocecum preparation revealed maximal increase in the peripheral resistance unit (PRU) for the human red cells followed by the rat and mouse red cells, respectively at a hematocrit (Hct) of 40% . Further, measurements of red cell velocities (Vrbc) in single arterioles of the mesocecum vasculature, during sustained perfusion with washed red cell suspensions, showed that at any given perfusion pressure (Pa), Vrbc for both mouse and rat red cells was higher than that for human red cells, while Vrbc for mouse red cells was higher than that for the rat . These results demonstrate that the microvascular flow behavior of these red cells is likely to be influenced by both physical and rheologic characteristics. Neuroscience, 1994 Jan, 58(2), 359 - 69 Reduction of voluntary alcohol intake in the rat by modulation of the dopaminergic mesolimbic system: transplantation of ventral mesencephalic cell suspensions; Lanca AJ; The dopaminergic mesolimbic system plays a major role in the mechanisms of reward and positive reinforcement, and is also known to be a primary target for the action of substances that are self-administered and are considered drugs of abuse . Even though alcohol administration has been shown, by physiological and pharmacological manipulations, to cause changes in the mesolimbic dopaminergic system, it has not yet been determined whether, conversely, experimentally induced changes in this system are effective in regulating the voluntary intake of ethanol . In the present study we assessed the effects of the intrastriatal transplantation of fetal dopaminergic grafts on the regulation of voluntary alcohol intake in the rat . Fetal dopaminergic transplants from ventral mesencephalon--but not dopamine-poor transplants or sham-operated animals--reduced the voluntary intake of ethanol by about 40-50% . These results indicate that the effects obtained are due to the dopaminergic nature of the grafts, and not the consequence of a non-specific effect of the graft, or of the surgical procedure itself . These results support the hypothesis that the dopaminergic mesolimbic system plays an important role in the regulation of the voluntary intake of ethanol. J Rheumatol, 1994 Jan, 21(1), 55 - 8 The effect of methotrexate on ex vivo lipoxygenase metabolism in neutrophils from patients with rheumatoid arthritis; Hawkes JS et al.; OBJECTIVE . To examine the effect of methotrexate (MTX) administered in vivo on the production of the 5-lipoxygenase (5-LO) metabolites of arachidonic acid by neutrophils from subjects with rheumatoid arthritis . METHODS . Neutrophils were isolated from peripheral blood samples taken 12 h before and 12 h after the ingestion of an oral dose of MTX and stimulated in vitro by calcium ionophore A23187 . Lipid extracts of cell suspensions were assayed for leukotriene B4 (LTB4), the all-trans isomers of LTB4, 20-hydroxy LTB4 and 5-hydroxyeicosatetraenoic acid by high pressure liquid chromatography . RESULTS . An increase in the production of all measured 5-LO metabolites was seen between the pre and postdose assessments . CONCLUSION . Our results do not support the putative inhibitory effect of MTX on 5-LO metabolism. J Rheumatol, 1994 Jan, 21(1), 10 - 6 Synovial tissue implants from patients with rheumatoid arthritis cause cartilage destruction in knee joints of SCID.bg mice; Sack U et al.; OBJECTIVE . To establish in SCID.bg mice a model in which joint destruction is initiated by human inflammatory cells from patients with rheumatoid arthritis (RA) . METHODS . Development of a surgical technique and immunohistologic analysis . RESULTS . Initial experiments with single cell suspensions failed because more than 70% of the cells injected intraarticularly left the mouse knee joint within 16 h without causing destruction . This was observed with peripheral blood mononuclear cells, T cell lines reactive to mouse or rat collagen type II, and synovial mononuclear cells . Cell immigration was reduced but not prevented by preactivation with mitogens . In contrast, small tissue implants from human synovial membrane which were transferred by surgical intervention into mouse knee joints remained at the site of injection and could be easily localized within the mouse joint (observation period up to 8 weeks) . The human synovial membrane implants induced pannus formation and erosion of cartilage and bone while only a mild and transient synovitis was observed with normal synovial membrane and control tissues like human thymus . The predominant cells at the site of destruction were human (CD68+) and murine (Mac-2+) monocytes/macrophages . CONCLUSION . The human/murine SCID arthritis is a useful model for studying pathogenetic aspects of joint destruction as well as effects of new drugs or novel treatment strategies. Biotech Histochem, 1994 Jan, 69(1), 38 - 44 A variant of a slam freezing device for electron microscopy; Lemke C et al.; A home-made slam freezing device is presented that allows reproducible results in freezing various unfixed tissues . The heart of the device is an aluminum socket, which harbors a plunger that is set in motion by a spring . At the end of the plunger there is an electromagnet which holds the sample on a sheet metal planchette . During stop freezing the electrical contacts are interrupted and the plunger can be withdrawn leaving the specimen on the cooled copper block . This guarantees freezing of not only solid tissues, but also cell suspensions, such as blood or bone marrow. Z Naturforsch {C}, 1994 Jan-Feb, 49(1-2), 26 - 32 Purification and properties of acridone synthase from cell suspension cultures of Ruta graveolens L; Baumert A et al.; Acridone synthase has been purified from cell suspension cultures of Ruta graveolens using a combination of gel filtration and ion exchange chromatography . The purified enzyme has an apparent molecular weight of 69 kDa on gel filtration and a subunit structure on SDS-PAGE of 40 kDa . The apparent Km-values are 10.64 microM and 32.8 microM for N-methylanthraniloyl-CoA and malonyl-CoA, respectively . Tryptic digestion of the homogeneous acridone synthase was