Enhanced characterization of serum autoantibody reactivity following HSP 60 immunization in a rat model of experimental autoimmune glaucoma.

PURPOSE: Antibodies against heat shock proteins have been identified in sera of human glaucoma patients in several studies and immunization with heat shock protein 60 (HSP 60) causes retinal ganglion cell (RGC) loss in an animal model of experimental autoimmune glaucoma. The aim of this study was to observe the time course of increased anti-retina antibody appearance in the serum and characterize the identification of prominent autoantibodies that accompany HSP 60 immunization in a rat model of experimental autoimmune glaucoma. METHODS: Eight weeks after immunization with HSP 60 retinal flatmounts were prepared and RGCs were counted in eight predefined areas and compared to controls. Serum collected before, as well as four and eight weeks after, immunization was used to detect antibody patterns against bovine retinal antigens using Western blotting techniques. These patterns were analyzed by multivariate statistical methods. Autoantibodies that were prominently increased were further identified through mass spectrometry. Intraocular pressure was measured throughout the study. RESULTS: After eight weeks, animals immunized with HSP 60 showed significant RGC loss of retinal flatmounts (P = 0.02), which was intraocular pressure independent. Early changes in antibody profiles, many of them significant upregulations, were detected. Antigens with significantly upregulated antibody reactivity after four weeks were identified as histone H2B type 1, S-arrestin, glial fibrillary acidic protein, vimentin, and heat shock protein 60. These upregulated autoantibodies returned to normal levels four weeks following their initial upregulation. Antibodies against retinaldehyde binding protein 1 on the other hand became upregulated after eight weeks. CONCLUSION: This is the first study to identify the appearance and disappearance of retinal autoantibodies in the serum of rats at several time-points following their initial upregulation in response to HSP 60 immunization in a model of experimental autoimmune glaucoma.

Functional characterization of a human aquaporin 0 mutation that leads to a congenital dominant lens cataract.

The aquaporin (AQP) transmembrane proteins facilitate the movement of water across the plasma membrane. In the lens, AQP0 is expressed in fiber cells and AQP1 in the epithelium. Recently, two individuals were identified with congenital polymorphic autosomal dominant cataract, due to a single nucleotide base deletion mutation in the lens AQP0. The deletion modified the reading frame resulting in the addition of a premature stop codon. In the present study, we examined the water permeability properties, trafficking and dominant negative effects as well as cytotoxicity due to the mutant AQP0 (Delta213-AQP0) protein. The membrane water permeability (P(w)) of Delta213-AQP0 expressing oocytes (14+/-1 microm/s) was significantly lower than those expressing WT-AQP0 (25+/-3 microm/s). P(w) of water injected control oocytes was 13+/-2 microm/s. Co-expression of WT-AQP0 with Delta213-AQP0 significantly lowered the P(w) (18+/-3 microm/s) compared to WT-AQP0. With or without the EGFP tag, WT-AQP0 protein localized in the plasma membranes of oocytes and cultured cells whereas Delta213-AQP0 was retained in the ER. Forster Resonance Energy Transfer (FRET) showed that WT-AQP0 partly localized with the co-expressed Delta213-AQP0. Co-localization studies suggest that the mutant AQP0 gained its dominant function by trapping the WT-AQP0 in the ER through hetero-oligomerization. Incubating the cells with chemical chaperones, namely, TMAO and DMSO, did not correct the folding/trafficking defects. Cell death in the Delta213-AQP0 expressing cells was due to necrosis caused by the accumulation of Delta213-AQP0 protein in the ER in cytotoxic proportions. The data show that replacement of the distal end of the 6th TM domain and the C-terminal domain of AQP0 due to the deletion mutation resulted in the impairment of cell membrane P(w), localization of the mutant protein in the ER without trafficking to the plasma membrane, and cytotoxicity due to the accumulation of the mutant protein. Cataracts in patients with this mutation might have resulted from the above mentioned consequences.

