J Bone Miner Res, 1996 Dec, 11(12), 1926 - 34 Lack of evidence for an increase in interleukin-6 expression in adult murine bone, bone marrow, and marrow stromal cell cultures after ovariectomy; Vargas SJ et al.; Interleukin-6 (IL-6) has been implicated as a mediator of postmenopausal bone loss . In vitro studies of bone and bone marrow cells have suggested that estrogen regulates bone turnover by controlling the production of IL-6, a potent stimulator of osteoclastogenesis and bone resorption . To investigate this hypothesis in an in vivo model, we examined the effect of ovariectomy or estrogen replacement on IL-6 mRNA and protein expression in adult mouse bone and bone marrow in vivo and in marrow stromal cell cultures . Eight-week-old CD-1 mice were sham-operated (SHAM), ovariectomized (OVX), or ovariectomized and subcutaneously implanted with 21-day slow-release pellets containing 10 micrograms of 17 beta-estradiol (O + E) . Placebo pellets were implanted in the SHAM and OVX mice . Uterine weights at 1, 2, or 3 weeks after surgery were significantly decreased (68-76%) in OVX animals compared with SHAM or O + E . In mice sacrificed at 1 or 3 weeks after surgery, we found by nonquantitative reverse transcribed polymerase chain reaction (RT-PCR), that SHAM, OVX, and O + E calvariae (CALV) constitutively expressed IL-6 mRNA . In contrast, IL-6 mRNA was either barely detectable or absent in the tibia (TIB) and bone marrow (BM) . In the mice sacrificed 3 weeks after surgery, we determined by quantitative RT-PCR that IL-6 mRNA in the CALV from the OVX and O + E groups were decreased by 81 and 92%, respectively, compared with SHAM . IL-6 protein levels in the flushed bone marrow (BMSups) were detectable and were not significantly different among the groups . In bone marrow cells that were cultured for 1 week, basal levels of IL-6 protein were low and did not differ significantly among the SHAM, OVX, or O + E groups sacrificed 1, 2, or 3 weeks after surgery . After the addition of hrIL-1 alpha, IL-6 protein levels increased 100- to 1300-fold over control . IL-6 levels in cells from animals sacrificed 2 weeks after surgery were significantly lower in the hrIL-1 alpha-stimulated OVX and O + E groups than in hrIL-1 alpha-stimulated SHAM cell cultures . In conclusion, in this model we could find no increase in IL-6 production with in vivo estrogen withdrawal in calvaria, long bones, bone marrow, or marrow stromal cell cultures . If increases in IL-6 expression are involved in the effects of estrogen withdrawal on bone, the magnitude of these changes are relatively small and below the limits of detection of the assays that we employed. J Bone Miner Res, 1996 Dec, 11(12), 1889 - 96 Inositol trisphosphate receptor gene expression and hormonal regulation in osteoblast-like cell lines and primary osteoblastic cell cultures; Kirkwood KL et al.; The inositol trisphosphate receptor (IP3R) is an intracellular calcium channel that mediates the cellular actions of a wide variety of hormones, growth factors, and cytokines . In osteoblastic cell cultures, many bone resorbing hormones increase phosphoinositide turnover, inositol trisphosphate production, mobilization of intracellular calcium, and the secretion of osteoclast recruitment and activating factors . In this study, the effects of 17 beta-estradiol, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), phrobol ester, and serum on IP3R mRNA levels were evaluated in osteogenic-osteosarcoma cells and in primary osteoblastic cultures derived from neonatal rat calvaria . Type-specific reverse transcription polymerase chain reaction (RT-PCR) indicated that all cell types evaluated (G-292, U-2 OS, Saos-2, MC3T3-E1, UMR-106, and calvarial osteoblastic cells) express IP3R mRNA type I; G-292, U-2 OS, MC3T3-E1, and calvarial osteoblastic cells also express type II IP3R mRNA; and UMR-106 and the calvarial osteoblastic cells express type III IP3R mRNA . Northern blot and RT-PCR analyses of human G-292 osteosarcoma cells and rat calvarial osteoblastic cells showed that phorbol ester and serum increase IP3R mRNA levels, whereas 17 beta-estradiol and 1,25(OH)2D3 decrease these levels . In G-292 cells, the effect of 17 beta-estradiol was not due to accelerated IP3R mRNA degradation and required continued protein synthesis . The results show that multiple IP3R types are expressed in osteoblasts and osteoblastic osteosarcoma cells and that this expression is regulated by 17 beta-estradiol and other osteoporotic and antiosteoporotic hormones . These findings indicate that hormonal control of IP3R expression may be relevant in the chronic regulation of osteoblast secretory activity. J Pharmacol Exp Ther, 1996 Dec, 279(3), 1520 - 6 Effects of silibinin and antioxidants on high glucose-induced alterations of fibronectin turnover in human mesangial cell cultures; Wenzel S et al.; To elucidate the primary mechanism of high glucose cytotoxicity, the cytoprotective properties of antioxidants against metabolical disorders were assessed in human mesangial cell (HMC) cultures . An 8-day incubation of HMC with high glucose concentration (30 mM) resulted in an extracellular accumulation of the matrixprotein fibronectin (FN), owing to both an expansion of the matrix-associated pericellular FN and a 60% increase of the soluble molecule in the culture medium . The high glucose-induced FN alterations were not due to osmotical effects, as assessed by an iso-osmotic mannitol control . Rather, they are mediated by oxygen-free radicals because the combined treatment of HMC with high glucose and either the antioxidative flavonoid silibinin (given as the water soluble derivative silibinin-C-2,3-dihydrogensuccinate disodium salt) or a radical scavenger cocktail totally prevented the extracellular FN accumulation . This is corroborated further by the determination of malondialdehyde, a product of lipid peroxidation . Incubation of HMC with high glucose resulted in an increase of malondialdehyde in cell homogenates which was completely counteracted by either silibinin or a radical scavenger cocktail . Silibinin alone had no effects on protein synthesis and culture growth . The data presented are compatible with oxidative stress induced by high glucose concentration in HMC cultures . The study further substantiates the proposed role of silibinin in the amelioration of glucose cytotoxicity in renal cells. J Pharmacol Exp Ther, 1996 Dec, 279(3), 1268 - 73 The role of interferon-gamma in antiretroviral activity of methionine enkephalin and AZT in a murine cell culture; Sin JI et al.; The ability of spleen cells treated with methionine enkephalin (Met-ENK) in the presence of 3'-azido-3'-deoxythymidine (AZT) to produce cytokines and inhibit Friend leukemia virus (FLV) replication in Mus dunni cell cultures was investigated . In the presence of murine spleen cells, combination treatments using AZT plus Met-ENK or concanavalin A reduced FLV replication by 63% and 84%, respectively, as compared with 47% for AZT alone . When interleukin (IL)-2, IL-4 and interferon (IFN gamma) levels were measured in FLV-infected cell cultures, both AZT and Met-ENK treatments induced a higher production of IFN gamma and a slight increase in IL-2 and IL-4, as compared with either treatment alone . Subsequent treatment of FLV-infected cells with concanavalin A-stimulated cell supernatants, containing approximately 10 U/ml each of IFN gamma and IL-2, resulted in inhibition of viral replication . Thus, in the absence of spleen cells, IFN gamma was added to cell cultures to determine whether this cytokine contributed to combination antiviral effects . Results show that addition of IFN gamma alone results in a slight suppression of FLV expression, whereas treatment with both AZT and IFN gamma inhibits FLV replication significantly . Subsequently, addition of anti-IFN gamma antibody to cell cultures treated with Met-ENK blocked antiviral effects due to this neuropeptide . Thus anti-FLV effects of spleen cells treated with Met-ENK in combination with AZT are mediated to a large degree by IFN gamma. Antiviral Res, 1996 Dec, 33(1), 21 - 31 Inhibition of bovine immunodeficiency virus by anti-HIV-1 compounds in a cell culture-based assay; Tobin GJ et al.; The bovine immunodeficiency virus (BIV) and human immunodeficiency virus types 1 and 2 (HIV-1 and -2) are members of the lentivirus genus of retroviruses . Although DNA sequences of these viruses have diverged considerably, the BIV genome organization, function of structural and regulatory genes, and replication cycle are very similar to that of HIV-1, making BIV a potentially useful model to study compounds with anti-HIV-1 activity . A cell culture-based antiviral assay was developed to test compounds for inhibition of BIV replication . The assay uses an embryonic rabbit epithelial (EREp) cell line that is highly sensitive to BIV infection and cytopathology . The 50% effective concentrations (EC50) at which the virus was inhibited in EREp cells were determined for 13 nucleoside analog, non-nucleoside, tumor-suppressive, or membrane-surface inhibitory compounds . The nucleoside analogs (3'-azido-2',3'-dideoxythymidine, 2',3'-dideoxyinosine and 2',3'-dideoxycytosine), surface-membrane inhibitors (dextran sulfate, hypericin, Chicago Sky Blue and quinobene), the nucleoside reductase inhibitor (hydroxyurea), and a tumor-suppressive phorbol ester (prostratin) inhibited BIV with EC50 values similar to those derived in HIV-1 lymphocyte (CD4+)-based assays . BIV was markedly more resistant to inhibition with HIV-1-specific non-nucleoside reverse transcriptase inhibitors (NNRTIs) (thiazolobenzimidazole, oxathiin carboxanilide and thiocarbamate) than was HIV-1, which parallels results with NNRTIs in HIV-2 assays. J Cell Physiol, 1996 Dec, 169(3), 411 - 9 Effects of cell culture time and bone matrix exposure on calmodulin content and ATP-dependent cell membrane acid transport in avian osteoclasts and macrophages; Williams JP et al.; Osteoclasts mediate bone resorption by secretion at the site of bone attachment . This process depends on calmodulin concentrated at a specialized acid-secreting membrane . We hypothesized that increased calmodulin and bone attachment were required for acid secretion . We tested this by studying calmodulin, bone attachment, and membrane acid transport in osteoclasts and their precursor mononuclear cells . Osteoclasts and macrophages were isolated from medullary bone of hens; cell fractions were prepared after culturing cells with or without bone . Calmodulin was visualized by Western analysis; calmodulin mRNA was determined by Northern hybridization, and ATP-dependent membrane acid transport was assayed by acridine orange uptake . Calmodulin decreased in osteoclasts cultured without bone . Calmodulin in isolated macrophages was approximately 25% of osteoclast levels, but increased several fold by 5 days . Bone had no effect . Calmodulin mRNA was similar in osteoclasts with or without bone . However, only osteoclasts cultured with bone retained acid transport capacity . Macrophage calmodulin mRNA was not affected by bone, but increased three fold by day 5, paralleling protein production . Macrophages developed acid transport capacity at 3-5 days, but at lower levels than osteoclasts, and bone had no measurable effect . Chicken cells express 1.6 kb and inducible 1.9 kb calmodulin transcripts; in macrophages and osteoclasts, the 1.9 kb transcript predominated . We conclude that, following isolation, calmodulin levels decline in osteoclasts via a post-transcriptional mechanism . In cultured macrophages, by contrast, calmodulin mRNA, protein, and acid secretion increase with time independently of bone substrate, possibly reflecting differentiation in vitro . Increased calmodulin correlated with membrane acid transport capacity in both cell types . The macrophage findings indicate that stimuli other than bone influence acid transport capacity in this family of cells. J Infect Dis, 1996 Dec, 174(6), 1324 - 7 Contamination of commercially available fetal bovine sera with bovine viral diarrhea virus genomes: implications for the study of hepatitis C virus in cell cultures; Yanagi M et al.; The establishment of cell cultures for hepatitis C virus (HCV) is important for its study as a human pathogen . However, in reported cell lines, HCV demonstrates low levels of replication detected primarily by reverse transcription-polymerase chain reaction (RT-PCR) assays . In attempts to culture HCV, an additional complication was observed . From mock-infected cultures, cDNA of appropriate size was obtained by RT-PCR with primers deduced from conserved domains of the 5' noncoding region of HCV . However, sequence analysis revealed that the cDNA was amplified from bovine viral diarrhea virus (BVDV) . All of 7 bovine sera tested were contaminated with BVDV . In conclusion, most commercially available bovine sera are contaminated with BVDV and, although there is no evidence that the virus is infectious, bovine sera should be screened for this virus by RT-PCR when used in conjunction with HCV or for the development or production of vaccine. J Urol, 1996 Dec, 156(6), 2067 - 72 Ureteral cell cultures II: Collagen production and response to pharmacologic agents; Wolf JS Jr et al.; PURPOSE: To investigate the in vitro response of ureteral cells to potentially anti-fibrotic agents . MATERIALS AND METHODS: Cultured human uroepithelial cells, smooth muscle cells, and myofibroblasts were assayed for proliferation and production of collagen types I and III, with and without the presence of hydrocortisone, colchicine, retinol, verapamil, and D-penicillamine . RESULTS: Hydrocortisone stimulated the proliferation of all three cell types and reduced the type I and type III collagen production by myofibroblasts and smooth muscle cells, respectively . Verapamil enhanced the growth of uroepithelial cells and decreased collagen III production