performed . Seven of the peptides were chosen for microsequencing . The homology of the amino acid sequences from this particular polypeptide and corresponding peptides from chalcone synthase 3 from garden pea amounted to 76%. Ann Clin Lab Sci, 1994 Jan-Feb, 24(1), 6 - 11 The role of flow cytometry in the diagnosis of lymphoma: a critical analysis; Morse EE et al.; Flow cytometry, now used routinely to aid in the classification of leukemias, is increasingly being evaluated as a rapid technique for determination of surface antigens on the cells teased from lymph nodes and other masses with suspected lymphoma . The present study reviews biopsy specimens from patients examined during a two year period which were sent for flow cytometry with a diagnosis of suspected lymphoma . Sixteen of 25 samples (64 percent) produced cell suspensions of sufficient quantity and quality to be diagnostically helpful . Results showed that in 9/16 (56 percent) the diagnosis of lymphoma or cancer could be suspected by flow cytometry alone, while 4/16 were consistent with the final tissue diagnosis of normal or reactive hyperplasia . Three samples that came from patients who had morphologic evidence of malignant disease on biopsy (two Hodgkin's disease and one large cell lymphoma) had flow cytometry results that were interpreted as normal . Flow cytometry is rapid and appears to be virtually diagnostic of non-Hodgkin's lymphoma when a majority of cells are B cells with an abnormal kappa/lambda ratio (> 4.0 or < 0.25) . Nonhematologic malignancy can be suspected if less than 75 percent of the cells show CD45 (common leukocyte antigen) . Hodgkin's disease cannot be detected by flow cytometry as it is currently used, and as many as 15 percent (1/6 in this study) of lymphomas may show normal results . It is extremely helpful when the biopsy sample actually contains the cells of interest in large proportion . Loss of architectural relationships in the course of processing specimens for flow cytometry is a major disadvantage when small foci of lymphoma or tumor cells exist together with large amounts of stroma or normal lymphocytes. J Steroid Biochem Mol Biol, 1994 Jan, 48(1), 47 - 54 Modulation of calcium signaling and LH secretion by progesterone in pituitary gonadotrophs and clonal pituitary cells; Ortmann O et al.; In estradiol-treated pituitary cells, progesterone enhances gonadotropin-releasing hormone (GnRH)-induced LH secretion from cultured rat pituitary cells during short-term treatment but attenuates this response during prolonged treatment . In the present study, the effects of gonadal steroids on GnRH-induced cytoplasmic calcium ({Ca2+}i) responses in gonadotrophs were analyzed in rat pituitary cells and immortalized (alpha T3-1) murine gonadotrophs . Ca2+ responses were measured in cell suspensions and single gonadotrophs, loaded with Fura-2 or Indo-1, respectively, and pretreated for 48 h with 1 nM estradiol with or without 100 nM progesterone, or for 48 h with 1 nM estradiol and then for 3 h with 100 nM progesterone . In cells of the alpha T3-1 gonadotroph lineage, GnRH elicited biphasic Ca2+ signals composed of an initial peak response followed by a prolonged plateau phase . The amplitudes of both the extracellular Ca(2+)-independent spike phase and the extracellular Ca(2+)-dependent plateau phase were enhanced or inhibited by short- or long-term progesterone treatment, respectively . In single pituitary gonadotrophs, GnRH (0.5 nM) elicited oscillatory responses due to intermittent release and uptake of Ca2+ from intracellular stores . Treatment with progesterone shifted the oscillatory signal toward biphasic (3 h) or subthreshold (48 h) response profiles, revealing a steroid-induced change in the pattern of Ca2+ mobilization . In addition to these agonist-induced responses, the transient {Ca2+}i responses of pituitary cells and individual gonadotrophs to high K+ were enhanced or inhibited after short- or long-term progesterone treatment, respectively . These actions were correlated with the effects of progesterone on K(+)-induced LH secretion . The {Ca2+}i and LH secretory responses to phorbol ester treatment were also enhanced by short-term exposure of the cells to progesterone . The results demonstrate that the stimulatory and inhibitory effects of progesterone on agonist-induced Ca2+ signaling result from changes in Ca2+ mobilization and entry, and contribute to the modulatory actions of the steroid on GnRH-induced LH secretion. Arzneimittelforschung, 1994 Jan, 44(1), 36 - 40 Effect of vintoperol on platelet aggregation and experimental thrombosis; Csomor K et al.; The platelet aggregation inhibitory and antithrombotic effect of the new peripheral circulation enhancing compound vintoperol (RGH-2981, CAS 106498-99-1) was studied . In vitro, vintoperol inhibited the aggregation response to collagen in platelet-rich plasma from mice, rats, rabbits and dogs . It was found to be highly effective in preventing mice from acute pulmonary thromboembolic death induced by adenosine diphosphate (ADP) or collagen . After the oral dose of 10 mg/kg the percentage of survivors increased from 9 to 60% and from 13 to 73%, respectively . In the "mouse antithrombotic assay" it was protective only at the 3 mg/kg dose . Sudden death of mice evoked by hardened red blood cell suspension was not protected by vintoperol . In mice receiving 30 mg/kg vintoperol orally, the inhibition of aggregation response to collagen, ADP and ADP/epinephrine by 15, 33 and 37%, respectively, was associated with a substantial increase in bleeding time . In a rat multifactorial thrombosis model the 10 mg/kg p.o . dose was also sufficient to obtain significant antithrombotic effect (p < 0.01) . Results of these experiments indicate that vintoperol interferes with platelet aggregation both in vitro and in vivo and possesses potent antithrombotic effects in thrombosis models in which platelet activation is mainly involved. Anal Cell Pathol, 1994 Jan, 6(1), 23 - 36 Inter-institutional reproducibility of flow cytometric DNA-analysis in breast carcinomas; Risberg B et al.; In