Antibodies to lens epithelium-derived growth factor (LEDGF) kill epithelial cells of whole lenses in organ culture.
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Animals↗
Biomedical subjects
L T Chylack
Publications and source records attributed to L T Chylack.
Lens autofluorescence and light scatter in relation to the lens opacities classification system, LOCS III.
PURPOSE: To compare values of the human lens autofluorescence and back light scatter measurements with the improved Lens Opacities Classification System, LOCS III. METHODS: We measured autofluorescence and back light scatter of the lens from 122 smoking males aged 57 to 76 years who participated in a cancer prevention study. The retroillumination and slit-lamp photographs of the lenses were graded according to LOCS III by the Center for Ophthalmic Research in Boston. Lens fluorometry was carried out with a previously described technique using blue-green (495 nm/520 nm) autofluorescence range. Interzeag Lens Opacity Meter 701 was used for light scatter measurements. RESULTS: LOCS III nuclear opalescence and color grades were statistically significantly correlated with lens autofluorescence as well as with light scatter values. The lens transmission index of autofluorescence measurements showed the highest correlation with the nuclear color (r = -0.71; p < 0.0001) and the light scatter value with nuclear opalescence (r = 0.64; p < 0.0001). There was no correlation between autofluorescence measurements and LOCS III grades of cortical or posterior subcapsular cataract. A weak relation could be found between the grades of cortical cataract and light scatter values. CONCLUSIONS: The lens fluorometry provides a practical clinical technique to evaluate the yellow coloration and opalescence of the human lens nucleus. It may be a useful additional tool together with a subjective grading system in the follow-up of optical changes occurring in the nuclear region of the lens.
Aged↗
Antibodies to a microbial peptide sharing sequence homology with betaA3-crystallin damage lens epithelial cells in vitro and in vivo.
Circulating auto-antibodies (Abs) against lens antigens (Ags) are highly prevalent in patients with cataract, but their origin and pathogenic significance are unknown. We hypothesized that Abs raised after exposure to infectious microbes could cross-react with lens Ags. To test this hypothesis, we generated a monoclonal Ab to human betaA3-crystallin. Epitope analysis indicated that the ETQAE sequence in the N-terminus region of betaA3-crystallin was critical for mounting a humoral response. Similar sequences were found in three microbial Ags. Mice injected with a microbial oligopeptide containing ETQAE emulsified with complete Freund's adjuvant (CFA) raised Abs which cross-reacted with betaA3-crystallin and developed lens epithelial cell (LEC) damage in vitro. We also genetically engineered an betaA3-crystallin-expressing E. coli. Mice immunized with the recombinant E. coli developed LEC damage. These results support the hypothesis that exposure to microbes having Ags homologous to self Ags can trigger a humoral immune response that leads to LEC damage in mice.
Amino Acid Sequence↗
Lens epithelium-derived growth factor: increased resistance to thermal and oxidative stresses.
PURPOSE: To investigate the role of lens epithelium- derived growth factor (LEDGF) in lens epithelial cells subjected to heat or oxidative stress and to determine the localization of LEDGF in lens epithelial cells at different temperatures. METHODS: Mouse lens epithelial cells overexpressing a fusion protein between a green fluorescent protein (GFP) and LEDGF (GFP-LEDGF) were cultured for up to 7 days at various temperatures or for 24 hours in the presence of hydrogen peroxide. Translocation of GFP-LEDGF in the lens epithelial cells was monitored with a fluorescence microscope. Cell survival was determined with the trypan blue dye exclusion test. Expression of heat shock proteins (Hsps) was studied with protein blot analyses using antibody probes. RESULTS: LEDGF was found in the cytosol of lens epithelial cells at 4 degrees C, 15 degrees C, and 28 degrees C, and in the nucleus and nucleolus at 37 degrees C. At 41 C, it was apparent at higher levels in the cytosol, nucleus, and nucleolus. Lens epithelial cells overexpressing LEDGF manifested resistance to thermal and oxidative stress. Levels of Hsp 27, and alphaB-crystallin were elevated in these cells. Depriving lens epithelial cells of LEDGF with anti-LEDGF antibodies reduced nuclear localization of LEDGF and induced cell death. CONCLUSIONS: The overexpression of LEDGF in lens epithelial cells conferred resistance to thermal and oxidative stress. The mechanism of this resistance appears to involve the increased expression of Hsp 27 and alphaB-crystallin.
