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M B Wax

Publications and source records attributed to M B Wax.

At least 37 records · Page 2Linked to original sources
PubMed2000

Responses of different cell lines from ocular tissues to elevated hydrostatic pressure.

BACKGROUND/AIMS: Mechanical forces are thought to induce cellular responses through activation of signalling pathways. Cells within the intraocular environment are exposed to constant changes in the levels of intraocular pressure. In this study, an attempt was made to determine the acute effects of elevated hydrostatic pressure on different intraocular cells grown in culture. METHODS: Different cell lines derived from ocular tissues including non-pigmented and pigmented ciliary epithelium, trabecular meshwork, retina, and lamina cribrosa were incubated in a pressurised chamber at 50 mm Hg in a culture incubator at 37 degrees C for up to 6 hours. Control cells were incubated at atmospheric pressure. The viability of the cells was examined using their intracellular esterase activity. The morphology and cytoskeleton of the cells were investigated using microscopy and phalloidin staining. Adenylyl cyclase activity was assessed by measuring the conversion of [(3)H]-cAMP from [(3)H]-ATP in response to elevated hydrostatic pressure for 1-6 hours. In addition, at the end of incubation period under elevated hydrostatic pressure the recovery of adenylyl cyclase activity to control levels was examined. RESULTS: Cell viability did not change following exposure to elevated hydrostatic pressure for 6 hours. Cells subjected to elevated hydrostatic pressure demonstrated morphological differences characterised by a more rounded shape and a redistribution of actin stress fibres that was most prominent in lamina cribrosa astrocytes. A time dependent increase in basal adenylyl cyclase activity, and a decrease in maximum forskolin stimulated activity were observed in all cell lines following exposure to elevated hydrostatic pressure. CONCLUSION: These observations demonstrate that cell lines from different ocular tissues are sensitive to changes in external pressure in vitro. They exhibit morphological and cytoskeletal changes as well as significant alterations of intracellular adenylyl cyclase activity following exposure to acute and sustained levels of elevated hydrostatic pressure of up to 6 hours' duration.

Actins↗
PubMed2000

Molecular identification of functional water channel protein in cultured human nonpigmented ciliary epithelial cells.

PURPOSE: Water channel proteins are important pathways for water movements across cell membranes, including those in the ciliary epithelium, which is the major site of aqueous humor secretion. In this study, we aimed to demonstrate the expression of functionally active aquaporin-1 (AQP1) water channels in cultured human ciliary epithelial cells. METHODS: Poly A(+) RNA was isolated from cell cultures of Simian Virus 40 (SV-40) transformed human nonpigmented ciliary epithelium (NPE) subjected to RT-PCR reaction using primers specific to AQP1. Northern analysis was used to define the expression of AQP1 in NPE cells. Western immunoblotting with polyclonal antibody raised against AQP1 was used to evaluate the AQP1 protein expression in the plasma membranes of human NPE cells. Light scattering method was used to determine the osmotic water permeability in the suspension of NPE cells. RESULTS: RT-PCR using specific primers for AQP1, Northern analysis and Western immunoblot using AQP1 specific antibody demonstrated the expression of AQP1 in the plasma membranes of NPE cells. Osmotic water permeability (P( f)) measurements confirmed that functional AQP1 water channels are expressed in human NPE cells and the P(f) for these cells was 9.8 x 10( -3) cm/s at 10 degrees C. CONCLUSIONS: The presence of AQP1 in human NPE cells suggests that it may have a role in the fluid flow across epithelial membranes. In addition, the existence of AQP1 in the human NPE cells provide an excellent in vivo model to study the regulation of aquaporins and their possible role in the aqueous humor secretion.

Animals↗
PubMed1999

Serum autoantibodies to optic nerve head glycosaminoglycans in patients with glaucoma.