order to study interinstitutional reproducibility of flow cytometric DNA-analysis (DNA-FCM), frozen pieces from 30 consecutive breast carcinomas were analysed by 5 laboratories . Different instruments, preparation and DNA staining methods were used . A concordance in DNA-ploidy status was obtained in 26 of the 30 tumours . The discrepancy can mainly be explained by intratumoural DNA-heterogeneity since a complete agreement in ploidy status was obtained when four of the laboratories analysed the same cell suspension, where solid bits showed differing results . The sampling method seems therefore to be a crucial step for the results and needs further studies . As far as the estimation of S-phase fraction was concerned, one laboratory obtained significantly higher values compared to the other four . The correlation between the other four laboratories varied between r = 0.66-0.92. Lasers Surg Med, 1994, 14(1), 34 - 9 Low intensity laser irradiation inhibits tritiated thymidine incorporation in the hemopoietic cell lines HL-60 and U937; O'Kane S et al.; The purpose of this study was to determine the effect of low intensity laser irradiation (660 nm, 12 mW, 5 kHz) on tritiated thymidine incorporation in two hemopoietic cell lines, HL-60 and U937 . Cells were suspended at a concentration of 1 x 10(6)/ml in their respective serum-free media and irradiated at energy densities from 1.0 to 11.5 J/cm2 . Twenty-four hours after irradiation the cells were assayed for their ability to incorporate tritiated thymidine (3H-TdR) in comparison with nonirradiated cells . Analysis by two-way analysis of variance (ANOVA) for unrelated groups showed that laser irradiation at all energy densities > or = 5.8 J/cm2 produced a significant decrease in 3H-TdR incorporation (P < 0.05) into HL-60 cells . In U937 cells, irradiation at energy densities of 5.8, 7.2, and 11.5 J/cm2 caused a similar reduction in 3H-TdR incorporation (P < 0.01), although not at 8.6 and 9.6 J/cm2 . The temperature of each cell suspension was recorded both during and immediately postirradiation, and no significant thermal changes were observed . These findings demonstrate a direct photobiological effect of laser irradiation on these two cell lines . The precise mechanism for this effect is unknown but may have significance in understanding the biological action of laser's known therapeutic effectiveness in promoting wound repair. Plant Mol Biol, 1994 Jan, 24(2), 401 - 5 Stable transformation and long-term expression of the gusA reporter gene in callus lines of perennial ryegrass (Lolium perenne L.); van der Maas HM et al.; Stable transformation of perennial ryegrass (Lolium perenne L.) was achieved by biolistic bombardment of a non embryogenic cell suspension culture, using the hpt and gusA gene . The transformation yielded on the average 5 callus lines per bombardment (1.4 x 10(6) cells) . Stable integration of the genes into the plant genome was demonstrated by Southern analysis of DNA, isolated from hygromycin-resistant callus lines . The gusA reporter gene, which was regulated by the constitutive promoter of the rice gene GOS2, was expressed in both transient and stable transformation assays, indicating that this promoter is suitable for expression of a transferred gene in perennial ryegrass . Long-term GUS expression was observed in ca . 40% of the callus lines, whereas the other callus lines showed instability after 6 months and 1 year of culture. Vox Sang, 1994, 66(4), 276 - 9 Microwave elution of red cell antibodies; Torloni AS et al.; Using a commercial microwave oven (750 W, 2,450 MHz), we compared the use of microwave energy to the commonly used method of acid-glycine elution (Elu-Kit II, Gamma Biologicals, Houston, Tex., USA) to elute antibodies from direct antiglobulin test positive red blood cells (RBC) . Using a 33% suspension of RBCs in cold saline (1-6 degrees C) in a polypropylene test tube, the microwave technique was comparable for eluting Rh (except anti-c and anti-e) and Kell and superior for eluting Duffy, Kidd antibodies while acid-glycine was superior in eluting S and s antibodies . Consistent results were obtained using the microwave technique when the RBC suspension reached temperature of 57-64 degrees C . Heat or the rate of temperature rise appears to play an important role in the microwave elution process although other not yet identified microwave properties may also be a factor . Microwaves are a quick (8 s) alternative to well-established methods of elution and have shown to give consistent results in our study . Commercially available microwave ovens vary substantially in power, making it necessary to perform an initial calibration of the microwave in order to determine a location within the microwave cavity where exposure to microwaves will consistently achieve temperatures of 57-64 degrees C in all cell suspensions. Cell Motil Cytoskeleton, 1994, 27(4), 327 - 36 Two-step mechanism for actin polymerization in human erythroleukemia cells induced by phorbol ester; Niu MY et al.; Human erythroleukemia (HEL) cells grow in suspension, but after treatment with nM PMA the cells adhere and spread on glass or fibronectin {Jarvinen et al., 1987: Eur . J . Cell Biol . 