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Stem Cells of the Meibomian Gland may play a Role in the Mucocutaneous Junctional Epithelium (MCJ) of the Eyelid
TEAR FILM & OCULAR SURFACE LACK OF TRKA EXPRESSION IN KERATOCONUS: MOLECULAR AND BIOCHEMICAL EVIDENCE OF A POTENTIAL PATHOGENETIC ROLE FOR NERVE GROWTH FACTOR (NGF). Alessandro Lambiase,1 Daniela Merlo,2 Cristiana Mollinari,1 Anna Maria Rinaldi,2 Stefano Bonini.1 Ophthalmology, University of Rome "Campus Bio-Medico", Italy;1 Istituto Superiore di Sanità, Dept. Cell Biology and Neuroscience, Rome, Italy.2 Purpose. To evaluate the involvement of nerve NGF in the pathogenesis of keratoconus. Methods. We investigated NGF/TrkANGFR/p75NTR mRNA and protein expression levels, in 10 corneas obtained at the time of penetrating keratoplasty for keratoconus, in 2 corneas with keratoconus secondary to refractive surgery, in 5 corneas with bullous keratopathy, in 3 corneas with Fuchs' dystrophy, in 3 corneas with post-traumatic leukoma and in 10 normal central corneas obtained by the local eye bank (Banca degli occhi di Roma). After surgical dissection, the corneas were processed for semi-quantitative RT-PCR to evaluate NGF, TrkANGFR and p75NTR mRNAs expression levels and immunohistochemistry, ELISA and Western Blotting to evaluate expression of NGF, TrkANGFR and p75NTR proteins, were performed on the same cornea samples. Sp3 expression was evaluated by Western Blotting. The effect of Sp3 on TrkA expression was investigated by transient transfection of SP3-short construct in human keratocytes and in HACAT keratinocyte. Indeed, in HACAT cells a transient tansfections of SP3-short construct and Human TrkA 5'-proximal promoter region was performed and TrkA gene reporter assays was evaluated by the DualLuciferase Reporter Assay System. Results. We have found the lack of TrkANGFR expression in corneas from patients affected by keratoconus as well as nerve growth factor (NGF) and p75NTR decreased expression levels, when compared to healthy corneas. The absence of TrkA expression is associated with a marked increase of the Sp3 short form (Sp3s) expression and the lack of the Sp3 long form. We demonstrated that Sp3s directly downregulated the TrkA expression. Conclusion. This is the first demonstration of the tissue specific absence of TrkANGFR expression in a human disease. Indeed, these findings demonstrating the specific involvement of TrkA in keratoconus, suggest a new diagnostic marker and new potential therapeutic targets. GLUCOSE CONCENTRATIONS IN TEARS VIA NOVEL MICROANALYTICAL METHODS. Jennifer Lane,1 David Krumholz,2 Robert Sack,2 Carol Morris.3 CIBA Vision Corporation, Duluth, GA;USA1 State University of New York State College of Optometry, New York, NY, USA;2 Southern Cross University, NSW, Australia.3 Purpose. To develop a more reliable and reproducible methodology for detecting glucose in tears, a HPLC based method using pulsed amperometric detection was compared with two commercially available glucose microassays. Methods. The HPLC used in this study was a 817 Bioscan system with a Carbosep-1 column both by Metrohm Peak. The glucose samples were diluted with a 10mM HEPES buffer, pH 7.0 containing 10ppm kathon and 15uM fucose. The Amplex Red Glucose Assay kit was bought directly from Molecular Probes and the Trinder reagent kit (spectrophotometric)was bought directly from Sigma and both were used according to the manufacturer’s instructions. Results. Linearity in all assays ranged from R2 values of 0.987-0.999. Precision was measured by calculating the response factor of the glucose standard concentrations, the %RSD observed in the response factors ranged from 9-25% among the assays studied here. Percent recovery was measured as a indicator of accuracy and ranged from 75-109% with %RSD ranging from 3.5-24.5%. The practical limit of quantitation was roughly 1mg/dL glucose. Conclusions. The smallest sample size we were able to analyze reliably was 1uL, with both the HPLC based assay and the Amplex assay. The most robust assay used was the HPLC based assay followed by the Amplex assay and finally the Trinder Assay. The Amplex and Trinder assays are both enzymatically controlled reactions with no end point, this can have some effect on the day to day reproducibility of these assays. In this study, Amplex and Trinder assays both exhibit some matrix effects which affected the limit of detection in clinical samples. The HPLC assays did not exhibit observable matrix effects due to its inherent separation properties and specific detection for polysaccharides.For high sample throughput capabilities, the microplate assays, which can run 96 samples in less than an hour, out perform the HPLC based assay which requires a 20 minute run time per individual analysis.
