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PubMed1998

Effect of epidermal growth factor upon morphological changes of human lens epithelial cells.

Phase-contrast and transmission electron microscopic studies were performed to evaluate the effect of epidermal growth factor (EGF) upon the morphology of lens epithelial cells (LECs) obtained from human cataractous lenses, cultured on human anterior lens capsules, and divided into the following three groups: group 1 receiving no EGF supplement, group 2 supplemented with 1 ng/ml EGF, and group 3 supplemented with 10 ng/ml EGE Phase-contrast microscopy revealed that the cells in the group supplemented with 1 ng/ml EGF concentration had elongated, and those supplemented with 10 ng/ml EGF disclosed changes indicating fibroblast-like cells. Under transmission electron microscopy, the cells in the group supplemented with 1 ng/ml EGF concentration had become multilayered and a few cells had a ball-and-socket junction structure and nucleolar chromatin condensation, while those supplemented with 10 ng/ml EGF showed changes not only in the cells but also in the anterior lens capsules to present numerous microfibers. This is the first study to confirm the effect of EGF upon LECs cultured on anterior lens capsules, a condition closely resembling the intraocular environment, and these findings are significant.

Aged↗
PubMedDate not supplied

Effects of isopropyl unoprostone ophthalmic solution on cultured rabbit corneal epithelial cells.

PURPOSE: To investigate the effects of isopropyl unoprostone (referred to as unoprostone) ophthalmic solution on the barrier function of cultured rabbit corneal epithelium grown on permeable supports. METHODS: Rabbit corneal epithelial cells cultured on collagen-coated filter inserts were administered one of the following for 30 min: unoprostone in vehicle solution (polysorbate 80), unoprostone in vehicle solution with a preservative (benzalkonium chloride), preservative only or vehicle only. For a control, no chemicals were added to the medium. After administration, the transepithelial electrical resistance (TER) measurement, a sensitive method by which to investigate the barrier function, and morphological observation using phase-contrast microscopy were performed before exposure and at 0.5, 1, 3, 6, 12, 24, 48 and 72 h after exposure. The transmission electron-microscopic observation was performed before and 72 h after exposure in all experimental conditions. RESULTS: The cells exposed to unoprostone with the preservative showed a significant decrease in the TER, although no morphological changes were observed. The corneal epithelial cells exposed to unoprostone without preservative, the vehicle only or the preservative only did not show any differences from the control group at any measurements. CONCLUSION: The corneal barrier function is damaged by a combined solution of unoprostone and preservative, but not by a single solution of unoprostone, in vitro.

Animals↗
PubMed1998

D2 dopamine-receptor-mediated inhibition of proliferation of rat lactotropes in culture is accompanied by changes in cell shape.

Dopaminergic agonists are effective in vivo in inhibiting lactotrope proliferation and prolactin (PRL)-secreting pituitary tumors. The purpose of the present study was to demonstrate in vitro actions of dopaminergic agents on proliferation and cell shape of rat lactotropes. Anterior pituitary cells cultured with serum-free, chemically defined medium were treated with dopaminergic agents and were labeled with 5-bromo-2'-deoxyuridine (BrdU) for 3 h before the end of culture. BrdU-labeling indices indicative of the proliferation rate of lactotropes were determined by double immunofluorescence staining for BrdU and PRL. Treatment with dopamine for 21 h decreased BrdU-labeling indices of lactotropes in a dose-dependent manner with a nadir at 3 x 10(-7) M. The inhibitory action of 10(-5) M dopamine appeared 15 h after the initiation of treatment and became pronounced with time up to 33 h. The dopamine action was mimicked by treatment with the D2 receptor agonist bromocriptine at concentrations over 10(-9) M. Phase-contrast microscopy revealed that the flat polygonal cell shape of cultured lactotropes had changed to a round refractive cell shape after treatment with dopamine or bromocriptine, and that these changes in cell shape exactly paralleled those in the BrdU-labeling index. The changes in cell shape of lactotropes were accompanied by changes in subcellular distribution of actin filaments. Pretreatment with 10(-7) M eticlopride, a D2 receptor antagonist, blocked the dopamine- or bromocriptine-induced changes in both BrdU-labeling index and cell shape. These results suggest that (1) the in vitro experimental system established in the present study is a good model for studying the mechanism of the antiproliferative action of dopamine and (2) D2-receptor-mediated inhibition of proliferation of lactotropes in serum-free culture is closely related to changes in actin organization and cell shape.