by both uroepithelial and smooth muscle cells . D-penicillamine increased the proliferation of uroepithelial and smooth muscle cells, and inhibited collagen type III production by all three cell types . CONCLUSIONS: In vitro evidence suggests that hydrocortisone, verapamil, and D-penicillamine have effects that could favorably alter the healing of endoscopic ureteral incisions. J Biol Chem, 1996 Nov 22, 271(47), 29612 - 8 The bZIP transcription factor Nrl stimulates rhodopsin promoter activity in primary retinal cell cultures; Kumar R et al.; In vitro DNA binding assays and transient transfection analysis with monkey kidney cells have implicated Nrl, a member of the Maf-Nrl subfamily of bZIP transcription factors, and the Nrl response element (NRE) in the regulation of rhodopsin expression . We have now further explored the role of the NRE and surrounding promoter elements . Using the yeast one-hybrid screen with integrated NRE and flanking DNA as bait, the predominant clone obtained was bovine Nrl . Recovery of truncated clones in the screen demonstrated that the carboxyl-terminal half of Nrl, which contains the basic and leucine zipper domains, is sufficient for DNA binding . To functionally dissect the rhodopsin promoter, transient expression studies with primary chick retinal cell cultures were performed . Deletion and mutation analyses identified two positive regulatory sequences: one between -40 and -84 base pairs (bp) and another between -84 and -130 bp . Activity of the -40 to -84 region was shown to be largely due to the NRE . On co-transfection with an NRL expression vector, there were 3-5-fold increases in the activity of rhodopsin promoter constructs containing an intact NRE but little or no effect with rhodopsin promoters containing a mutated or deleted NRE . Nrl was more effective than the related bZIP proteins, c-Fos and c-Jun, in stimulating rhodopsin promoter activity . The -84- to -130-bp region acted synergistically with the NRE to enhance both the level of basal expression and the degree of Nrl-mediated trans-activation . These studies support Nrl as a regulator of rhodopsin expression in vivo, identify an additional regulatory region just upstream of the NRE, and demonstrate the utility of primary retinal cell cultures for characterizing both the cis-acting response elements and trans-acting factors that regulate photoreceptor gene expression. J Clin Invest, 1996 Nov 15, 98(10), 2346 - 50 Human primary myoblast cell cultures from non-diabetic insulin resistant subjects retain defects in insulin action; Thompson DB et al.; Insulin resistance is a predictor of the development of noninsulin-dependent diabetes mellitus (NIDDM) in humans . It is unclear whether insulin resistance is a primary defect leading to NIDDM or the result of hyperinsulinemia and hyperglycemia . To determine if insulin resistance is the result of extrinsic factors such as hyperinsulinemia primary skeletal muscle cell cultures were established from muscle biopsies from Pima Indians with differing in vivo insulin sensitivities . These cell cultures expressed a variety of muscle-specific phenotypes including the proteins alpha-actinin and myosin, muscle-specific creatine kinase activity, and RNA encoding GLUT4, MYF5, MYOD1, and MYOGENIN . Labeled glucose was used to measure the insulin-stimulated conversion of glucose to glycogen in these cultures . The in vivo rates of insulin-stimulated glycogen production (insulin resistance) were correlated with in vitro measures of glycogen production (P = 0.007, r = 0.58) . This defect in insulin action is stable in a uniform culture environment and is retained over time . The retention of insulin resistance in myoblast derived cell cultures is consistent with the expression of an underlying biochemical defect in insulin resistant skeletal muscle. Indian J Exp Biol, 1996 Nov, 34(11), 1169 - 71 Direct detection of bovine herpes virus-1 DNA from cell culture fluids using polymerase chain reaction; Sreenivasa BP et al.; A pair of oligomers of 20 and 23 bp were designed for amplifying a 381 bp sequence from glycoprotein IV gene of bovine herpesvirus 1 . The primer pairs were used for amplifying genomic DNA of BHV-1 directly from cell culture fluids under different experimental conditions such as, untreated cell culture fluid, thermal denaturation and proteinase K treatment in presence of detergent . The results reveal that direct thermal denaturation of cell culture fluid is sufficient to detect the virus by polymerase chain reaction. Biofizika, 1996 Nov-Dec, 41(6), 1284 - 8 {The use of 1H-NMR spectroscopy for the study of cell cultures}; Iurkevich DI et al.; Production of lactate by the HSR-1, HSR-8, HET-SR fibroblasts have been investigated by 1H-NMR method . Were investigated both monolayer cell cultures and cells immobilized in collagen lattice . Represented data demonstrate the possibility of the NMR-spectroscopy to investigate growth's processes in the cell cultures. Int J Dev Neurosci, 1996 Nov, 14(7-8), 823 - 39 Characterization of olfactory receptor neurons and other cell types in dissociated rat olfactory cell cultures; Pixley SK; In dissociated cell cultures, control over the cellular environment facilitates study of the differentiation of mature cellular phenotypes . Central to this approach is a rigorous characterization of the cells that reside in culture . Therefore, we have used a battery of cell type-specific antibody markers to identify the cell types present in dissociated cultures of olfactory mucosal cells (containing cells from both the epithelium and lamina propria) . To identify olfactory receptor neurons in the cultures, staining with antibodies against neuron-specific tubulin was compared to staining with antibodies to neuron-specific enolase, the neural cell adhesion molecule, N-CAM, and the adhesion molecule, LI . Staining of mature olfactory neurons in culture, with an antibody against the olfactory marker protein, was compared to staining with antibodies to carnosine . In contrast to tissue section staining, the overlap between carnosine and olfactory marker protein staining was not complete . Olfactory nerve glial cells were immunoreactive for the S100 beta protein and nestin, an intermediate filament found in early neuronal progenitor cells and Schwann cells . Antibodies to nestin did not label olfactory neurons or progenitor cells . An antibody to an oligodendrocyte-Schwann cell enzyme, 2',3'-cyclic nucleotide 3'-phosphodiesterase, did not label olfactory glia, but did label oligodendrocyte-like cells that appeared to be derived from the CNS glial feeder layer . An antibody against the heavy (200 kDa) neurofilament protein stained a minor subset of cells . The cultures also contained muscle cells, cartilage cells and macrophages (and/or microglia) . These results demonstrate that multiple cell types either maintain or re-establish differentiated, cell type-specific phenotypes in dissociated olfactory cell cultures. J Math Biol, 1996 Nov, 35(1), 97 - 113 Modeling tendon morphogenesis in vivo based on cell density signaling in cell culture; Schwarz RI; A mathematical model of tendon morphogenesis is presented that is consistent with the dramatic transitions seen in this tissue as it progresses from rapid growth early in development to no growth in the adult . To accomplish this change, the embryonic chick tendon is hypercellular with each cell dedicating half of its protein production to procollagen but over time, as growth subsides, the tissue gradually becomes hypocellular with each cell producing only about 1% procollagen . Making this transition from the embryonic to the adult state, forming a roughly cylindrical tissue composed of approximately 90% collagen, and linking the correct muscle to the right bone, is a complex task . The proposed solution requires only two factors: an activator of growth and an inhibitor complex, composed of the activator and another molecule that modifies the activity of the activator . From a diverse set of cell culture observations, these two factors were deduced as the primary components of the mechanism that allows cells to signal their presence to their neighbors . Since cell density signaling is the principal regulator of both collagen synthesis and cell proliferation, its components should play the key role in tendon development . A mathematical model based on the changes in the concentrations of these factors with cell density correlates well with the transitions observed in vivo . Furthermore, the model predicts that in the maturing chicken there should be a high cell density region at the muscle tendon interface . Experimental observations of frozen sections of tendon from a 4 month old chicken confirm this prediction. Acta Radiol, 1996 Nov, 37(6), 923 - 6 Toxicity of ethanol in low concentrations . Experimental evaluation in cell culture; Tapani E et al.; PURPOSE: To define the threshold ethanol concentration that is toxic to cultured cells . METHODS: Three malignant cell lines and freshly isolated normal rat hepatocytes were exposed to 0-50% (vol.) ethanol (concentrations used were 0, 5, 10, 15, 20, 25, 30, 40 and 50%) on tissue culture plates for 0.25-60 min (exposure times used were 0.25, 5, 10, 20, 30, 40 50 and 60 min) . Cytotoxicity was estimated by trypan blue exclusion test and from 3H-thymidine incorporation . RESULTS: All cells were killed by a 15-s exposure to 30-40% ethanol while a concentration as low as 15-20% gave a total response after 5-10-min exposures . After a one-hour exposure of F9 carcinoma cells and hepatocytes, a total or nearly total response was achieved with 10% ethanol . The cytotoxic effect was thus dependent both on the exposure time and on the concentration of ethanol . There were no significant differences in ethanol tolerance among the cell types . CONCLUSION: Ethanol seemed to kill cells in the cell culture effectively in much lower concentrations than those currently used in tumour ablation. Trop Anim Health Prod, 1996 Nov, 28(4), 289 - 92 Theileria annulata schizonts found in calf kidney cell culture; Abd el Raouf Y et al.; Mononuclear cells infected with Theileria annulata schizonts were isolated at 4 different times from monolayer cultures of primary kidney cells derived from apparently healthy, 2 to 6 weeks old zebu calves . The presence of such an organism could interfere with the manufacturing procedure of rinderpest tissue culture vaccine if it is to be carried out in calf kidney cell culture. Acta Otolaryngol, 1996 Nov, 116(6), 805 - 11 Establishment of primary cell culture from stria vascularis explants . Morphological and functional characterization; Kim HN et al.; To provide the prerequisite for long-term study of the inner ear related to structural and functional integrity, tissue of stria vascularis with spiral ligament was isolated from Wistar rat cochleas and cultured using the explant-culture technique . The following culture media were used: EMEM with Hepes buffer, hydrocortisone (400 ng/ml), transferrin (5 micrograms/ml) . triiodothyronine (10(-9) M), cholera toxin (10(-10) M), insulin (5 micrograms/ml), and epidermal growth factor (10 ng/ml) . To characterize the cells growing out from the explant, immunofluorescence with cytokeratin (cytokeratin 18) and ultrastructural examination with SEM and TEM were performed . The marginal cell function was investigated by expression of Na+, K(+)-ATPase antisera against beta 2 subunit of rat Na+, K(+)-ATPase and P-NPPase . We were able to maintain the cultured cells for 3 weeks or more . Monolayered marginal cells were observed beyond 14 days in vitro and the expression of cytokeratin 18 was especially enhanced . The cultured marginal cells were almost identical to in vivo cells both as regards ultrastructural features and Na+, K(+)-ATPase activity . The present results suggest that the primary explant culture technique is a reliable in vitro model of strial marginal cells . However, establishment of the cell line is needed for long-term study. Ren Fail, 1996 Nov, 18(6), 867 - 82 Cytotoxicity, zinc protection, and stress protein induction in rat proximal tubule cells exposed to cadmium chloride in primary cell culture; Liu J et al.; Primary cell culture was utilized to study the relationships between stress protein induction by zinc in vivo and cadmium toxicity in vitro . Effects of cadmium on cell viability were evaluated by the alamar blue assay, in conjunction with the ultrastructural morphology of cells by transmission electron microscopy . The expression of stress protein gene products was evaluated by 35S two-dimensional gel electrophoresis . The results showed cytotoxicity of CdCl2 at and above 129 microM (14.55 micrograms cadmium/mL medium) following 4 h of exposure . Prior zinc administration (20 mg zinc/kg, s.c., two daily doses) in vivo significantly protected the cells in vitro as demonstrated by improved cell viability . The 35S labeling of proteins induced by CdCl2 exposure clearly demonstrated for the first time that gene product of the 70-kDa family was induced in cultured rat proximal tubule cells which are the target cells for cadmium toxicity in vivo . Zinc in vivo pretreatment of animals induced proteins in the 90-, 70-, and 38-kDa families, which may act together with metallothionein to protect cells against cadmium toxicity . The results also indicate that the protective effect of zinc remains after the cells have been put in culture and thus provides a system in which we can study the changes that occur as a result of zinc exposure that decreases cadmium toxicity. Am J Clin Pathol, 1996 Nov, 106(5), 634 - 9 Quality control of proliferation marker (MIB-1) in image analysis systems utilizing cell culture-based control materials; Ruby SG et al.; Standard controls for quality control of cell growth (proliferation) assays in image analysis systems are not currently available . The authors have developed a system of