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PubMed2004

Proliferative response of corneal endothelial cells from young and older donors.

PURPOSE: To compare the effect of epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor-BB (PDGF-BB), bovine pituitary extract, and fetal bovine serum (FBS), alone or in combination, on proliferation of human corneal endothelial cells (HCEC) cultured from young (<30 years old) and older donors (>50 years old). METHODS: Corneas from donors 2 to 79 years old were obtained from the National Disease Research Interchange. Descemet's membrane with intact endothelium was dissected. Cells were isolated by EDTA treatment and cultured to confluence. The HCEC marker, antibody 9.3.E, tested for pure endothelial populations. Antibody Ki67 and ZO-1 tested either before or after cultured cells reached confluence to indicate cell proliferation and cell-cell contact formation. Cell morphology was documented by inverted phase-contrast microscopy. Passages I through VII were used to test the effect of various factors on cell proliferation. For each study, equal numbers of cells were seeded, maintained overnight in 4% FBS to permit cell attachment, washed, and incubated for up to 3 weeks in one of the following: modified Eagle's Minimum Essential Medium (Opti-MEM-I) alone; Opti-MEM-I plus EGF, NGF, PDGF-BB, bovine pituitary extract, or FBS; or a combination of factors. At various times after seeding, cell numbers were determined by electronic cell counter. For each condition, three separate wells were tested and each sample was counted three times. Studies were repeated at least twice using cells from different donors and age groups. Within each study, a one-way ANOVA test was performed to analyze statistical significance. RESULTS: Cells stained positively with antibody 9.3.E, indicating isolation of HCEC and lack of contamination with epithelial cells or keratocytes. Positive staining of Ki67, indicating cycling cells, was found in subconfluent cultures. Plasma membrane-associated ZO-1 staining and lack of Ki67 staining indicated that cultured cells formed a contact-inhibited monolayer. Cultured cells decreased in density, increased in size, and became more heterogeneous depending on donor age and on the number of passages. Incubation in OptiMEM-I promoted attachment and induced a moderate proliferative response above that of MEM (P < 0.001). In general, proliferative responses to growth stimuli were relatively slow, with cell counts generally plateauing 10 to 14 days after exposure to growth-promoting agents. EGF yielded a broad, dose-dependent effect and, at 5-50 ng/mL, peak cell counts were significantly higher (P < 0.001) than basal levels. EGF consistently stimulated proliferation in cells from younger donors, but was less effective in stimulating growth of cells from older donors. NGF did not show a consistent significant stimulatory effect at any concentration tested. PDGF-BB (25 ng/mL) tended to stimulate growth to a greater extent than EGF (P < 0.05) in cultures from the same donor. Pituitary extract significantly increased counts at 1.0 (P < 0.05) to 100 ug/mL (P < 0.001). PDGF-BB plus pituitary extract demonstrated greater stimulation than pituitary extract (P < 0.01) or PDGF-BB alone (P < 0.01). FBS (1%-8%) increased cell numbers in a dose-dependent manner, and, at 4%-8%, yielded counts significantly higher (P < 0.001) than that of any single growth-promoting agent tested. CONCLUSIONS: HCEC from both young and older donors can proliferate in vitro in response to growth-promoting agents. Proliferation in the presence of multiple mitogens ceased when confluence was reached, indicating the formation of a contact-inhibited monolayer. In general, cells cultured from young donors were more responsive to the agents tested, but the relative response of HCEC to these agents was similar, regardless of donor age. The relative difference in the extent of the response of the same cell population to different mitogens suggests that these mitogens induce different downstream signals. The relatively robust proliferative response of HCEC to FBS may involve stimulation of multiple downstream signaling pathways may involve stimulation of multiple downstream signaling pathways and/or induce more sustained downstream signaling than the other growth-promoting agents tested.

Adolescent↗
PubMed2001

HpaA shows variable surface localization but the gene expression is similar in different Helicobacter pylori strains.

