Effects of iron ions, protons and X rays on human lens cell differentiation.

We have investigated molecular changes in cultured differentiating human lens epithelial cells exposed to high-energy accelerated iron-ion beams as well as to protons and X rays. In this paper, we present results on the effects of radiation on gene families that include or are related to DNA damage, cell cycle regulators, cell adhesion molecules, and cell cytoskeletal function. A limited microarray survey with a panel of cell cycle-regulated genes illustrates that irradiation with protons altered the gene expression pattern of human lens epithelial cells. A focus of our work is CDKN1A (p21(CIP1/WAF1)), a protein that we demonstrate here has a role in several pathways functionally related to LET-responsive radiation damage. We quantitatively assessed RNA and protein expression in a time course before and after single 4-Gy radiation doses and demonstrated that transcription and translation of CDKN1A are both temporally regulated after exposure. Furthermore, we show qualitative differences in the distribution of CDKN1A immunofluorescence signals after exposure to X rays, protons or iron ions, suggesting that LET effects likely play a role in the misregulation of gene function in these cells. A model of molecular and cellular events is proposed to account for precataractous changes in the human lens after exposure to low- or high-LET radiations.

[Antioxidant micronutrients and cataract. Review and comparison of the AREDS and REACT cataract studies]. / Antioxidative Mikronährstoffe und Katarakt. Ein Uberblick und ein Vergleich der Kataraktstudien "AREDS" und "REACT"

Age-related cataract remains the major cause of preventable blindness throughout the world. It has long been realized that one of the important etiological factors for this disease is oxidative and in particular photooxidative damage to the lens. Therefore, the antioxidant micronutrients, vitamins C and E and the carotenoids, in particular beta-carotene, have been discussed as factors that could reduce the risk for this disease. The present article reviews what is known about the transport of these substances to the lens, their accumulation, and their concentrations in the lens. Furthermore, the available epidemiological literature is briefly mentioned, but more emphasis has been placed on a description and discussion of major clinical intervention studies. Finally, the design and results of two of those trials using antioxidant micronutrients, the Age-Related Eye Disease Study (AREDS) and the Roche European American Cataract Trial (REACT), are compared. The AREDS trial did show a positive effect only for age-related macular degeneration but not for cataract, while the REACT trial demonstrated a small but statistically significant deceleration of cataract progression. The techniques for following the course of a cataract in the REACT study were more sensitive to subtle changes than those used in the AREDS study, and this may have been one important factor accounting for the differences. The authors' detailed comparison of these studies, however, suggests that even more important may have been the fact that in the REACT study intervention started earlier in the disease process,with higher doses of vitamins C and E and beta-carotene and consequently with larger plasma concentrations of these antioxidant micronutrients. The REACT trial results support the early complementation of a diversified diet with supplements containing vitamins C and E and beta-carotene as well as other carotenoids. The authors also believe that it is reasonable to include these micronutrients in the therapeutic armamentarium of general ophthalmological practice.

Transactivation of involucrin, a marker of differentiation in keratinocytes, by lens epithelium-derived growth factor (LEDGF).

Human involucrin (hINV), first appears in the cytosol of keratinocytes and ultimately cross-linked to membrane proteins via transglutaminase and forms a protective barrier as an insoluble envelope beneath the plasma membrane. Although the function and evolution of involucrin is known, the regulation of its gene expression is not well understood. An analysis of the hINV gene sequence, upstream of the transcription start site (-534 to +1 nt) revealed the presence of potential sites for binding of lens epithelium-derived growth factor (LEDGF); stress response element (STRE; A/TGGGGA/T) and heat shock element (HSE; nGAAn). We reported earlier that LEDGF activates stress-associated genes by binding to these elements and elevates cellular resistance to various stresses. Here, gel-shift and super-shift assays confirm the binding of LEDGF to the DNA fragments containing HSEs and STREs that are present in the involucrin gene promoter. Furthermore, hINV promoter linked to CAT reporter gene, cotransfected in human corneal simian virus 40-transformed keratinocytes (HCK), was transactivated by LEDGF significantly. In contrast, the activity of hINV promoter bearing mutations at the WT1 (containing HSE and STRE), WT2 (containing STRE) and WT3 (containing STRE) binding sites was diminished. In addition, in HCK cell over-expressing LEDGF, the levels of hINV mRNA and hINV protein are increased by four to five-fold. LEDGF is inducible to oxidants. Cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), known to stimulate production of H(2)O(2), showed higher levels of LEDGF mRNA. Furthermore, our immunohistochemical studies revealed that hINV protein is found in the cytoplasm of HCK cells over-expressing LEDGF, but not detectable in the normal HCK cells or HCK cells transfected with vector. This regulation appears to be physiologically important, as over-expression of HCK with LEDGF increases the expression of the endogenous hINV gene and may provide new insight to understand the molecular mechanism of transcriptional regulation of this gene. LEDGF may play an important role in establishing an important barrier in corneal keratinocytes by maintaining epidermal turn-over rate, and protecting HCKs against stress.