Alterations in the morphology of lamina cribrosa pores in glaucomatous eyes.

AIMS: To determine alterations which occur in the size and shape of lamina cribrosa (LC) pores in glaucomatous eyes over a period of time. METHODS: Baseline and follow up optic disc photographs were retrospectively studied in 39 eyes of 39 patients with glaucoma. Only eyes with a vertical cup to disc ratio equal to or greater than 0.6 were included in the study. In addition, all selected eyes had to have serial optic disc photographs obtained at least 3 years apart allowing clear visualisation of LC surface. The association of the alterations in LC surface morphology with patient specific and eye specific characteristics was statistically analysed. RESULTS: During a mean study period of 3.90 (SD 0.7) years, individual pore size (mean pore area to disc area ratio) exhibited a significant decrease between baseline and follow up measurements of each eye (p<0.0001). However, during the study period, total pore area to disc area ratio did not change (p>0.05), and the change in pore shape in some eyes (from circular to more oval and elongated) was statistically insignificant (p = 0.12). Although a relation was detectable between the optic disc and lamina cribrosa parameters at a given time, which reflects cumulative effects, during the study period, there was no significant association between the changes of the LC parameters and neural tissue damage. The rate and the magnitude of the changes in individual pore size during the study period were not significantly different among the eyes exhibiting progressive neural rim damage and those staying stable (p>0.05). CONCLUSION: These findings demonstrate that the LC surface morphology exhibits changes along with the glaucomatous optic disc damage. However, the clinical appearance of LC surface in glaucomatous eyes may continue to change, even when the neural rim damage is clinically stable. These findings are probably associated with the chronic cellular events of tissue remodelling that occur in the glaucomatous optic nerve head.

Fluid transport by human nonpigmented ciliary epithelial layers in culture: a homeostatic role for aquaporin-1.

We report for the first time that cultured nonpigmented human ciliary epithelial (NPE) cell layers transport fluid. Cells were grown to confluence on permeable membrane inserts, and fluid transport across the resulting cell layers was determined by volume clamp at 37 degrees C. These cell layers translocated fluid from the apical to the basal side at a steady rate of 3.6 microl x h(-1) x cm(-2) (n = 4) for 8 h. This fluid movement was independent of hydrostatic pressure and was completely inhibited by 1 mM ouabain, suggesting it arose from fluid transport. Mercuric chloride, a nonspecific but potent blocker of Hg(2+)-sensitive aquaporins, and aquaporin-1 antisense oligonucleotides both partially inhibited fluid transport across the cell layers, which suggests that water channels have a role in NPE cell homeostasis. In addition, these results suggest that of the two ciliary epithelial layers in tandem, the NPE layer by itself can transport fluid. This cultured layer, therefore, constitutes an interesting model that may be useful for physiological and pharmacological characterization of ciliary epithelial fluid secretion.

TNF-alpha and TNF-alpha receptor-1 in the retina of normal and glaucomatous eyes.

PURPOSE: To determine the expression and localization of tumor necrosis factor (TNF)-alpha and TNF-alpha receptor-1 in the retina of normal and glaucomatous eyes. METHODS: Using immunohistochemistry and in situ hybridization, retinal expression and localization of TNF-alpha and TNF-alpha receptor-1 were studied in retina sections from 20 eyes of donors with glaucoma, and 20 eyes of age-matched normal donors. RESULTS: According to immunohistochemistry, the intensity of the immunostaining and the number of labeled cells for TNF-alpha or its receptor were greater in retina sections of glaucomatous eyes than in control eyes of age-matched normal donors. In situ hybridization showed that mRNA signals for TNF-alpha or TNF-alpha receptor-1 were similarly more intense in glaucomatous eyes than in age-matched control eyes. Both protein and mRNA of TNF-alpha or TNF-alpha receptor-1 were predominantly localized to the inner retinal layers. Double-immunofluorescence labeling demonstrated that retinal immunostaining for TNF-alpha was predominantly positive in the glial cells, whereas immunostaining for TNF-alpha receptor-1 was mainly positive in the retinal ganglion cells. CONCLUSIONS: Upregulation of TNF-alpha and its receptor-1 in glaucomatous retina suggest that TNF-alpha-mediated cell death is involved in the neurodegeneration process of glaucoma.

