Differential effects of gamma interferon on Chlamydia trachomatis growth in polarized and nonpolarized human epithelial cells in culture

The effects of gamma interferon (IFN-gamma) on Chlamydia trachomatis growth in polarized epithelial cells were examined. The range of IFN-gamma concentrations causing aberrant chlamydial growth was wider in polarized than in nonpolarized cultures. Results indicate that chlamydial growth modulation in polarized cells readily leads to persistence and better reflects in vivo conditions.     

Evidence for synthesis and release of catecholamines by human amniotic epithelial cells

The present study investigated the presence, possible synthesis and release of catecholamines (CA) by human amniotic epithelial cells (HAEC) using HPLC with electrochemical detection. The presence of CA was indicated by the detection of norepinephrine (NE), dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in extracts of cultured HAEC. Incubation of HAE cells in medium supplemented with 1-tyrosine (CA precursor) and tetrahydrobiopterin (tyrosine hydroxylase cofactor) significantly increased the production of catecholamines, suggesting CA synthesis by HAEC. In contrast, pharmacological inhibition of tyrosine hydroxylase by alpha-methyl-p-tyrosine (MPT) significantly reduced CA production, further confirming CA synthesis by HAEC. Catecholamines were also detected in the cell incubation media, demonstrating the ability of HAEC to spontaneously secrete CA. Moreover, incubation of cells with 50 mM K+ for 10 min increased the amount of CA released into the medium. Additionally, the detection of DOPAC, a primary metabolite of DA, in HAEC strongly indicates that these cells contain DA metabolizing enzymes. The present results suggest that HAEC synthesize and release CA. These cells may be a possible candidate for transplantation therapy of neurodegenerative diseases such as Parkinson's disease and also may serve as a model to study the aspects of catecholaminergic activity.     

Stimulation of tetrapyrrole synthesis in mammalian epithelial cells in culture by exposure to aminolaevulinic acid

Tetrapyrrole synthesis in CNCM-1221 cells exposed to 0.6 mM aminolaevulinic acid (ALA) was found to be approximately linear over a 6-h period of incubation. The rate was not significantly affected by cell density over a range of 0.015 to 0.15 x 10(6) cells cm(-2) (final cell density). Tetrapyrrole synthesis was not affected by GABA or glutamic acid in concentrations up to 6 mM and 2.72 mM respectively, suggesting that these amino acids, which are similar in structure to ALA, do not competitively inhibit the ALA uptake pathway in these cells. Pre-exposure to haem arginate (up to 100 microM) was inhibitory, presumably by suppression (through the inhibition of ALA synthase) of an endogenous component of the response. The ALA-stimulated response was not modified by co-exposure to AIA (up to 100 mg ml(-1)). Despite significant reduction of protein synthesis, the porphyrinogenic response of cells exposed to ALA was unaffected by cycloheximide (10 microg ml(-1)) or actinomycin D (10 microg ml(-1)) even when cells were preincubated with these agents for 3 h before ALA exposure. Fetal bovine serum (10%) inhibited tetrapyrrole synthesis by 30% but increased the rate of porphyrin export by cells by a factor of 1.5. The uptake of [14C]ALA was shown to be strongly influenced by the density of the cultures. In dense cultures (final cell density of approximately 0.15 x 10(6) cells cm(-2)), the ALA uptake rate was less than 0.8 compared with a maximum rate of 4.2 fmol per cell h(-1) at a cell density of 0.02 x 10(6) cells cm(-2). Since tetrapyrrole synthesis is less affected than ALA uptake by cell density, the resultant discrepancy in ALA incorporation occurring in dense cultures implies that endogenous ALA synthesis is induced in these cells. ALA uptake was not affected by cycloheximide or actinomycin D in serum-free conditions. However, fetal bovine serum decreased external ALA uptake by about 50%. This effect was abrogated by preincubation with cycloheximide.     