Actins↗
PubMed1998

The effect of minoxidil on keratocyte proliferation in cell culture.

PURPOSE: To determine if minoxidil inhibits keratocyte proliferation in a nontoxic manner. METHODS: Rabbit keratocytes were cultured in Eagle's minimum essential medium supplemented with fetal bovine serum. Minoxidil varying in concentration from 10(0) to 10(3) micrograms/ml was added to the culture medium and incubated for 7 days. The cultures were inspected for morphologic appearance and the cell number was determined at 1, 3 and 7 days after the addition of minoxidil. After 7 days of incubation, minoxidil was withdrawn from the cell culture medium and the cells were examined 3 and 7 days thereafter. In addition, a nonradioactive cytotoxic assay was performed to determine if toxicity is associated with the presence of minoxidil. RESULTS: Minoxidil inhibited keratocyte proliferation in a dose-dependent fashion. 29% of control growth was achieved when keratocytes were cultured for 7 days in 10(3) micrograms/ml, whereas 82% control growth was achieved when keratocytes were cultured in 10(2) micrograms/ml of minoxidil. Intermediate concentrations between 10(2) and 10(3) micrograms/ml produced a linear decline in cell counts in a dose-dependent fashion. The concentration of minoxidil required for 50% control growth at 7 days extrapolated from the dose-response curve was 600 micrograms/ml. Upon withdrawal of minoxidil, cell counts returned to baseline for concentrations of 10(2) micrograms/ml or less. Phase contrast microscopy revealed that the presence of minoxidil was associated with intercellular separation, enlargement of cell bodies and elongated processes. After the withdrawal of minoxidil, the cells in all media reassumed the morphological features of normal keratocytes which included a regular fusiform shape and extensive intercellular contact. The nonradioactive cytotoxic assay revealed the lack of cytotoxicity at all concentrations of minoxidil based on a lack of lactate dehydrogenase release. CONCLUSIONS: Minoxidil inhibits keratocyte proliferation by a nontoxic mechanism. It might be particularly useful for modulating corneal wound healing following excimer laser photorefractive keratectomy.

Animals↗
PubMed1979

NGF and anti-NGF: evidence against effects on fiber growth in locus coeruleus from cultures of perinatal CNS tissues.

The present study examines whether the developing noradrenergic neurons of locus coeruleus depend on endogenous nerve growth factor (NGF) for nerve fiber production and if exogenous NGF stimulates fiber growth in this nucleus, using a collagen gel tissue culture technique. Lucus coeruleus from perinatal rat brain was used in three culture experiments: (1) lucus coeruleus, parietal cerebral cortex, and the superior cervical ganglion, prepared from newborn rats and cultured in different sectors of the same dishes; (2) locus coeruleus and parietal cerebral cortex from 17-day-old rat fetuses cultured in the same manner, and (3) locus coeruleus from 17-day-old rat fetuses co-cultured with spinal, sympathetic and ciliary ganglia from 8-day chick embryos. Experiments 1 and 2 were run with and without addition of NGF and anti-NGF, experiment 3 with and without anti-NGF. Total fiber production in all cultured tissues was evaluated daily by dark field and phase contrast microscopy during 4 days. Adrenergic nerve fiber production was then studied in the same locus coeruleus and superior cervical ganglia from the rats by Falck-Hillarp fluorescence histochemistry. Locus coeruleus and cortex cerebri from fetal rats produced dense fiber halos in culture. Locus coeruleus from newborn rats produced considerably less fibers, newborn cortex only few fibers. Superior cervical ganglia from the same newborn animals produced no or almost no fibers. Addition of NGF was not able to stimulate fiber growth in locus coeruleus nor in cortex cerebri as observed both in the living cultures and by fluorescence microscopy. Likewise, addition of anti-NGF did not affect fiber production in the CNS areas. The negative results with NGF on newborn locus coeruleus and cortex cerebri were in sharp contrast to the strong, highly significant fiber growth response demonstrated by the superior cervical ganglion from the same animals cultured in the same dishes. The third experiment tested whether locus coeruleus in tissue culture contained or produced nerve growth factor or any one of the three chick embryo ganglia. No response whatsoever in these three ganglia was observed. It is concluded that the developing locus coeruleus area does not contain or produce NGF, does not depend on NGF for fiber production, and is not stimulated by exogenous NGF.