Due to earlier contradictory results regarding the localization of the putative Helicobacter pylori adhesin A (HpaA), we aimed to compare the gene and protein expression and surface localization of HpaA in different H. pylori strains. Five H. pylori strains were cultivated for 11 days and analysed by Northern blot analysis, flow cytometry (FCM), semi-quantitative dot blot, colony blot, immuno-electron microscopy (IEM), and phase-contrast microscopy. The highest transcriptional activity of the hapA gene as observed after 3-4 days of cultivation and two mRNA transcripts of 1600 and 3100 nucleotides, respectively, were detected in all five strains with the hpaA probe. We also showed by reverse transcription-polymerase chain reaction (RT-PCR) that the hpaA gene is co-transcribed with the downstream omp18 gene. The highest total HpaA protein production in bacteria occurred between day 3 and 7, as determined by semi-quantitative dot blot, and was similar in the different strains. The maximal proportion of cells with HpaA on the bacterial surface, detected by FCM, was for strain SS1, 90%; Hel 344, 60%; CCUG 17875, 61%; CCUG 17874, 86% and for strain AH 244 only 35%. By IEM HpaA was detected in all strains both on the bacterial surface and on the flagellar sheath.

Adhesins, Bacterial↗
PubMed1979

Trichomonas vaginalis infections in Sydney: laboratory diagnosis and prevalence.

The prevalence of trichomoniasis in Sydney was determined by examining 1070 sexually active females in three different practices. Infection rates ranged from 17.8% in a sympatomatic venereal-disease clinic group to 0.5% in non-promiscuous family-planning clinic patients. In each of these categories, the infestation rate, when compared with overseas reports, was lower but no explanation for this difference is apparent. Of the various laboratory methods used to detect Trichomonas vaginalis, the most sensitive was a culture method. The most convenient and reliable microscopy technique was phase-contrast microscopy which detected 80% of those with positive culture results. Stuart's Transport Medium will maintain trichomonad's viability for at least 24 hours and allow vaginal specimens to be reliably transported for examination.

Australia↗
PubMed1999

Characterization of a stromal cell model of the human benign and malignant prostate from explant culture.

PURPOSE: There is a lack of suitable in vitro models for the human prostate. To study stromal-epithelial interactions, we established stromal cells in cultures from benign and malignant prostate tissue that resemble more closely the in vivo conditions of the human prostate. MATERIALS AND METHODS: Stromal cells were obtained from explant primary culture, established in DU145 cell conditioned medium and maintained in RPMI-fetal bovine serum (FBS) supplemented with insulin, transferrin and selenium (ITS). Proliferation studies to compare different media were performed using a 3[H]thymidine assay. Stromal cells were characterized by immunocytochemistry using epithelial and mesenchymal markers. Morphology was evaluated by electron microscopy, light and phase-contrast microscopy. Androgen receptor (AR) mRNA expression was measured by polymerase-chain-reaction (PCR). The response to different concentrations of dihydrotestosterone (DHT) and the antihormones flutamide and hydroxyflutamide was tested by 3[H] thymidine assay. RESULTS: Microscopic evaluation revealed typical stromal morphology with elongated cell shapes, cilia, collagen and microfilaments. Immunocytochemical characterization revealed typical fibroblastic and smooth muscle differentiation. ITS supplemented in RPMI-FBS showed the best growth stimulation compared with other serum-free media (p <0.05) and became our basal medium. The presence of DU145 cell conditioned medium in this basal medium showed a significant increase in cell proliferation in stromal cells. Stromal cells maintained AR mRNA expression and significant DHT dose dependent growth stimulation in up to 10 passages. Both the antiandrogens flutamide and hydroxyflutamide counteracted the DHT effect (p <0.05). CONCLUSIONS: This stromal cell model maintains many cellular and functional properties of the human prostate, which may enable us to study growth factor modulation, drug and hormone metabolism in stromal-epithelial interaction with emphasis on the pathogenesis of BPH and prostate cancer.

Cell Division↗
PubMed2000

Fas- and interferon gamma-induced apoptosis in Chang conjunctival cells: further investigations.