Proliferation and agglutinability of primary and transformed human epithelial cells in culture

Primary epithelial populations (HAM) were obtained by dissociation of the amniotic membrane stripped from human placentae. Agglutinability of cells from such normal populations and of cells from the transformed epithelial line WISH was then compared using concavanalin A as mediator. Extensive similar studies have previously been reported with cell strains isolated from other species. Freshly dissociated HAM cells from primary cultures agglutinated much less readily than did cells from WISH populations. Furthermore, the former exhibited a drastic decline in agglutinability as a function of time in suspension culture after trypsinization. Short-term exposure (60 h) of HAM cells in monolayer culture to 5-bromodeoxyuridine (BrdU) elicited heightened agglutinability detectable through 22 days in vitro. Addition of the protease inhibitors n-tosyl-L-lysyl-chloromethyl ketone (TLCK) or p-tosyl-L-arginine-methyl ester (TAME) to the culture medium inhibited proliferation of the WISH line by 40--50% while effecting only a 10-15% inhibition of HAM cells. These results also confirm data with other cell species indicating that high proteolytic activity at the surface of transformed cells may be related to the rapid proliferation rate.     

Expression and function of Fas (CD95) on human renal tubular epithelial cells

Renal transplant rejection is characterized by an influx of mononuclear cells in the tubulointerstitial area. Recent studies indicate that tubular damage during graft rejection is dependent, at least in part, on apoptosis. It is thought that apoptosis may be induced by the mononuclear cell infiltrate via the perforin/granzyme or the Fas/Fas ligand pathway. Fas is a 43-kD member of the tumor necrosis factor receptor family, and ligation results in apoptosis of the Fas-bearing cell. The present study analyzes whether Fas is expressed on human tubular epithelial cells in situ and in vitro. It was found that 50 to 70% of the tubules in renal tissue exhibited a positive staining for Fas. To further study the occurrence of Fas on tubular cells, eight different primary proximal tubular epithelial cell (PTEC) lines were analyzed for Fas expression. More than 90% of the PTEC were positive for Fas, and treatment with IFN-gamma resulted in an even higher expression. To determine whether Fas ligation resulted in apoptosis of PTEC in culture, PTEC were incubated with two different anti-Fas antibodies, which were able to induce apoptosis in Jurkat cells. No apoptotic PTEC were observed after Fas ligation, as determined by morphological staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis. Simultaneous CD40 and Fas ligation, or treatment with IFN-gamma before Fas ligation, also did not result in the induction of apoptosis. Fas ligation did not result in proliferation or activation of PTEC, as measured by interleukin-8 production. Apoptotic PTEC could only be detected when the cells were incubated with both anti-Fas antibodies and cycloheximide, resulting in up to 92% apoptotic cells. This study demonstrates that although renal tubular epithelial cells express Fas, they appear to be resistant to Fas-mediated apoptosis, suggesting that Fas-mediated apoptosis does not play a role in the induction of apoptosis during renal transplant rejection.     

Primary culture and transfection of epithelial cells of human small intestine

OBJECTIVE: To evaluate perioperative and long-term morbidity in patients undergoing selective evaluation of coronary artery disease prior to abdominal aortic aneurysm (AAA) repair. DESIGN: Case series. SETTING: University and Veterans' Administration medical centers. PATIENTS: One hundred eighty-nine consecutive patients undergoing AAA repair between January 1989 and September 1996 were selectively evaluated for coronary artery disease and assigned to 1 of 3 groups: group 1, no abnormal cardiac history, normal electrocardiogram; group 2, minimal symptoms, history of myocardial infarction (MI), older than 70 years, diabetes mellitus, or congestive heart failure; or group 3, severe or unstable angina, ventricular dysfunction. INTERVENTIONS: Group 1 patients proceeded to AAA repair without further workup. Group 2 patients underwent pharmacologic or exercise stress testing followed by coronary angiography and intervention as required. Group 3 patients went directly to coronary angiography and intervention as needed. MAIN OUTCOME MEASURES: Perioperative MI, arrhythmias, or death. Long-term follow-up measures included MI and death. RESULTS: Adequate documentation was available on 171 patients. Twenty-four patients (14%) were in group 1. Of 136 patients (79.5%) in group 2, coronary angiography was performed in 36 (26%), followed by percutaneous transluminal coronary angioplasty (PTCA) in 9 (7%) and coronary artery bypass (CAB) in 5 (4%). Of 11 patients in group 3, 3 (27%) each received PTCA and CAB. Remote CAB or PTCA had been performed in 32 (19%) and 12 (7%) patients, respectively. Two perioperative deaths (1.1%) occurred in the 189 patients, one due to MI in a group 2 patient. There were 2 (1%) nonfatal MIs, both in group 2 patients who had no preoperative intervention. Arrhythmias and/or congestive heart failure occurred in 17 (9%) cases, 7 (39%) having had recent coronary revascularization (P = .001). By univariate analysis, only preoperative renal dysfunction predicted perioperative complications (P = .03) Overall survival by lifetable analysis was 87.9% and 69.7% at 3 and 5 years, respectively. CONCLUSION: Coronary artery disease is common in patients undergoing AAA repair, with 35.7% having preoperative coronary revascularization at some point. Selective preoperative coronary artery disease screening achieves excellent perioperative and late results in this population.     