controls, based on cultured and harvested human cell lines, that can mimic tissue sections . These controls help ensure quality control for the entire cell proliferation analysis process, from the initial cutting of the paraffin block through fixation, immunohistochemical staining, and interactive image analysis . The use of cell line controls is advantageous because of the greater cell population homogeneity and the volume of uniform slides that are obtainable, as opposed to the use of a heterogeneous tissue sample . This system provides an excellent means of evaluating the day-to-day performance of cell proliferation analysis and may also be adapted for use as a method of multi-institutional proficiency testing. Toxicol Lett, 1996 Nov, 88(1-3), 35 - 7 Tumor necrosis factor alpha stimulates arachidonic acid metabolisms and mucus production in rat tracheal epithelial cell cultures; Nettesheim P et al.; Air-liquid interface (ALI) cultures of rat tracheal epithelial (RTE) cells were used to study the response of differentiated airway epithelial cells to the inflammatory cytokine, tumor necrosis factor alpha (TNF alpha) . We found that the cultures expressed low levels of TNF alpha receptors . TNF alpha stimulated the AA-cascade: cytoplasmic phospholipase A2 (cPLA2) and prostaglandin H synthase 2 (PGHS2) were upregulated; as a result prostaglandin E (PGE2) secretion was increased . Subsequent to the increase in PGE2 mucus secretion increased, suggesting that PGE2 may act as an autocrine regulator of mucus secretion. J Bone Miner Res, 1996 Nov, 11(11), 1694 - 702 The mechanism of beta-glycerophosphate action in mineralizing chick limb-bud mesenchymal cell cultures; Boskey AL et al.; Differentiating chick limb-bud mesenchymal cells plated in micromass culture form a cartilage matrix that can be mineralized in the presence of 4 mM inorganic phosphate (Pi), and 1 mM calcium . Previous studies showed that when beta-glycerophosphate (beta GP) is used in place of Pi, the mineral crystals formed are larger and differ in distribution . The present study shows that the difference in distribution is not associated with alterations in cell proliferation, protein synthesis, or with collagen, proteoglycan core protein, or alkaline phosphatase gene expression . Cultures with 2.5, 5, and 10 mM beta GP did show different levels of alkaline phosphatase activity, and in the presence of low (0.3 mM) Ca had different Pi contents (4, 6 and 9 mM, respectively), indicating that the increase in CaxP product may in part be responsible for the altered pattern of mineralization . However, cultures with beta GP in which alkaline phosphatase activity was inhibited with levamisole still had an altered mineral distribution as revealed by Fourier transform-infrared (FT-IR) microspectroscopy . The presence of a casein kinase II-like activity in the mineralizing cultures, the ability of specific inhibitors of this enzyme to block mineralization, and the known ability of beta GP to block phosphoprotein phosphatase activity suggests that altered patterns of matrix protein phosphorylation may influence mineral deposition in these cultures. Glia, 1996 Nov, 18(3), 211 - 23 Aged median eminence glial cell cultures promote survival and neurite outgrowth of cocultured neurons; Chauvet N et al.; We have recently shown that tanycytes, a particular type of glial cell that has morphological and biochemical similarities with radial glial cells, constitute a preferential support for the regeneration of lesioned neurohypophysial axons . The present study was designed to explore the possible neurotrophic role of tanycytes in vitro . Glial cells derived from the median eminence or from the cerebral cortex of 10-day-old rats were cultured for 4-7 weeks . At these times the majority of the cells identified in the median eminence cultures exhibited immunostaining patterns of tanycytes, as detected in the mediobasal hypothalamus of 10-day-old and adult rats, i.e., they were immunoreactive to vimentin (VIM), to DARPP-32 (a dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein), and to a lesser extent to glial fibrillary acidic protein (GFAP) antibodies . On the other hand, the majority of cells in cortex cultures showed immunostaining patterns of astrocytes, i.e., they were intensely immunoreactive to GFAP and VIM antibodies but negative to DARPP-32 . Cells obtained from the dissociation of 3-day-old rat mesencephalon, cortex, and hypothalamus were cocultured on these glial monolayers, and the number of surviving neurons and their neurite length were quantified after 8 days . Our data showed that, when compared with astrocytes, tanycytes greatly improved both survival (six-to ten-fold higher) and neurite outgrowth (two- to five-fold longer) of cocultured neurons whatever their origin . Experiments performed by coculturing neurons on millicell inserts placed above the glial monolayers showed that diffusible factors from median eminence glial cells slightly increased survival (1.7-fold higher) of cocultured neurons but had no significant effect on neurite outgrowth . These observations indicate: 1) that aged tanycytes have a capacity to support survival and neurite outgrowth for a variety of postnatal neurons; and 2) that this neurotrophic effect is exerted mainly by means of specific molecules bound to the tanycytic plasmalemma limiting membrane and/or to the extracellular matrix. Am J Vet Res, 1996 Nov, 57(11), 1594 - 8 Attempted transmission of Ehrlichia canis by Rhipicephalus sanguineus after passage in cell culture; Mathew JS et al.; OBJECTIVES: To compare the transmissibility by the brown dog tick, Rhipicephalus sanguineus, of a recent isolate of Ehrlichia canis (Ebony) with that of another isolate (Oklahoma) that had been passaged in cell culture, and to assess the genetic similarity of the 2 isolates as reflected in the nucleotide (NT) sequence of 16S rDNA . ANIMALS: 13 healthy dogs of various ages and breeds . PROCEDURE: Larval and nymphal ticks were acquisition fed on acutely infected dogs, and, after molting, they were transmission fed as nymphs and adults, respectively, on Ehrlichia-naive dogs . All dogs were monitored daily by blood smear evaluation for evidence of parasitized leukocytes and by physical examination for clinical signs of ehrlichiosis . Serologic and hematologic values were measured weekly . Using a nested polymerase chain reaction, the 16S rDNA was amplified, and the NT sequence of the template DNA was determined . RESULTS: The Ebony isolate of E canis was successfully transmitted to dogs by nymphal and adult ticks . In contrast, no ticks that fed on dogs harboring the cell-cultured isolate (Oklahoma) transmitted it to dogs . On the basis of 16S rDNA sequence, the 2 isolates were 99.9% similar, with only 1 NT difference . CONCLUSIONS: These results reconfirm the vector potential of R sanguineus for E canis . Passage of the Oklahoma isolate of E canis in cell culture apparently adversely affected its transmissibility by ticks, raising the possibility that cell-cultured isolates of this rickettsia may lose their affinity for ticks . Determination of 16S rDNA sequence suggests minor strain variation within the species E canis. Arch Dermatol, 1996 Nov, 132(11), 1323 - 9 Enhanced interaction of patients' lymphocytes with human dermal microvascular endothelial cell cultures in active Adamantiades-Behçet disease; Treudler R et al.; BACKGROUND AND DESIGN: To elucidate the role of lymphocyte/endothelial cell interactions in patients with Adamantiades-Behcet disease (ABD), we studied 16 patients of German and Turkish nationality (aged 18-57 years), all with active ABD, and 12 healthy volunteers (controls) of similar age and nationality . Peripheral blood lymphocytes (PBL) of patients were coincubated with human dermal microvascular endothelial cells (HDMEC) and human keratinocytes (HK) in vitro; interactions of PBL with HDMEC and HK were investigated using an established fluorometric assay . Interactions of patients' PBL with HDMEC, HK, or both were the main outcome measures . RESULTS: A significant increase of fluorescence with increasing PBL/HDMEC ratios was seen in patients and controls (P < .001); patients showed a significantly higher increase of fluorescence at higher PBL/HDMEC ratios (P < .05) . The PBL/HK coincubation did not show significant alterations compared with the basal fluorescence signals of HK monolayers alone . Peripheral blood lymphocyte and HDMEC fluorescence values that were more than 2 SDs of controls (defined as positive result of assay) were found in a significantly higher number of patients with 2 or more active symptoms at the time of investigation (83%) compared with patients with only 1 active symptom (10%) (P = .008) . Other clinical data did not correlate with the results of the PBL/HDMEC coincubation assay . CONCLUSIONS: Our results indicate enhanced in vitro interaction of PBL from patients with ABD with HDMEC, which was additionally shown to be a marker of the activity of the disease. J Nutr, 1996 Nov, 126(11), 2831 - 42 7S globulin from soybean is metabolized in human cell cultures by a specific uptake and degradation system; Lovati MR et al.; We examined the biological fate of 7S globulin from soybean in a hepatoma cell line (Hep G2) and in human skin fibroblasts (HSF) to gain new insights into the 7S globulin cell process, the final effect of which is an enhanced expression of the LDL-receptor . The ability of 7S globulin to bind and to be internalized and degraded by both cell types was investigated under different experimental conditions . In all cases, specific uptake (binding + internalization) and degradation of 125I-7S globulin were curvilinear functions of substrate concentration at 37 degrees C . The two processes were saturated at around 80 mg/L, a concentration at which an up-regulation of LDL-receptor was previously reported . The specific uptake of 125I-7S globulin at 37 degrees C was a curvilinear function of time, and achieved equilibrium after 6 and 12 h in HSF and Hep G2 cells, respectively . Binding experiments, conducted at 4 degrees C in Hep G2 cells, showed a specific and saturable association of 7S globulin to the cell membrane . Linear Scatchard analysis demonstrated a single population of binding sites . The amount of 7S globulin bound at saturation (Bmax) was about 2.73 mg/L, with an apparent Kd of 21 micromol/L, assuming 175 kDa as the 7S globulin molecular weight . SDS-PAGE of Hep G2 membrane proteins incubated with 125I-7S globulin revealed a specific interaction of 7S globulin with a cell protein component with molecular weight between 14 and 21 kDa . Further studies are needed to ascertain whether this interaction is directly or indirectly related to the observed stimulation of the LDL-receptor. J Cell Physiol, 1996 Nov, 169(2), 269 - 80 Differential growth factor responses of epithelial cell cultures derived from normal human prostate, benign prostatic hyperplasia, and primary prostate carcinoma; Chopra DP et al.; Because of a lack of information of the optimum nutritional requirements, epithelial cells derived from normal human prostate and prostate tumors have been difficult to propagate in vitro, which hinders research in prostate carcinogenesis . In an effort to establish optimum nutritional conditions and differences in growth characteristics of normal human prostate (NP), benign prostatic hyperplasia (BPH), and prostatic carcinoma (PCA), we have compared the effects of several growth factors on cell proliferation and elucidated growth properties of low passage epithelial cells derived from NP, BPH, and PCA of an African-American patient . Primary and low passage cultures were propagated in serum-free keratinocyte basal medium (KBM) supplemented with insulin (5 micrograms/ml), hydrocortisone (0.5 microgram/ml), epidermal growth factor (EGF, 10 ng/ml), bovine pituitary extract (BPE; 50 micrograms/ml), cholera toxin (10 ng/ml), and antibiotics . Almost all NP, BPH, and PCA cells were positive for cytokeratins and prostate-specific antigen (PSA) . The NP, BPH, and PCA cells were essentially diploid and lacked mutations in c-K-ras and c-Ha-ras oncogenes, and p53 tumor suppressor gene . However, they exhibited progressively accelerating growth parameters . The population doubling times of NP, BPH and PCA were 51 hr, 37 hr, and 29 hr, respectively; their saturation densities were 2.9 x 10(4)/cm2, 3.3 x 10(4)/cm2, and 7.2 x 10(4)/cm2, respectively . The NP and BPH cells required all of the growth factors in the medium, as deletion of any one of the above factors strongly inhibited their growth . The PCA cells, however, were independent of EGF and hydrocortisone . PC-3, an established human prostate cancer cell line, was independent of the growth factors tested . Fetal bovine serum (FBS) inhibited the growth of NP, BPH and PCA cells . In contrast, FBS stimulated the growth of the PC-3 cells in a concentration-dependent manner . These results indicate that in the absence of any apparent karyotype alterations and mutations in c-K-ras, c-Ha-ras and p53 genes, epithelial cells derived from NP, BPH, and PCA exhibit significant differences in their growth properties and responses to growth factors . These variations may represent early changes involved in prostate cancer, while gene mutations and cytogenetic alterations occur in advanced and/or metastatic tumors. Br J Haematol, 1996 Nov, 95(2), 354 - 63 A role for paclitaxel in the combination chemotherapy of acute myeloblastic leukaemia: preclinical cell culture studies; Curtis JE et al.; Paclitaxel dose responses in culture have been investigated alone and in association with cytosine arabinoside (ARA-C) and all-trans retinoic acid (ATRA), with the objective of identifying a role for paclitaxel in the treatment of acute myeloblastic leukaemia (AML) . Initial studies were done to determine if paclitaxel dose responses of AML blast cell precursors were