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Biomedical subjects

Stephen C Pflugfelder

Publications and source records attributed to Stephen C Pflugfelder.

At least 19 recordsLinked to original sources
PubMed2006

Targeted inhibition of p57 and p15 blocks transforming growth factor beta-inhibited proliferation of primary cultured human limbal epithelial cells.

PURPOSE: To evaluate the role of cyclin-dependent kinase inhibitors p57 and p15 in transforming growth factor (TGF)-beta1 or TGF-beta2 inhibited proliferation of primary cultured human limbal epithelial cells using short interfering RNA (siRNA). METHODS: Primary cultured human limbal epithelial cells were treated with TGF-beta1 or TGF-beta2 for 6 and 24 h, and total RNA extracted for RT-PCR and real-time PCR using primers for p21, p27, and p57 (CipP/Kip family) and p15 and p19 (INK4 family). Proteins were extracted for western blot analysis of p57 and p15. For RNA interference, primary cultured human limbal epithelial cells were transfected with annealed double-stranded siRNA (67 nM) specific for p57, p15, or siRNA-Fluorescein (siRNA-F; as a negative control) followed by treatment with TGF-beta1 or TGF-beta2 at 1 ng/ml. P57 and p15 were quantitatively detected by real-time PCR and western blot; and immunolocalized by immunofluorescent staining. The effects of TGF-beta1 or TGF-beta2 on cell proliferation were evaluated by BrdU incorporation and MTT assay. RESULTS: TGF-beta1 or TGF-beta2 significantly inhibited primary cultured human limbal epithelial cell proliferation measured by BrdU incorporation and MTT assay. TGF-beta1 or TGF-beta2 upregulated the expression of p57 and p15 mRNA and protein, but did not effect the expression of p19, p21, or p27. The siRNA transfection efficiency of these cells was 75% and no cellular toxicity was observed by 24 h. The TGF-beta1 or TGF-beta2 stimulated expression of p57 and p15 mRNA were markedly blocked by siRNA-p57 or siRNA-p15, respectively, but not by siRNA-F. The TGF-beta1 or TGF-beta2 suppression of epithelial proliferation measured by BrdU incorporation and MTT generation was increased to near normal levels by siRNA-p57 or siRNA-p15. Western blot and immunofluorescent staining showed that levels of p57 and p15 proteins were equally reduced in the cytoplasm and nucleus. CONCLUSIONS: These findings demonstrate that TGF-beta1 and/or TGF-beta2 inhibit proliferation of primary cultured human limbal epithelial cells and that p57 and p15 play roles in this process.

Cell Proliferation↗
PubMed2006

Improved transduction of human corneal epithelial progenitor cells with cell-targeting adenoviral vectors.

The development of vectors and techniques to transfer therapeutic genes to corneal epithelium has broad clinical applications. To determine if adenoviral (Ad5) vectors could be tailored to increase transduction of corneal epithelial progenitor cells expressing epidermal growth factor receptor (EGFR), the feasibility of targeting gene therapy vectors to genetically modify primary cultured human corneal epithelial cells (PHCEC) was evaluated. PHCECs were cultured from human limbal explants and transduced with Ad5 vectors containing the enhanced green fluorescent protein (GFP) reporter cassette to mediate gene transfer. The efficiencies of transduction with different Ad5 dosages and different time periods of exposure were compared. Metabolically biotinylated Ad5 vectors were retargeted to PHCECs using biotinylated epidermal growth factor (EGF) as a cell-targeting ligand. Phenotypes and function assays of transduced cells were determined by real-time PCR and BrdU incorporation. Ad5 vectors transduced approximately 50-93% of PHCEC at 10-100 PFU/cell in a dose-dependent manner and the transgene persisted for more than 2 weeks in vitro. Retargeting of biotinylated Ad5 with EGF increased transduction of EGFR and ABCG2-expressing corneal epithelial progenitor cells up to nine-fold and reduced transduction of K12 and involucrin-expressing differentiated corneal epithelial cells and had higher BrdU incorporation indexes. These data provide proof of principle that ligand-bearing modified Ad5 vectors can target a population of corneal epithelial progenitor cells for corneal gene therapy.