A simplified cataract grading system.

A simplified method for grading the presence and severity of different cataract types is needed for field use in assessment of the magnitude of the cataract problem. A cataract grading system was developed by a panel of experts with the objective of making available a simple system for use with a slit lamp to allow for the reliable grading of the most common forms of cataract by relatively inexperienced observers. Three levels, reflecting progressive severity, for grading of nuclear, cortical and posterior subcapsular (PSC) cataract were included in the classification; three standard photos were used for grading nuclear cataract. Field evaluation from four different sites indicated very good to fair interobserver agreement with the use of this system following minimal training of residents in ophthalmology at each site. Further testing of this system is warranted. The WHO simplified cataract grading system should allow for the obtaining of comparable data across countries based on field assessment of the most common forms of cataract.

Transcriptional regulation of the antioxidant protein 2 gene, a thiol-specific antioxidant, by lens epithelium-derived growth factor to protect cells from oxidative stress.

Antioxidant protein 2 (AOP2), a member of the newly defined family of thiol-specific antioxidant proteins, has been shown to remove H(2)O(2) and protect proteins and DNA from oxidative stress. Here we report that LEDGF is one of the regulatory factors for the AOP2 gene. We found that LEDGF bound to the heat shock element and to stress-related elements in the AOP2 promoter. It trans-activated expression of AOP2-CAT in COS-7 cells and lens epithelial cells overexpressing LEDGF. Mutations in the heat shock element and stress-related elements of the AOP2 promoter reduced LEDGF-dependent trans-activation. Lens epithelial cells showed a higher level of AOP2 mRNA in the presence of LEDGF. Cells overexpressing LEDGF exhibited a higher level of AOP2 protein, the level of which was directly related to the increase in cellular protection. Thus, LEDGF, by activating the AOP2 gene, protected and enhanced the survival of cells under oxidative stress.

Spatial and temporal dynamics of two alternatively spliced regulatory factors, lens epithelium-derived growth factor (ledgf/p75) and p52, in the nucleus.

Regulatory factors, lens epithelium-derived growth factor (LEDGF)/p75 and p52, are generated from a single LEDGF gene by alternative splicing. They have identical amino acid residues between positions 1-325, but 205 and 8 of the remaining residues are different in LEDGF and p52, respectively. LEDGF promotes growth and survival of many cell types. It has an antiapoptotic function and is a weak general transcriptional co-activator. p52 is a transcriptional activator and an essential splicing factor. We investigated the spatial and temporal dynamics of LEDGF/p75 and p52, each being tagged with a fluorescent protein, during the cell cycles of CHO-K1, MCDK, and NRK cells in culture. Both LEDGF/p75 and p52 were localized predominantly in the nucleus. LEDGF/p75 was distributed diffusely in the nucleoplasm in the G1-phase and attached to chromatin heterogeneously during the G2 and M-phases of cells. In contrast, p52 was localized in the nuclear periphery during the G1-phase and formed a speckle pattern at the S-phase. It formed a cylindrical pattern around the chromosomes during the M-phases of cells. LEDGF and p52 on sister chromatids migrated into daughter cells. Thus, LEDGF/p75 and p52 are localized in distinct nuclear compartments where they can activate transcription or splicing of pre-mRNAs.

Long-term nutrient intake and early age-related nuclear lens opacities.