altered by regulatory compounds known to modify the dose responses of ARA-C . In contrast to ARA-C, paclitaxel dose responses were independent of cell culture method, the growth factors G-CSF and GM-CSF, and the ligands all-trans retinoic acid (ATRA) and hydrocortisone . Most blast cell populations were sensitive to paclitaxel; compared with normal marrow progenitors the dose responses were markedly heterogenous with some more, and others less, sensitive . Remission marrow progenitor paclitaxel responses resembled those of AML blasts in heterogeneity . The cell culture model tested the effect of pacliataxel and ATRA on the ARA-C dose responses of OCI/ AML-5; paclitaxel exposure was either before or after ARA-C to test for an effect of schedule; ATRA was added to the MEC cultures after paclitaxel and ARA-C . Repeat experiments were done to test three dose levels each of paclitaxel and ATRA . When paclitaxel was given after ARA-C, synergism was found for all but one of the dose combinations tested; only three examples of synergy were seen when paclitaxel preceded ARA-C . The studies justify trials combining ARA-C, paclitaxel and ATRA using a schedule suggested by the cell culture findings. J Neuroimmunol, 1996 Nov, 70(2), 123 - 9 Reduction of the microglial cell number in rat primary glial cell cultures by exogenous addition of dibutyryl cyclic adenosine monophosphate; Dalmau I et al.; The present work examined the effects induced by dibutyryl cyclic adenosine monophosphate (dB-cAMP) on microglial cells in primary glial cell cultures from newborn rats . Microglial cells were identified by OX42 immunohistochemistry and nucleoside diphosphatase histochemistry . Double staining for astrocytes was carried out by combination with glial fibrillary acidic protein immunolabeling . Addition of 0.25 mM dB-cAMP to the cultures decreased the microglial cell number about sixfold . The findings suggest that the effect of dB-cAMP on the microglial cells might be either a direct action of dB-cAMP on the microglial cells or an indirect effect mediated by the astroglial cells. J Biomed Mater Res, 1996 Nov, 32(3), 333 - 40 In vitro bone formation by rat marrow cell culture; Ohgushi H et al.; Fresh marrow cells were obtained from the femora Fischer rats and cultured in a medium containing 15% fetal calf serum (FCS) to leach confluent . After trypsinization, cells were subcultured at a cell density of 100 x 10(3)/35 mm well in the presence of FCS, 10 mM beta-glycerophosphate, 82 micrograms/mL ascorbic acid phosphate, and 10(-8)M dexamethasone (Dex) . Osteoblastic cells and microscopic mineralized nodules began to appear at about 1 week after the subculture, and at 2 weeks many macroscopic nodules that showed high alkaline phosphatase activity (ALP) and appearance of bone Gla protein (BGP) mRNA were evident . As demonstrated by in situ hybridization, the mRNA was manifested by cuboid-shaped cells (osteoblastic cells) . X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FTIR) showed the mineralization of fine crystals of hydroxyapatite comparable to natural rat bone mineral . In contrast to these findings, subculture done under the same conditions except for the lack of Dex did not show mineralized nodules, nor did they show the osteoblastic phenotype expression . These analyses indicate that Dex-induced mineralization using rat bone marrow cell culture is an in vitro counterpart of bone formed in vivo . Such a culture is useful for investigating materials/ osteogenic cells interactions. Endocrinology, 1996 Nov, 137(11), 4665 - 70 Autocrine down-regulation of collagenase-3 in rat bone cell cultures by insulin-like growth factors; Delany AM et al.; Insulin-like growth factors (IGF)-I and -II are presumed to act as autocrine regulators of bone formation . Recently, we demonstrated that IGF-I and -II inhibit bone collagen degradation and collagenase-3 synthesis in osteoblast cultures . Therefore, we tested the autocrine role of IGFs in the endogenous expression of collagenase-3 in cultures of osteoblast-enriched cells from 22-day fetal rat calvariae (Ob cells) . Steady-state messenger RNA (mRNA) levels were determined by Northern blot analysis and collagenase concentrations in the culture medium were determined by Western immunoblot . Basal level collagenase-3 transcripts decreased in Ob cell cultures, coinciding with an increase in IGF-I and -II protein levels . Removal of the conditioned medium modestly increased collagenase-3 mRNA levels and restored the ability of exogenously added IGF-I to repress collagenase-3 transcripts . IGF neutralizing antibodies and IGF binding proteins-2 and -3 in excess increased and sustained collagenase mRNA, heterogeneous nuclear RNA, and protease levels in Ob cell cultures . In conclusion, IGF-I and -II are autocrine repressors of collagenase-3 synthesis, and this effect may contribute to their actions on the maintenance of a normal bone collagen matrix. Transplantation, 1996 Oct 27, 62(8), 1085 - 9 Comparison of adenovirus gene transfer to vascular endothelial cells in cell culture, organ culture, and in vivo; Merrick AF et al.; A replication-defective adenovirus 5 vector carrying the beta-galactosidase reporter gene was tested for its efficiency for gene delivery to vascular endothelial cells in various situations . Both porcine and human primary vascular endothelial cell cultures were very efficiently infected (>90%) at adenovirus concentrations of 10(10) pfu/ml or higher . Cultured rat fibroblasts and keratinocytes were even more readily infected, with >90% infection with adenovirus titers of 10(8) pfu/ml or higher . However, nondividing vascular endothelium in situ was very poorly transduced . Pieces of aorta from adult pigs, sheep, rabbit and rat, and pieces of human umbilical artery and vein were studied in organ culture . These showed only occasional positive vascular endothelial cells when exposed to the adenovirus vector at concentrations up to 5x10(11) pfu/ml . Kidney perfusion studies in rats and pigs gave similar results . The only exception to the above findings was in very young (3-4 day old) piglets, which showed excellent (>90%) infection of vascular endothelium with the adenovirus vector at titers of 10(10) pfu/ml . Our data suggest that adenovirus vectors will not be of value for gene delivery to uninjured vascular endothelium in situ, and are therefore unsuited for ex vivo genetic manipulation of vascular endothelium in organs for transplantation. Biomed Tech (Berl), 1996 Oct, 41(10), 278 - 83 {A new osteoblast cell culture system for standardized testing of biomaterials}; Hendrich C et al.; For in vitro testing of new biomaterials cultured fibroblasts are employed . In the case of the agar diffusion test survival of cells is involved in the presence of the material to be tested . Further statements on the biological effects of a biomaterial require the use of cell cultures adapted to the tissue concerned and the underlying problem being investigated . In the present study, an osteoblast cell culture system with which implant surfaces in contact with bone can be tested as required by the relevant standards is described . Test bodies made of titanium, polystyrene or copper were used in the conventional agar diffusion test, and were also overgrown with fibroblasts or a cell line of foetal human osteoblasts . For the agar diffusion test, the test criterion was the extent of the inhibition area on staining with neutral red, while for the overgrowth, the mean cell diameter and the number of cells was employed . The phenotype of the osteoblast cell line was determined immunohistochemically by means of alkaline phosphatase or immunohistologically by means of collagen I and osteocalcin . Calcification was demonstrated using the v . Kossa stain . In the case of the osteoblasts, a differentiation of a collagen I and alkaline phosphatase-positive phenotype over an osteocalcin-positive phenotype to an increase in calcium deposition was shown . As in the case of the agar diffusion test, direct overgrowth also revealed no cytotoxic effect for titanium and polystyrene . In contrast, a cytotoxic effect consisting in a decrease in the number of cells and also a left shift in the size distribution was observed for copper . The standard deviations of the individual tests were less for overgrowth than for the agar diffusion test . The culture system for osteoblast cells thus meets the criteria of the EN/DIN 30993-5 in terms of the quality and accuracy of the results obtained . In addition to excluding direct cytotoxicity, this test system offers a new possibility of examining the influence of the material on cell growth . Consequently, it permits a repeatable examination of proliferation and differentiation of the osteoblasts on each material surface. Trends Biotechnol, 1996 Oct, 14(10), 388 - 96 Hematopoietic cell culture therapies (Part II): Clinical aspects and applications; McAdams TA et al.; High-dose chemotherapy, followed by hematopoietic stem cell transplantation, holds significant promise for increasing the probability of long-term remission and possibly cure in a variety of cancers . Hematopoietic cell culture, or ex vivo expansion of hematopoietic cells, may play a significant role in reducing the danger and expense associated with the transplantation procedure . Phase I clinical trials have shown that ex vivo expanded cells have no significant toxicities, and some benefits . Ex vivo expansion of hematopoietic cells is likely to find other applications in gene therapy, tumor purging, production of dendritic cells for immunotherapy and the production of mature blood cells for transfusion therapies. Int J Dev Neurosci, 1996 Oct, 14(6), 785 - 95 Neurotrophic effects of transferrin on embryonic chick brain and neural retinal cell cultures; Bruinink A et al.; The viability and differentiation promoting effects of various transferrins {iron-saturated (holo) and iron-depleted (apo) human and chick ovo (conalbumin)-transferrins, and bovine apo-transferrin} were studied, using serum-free, flat-sedimented cell cultures of embryonic chick brain and neural retina . The effects of transferrin (Tf) on the cell cultures depended on the type of Tf used and the parameter measured . Significant differences between brain and neural retina cultures in the effects of apo-ovoTf and iron {supplemented as ammonium-iron (III) citrate} were detected . Maximal levels of mitochondrial activity were observed in the presence of 2 mg/l apo-ovoTf in neural retina cell cultures . In brain cell cultures, 40 mg ovoTf/l were needed to achieve maximal levels . In brain, but not in neural, retina cell cultures ovoTf and optimal concentrations of Fe3+ exhibited similar effects on biochemical parameters of cell function and differentiation . Although, in the absence of ovoTf, neuronal outgrowth on areas not covered by glial cells was inhibited in both cell cultures, the differences were more prominent in neural retina cell cultures . Our data strongly suggest that Tf plays a key role in processes not connected directly with its iron transport capability. Histochem J, 1996 Oct, 28(10), 671 - 80 S-100 protein subunits in bovine oviduct epithelium: in situ distribution and changes during primary cell culture; Walter I et al.; Cultures of bovine oviduct epithelial cells are widely used in co-culture systems to improve the results of in vitro fertilization . The aim of the present study was to evaluate the suitability of S-100 protein as a differentiation marker for bovine oviduct epithelial cells in vitro . The distribution of S-100 alpha and S-100 beta was examined immunohistochemically in bovine oviduct epithelium in situ and in primary cell cultures derived from it . Three segments of the Fallopian tube (isthmus, ampulla and fimbriae) were compared and analysed during different stages of the oestrus cycle (luteal phase and follicular phase) . Ciliated and non-ciliated cells of the epithelium reacted with anti-S-100 alpha, S-100a (alpha beta) and S-100 beta antibodies, except for isthmic non-ciliated cells, which did not bind anti-S-100 beta or anti-S-100a (alpha beta) . In addition, basal cells never showed immunoreactivity for S-100 . In confluent monolayers of cultured oviduct epithelial cells, disappearance of reactivity for S-100 paralleled morphological signs of dedifferentiation (loss of cilia, cytoplasmic vacuolization) . Free-floating oviduct epithelial cells, in contrast, retained morphological differentiation and still expressed S-100 antigen even after seven days in vitro . The immunohistochemical findings were confirmed by polyacrylamide gel electrophoresis and Western blotting . The results indicate that the presence of S-100 is closely connected to morphological differentiation and to the specific functional condition of bovine oviduct epithelial cells. J Microsc, 1996 Oct, 184 ( Pt 1), 22 - 34 Use of primary cell cultures and intact isolated glandular epithelia for X-ray microanalysis; Hongpaisan J et al.; Changes in the elemental composition of cells during isolation of glandular epithelia were studied by electron probe X-ray microanalysis . Fine chopping of rat submandibular gland followed by enzymatic treatment for 15 min caused marked increases in Na and Cl and a decrease in K concentrations in acinar cells . After enzymatic treatment for 50 min, Na, Cl and K concentrations returned to close to the control level . Mechanical disaggregation of the acinar clumps following enzymatic treatment resulted again in minor increases in Na and Cl and a marked decrease in K concentration . Exposure of isolated acini to cholinergic stimulation in vitro resulted in secretion of Cl and K from the acinar cells . Dissection of the sweat gland from human skin caused a decrease in the K/Na ratio . Incubation of the gland for 30-45 min with collagenase gave rise to a gradual decrease in the K/Na ratio . After mechanical separation of the gland