BACKGROUND: Serum autoantibodies that cross-react with glycosaminoglycans have been proposed to play a significant role in specific tissue injury in patients with systemic autoimmune diseases. OBJECTIVE: To investigate whether serum immunoreactivity to glycosaminoglycans is present in patients with glaucoma who have aberrant serum autoantibodies to DNA, RNA, nuclear proteins, or retinal proteins, as proteoglycans and their glycosaminoglycan side chains are important components of the optic nerve head and its vasculature. METHODS: We performed Western blotting using patient serum samples and human optic nerve head homogenates that were treated with or without specific glycosaminoglycan degrading enzymes. Monoclonal antibodies that recognize different determinants of glycosaminoglycans were used to identify specific substrate antigenicity. We compared the serum immunoreactivity to glycosaminoglycans in 60 age-matched patients with normal-pressure glaucoma, 36 patients with primary open-angle glaucoma, and 20 control subjects by enzyme-linked immunosorbent assay. In addition, immunohistochemistry was performed to compare the distribution patterns of glycosaminoglycans in the optic nerve head of postmortem eyes of age-matched patients with normal-pressure glaucoma, primary open-angle glaucoma, and control subjects. RESULTS: Western blotting demonstrated that serum samples from patients with glaucoma who have circulating autoantibodies can recognize optic nerve head proteoglycans, including chondroitin sulfate and heparan sulfate. The level of serum autoantibodies binding purified chondroitin sulfate and heparan sulfate glycosaminoglycans in an enzyme-linked immunosorbent assay was approximately 100% higher in patients with normal-pressure glaucoma than that in control subjects and approximately 50% higher than that in patients with primary open-angle glaucoma. We also observed increased immunostaining of glycosaminoglycans in the optic nerve head of eyes with glaucoma, particularly those with normal intraocular pressure, compared with control eyes. CONCLUSION: There are increased levels of autoantibodies recognizing glycosaminoglycans of the optic nerve head in the serum samples of some patients with glaucoma. CLINICAL RELEVANCE: These autoantibodies may increase the susceptibility of the optic nerve head to damage in these patients by changing the functional properties of the lamina cribrosa, its vasculature, or both.

Antibodies, Monoclonal↗
PubMed1999

Effects of methotrexate treatment on serum immunoreactivity of a patient with normal-pressure glaucoma.

PURPOSE: Increased serum immunoreactivity to retinal proteins may have a role in the disease process of some glaucoma patients. We describe a patient with normal-pressure glaucoma whose serum immunoreactivity to retinal proteins regressed after methotrexate treatment for rheumatoid disease. METHOD: Case report. RESULTS: In a 66-year-old white female with normal-pressure glaucoma and rheumatoid disease, sequential Western blots using patient sera against retinal proteins demonstrated a decrease in the immunoreactive bands after treatment. During the treatment period of 3 years, her visual fields appeared to have improved. Optic disk examination during the short periods without treatment, however, disclosed new, bilateral splinter hemorrhages on the optic disks. CONCLUSION: These observations suggest a potential role for immune-based intervention in similar patients.

Aged↗
PubMed1999

Density-dependent resistance to apoptosis in retinal cells.

PURPOSE: To study effects of cell density on retinal cell survival. METHODS: Apoptotic cell death was induced in cultured retinal cells seeded at higher or lower density by various stimuli including simulated ischemia, excitotoxicity and antibody against heat shock protein 27 (hsp27). Quantitative analysis of apoptotic cells was performed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling technique and flow cytometry. Cytoskeleton was examined using immunocytochemistry and specific staining of actin by phalloidin and DNase I. In addition, alterations in the cytoskeletal proteins, bcl-2 family of proteins and hsp27 were studied using western blotting. RESULTS: Incubation of the cells under apoptotic stimuli caused higher rates of apoptosis in lower density cultures as determined by TUNEL technique and flow cytometric analysis. Both morphologic examination of cytoskeleton and western blotting revealed that after incubation with various stressors, degradation of actin and tubulin was more prominent in lower density cultures compared to higher density cultures. The expression of bcl-2 and bcl-xL was higher and the expression of bax was lower in lower density cultures compared to higher density cultures at basal condition. After incubation with stressors, bcl-2 and bcl-xL expressions decreased and bax expression increased in both lower and higher density cultures. However, we observed that the expression of hsp27 was higher in higher density cultures than in lower density cultures in the presence or absence of apoptotic stimuli. CONCLUSIONS: These findings demonstrate that retinal cells are more resistant to apoptosis in higher density cultures, independent of the inducer. This might be partly due to protective activity of endogenous hsp27 in the cells at higher density, which contributes to cytoskeletal integrity in response to apoptotic stimuli.