Restoration of down-regulated PDGF receptors by TGF-beta in human embryonic fibroblasts. Enhanced response during cellular in vitro aging

The study of [125I]PDGF-BB binding to normal human embryonic lung fibroblasts, quiescent when cultured at sparsity in the presence of minute concentrations of homologous PDS, reveals approximately 2 x 10(5) binding sites for PDGF per cell; this number significantly increases during prolonged quiescence of the culture. As late as 48 h after down-regulation of PDGF receptors, the cells restore only partially their capacity to bind PDGF, with aged cells (above CPD 45) responding more rapidly and efficiently than younger ones. TGF-beta significantly enhances restoration of PDGF receptors and, in aged cells in particular, its presence results in total receptor recovery within 24 h, suggesting a concerted action of PDGF and TGF-beta regulating the proliferation of human fibroblasts in tissue regeneration.     

Characterization of CD44-mediated hyaluronan binding by renal tubular epithelial cells

Sedimentation equilibrium studies of dilute solutions of tryptophan synthase reveal dissociation from the holoenzyme form, alpha 2 beta 2, into mixtures of alpha beta 2, small amounts of beta 2, and alpha as well as the original alpha 2 beta 2 holoenzyme. The holoenzyme form is stabilized by pyridoxal 5'-phosphate. A new sedimentation equilibrium analytical procedure shows the dissociation of the second alpha subunit to be negatively cooperative. The analytical procedure calculates theoretical error profiles with assumed values of the dissociation constant, k, and a cooperativity parameter until a match is made between one of the theoretical profiles and that computed from experimental data. The latter profile is calculated with an experimentally determined k and assumed values of the cooperativity parameter.     

TGF-beta1 effects on proliferation of rat intestinal epithelial cells are due to inhibition of cyclin D1 expression

Transforming growth factor-beta 1 (TGF-beta1) arrests intestinal epithelial cells (RIE-1 and IEC-6) in the G1 phase of the cell cycle and inhibits cyclin D1 expression. This report describes experiments designed to elucidate the mechanism of cyclin D1 inhibition and to determine whether inhibition of cyclin D1 expression is the cause, rather than the result, of TGF-beta1-mediated cell cycle arrest. TGF-beta1 inhibition of IEC-6 cell proliferation was associated with a decrease in the abundance of cyclin D1/Cdk4 complexes and a corresponding decrease in Cdk4-dependent phosphorylation of the retinoblastoma protein. Metabolic labeling studies indicated that TGF-beta1 inhibited cyclin D1 synthesis without altering the rate of cyclin D1 protein degradation. Cyclin D1 antisense oligonucleotides blocked serum-stimulated induction of cyclin D1 and DNA synthesis, whereas cyclin D1 sense oligonucleotides had no effect. RIE-1 cells were engineered to overexpress human cyclin D1 under the control of a tetracycline-repressible promoter. These cells entered S phase in the presence of TGF-beta1 only when human cyclin D1 was derepressed by the withdrawal of tetracycline. These data indicate that TGF-beta1 inhibits the synthesis of cyclin D1 in gut epithelial cells and that this inhibition is the cause, rather than the result, of TGF-beta1-mediated arrest of intestinal epithelial cell proliferation.     