Animals↗
PubMedDate not supplied

Growth characteristics of cultured human macrovascular venous and arterial and microvascular endothelial cells.

The morphological and growth characteristics of human macrovascular endothelial cells (ECs) from venous and arterial umbilical cord vessels and microvascular ECs from foreskin were compared during cultivation. By means of time-lapse microcinematography and phase-contrast microscopy, differences in cell morphology and migratory activity between the different types of ECs were found. Growth characteristics were dependent on the type of EC, the nature of the substrates on which the ECs were grown and the presence of growth factors. For all types of ECs optimal growth and formation of a monolayer were observed when the ECs were cultured on fibronectin or gelatin substrates in the presence of EC growth factor and heparin. Under these conditions confluent cultures of macrovascular ECs reached maximal cell densities of 1,400-1,900 ECs/mm2, whereas microvascular ECs reached maximal cell densities of about 700-900 ECs/mm2. The cell cycle times calculated from the population-doubling time and the stathmokinetic index, respectively, amounted to 63 and 83 h for microvascular ECs, 33 and 35 h for venous macrovascular ECs, and 29 and 35 h for arterial macrovascular ECs.

Capillaries↗
PubMed1997

Is the red cell morphology really useful to detect the source of hematuria?

Morphological analysis of urinary red blood cells by phase-contrast microscopy to identify the source of bleeding was, and still is, widely used also as a starting point for workup. To evaluate the reliability of this approach, we studied 129 outpatients presenting with persistent isolated microhematuria; 31 subjects also had mild proteinuria (1 g/day), while 21 had pathological albumin levels. All patients were followed for a period of 6 years. During this time, 6 patients underwent renal biopsy for the onset of macrohematuria episodes and proteinuria of 2-3 g/day. Glomerular bleeding was identified in only 14.7% of the patients, despite the persistent microhematuria and the presence of proteinuria or microalbuminuria. The renal origin of the urinary erythrocytes correlated with histological findings in only 2 of 6 patients with dysmorphic erythrocytes who developed proteinuria (exceeding 1 g/day), and none with isomorphic erythrocytes showed urological abnormalities. These results challenge the validity and reliability of morphological analysis to identify the source of bleeding along the urinary tract.

Adult↗
PubMedDate not supplied

Ototoxicity of the new platinum anticancer agent TRK-710 in comparison with cisplatin.

The pharmacokinetics and ototoxicity of the new platinum analogue TRK-710 (3, 9, 15 mg/kg x 3 days) were compared with those of cisplatin (1, 3, 5 mg/kg x 3). The perilymphatic concentration of TRK-710 was one seventh of that of cisplatin even 1 h after the administration. The N1 threshold of the compound action potential was elevated dose-dependently in both groups with a similar degree of hearing impairment. Morphological observation using phase contrast microscopy and scanning electron microscopy revealed the damage of the outer hair cells to almost the same degree mainly in the basal and second turns. Despite its usefulness against cisplatin-resistant tumors and a lesser degree of nephrotoxicity and myelosuppression, TRK-710 should be clinically used with caution similar to cisplatin.