Adenoviridae↗
PubMed2006

Corticosteroid and doxycycline suppress MMP-9 and inflammatory cytokine expression, MAPK activation in the corneal epithelium in experimental dry eye.

We investigated the effects of corticosteroid and doxycycline on expression of matrix metalloproteinase (MMP)-9 and inflammatory cytokines and activation of mitogen-activated protein kinase (MAPK) signaling pathways, c-jun N-terminal kinases (JNK), extracellular-regulated kinases (ERK) and p38, in experimental murine dry eye. Experimental dry eye (EDE) was created in C57BL6 mice, with or without or topical treatment consisting of 1% methylprednisolone, 0.025% doxycycline or balanced salt solution four times per day. MMP-9 expression in the cornea epithelia was evaluated by laser scanning confocal microscopy. Gelatinase activity in the cornea was evaluated by in situ zymography and MMP-9 activity in tear washings was evaluated by gelatin zymography. Total and phosphorylated MAPKs (JNK1/2, ERK1/2, p38) were detected by Luminex immunobead assay. Levels of MMP-9, interleukin (IL)-1alpha, IL-1beta and tumor necrosis factor (TNF)-alpha RNA transcripts were evaluated by real-time PCR. MMP-9 immunoreactivity was localized to the apical corneal epithelial cell membranes in normal control eyes. Desiccating stress significantly increased production of MMP-9 by the corneal epithelium and increased its activity in the corneal epithelium and tear fluid. Dryness also increased expression of IL-1alpha, IL-1beta and TNF-alpha mRNA and stimulated phosphorylation of JNK1/2, ERK1/2 and p38 MAPKs in the corneal epithelium. Both methylprednisolone and doxycycline reduced expression and activity of MMP-9, decreased levels of inflammatory cytokines transcripts and reduced activation of MAPKs in the corneal epithelium in response to EDE. Desiccating stress stimulates expression of MMP-9, IL-1alpha, IL-1beta and TNF-alpha mRNA , as well as activates MAPK signaling pathways in the corneal epithelium. Both corticosteroid and doxycycline suppressed this molecular stress response.

Animals↗
PubMed2006

Desiccating stress induces T cell-mediated Sjögren's Syndrome-like lacrimal keratoconjunctivitis.

Chronic dry eye syndrome affects over 10 million people in the United States; it is associated with inflammation of the lacrimal gland (LG) and in some cases involves T cell infiltration of the conjunctiva. We demonstrate that environmental desiccating stress (DS) elicits T cell-mediated inflammation of the cornea, conjunctiva, and LG, but not other organs in mice. The lacrimal keratoconjunctivitis (LKC) was mediated by CD4(+) T cells, which, when adoptively transferred to T cell-deficient nude mice, produced inflammation in the LG, cornea, and conjunctiva, but not in any other organ. Adoptively transferred CD4(+) T cells produced LKC even though recipients were not exposed to DS. LKC was exacerbated in euthymic mice depleted of CD4(+)CD25(+)forkhead/winged helix transcription factor(+) regulatory T cells. The results suggest that DS exposes shared epitopes in the cornea, conjunctiva, and LG that induce pathogenic CD4(+) T cells that produce LKC, which under normal circumstances is restrained by CD4(+)CD25(+)forkhead/winged helix transcription factor(+) regulatory T cells.

Animals↗
PubMed2006

Gap junction protein connexin 43 serves as a negative marker for a stem cell-containing population of human limbal epithelial cells.