Apoptosis of tubular epithelial cells in familial juvenile gouty nephropathy

Chronic ruptures of the patellar tendon are uncommon injuries. They are technically difficult to repair because of scar formation, poor quality of the remaining tendon, and quadriceps muscle atrophy and contracture. We report the reconstruction of a chronic patellar tendon rupture with an interesting complication, a tibial stress fracture. The reconstruction was performed 3 months after the injury using an Achilles tendon-bone allograft and reinforcing suprapatellar wire. At 2 weeks postoperatively, the patient had attained full extension and 90 degrees of flexion. Ten months after the index procedure, the patient had range of motion 0 degrees to 120 degrees and was diagnosed with a healing tibial stress fracture. At 17 months postoperatively, the patient had attained full extension, 120 degrees of flexion, and 85% quadriceps strength. The preoperative goals of attaining full range of motion, improving quadriceps strength, obtaining anatomic patellar alignment, and restoring function were obtained despite the complication of a tibial stress fracture. Although this reconstructive procedure is technically demanding, with potential complications, the functional results obtained can be excellent.     

Anandamide hydrolysis by human cells in culture and brain

Anandamide (arachidonylethanolamide; AnNH) has important neuromodulatory and immunomodulatory activities. This lipid is rapidly taken up and hydrolyzed to arachidonate and ethanolamine in many organisms. As yet, AnNH inactivation has not been studied in humans. Here, a human brain fatty-acid amide hydrolase (FAAH) has been characterized as a single protein of 67 kDa with a pI of 7.6, showing apparent Km and Vmax values for AnNH of 2.0 +/- 0.2 microM and 800 +/- 75 pmol.min-1.mg of protein-1, respectively. The optimum pH and temperature for AnNH hydrolysis were 9.0 and 37 degreesC, respectively, and the activation energy of the reaction was 43.5 +/- 4.5 kJ.mol-1. Hydro(pero)xides derived from AnNH or its linoleoyl analogues by lipoxygenase action were competitive inhibitors of human brain FAAH, with apparent Ki values in the low micromolar range. One of these compounds, linoleoylethanolamide is the first natural inhibitor (Ki = 9.0 +/- 0.9 microM) of FAAH as yet discovered. An FAAH activity sharing several biochemical properties with the human brain enzyme was demonstrated in human neuroblastoma CHP100 and lymphoma U937 cells. Both cell lines have a high affinity transporter for AnNH, which had apparent Km and Vmax values for AnNH of 0.20 +/- 0.02 microM and 30 +/- 3 pmol.min-1.mg of protein-1 (CHP100 cells) and 0.13 +/- 0.01 microM and 140 +/- 15 pmol.min-1.mg of protein-1 (U937 cells), respectively. The AnNH carrier of both cell lines was activated up to 170% of the control by nitric oxide.     

Biological characterization of human epithelial ovarian carcinoma cells in primary culture: the insulin-like growth factor system

Current hypotheses for explaining which sex cares for offspring assume a relationship between the mode of fertilization and the sex showing parental care. In general, it is hypothesized that maternal care should be concentrated in taxa with internal fertilization, and paternal care should be concentrated in taxa with external fertilization. Studies have supported this relationship; however, new comparative techniques and new data on parental care and the frequency of internal fertilization in anurans suggest that the relationship should be re-evaluated. I examined the relationship between mode of fertilization and sex providing parental care in 334 taxa of anurans using concentrated changes tests and in 396 taxa of anurans using a method developed by Ridley (1983. The Explanation of Organic Diversity. The Comparative Method and Adaptations of Mating. Oxford: Clarendon). The results of the concentrated changes tests showed that both female and male parental care are randomly distributed among taxa with respect to mode of fertilization. However, using Ridley's method, I found significant relationships between mode of fertilization and sex providing parental care. The observed and expected numbers of transitions from external fertilization and no parental care to external fertilization and male parental care or to internal fertilization and female parental care are not qualitatively different. Therefore, the results of my analyses suggest that current hypotheses for explaining the occurrence of maternal versus paternal care in anurans should be reconsidered. Copyright 1998 The Association for the Study of Animal Behaviour.     

Molecular mechanism of the regulation of glutathione synthesis by tumor necrosis factor-alpha and dexamethasone in human alveolar epithelial cells