44:238-246} . We observed an early (20-30 min) stage of spreading in which F-actin was organized into peripheral arcs near the spreading margin and vinculin was localised to the cell's periphery at the ends of these arcs . By 1 h the cells were well spread with straight actin bundles many of which ended at more central sites terminating on patches containing vinculin and talin; thus the cells assemble typical stress fibers but do not appear to polarize . The cells also spread on RGD polymer . DiC8 (1,2-dioctanoyl-sn-glycerol, C8:0, Sigma Chemical Co., St . Louis, MO) induced spreading but only if DAG kinase inhibitor and A-23187 were also present; in their absence cells adhered but did not spread . Spreading was approximately 85% inhibited by 100 nM staurosporine . PKC-beta was shown to be present in the cells by immunoblotting . In cells spread for 1 h with PMA, F-actin increased to 180% of control levels as measured by RP binding and the actin sequestering complex of G-actin-thymosin beta 4 decreased significantly . To determine whether the F-actin increase required adhesion, we inhibited cell attachment to the substratum by adding RGDS, by coating glass surfaces with hemoglobin, or by a combined treatment . Under these conditions PMA-treated suspended cells still increased their F-actin to 126-137% of controls, a significant increase over control levels . Staurosporine inhibited F-actin increases under all the conditions studied . Permeabilized cell suspensions, incubated with rhodamine labelled G-actin, incorporated the labelled actin along cell membranes at a low level . A few minutes preincubation with either diC8 plus DAG kinase inhibitor or with PMA strongly increased the incorporation . This increased incorporation was reduced to below control levels by either staurosporine (100 nM) or cytochalasin D (1 microM) . We conclude that both suspended and spreading HEL cells can be stimulated to polymerize actin by a mechanism dependent on PKC or a PKC-like molecule . In suspended cells, the polymerization occurs along the membrane . When cells spread, F-actin increased to a significantly greater extent . This second step could involve additional polymerization, perhaps at the observed adhesion sites, decreased turnover of the actin bundles, or a combined effect of both mechanisms. Dev Comp Immunol, 1994 Jan-Feb, 18(1), 45 - 56 Characterisation of immunoglobulin-binding leucocytes in carp (Cyprinus carpio L.); Koumans-van Diepen JC et al.; This study demonstrates the immunoglobulin(Ig)-binding capacity of Ig-positive carp macrophages employing immunofluorescence and immunogold methods . These methods allow for the characterisation of the Ig-binding cells . After internalisation of fluorescent- or gold-labelled Ig (30 min at room temperature), most macrophages from the hindgut were able to bind added purified carp Ig, which could be demonstrated clearly with a second fluorescent or gold label . In pronephros, an important haemopoietic organ in fish, a limited number of monocyte-like cells also showed Ig binding . Pronephros macrophages and neutrophilic granulocytes appeared to be Ig-negative . The use of goat anti-mouse Ig gold particles bound by carp anti-goat antibodies revealed that, in addition to hindgut macrophages and pronephric monocyte-like cells, some lymphoid cells in both hindgut and pronephros cell suspensions were also able to bind Ig . The classic erythrocyte-antibody rosette assay resulted in a limited number of small rosettes in cell suspensions from both organs. Microbios, 1994, 78(315), 91 - 101 Modulation of pokeweed mitogen-induced B cell differentiation by polymorphonuclear cells: effects of bacterial lipopolysaccharides; Tortorella C et al.; The capacity of polymorphonuclear (PMN) cells to release several cytokines stresses the potential immunomodulatory role of these cells . The effects mediated by purified PMN cell suspensions on pokeweed mitogen (PWM)-driven B cell differentiation was investigated . Results showed that the addition of increasing concentrations of resting PMN cells to peripheral blood mononuclear cell (PBMC) cultures gave rise to inhibition of immunoglobulin (Ig) production . At the same time, similar results were obtained using lipopolysaccharide (LPS)-pretreated PMN cells . In contrast, when LPS, at different concentrations, and PMN cells were both added to PBMC cultures an enhancement of IgG or IgM release in comparison with cultures treated with PMN cells only occurred at low PMN cell/PBMC ratios (1:20 and 1:10), which was maximal in the presence of 10 or 100 ng/ml LPS . This effect was probably mediated by LPS-induced monocyte stimulation, since the supplementation of LPS-activated monocyte supernatants to PMN cell/PBMC cocultures led to an Ig synthesis which mimicked that seen in similarly-treated PBMC cultures . These data suggest the occurrence of various in vitro modulatory effects in the interactions between PMN, LPS and lymphocytes in a PWM-induced B cell polyclonal responsiveness system. Vox Sang, 1994, 66(3), 153 - 60 Relative efficiency of leucocyte removal procedures for the production of leucocyte-poor red cell concentrates assessed by flow cytometry; Farrugia A et al.; Flow cytometry was used to: (1) determine residual leucocyte numbers in red cell suspensions following the range of leucocyte depletion procedures used in our organisation, and (2) to characterize phenotypically the leucocytes using direct immunofluorescence with monoclonal antibodies to cell surface receptors . Under the conditions used, a lower limit of detection of 2.5 leucocytes per microliter (equivalent to 3.43 log10 or 99.96% removal) could be achieved . Filtration through polyester filters was found to remove up to > 99.96% of the initial leucocytes; however, a significant differential efficacy was observed between filters from different manufacturers even when filters with similar costs were compared . The order of filter brands with respect to leucocyte removal found was Pall BPF4 = Erypur Optima G-O > Sepacell R500 > Pall RC50 . Phenotyping revealed that increasing filtration efficacy was associated with a preferential removal of lymphocytes; conversely, a second filtration over one brand of filter allowed proportionately more lymphocytes to pass through compared with the first filtration . A saline wash following filtration removed a further 0.5% of the initial leucocyte content, and was associated with a preferential loss of granulocytes . Freeze-thawing the red cell suspension removed fewer leucocytes (96.3%) than did filtration (98.74% to > 99.6%) or filtration followed by washing (99.22%), and also led to preferential loss of granulocytes . Flow cytometry provides a reliable tool for the quality control of leuco-depleted red cells, and allows a qualitative assessment of the residual leucocytes . This information is of value in choosing procedures aimed at decreasing the risk of alloimmunisation and post-transfusion reactions. Mycopathologia, 1994 Jan, 