PURPOSE: Previously interferon (IFN)gamma-induced apoptosis and expression of inflammation-related proteins in a human conjunctival cell line were demonstrated. The aim of this study was to further investigate the mechanisms of IFNgamma-, Fas-, and cycloheximide (CHX)-induced programmed cell death, with special attention to the role of transcriptional factors NF-kappaB and STAT1. METHODS: In a human conjunctival cell line (Chang conjunctival cells) apoptosis was induced with 500 ng/ml anti-Fas antibody (anti-Fas ab) alone (24 or 48 hours) or, as previously reported, with 300 U/ml of human recombinant IFNgamma alone (48 hours). To study the role of IFNgamma on Fas-induced apoptosis, cells were treated first with IFNgamma at 30 U/ml during 24 hours (nontoxic dose), and then anti-Fas ab was applied for 24 hours. Moreover, to study the influence of CHX on Fas- and IFNgamma-induced apoptosis, cells were treated for 24 hours with 300 U/ml IFNgamma together with a nontoxic concentration (1 microg/ml) of CHX, or with 500 ng/ml anti-Fas ab together with 1 microg/ml CHX (24 hours). After treatment, cell viability (neutral red assay), mitochondrial membrane potential (rhodamine 123 assay), chromatin condensation (Hoechst 33342 assay), and the index Hoechst/neutral red were studied by cold light microplate cytometry. The apoptotic process was sought for by contrast phase microscopy and DAPI staining and was confirmed by immunoblotting of PARP. Activation of caspase-3 (CPP32) and caspase-8 were investigated by Western blot analysis. NF-kappaB and STAT DNA-binding activities were studied by electrophoretic mobility shift assays (EMSA). RESULTS: After 24 and 48 hours of treatment with anti-Fas ab alone, 15% to 20% and 30%, respectively, of apoptotic cells were observed. When anti-Fas sera were applied after IFNgamma pretreatment or together with CHX, 50% to 80% of cells demonstrated morphologic characteristics of programmed cell death. Apoptosis was confirmed by a cleavage of PARP and CPP32, by caspase-8 activation, and by an index Hoechst/neutral red greater than one. All these modifications were preceded by a decrease in mitochondrial membrane potential. EMSA revealed that NF-kappaB was activated after IFNgamma and anti-Fas ab treatments and inhibited after CHX treatment. STAT1 was strongly activated after IFNgamma treatment and only in a minor degree after anti-Fas ab treatment. STAT1-binding activity persisted after CHX treatment. CONCLUSIONS: The relative resistance of Chang cells toward Fas-induced apoptosis could be related to the activation of NF-kappaB. IFNgamma-induced programmed cell death preferentially involves the activation of STAT1 that counterbalances NF-kappaB antiapoptotic effects. In fact, Fas-induced apoptosis was potentiated by IFNgamma or CHX treatments. These results suggest that NF-kappaB activation could maintain cell viability as well as participate in IFNgamma-induced inflammatory modifications, whereas STAT1 activation could provide, in this model, a proapoptotic signal.

Antibodies, Monoclonal↗
PubMed2005

[Effects of hepatocyte growth factor on IL-1alpha triggered tubular epithelial-myofibroblast transdifferentiation and fibronectin secretion in vitro].