Clinical factors associated with progression of glaucomatous optic disc damage in treated patients.

BACKGROUND: Reducing intraocular pressure (IOP) in glaucomatous eyes does not always prevent disease progression. OBJECTIVE: To determine the clinical factors associated with progressive optic disc damage in glaucomatous eyes receiving treatment to reduce IOP. METHODS: Baseline and follow-up optic disc photographs as well as demographic and clinical data were retrospectively studied in 186 eyes of 93 patients with primary open-angle glaucoma, and in 138 eyes of 69 patients with normal-pressure glaucoma. The patients with primary open-angle glaucoma were included in the study only if their treated IOPs during a follow-up period of 5 years were less than 21 mm Hg. The patients with normal-pressure glaucoma were included only if their IOPs were reduced by at least 20% during the follow-up period. The association of progressive optic disc damage with patient- and eye-specific characteristics was examined using multivariate analysis. RESULTS: During the 5-year study period, 141 (43.5%) of the 324 eyes exhibited progressive optic disc damage defined by at least a 5% decrease in the neural rim area-to-disc area ratio. Using multivariate analysis, the following were found to be strongly associated with progressive neural rim damage: a baseline smaller neural rim area-disc area ratio (P<.001); a baseline larger zone beta area-disc area ratio (P =.04); a baseline larger parapapillary atrophy length-disc circumference ratio (P =.05); a diagnosis of normal-pressure glaucoma (P =.01); and combined medical and surgical treatment prior to the study period (P =.01). CONCLUSIONS: Clinical factors other than IOP may be important indicators of subsequent progression of glaucomatous optic disc damage. Our findings suggest that eyes with advanced glaucomatous optic disc damage and normal-pressure glaucoma are more likely to progress despite receiving treatment to reduce IOP.

Serum autoantibody against glutathione S-transferase in patients with glaucoma.

PURPOSE: To identify retinal proteins that are the targets of serum autoantibodies in patients with glaucoma. METHODS: To identify retinal antigens that are recognized by the sera of patients with glaucoma, immunoreactive bands were separated, by using two-dimensional gel electrophoresis of the bovine retinal soluble fraction. A 29-kDa band was then selected for further analysis. Tryptic peptides of the 29-kDa band were analyzed using electrospray mass spectrometry to identify the protein. After protein identification, immunoreactivity against this newly identified protein was studied by Western blot analysis using sera from 65 patients with glaucoma (25 with primary open-angle glaucoma [POAG]; 40 with normal-pressure glaucoma [NPG]) and 25 age-matched healthy subjects. In addition, serum antibody titers were compared in these groups, by using a specific enzyme-linked immunosorbent assay (ELISA). RESULTS: The 29-kDa band was identified as glutathione S-transferase (GST). Western blot analysis revealed that serum antibodies against GST antigen were recognized in 34 (52%) of 65 patients with glaucoma (22 of NPG and 12 of POAG) and 5 (20%) of 25 age-matched control subjects (chi(2) test, P < 0.05). By ELISA, it was also found that patients with glaucoma had higher titers of anti-GST antibody, compared with the control group (Mann-Whitney test; NPG versus control, P = 0.013; POAG versus control, P = 0.0006). CONCLUSIONS: These findings indicate that GST is one of the retinal antigens targeted by the serum antibodies detected in some patients with glaucoma.

In vitro evaluation of reactive astrocyte migration, a component of tissue remodeling in glaucomatous optic nerve head.