125(1), 19 - 22 Epidemiological study of sporotrichosis and histoplasmosis in captive Latin American wild mammals, São Paulo, Brazil; Costa EO et al.; Sporotrichosis and histoplasmosis are deep mycosis with a high incidence in human beings in Brazil . In domestic animals histoplasmosis has been described only in dogs, but the occurrence of sporotrichosis among domestic animals in Brazil has been described in dogs, cats, mules and asses . There is also a case of this disease reported in a chimpanzee (Pan troglodites) . The purpose of this research was to perform an epidomiological study of these mycoses using delayed hypersensitivity tests (histoplasmin and sporotrichin) in Latin American wild mammals . This research was assayed using 96 healthy animals at Parque Zoologico de Sao Paulo, Brazil: Primates: 33 Cebus apella--weeping-capuchin and 16 Callithrix jacchus--marmoset; Procyonidae: 37 Nasua nasua--coatimundi and 10 Felidae (Panthera onca--jaguar; Felis pardalis--ocelot Felis wiedii--margay; Felis tigrina--wild cat) . For intradermic tests, the following antigens were used: Sporothrix schenkii cell suspension (sporotrichin, histoplasmin-filtrate), Histoplasma capsulatum cell suspension (histoplasmin), and Histoplasma capsulatum (polysaccharide) . The positivity to histoplasmin was 44.79% (Cebidae 15.15%; Callithricidae 6.25%; Procyonidae 86.49% and Felidae 50.00%, respectively) . With respect to sporotrichin, 30.21% (Cebidae 6.06%, Callithricidae 0.0%; Procyonidae 64.86% and Felidae 30.00% respectively) . The pattern of infection is similar to that shown by human beings and this may suggest that these animals could be involved in the epidemiologic chain of sporotrichosis and histoplasmosis, the second most prevalent human deep mycoses in Brazil . It is important to point out the absence of similar studies in Latin American wild animals. Ultrasound Med Biol, 1994, 20(2), 187 - 93 Cavitation dosimetry: estimates for single bubbles in a rotating-tube exposure system; Miller DL et al.; Cell lysis and hydrogen peroxide production from cavitation in a 60 rpm rotating-tube exposure system were observed for 2.17 MHz ultrasound at 0.8 MPa peak negative pressure amplitude . Synchronized 10 ms burst mode exposure was utilized to emphasize the phenomenon of bubble cycling each half rotation . Low cell numbers and inhibition of H2O2-consuming enzymes allowed measurement of the residual hydrogen peroxide in exposed cell suspensions . Canine red blood cells (RBCs) or Chinese hamster ovary cells (CHO) suspended at 1 x 10(6) mL-1 in phosphate buffered saline were lysed exponentially with the number of bursts . The CHO cells were lysed faster than RBCs . The H2O2 increased approximately in proportion to the number of bursts . Longer bursts (100 ms) or continuous exposure produced similar trends, but were less effective per unit of total on-time . The number of bubbles per 10 ms burst was estimated from a simple model to be about 7100 . The faster lysis of the CHO cells could be explained mostly by the larger size of these cells, which makes them more likely to meet a bubble . The H2O2 production gave concentrations of about 93-155 fM per bubble per burst . Similar calculations gave estimates of 178 fM per bubble for 100 ms bursts and 150 fM per bubble for continuous exposure . The rate of H2O2 production was roughly 500 fmole s-1 while a bubble crossed the tube . This sonochemical yield could be biologically significant under favorable circumstances. Microvasc Res, 1994 Jan, 47(1), 126 - 39 Modified cell-flow microchannels in a single-crystal silicon substrate and flow behavior of blood cells; Kikuchi Y et al.; Previously reported cell-flow microchannels in a single-crystal silicon substrate (Microvasc . Res . 44, 226-240, 1992) have been modified, and flow behavior of blood cells is described using flow rate-time curves and video pictures . The principal structure (2600 identically sized channels in parallel) was retained to give the same simple quantitative measure of the total flow rate for blood cell suspensions under constant suction . Level areas (terraces) were placed at the entrance and exit sides of the parallel channels level with the channel depth (4.5 microns) so that blood cells just entering into and flowing out of the channels could be more clearly observed under reflecting illumination . Three lengths (10, 20, and 100 microns) of channel were used each with a terrace width of 30 microns . In agreement with calculated values, the resistance to flow at the terrace portion was shown to be nearly equal to that per 10 microns of the channel portion . Clearer pictures were obtained of channel blocking by activated leukocytes and platelet aggregates after addition of each stimulant . Erythrocyte aggregates showed easy transit even through the 100-microns-long channels and through narrow spaces, including gaps probably narrower than 2 microns, which were formed between plugging leukocytes at the terrace portion. Mol Membr Biol, 1994 Jan-Mar, 11(1), 39 - 44 Rapid determination of the transbilayer distribution of NBD-phospholipids in erythrocyte membranes with dithionite; Pomorski T et al.; The assessment of the transverse distribution and mobility of NBD-labelled phospholipid analogues in biological membranes by selective chemical destruction of fluorescent label in the outer monolayer with dithionite has been investigated using resealed erythrocyte ghosts as a model system . The distribution of those analogues can be determined in < 30 s directly in the cell suspension provided the permeation of dithionite across the membrane is suppressed . The results were compared with data on translocation of either NBD- or spin-labelled phospholipid analogues obtained with the technique of back exchange to BSA . It is shown that the passage of dithionite can be mediated by anion-transport systems such as band 3 which is inhibited by DIDS . Appropriate conditions for the applicability of the assay were elucidated also using resealed ghosts having fluorescent NBD-taurine in the intracellular lumen . The application of the assay to