OBJECTIVE: To observe the effects of hepatocyte growth factor (HGF) on tubular epithelial-myofibroblast transdifferentiation (TEMT) triggered by IL-1alpha and the fibronectin secretion of TEMT. METHODS: The normal rat kidney tubular epithelial cell line (NRK52E) was cultured for six days on plastic or collagen type I-coated plates in the presence or absence of HGF or IL-1alpha. The morphology of transdifferentiation tubular cells was observed by scanning electron microscopy (SEM) and phase-contrast microscopy. The number of alpha-SMA+ cells, the percentage of alpha-SMA+ cells and the mean channel fluorescence (MCF) were assessed by immunohistochemistry and flowcytometry. The level of fibronectin in supernatant was measured by ELISA. RESULTS: The NRK52E cells triggered by IL-1alpha became fibroblast-like morphologically, and strong alpha-SMA immunostaining of those cells was seen. The level of FN in the culture supernatant, the percentage of alpha-SMA+ cells and the MCF of cells triggered by IL-1alpha were obviously higher than those of blank control group (P<0.05). In the groups with IL-1alpha and different doses of HGF, the transdifferentiation of NRK52E cells was inhibited. With the increase of HGF dose, the percentage of alpha-SMA+ cells and the level of FN showed a tendency to decrease. There was no significant difference between the groups treated with only HGF at different dose levels and the blank control group (P>0.05). CONCLUSION: IL-1alpha can induce tubular epithelial cell to transdifferentiate to myofibroblast and increase the secretion of FN. These results suggest that TEMT may play an important role in the pathogenesis of renal fibrosis. HGF could block the transdifferentiation of tubular epithelial cell and inhibit the secretion of FN. These would provide a novel therapeutic strategy for the treatment of renal interstitial fibrosis and end stage renal disease.

Actins↗
PubMed1984

Influence of cefmenoxime, ceftriaxone, latamoxef, and ceftazidime on the lysis of Klebsiella pneumoniae: a light and electron microscopic study.

The influence of antibiotics on the morphologic alterations of the cell wall of Klebsiella pneumoniae serotype K 17 has been studied by electron microscopy as well as by phase-contrast microscopy. Ceftazidime, cefmenoxime, ceftriaxone, and latamoxef were the antibiotics used. The effect of each concentration between 0.03 and 1,024 mg/liter was investigated on 5 X 10(7) bacteria per milliliter. Two different reactions could be detected: ceftazidime and latamoxef produced at first long forms and suddenly lysis; cefmenoxime and ceftriaxone produced at first long forms, later spherical forms, and then gradually lysis.

Bacteriolysis↗
PubMed1984

The morphology of hematopoietic layers in long-term cultures of mouse bone marrow.

Mouse bone marrow cells in long-term culture were examined with scanning electron microscopy during the first 10 days of growth and with phase contrast microscopy during the first 4 weeks. The development of stroma and hematopoiesis was studied, and phase microscopic observation was used in order to achieve positive cell identifications with scanning EM. We analyzed those cell populations that could not be washed away from the adherent culture layer. These adherent cells in 24-hour cultures contain the full potential of hematopoietic long-term production. Stromal cells started to spread almost immediately and by 5 days had established several layers. Although in early cultures hematopoietic cells were found resting on the surfaces of stromal cells, they were later packed between stromal layers. The blast cells, especially, were usually buried under and between thin sheets of reticulum cells. The study confirms the three-dimensional nature of bone marrow in culture and points to close correspondence with bone marrow structure as studied by others in vivo.

Animals↗
PubMed1977

Reorientation of myofilaments during contraction of a vertebrate smooth muscle.

The purpose of the investigation was to determine whether filaments within smooth muscle cells changed their orientation (with respect to the main axis of the cell) during contraction. The stomach muscle of Bufo marinus was used, since its cells may be easily isolated, enabling direct observation in living cells. In addition to still micrography, cinemicrography was used to record continuously during contraction. Polarization microscopy revealed a change in birefringence after contraction, with relaxed cells exhibiting uniform birefringence while contracted cells displayed a discontinuous pattern. Movies revealed a progressive change in orientation of birefringent elements from nearly parallel to the cell's main axis in relaxed cells to increasingly larger angles to the cell's axis as contraction progressed. Phase-contrast microscopy revealed a change in filamentous components, from being parallel to the cell's axis in relaxed cells to being in an undulating or helical pattern during concentration. Cell shape tended to follow the configuration of the filamentous component. Electron microscopy of muscle strips corroborated the observations of living cells and substantiated the conclusion that filaments change their orientation from parallel to oblique (with respect to the cell's axis) during shortening with an undulating or helical pattern of filaments in shortened muscles.

Animals↗