Action Potentials↗
PubMed2003

In vitro study on the closure of posterior capsulorrhexis in the human eye.

PURPOSE: An unexplained clinical observation is the development of posterior capsular opacification (PCO), even when the central part of the posterior capsule has been removed. The purpose of this study was to investigate in vitro the mechanisms involved in the closure of the posterior capsulorrhexis in a capsular bag model. METHODS: A sham extracapsular cataract extraction was performed in 71 human donor eyes, followed by a central posterior capsulorrhexis 3 to 4 mm in diameter. Each capsular bag was pinned to a PMMA ring with a central hole of 5 mm and placed in a Petri dish. The capsular bags were cultured and monitored for 3 to 7 weeks by phase-contrast microscopy, after which they were prepared for light, transmission, and scanning electron microscopy. RESULTS: Proliferation of lens epithelial cells (LECs) within the posterior rhexis area was found in 22 cases (31%) of which 3 had a complete closure. In the absence of the posterior capsule, a monolayer of LECs was observed growing on a basal lamina, consisting of loosely arranged fibers. Further observations on noncultured capsular bags revealed that this basal lamina corresponds to the anterior hyaloid membrane. CONCLUSIONS: This study corroborates the clinical observation that LECs that remain after cataract extraction have the potential to proliferate, in the absence of their natural substrate, on a basal lamina of vitreous origin and are able to close the posterior capsulorrhexis partially or totally in approximately one third of cases.

Adolescent↗
PubMed2003

Human keratocytes cultured on amniotic membrane stroma preserve morphology and express keratocan.

PURPOSE: To develop a new method of expanding human corneal keratocytes in serum while maintaining their characteristic morphology and keratocan expression. METHODS: Human keratocytes were isolated from central corneal buttons by digestion in 1 mg/mL of collagenase A in DMEM and seeded on plastic or the stromal matrix of human amniotic membrane (AM) in DMEM with different concentrations of FBS. On confluence, cells on AM were continuously subcultured for six passages on AM or plastic. In parallel, cells cultured on plastic at passages 3 and 11 were reseeded on AM. Cellular morphology and cell-cell networks were assessed by phase-contrast microscopy and a cell viability assay, respectively. Expression of keratocan was determined by RT-PCR and Western blot analysis. RESULTS: Trephined stroma yielded 91,600 +/- 26,300 cells (ranging from 67,000 to 128,000 cells per corneal button). Twenty-four hours after seeding, cells appeared dendritic on AM, even in 10% FBS but fibroblastic on plastic. Such a difference in morphology correlated with expression of keratocan assessed by RT-PCR and Western blot, which was high and continued at least to passage 6 on AM, even in 10% FBS, but was rapidly lost each time when cells on AM were passaged on plastic. Fibroblasts continuously cultured on plastic to passages 3 and 11 did not reverse their morphology or synthesize keratocan when reseeded on plastic in 1% FBS or on AM. CONCLUSIONS: Human keratocytes maintain their characteristic morphology and keratocan expression when subcultured on AM stromal matrix even in the presence of high serum concentrations. This method can be used to engineer a new corneal stroma.

Amnion↗
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↗
PubMed2005

Soluble CD44 is cytotoxic to trabecular meshwork and retinal ganglion cells in vitro.