Animals↗
PubMed1999

Epitope mapping of anti-rhodopsin antibodies from patients with normal pressure glaucoma.

PURPOSE: The presence of anti-rhodopsin antibodies in patients with normal pressure glaucoma (NPG) has been previously demonstrated with western blot analysis and enzyme-linked immunosorbent assay. To learn more about the characteristics, origin, and possible significance of these antibodies, the epitopic specificity of the anti-rhodopsin antibodies was examined in four NPG patients. METHODS: Antibodies in patient sera were assayed by western blot analysis against purified bovine rhodopsin. Peptides derived from particular segments of the rhodopsin sequence were tested for activity in competing for rhodopsin-antibody binding. RESULTS: Of a series of nine peptides that constitute most of the hydrophilic regions of rhodopsin, only one, consisting of the C-terminal 25 amino acids, prevented binding of the patient antibodies to rhodopsin. Higher resolution mapping using a set of dodecamers of overlapping sequences from the C-terminal region demonstrated that antibody binding is completely dependent on the last two amino acids. Removing the C-terminal alanine alone, or amidating the C terminus carboxyl group, also eliminated antibody binding. CONCLUSIONS: Because four of four patient antibodies examined exhibited the identical epitopic specificity, it is likely that a common mechanism underlies their generation. This may indicate that molecular mimicry has occurred, because several pathogens contain similar C-terminal sequences. Although they may serve as diagnostic markers, and provide evidence that there is an autoimmune component in some patients with glaucoma, the role, if any, that these antibodies play in the pathogenesis of the disease remains unknown.

Animals↗
PubMed1999

Inhibition of caspase activity in retinal cell apoptosis induced by various stimuli in vitro.

PURPOSE: Although recent work implicates a decisive role for a family of cysteine aspartic acid proteases, termed caspases, as mediators of neuronal apoptosis, little is known about caspase activation that accompanies apoptosis in the retina. The purpose of this study was to investigate caspase activation in retinal cell apoptosis induced by various stimuli, including simulated ischemia, excitotoxicity, and antibody to heat shock protein 27 (hsp27), and to assess whether the inhibition of caspases can block apoptosis in retinal cells induced by different stimuli. METHODS: Apoptotic cell death induced in cultured retinal cells by simulated ischemia, excitotoxicity, or hsp27 antibody was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique. Changes in the caspase activity were studied using western blot analysis and a fluorometric protease activity assay in the presence or absence of caspase inhibitors. In addition, changes in the expression of bcl-2 and bax were examined by western blot analysis. RESULTS: The authors' in vitro observations revealed that the apoptotic process in retinal cells induced by different stimuli share a common executioner proteolysis cascade, including caspase-3 and poly(ADP ribose) polymerase cleavage. One exception, however, was that caspase-8 activation was only observed during the apoptosis induced by hsp27 antibody. In retinal cells going to apoptosis regardless of the stimulus, bcl-2 expression was decreased and bax expression was increased. Furthermore, the authors observed that treatment of retinal cells with inhibitors of caspases, including B-D-FMK and Z-IETD-FMK, blocked the apoptotic cell death induced by different stimuli. CONCLUSIONS: The authors' observations provide a better understanding of the apoptotic process in retinal cells at molecular level and demonstrate an effective blockade of caspase activation with specific inhibitors. These findings may have therapeutic implications in the treatment of neuroretinal diseases, which are characterized by apoptotic cell death.

Animals↗
PubMed1998

Regulation of aquaporin-4 water channels by phorbol ester-dependent protein phosphorylation.