Glutathione (GSH) is an important physiological antioxidant in lung epithelial cells and lung lining fluid. We studied the regulation of GSH synthesis in response to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and the anti-inflammatory agent dexamethasone in human alveolar epithelial cells (A549). TNF-alpha (10 ng/ml) exposure increased GSH levels, concomitant with a significant increase in gamma-glutamylcysteine synthetase (gamma-GCS) activity and the expression of gamma-GCS heavy subunit (gamma-GCS-HS) mRNA at 24 h. Treatment with TNF-alpha also increased chloramphenicol acetyltransferase (CAT) activity of a gamma-GCS-HS 5'-flanking region reporter construct, transfected into alveolar epithelial cells. Mutation of the putative proximal AP-1-binding site (-269 to -263 base pairs), abolished TNF-alpha-mediated activation of the promoter. Gel shift and supershift analysis showed that TNF-alpha increased AP-1 DNA binding which was predominantly formed by dimers of c-Jun. Dexamethasone (3 microM) produced a significant decrease in the levels of GSH, decreased gamma-GCS activity and gamma-GCS-HS mRNA expression at 24 h. The increase in GSH levels, gamma-GCS-HS mRNA, gamma-GCS-HS promoter activity, and AP-1 DNA binding produced by TNF-alpha were abrogated by co-treating the cells with dexamethasone. Thus these data demonstrate that TNF-alpha and dexamethasone modulate GSH levels and gamma-GCS-HS mRNA expression by their effects on AP-1 (c-Jun homodimer). These data have implications for the oxidant/antioxidant balance in inflammatory lung diseases.     

Phytoestrogen concentration determines effects on DNA synthesis in human breast cancer cells

FAUSTLOS is a curriculum that has been developed for the prevention of aggression and potentially violent behavior in children in nursery and primary school. A lack of social skills is regarded as one of the fundamental causes that deteriorates problem and conflict solving. FAUSTLOS is the German version of an American program called Second Step that has been developed by the Committee for Children in Seattle and has been successfully put into practice in several American states over the last eight years. The project "Kinder und Gewalt" has adapted it for the German speaking countries. FAUSTLOS is at present in its pilot phase. The following is a general survey of the inception, contents and methods of the curriculum and the planning and execution of the pilot phase.     

1,25-dihydroxycholecalciferol enhances the expression of MHC class II antigens and intercellular adhesion molecule-1 by human renal tubular epithelial cells

PURPOSE: Beside its role in calcium and phosphorus metabolism, 1,25-dihydroxycholecalciferol (1,25-D3) exerts multiple effects on cytokine and major histocompatibility complex (MHC) class II expression in monocytes and lymphocytes. In different renal diseases tubular epithelial cells express MHC class II molecules and cell adhesion molecules,, such as intracellular adhesion molecule-1 (ICAM-1). Therefore, modulation of MHC class II and ICAM-1 expression in renal tubular epithelial cells by 1,25-D3 may be relevant to lymphocyte adhesion to tubular epithelial cells and immune mediated renal injury. However, the expression of MHC class II antigens and cellular adhesion molecules by renal tubular epithelial cells in response to 1,25-D3 has not been investigated. MATERIALS AND METHODS: We generated human renal tubular epithelial cells and SV40 transfected tubular epithelial cells to investigate immune modulation of 1,25 on renal epithelial cells. Major histocompatibility complex class II molecules and ICAM-1 were detected by a specific enzyme linked immunoassay. RESULTS: We found a dose-dependent increase of both constitutive and induced MHC class II and ICAM-1 expression in tubular epithelial cells stimulated with 1,25-D3. Dose-dependent stimulation of MHC class II and ICAM-1 expression was not restricted to primary human renal tubular epithelial cells but was also detected in SV40 transfected cells. CONCLUSIONS: Expression of MHC class II and ICAM-1 is crucial for antigen presentation by and lymphocyte adhesion to renal tubular epithelial cells. Modulatory effects of 1,25-D3 on immune accessory function of renal tubular epithelial cells may be of clinical significance in renal diseases.     

Erythropoietin production by human renal carcinoma cells in culture

Cells from human renal tumors were grown in monolayer cultures, and the media obtained at each medium change were assayed for erythropoietin activity. The medium from carcinoma I (a granular cell tumor) contained a high level of activity initially. The concentration of erythropoietin activity decreased with time in culture, but was significantly higher than that in controls after four months in vitro. There was , in addition, evidence of an inhibitory material present in the culture media. The activity formed by the tumor cells could be neutralized by an antibody to human urinary erythropoietin. The difference between activity measured in marrow cell cultures and that found by in vitro assay, and the chromatographic properties of the active preparation, suggest that the tumor-derived activity may be largely asialoerthropoietin. Two other renal carcinomas, of a different cellular type, produced significant erythropoietic activity.