This study evaluated whether the gap junction protein connexin (Cx) 43 could serve as a negative cell surface marker for human corneal epithelial stem cells. Cx43 expression was evaluated in corneo-limbal tissue and primary limbal epithelial cultures. Immunofluorescent staining and laser scanning confocal microscopy showed that Cx43 was strongly expressed in the corneal and limbal suprabasal epithelial cells, but the basal cells of the limbal epithelium were negative. Cx43 antibody stained mainly large cells but not small cells in primary limbal epithelial cultures. As determined by semiquantitative reverse transcription polymerase chain reaction (PCR) and real-time PCR, Cx43 mRNA was more abundant in the corneal than limbal epithelia, and it was expressed in higher levels in mature limbal epithelial cultures. Using GAP11, a rabbit polyclonal antibody against the Cx32 extracellular loop 2 (151-187), a sequence that is highly homologous in Cx43, the Cx43(dim) and Cx43(bright) cells were selected from primary limbal epithelial cultures by fluorescence-activated cell sorting and were evaluated for stem cell properties. These Cx43(dim) and Cx43(bright) cells were confirmed by their expression levels of Cx43 protein and mRNA. The Cx43(dim) cells were found to contain higher percentages of slow-cycling bromodeoxyuridine (BrdU)-label retaining cells and the cells that were positive for stem cell-associated markers p63, ABCG2, and integrin beta1 and negative for differentiation markers K3 and involucrin. The Cx43(dim) cells possessed a greater proliferative potential than Cx43(bright) cells and nonfractionated cells as evaluated by BrdU incorporation, colony-forming efficiency, and growth capacity. Our findings suggest that human limbal basal cells do not express connexin 43, which could serve as a negative cell surface marker for the stem cell-containing population of human limbal epithelial cells.

Adult↗
PubMed2006

The incidence and risk factors for developing dry eye after myopic LASIK.

PURPOSE: To determine the incidence of dry eye and its risk factors after myopic laser-assisted in situ keratomileusis (LASIK). DESIGN: Single-center, prospective randomized clinical trial of 35 adult patients, aged 24 to 54 years, with myopia undergoing LASIK. METHODS: setting and study population: Participants were randomized to undergo LASIK with a superior or a nasal hinge flap. They were evaluated at 1 week and 1, 3, and 6 months after surgery. intervention: Bilateral LASIK with either a superior-hinge Hansatome microkeratome (n = 17) or a nasal-hinge Amadeus microkeratome (n = 18). main outcome measures: The criterion for dry eye was a total corneal fluorescein staining score > or =3. Visual acuity, ocular surface parameters, and corneal sensitivity were also analyzed. Cox proportional-hazard regression was used to assess rate ratios (RRs) with 95% confidence intervals. RESULTS: The incidence of dry eye in the nasal- and superior-hinge group was eight (47.06%) of 17 and nine (52.94%) of 17 at 1 week, seven (38.89%) of 18 and seven (41.18%) of 17 at 1 month, four (25%) of 16 and three (17.65%) of 17 at 3 months, and two (12.50%) of 16 and six (35.29%) of 17 at 6 months, respectively. Dry eye was associated with level of preoperative myopia (RR 0.88/each diopter, P = .04), laser-calculated ablation depth (RR 1.01/microm, P = 0.01), and combined ablation depth and flap thickness (RR 1.01/microm, P = 0.01). CONCLUSIONS: Dry eye occurs commonly after LASIK surgery in patients with no history of dry eye. The risk of developing dry eye is correlated with the degree of preoperative myopia and the depth of laser treatment.

Adult↗
PubMed2006

Effect of topical ophthalmic epinastine and olopatadine on tear volume in mice.

PURPOSE: To investigate the effects of topical epinastine and olopatadine on tear volume by using a mouse model. METHODS: Eighty-five C57BL6 mice (170 eyes) were treated twice daily with topical ophthalmic epinastine 0.05%, olopatadine 0.1%, or atropine 1% or served as untreated controls. A thread-wetting assay was used to measure tear volume at baseline and 15, 45, 90, 120, and 240 minutes after the last instillation of the drug on days 2 and 4 of treatment. RESULTS: After 2 days of treatment, epinastine-treated mice showed greater mean tear volumes than olopatadine-treated mice did at 15, 45, 90, and 240 minutes, with statistical significance at 15 and 45 minutes (P<0.001). Olopatadine significantly reduced tear volume versus untreated controls at 15 and 45 minutes (P<0.001). After 4 days, tear volumes with epinastine treatment exceeded those with olopatadine treatment at all time points, with statistical significance at 45 minutes (P<0.05). Atropine rendered tears undetectable at 15, 45, and 90 minutes; tear volume returned to baseline levels at 240 minutes. CONCLUSIONS: Topical epinastine did not inhibit tear secretion, whereas olopatadine caused a significant decrease in tear volume. Because of its neutral impact on the lacrimal functional unit, epinastine may be an especially good choice for the treatment of allergic conjunctivitis in patients with dry eye disease or in those who are at risk for developing dry eye.