THE DURATION AND EFFECT OF ARTIFICIAL TEARS ON VISUAL ACUITY. Robert Latkany, Mohammad Nilforoushan, Mark Speaker. New York Eye & Ear Infirmary, Center for Ocular Tear Film Disorders, New York, NY USA. Purpose. To assess the change in both near and distant corrected and uncorrected visual acuity after the application of one drop of preservative free Carboxymethylcellulose (CMC) sodium 0.5%. Methods. 50 eyes of 25 patients were prospectively tested. Each patient’s visual acuity was tested for both near and distance using a near Snellen card at 16 inches and the Early Treatment Diabetic Retinopathy Study (ETDRS) vision chart respectively. As a control, the vision was retested after blink alone. After this data was documented, the patient was provided with a drop of preservative free artificial tears and their visual acuity was reassessed for both near and distance. The data was analyzed comparing the results of change in vision with blink alone to change in vision with artificial tears. These patients were also tested at 1 minute intervals until their vision returned to their baseline visual acuity and this time was recorded. All patients had a Slit Lamp examination and had to answer a Ocular Surface Disease Index (OSDI) questionnaire to determine their degree of Dry Eyes. Result. A pair T-test was used to statistically compare the change in distant visual acuity between the control and test group. A chi-square method was used to compare the change in near visual acuity between the control and test group. Both test subjects showed statistical significant difference when compared to controls. Conclusion. CMC temporarily improves near and distance visual acuity. Support: None STEM CELLS OF THE MEIBOMIAN GLAND MAY PLAY A ROLE IN THE MUCOCUTANEOUS JUNCTIONAL EPITHELIUM (MCJ) OF THE EYELID. Robert M. Lavker,1 James Treet,2 Tung-Tien Sun.3 Departments of Dermatology, Northwestern University, Chicago IL;1 University of Pennsylvania, Philadelphia, PA,2 New York University Medical Center, New York, NY.3 Purpose. As stem cells are targets of carcinogenic agents, knowledge of their location within the meibomian gland and the MCJ epithelium is important for understanding the etiology of the unusually aggressive mucoepidermoid and squamous cell carcinomas that arise in these regions. Thus we investigated the cell kinetic properties of epithelial cells from these two regions. Methods. To identify slow cycling cells (label-retaining cells; LRCs), we injected neonatal mice (p3) subcutaneously with 5-bromo-2deoxyuridine (BrdU) twice daily for 3 days. After a 6-8 week chase, mice were sacrificed and the BrdU-positive cells within the MCJ epithelium and the meibomian gland were visualized. We identified rapidly cycling cells by a single subcutaneous injection of either BrdU or tritiated thymidine (3HTdR), one hour before sacrifice. Results. LRCs were concentrated in the ductal epithelium of the meibomian gland, with few if any LRCs in the glands. Many of the cells in the uppermost portion of the meibomian gland ductal epithelium contained red-speckled nuclei, reflecting the dilution of the BrdU-label by cell division. Under normal conditions, most of the rapidly cycling cells were seen in the basal sebocytes of the meibomian gland. The MCJ epithelium contained more rapidly proliferating cells and displayed a much higher proliferative rate than other zones of the conjunctival epithelium. Despite its high proliferative rate, LRCs were rarely observed in the MCJ epithelium. Upon stimulation of the ocular surface with phorbol myristate, the meibomian gland LRCs divided and their progeny migrated towards the MCJ epithelium of the eyelid. Conclusions. These findings indicate that the meibomian gland ductal epithelium may be a region enriched in epithelial stem cells. The relationship between the meibomian gland and the MCJ epithelium bears striking similarities to the relationship between the hair follicle and the epidermis. In the latter instance, progeny of the hair follicle stem cells have been demonstrated to give rise to the epidermis. We hypothesize that the meibomian gland ductal epithelium may serve as a source of cells that help maintain its overlying MCJ epithelium.