In order to improve understanding of remodeling events in the glaucomatous optic nerve head, the migration of optic nerve head astrocytes was studied in vitro. Since elevated intraocular pressure is an important stress factor identified in glaucomatous eyes, optic nerve head astrocytes were incubated under physical stress created by elevated hydrostatic pressure. In addition, they were incubated in the presence of a chemical stimulus, lipolysaccharide (LPS). Migration of reactivated astrocytes in the presence of these stressors was examined using chambers in which cell migration through extracellular matrix-coated pores is only possible following proteolytic digestion of the matrix. We observed that the migratory ability of optic nerve head astrocytes was approximately 4-6 times greater following exposure to elevated hydrostatic pressure or LPS for up to 48 h. Phosphoinositide 3-kinase, protein kinase C, and tyrosine kinase were found to be involved in the signal transduction for activated migration of optic nerve head astrocytes in response to elevated hydrostatic pressure or LPS. In addition, we observed that the stress-induced migration of optic nerve head astrocytes, which is accompanied by proteolytic degradation, resulted in the formation of culture cavities containing mucopolysaccharides. These in vitro findings provide a clearer understanding of the pathophysiologic mechanisms of characteristic tissue remodeling events that occur, in vivo, in the glaucomatous optic nerve head.

In situ detection of apoptosis in normal pressure glaucoma. a preliminary examination.

Retinal ganglion cell (RGC) neurons are believed to die via apoptosis in human primary and secondary open-angle glaucoma. Previous studies have relied solely on the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate [UTP]-biotin nick end-labeling) method of detecting DNA fragmentation to identify apoptotic nuclei. However, it is now clear that the TUNEL method cannot distinguish between the single- and double-strand DNA breaks that can be a feature of both apoptosis and necrosis. We have developed a double fluorescent labeling method that simultaneously combines in situ end-labeling (ISEL) to detect DNA fragmentation followed by staining with a cyanine dye, YOYO-1, to visualize apoptotic chromatin condensation. This allows for the unequivocal identification of an apoptotic nucleus. Our preliminary data obtained from one case of normal pressure glaucoma suggests that RGC neurons may die via apoptosis when intraocular pressure is not elevated.

T cell subsets and sIL-2R/IL-2 levels in patients with glaucoma.

PURPOSE: We hypothesize that cellular immunity may have a previously unrecognized role in glaucomatous optic neuropathy. The purpose of this study is to analyze subsets of T cells and the levels of cytokine IL-2 and the soluble IL-2 receptor in peripheral blood from patients with normal pressure glaucoma (NPG) or primary open angle glaucoma (POAG) in comparison to age-matched control subjects. METHODS: In this study, 38 patients (20 NPG; 18 POAG) and 19 controls were included. sIL-2R and IL-2 were assayed by ELISA. T cell subsets were analyzed by flow cytometry and lymphocyte proliferation was used to measure the reactive ability of T cells to phytohemagglutinin (PHA). RESULTS: The frequency of CD8(+)HLA-DR(+) lymphocytes were increased in patients with NPG (P = 0.008), and CD3(+)CD8(+) lymphocytes increased in both NPG (P = 0.03) and POAG patients (P = 0.0004). CD5(+) lymphocytes were higher only in POAG patients (P = 0.0012). In comparison to controls, the ratio of CD4(+)/CD8(+) lymphocytes was similar in both groups. The mean concentrations of sIL-2R in NPG (P = 0.011) and POAG (P = 0.0023) patients were higher than that found in control subjects although IL-2 concentrations were similar in these groups. In addition, the reactive ability of T lymphocytes to the non-specific reagent (PHA) was reduced significantly in NPG (P = 0.02) and POAG patients (P=0.04). CONCLUSION: The alterations of the cellular immune system in patients with glaucoma support our hypothesis that the immune system may play an important role in the initiation and/or sustainment of glaucomatous optic neuropathy in some patients.

Induction of HLA-DR expression in human lamina cribrosa astrocytes by cytokines and simulated ischemia.