into the secretory coil and reabsorptive duct, a further reduction of the K/Na ratio was seen . However, the duct cells had a much lower K/Na ratio and higher Ca concentration than the coil cells . In primary cultures, the K/Na ratios of the coil and duct cells returned to the in situ level . The elemental composition of sweat gland cells incubated in collagenase-containing medium was no different from that in cells incubated in collagenase-free medium . In the intact collagenase-isolated tissue, Cl- secretion in the coil was elicited by carbachol but not by cAMP, whereas in the duct cells the reverse was the case . In primary cell cultures, Cl- efflux in both coil and duct cells could be elicited by both carbachol and cAMP . In conclusion, although changes in elemental composition of gland cells during the isolation procedure occur, physiological responses can be detected . When primary cell cultures are used, it should be borne in mind that cultured cells may have physiological properties different from those of the intact tissue. J Dermatol Sci, 1996 Oct, 13(1), 25 - 9 Non-disulfided pro alpha 1(IV) chain in B16 melanoma cell culture; Tajima S et al.; We have previously reported that two species of pro alpha 1(IV) collagen chains, disulfided (500 kDa) and non-disulfided (180 kDa), were produced by B16 melanoma cells (J Biochem (1994) 116; 1039-1043) . The mechanism by which the non-disulfided pro alpha 1(IV) chain is produced was studied . No significant difference in prolyl hydroxylation in both polypeptides was observed . When the culture was treated with alpha, alpha 'dipyridyl', a potent inhibitor for hydroxylation of collagen, the secretion of the disulfided alpha 1(IV) chain was inhibited, but non-disulfided alpha 1(IV) chain secretion was not affected . Short pulse and pulse-chase experiments demonstrated that both chains appeared at the same time in the culture medium and the relative amounts of both chains in the medium were unaltered with increasing chase periods . These results indicate that the non-disulfided alpha 1(IV) chain is fully hydroxylated and that it's secretion is independent of it's hydroxylation level . A marked susceptibility of the 180 kDa alpha 1(IV) chain to pepsin at 4 degrees C suggests that the non-disulfided chain may be present in a denatured form. J Parasitol, 1996 Oct, 82(5), 769 - 77 In vitro destruction of nerve cell cultures by Acanthamoeba spp.: a transmission and scanning electron microscopy study; Pettit DA et al.; Trophozoites of 4 species of Acanthamoeba were cytopathic for cultured rat B103 neuroblastoma cells . Cytopathogenicity was evaluated by a chromium release assay and by transmission and scanning electron microscopy . Acanthamoeba culbertsoni, Acanthamoeba castellanii, and Acanthamoeba polyphaga destroyed B103 target cells at 37 C as evidenced by the release of radiolabel . Acanthamoeba astronyxis did not produce cytopathology at 37 C but destroyed nerve cells at 25 C . Transmission and scanning electron microscopy of cocultures maintained at different time periods revealed that all species of Acanthamoeba exhibited long cylindrical structures, termed digipodia, which made contact with target cells . Following this effector cell-target cell contact, membrane blebbing on the nerve cells was observed . These events were followed either by lysis of target nerve cells or ingestion of the target cells via food-cups and their subsequent channeling into intracytoplasmic food vacuoles . Use of the TUNEL (TdT-mediated dUTP nick end labeling) technique indicated that approximately 40% of B103 cells incubated with A . culbertsoni, 20% of B103 cells cocultured with A . castellanii or with A . polyphaga, and less than 1% of B103 cells incubated with A . astronyxis at 37 C were apoptotic after 24 hr of coculture . Studies using electron microscopy indicated that Acanthamoeba trophozoites destroyed nerve cells both by cytolysis and by ingestion of whole nerve cells via food-cups. Endocrinology, 1996 Oct, 137(10), 4451 - 9 Body temperature (37 C) specifically down-regulates the messenger ribonucleic acid for the major sperm surface antigen CD52 in epididymal cell culture; Pera I et al.; To study the role of scrotal vs . body temperature in epididymal function we established a simplified cell culture system from the dog epididymis in which the cells showed a pattern of gene expression similar to that in the human epididymis and retained many characteristics of epididymal epithelial cells . The cultured cells had an epithelial-type cytoskeleton, nuclear androgen receptor protein, and a striking temperature responsiveness . Exposure of the cells to a culture temperature of 37 C, compared to 33 C, had a fast and irreversibly suppressive effect on the levels of an abundant epididymal messenger RNA (mRNA), CE5, which represents the canine counterpart of the human CD52/HE5 mRNA, encoding a major glycosylphoshatidylinnositol (GPI)-anchored sperm membrane glycopeptide . The temperature effect on the mRNA was a direct and specific one, not mediated by temperature influences on the testis and not affecting other epididymal mRNAs . Exposure of the cells to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (25 micrograms/ml culture medium) and cycloheximide (2 micrograms/ml) suggested that the steady state levels of CD52/CE5 mRNA may be controlled posttranscriptionally by changing the half-life of this specific mRNA in response to an extracellular temperature stimulus. Endocrinology, 1996 Oct, 137(10), 4115 - 9 Expression and growth factor regulation of platelet-derived growth factor B transcripts in primary osteoblast cell cultures; Rydziel S et al.; Platelet-derived growth factor (PDGF), an important bone cell mitogen, exists as a homo- or heterodimer product of the PDGF-A and -B genes . Normal unstimulated cells of the osteoblast lineage express the PDGF-A gene, but it is not known whether they express the PDGF-B gene . We examined the expression of PDGF-B messenger RNA (mRNA) levels in cultures of osteoblast-enriched cells from 22-day-old fetal rat calvariae (Ob cells) and determined whether they were modified by transforming growth factor-beta 1 (TGF beta 1), basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF-I), and PDGF-BB . Ob cells expressed PDGF-B transcripts of 3.5 kilo-bases, as determined by Northern blot analysis . Treatment of Ob cells with TGF beta 1 at 0.01-1.2 nM caused a dose-dependent increase in steady state PDGF-B mRNA, an effect that was initially observed after 2 h and was maximal after 6h . Cycloheximide induced PDGF-B transcripts and decreased the effect of TGF beta 1 . TGF beta 1 did not modify the half-life of PDGF-B mRNA in transcriptionally arrested Ob cells and increased the rate of PDGF-B gene transcription in nuclear run-on assays . In contrast, treatment with PDGF-BB at 3.3 nM, bFGF at 6 nM, or IGF-I at 100 nM for 2-24 h did not modify PDGF-B mRNA levels in Ob cells . In conclusion, normal Ob cells express the PDGF-B gene, and TGF beta 1 induces its transcription, whereas bFGF, IGF-I, and PDGF-BB do not enhance the levels of PDGF-B mRNA . PDGF-BB may act not only as a systemic but also as a local regulator of bone cell function. Mol Gen Genet, 1996 Sep 25, 252(4), 465 - 9 Comparable processing of beta-lactoglobulin pre-mRNA in cell culture and transgenic mouse models; Donofrio G et al.; Eukaryotic pre-mRNAs undergo a variety of post-transcriptional modifications, including the removal of intronic sequences by splicing, leading to creation of a functional mRNA . We have compared the processing of transcripts generated from ovine beta-lactoglobulin gene constructs in stably transfected cells and in transgenic mice . In both the in vitro and in vivo model systems the removal of the middle two introns resulted in the inefficient splicing of the downstream, terminal intron . This intron-containing transcript was detected in the cytoplasmic RNA fraction . Thus, the initial in vitro analysis in cell lines of minigene constructs destined for expression in transgenic animals may provide a rapid and reliable indicator of the processing efficiency of the pre-mRNA produced by the construct in vivo . This is in contrast to the apparent limitations of in vitro systems in the analysis of transcription regulatory elements required for transgene expression. Hear Res, 1996 Sep 15, 99(1-2), 71 - 8 Characterization and development of an inner ear type I fibrocyte cell culture; Gratton MA et al.; A method has been developed that allows successful maintenance of secondary cell cultures derived from explants of the cochlear lateral wall of young adult gerbils . The secondary cultures were characterized morphologically with light and transmission electron microscopy and immunocytochemically with protein markers specific to various lateral wall cell types . Structural studies revealed fusiform-shaped cells with a paucity of cytoplasm surrounding the nucleus and slender processes . The cells showed little evidence of intercellular contact even when confluent . The cultures were immunopositive for vimentin, carbonic anhydrase isozyme II, creatine kinase isozyme BB and smooth endoplasmic reticulum Ca-ATPase, but lacked reactivity for cytokeratins and Na,K-ATPase . The results indicate that the cultures are comprised of type I fibrocytes from the spiral ligament . These findings are the first to demonstrate that inner ear spiral ligament cells can be isolated and maintained in secondary culture while retaining many of their in vivo characteristics . Based upon their location and content of ion transport enzymes, type I fibrocytes are thought to be involved in the recycling of potassium from perilymph into the stria vascularis . The establishment of this cell line provides a means to analyze the role of spiral ligament fibrocytes in maintenance of inner ear homeostasis. Virology, 1996 Sep 15, 223(2), 409 - 12 A hyperimmune serum against a synthetic peptide corresponding to the hypervariable region 1 of hepatitis C virus can prevent viral infection in cell cultures; Shimizu YK et al.; To investigate whether a principal neutralization epitope exists in hypervariable region 1 (HVR1) within the putative envelope of hepatitis C virus (HCV), we generated a hyperimmune rabbit serum against a synthetic peptide corresponding to HVR1 of HCV isolate H77 . The reactivity of the serum in the enzyme-linked immunosorbent assay was correlated with the 13 amino acids (position 398-410) in HVR1 . The serum prevented infection with H77 virus in cell cultures but did not prevent infection with H90 virus, a genetically divergent isolate from the same patient . The study demonstrated that neutralization of HCV was mediated, in part, by isolate-specific antibody recognizing HVR1. Toxicology, 1996 Sep 2, 112(3), 219 - 26 Cadmium-induced production of superoxide anion and nitric oxide, DNA single strand breaks and lactate dehydrogenase leakage in J774A.1 cell cultures; Hassoun EA et al.; The involvement of reactive oxygen species in the toxicity of cadmium (Cd) has been proposed . We have, therefore, examined the effects of this cation on the production of superoxide anion and nitric oxide and DNA single strand breaks in J774A.1 macrophage cells in culture as well as the effects on lactate dehydrogenase (LDH) leakage and cell viability . Following a 48-h incubation, over 2-fold increases in superoxide anion and nitric oxide (NO) production were observed at a Cd concentration of approximately 0.60 microM, while a 50% decrease in viability was observed at this concentration . LDH leakage paralleled the superoxide anion and nitric oxide production . Concentration-dependent increases in DNA single strand breaks (SSB) were observed after incubation with Cd with a maximum increase occurring at a concentration of approximately 0.40 microM . The results indicate that Cd is toxic to the J774A.1 cell line, and support the hypothesis that the toxicity may be due at least in part to an oxidative stress induced by the production of reactive oxygen species following exposure to this cation. J Mol Recognit . 