The molecular mechanisms for regulating water balance in many tissues are unknown. Like the kidney, the eye contains multiple water channel proteins (aquaporins) that transport water through membranes, including two (AQP1 and AQP4) in the ciliary body, the site of aqueous humor production. However, because humans with defective AQP1 are phenotypically normal and because the ocular application of phorbol esters reduce intraocular pressure, we postulated that the water channel activity of AQP4 may be regulated by these agents. We now report that protein kinase C activators, phorbol 12,13-dibutyrate, and phorbol 12-myristate 13-acetate strongly stimulate the phosphorylation of AQP4 and inhibit its activity in a dose-dependent manner. Phorbol 12,13-dibutyrate (10 microM) and phorbol 12-myristate 13-acetate (10 nM) reduced the rate of AQP4-expressing oocyte swelling by 87 and 92%, respectively. Further, phorbol 12,13-dibutyrate significantly increased the amount of phosphorylated AQP4. These results demonstrate that protein kinase C can regulate the activity of AQP4 through a mechanism involving protein phosphorylation. Moreover, they suggest important potential roles for AQP4 in several clinical disorders involving rapid water transport such as glaucoma, brain edema, and swelling of premature infant lungs.

Animals↗
PubMed1998

Clinical and ocular histopathological findings in a patient with normal-pressure glaucoma.

OBJECTIVE: To study the histopathological changes of eyes from a patient with normal-pressure glaucoma whose clinical and laboratory findings were well documented. METHODS: Postmortem histopathological findings in a patient with normal-pressure glaucoma who had monoclonal gammopathy and serum immunoreactivity to retinal proteins were examined in comparison with those of an age-matched control subject. Clinicopathological correlations to laboratory findings were examined. RESULTS: Clinical and histopathological findings of the patient, including cavernous degeneration of optic nerve and characteristic optic nerve cupping, were similar to those in patients with glaucoma who had high intraocular pressure. In addition, we found immunoglobulin G and immonuglobulin. A deposition in the ganglion cells, inner and outer nuclear layers of the retina, and evidence of apoptotic retinal cell death using terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling technique. CONCLUSIONS: Serum antibodies to retinal proteins and retinal immunoglobulin deposition constitute novel findings in a patient with normal-pressure glaucoma and may contribute to better understanding of the mechanisms underlying glaucomatous optic neuropathy in this disorder.

Aged↗
PubMed1998

Latanoprost treatment for glaucoma: effects of treating for 1 year and of switching from timolol. United States Latanoprost Study Group.

PURPOSE: To determine the efficacy and safety of latanoprost treatment for 1 year in glaucoma patients, and to evaluate the effects of switching from timolol to latanoprost therapy. METHODS: Latanoprost 0.005% was topically applied once daily without masking for 6 months in 223 patients with elevated intraocular pressure after previous treatment with latanoprost once daily or 0.5% timolol twice daily for 6 months in a multicenter, randomized, double-masked, parallel group study. RESULTS: Compared with baseline values before treatment, a significant (P < .0001) diurnal reduction in intraocular pressure of 6 to 8 mm Hg was maintained with minimal fluctuation for the duration of treatment. When treatment was switched from timolol to latanoprost, intraocular pressure was reduced by 1.5 +/- 0.3 mm Hg (mean +/- SEM; 8% change in intraocular pressure; 31% of the intraocular pressure reduction produced by timolol; P < .001) compared with the change in intraocular pressure in patients remaining on latanoprost therapy. Of the patients initially enrolled, 95% successfully completed treatment. There was a slight overall increase in conjunctival hyperemia in patients who switched from timolol to latanoprost, but no change in those who continued latanoprost. The timolol-induced reduction of resting heart rate returned to baseline levels after switching to latanoprost. Of the 247 patients treated with latanoprost during the masked and/or open-label studies, 12 (5%) demonstrated a definite (n = 4) or possible (n = 8) increase in iris pigmentation. CONCLUSIONS: Latanoprost is a well-tolerated ocular hypotensive agent that appears to be more effective than timolol in reducing intraocular pressure. The increase in iris pigmentation appears to be harmless but requires further investigation.

Adult↗
PubMed1998

Anti-Ro/SS-A positivity and heat shock protein antibodies in patients with normal-pressure glaucoma.

