AIM1 and LINE-1 epigenetic aberrations in tumor and serum relate to melanoma progression and disease outcome.

Aberrations in the methylation status of noncoding genomic repeat DNA sequences and specific gene promoter region are important epigenetic events in melanoma progression. Promoter methylation status in long interspersed nucleotide element-1 (LINE-1) and absent in melanoma-1 (AIM1; 6q21) associated with melanoma progression and disease outcome was assessed. LINE-1 and AIM1 methylation status was assessed in paraffin-embedded archival tissue (PEAT; n = 133) and in melanoma patients' serum (n = 56). LINE-1 U-Index (hypomethylation) and AIM1 were analyzed in microdissected melanoma PEAT sections. The LINE-1 U-Index of melanoma (n = 100) was significantly higher than that of normal skin (n = 14) and nevi (n = 12; P = 0.0004). LINE-1 U-Index level was elevated with increasing American Joint Committee on Cancer (AJCC) stage (P<0.0001). AIM1 promoter hypermethylation was found in higher frequency (P = 0.005) in metastatic melanoma (65%) than in primary melanomas (38%). When analyzed, high LINE-1 U-Index and/or AIM1 methylation in melanomas were associated with disease-free survival (DFS) and overall survival (OS) in stage I/II patients (P = 0.017 and 0.027, respectively). In multivariate analysis, melanoma AIM1 methylation status was a significant prognostic factor of OS (P = 0.032). Furthermore, serum unmethylated LINE-1 was at higher levels in both stage III (n = 20) and stage IV (n = 36) patients compared with healthy donors (n = 14; P = 0.022). Circulating methylated AIM1 was detected in patients' serum and was predictive of OS in stage IV patients (P = 0.009). LINE-1 hypomethylation and AIM1 hypermethylation have prognostic utility in both melanoma patients' tumors and serum.

Immunochemical detection of glycated lens crystallins and their circulating autoantibodies in human serum during aging.

PURPOSE: The aim of this investigation was to exploit lens-specific glycated crystallins as an immunogen to detect human glycated crystallins and their circulating autoantibodies in human serum during aging in relation to the development of cataract. METHODS: Polyclonal antibodies were produced against human total lens proteins (40-80 years) in rabbits. The specificity of the antibodies produced were determined by antibody capture assay using purified human lens crystallins (high molecular weight fraction [HMW]+alpha, HMW+alpha-glycated, beta, beta-glycated, gamma, and gamma-glycated) as antigens. The cross-reactivity of these lens specific antibodies against rat beta-, beta-glycated, gamma-, and gamma-glycated lens crystallins was also analyzed. A non-competitive enzyme linked immunosorbent assay (ELISA) methodology was developed for the detection of circulating lens crystallins in human sera using HMW+alpha, HMW+alpha-glycated, beta-, and beta-glycated crystallins from humans and gamma- and gamma-glycated crystallins from rats as immobilized antigens. Circulating autoantibodies were also detected in human sera by antibody capture assay. The methodology was validated by evaluating 60 human serum samples collected from cataract patients and 30 human serum samples from apparently normal subjects belonging to the same age group. RESULTS: The polyclonal antibodies raised against human total lens proteins showed 90% and 65% cross-reactivity with rat gamma- and beta-crystallins, respectively, by ELISA. Further, these polyclonal antibodies were capable of detecting both native and in vitro synthesized glycated crystallins. Their IC50 values were observed to be (i) human total lens proteins (55 ng), (ii) human HMW+alpha (16.45 ng), (iii) human HMW+alpha-glycated (273 ng), (iv) human beta- (37.82 ng), (v) human beta-glycated (260 ng), (vi) rat gamma- (105.34 ng), and (vii) rat gamma-glycated (313 ng). The immunochemical analysis of human serum indicated a significant change (p<0.001) in the levels of circulating beta-glycated and gamma-glycated crystallins in the age group of 40-80 years with respect to their control groups. However, there was no statistically significant change in the levels of HMW+alpha-glycated crystallins in the age group of 40-80 years as compared to their age-matched controls. Notably, the levels of serum gamma-glycated crystallins were found to be threefold higher than that of HMW+alpha-glycated and beta-glycated crystallins in the age group of 70-80 years. Circulating autoantibodies to HMW+alpha-glycated, beta-glycated, and gamma-glycated crystallins were detected in the serum of both apparently normal and cataract patients in the age group of 40-80 years by antibody capture assay. The levels of these autoantibodies were significantly higher at every time point compared to their respective controls. Autoantibodies to gamma-glycated crystallins were found to be twofold and 3.2 fold higher as compared to the levels of autoantibodies to beta-glycated and HMW+alpha-glycated crystallins, respectively. Western blot and immunohistochemical analysis substantiated the observations made in non-competitive ELISA. CONCLUSIONS: During the course of aging, leakage of lens crystallins (HMW+alpha, HMW+alpha-glycated, beta, beta-glycated, gamma, and gamma-glycated) elicit an immune response resulting in the formation of autoantibodies in cataract patients (40-80 years) as compared to age matched controls. This is the first experimental report where polyclonal antibodies raised against lens-specific glycated crystallins were capable of detecting the early leakage of glycated crystallins in human subjects. This immunochemical approach has implications in the early detection of senile cataract.