1996 Sep-Dec;9(5-6):747. Purification of an expressed insect transferrin from cell culture media using high-capacity Ni(2+)-dipicolylamine gel; Winzerling JJ et al.; Vertebrate transferrin is a well characterized iron transport protein . In contrast, little is known concerning the role of transferrin in insects . Yet, study of iron metabolism in insects could give insights into strategies for insect control, particularly for insects that transmit disease. Toxicol Ind Health, 1996 Sep-Oct, 12(5), 683 - 96 An in vitro model for toxicological investigations of environmental neurotoxins in primary neuronal cell cultures; Schmuck G et al.; Currently, most neurotoxicological investigations are still conducted using various animal models (e.g . chickens, rodents) . In this report, alternative strategies of testing were examined to detect the neurotoxic potency of foreign compounds . Primary neuronal cell cultures from fetal rats are already an accepted model for mechanistic and pharmacological studies in drug research . Their suitability for neurotoxicological studies was examined by using industrial model compounds, which are well-known inductors of neuropathies: acrylamide, hexachlorophene, paraquat, n-hexane, and its neurotoxic metabolites acetylaceton and 2,5-hexandione . As a control compound, the nonneurotoxic solvent n-heptane was used . General cytotoxicity and the intracellular content of glial fibrillary acid protein, neuron-specific enolase, and neurofilaments were measured . n-Heptane induced an acute cytotoxicity and acrylamide and 2,5-hexandione produced a delayed cytotoxicity in primary neuronal cells, whereas the others showed no cytotoxic potency in the tested concentration range . These results were in agreement with the quantification of neurons by neuron-specific enolase . In contrast, with the exception of acetylaceton, glia cells were significantly affected by all neurotoxins at the later time . Signs of axonopathies were demonstrated for acrylamide, n-hexane and its metabolites, as well as for hexachlorophene and paraquat in vitro, by determining the intracellular neurofilament level . Therefore, the determination of cell-specific end points is necessary to detect the neurotoxic potency and quality of a compound, whereas the cytotoxicity assay limited the tested concentration range. J Pathol, 1996 Sep, 180(1), 95 - 101 Cytokine production by cell cultures from bronchial subepithelial myofibroblasts; Zhang S et al.; Myofibroblasts have been previously described beneath the bronchial epithelium and were found to increase in number proportional to the accumulation of extracellular matrix in the bronchial lamina reticularis in asthma . The aim of this study was to assess further the contribution of these structural cells to allergic inflammation in the bronchial mucosa through their cytokine expression . Cell cultures were established from the lamina reticularis of human bronchial biopsies from asthmatic and non-asthmatic subjects . Cytokine secretion was measured by ELISA in supernatants of cultures with or without tumour necrosis factor-alpha (TNF-alpha) . The mRNA levels for granulocyte-macrophage colony-stimulating factor (GM-CSF) in the cultures were examined by ribonuclease protection assays (RPAs) . Bronchial myofibroblasts grown from bronchial biopsies were capable of producing GM-CSF, interleukin-6 (IL-6), interleukin-8 (IL-8), and stem cell factor (SCF) constitutively . The GM-CSF production by myofibroblasts was significantly increased in response to TNF-alpha simulation with a corresponding increase in GM-CSF mRNA expression . The enhancement of GM-CSF production by TNF-alpha in myofibroblasts was blocked by the inhibition of RNA synthesis . Prednisolone abolished the GM-CSF production . This study provides evidence for the role of bronchial myofibroblasts in the regulation of inflammatory cell recruitment and activation by interaction in the cytokine network in the bronchial mucosa. Hum Reprod, 1996 Sep, 11(9), 1966 - 74 The influence of Vero cell culture on human embryo development and chorionic gonadotrophin production in vitro; Turner K et al.; Co-culture of human embryos with cell layers has generally shown that blastocyst formation rates are improved compared to routine culture in medium alone . In order to assess this further, we have additionally classified resulting blastocysts according to their morphology and secretion of human chorionic gonadotrophin (HCG) . A total of 70 supernumerary human embryos from 15 patients were divided equally and randomly between two culture conditions: (i) co-culture with Vero cells; and (ii) culture in our routine medium . Embryo development and morphology were recorded for up to 14 days in culture . The results showed that embryos on Vero cells had a significantly higher blastocyst formation rate (P < 0.02) by or on day 6 of development than those in routine culture medium alone (77 and 46% respectively) . For HCG analysis, the culture medium was changed in both culture systems on days 5, 7, 9, 12 and 14 of embryo development and analysed . Most embryos began to produce HCG between days 7 and 9, with HCG secretion being significantly higher from embryos on Vero cells between days 9 and 12 than from embryos in routine culture (P < 0.03) . The morphology of the blastocysts obtained was related to their ability to hatch and produce HCG but was not significantly better for one type of culture system than for the other. Acta Otolaryngol, 1996 Sep, 116(5), 690 - 6 Auditory cortical neurons in vitro: cell culture and multichannel extracellular recording; Gopal KV et al.; Self organization, pattern generation, and pattern processing in local cortical circuits are difficult to study in vivo . The complexities of cortical circuits require simplified systems for study . We have developed a simplified model of auditory cortical neurons growing as monolayer networks in culture . Neurons dissociated from auditory cortex of 14-day mouse embryos were grown on photoetched microelectrode array containing 64 transparent indium-tin oxide electrodes . Cultures were maintained in incubators for up to 113 days . Neurons developed processes and made synaptic connections . All cultures were spontaneously active and exhibited complex temporal burst patterns . In a data set of 12 cultures, the number of active channels varied from culture to culture and ranged from 6-17 . Signal/noise ratios ranged from 3:1 to a maximum of 16:1 . No significant correlations were found between age of the culture and number of active channels, or signal/noise ratios . Spontaneous firing patterns recorded from various channels showed complex bursting patterns in all cultures . Within a culture, coordinated synchronous bursting were found among some channels, and independent bursting on others . Preliminary histological analysis of cultures using the Loots-modified Bodian stain showed neurons with axonal and dendritic profiles growing extensively on top of the glial carpet . Neuronal processes crossing the electrodes singly or in small groups were also observed . Pyramidal and non-pyramidal cells could be identified . In a pool of 2,093 neurons in a 49-day-old culture, the average size of the somata was found to be 16 microns, with a mode of 12 microns. In Vivo, 1996 Sep-Oct, 10(5), 515 - 26 Cell culture observations of human postnatal thymic epithelium: an in vitro model for growth and humoral influence on intrathymic T lymphocyte maturation; Bodey B et al.; Sixteen postnatal human thymuses were obtained at the time of corrective cardiovascular surgery and maintained in vitro as separate cultures of thymocytes and reticulo-epithelial (RE) cells . The stages of differentiation of the thymocytes were investigated in situ with a library of 10 monoclonal antibodies (MoABs) directed against human lymphocyte differentiation antigens . Employing immunofluorescence staining and flow cytometric (FACS) analysis, in vitro immunophenotype (IP) changes were demonstrated, which appeared after use of a combination of mitogen (PHA), recombinant interleukin-2 (rIL-2) and autologous thymic RE cell culture supernatants . RE cell supernatant participated in increasing the expression of the IL-2 receptor (IL-2R) during combined stimulation with phytohaemagglutinin (PHA) and rIL-2 . Thymocyte proliferation was measured in 4 hour tritiated-thymidine (3H-TdR) incorporation (proliferation) assay . We were able to isolate the thymic nurse cells (TNC) with and without enzymatic tissue digestion . TNCs were separated from accompanying thymocytes and cultured . They grew as large, sometimes connected cells, but did not display the epithelial type of tissue organization . After in vitro culturing, the cytoskeleton of TNCs expressed high molecular weight cytokeratin and vimentin and intracytoplasmic tonofilaments, characteristic of epithelium . Whole thymic tissue pieces were cultured with and without previous trypsinization . The initial outgrowth of the cuboidal epithelial tissue layer occurred within 24-48 hours, and the RE cells remained functionally active for at least 15 days . RE cell supernatants were collected daily for two weeks and used in thymocyte differentiation experiments . The results indicated that thymic humoral factors contribute to a select, not fully understood differentiation pathway of thymocytes: a) more mature immunophenotype (IP) characterized by CD3 expression; b) de novo synthesis of interleukin-2 receptor (IL-2R); and; c) differentiation of the CD8+ subpopulation, identifying regulatory cells within the two major CD8+ and CD4+ subsets . Use of mitogenic (PHA) stimulation, after 5 days in vitro, resulted in a T helper (CD4+) oriented differentiation pathway of cortical thymocytes . At the same time, the cultured thymocytes expressed CD11 de novo, an early thymocyte differentiation antigen, and CD7, a marker not present on mature peripheral lymphocyte subsets (the IP changes demonstrated a dedifferentiation) . Our overall impression, following the studies with the proliferation assays, was that in our experimental in vitro model, thymic hormones did not contribute to the induction of generalized thymocyte proliferation. Bone Marrow Transplant, 1996 Sep, 18 Suppl 1, S18 - 20 Detection of cancer cells in peripheral blood stem cells of women with breast cancer by RT-PCR and cell culture; Kruger WH et al.; The benefit of high-dose therapy and blood stem cell reinfusion for women with high-risk breast cancer is currently under investigation . Contaminations of autologous blood stem cells with cancer cells have been described . Cancer micrometastases may be detected by immunocytochemistry, culture techniques and cytokeratin-19 mRNA reverse transcriptase PCR . Women with breast cancer received adjuvant HD-CTM with peripheral blood stem cell (PBSC) support after surgical therapy and 4 cycles conventional chemotherapy . Peripheral blood stem cells were mobilised by G-CsF and harvested after the third or fourth cycle of standard therapy . Aliquots of PBSC-collections (10(7)-2*10(7) cells) were subjected to CK19-mRNA reverse transcriptase PCR . RNA was extracted by standard methods and reverse transcription was performed with MMV-RT . Integrity of RNA was checked by coamplification of housekeeping sequences . Aliquots of the RT-mix were subjected to PCR-amplification with outer and inner primer pairs, subsequently . A second aliquot of 2*10(7) cells was cultured over 42 days in liquid culture . Cytospins were prepared weekly from cultured cells and evaluated by light microscopy with or without prior immunocytochemistry . Ten leukaphereses from 6 women were available for PCR-analysis and cell culture . Six leukaphereses were negative for CK19-mRNA and for detection of cancer cells by culture technique, two samples were positive for CK19-mRNA and culturally enriched cells and two samples were positive for CK19-mRNA and negative for cultured cancer cells . No sample was positive for cultured cells and negative for CK19-mRNA . Overall, the results corresponded in 80% . Two sensitive techniques for the detection of cancer micrometastases were applied to aliquots from 10 leukaphereses of six breast cancer patients with corresponding results in 80% . PCR-mediated detection of cancer cells was confirmed by culture technique and light microscopy, however, further comparison of CK19-PCR with standard techniques like cell culture and immunocytochemistry is still necessary. Keio J Med, 1996 Sep, 45(3), 200 - 6 Human brain microvessel endothelial cell culture as a model system to study vascular factors of ischemic brain; Nagy Z et al.; Cerebral ischemia is caused by reduced blood supply at the microcirculatory level . In the microvessels, the main elements of the reperfusion injury following brain ischemia are the transformation of endothelial cell-surface from anticoagulant to procoagulant property, leukocyte adhesion, sludge or clot formation . There is a paucity of information on how hemostatic factors, cytokines, lipoprotein(a) (Lp(a)) and endothelin-1 (ET-1), being responsible for ischemic/reperfusion injury, interact with human brain microvessel endothelium (HBEC) . There are no data furthermore about the expression of complement proteins of HBEC influenced by cytokines or fibrinolytic factors . Previously we established optimal conditions for culturing HBEC . Cell contraction induced by thrombin, plasmin, miniplasmin was recorded . The reassembly of F-actin was observed after thrombin treatment . ICAM-1 upregulation was measured following TNF-alpha, IL-1-alpha and thrombin incubation . Plasmin and miniplasmin downregulated the ICAM-1 in our cell culture system . Lp(a) modulated the thromboresistant cell-surface by reduction of t-PA and u-PA, but PAI-1 remained unchanged . Lp(a) modulated the ET-1 production by early increasing and late decreasing, in a bimodal manner . The increased secretion of ET-1 by cytokines (TNF-alpha, IL-1-alpha) was reduced in the presence of Lp(a) . Gradual increase of complement proteins (factor H, factor B, C4) was induced by cytokines . Plasmin and miniplasmin augmented a rapid increase of C4 . Some factors of complex relationship between regulators and modulators of endothelial adhesion molecules have been demonstrated in a human cell culture system prepared from brain microvessel endothelium . A unified concept of sequential events of ischemia/reperfusion in the brain has not yet developed. Toxicon, 1996 Sep, 34(9), 1054 - 7 Neuroblastoma cell culture assay shows that Carcinoscorpius rotundicauda haemolymph neutralizes tetrodotoxin; Yeo DS et al.; Veratridine facilitates the influx of Na+ into Neuro-2A cells and this process is exacerbated by the presence of a Na+/K(+)-ATPase inhibitor, ouabain, leading to a reduction in cell viability . However, tetrodotoxin neutralizes the cytotoxic effects of veratridine, hence sustaining the viability of Neuro-2A cells . This neutralizing ability was negated when TTX was first reacted with cell-free haemolymph of Carcinoscorpius rotundicauda, resulting in reduced cell viability . These results therefore indicate a bona fide effect of the cell-free haemolymph against tetrodotoxin as demonstrated by in vitro cell culture technique. J Neurophysiol, 1996 Sep, 76(3), 2111 - 4 Long-term depression of Aplysia sensorimotor synapses in cell culture: inductive role of a rise in postsynaptic calcium; Lin XY et al.; 1 . Activation of sensory neurons at 2 Hz for 15 min induces long-term depression (LTD) of isolated Aplysia sensorimotor synapses in cell culture . 2 . Prior infusion of the Ca2+ chelator 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) into the postsynaptic motor neuron blocks the induction of LTD, but not short-term synaptic depression . 