PURPOSE: To describe the clinical and laboratory findings in 10 patients with normal-pressure glaucoma and anti-Ro/SS-A positivity by enzyme-linked immunosorbent assay (ELISA) and to determine whether that positivity may be related to an autoimmune mechanism for the optic neuropathy. METHODS: In this prospective study, we evaluated ocular and systemic clinical findings of 10 patients with normal-pressure glaucoma and anti-Ro/SS-A positivity by ELISA, including sicca complex features. Oüchterlony immunodiffusion was performed to confirm the presence of antibodies for Ro/SS-A, and the presence of other serum antibodies and their possible cross-reactivities with Ro/SS-A were investigated. RESULTS: None of the 10 patients with normal-pressure glaucoma and anti-Ro/SS-A positivity (by ELISA) had clinical or laboratory signs of Sjögren syndrome or other connective tissue diseases. Only one of 10 patients had evidence of anti-Ro/SS-A antibodies by Oüchterlony immunodiffusion. All patients demonstrated serum immunoreactivity to bacterial heat shock protein 60 (hsp60) by Western blotting. Cross-reactivity between bacterial hsp60 and Ro/SS-A was demonstrated by Western blotting. Immunoreactivity to bacterial hsp60 by ELISA was significantly elevated in the sera of patients with normal-pressure glaucoma. Furthermore, patients with either normal-pressure or primary open-angle glaucoma had increased serum immunoreactivity to human hsp60. CONCLUSIONS: Anti-Ro/SS-A positivity by ELISA in 10 patients with normal-pressure glaucoma was associated with a high level of serum immunoreactivity to bacterial hsp60, which may indicate that their glaucomatous optic neuropathy involves an as yet unidentified autoimmune mechanism. The identification of autoantibodies that react with human hsp60 in patients with normal-pressure and primary open-angle glaucoma may signify a common finding associated with the glaucomatous optic neuropathy process in some patients and appears to be unrelated to intraocular pressure levels.

Adult↗
PubMed1998

Late removal of releasable sutures after trabeculectomy or combined trabeculectomy with cataract extraction supplemented with antifibrotics.

PURPOSE: Releasable scleral flap sutures have been effectively used in trabeculectomy. The suggested time for suture removal is within the first two postoperative weeks. The authors wished to determine whether the use of intraoperative antifibrotics alters the time of suture removal and the results of surgery. METHODS: The charts of 324 patients (388 eyes) undergoing trabeculectomy and 141 patients (174 eyes) undergoing combined trabeculectomy with cataract extraction with a minimum follow-up of 12 months were retrospectively reviewed. All eyes received intraoperative antifibrotics; mitomycin C in 534 eyes and 5-fluorouracil in 29 eyes. Two releasable scleral flap sutures were used in all of the eyes. RESULTS: The removal time of at least one of the two releasable sutures was later than 21 days postoperatively in 89 eyes of 83 patients with trabeculectomy (22.9%) and in 58 eyes of 55 patients with combined surgery (33.3%). The suture removal was deferred in 39 eyes because of an early complication such as hypotony (intraocular pressure (IOP) < or = 5 mm Hg), bleb leak, or shallow or flat anterior chamber. There was no complication in the other 108 eyes, but suture removal was delayed until the IOP was more than 10 mm Hg. Immediate IOP reduction was (mean +/- standard deviation) 6.3 +/- 2.8 mm Hg when suture removal was performed beyond 3 weeks. There was a decreased response as the postoperative time until suture removal increases (r = -0.57). No serious complication associated with late suture removal was noted during the follow-up period (mean +/- standard deviation, 19.8 +/- 10.1 months). CONCLUSION: Intraoperative pharmacologic modulation of wound healing in trabeculectomy and combined trabeculectomy with cataract extraction extends the period that releasable suture removal is clinically effective. However the response decreases with a longer interval to releasable suture removal.

Aged↗
PubMed1998

Autoantibodies to small heat shock proteins in glaucoma.