Animals↗
Risk factors for nuclear opalescence in a longitudinal study. LSC Group. Longitudinal Study of Cataract.
This study evaluated risk factors for increases in nuclear opacification of the lens in the Longitudinal Study of Cataract (1989-1993; Boston, Massachusetts), which included 764 participants. Baseline data on demographic, medical, and other risk factors were available from an earlier case-control study; follow-up visits were completed yearly over a 4-year period. The lens photographs taken at baseline and at each follow-up visit were graded using the Lens Opacities Classification System III protocol. Analyses evaluated which risk factors collected at baseline were related to increased nuclear opacification at follow-up. The MULCOX2 method, an extension of Cox regression for nested event-time data, was used to estimate the effects of the risk factors. This method accounted for the correlation between fellow eyes. Results showed that the risk of nuclear opacification increased with each year of age (relative risk (RR) = 1.07), white race (RR = 2.94), lower education (RR = 1.50), use of gout medications (RR = 2.32), current smoking (RR = 1.58), family history of cataract (RR = 1.39), and preexisting posterior subcapsular opacities (RR = 6.67). An association with early use of eyeglasses was also suggested (RR = 1.37). In conclusion, nuclear opacification was related to demographic and other variables, including potentially modifiable factors such as current smoking and use of gout medications. Most risk factors identified by this longitudinal study confirm those found by the original case-control study. The increased risk of nuclear opacities in whites appears to be a new finding.
Adult↗
Antioxidant vitamins and nuclear opacities: the longitudinal study of cataract.
OBJECTIVE: The association of antioxidant nutrients and risk of nuclear opacification was evaluated in the Longitudinal Study of Cataract. DESIGN: Nutritional data were collected at baseline on the 764 participants, which included assessment of dietary intake, use of vitamin supplements, and plasma levels of vitamin E. Ophthalmologic and other data were collected at baseline and at yearly follow-up visits, including lens photographs, which were graded using the Lens Opacities Classification System III protocol. MAIN OUTCOME MEASURES: Analyses examined whether the nutritional factors at baseline were related to increases in nuclear opacification at follow-up. The MULCOX2 approach, an extension of the Cox regression model, was used. Results are presented as relative risks (RRs) and 95% confidence intervals. INTERVENTION: Intervention was not applicable. RESULTS: The risk of nuclear opacification at follow-up was decreased in regular users of multivitamin supplements (RR = 0.69; 0.48-0.99), vitamin E supplements (RR = 0.43; 0.19-0.99), and in persons with higher plasma levels of vitamin E (RR = 0.58; 0.36-0.94). CONCLUSIONS: In regular users of multivitamin supplements, the risk of nuclear opacification was reduced by one third; in regular users of vitamin E supplements and persons with higher plasma levels of vitamin E, the risk was reduced by approximately half. These results are similar to those obtained in our earlier case-control study. Because these data are based on observational studies only, the results are suggestive but inconclusive. The possible effect of nutritional supplements on the lens requires confirmation by ongoing clinical trials.
Adult↗
Fluorescence and immunochemical studies of advanced glycation-related lens pigments.
PURPOSE: To establish whether advanced glycation is the major mechanism for yellowing of lens proteins. METHODS: Synchronous fluorescence (SF) and immunochemical assays were used to study glycation in vitro and in vivo. In the in vitro study, advanced glycation end products (AGEs) were prepared and used as antigens to induce antibodies to AGEs. The in vitro AGEs and classified nuclear cataracts were analyzed by SF and immunochemical assays. RESULTS: In vitro AGEs generated from various glycating agents and carrier proteins displayed strong SF above 350 nm; the spectra were well resolved with major bands at 380 nm and 420 nm. Samples from human lenses manifested a band at 395 nm in addition to the two bands shown by in vitro AGEs. SF intensity is greater for the water-insoluble (WI) than water-soluble (WS) fraction, but both increased with increasing nuclear color. The immunoreactivity data also showed that the WI fraction contained more AGEs than the WS fraction and that the amount of AGEs increased with increasing nuclear color. CONCLUSIONS: Fluorescence and immunoassays indicated that pigmented AGEs contributed to yellowing of the crystalline lens nucleus.