measure fast translocation processes, e.g . those mediated by the aminophospholipid translocase, is described. Biosens Bioelectron, 1994, 9(2), 91 - 103 Harmonic generation in "non-linear" biological systems; Hutchings MJ et al.; We describe a high performance, low frequency Fourier Transform based spectrum analyser, with design features particularly suited to the harmonic analysis of the non-linear amplitude transfer functions of various biological systems . A previous published method, using general purpose systems to produce reference and signal plus reference "power" spectra, was susceptible to ambiguous interpretation of results . Unlike that design, the present spectrometer derives quantitative complex voltage (amplitude and phase) harmonics of sampled voltage data, maintaining potentially important information . Based on the use of a basic IBM or compatible PC, it features high sensitivity, high speed data processing and large dynamic range, is compact, easy to use, flexible in operation and low in cost . Its precision current signal source eliminates harmonics generated at the drive electrodes from the measurement . Use of the instrument leads us to conclude that, contrary to previous assertions, no repeatable dielectric or conductive non-linearity is exhibited in the bulk cell suspensions tested under the field and frequency conditions reported. Biochem Mol Biol Int, 1994 Jan, 32(1), 167 - 71 Interferon-beta endogenously produced by intratumoral injection of cationic liposome-encapsulated gene: cytocidal effect on glioma transplanted into nude mouse brain; Yagi K et al.; The cytocidal effect of endogenously produced human interferon-beta was tested on glioma transplanted into the brain of nude mice by intracranial injection of a cell suspension of human glioma cell line U251-SP . When plasmid pSV2IFN-beta, bearing the human interferon-beta gene, was encapsulated into cationic multilamellar liposomes composed of N-(alpha-trimethylammonioacetyl)-didodecyl-D-glutamate chloride, dilauroyl phosphatidylcholine, and dioleoyl phosphatidylethanolamine in a molar ratio of 1:2:2 and injected intratumorally, human interferon-beta was produced in the tumor . The tumor completely disappeared if the injection was done shortly after the transplantation . Even when the tumor did not disappear, survival of the tumor-bearing nude mice was markedly prolonged . In control experiments made with normal brain having no glioma, interferon-beta was not detected. Acta Oncol, 1994, 33(7), 813 - 7 The response of quiescent cell populations in murine solid tumors to irradiation with fast neutrons; Masunaga S et al.; 5-bromo-2'-deoxyuridine (BUdR) was injected into SCC VII tumor-bearing mice intraperitoneally to label all proliferating tumor cells . The mice were irradiated with fast neutrons or x-rays . Immediately, or 24 h after irradiation, the tumors were excised, minced and trypsinized . The tumor cell suspensions were incubated with cytochalasin-B (a cytokinesis blocker) . The micronucleus frequency was determined using immunofluorescence staining to BUdR . The cells that were not labeled with BUdR could be regarded as the quiescent cells . The micronucleus frequency in total tumor cells was determined from the irradiated tumors that were not pretreated with BUdR . The difference in radiosensitivity between total and quiescent cells was markedly reduced with fast neutrons, especially at higher doses of radiation . Potentially lethal damage repair by total and quiescent cells was inhibited more strongly with neutrons than with x-rays . When using fast neutrons, the radiosensitivity of solid tumors depends on their heterogeneity less critically than for x-rays. Peptides, 1994, 15(5), 791 - 7 Lymphoid cell subpopulations containing vasoactive intestinal peptide in the rat; Leceta J et al.; In the present study we describe the cell types containing immunoreactive vasoactive intestinal peptide (IR-VIP) in rat thymus, spleen, and lymph nodes . Indirect immunofluorescence staining and flow cytometry indicated that all lymphoid organs studied contained VIP-positive cells, with the spleen and lymph nodes having a higher proportion than the thymus . Vasoactive intestinal peptide was found in both lymphocytes and nonlymphoid cells, lymphocytes predominating among VIP-positive cells . Double immunofluorescent staining and flow cytometry showed that all lymphoid subpopulations identified contained variable proportions of VIP-positive lymphocytes . Immunocytochemical staining of cell suspensions for both light and electron microscopy showed the cytoplasmic localization of the IR-VIP . These findings, coupled to our previous results, are consistent with the idea that VIP may have a lymphoid origin and could be active in local immune responses. Urol Int, 1994, 53(1), 12 - 7 In vitro effects of high energy shock wave alone and combined with anticancer drugs on human bladder cancer cells; Rahman M; We investigated the in vitro effects of high energy shock wave alone or combined with anticancer drug treatment on an established human bladder cancer cell line, KK-47 cells . Exposure of the cell suspensions to 500-2,500 shock waves, generated with the EDAP lithotriptor LT01, resulted in reduced cell viability with an increasing number of shock doses . DNA flow cytometric analysis revealed a marginal effect on the G2+M and S phases of the cell cycle . When shock wave-treated cells were exposed to mitomycin C, cisplatin, methotrexate or adriamycin, an additive cytotoxic effect was obtained at all shock doses . Adriamycin showed the highest additive effect of the drugs tested . Significant increases in glutamic oxaloacetic transaminase, alkaline phosphatase, creatine phosphokinase and lactate dehydrogenase concentrations in the nutrient medium were observed at over 400 shock doses, reflecting damage to the cell membrane . These data suggest that high energy shock wave may be applicable in combination with some anticancer drugs in the treatment of transitional cell carcinoma. Retina, 1994, 14(3), 264 - 9 Establishment of pigmented choroidal melanomas in a rabbit