THE OCULAR SURFACE / JANUARY, 2005, VOL. 3, NO. 1 / SUPPLEMENT
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THE DURATION AND EFFECT OF ARTIFICIAL TEARS ON VISUAL ACUITY. Robert Latkany, Mohammad Nilforoushan, Mark Speaker. New York Eye & Ear Infirmary, Center for Ocular Tear Film Disorders, New York, NY USA. Purpose. To assess the change in both near and distant corrected and uncorrected visual acuity after the application of one drop of preservative free Carboxymethylcellulose (CMC) sodium 0.5%. Methods. 50 eyes of 25 patients were prospectively tested. Each patient’s visual acuity was tested for both near and distance using a near Snellen card at 16 inches and the Early Treatment Diabetic Retinopathy Study (ETDRS) vision chart respectively. As a control, the vision was retested after blink alone. After this data was documented, the patient was provided with a drop of preservative free artificial tears and their visual acuity was reassessed for both near and distance. The data was analyzed comparing the results of change in vision with blink alone to change in vision with artificial tears. These patients were also tested at 1 minute intervals until their vision returned to their baseline visual acuity and this time was recorded. All patients had a Slit Lamp examination and had to answer a Ocular Surface Disease Index (OSDI) questionnaire to determine their degree of Dry Eyes. Result. A pair T-test was used to statistically compare the change in distant visual acuity between the control and test group. A chi-square method was used to compare the change in near visual acuity between the control and test group. Both test subjects showed statistical significant difference when compared to controls. Conclusion. CMC temporarily improves near and distance visual acuity. Support: None STEM CELLS OF THE MEIBOMIAN GLAND MAY PLAY A ROLE IN THE MUCOCUTANEOUS JUNCTIONAL EPITHELIUM (MCJ) OF THE EYELID. Robert M. Lavker,1 James Treet,2 Tung-Tien Sun.3 Departments of Dermatology, Northwestern University, Chicago IL;1 University of Pennsylvania, Philadelphia, PA,2 New York University Medical Center, New York, NY.3 Purpose. As stem cells are targets of carcinogenic agents, knowledge of their location within the meibomian gland and the MCJ epithelium is important for understanding the etiology of the unusually aggressive mucoepidermoid and squamous cell carcinomas that arise in these regions. Thus we investigated the cell kinetic properties of epithelial cells from these two regions. Methods. To identify slow cycling cells (label-retaining cells; LRCs), we injected neonatal mice (p3) subcutaneously with 5-bromo-2deoxyuridine (BrdU) twice daily for 3 days. After a 6-8 week chase, mice were sacrificed and the BrdU-positive cells within the MCJ epithelium and the meibomian gland were visualized. We identified rapidly cycling cells by a single subcutaneous injection of either BrdU or tritiated thymidine (3HTdR), one hour before sacrifice. Results. LRCs were concentrated in the ductal epithelium of the meibomian gland, with few if any LRCs in the glands. Many of the cells in the uppermost portion of the meibomian gland ductal epithelium contained red-speckled nuclei, reflecting the dilution of the BrdU-label by cell division. Under normal conditions, most of the rapidly cycling cells were seen in the basal sebocytes of the meibomian gland. The MCJ epithelium contained more rapidly proliferating cells and displayed a much higher proliferative rate than other zones of the conjunctival epithelium. Despite its high proliferative rate, LRCs were rarely observed in the MCJ epithelium. Upon stimulation of the ocular surface with phorbol myristate, the meibomian gland LRCs divided and their progeny migrated towards the MCJ epithelium of the eyelid. Conclusions. These findings indicate that the meibomian gland ductal epithelium may be a region enriched in epithelial stem cells. The relationship between the meibomian gland and the MCJ epithelium bears striking similarities to the relationship between the hair follicle and the epidermis. In the latter instance, progeny of the hair follicle stem cells have been demonstrated to give rise to the epidermis. We hypothesize that the meibomian gland ductal epithelium may serve as a source of cells that help maintain its overlying MCJ epithelium.
THE OCULAR SURFACE / JANUARY, 2005, VOL. 3, NO. 1 / SUPPLEMENT
S85