Animals↗
Cataracts and inhaled corticosteroids.
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Administration, Inhalation↗
Expression of extracellular calcium-sensing receptor by human lens epithelial cells.
The extracellular calcium-sensing receptor (CaR) confers the capacity to sense small changes in the extracellular Ca2+ concentration (Ca2+o) not only upon cells involved in maintaining systemic Ca2+ homeostasis but also upon those not directly involved in this process. Since high Ca2+o is known to affect various physiological processes in lens epithelium both in health and in disease states (e.g., the formation of cataracts in hypocalcemic states), we investigated the expression and function of the CaR in these cells. By RT-PCR and immunocytochemistry the CaR is expressed in human lens epithelial cells in culture. In addition, the open state probability of a Ca(2+)-activated potassium (K+) channel with a conductance of 82 +/- 3 pS is significantly increased by elevating Ca2+o to 3.0 mM or by application of 100 microM neomycin, both effective CaR agonists. Therefore, our data suggest that human lens-epithelial cells express the CaR, which may be functionally linked to Ca(2+)-activated K+ channels and, perhaps, to other ion channels involved in ionic homeostasis in the lens.
Base Sequence↗
Anti-beta-crystallin antibodies (mouse) or sera from humans with age-related cataract are cytotoxic for lens epithelial cells in culture.
Circulating autoantibodies against lens antigens are prevalent in patients with age-related cataract (ARC), but their pathogenic significance is unknown. We hypothesized that these autoantibodies are cytotoxic for lens epithelial cells (LECs). To test this hypothesis. We incubated LECs with mouse polyclonal or monoclonal antibodies against beta-crystallin (anti-beta) in the presence or absence of guinea pig complement. We found that anti-beta in the presence of the complement bound to and killed mouse LECs (MLECs) and human LECs (HLECs). Sera obtained from patients with ARC also were cytotoxic to both HLECs and MLECs in culture. Heat-inactivated human sera were not cytotoxic to LECs in the absence of the complement, but were cytotoxic to both HLECs and MLECs in the presence of additional complement. These results support the hypothesis that autoantibodies against lens antigens are cytotoxic to LECs, and that cell death may involve complement-mediated pathways.
Animals↗
Oral administration of lens homogenate suppresses antibody production in mice injected with beta-crystallin emulsified in CFA.
Auto-antibodies (Abs) against lens antigens (Ags) are present in most patients with age-related cataract, and with complement they kill lens epithelial cells (LECs) in vitro. We studied, in an animal model, whether cytotoxic Abs against lens Ags can be suppressed by oral administration of the Ags. Mice were fed calf lens homogenate, 4 mg/mouse, every 4 days for 4-5 weeks, or bovine serum albumin (BSA) before and after immunisation with beta-crystallins emulsified in complete Freund's adjuvant (CFA). Sera from these animals were analysed for Abs to beta-crystallins by enzyme-linked immunosorbent assay (ELISA) and protein blot analysis. In addition, we studied the proliferative response of T-lymphocytes to beta-crystallins. The titer of anti-beta-crystallin Abs in the control animals fed BSA gradually increased to 1.5 x 10(-6) by the 5th week after the first injection. In contrast, the titer of anti-beta-crystallin Abs in animals fed calf lens homogenate was reduced to 30-70% of the control. Feeding lens homogenate prior to or concomitant with beta-crystallins immunization, was more effective than feeding after immunization (65% suppression vs. 30% suppression, respectively). Also the proliferative response of T-lymphocytes to beta-crystallins in mice fed homogenate was suppressed significantly. Thus, oral administration of lens homogenate is a specific and nontoxic method of suppressing anti-beta-crystallin Ab production in mice. We are exploring the therapeutic value of oral administration of lens proteins in age-related cataract.
Animals↗
New non-contact specular microscope for lens epithelial cell visualization.
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Adult↗
Incidence and progression of cortical and posterior subcapsular opacities: the Longitudinal Study of Cataract. The LSC Group.