PURPOSE: Current glaucoma research targets neuroprotective therapies for retinal ganglion cells (RGCs) in primary open-angle glaucoma (POAG). The purpose of this study was to determine whether the 32-kDa ectodomain fragment of CD44-soluble CD44 (sCD44)-which is increased in the aqueous of patients with POAG, affects RGC and trabecular meshwork (TM) cell survival in vitro. METHODS: sCD44 was isolated from human or fetal calf serum (FCS) by urea solubilization and immunoprecipitation. A transformed rat RGC-like cell line (RGC-5), human and bovine TM cells, and control cells were grown in Dulbecco's modified Eagle's medium containing 10% FCS until confluent and then were incubated in medium containing 0.1% FCS and treated with various doses of purified sCD44 and 17-alpha-methyl testosterone (17-alpha-MT). The cytotoxicity of sCD44 was verified by heat-inactivation, pretreatment with a pan-caspase inhibitor, and coadministration of anti-CD44 neutralizing antibody or hyaluronic acid (HA). Cell viability was assessed by trypan blue staining, cell counting, and phase-contrast microscopy. RESULTS: There was a statistically significant dose- and time-dependent decrease in the number of cells and viability in the RGC-5 and TM cells treated with sCD44. Within 12 hours of sCD44 treatment, RGC-5 and TM cells displayed cell rounding, detachment, and swelling. sCD44-induced cell death was cell specific. Smooth muscle cells were resistant to sCD44, whereas human cortical neuronal-like cells were susceptible to sCD44 after 24 hours, but recovered. The cytotoxicity of sCD44 was blocked by heat-inactivation, pretreatment with a pan-caspase inhibitor, or coadministration of anti-CD44 antibody or HA. 17-alpha-MT prevented sCD44 cytotoxicity in both RGC-5 and TM cells. CONCLUSIONS: The results indicate that exogenous sCD44 adversely affects RGC-5 and TM cell survival in vitro by activating proapoptotic pathways.

Animals↗
PubMed2005

The peptides ADNF-9 and NAP increase survival and neurite outgrowth of rat retinal ganglion cells in vitro.

PURPOSE: Recent studies demonstrated that short peptides derived from activity-dependent neurotrophic factor (ADNF) and activity-dependent neuroprotective protein (ADNP) are neuroprotective at femtomolar concentrations. We evaluated these findings in cultures of purified rat retinal ganglion cells (RGCs) using two such peptides: ADNF-9 and NAP. In a second step, the influence of these peptides on neurite outgrowth in retinal explants was investigated. METHODS: Retinal ganglion cells (RGCs) were purified from newborn (postnatal day [P]0-P2) rat retina by immunopanning with antibodies against Thy1.1 and were cultured in serum-free N2 medium for 2 days. RGCs were treated with ADNF-9 and NAP at concentrations ranging from 10(-18) to 10(-10) M. Survival was quantified by counting viable cells by phase-contrast microscopy. Retinal explants from postnatal (P9-P11) rats were cultured in three-dimensional fibrin clots in serum-free medium for 3 days. Explants were treated with 1 microM NAP or 1 microM ADNF-9. Neurite outgrowth was visualized by staining with Sudan black and quantified by measuring axonal length. RESULTS: Both peptides enhanced survival of RGCs in a dose-dependent manner. ADNF-9 showed a maximum effect at 0.1 pM with an increase in survival to 177% (95% confidence interval: 149-204) of the control level. The EC(50) was 10.9 fM. NAP showed a maximum effect at 5 pM with an increase in survival to 167% (146-189) and an EC(50) of 6.1 fM. In the explants, 1 microM ADNF-9 enhanced axonal outgrowth to 126% (118-133) and 1 microM NAP to 117% (98-137) compared with the control. CONCLUSIONS: Both peptides, ADNF-9 and NAP, not only increase RGC survival in vitro but also support neurite outgrowth in retinal explants. These peptides deserve further attention as potential neuroprotective compounds in retinal and optic nerve diseases.

Animals↗
PubMed2005

Upregulation of heat shock protein expression by proteasome inhibition: an antiapoptotic mechanism in the lens.