3 . Invertebrate central synapses possess the capacity for LTD . This form of long-term synaptic plasticity may play an important role in learning in Aplysia. J Neurosci Methods, 1996 Sep, 68(1), 49 - 53 Biochemical characteristics of a primary blood-brain barrier cell culture system as a function of the activity of the proteases used in tissue disaggregation; Ng KY et al.; Utilization of primary cultured brain capillary endothelial cells (BCECs) as an in vitro model of the blood-brain barrier (BBB) depends on the extent to which cultured BCECs retain the in vivo characteristics . Recently, we have reported that consistent isolation of BCECs that mimic the in vivo BBB depends on whether a specific ratio between the weight of the isolation enzyme (collagenase/dispase) and the weight of the capillaries present during the isolation is used . Since it is possible for the same weight of an enzyme to possess different activity levels, it is felt that activity rather than weight of an enzyme should be used in arriving at the above ratio . Therefore, using bovine brain as the source of BCECs, we have quantified the amount of collagenase/dispase needed for optimal isolation of BCECs and retention of their phenotypic properties in terms of collagenase/dispase activity per g of capillaries . Monolayers of bovine BCECs isolated at 0.15 or 0.30 units of collagenase and 2.06 or 4.12 units dispase per g of capillaries gave the best overall quality as judged by their permeability characteristics and the activities of angiotensin converting enzyme, alkaline phosphatase and gamma-glutamyl transpeptidase. Eur Respir J, 1996 Sep, 9(9), 1839 - 46 Cell cultures from bronchial subepithelial myofibroblasts enhance eosinophil survival in vitro; Zhang S et al.; Mechanisms of eosinophil accumulation and activation in the bronchial mucosa are crucial for the pathogenesis of asthma . The location of specialized fibroblasts, myofibroblasts, beneath the bronchial basement membrane and their proximity to infiltrating eosinophils potentially enable the myofibroblasts to modulate eosinophil survival and function in asthma . The aim of this study was to investigate the effects of bronchial myofibroblasts on eosinophil survival in vitro . Eosinophils from human peripheral blood were exposed to cell cultures from bronchial myofibroblasts and to myofibroblast-conditioned media . Eosinophil viability was assessed and granulocyte/macrophage colony-stimulating factor (GM-CSF) production was examined in co-culture supernatants and as messenger ribonucleic acid (mRNA) in myofibroblasts . Eosinophil survival was significantly increased and eosinophil apoptosis was inhibited by co-culture with myofibroblasts . Conditioned medium from tumour necrosis factor-alpha (TNF-alpha)-stimulated myofibroblasts also prolonged eosinophil survival . This effect could be blocked by GM-CSF antibody . GM-CSF mRNA and secretion from myofibroblasts were increased in co-cultures and by eosinophil-conditioned medium . Addition of antibodies to TNF-alpha and interleukin-1 alpha (IL-1 alpha) to co-cultures resulted in significant reduction both in eosinophil survival and GM-CSF levels . Blocking of fibronectin in the co-cultures did not affect the eosinophil survival enhancing activity . Prednisolone inhibited the eosinophil survival enhancing activity of the co-cultures by suppression of GM-CSF production . Soluble eosinophil-derived cytokines are involved in the interaction of eosinophils with myofibroblasts, which results in a tumour necrosis factor-alpha/interleukin-1 alpha mediated release of granulocyte/macrophage colony-stimulating factor from myofibroblasts . Bronchial myofibroblasts can, thereby, contribute to allergic inflammation by granulocyte/macrophage colony-stimulating factor-mediated inhibition of eosinophil apoptosis. Biotechnol Prog, 1996 Sep-Oct, 12(5), 700 - 2 Photoimmobilization of insulin onto polystyrene dishes for protein-free cell culture; Ito Y et al.; Photoreactive insulin was synthesized by coupling with azidobenzoic acid . The insulin derivative was immobilized onto the wells of polystyrene culture plates by photoir-radiation . The photoimmobilized insulin enhanced the growth of anchorage-dependent cells such as Chinese hamster ovary CHO-K1 and mouse fibroblast STO cells by more than native or azidophenyl-derivatized insulin . A small amount of photoimmobilized insulin (1-10% of the amount of the native or derivatized insulin) enhanced the growth of CHO-K1 and STO cells . In addition, the maximal mitogenic effect of the immobilized insulin was greater than that of native or derivatized insulin . Photoimmobilization could be a universal means of immobilizing growth factors onto the surface of materials devoid of chemical functional groups scaffolding growth factors and providing a new protein-free cell culture system or tissue engineering materials. J Neuroendocrinol, 1996 Sep, 8(9), 687 - 93 Identification and characterization of angiotensinIV binding sites in rat neurone and astrocyte cell cultures; Greenland K et al.; This study demonstrates the existence of the putative receptor for the hexapeptide (3-8) fragment of angiotensin II (AngIV) on rat astrocytes and neurons grown in cell culture . Binding of 125I-AngIV was saturable and distinct from that of the AngII receptor subtypes . Equilibrium binding was attained in 15 min in astrocytes and 75 min in neurons at 22 degrees C . The bound peptide was confirmed by HPLC to be intact AngIV while the bound peptide was substantially degraded, even in the presence of peptidase inhibitors . Scatchard analysis of equilibrium binding was consistent with a two binding site model, revealing a high affinity and a low affinity binding site in both cell types . In neurons, the respective association constants (Ka) were 2.72 +/- 0.23 nM-1 and 727 +/- 354 nM-1, with associated receptor densities of 109.30 +/- 58.87 and 1723 +/- 1167 fmol/mg protein . Similar analyses in astrocytes gave Kas of 5.71 +/- 2.85 nM-1 and 277 +/- 205 nM-1, and respective densities of 191.1 +/- 90.1 and 1425 +/- 1250 fmol/mg protein . However, the quantitative reliability of these binding isotherms may be influenced by the degration of unbound peptide . Competitive binding analysis was used to determine the specificity of the receptor site, with the relative order of affinities being AngIV > AngIII > AngII(4-8), and no displacement by AngII, Iosartan and PD123319 in either neurons or astrocytes . Autoradiography with 125I-AngIV performed on neuronal cultures demonstrated that binding was confined to a subpopulation of the total cells . These data support the existence of a specific binding site for AngIV in both neurons and astrocytes, consistent with the properties of binding reported previously in the brain, and distinguish this site from the AngII receptor subtypes. J Neurosci Res, 1996 Sep 1, 45(5), 631 - 6 Selective toxicity of the general anesthetic propofol for GABAergic neurons in rat brain cell cultures; Honegger P et al.; The intravenous, short-acting general anesthetic propofol was applied to three-dimensional (aggregating) cell cultures of fetal rat telencephalon . Both the clinically used formulation (Disoprivan, ICI Pharmaceuticals, Cheshire, England) and the pure form (2,6-diisopropylphenol) were tested at two different periods of brain development: immature brain cell cultures prior to synaptogenesis and at the time of intense synapses and myelin formation . At both time periods and for clinically relevant concentrations and time of exposure (i.e., concentrations > or = 2.0 micrograms/ml for 8 hr), propofol caused a significant decrease of glutamic acid decarboxylase activity . This effect persisted after removal of the drug, suggesting irreversible structural changes in GABAergic neurons . The gamma-aminobutyric acid type A (GABAA) blocking agents bicuculline and picrotoxin partially attenuated the neurotoxic effect of propofol in cultures treated at the more mature phase of development . This protective effect was not observed in the immature brain cells . The present data suggest that propofol may cause irreversible lesions to GABAergic neurons when given at a critical phase of brain development . In contrast, glial cells and myelin appeared resistant even to high doses of propofol. Mol Mar Biol Biotechnol, 1996 Sep, 5(3), 167 - 74 Transient expression of luciferase reporter gene after lipofection in oyster (Crassostrea gigas) primary cell cultures; Boulo V et al.; Transient expression of the luciferase gene, under transcriptional control of several heterologous promoters, was obtained in heart primary cell cultures of the Pacific oyster, Crassostrea gigas . Drosophila heat shock protein 70 promoter (hsp70), cytomegalovirus, and simian virus early promoters, controlling the luciferase gene, were transfected into the cell cultures using liposomes . Two culture media were used to establish primary cell cultures and tested as transfection media . Parameters such as the quantity of DNA and the ratio of DNA to liposome were analyzed to define the best transfection conditions . In oysters, the Drosophila inducible hsp70 promoter behaved in a way similar to that observed in other animal species . Moreover, for this study, hsp70 was more efficient than the cytomegalovirus and simian virus promoters. J Gen Virol, 1996 Sep, 77 ( Pt 9), 2277 - 85 Glycoprotein gH of pseudorabies virus is essential for penetration and propagation in cell culture and in the nervous system of mice; Babic N et al.; Glycoprotein H (gH) of pseudorabies virus (PrV) is a structural component of the virion and forms a complex with another glycoprotein, gL . For a detailed analysis of the function of PrV gH, we isolated a gH-deficient mutant on trans-complementing gH-expressing cells after insertion of a beta-galactosidase expression cassette into a partially deleted gH gene . The absence of gH did not affect primary or secondary attachment of PrV but the mutant was not infectious . The defect in infectivity could partially be overcome by experimentally induced membrane fusion using PEG, which suggests that gH was necessary for fusion between virion and cellular membranes . After intranasal inoculation into mice, the LD50 of complemented gH- PrV was more than four orders of magnitude higher than that of wild-type PrV . Infection of the respiratory epithelium was much less efficient with complemented gH- PrV as compared with rescued PrV, reflecting the lack of direct cell-to-cell spread . Complemented gH- PrV was able to penetrate into a few trigeminal and sympathetic first order neurons accessible from the nasal cavity, whereas transneuronal transfer in the second order neurons was not observed . In summary, gH is essential for entry and cell-to-cell spread in cell culture, and for propagation in the nervous system of mice . This substantiates the hypothesis that transneuronal spread in vivo and direct cell-to-cell spread in cell culture are governed by similar mechanisms. J Gen Virol, 1996 Sep, 77 ( Pt 9), 2067 - 71 Cell culture isolation of piscine neuropathy nodavirus from juvenile sea bass, Dicentrarchus labrax; Frerichs GN et al.; A virus causing a vacuolating encephalopathy and retinopathy in juvenile sea bass, Dicentrarchus labrax, was isolated from brain tissue in a fish cell line (SSN-1) derived from striped snakehead, Channa striatus . The isometric, non-enveloped, 30 nm diameter virus particles were resistant to pH 2-9 and heating at 56 degrees C for 30 min . Infectious particles had a buoyant density of approximately 1.31 g/cm3 in CsCl . Two structural polypeptides of molecular mass 40 and 42 kDa were identified and the ssRNA consisted of two fragments of molecular mass 1.10 and 0.51 x 10(6) Da . From these characteristics the virus was identified as a nodavirus . Due to the broad range of susceptible fish hosts and the consistent neuropathology of the disease condition, the generic term piscine neuropathy nodavirus (PNN) is proposed for this infectious agent. Endocrinology, 1996 Sep, 137(9), 3802 - 7 Corticosterone selectively increases follicle-stimulating hormone beta-subunit messenger ribonucleic acid in primary anterior pituitary cell culture without affecting its half-life; Kilen SM et al.; We demonstrated previously that glucocorticoids differentially affect the levels of the two pituitary gonadotropins, LH and FSH, both in vivo and in vitro . In vivo, the effect of glucocorticoids is GnRH independent, indicating a direct action on the gonadotrope, and it leads to selective up-regulation of the pituitary content of FSH and FSH beta-subunit messenger RNA (mRNA) . The objective of the present study was to confirm the direct action of corticosterone (B) on FSH beta-subunit mRNA in primary anterior pituitary cell culture and to assess whether the selective B-induced rise in FSH beta mRNA is mediated through altered stability of the FSH beta transcript . Anterior pituitary glands collected from randomly cycling female rats were dissociated with trypsin . Cells were incubated at 37 C for 48 h and subsequently exposed to vehicle or B (1.7 microM) for an additional 42 h . At the end of the incubation, media were sampled for FSH and LH, cells were lysed, and total RNA was isolated for Northern blot analysis . Exposure to B for 42 h caused direct and selective upregulation of FSH release, FSH content, and FSH beta mRNA; decreased alpha-subunit mRNA; and had no significant effect on LH release, LH content, or LH beta mRNA . To evaluate the mRNA stability of the three subunits, cells were exposed to the transcription blocker actinomycin D (act D; 5 micrograms/ml) for an additional 6 h . The combined 6-h treatment with B and act D slightly, but significantly, suppressed alpha-subunit mRNA and did not change LH beta mRNA, confirming a long half-life of the two gonadotropin subunit mRNAs . In contrast, FSH beta mRNA was significantly suppressed by act D to the same level in vehicle- and