PURPOSE: Recent evidence strongly suggests that activated immunity occurs during the neurodegenerative process of glaucomatous optic neuropathy. Although activation of lamina cribrosa astrocytes has been identified in glaucomatous optic nerve head, their role on the activated immune responses seen in glaucoma patients is unknown. Here, the authors aimed to study the potential role of lamina cribrosa astrocytes as a component of activated immune responses seen in glaucoma patients. METHODS: Expression of HLA-DR in optic nerve head astrocytes was studied using immunohistochemistry in postmortem eyes of patients with glaucoma and normal donors. Serum cytokine levels of patients with glaucoma and control subjects were measured using enzyme-linked, immunosorbent assay. In addition, in vitro experiments were performed using astrocyte cultures derived from human optic nerve head or fetal human brain. The cultured astrocytes were incubated under selected stress conditions such as exposure to cytokines, IFN-gamma and IL-10, or simulated ischemia for up to 48 hours. The expression of HLA-DR was studied in these cells using flow cytometry and immunocytochemistry. RESULTS: Immunohistochemistry demonstrated an upregulation of the HLA-DR expression in the optic nerve head astrocytes in glaucoma. In addition, serum levels of IL-10 was higher in the patients with normal pressure glaucoma compared to age-matched control subjects (P: = 0.001). Regarding in vitro experiments, unlike brain astrocytes, the percentage of cells expressing HLA-DR was approximately 3 times higher in the cultures of optic nerve head astrocytes exposed to simulated ischemia compared to cultures incubated under normal conditions (P: = 0.09). Incubation with IFN-gamma induced HLA-DR expression in brain and lamina cribrosa astrocytes, up to 25-fold, (P < 0.001) either in the absence or presence of simulated ischemia. Induction of HLA-DR expression by IL-10 was approximately 6 times higher in lamina cribrosa astrocytes incubated under simulated ischemia compared to that incubated under normal condition (P: = 0.004) and was not prominent in brain astrocytes. CONCLUSIONS: These findings suggest that optic nerve head astrocytes function as antigen-presenting cells and that their immunogenic capacity is more sensitive to ischemia than brain astrocytes. Taken together, these findings provide novel evidence that regulation of immunogenic capacity of optic nerve head astrocytes by cytokines or ischemic stress may have a role during the neurodegeneration process in patients with glaucoma.

Serum autoantibodies to heat shock proteins in glaucoma patients from Japan and the United States.

OBJECTIVE: To study serum titers of antibodies against heat shock proteins in glaucoma patients from two different ethnic populations and to examine the relationship between serum antibody titers and glaucomatous damage. STUDY DESIGN: Comparative, cross-sectional study. PARTICIPANTS: Twenty-seven age-matched patients with primary open-angle glaucoma, 28 patients with normal pressure glaucoma, and a control group of 26 healthy subjects from Japan; and 21 age-matched patients with primary open-angle glaucoma, 40 patients with normal pressure glaucoma, and a control group of 20 healthy subjects from the United States. MAIN OUTCOME MEASURES: Measurement of serum antibody titers and examination of optic disc and visual field parameters. METHODS: Serum immunoreactivity against heat shock proteins, including hsp27, alphaB-crystallin, and human and bacterial hsp60, was studied by enzyme-linked immunosorbent assay (ELISA). The relationship of serum antibody titers to glaucoma parameters, including mean deviation, neural rim area-to-disc area ratio, and peripapillary atrophy area-to-disc area ratio was examined. RESULTS: The serum ELISA titers of antibodies, including hsp27, alphaB-crystallin, human hsp60, and bacterial hsp60 antibodies, were higher in glaucoma patients compared with control subjects in both the Japanese and American groups (P: < 0.05). There was no association between the serum antibody titers and optic disc parameters in either group from Japan or the United States (P: > 0.05). CONCLUSIONS: These findings suggest that increased titers of circulating antibodies against heat shock proteins occur in both Japanese and American patients with glaucoma. The lack of a relationship between the level of serum antibody titers and the degree of glaucomatous damage in either ethnic group suggests that increased antibodies to heat shock proteins do not occur as an epiphenomenon of the glaucomatous neurodegeneration process.