PURPOSE: Studies have shown that proteasome inhibition protects lens epithelial cells (LECs) against interferon (IFN)-gamma-induced apoptosis. The present study was conducted to test the hypothesis that proteasome inhibition can protect lens cells against apoptosis by upregulating heat shock protein (HSP) expression. METHODS: Murine lens epithelial alphaTN4-1 cells were treated with combinations of 100 U/mL IFN-gamma, 10 muM MG132 (proteasome inhibitor), and 100 muM quercetin (HSP inhibitor). mRNA and protein expression were observed by RT-PCR and Western blot analysis, respectively. Caspase activities were measured by using cleavage of colorimetric substrate. Apoptosis was measured by phase-contrast microscopy and flow cytometry. RESULTS: At the mRNA level, the proteasome inhibitor, MG132, caused a >10-fold increase in HSP27 and a small increase (1.2- to 1.6-fold) in alphaB-crystallin but no change in HSP70 or -90. At the protein level, a more than twofold increase in HSP27 and -90, a marked increase in HSP70, but no significant change in alphaB-crystallin, was observed. Downregulation of alphaA-crystallin by MG132 was observed at both the mRNA and protein levels. MG132 caused no significant change in heat shock factor (HSF)-1, but a more than twofold increase in HSF2 and -4 protein expression. MG132 prevented the IFN-gamma-induced increase in caspase-1, -6, and -8 activities. Quercetin decreased MG132-induced expression of HSP27, -70, and -90 by more than 70%, and heat shock factors HSF2 and -4 by more than 65%. Quercetin pretreatment significantly reversed the decrease in caspase-1, -6, and -8 activities and the antiapoptotic effect of MG132 on IFN-gamma-treated LECs. CONCLUSIONS: The antiapoptotic effect of proteasome inhibition of IFN-gamma-induced apoptosis in LECs correlates with increased expression of HSPs and inhibition of caspase activities. Inhibition of HSP expression restores caspase activities and abolishes the antiapoptotic effect of proteasome inhibition, implicating HSPs as mediators of the protective effect of proteasome inhibition.

Animals↗
PubMed2005

The heterogeneous murine corneal stromal cell populations in vitro.

PURPOSE: To demonstrate that the murine corneal stroma is inhabited by heterogeneous cell populations that include cells expressing nestin. METHODS: Collagenase-isolated corneal stroma cells obtained from newborn and adult mice (2nd and 12th postnatal weeks, respectively), were seeded at low (5 cells/mm2), intermediate (50 cells/mm2), and high (500 cells/mm2) densities in DMEM/F12 containing insulin, transferrin, selenium, and 1% nonessential amino acids. Corneal stroma cells cultured at 500 cells/mm2 were treated with 10 ng/mL human recombinant transforming growth factor (TGF)-beta1 for 5 days. Cell morphology and expression of alpha-smooth muscle actin, choline acetyltransferase, CD45, glial fibrillary acidic protein (GFAP), keratocan, nestin, neurofilaments, protein gene product 9.5, tyrosine hydroxylase, and vimentin were examined. RESULTS: Phase-contrast microscopy demonstrated that freshly isolated corneal stromal cells are heterogeneous in morphology and include dendritic, stellate, neuronal, and small polyhedral cells. Immunostaining of primary cultures of 2- and 12-week-old mice, 24 hours after seeding at the intermediate density, showed that 100% of cells expressed vimentin and 97.7% +/- 2.7% expressed keratocan. alpha-Smooth muscle actin was expressed by 0.2% +/- 0.05% of cells in the 2-week-old group and 0.1% +/- 0.07% in 12-week-old group. Neurofilament was expressed by 0.5% +/- 0.03% and 0.7% +/- 0.03% of cells in the 2- and 12-week-old groups, respectively. No cell expressed GFAP or nestin. After 5 days in culture, cells seeded at high density aggregated as clusters that were immunoreactive to nestin in both groups. Cell clusters and migrating cells reacted to pgp 9.5, and migrating cells, but not the cell clusters, reacted to tyrosine hydroxylase. Cell cluster formation and nestin expression were abolished by culturing in the presence of TGF-beta1. CONCLUSIONS: Normal murine corneal stroma contains heterogeneous cell populations including cells with the potential to form clusters and express the progenitor marker nestin. This potential is disrupted by the addition of TGF-beta1 to the culture medium.

Actins↗
PubMed1993

An in vivo study of the influence of the surface roughness of implants on the microbiology of supra- and subgingival plaque.