Administration, Topical↗
PubMed2006

Dysfunctional tear syndrome: a Delphi approach to treatment recommendations.

PURPOSE: To develop current treatment recommendations for dry eye disease from consensus of expert advice. METHODS: Of 25 preselected international specialists on dry eye, 17 agreed to participate in a modified, 2-round Delphi panel approach. Based on available literature and standards of care, a survey was presented to each panelist. A two-thirds majority was used for consensus building from responses obtained. Treatment algorithms were created. Treatment recommendations for different types and severity levels of dry eye disease were the main outcome. RESULTS: A new term for dry eye disease was proposed: dysfunctional tear syndrome (DTS). Treatment recommendations were based primarily on patient symptoms and signs. Available diagnostic tests were considered of secondary importance in guiding therapy. Development of algorithms was based on the presence or absence of lid margin disease and disturbances of tear distribution and clearance. Disease severity was considered the most important factor for treatment decision-making and was categorized into 4 levels. Severity was assessed on the basis of tear substitute requirements, symptoms of ocular discomfort, and visual disturbance. Clinical signs present in lids, tear film, conjunctiva, and cornea were also used for categorization of severity. Consensus was reached on treatment algorithms for DTS with and without concurrent lid disease. CONCLUSION: Panelist opinion relied on symptoms and signs (not tests) for selection of treatment strategies. Therapy is chosen to match disease severity and presence versus absence of lid margin disease or tear distribution and clearance disturbances.

Algorithms↗
PubMed2006

Expression and regulation of cornified envelope proteins in human corneal epithelium.

PURPOSE: Stratified squamous epithelial cells assemble a specialized protective barrier structure on their periphery, termed the cornified envelope. The purpose of this study was to evaluate the presence and distribution of cornified envelope precursors in human corneal epithelium, their expression in human corneal epithelial cell cultures, and the effect of ultraviolet radiation (UVB) and transglutaminase (TG) inhibition on their expression. METHODS: Tissue distribution of small proline-rich proteins (SPRRs) and filaggrin and involucrin was studied in human cornea sections by immunofluorescence staining. Primary human corneal epithelial cells (HCECs) from limbal explants were used in cell culture experiments. A single dose of UVB at 20 mJ/cm2 was used to stimulate these cells, in the presence or absence of mono-dansyl cadaverine (MDC), a TG inhibitor. SPRR2 and involucrin protein levels were studied by immunofluorescence staining and Western blot analysis. Gene expression of 12 proteins was investigated by semiquantitative reverse transcription-polymerase chain reaction. RESULTS: In human cornea tissue, SPRR1, SPRR2, filaggrin, and involucrin protein expression were detected in the central and peripheral corneal and limbal epithelium. In HCECs, SPRR2 and involucrin proteins were detected in the cytosolic fraction, and involucrin levels increased after UVB. Both SPRR2 and involucrin levels accumulated in the presence of MDC. Nine genes including involucrin, SPRR (types 1A, 1B, 2A, 2B, and 3), late envelope protein (LEP) 1 and 16, and filaggrin were expressed by HCECs. SPRR 4, loricrin, and LEP 6 transcripts were not detected. UVB downregulated SPRR (2A, 2B) and LEP 1 transcripts. CONCLUSIONS: Various envelope precursors are expressed in human corneal epithelium and in HCECs, acute UVB stress differentially alters their expression in HCECs. The expression of envelope precursors and their rapid modulation by UVB supports the role of these proteins in the regulation of ocular surface stress. TG function may be relevant in the regulation of soluble precursors in UVB-stimulated corneal epithelium.

Adolescent↗
PubMed2006

Apical corneal barrier disruption in experimental murine dry eye is abrogated by methylprednisolone and doxycycline.