Is there a role for the immune system in glaucomatous optic neuropathy?

Glaucoma and immunity are not traditionally perceived as being causally related. Recently, however, compelling observations have provided insight into a potential role for the immune system in the development of glaucomatous optic neuropathy. In this article, it is proposed that the role of the immune system in glaucoma is two-fold. In some patients, there is evidence that an autoimmune mechanism may be responsible for eliciting damage to the optic nerve, resulting in glaucomatous injury. Autoimmune damage to the optic nerve may occur directly by autoantibodies, or indirectly by way of a "mimicked" autoimmune response to a sensitizing antigen which, in turn, injuries retinal ganglion cells. We suggest that autoimmune-mediated glaucoma injury occurs most often, but not exclusively, in patients in whom the intraocular pressure has never been found to be elevated. A second role of the immune system in glaucoma is likely one of surveillance, in which signal pathways of the immune system regulate cell death in response to conditions that stress retinal neurons in glaucoma. These might include mechanical stress from high intraocular pressure, ischemia, excessive excitatory amino acids, or toxic products resulting from excessive nitric oxide synthase production in either neurons or glial fibers that surround the optic nerve as it exits the eye. In these cases, we propose that the immune system acts as an "arbiter" to help determine whether a neuronal cell will ultimately survive, or succumb to, those stressors that are perceived as injurious. It is conceivable that such surveillance and cell death regulation by the immune system is important in determining the fate of retinal neurons in both the more common "high-pressure" forms of glaucoma, such as primary open-angle glaucoma, and in cases in which the intraocular pressure appears within normal range.

Concordance of parapapillary chorioretinal atrophy in ocular hypertension with visual field defects that accompany glaucoma development.

OBJECTIVE: To determine whether the extent and location of progressive parapapillary chorioretinal atrophy noted in some patients with ocular hypertension are correlated with the extent and location of visual field defects that occur with progression to glaucoma. STUDY DESIGN: Retrospective cohort study. PARTICIPANTS: Thirty patients with ocular hypertension who had progressive changes of parapapillary atrophy develop before clinically detectable optic disc or visual field damage. MAIN OUTCOME MEASURES: Assessment of changes in the parapapillary atrophy and visual field parameters. METHODS: Baseline and follow-up optic disc photographs and visual field test results were retrospectively analyzed. The relationship between the extent of parapapillary atrophy observed during the ocular hypertension period and initial visual field abnormalities detected after glaucoma development, as well as their spatial relationship, was statistically analyzed. RESULTS: The extent of progressive changes of the parapapillary atrophy detected during the ocular hypertension period was correlated with the extent of changes in the visual field parameters, including corrected pattern standard deviation and mean deviation measured after glaucoma development (Mantel-Haenszel chi-square test, P = 0.026, P = 0.037, respectively). In addition, the visual field abnormalities occurred in the corresponding quadrants of the progressive parapapillary atrophy. Analysis of the spatial relationship revealed that the location of progressive changes of the parapapillary atrophy was concordant with the location of visual field abnormalities in 78% of the quadrants (94 of 120 quadrants) (chi-square test, P = 0.001). CONCLUSIONS: The extent and location of visual field abnormalities that develop in ocular hypertensive eyes with progression to glaucoma exhibit a concordance with the extent and location of progressive parapapillary atrophy noted in the ocular hypertension period. This suggests the importance of detailed examination of the parapapillary area in ocular hypertensive eyes.

Matrix metalloproteinases and tumor necrosis factor alpha in glaucomatous optic nerve head.