model; Hu LK et al.; PURPOSE: To establish an animal model of pigmented choroidal melanoma . METHODS: Four melanoma cell lines originally isolated from melanotic tumors (B16F10, RPMI 1846, OCM 1, and IIB) were used to establish choroidal melanomas in 105 rabbits; 88 animals were immunosuppressed with cyclosporine . Tumor cells were implanted transclerally and examined with indirect ophthalmoscopy, ultrasound, and photography . RESULTS: Characteristic growth patterns were noted for each cell line . Animal cell lines typically produced choroidal tumors 3 to 4 mm in height within 2 weeks; human cell lines took an additional 7 to 10 days to achieve tumors of similar height . Tumors of heaviest pigmentation were generated consistently with the B16F10 cells, and with the other three cell lines only mild pigmentation was observed . Tumor shape varied depending on the source of implantation: diffuse, flat tumors were observed when cell suspensions were implanted, and nodular tumors were obtained with tumor fragments . Histopathologically, lesions were highly cellular, with rich vascularity and large numbers of mitotic figures . CONCLUSION: As the majority of human uveal melanomas are pigmented, the added feature of pigmentation associated with this model makes it more suitable for evaluating the role of newly developed phototherapies in the management of uveal melanoma. Eur J Cancer, 1994, 30A(7), 1022 - 6 In vitro activity of 2-chlorodeoxyadenosine (CdA) in primary cultures of human haematological and solid tumours; Larsson R et al.; 2-Chlorodeoxyadenosine (CdA) is a deaminase-resistant purine analogue which has shown clinical activity against various haematological tumours, and is currently undergoing phase II trials . In the present study, the semiautomated fluorometric microculture cytotoxicity assay (FMCA) was used for in vitro evaluation of CdA activity in cell suspensions from both haematological and solid tumours . A total of 133 samples from various diagnoses were successfully tested with continuous drug exposure . CdA showed high in vitro activity against samples from chronic and acute lymphocytic leukaemia and acute myelocytic leukaemia, but little or no response was observed in the solid tumour groups . Cross-resistance analysis with standard drugs revealed the following rank order of correlation coefficients: cytosine arabinoside (AraC) > daunorubicin > doxorubicin > vincristine > prednisolone > 4-hydroperoxycyclophosphamide > etoposide > cisplatin . The high correlation between CdA and AraC was maintained even if the analysis was based only on the haematological tumours . The results indicate that CdA is differentially active against haematological tumours with little or no activity against solid tumours . CdA also appears highly cross resistant with AraC . If this disease-specific information is substantiated in further clinical trials and extended to other phase I-II drugs, non-clonogenic drug resistance assays such as the FMCA may become useful in new drug evaluation, and in targeting specific diagnoses and patients for phase II trials. J Clin Pathol, 1994 Jan, 47(1), 18 - 22 MIB-1, Ki67, and PCNA scores and DNA flow cytometry in intermediate grade malignant lymphomas; Pich A et al.; AIMS--To verify the correlation between MIB-1, Ki67, and proliferating cell nuclear antigen (PCNA-PC10) scores and S-phase fraction in intermediate grade non-Hodgkin's lymphomas (Working Formulation F); and their reliability in differently processed tissues . METHODS--Forty one non-Hodgkin's lymphomas were classified as (F) intermediate grade malignant lymphomas according to the Working Formulation; mitotic counts and percentage of large cells were assessed for each case . Sections from formalin fixed, paraffin wax embedded tissues were stained with anti MIB-1 monoclonal antibody, after microwave oven processing, and anti-PCNA (PC10) monoclonal antibody using an avidin-biotin immunoperoxidase (ABC) method . One thousand cells from 10 representative fields were scored . Frozen sections from surgical specimens were stained with Ki67 monoclonal antibody using the ABC method; the fraction of Ki67 positive cells was calculated scoring 1000 cells . Flow cytometry analysis (FCM) was performed on cell suspensions from fresh tissues . Correlations between data were estimated using linear regression . RESULTS--A linear correlation was found between MIB-1 and Ki67 scores (r = 0.92; p < 0.00001); between MIB-1 and PCNA scores (r = 0.79; p < 0.00001); and between MIB-1 score and S-phase fraction (r = 0.51; p = 0.0006) . A linear correlation was also found between Ki67 and PCNA scores (r = 0.85; p < 0.00001); between Ki67 score and S-phase fraction (r = 0.6; p = 0.0002); and between PCNA score and S-phase fraction (r = 0.74; p < 0.00001) . A correlation was found between mitotic counts and MIB-1 (r = 0.56; p = 0.0001), PCNA (r = 0.51; p = 0.0007), or Ki67 scores (r = 0.47; p = 0.002); between the percentage of large cells and MIB-1 (r = 0.49; p = 0.0009), PCNA (r = 0.6; p = 0.00003), and Ki67 scores (r = 0.53; p = 0.0003) and S-phase fraction (r = 0.55; p = 0.0002) . CONCLUSION--MIB-1, Ki67, and PCNA (PC10) scores and S-phase fraction are highly correlated and equally well represent the proliferative activity of intermediate grade non-Hodgkin's lymphomas in differently processed material . MIB-1 and PCNA stains can be applied even on small biopsy specimens . MIB-1 produces homogenous staining without background; it also strongly stains mitotic figures . It can be performed on routinely processed tissues, permitting the simultaneous evaluation of the morphology and tumour cell kinetics . The wide standard deviations of the proliferative indices found for intermediate grade NHL suggest that this category probably includes various degrees of malignancy. Exp Brain Res, 1994, 102(1), 10 - 20 Regulation of dopamine levels in intrastriatal grafts of fetal mesencephalic cell suspension: an in vivo voltammetric approach; Moukhles H et al.; An in vivo voltammetric technique was used to monitor dopamine (DA) release in the 6-hydroxydopamine (6-OHDA)-lesioned rat striatum reinnervated by grafts of ventral mesencephalon containing DA neurons . Extracellular levels of DA were measured during the administration of D1 or D2 DA receptor antagonists . In addition, changes in DA levels induced by agonists and antagonists of excitatory amino acid (EAA) receptors were studied to verify the possible existence of a host glutamatergic control on the grafted DA cells in the 'transplanted' rats . Two months after the grafts were performed, the voltammetric signal measured under baseline conditions in the grafted striata was found to be almost similar to that recorded on the contralateral control side . Likewise, in another group of transplanted rats, the turnover of the amine, as expressed by the DO-PAC/DA tissue level ratio, was found to have become "normalized" after grafting, compared with the lesion-only group . The increase in the voltammetric signal observed after administering the D2 antagonist sulpiride (100 mg/kg i.p.) was lower in the grafted striata than on the contralateral side, however . This suggests that some D2 autoreceptor subsensitivity may have helped to maintain the baseline level of dopaminergic transmission . Adaptive processes of this kind might compensate for the partial DA reinnervation of the host striatum found to occur on the basis of the tyrosine hydroxylase immunostaining patterns . After administration of either the D1 antagonist SCH 23390 (0.1 mg/kg s.c.), or injection of EAA receptor agonists--1-glutamate, quisqualate and N-methyl-D-aspartate (all 10 nmol i.c.v.)--and antagonists--amino-phosphono-valeric acid (10 nmol i.c.v.) and dizocilpine (MK801, 0.2 mg/kg i.p.)--no significant differences between the two striata were detected in the voltammetric signals . These results suggest that, in the grafted rats, neurons belonging to the host population, such as the striatal cells bearing D1 receptors or the corticostriatal afferents presumed to contain glutamate, might modulate the DA levels, as was found to occur under normal conditions. Folia Parasitol (Praha), 1994, 41(3), 173 - 6 Response of Ig-positive cells to Goussia carpelli (Protozoa: Apicomplexa) infections in carp (Cyprinus carpio L.); Steinhagen D et al.; The kinetics of Ig-positive cell populations in carp tissues was followed during an infection with the gut dwelling coccidian Goussia carpelli Leger et Stankovich, 1921 . In cell suspensions of the anterior and posterior sections of the intestine, the proportion of Ig-positive cells increased with the development of the coccidia and peaked during oocyst formation at day 15 post exposure . These results suggest a reaction of the local mucosal immune system . In cell suspensions of pronephros the proportion of Ig-positive cells increased as well, indicating that a systemic immune response was also induced against this intestinal coccidian parasite of carp. Breast Cancer Res Treat, 1994, 31(2-3), 285 - 99 Metabolism of breast cancer cells as revealed by non-invasive magnetic resonance spectroscopy studies; Kaplan O et al.; The basis for the use of nuclear magnetic resonance (NMR) spectroscopy as a tool to study the metabolism of breast cancer cells is described . The differences between proton (1H), carbon (13C), and phosphorus (31P) NMR methods is explained, and the techniques of cell extracts, cell suspensions and perfusion methods for cells are detailed . In order to perfuse cells they are preferably trapped in a gel matrix, either in the form of a thread or a bead . The gel must have appropriate properties that enables efficient oxygenation and availability of nutrients and drugs . The metabolic effects of perfusion of breast cancer cells with nutrients, drugs, and hormones are reported, and the clinical relevance of these results and methods are outlined. Adv Neuroimmunol, 1994, 4(3), 261 - 4 In vivo model of HIV infection of the human brain; Achim CL et al.; Approximately one quarter of AIDS patients develop neurologic symptoms attributable to HIV infection within the brain . Previous studies suggest that HIV associated neurologic damage may be mediated by immune factors secreted by activated/infected CNS macrophages . We developed an in vivo system in which human embryonic brain tissue can be infected with HIV and the associated pathology monitored . In this model, dissociated human brain tissue is grown in vitro as single cell suspension in serum free medium . Fetal neural cells aggregate and form "brain microspheres" that are then transplanted into SCID mice . Pilot studies suggest that brain microspheres injected in the fat pad of SCID mice differentiate and survive for several months in vivo . Study of these grafts shows presence of functional neural cells and vascular organization suggesting a blood-brain barrier . When brain microspheres are co-cultured in vitro with HIV-infected human macrophages, virus is detected inside the human neural tissue grafts in SCID mice and measurements of viral and immune factors can be performed . To promote physiologic neuronal differentiation within the human grafts, implantation in the brain of SCID mice is being tested at the present time. Methods Cell Biol, 1994, 41, 297 - 316 Immunochemical quantitation of bromodeoxyuridine: application to cell-cycle kinetics; Dolbeare F et al.; We have described several laboratory procedures for the immunochemical staining of the halopyrimidines, BrdUrd and IdUrd, in cell suspensions for flow cytometry and a method for staining histological sections on slides . Halogenated pyrimidine quantitation allows cell-cycle parameters, including total cell-cycle time, phase durations, and growth fraction to be determined . We have presented some flow cytometric data to demonstrate the use of these methods in determining bivariate BrdUrd/DNA histograms with CHO cells and in kinetic studies with the brown Norway rat myeloid leukemia model. Exp Brain Res, 1994, 101(3), 353 - 64 Synaptic connectivity of serotonin graft efferents in the suprachiasmatic and supraoptic nuclei of the hypothalamus; Boulaich S et al.; We have previously reported that a cell suspension</