PURPOSE: To evaluate the mechanism of apical corneal epithelial barrier disruption in response to experimental ocular surface desiccation and the effects of two anti-inflammatory agents (methylprednisolone and doxycycline) on this process. METHODS: Experimental dry eye (EDE) was created in C57BL/6 mice, without or with topical therapy, 1% methylprednisolone, 0.025% doxycycline, or physiologic saline solution (PSS) four times per day. Corneal smoothness and Oregon green dextran (OGD) permeability were assessed. Desquamation of and cornified envelope protein (involucrin and small proline-rich protein [SPRR]-2) expression by the corneal epithelium was evaluated by laser scanning confocal microscopy. Levels of cornified envelope proteins mRNA were measured by real-time PCR. RESULTS: Corneal OGD permeability, surface irregularity, and the number of desquamating apical corneal epithelia significantly increased in EDE. Desiccating stress significantly increased expression of involucrin and SPRR-2 in the corneal epithelia. Treatment of EDE with methylprednisolone or doxycycline reduced corneal permeability to OGD, improved corneal smoothness, and decreased involucrin and SPRR-2 immunoreactivity compared with EDE+PSS. CONCLUSIONS: Disruption of apical corneal epithelial barrier function in dry eye is accompanied by increased apical desquamation and increased expression of cornified envelope proteins. Topical treatment of EDE with the anti-inflammatory agents methylprednisolone or doxycycline preserves apical corneal barrier function.

Animals↗
PubMed2006

Desiccating stress stimulates expression of matrix metalloproteinases by the corneal epithelium.

PURPOSE: To determine the effects of experimental dryness on production of matrix metalloproteinases (MMPs) and their physiological inhibitors (TIMPs) by the corneal epithelium. METHODS: Experimental dry eye (EDE) was created in two strains of mice: BALB/c and C57BL/6. Real-time PCR was performed with MMP and TIMP probes, and the results were analyzed by the comparative C(T) method, selecting the relative mRNA levels in untreated control samples as calibrator. Immunofluorescent staining with specific antibodies immunolocalized MMP proteins in situ. MMP enzymatic activity was evaluated in tears and corneal lysates. Corneal permeability to Oregon green dextran (OGD) and sodium fluorescein was measured. Corneal smoothness was evaluated by graded regularity of a ring reflected off the corneal surface. RESULTS: Desiccating stress significantly increased levels of MMP-1, -3, -9, and -10 transcripts in the corneal epithelium in C57BL/6 mice, but had no effect on levels of MMP transcripts in the corneal epithelium of BALB/c mice. There was no change in levels of TIMP transcripts except for TIMP-4 which significantly increased on day 10 in C57BL/6 mice. The MMP-1, -3, and -9 concentration in tears significantly increased compared with control levels after EDE for 4 and 6 days, respectively, in C57BL/6 and BALB/c. Changes in MMP protein expression detected by immunofluorescent staining were similar to changes in gene transcripts for most MMPs. EDE increased corneal permeability to OGD and fluorescein and corneal surface irregularity. CONCLUSIONS: Corneal dryness stimulates production of certain MMPs in a strain-dependent fashion and causes the disruption of the corneal barrier, thus increasing permeability and corneal irregularity.

Animals↗
PubMed2006

Transglutaminase participates in UVB-induced cell death pathways in human corneal epithelial cells.

PURPOSE: Ultraviolet light (UVB) is known to cause apoptosis in human corneal epithelial cells. This study evaluates the role of transglutaminase in regulating tumor necrosis factor (TNF) receptor clustering as well as caspase activation in UVB-induced apoptosis in human corneal epithelial cells. METHODS: A human corneal epithelial cell line was used. A single dose of UVB (20 mJ/cm2) was used as a stimulus. Cell viability and cell death were investigated by MTT, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL), and caspase-3 assays. Immunofluorescent staining was used to investigate TNF receptor-I clustering at various time intervals after UVB. Short interfering RNA was used to knock down transglutaminase-2 expression. Fluorescein-cadaverine uptake was used to assess transglutaminase activity. A noncovalent peptide delivery system was used to transfect guinea pig liver transglutaminase into corneal epithelial cells. RESULTS: UVB increased transglutaminase activity, reduced cell viability, and increased TUNEL staining. UVB or TNF-alpha promoted TNF-receptor-I clustering, a process inhibited by the transglutaminase inhibitor, mono-dansyl cadaverine. UVB also increased activated caspase-3, in a manner suppressible by mono-dansyl cadaverine. Intracellular delivery of exogenous transglutaminase markedly increase caspase-3 activation compared with the vehicle control. CONCLUSIONS: Transglutaminase enzymatic activity is involved in corneal epithelial cell death after UVB and appears to participate in two steps regulating this process, clustering of TNF receptor-I and caspase-3 activation.