OBJECTIVE: To study expression and location of matrix metalloproteinases (MMPs) and tumor necrosis factor alpha (TNF-alpha) in glaucomatous optic nerve heads, which are known to be secreted in response to a variety of neuronal injury. METHODS: Four postmortem eyes from patients with primary open-angle glaucoma, 7 eyes from patients with normal-pressure glaucoma, and 4 eyes from age-matched normal donors were studied by immunohistochemistry. The sections of the optic nerve heads were examined after immunostaining with antibodies to MMPs (MMP-1, MMP-2, and MMP-3), TNF-alpha, or TNF-alpha receptor 1. RESULTS: The intensity of the immunostaining and the number of stained cells for MMPs, TNF-alpha, or TNF-alpha receptor 1 were greater in the glaucomatous optic nerve heads, particularly in eyes with normal-pressure glaucoma compared with age-matched controls. Positive immunostaining was observed in all regions of the glaucomatous optic nerve heads, but most prominently in the postlaminar region. Immunostaining was observed mainly in glial cells and their processes around the axons and blood vessels and in pial septae. CONCLUSION: There is increased immunostaining for MMPs, TNF-alpha and TNF-alpha receptor 1 in the glaucomatous optic nerve head, which suggests increased expression of these proteins in glaucoma and thereby implies a role in the tissue remodeling and degenerative changes seen in glaucomatous optic nerve heads. CLINICAL RELEVANCE: The MMPs and TNF-alpha may be components of astroglial activation that occurs in glaucomatous optic nerve heads. The biological alterations in the expression of these proteins may play a role in the progression of glaucomatous optic neuropathy.

The mechanisms of hsp27 antibody-mediated apoptosis in retinal neuronal cells.

Although elevated titers of serum antibodies to hsp27 accompany human diseases such as cancer and glaucoma, evidence of their pathogenic effects is lacking. Here we present novel evidence that exogenously applied hsp27 antibody enters neuronal cells in human retina by an endocytic mechanism. Subsequent to internalization, hsp27 antibody facilitates apoptotic cell death as characterized by morphological assessment, DNA fragmentation, and the activation of cysteine aspartic acid proteases. In addition, we demonstrate that after internalization, hsp27 antibody is detected in discrete cytoplasmic and nuclear structures and colocalizes to actin cytoskeleton. Hsp27 antibody binding to actin results in depolymerization and proteolytic cleavage of actin in a dose-dependent manner. These results suggest that exogenous hsp27 antibody may induce neuronal apoptosis by inactivating or attenuating the ability of native hsp27 to stabilize actin cytoskeleton, thereby providing a novel mechanism by which autoantibodies to hsp27 may impair cell survival in selective human diseases.

Immunostaining of heat shock proteins in the retina and optic nerve head of normal and glaucomatous eyes.

PURPOSE: To examine immunostaining of 60-kd and 27-kd heat shock proteins (HSP 60 and HSP 27), which are known to increase cell survival in response to stress, in glaucomatous retina and optic nerve head. METHODS: Six postmortem eyes from patients with primary open-angle glaucoma, 6 eyes from patients with normal-pressure glaucoma, and 6 eyes from age-matched normal subjects were studied by immunohistochemistry. The sections of the retina and optic nerve head were examined after immunostaining with antibodies to HSP 60 and HSP 27. RESULTS: The intensity of the immunostaining and the number of labeled cells for heat shock proteins (HSPs) were greater in retina sections from glaucomatous eyes than in sections from normal eyes from age-matched donors. Retinal immunostaining of HSP 60 was prominent in the retinal ganglion cells and photoreceptors, whereas immunostaining of HSP 27 was prominent in the nerve fiber layer and ganglion cells as well as in the retinal vessels. In addition, retinal immunostaining of these HSPs exhibited regional and cellular differences. Optic nerve heads of glaucomatous eyes exhibited increased immunostaining of HSP 27, but not HSP 60, which was mostly associated with astroglial cells in the lamina cribrosa. CONCLUSION: The increased immunostaining of HSP 60 and HSP 27 in the glaucomatous eyes may reflect a role of these proteins as a cellular defense mechanism in response to stress or injury in glaucoma. CLINICAL RELEVANCE: These findings suggest that immunoregulation is an important component of glaucomatous optic neuropathy.

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.