OBJECTIVE: To assess the relation between usual nutrient intake and subsequently diagnosed age-related nuclear lens opacities. SUBJECTS: Four hundred seventy-eight nondiabetic women aged 53 to 73 years from the Boston, Mass, area without previously diagnosed cataracts sampled from the Nurses' Health Study cohort. METHODS: Usual nutrient intake was calculated as the average intake from 5 food frequency questionnaires that were collected during a 13- to 15-year period before the evaluation of lens opacities. The duration of vitamin supplement use was determined from 7 questionnaires collected during this same period. We defined nuclear opacities as a nuclear opalescence grade of 2.5 or higher using the Lens Opacification Classification System III. RESULTS: The prevalence of nuclear opacification was significantly lower in the highest nutrient intake quintile category relative to the lowest quintile category for vitamin C (P<.001), vitamin E (P =.02), riboflavin (P =.005), folate (P =.009), beta-carotene (P =.04), and lutein/zeaxanthin (P =.03). After adjustment for other nutrients, only vitamin C intake remained significantly associated (P =.003 for trend) with the prevalence of nuclear opacities. The prevalence of nuclear opacities was significantly lower (P<.001) in the highest vitamin C intake quintile category relative to the lowest quintile category (odds ratio, 0.31; 95% confidence interval, 0.16-0.58). There were also statistically significant trends of decreasing prevalence of nuclear opacities with increasing duration of use of vitamin C (P =.004 for trend), vitamin E (P =.03 for trend), and multivitamin (P =.04 for trend) supplements, but only duration of vitamin C supplement use remained significantly associated with nuclear opacities after mutual adjustment for use of vitamin E (P =.05 for trend) or multivitamin (P =.02 for trend) supplements. The prevalence of nuclear opacities was significantly lower (P =.004) for women who used a vitamin C supplement for 10 or more years relative to women who never used vitamin C supplements (odds ratio, 0.36; 95% confidence interval, 0.18-0.72). Plasma measures of vitamins C and E taken at the eye examination were also inversely associated with the prevalence of nuclear opacities. CONCLUSION: These results provide additional evidence that antioxidant nutrients play a role in the prevention of age-related nuclear lens opacities.

LEDGF binds to heat shock and stress-related element to activate the expression of stress-related genes.

We have investigated the mechanism by which LEDGF protects cells against environmental stress. Our earlier report showed that a low level of LEDGF was present in the nucleus of most cell types and significant elevation of LEDGF level was induced by heat and oxidative stress. The cells overexpressing LEDGF-activated expression of heat shock proteins and enhanced survival of many cell types. Here we show that LEDGF binds to heat shock element (HSE) and stress-related regulatory element (STRE) to activate the expression of stress-related genes (Hsp27 and alphaB-crystallin). Apparently, HSE and STRE are present in promoters of many stress-related genes. Elevation of many stress-related proteins (STRPs) induced by LEDGF may protect cells against environmental stress. In yeast, it has been demonstrated that a single stress can activate the expression of multiple STRPs. This is known as "cross-protection," and now similar mechanism has been found in mammalian cells and LEDGF plays a vital role in it.

Lens epithelium-derived growth factor promotes photoreceptor survival in light-damaged and RCS rats.