OBJECTIVE: The purpose of the study is to estimate incidence and progression rates of cortical and posterior subcapsular (PSC) opacities in the Longitudinal Study of Cataract (LSC). DESIGN: An epidemiologic study of the natural history of lens opacities in a clinic-based population. PARTICIPANTS: The LSC was based on 764 participants in an earlier case-control study of lens opacities. MAIN OUTCOME MEASURES: Baseline data, collected until 1988, included color slit and retroillumination photographs. The same data were collected at follow-up visits from 1989 to 1993. The Lens Opacities Classification System III (LOCS III) was used to assess lens changes between baseline and follow-up photographs. The product-limit method was used to estimate the incidence and progression rates. RESULTS: After 5 years of follow-up, the incidence rates for developing cortical and PSC opacities were 7.7% and 4.3%, respectively. The progression rate of pre-existing cortical opacities was 16.2% after 5 years, and was twice as high as the incidence rate. The progression of pre-existing PSC opacities was much higher, and reached 55.1% after 5 years of follow-up. The incidence of newly developed cortical or PSC opacities increased with age. The incidence of PSC opacities also increased when coexisting opacities were present at baseline. CONCLUSIONS: After 5 years, 1 in every 13 patients developed new cortical opacities, and 1 in 24 developed new PSC opacities. The 5-year progression rates for cortical and PSC opacities were much higher than the incidence rates. These results can be used to estimate the rate of cortical and PSC changes in similar populations.
Aged↗
Incidence and progression of nuclear opacities in the Longitudinal Study of Cataract.
PURPOSE: To estimate incidence and progression rates of nuclear opacities in the Longitudinal Study of Cataract, an epidemiologic study of the natural history of all types of lens opacities. METHODS: The Lens Opacities Classification System III was used to assess longitudinal changes between baseline and follow-up lens photographs for the 764 Longitudinal Study of Cataract participants. Baseline data, collected until December 1988 as part of a case-control study, included color slit, retroillumination, and Scheimpflug photographs. The same data were collected by the longitudinal Study of Cataract at four subsequent visits at yearly intervals. RESULTS: Among patients free of nuclear opacities at baseline, the incidence of new opacities was 6% after 2 years and 8% after 5 years of follow-up. The progression of pre-existing nuclear opacities was much higher. After 2 years, nuclear opacities had progressed in more than one third of the patients with pre-existing opacities; after 5 years, almost half had progressed. Older age was significantly related to higher incidence of new nuclear opacities, but not to progression of pre-existing opacities. Patients with other opacity types had higher nuclear incidence and progression rates. CONCLUSIONS: In this clinic-based, older-patient population, new nuclear opacities developed in less than one tenth of the patients after 5 years of follow-up. In contrast, almost one half of the patients with pre-existing opacities had worsened after 5 years. These estimated rates can be used to plan intervention or other studies of nuclear changes in similar populations.
Aged↗
Biochemical factors in the lens opacities. Case-control study. The Lens Opacities Case-Control Study Group.
OBJECTIVE: To evaluate associations with biochemical indicators of nutritional and other risk factors in the Lens Opacities Case-Control Study. DESIGN: Case-control study. SETTING AND PARTICIPANTS: The Lens Opacities Case-Control Study determined risk factors for cortical, nuclear, and posterior subcapsular opacities among 1380 participants aged 40 to 79 years. DATA COLLECTION: Vitamin E, selenium, and biochemistry profile determinations were performed on all patients; red blood cell enzymes and amino acids were measured in systematic samples of about 25% of the Lens Opacities Case-Control Study population. OUTCOME: Laboratory test values in cases and controls were compared and expressed as odds ratios and 95% confidence intervals. RESULTS: In polychotomous logistic regression analyses controlling for age and sex, the risk of opacities was reduced to less than one half in persons with higher levels of vitamin E (odds ratio, 0.44 for nuclear opacities), albumin-globulin ratio (odds ratio, 0.41 for mixed opacities), or iron (odds ratio, 0.43 for cortical opacities); higher uric acid levels increased risk (odds ratio, 1.74 for mixed opacities). Persons with opacities were twice as likely to have high glutathione reductase activity (with flavin adenine dinucleotide), which suggests low riboflavin status (odds ratio, 2.13). Most odds ratios for amino acids were under unity and were significantly decreased for glycine (0.36) and aspartic acid (0.31). CONCLUSIONS: Lens opacities were associated with lower levels of riboflavin, vitamin E, iron, and protein nutritional status. Higher levels of uric acid increased risk of mixed opacities. The findings for riboflavin, vitamin E, iron, and uric acid are compatible with the dietary intake and medical history results of the Lens Opacities Case-Control Study.