In nine patients with fixed prostheses supported by endosseous titanium implants, 2 titanium abutments (transmucosal part of the implant) were replaced by either an unused standard abutment or a roughened titanium abutment. After 3 months of habitual oral hygiene, plaque samples were taken for differential phase-contrast microscopy, DNA probe analysis, and culturing. Supragingivally, rough abutments harbored significantly fewer coccoid micro-organisms (64 vs. 81%), which is indicative of a more mature plaque. Subgingivally, the observations depended on the sampling procedure. For plaque collected with paper points, only minor qualitative and quantitative differences between both substrata could be registered. However, when the microbiota adhering to the abutment were considered, rough surfaces harbored 25 times more bacteria, with a slightly lower density of coccoid organisms. The presence and density of periodontal pathogens subgingivally were, however, more related to the patient's dental status than to the surface characteristics of the abutments. These results justify the search for optimal surface smoothness for all intra-oral and intra-sulcular hard surfaces for reduction of bacterial colonization and of periodontal pathogens.

Bacterial Adhesion↗
PubMedDate not supplied

Glutamine preserves cardiomyocyte viability and enhances recovery of contractile function after ischemia-reperfusion injury.

BACKGROUND: Glutamine has been shown to protect against cellular injury in in vitro gut epithelial cells and in vivo in the septic rat. Glutamine's effect on the cardiomyocyte has not been explored. We tested the hypothesis that glutamine can enhance heat shock protein 72 (HSP 72) expression, attenuate intracellular oxidant generation, and protect cardiomyocytes against ischemia/reperfusion (I/R) injury. METHODS: Chicken cardiomyocytes were supplemented with glutamine (10 mmol/L) or were controls (0 mmol/L). Cells underwent I/R, and HSP 72 content was evaluated using Western blotting. Reactive oxygen species generation was quantified through 2'-7'-dichlorofluorescin diacetate oxidation. Cell viability was quantified using propidium iodide staining. Return of contractile function was analyzed through phase contrast microscopy. RESULTS: Glutamine significantly increased cardiomyocyte HSP 72 expression and markedly reduced cell death after I/R injury. Glutamine did not significantly decrease intracellular oxidant generation. Contractile function returned in all glutamine-treated cells versus none of the control cells postreperfusion. CONCLUSIONS: Glutamine significantly increases cardiomyocytes survival and recovery of contractile function after I/R injury. This protection is associated with enhanced HSP 72 expression. These observations suggest that glutamine may prove beneficial as a protective therapy in patients at risk for cardiac ischemia and reperfusion injury, such as patients undergoing procedures requiring cardiopulmonary bypass and patients with coronary artery disease.

Animals↗
PubMed1998

Leukotrienes C4 and D4 increase the ciliary beat frequency in human upper airway mucosa in vitro.

It has been suggested that leukotrienes C4 (LTC4) and D4 (LTD4) released from upper respiratory mucosa influence mucociliary transport during allergic reactions. We studied the in vitro effects of leukotrienes C4 and D4 on the ciliary beat frequency (CBF) of human adenoid explants over a 5-hour period. Tissue explants were cultured at 35 degrees C in Minimum Essential Medium Eagle (MEM). The CBF was measured using phase contrast microscopy and microphotometry. Measurements of CBF were recorded in medium alone and in medium containing LTC4 or LTD4 at concentrations of 10(-8) and 10(-6) M. LTC4 and LTD4 increased CBF at concentrations of 10(-8) and 10(-6) M with increases of 20.51% +/- 2.69% and 29.84% +/- 4.06%, respectively. To determine the specificity of the LTC4 and LTD4 effects, the ciliated epithelium was treated with the specific leukotriene receptor antagonist LY-171,883 before administration of LTC4 and LTD4. LY-171,883 (10(-6) M) significantly inhibited the ciliostimulatory effects of both leukotrienes. Our findings indicate that LTC4 and LTD4 increase CBF in vitro by activation of the LTD4 receptor.

Adenoids↗