PURPOSE: To investigate possible protective effects of lens epithelium-derived growth factor (LEDGF) against photoreceptor death in light-damaged, Royal College of Surgeons (RCS) and P23H rhodopsin transgenic rats. METHODS: Twelve-week-old Sprague-Dawley (SD), 6-week-old RCS, and 10-day-old P23H (line 1, heterozygote) rats received an intravitreal injection of LEDGF fused with glutathione-S-transferase (GST-LEDGF). Fellow eyes received vehicle and served as control specimens. Two days after the injections, the SD rats were exposed to light of 2000 lux for 48 hours. Corneal Ganzfeld ERGs were recorded 10 days after light damage, at 10 weeks of age in RCS rats, and at 4 weeks of age in P23H rats. The eyes were then processed for histologic analysis. Heat shock protein (hsp) content in the sensory retina was analyzed quantitatively by protein immunoblot. RESULTS: In light-damaged rats, the ERG indicated retinal protection in GST-LEDGF-injected eyes, with b-wave and STR thresholds being 1.14 +/- 0.50 (mean +/- SD) and 0.60 +/- 0.26 log candela (cd)/m2 lower, respectively, than in vehicle-injected eyes (P < 0.01). The GST-LEDGF-treated eyes had maximum b-wave amplitudes that were significantly larger (P < 0.0005), had more than twice as many remaining photoreceptors, and had better organized outer segments than the control eyes. In RCS rats, the treated eyes had 2.76 +/- 0.73 and 0.83 +/- 0.09 log cd/m(2) lower thresholds for the b-wave and STR, respectively (P < 0.005), and had significantly larger maximum b-wave amplitude (P < 0.0005). GST-LEDGF-treated eyes of RCS rats also had more photoreceptors remaining (P < 0.005) and a thinner debris layer than control eyes. In P23H rats, GST-LEDGF treatment did not protect either retinal function or structure. The retinas from GST-LEDGF-treated eyes of SD and RCS rats had higher levels of hsp25 and alphaB-crystallin than vehicle-injected eyes. CONCLUSIONS: GST-LEDGF protects photoreceptor structure and function in both light-damaged and RCS rats. The increased expression of hsp25 and alphaB-crystallin may play a role in this protection. The absence of rescue in P23H raises the possibility that some forms of inherited retinal degeneration may not be amenable to treatment by intraocular injection of LEDGF.

Activation of LEDGF gene by thermal-and oxidative-stresses.

LEDGF promotes survival of many cell types against a wide range of environmental stresses, and cells responding to those stresses expressed higher levels of heat shock proteins (Hsps). LEDGF/p75 is a weak coactivator of general transcription. We speculated that a stress signal may activate expression of the LEDGF gene, resulting in elevated levels of LEDGF which may activate expression of stress-related proteins to protect cells from stresses. Lens epithelial cells (LECs) and cos7 cells were cultured under heat- or oxidative-stress. After cells were cultured for defined time, we quantified LEDGF mRNA and LEDGF protein with RT-PCR, Northern blot, and protein blot analysis. Our results showed that higher levels of LEDGF were found in both cell types under heat- and oxidative-stress than that in cells cultured at nonstress condition. Thus, one of the primary events in the stress-related protective events is the activation of LEDGF, a regulatory protein.

Heparin's roles in stabilizing, potentiating, and transporting LEDGF into the nucleus.

PURPOSE: Lens epithelium-derived growth factor (LEDGF) is a 60-kDa protein that dramatically enhances cellular survival, growth, adhesiveness, and resistance to heat and oxidative stress. Full-size recombinant LEDGF is degraded during prokaryotic preparation. Heparin's capacity to stabilize recombinant LEDGF in the face of various stresses (heat, pH, proteolysis), to potentiate its growth-enhancing properties, and to enable transport of LEDGF into the nucleus of mouse lens epithelial cells has been characterized. METHODS: LEDGF-cDNA was cloned in a pGEX-2T expression vector to produce a fusion protein, GST-LEDGF. Porcine heparin was used to stabilize GST-LEDGF. Heparin-Sepharose was used to characterize heparin-GST-LEDGF binding, and GST-LEDGF or heparin-GST-LEDGF was used to quantitate heparin's stabilization of LEDGF in the face of heat, pH, and proteolytic stresses. Fluorescein isothiocyanate-labeled GST-LEDGF and heparin-GST-LEDGF were incubated with cultured mouse lens epithelial cells (LECs). Fluorescence microscopy and immunostaining techniques were used to monitor heparin's potentiation of LEDGF's growth stimulation and heparin's role in the translocation of GST-LEDGF from the extracellular space into the cytoplasm and nucleus. RESULTS: Heparin, at concentrations as low as 7.1 mg/ml, protected GST-LEDGF from degradation and increased the yield of the full-size fusion protein in a prokaryotic system. It also protected GST-LEDGF from heat, acid-base deactivation, and proteolytic degradation with trypsin and chymotrypsin and greatly potentiated LEDGF's enhancement of mouse LEC growth in culture. It also increased nuclear uptake of exogenous GST-LEDGF and endogenous LEDGF. CONCLUSIONS: Heparin protected GST-LEDGF from degradation under various stress conditions and facilitated transport of GST-LEDGF into the nucleus.