Apoptosis↗
PubMed2005

JNK and ERK MAP kinases mediate induction of IL-1beta, TNF-alpha and IL-8 following hyperosmolar stress in human limbal epithelial cells.

Hyperosmolarity has been recognized to be a pro-inflammatory stress to the corneal epithelium. The cell signalling pathways linking hyperosmolar stress and inflammation have not been well elucidated. This study investigated whether exposure of human limbal epithelial cells to hyperosmotic stress activates the mitogen-activated protein kinase (MAPK) pathways and induces production of pro-inflammatory cytokines, interleukin (IL) -1beta, tumor necrosis factor (TNF) alpha, and the C-X-C chemokine IL-8. Primary human limbal epithelial cultures in normal osmolar media (312 mOsM) were exposed to media with higher osmolarity (400-500 mOsM) by adding 50-90 mM NaCl, with or without SB202190, an inhibitor of c-Jun N-terminal kinases (JNK) pathway, PD 98059, an inhibitor of extracellular-regulated kinase (ERK) pathway, dexamethasone or doxycycline for different lengths of time. The conditioned media were collected after 24 hr of treatment for ELISA. Total RNA was extracted from cultures treated for 6 hr for semi-quantitative RT-PCR. Cells treated for 15-60 min were lysed in RIPA buffer and subjected to Western blot with phospho (p)-specific antibodies against p-JNK and p-ERK. The concentrations of IL-1beta, TNF-alpha and IL-8 proteins in 24 hr conditioned media of limbal epithelial cells progressively increased as the media osmolarity increased from 312 to 500 mOsM. Active p-JNK-1/p-JNK-2 and p-ERK-1/p-ERK-2 were detected by Western blot and peaked at 60 min in cells exposed to hyperosmolar media. The levels of p-JNK-1/p-JNK-2 and p-ERK1/p-ERK2 were positively correlated with the medium osmolarity. SB202190, PD98059 and doxycycline markedly suppressed the levels of p-JNK-1/p-JNK-2 and/or p-ERK1/p-ERK2, as well as IL-1beta, TNF-alpha and IL-8 mRNAs and proteins stimulated by hyperosmolar media. These findings provide direct evidence that hyperosmolarity induces inflammation in human limbal epithelial cells by increasing expression and production of pro-inflammatory cytokines and chemokines, a process that appears to be mediated through activation of the JNK and ERK MAPK signalling pathways. The efficacy of doxycycline in treating ocular surface diseases may be due to its ability to suppress JNK and ERK signalling activation and inflammatory mediator production in the limbal epithelium.

Adult↗
PubMed2005

Cell size correlates with phenotype and proliferative capacity in human corneal epithelial cells.

This study investigated whether cell size correlates with phenotype and proliferative capacity in human corneal epithelial cells. Primary cultured human corneal epithelial cells were sorted by flow cytometry based on forward scatter profile in comparison with the profile of beads of known size. Four fractions (A, B, C, and D) of cells ranging in size from 10 to 16, 17 to 23, 24 to 30, and >or=31 microm in diameter, respectively, were collected to evaluate their 5-bromo-2-deoxyuridine (BrdU) label retention properties, cell phenotype, and clonal growth capacity on a 3T3 fibroblast feeder layer. Among these four populations, cell size was shown to positively correlate with the expression of the differentiation markers keratin (K) 3, K12, and involucrin and inversely with the levels of stem cell-associated markers DeltaNp63 and ABCG2 and with colony-forming efficiency (CFE) and growth capacity. Population A with the smallest size, accounting for 11.0%+/-4.5% of the entire population, contained the greatest number of BrdU label-retaining slow-cycling cells, displayed the highest percentage of cells immunopositive to p63 and ABCG2 and negative to K3 and involucrin, expressed the highest levels of DeltaNp63 and ABCG2 mRNA and the lowest levels of K3, K12, and involucrin, and possessed the highest CFE and growth capacity. These findings suggest that cell size correlates with cell differentiation phenotypes and proliferative capacity in human corneal epithelial cells. The smallest cells in population A seem to be enriched for putative stem cells, and small cell size may represent one of the important properties of adult corneal epithelial stem cells.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2005

Partial enrichment of a population of human limbal epithelial cells with putative stem cell properties based on collagen type IV adhesiveness.