B-treated cells . The posttranscriptional decay rate was examined by sampling at 0, 1, 2, 3, and 6 h during the 6-h act D treatment period . Decay curves for FSH beta mRNA were parallel in vehicle- and B-treated cells, indicating that B did not alter FSH beta mRNA stability . We conclude that the selective B-induced rise in FSH beta mRNA is mediated at the level of transcription rather than mRNA stabilization. J Urol, 1996 Sep, 156(3), 1198 - 203 Ureteral cell cultures . I . Characterization and cellular interactions; Wolf JS Jr et al.; PURPOSE: To further understand the biology of ureteral cells, we studied the growth characteristics and in vitro cellular interactions of human ureteral uroepithelial cells, smooth muscle cells and myofibroblasts . MATERIALS AND METHODS: The proliferation, morphology and immunohistochemical characteristics of human ureteral cells grown in vitro were evaluated under varying conditions . RESULTS: The growth and morphology of ureteral cells were dependent upon media characteristics, especially the calcium concentration and presence of epidermal growth factor and bovine pituitary extract . Cells demonstrated specific stimulatory interactions via both soluble and insoluble factors . Most important, uroepithelial cells and smooth muscle cells displayed reciprocal enhancement of growth . CONCLUSIONS: The favorable interactions between ureteral cell types in vitro have implications for future work involving these cells. Cell Immunol, 1996 Aug 25, 172(1), 77 - 83 The effects of the HIV-1 envelope protein gp120 on the production of nitric oxide and proinflammatory cytokines in mixed glial cell cultures; Kong LY et al.; Although the neurotoxicity induced by the HIV envelope protein, gp120, has been demonstrated to require the presence of glial cells (microglia/astrocytes), the mechanisms for the gp120-induced neurotoxicity are not well understood . Moreover, the neurotoxic potencies of gp120s obtained from various HIV isolates are different . Since nitric oxide (NO) and proinflammatory cytokines (TNF-alpha, IL-1, IL-6) produced by glial cells have been involved in the neuropathogenesis of various diseases, this study examined the effects of gp120 obtained from two strains, HIV-1IIIB and HIV-1SF2, of the HIV-1 virus on the production of NO, TNF-alpha, IL-1 alpha, IL-1 beta, and IL-6 in murine primary mixed glial cell cultures . The glial cells exposed to HIV-1IIIB gp120 released NO, TNF-alpha, and IL-6 in a dose-dependent manner, whereas IL-1 alpha and IL-1 beta were undetectable . The cells exposed to HIV-1SF2 gp120 increased the release of IL-6 only . The gp120-induced effects were significantly enhanced by priming glial cells with IFN-gamma . To investigate the cellular sources and mechanisms of the gp120-induced IL-6 production, in situ hybridization with mRNA for IL-6 was performed in HIV-1IIIB gp120- or HIV-1SF2 gp120-stimulated microgliaenriched or astrocyte-enriched cultures . HIV-1IIIB gp120 or HIV-1SF2 gp120 induced the expression of IL-6 mRNA in both microglia-enriched and astrocyte-enriched cultures, indicating that both microglia and astrocytes produce IL-6, and that the transcriptional regulation is involved in the gp120-induced IL-6 production . Taken together, these results demonstrate that the production of NO, TNF-alpha, IL-1, or IL-6 from glial cells is differentially regulated by HIV-1IIIB gp120 and HIV-1SF2 gp120 . These results may provide insights into the roles of NO and proinflammatory cytokines in the neurotoxicity of gp120s and the neuropathology of different strains of HIV-1 viruses. Brain Res, 1996 Aug 19, 730(1-2), 67 - 74 Dexamethasone and forskolin synergistically increase {Met5}enkephalin accumulation in mixed brain cell cultures; McMillian MK et al.; Possible synergistic effects of the glucocorticoid dexamethasone (DEX, 10(-7) M) and the adenylate cyclase agonist forskolin (FSK, 10(-5) M) on {Met5}enkephalin (ME) accumulation were examined in enriched rat glial cultures and in mixed neuronal/glial cultures . In enriched glial cultures, DEX and FSK each stimulated the accumulation of ME 2-3-fold over basal media levels, but there was little additional stimulation when these agonists were combined . In contrast, mixed neuronal/glial cultures showed only weak responses to DEX or FSK alone, but the combination of these agonists produced a pronounced synergistic effect on media ME accumulation (6-10-fold over basal levels) . The DEX effect was mediated via a classical glucocorticoid receptor, since DEX was potent (acting over a concentration range of 10(-11)-10(-7) M), mimicked by corticosterone (10(-6) M), and blocked by the glucocorticoid receptor antagonist RU486 . There was a pronounced time lag (2 days) for the synergistic effects of DEX + FSK to develop . In situ hybridization and immunocytochemical studies suggested that astrocytes were the major source for the increased ME production in all mixed neuronal/glial cultures examined . Creating a mixed culture by plating fetal neurons onto confluent, enriched P7 glial cultures inhibited accumulation of ME in the media . DEX + FSK, but neither agonist alone, overcame this neuronal inhibition and increased accumulation of media ME to levels identical to levels in stimulated enriched glial cultures . The net effect was a 6-fold increase in ME accumulation in the mixed neuronal/glial cultures relative to a 2.5-fold increase in the enriched glial cultures . Neuronal inhibition of basal glial ME production could explain the similar synergistic effects of DEX + FSK observed in all mixed neuronal/glial cultures examined, and may be important in suppressing ME production by astrocytes in the brain. J Immunol, 1996 Aug 15, 157(4), 1415 - 21 Inhibition of transcription factor Stat1 activity in mononuclear cell cultures and T cells by the cyclic AMP signaling pathway; Ivashkiv LB et al.; Activation of T cells results in a cascade of gene activation and subsequent proliferation and differentiation into effector phenotypes . The regulation of transcription factors belonging to the signal transducer and activator of transcription (STAT) family was analyzed in PHA-activated mononuclear cells and in purified T cells activated by cross-linking cell surface CD3 . Cell activation resulted in a delayed induction of STAT DNA-binding activity, which was sustained for several days, was composed predominantly of Stat1 and Stat3, and was blocked by cycloheximide and actinomycin D . Increased Stat1 and Stat3 mRNA and protein levels were detected, respectively 4 and 24 h after activation . Stimulation of the cAMP signal transduction pathway, which skews cytokine production toward a Th2 pattern, resulted in the preferential suppression of Stat1 activity . cAMP inhibited the induction of expression of IL-2 receptor components, but did not inhibit IL-4 receptor alpha-chain and CD69 expression or the induction of activator protein 1 transcription factors . cAMP signaling inhibited Stat1 at several different levels, including suppression of DNA binding and down-regulation of Stat1 protein and mRNA levels . Our results demonstrate the regulation of STAT activity by a signaling pathway that regulates the T cell functional phenotype and is distinct from the cytokine-activated Janus kinase-STAT signaling pathway. J Immunol Methods, 1996 Aug 14, 194(2), 191 - 9 The protein hydrolysate, Primatone RL, is a cost-effective multiple growth promoter of mammalian cell culture in serum-containing and serum-free media and displays anti-apoptosis properties; Schlaeger EJ; The tryptic meat digest Primatone RL is a low-cost medium supplement of a complex nature which serves as a source of amino acids, oligopeptides, iron salts, some lipids and other trace low molecular weight substances . Its addition to mammalian and insect cell culture media significantly improves the cell growth properties of many cell lines . In this work the growth promoting effects of Primatone RL are described in more detail using different mouse hybridomas, a mouse myeloma cell line, and human promyelocytic leukemia HL-60 cells . The positive effects on cell growth induced by Primatone were observed in the presence of serum but were even more pronounced in serum-free culture . In addition the adaptation time from high serum to low (1%) or serum-free growth in the presence of Primatone is also significantly reduced . Primatone RL, when added to HL and DHI medium, improves cell growth under low serum or serum-free conditions by increasing the maximum cell numbers and in particular the viability of the culture . The observed decrease in cell death (apoptosis) induction leads to a significant improvement in antibody (recombinant protein) production by increasing the volumetric yields during long-term batch culture . The so-called anti-apoptotic effects of Primatone RL for mouse hybridomas, which is concentration dependent, is not fully understood. Proc Natl Acad Sci U S A, 1996 Aug 6, 93(16), 8584 - 9 Perturbations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia; Kuznetsov G et al.; The effects of ischemia on the maturation of secretory proteins are not well understood . Among several events that occur during ischemia-reperfusion are a rapid and extensive decrease in ATP levels and an alteration of cellular oxidative state . Since the normal folding and assembly of secretory proteins are mediated by endoplasmic reticulum (ER) molecular chaperones, the function of which depends on ATP and maintenance of an appropriate redox environment, ischemia might be expected to perturb folding of secretory proteins . In this study, whole animal and cultured cell models for the epithelial ischemic state were used to examine this possibility . After acute kidney ischemia, marked increases in the mRNA levels of the ER chaperones glucose-regulated protein (grp)78/immunoglobulin-binding protein (BiP), grp94, and ER protein (ERp)72 were noted . Likewise, when cellular ATP was depleted to less than 10% of control with antimycin A, mRNA levels of BiP, ERp72, and grp94 were increased in kidney and thyroid epithelial cell culture models . Since the signal for the up-regulation of these stress proteins is believed to be the accumulation of misfolded/misassembled secretory proteins in the ER, their induction after ischemia in vivo and antimycin treatment of cultured cells suggests that maturation of secretory proteins in the ER lumen might indeed be perturbed . To analyze the effects of antimycin A on the maturation of secretory proteins, we studied the fate of thyroglobulin (Tg), a large oligomeric secretory glycoprotein, the folding and assembly of which seems to require a variety of ER chaperones . Treatment of cultured thyroid epithelial cells with antimycin A greatly inhibited ( > 90%) the secretion of Tg . Sucrose density gradient analysis revealed that in antimycin A-treated cells Tg associates into large macromolecular complexes which, by immunofluorescence, appeared to localize to the ER . Furthermore, coimmunoprecipitation studies after antimycin A treatment demonstrated that Tg stably associates with BiP, grp94, and ERp72 . Together, our results suggest that a key cellular lesion in ischemia is the misfolding of secretory proteins as they transit the ER, and this leads not only to increased expression of ER chaperones but also to their stable association with and the subsequent retention of at least some misfolded secretory proteins. Cytokine, 1996 Aug, 8(8), 622 - 30 Inflammatory cytokines induce intercellular adhesion molecule-1 (ICAM-1) mRNA synthesis and protein secretion by human retinal pigment epithelial cell cultures; Nagineni CN et al.; Retinal inflammatory diseases in man are associated with an upregulation in the expression of intercellular adhesion molecule-1 (ICAM-1) in cells within the retina and with an increase in soluble ICAM-1 within the vitreous . These studies suggest that this protein may contribute to immunopathological processes within the eye . The effects of inflammatory mediators on the regulation of the expression and secretion of ICAM-1 by human retinal pigment epithelial cell cultures (HRPE) were investigated in order to identify the possible source of soluble ICAM-1 and the conditions which enhance its production . Immunofluorescence studies on TNF-alpha and/or IFN-gamma treated HRPE cells demonstrated cellular expression of ICAM-1 which was predominantly localized to intercellular junctions . Moreover, treatment of HRPE for 24 h with tumour necrosis factor alpha (TNF-alpha) (10 ng/ml), interferon gamma (IFN-gamma) (500 u/ml), interleukin 1 alpha (IL-1 alpha) (10 ng/ml) and IL-1 beta (10 ng/ml) results in the secretion of ICAM-1, ranging from 9 to 13 ng per 10(6) cells . IFN-gamma acts synergistically with (TNF-alpha) and IL-1 in the secretion of ICAM-1 by HRPE . Only 1.75 ng of soluble ICAM-1 was detected in untreated HRPE cells . In contrast, lipopolysaccharide (LPS), IL-6, IFN-alpha or TGF-beta did not exhibit any influence on ICAM-1 secretion by these cells . Northern blot analysis reveals an increased expression of ICAM-1 mRNA in HRPE stimulated with IFN-gamma, TNF-alpha or IL-1 for 24 h . In untreated cells, ICAM-1 mRNA is not detectable . There is a progressive increase in ICAM-1 mRNA levels in cytokine-treated HRPE, that reaches steady state by 12 h . Furthermore, a close correlation is noted between ICAM-1 mRNA levels and the secretion of ICAM-1 protein, suggesting regulation at the level of gene transcription . ICAM-1 secretion by RPE might actively participate in the immune reactions in the retina, by recruiting and activating lymphocytes, and contribute to the immunopathological processes in inflammatory diseases. J Dairy Sci, 1996 Aug, 79(8), 1353 - 60 Cell culture system for studying bovine neutrophil diapedesis; Smits E et al.; Neutrophils are the major defense against bacterial infection in the bovine mammary gland . Neutrophils migrate from blood into the lumen of