3-Hydroxykynurenine and 3-hydroxyanthranilic acid generate hydrogen peroxide and promote alpha-crystallin cross-linking by metal ion reduction.

The kynurenine pathway catabolite 3-hydroxykynurenine (3HK) and redox-active metals such as copper and iron are implicated in cataractogenesis. Here we investigate the reaction of kynurenine pathway catabolites with copper and iron, as well as interactions with the major lenticular structural proteins, the alpha-crystallins. The o-aminophenol kynurenine catabolites 3HK and 3-hydroxyanthranilic acid (3HAA) reduced Cu(II)>Fe(III) to Cu(I) and Fe(II), respectively, whereas quinolinic acid and the nonphenolic kynurenine catabolites kynurenine and anthranilic acid did not reduce either metal. Both 3HK and 3HAA generated superoxide and hydrogen peroxide in a copper-dependent manner. In addition, 3HK and 3HAA fostered copper-dependent alpha-crystallin cross-linking. 3HK- or 3HAA-modifed alpha-crystallin showed enhanced redox activity in comparison to unmodified alpha-crystallin or ascorbate-modified alpha-crystallin. These data support the possibility that 3HK and 3HAA may be cofactors in the oxidative damage of proteins, such as alpha-crystallin, through interactions with redox-active metals and especially copper. These findings may have relevance for understanding cataractogenesis and other degenerative conditions in which the kynurenine pathway is activated.

Review: Age-related cataract: immunity and lens epithelium-derived growth factor (LEDGF).

This short review summarizes our recent work and relevant publications on autoimmunity and cataract. A complete review of this subject is beyond the scope of this paper. Age-related cataract (ARC) is the leading cause of world blindness. In spite of more than fifty years of basic and clinical research, there is no nonsurgical intervention to prevent or treat ARC, but there is a better understanding of the manifold complexities of this age-related condition. ARC is a multifactorial condition in which incidence and progress are modified by factors such as age, sex, radiation [visible, ultraviolet (UV), and X-ray], oxidation, physical trauma, diet, and medications. The lens contains at least three different cell types: central epithelial cells, dividing germinative epithelial cells, and fiber cells. The central epithelial cells covering the anterior axial part of the lens do not divide but survive throughout life. The bulk of the lens comprises anucleate fiber cells, differentiated germinative epithelial cells, which have undergone an apoptosis-like change "diffoptosis" to become elongated, crystallin-rich, organelle-deficient, cells. The epithelial cells and their active transport mechanisms maintain lens homeostasis and clarity. The survival mechanisms of the central lens epithelial cells (LECs) are unknown. In other cells, growth or survival factors, when present, enhance survival and, when absent or deficient, induce programmed cell death "apoptosis". Many developing mammalian cells produce signal proteins, or require signal proteins from other cells, to avoid apoptosis. Although much is known about the role of growth factors in the lens, less is known about how such signals are involved in the survival and death of LECs. We have hypothesized that LECs, like other mammalian cells, use signal proteins to regulate growth, survival, and apoptosis, and we have begun a search for such molecules. Furthermore, we have hypothesized that such factors, if found, may also be involved in the death of LECs, the consequent alteration of lens homeostasis and, eventually, certain types of ARC.

Lens epithelium-derived growth factor (LEDGF/p75) and p52 are derived from a single gene by alternative splicing.

A human gene that encodes lens epithelium-derived growth factor (LEDGF) was isolated, and the DNA sequence and the exon/intron organization was determined. The gene contains at least 15 exons and 14 introns and encodes LEDGF mRNA and p52 mRNA. Exons 1-15 encode LEDGF mRNA, and exons 1-9, and a part of the ninth intron encode a splice variant (p52). Sequences of the exon/intron junctions of the gene have the highly conserved GT/AG rule. Most intron/exon junctions correspond to junctions of individual protein motifs. Almost equal amounts of LEDGF and p52 are expressed in lens epithelial cells in culture. The LEDGF gene is assigned to chromosome 9p22.2, which is adjacent to the major cell malignancy locus.