The concept that corneal epithelium stem cells reside in limbus has been recognized for more than a decade, but isolation of these stem cells has not been accomplished. This study was an initial attempt to isolate a population of human limbal epithelial cells enriched for certain putative stem cell properties based on their phenotype. Epithelial cells harvested from fresh human limbal rings and their primary cultures were allowed to adhere to collagen IV-coated dishes for 20 min and 2 hr, sequentially. The rapidly adherent cells (RAC), slowly adherent cells and non-adherent cells were evaluated for certain stem cell properties: (a) BrdU-label retention, (b) expression of basal cell (integrin beta1, p63, ABCG2) and differentiation (involucrin, keratin 12) markers, and (c) colony forming efficiency (CFE) and growth capacity on a 3T3 fibroblast feeder layer. Among unfractionated cells and the three selected populations, the RAC, accounting for about 10% of whole population, were enriched 5-fold in BrdU label-retaining cells, displayed the highest number of integrin beta1 and p63 positive and involucrin negative cells, expressed high levels of DeltaNp63 and ABCG2 mRNA, and lacked involucrin and K12 expression, and possessed the greatest CFE and growth capacity. These findings demonstrated for the first time that human limbal epithelial cells with stem cell properties can be partially enriched by their adhesiveness to collagen IV. The RAC population enriched for certain putative stem cell properties may prove useful in the future for transplantation to diseased and damaged corneas with limbal stem cell deficiency.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2005

Phase III safety evaluation of cyclosporine 0.1% ophthalmic emulsion administered twice daily to dry eye disease patients for up to 3 years.

OBJECTIVE: To evaluate cyclosporine 0.1% ophthalmic emulsion over a 1- to 3-year period in moderate to severe dry eye disease patients. DESIGN: Nonrandomized, multicenter, open-label clinical trial extending 2 ophthalmic cyclosporine phase III clinical trials. PARTICIPANTS: Four hundred twelve patients previously dosed for 6 to 12 months with cyclosporine 0.05% or 0.1% in prior phase III trials. INTERVENTION: Patients instilled ophthalmic cyclosporine 0.1% twice daily into both eyes for up to 3 consecutive 12-month extension periods. MAIN OUTCOME MEASURES: Corneal staining, Schirmer tests, and symptom severity assessments were conducted during the first 12-month extension, with a patient survey during the second 12-month extension. Biomicroscopy and visual acuity (VA) examinations, intraocular pressure (IOP) measurements, and adverse effects queries occurred at 6-month intervals. RESULTS: Mean duration of treatment was 19.8 months. Improvements in objective and subjective measures of dry eye disease were modest, probably because of prior treatment with cyclosporine. Most survey respondents said their symptoms began to resolve in the first 3 months of cyclosporine treatment during the previous phase III clinical trials. At study exit, VA decreased in 12.6% (93/738) and increased in 5.4% (40/738) of eyes by > or =2 lines; severity of biomicroscopy findings increased in 3.4% (chemosis; 26/760), 7.2% (conjunctival hyperemia; 55/760), or 8.5% (tear film debris; 64/756) of eyes; and mean IOP increased 0.18 mmHg relative to baseline. The most common treatment-related adverse events were burning (10.9% of patients [45/412]), stinging (3.9% [16/412]), and conjunctival hyperemia (3.4% [14/412]). No serious treatment-related adverse events occurred. Most patients (95.2% [140/147]) said they would continue cyclosporine therapy; 97.9% (143/146) would recommend it to other dry eye patients. CONCLUSIONS: Therapy of chronic dry eye disease with cyclosporine 0.1% ophthalmic emulsion for 1 to 3 years was safe, well tolerated, and not associated with systemic side effects. The results supplement the safety record of the commercially available cyclosporine 0.05% ophthalmic emulsion.

Adult↗