[Antibodies against lens proteins in the blood in patients with cataract]. / Przeciwciala przeciwko bialkom soczewki we krwi chorych z zacma.

Investigations were carried out to clarify the role of autoimmune phenomena in the pathogenesis of human cataract. We determined the antibodies to lens proteins of serum in the following groups of patients: patients with senile cataract, patients with diabetic cataract, patients with diabetes mellitus, dependent of insulin and without cataract, patients without cataract and without diabetes mellitus (healthy adults), using the plate gel with double diffusion method described by Ouchterlony. 98% patients with senile cataract, 100% patients with diabetic cataract and only 12% healthy adults showed positive reactions to the test. There is little evidence so far, to incriminate immunological mechanisms in the pathogenesis of cataract.

AlphaB-crystallin, a low-molecular-weight heat shock protein, acts as a regulator of platelet function.

It has recently been reported that alphaB-crystallin, a low-molecular-weight heat shock protein, may be released from cells by mechanical stretch. We investigated a physiological role of alphaB-crystallin in platelet function. AlphaB-crystallin inhibited platelet aggregation induced by thrombin or botrocetin in hamsters and humans. These platelets had specific binding sites for alphaB-crystallin. Moreover, alphaB-crystallin significantly reduced thrombin-induced Ca2+ influx and phosphoinositide hydrolysis by phospholipase C in human platelets. Additionally, plasma levels of alphaB-crystallin were markedly elevated in cardiomyopathic hamsters. Levels of alphaB-crystallin in vessel walls after endothelial injury were markedly reduced. Therefore, our results suggest that alphaB-crystallin, which is discharged from vessel walls in response to endothelial injury, acts intercellularly as a regulator of platelet function.

Effects of age, sex, cataract, and cataract surgery on serum gamma-crystallin concentration.

PURPOSE: To determine if measurement of lens protein in serum is a feasible means to gain information on the physiologic status of the lens in human subjects. METHODS: The gamma-crystallin concentration was measured by a sandwich radioimmunoassay in the sera of 280 subjects aged 25-94 years. Medical records were reviewed for diagnoses of cataract and aphakia. RESULTS: There was no effect of age or sex on the serum gamma-crystallin concentration. There were 57 subjects with cataract and 27 with aphakia. gamma-Crystallin was higher in all cataract groups and lower in aphakia. The mean gamma-crystallin concentrations for selected subject groups were as follows: clear lens 301 pg/ml; pure nuclear cataract 344 pg/ml; pure cortical cataract 439 pg/ml and aphakia 255 pg/ml. CONCLUSIONS: This is the first published report to show that lens protein is measurable in serum and to demonstrate the feasibility of using serum assays of lens proteins to gain information on the physiological status of the lens. Our results confirm the hypothesis that molecular and cellular events leading to cataract cause increased leakiness of lens cell membranes with release of lens proteins appearing in the blood. It is conceivable that measurement of lens proteins in serum might find future use in the evaluation of cataract risk, potentially cataractogenic and anticataractogenic agents, retained lens fragments after phacoemulsification, secondary cataract, phacolytic glaucoma, anaphylactic endophthalmitis, eye injuries, and other eye diseases.