Noncontact specular microscopy of human lens epithelium.

PURPOSE: To obtain in vivo specular images of human lens epithelial cells (LECs) from persons with or without age-related cataract (ARC); to identify features that describe individual aspects of these complex images; to develop feature scales to quantify the severity of each feature; and to study the association of these features with LEC count, age, Lens Opacity Classification System III (LOCS III) classifications and microscopic features of lens epithelium in ARC. METHODS: One hundred fifty-two individuals underwent ophthalmic examinations and LOCS III cataract classifications. Specular images of lenses were captured using a modified noncontact corneal specular microscope (SML-2; Konan, Hyogo, Japan). Enhanced images were graded in a masked fashion, and the presence or absence and severity of each of four features in the specular image ("columnar organization," "linear furrows," "puffy clouds," and "black holes") was graded on a four-step scale. The generalized linear model with intraclass correlation was used to ascertain the statistical significance of associations between age, sex, LOCS III grade, cell count, and feature grade. Capsulorrhexis specimens from 29 patients were studied with correlative light and electron microscopy. RESULTS: LEC density declined with age and was inversely correlated with the scalar grade for puffy clouds and for the size and number of black holes. The scalar grade for columnar organization was inversely associated with the severity of posterior subcapsular and nuclear cataracts, which was the only feature associated with the LOCS III grade of ARC. No statistically significant associations were found between average cell count and LOCS III grade. CONCLUSIONS: With the use of the corneal specular microscope excellent in vivo specular images of the LECs were obtained, the features in these images that correlated well with microscopic findings were classified, and cell density in vivo was estimated.

Lens epithelium-derived growth factor: effects on growth and survival of lens epithelial cells, keratinocytes, and fibroblasts.

We isolated a clone encoding a protein from a human lens epithelial cell (LEC) cDNA library with antibody (Ab) from a cataract patient and named it "lens epithelium-derived growth factor" (LEDGF). LEDGF is found to be identical to p75, a coactivator of both transcription (1) and pre-mRNA splicing (2). In serum-free medium LEDGF stimulated growth of LECs, cos7 cells, skin fibroblasts, and keratinocytes, and prolonged cell survival. Without LEDGF, the aforementioned cells did not survive. Also in serum-free medium, Ab to LEDGF neutralizing LEDGF blocked cell growth and caused cell death. Thus, LEDGF, a regulatory factor, may play an important role for growth and survival of a wide range of cell types.

LEDGF: survival of embryonic chick retinal photoreceptor cells.

PURPOSE: Lens epithelium-derived growth factor (LEDGF) is a novel adhesive, survival, and growth factor for lens epithelial cells, keratinocytes, fibroblasts, and cos7 cells. In the presence of LEDGF, these cells acquire resistance to environmental stresses, and in the absence of LEDGF they die. The effects of LEDGF on survival of embryonic chick retinal photoreceptor cells under serum starvation and heat stress were studied. METHODS: The expression pattern of LEDGF in embryonic chick retinal photoreceptor cells was investigated with protein blot analysis and immunohistochemistry using antibodies (Abs) to LEDGF. Retinal cells were cultured in serum-free medium for up to 6 days in the presence of varying amounts of LEDGF at 37 degrees or 41 degrees C. The photoreceptor cells were immunostained with Abs to arrestin and counted to evaluate the photoreceptor cell viability. Heat shock proteins in the cultured cells were quantified by protein blot analysis with Ab probes and semiquantitative reverse transcription-polymerase chain reaction analysis. RESULTS: LEDGF was found predominantly in the nucleus of neuroretinal cells, including photoreceptor cells. In the presence of LEDGF, photoreceptor cells manifested increased resistance to serum starvation and thermal stress and survived for a longer period. The levels of heat shock protein 90 were elevated in those cells. Most retinal cells died in the absence of LEDGF. CONCLUSIONS: LEDGF enhanced survival of retinal photoreceptor cells under serum starvation and heat stress. Thus, LEDGF has a potency to enhance survival of neuronal cell types against environmental stresses, and it may be applicable as a therapeutic agent for those cells.

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.

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.