Adult↗
Validation of methods for the assessment of cataract progression in the Roche European-American Anticataract Trial (REACT)
The Roche European-American Anticataract Trial (REACT) will assess the effect of antioxidants on progression of cataract in humans. This report evaluates the methods used in REACT. Seventy three subjects (139 eyes) with cortical (C), posterior subcapsular (P), nuclear (N) or mixed cataract were seen twice within two weeks for eye examinations, assessments of visual function, lens photographs and CCD images. The degree of cataract and nuclear color (NC) were assessed with subjective (LOCS III) and objective (computerized, CASE 2000 CCD) methods. Repeat visit values were used to calculate intraclass correlation coefficients (r1) and 95% tolerance limits (TL). A clinically significant change (CSC) was defined as one step in LOCS III. The relative power of each method to detect cataract change and sample sizes needed to achieve statistically significant results were calculated. The r1 values for visual function tests ranged from 0.76 to 0.88; if these tests of visual function were used to detect a clinically significant change in cataract severity, sample sizes of 840 to 2707 per group would be needed. The r1 values for LOCS III were 0.88 to 0.97, and sample sizes ranged from 50 to 135 per group. The r1 values for the CCD were 0.93 to 0.98, and sample sizes ranged from 1 to 42 with poorer values relating to measurement of P. We conclude that the methods used in REACT are reproducible. The analytical algorithms in the image analysis programs did not permit differentiation between C and P opacification; therefore, P cataract is best measured with LOCS III. REACT sample sizes are adequate to detect a difference of 0.2 LOCS III units/year between the mean rates of cataract progression in two groups.
Adult↗
Comparing self-reported and physician-reported medical history.
The authors compared self-reported medical history and medication use in a cataract case-control study of 1,380 persons (1985-1989) in Boston, Massachusetts, with information from the participants' physicians. Under- and overreporting varied by condition and type of medication. A self-reported history of hypertension had the highest sensitivity (91%), and diabetes history had the highest specificity (97%). Among different medications investigated, self-reported antihypertensive medication use was the most sensitive (88%), while self-reported use of insulin was the most specific (99%). Differences between patient- and physician-reported frequencies were very small, except for arthritis (15%) and regular aspirin use (21%). Results suggest an accurate recall of medical and drug usage history in well-defined chronic conditions.
Aged↗
Propagation and immortalization of human lens epithelial cells in culture.
PURPOSE: To establish primary and immortalized cell cultures of human lens epithelial cells for a model system investigating human lens epithelial physiology and cataract. METHODS: Human lens epithelial cells in culture were grown by isolating epithelium fragments from infant human lenses from patients who underwent treatment for retinopathy of prematurity and by allowing epithelial cells to grow from explants. To immortalize cells, the cultures were infected with an adenovirus 12-SV40 virus (Ad12-SV40). RESULTS: The primary cells from infant eyes proliferated for three passages before senescence was observed. However, the immortalized cells remained proliferative and retained the morphology of the primary cells. Immunohistochemical analysis demonstrated that these immortalized cells were SV40 large T antigen-positive and ceased to produce infectious virus after a few passages. Immortalized cells passaged to population doubling levels of 76 continued to form confluent cultures within 7 days of subculture. Analysis of proteins by SDS-PAGE and immunoblotting showed that immortalized cells produce a protein with molecular weight of about 25 kD, which reacted with an antibody to beta H-crystallin. CONCLUSIONS: This report constitutes the first successful immortalization of human lens epithelial cells. Currently, two cell lines have been created (B-3 and B-4) and passaged to population doubling levels of 76 and 52, respectively. These cells may provide an important human cell line specific to in vivo human lens epithelial cell physiology and would be of interest in establishing a human model to study lens cell differentiation and the etiology of cataract. These cells may also provide a constant and reproducible source of lens epithelial cells for eye-related toxicology studies and to assay inhibitory drugs for the prevention of cataracts and posterior capsular opacification observed after cataract extraction.
Cell Division↗