PURPOSE: To identify the low-molecular-weight retinal proteins that are the targets of serum autoantibodies in patients with glaucoma and to study the ability of these antibodies to induce retinal apoptosis. METHODS: Serum immunoreactivity against retinal proteins was examined in age-matched groups of 60 patients with normal-pressure glaucoma, 36 patients with high-pressure glaucoma, and a control group of 20 healthy subjects, by means of western blot analysis and enzyme-linked immunosorbent assay. The specificity of the immunoreactivity to small heat shock proteins, including alpha-crystallins and hsp27, was tested by immunoprecipitation of these proteins in retinal fractions. The direct effects of antibodies specific to small heat shock proteins were then studied in isolated intact human retina (ex vivo) and cultured rat retinal cells (in vitro) by immunocytochemistry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique in conjunction with fluorescence microscopy and confocal imaging. RESULTS: Serum immunoreactivity against retinal proteins with low molecular weight in patients with glaucoma was to small heat shock proteins, including alpha-crystallins and hsp27. In addition, patients with normal pressure glaucoma had a higher titer of autoantibodies to small heat shock proteins than did age-matched patients with high-pressure glaucoma or control subjects. It was observed that when antibodies against small heat shock proteins were applied directly to retina tissue or cells, they could trigger cell death through an apoptotic mechanism. CONCLUSIONS: These findings suggest that increased titers of circulating antibodies against retinal small heat shock proteins may have pathogenic significance in some patients with glaucomatous optic neuropathy.

Aged↗
PubMed1997

Regulation of water channel activity of aquaporin 1 by arginine vasopressin and atrial natriuretic peptide.

Aquaporin 1 (AQP1), a six-transmembrane domain protein that functions as a water channel, is present in many fluid secreting and absorbing tissues such as kidney, brain, heart, and eye. It is believed that among the five known mammalian aquaporins, kidney aquaporin (AQP2) is the only water channel that is regulated by arginine vasopressin (AVP). The present data suggest that AQP1 may also be regulated by AVP. The application of AVP to Xenopus oocytes injected with AQP1 cRNA increased the membrane permeability to water. In addition, our data reveal that atrial natriuretic peptide (ANP), a peptide hormone that plays an important role in the regulation of body fluid homeostasis, blocks the AQP1-mediated increase in water permeability. Incubation with 8-bromo-cAMP or direct 8-bromo-cAMP injection into oocytes expressing AQP1 cRNA significantly increased membrane permeability to water, suggesting that stimulation of AQP1 activity by AVP may involve a cAMP-dependent mechanism. Regulation of water permeability by AVP and ANP has potential relevance to active water transport in a variety of tissues that express AQP1 including kidney, brain, and eye.

Animals↗
PubMed1997

Vacuolar H+-ATPase in ocular ciliary epithelium.

The mechanisms controlling the production of aqueous humor and the regulation of intraocular pressure are poorly understood. Here, we provide evidence that a vacuolar H+-ATPase (V-ATPase) in the ocular ciliary epithelium is a key component of this process. In intracellular pH (pHi) measurements of isolated ciliary epithelium performed with 2',7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF), the selective V-ATPase inhibitor bafilomycin A1 slowed the recovery of pHi in response to acute intracellular acidification, demonstrating the presence of V-ATPase in the plasma membrane. In isolated rabbit ciliary body preparations examined under voltage-clamped conditions, bafilomycin A1 produced a concentration-dependent decrease in short-circuit current, and topical application of bafilomycin A1 reduced intraocular pressure in rabbits, indicating an essential role of the V-ATPase in ciliary epithelial ion transport. Immunocytochemistry utilizing antibodies specific for the B1 isoform of the V-ATPase 56-kDa subunit revealed localization of V-ATPase in both the plasma membrane and cytoplasm of the native ciliary epithelium in both rabbit and rat eye. The regional and subcellular distribution of V-ATPase in specific regions of the ciliary process was altered profoundly by isoproterenol and phorbol esters, suggesting that change in the intracellular distribution of the enzyme is a mechanism by which drugs, hormones, and neurotransmitters modify aqueous humor production.

Animals↗