Comparison of a novel whole blood transglutaminase-based ELISA with a whole blood rapid antibody test and established conventional serological celiac disease assays.

OBJECTIVES: Serum immunoglobulin A-class tissue transglutaminase (tTG-ab) and endomysial antibody (EMA) tests play a key role in the diagnostic evaluation of celiac disease. Recently, a novel whole blood rapid test based on self-tissue transglutaminase (tTG) was developed for celiac disease case finding. Based on the same principle, a whole blood self-tTG enzyme-linked immunosorbent assay (ELISA), especially applicable to large-scale screening of celiac disease, has been produced. We assessed the value of this new test in celiac disease antibody detection. PATIENTS AND METHODS: The new test uses endogenous tTG found in red blood cells of whole blood in IgA-class tTG-ab measurement by detecting tTG-tTG-ab complexes formed after hemolysis of the whole blood sample. Stored whole blood samples from 150 untreated celiac disease patients and 107 control individuals without celiac disease were evaluated, and the test results were compared with those of the whole blood rapid test, 2 conventional serum-based tTG-ab ELISA tests, and 2 EMA tests. RESULTS: A total of 15 whole blood samples were found to be clotted or dried after storage and were excluded from further evaluation. The whole blood ELISA test had a specificity (98%) comparable to that of the conventional serological tests, the sensitivity (91%) being slightly lower. The test was concordant with the whole blood rapid test in 92% of cases, with 2 serological ELISA tests in 91% and 94% of cases and with EMA tests in 94% and 93% of cases. CONCLUSIONS: Whole blood self-tTG-based testing is accurate in celiac antibody detection, also when an ELISA method is applied. The testing requires no serum separation or external tTG.

A structural model of 20S immunoproteasomes: effect of LMP2 codon 60 polymorphism on expression, activity, intracellular localisation and insight into the regulatory mechanisms.

The immunoproteasome subunit low molecular weight protein 2 (LMP2) codon 60 polymorphism has been associated with autoimmune diseases. It has also been demonstrated to influence susceptibility to TNF-alpha-induced apoptosis in blood cells and proteasome activity in aged human brain. In the present study, an in silico model of immunoproteasome was used to examine the effect of the R60H polymorphism in the LMP2 subunit. The investigation of immunoproteasome expression, activity and intracellular localisation in an in vitro cellular model, namely lymphoblastoid cell lines, showed no major variations in functionality and amount, while a significant difference in antibody affinity was apparent. These data were integrated with previous results obtained in different tissues and combined with a structural model of the LMP2 polymorphism. Accordingly, we identified three prospective mechanisms that could explain the biological data for the polymorphism, such as modulation of the binding affinity of a putative non-catalytic modifier site on the external surface of the immunoproteasome core, or the modification of any channel between alpha and beta rings.

Repeated exposure of human skin fibroblasts to UVB at subcytotoxic level triggers premature senescence through the TGF-beta1 signaling pathway.

Premature senescence of human diploid fibroblasts (HDFs) can be induced by exposures to a variety of oxidative stress and DNA damaging agents. In this study we developed a robust model of UVB-induced premature senescence of skin HDFs. After a series of 10 subcytotoxic (non-proapoptotic) exposures to UVB at 250 mJ/cm2, the so-called biomarkers of senescence were markedly expressed: growth arrest, senescence-associated beta-galactosidase activity, senescence-associated gene overexpression, deletion in mitochondrial DNA. A set of 44 stress- and senescence-associated genes were found to be differentially expressed in this model, among which clusterin/apolipoprotein J (apo J) and transforming growth factor-beta1 (TGF-beta1). Transfection of apo J cDNA provided protection against premature senescence-inducing doses of UVB and other stressful agents. Neutralizing antibodies against TGF-beta1 or its receptor II (TbetaRII) sharply attenuated the senescence-associated features, suggesting a role for TGF-beta1 in UVB-induced premature senescence. Both the latent and active forms of TGF-beta1 were increased with time after the last UVB stress. Proteasome inhibition was ruled out as a potential mechanism of UVB-induced stress-induced premature senescence (SIPS). This model represents an alternative in vitro model in photoaging research for screening potential anti-photoaging compounds.

Age-dependent protein modifications and declining proteasome activity in the human lens.

The proteasome is known to be the main enzymatic complex responsible for the intracellular degradation of altered proteins, and the age-related accumulation of modified lens proteins is associated to the formation of cataracts. The aim of this study was to determine whether the human lens proteasome becomes functionally impaired with age. The soluble and insoluble protein fractions of human lenses corresponding to various age-groups were characterized in terms of their levels of glyco-oxidative damage and found to show increasing anti-carboxymethyl-lysine immunoreactivity with age. Concomitantly, decreasing proteasome contents and peptidase activities were observed in the water-soluble fraction. The fact that peptidylglutamyl-peptide hydrolase activity is most severely affected with age suggests that specific changes are undergone by the proteasome itself. In particular, increasing levels of carboxymethylation were observed with age in the proteasome. It was concluded that the lower levels of soluble active enzymatic complex present in elderly lenses and the post-translational modifications affecting the proteasome may at least partly explain the decrease in proteasome activity and the concomitant accumulation of carboxymethylated and ubiquitinated proteins which occur with age.

Dysfunction of mitochondrial complex I and the proteasome: interactions between two biochemical deficits in a cellular model of Parkinson's disease.

Two biochemical deficits have been described in the substantia nigra in Parkinson's disease, decreased activity of mitochondrial complex I and reduced proteasomal activity. We analysed interactions between these deficits in primary mesencephalic cultures. Proteasome inhibitors (epoxomicin, MG132) exacerbated the toxicity of complex I inhibitors [rotenone, 1-methyl-4-phenylpyridinium (MPP+)] and of the toxic dopamine analogue 6-hydroxydopamine, but not of inhibitors of mitochondrial complex II-V or excitotoxins [N-methyl-d-aspartate (NMDA), kainate]. Rotenone and MPP+ increased free radicals and reduced proteasomal activity via adenosine triphosphate (ATP) depletion. 6-hydroxydopamine also increased free radicals, but did not affect ATP levels and increased proteasomal activity, presumably in response to oxidative damage. Proteasome inhibition potentiated the toxicity of rotenone, MPP+ and 6-hydroxydopamine at concentrations at which they increased free radical levels >/= 40% above baseline, exceeding the cellular capacity to detoxify oxidized proteins reduced by proteasome inhibition, and also exacerbated ATP depletion caused by complex I inhibition. Consistently, both free radical scavenging and stimulation of ATP production by glucose supplementation protected against the synergistic toxicity. In summary, proteasome inhibition increases neuronal vulnerability to normally subtoxic levels of free radicals and amplifies energy depletion following complex I inhibition.

Potentiation of tumor necrosis factor-induced NF-kappa B activation by deacetylase inhibitors is associated with a delayed cytoplasmic reappearance of I kappa B alpha.

Previous studies have implicated acetylases and deacetylases in regulating the transcriptional activity of NF-kappa B. Here, we show that inhibitors of deacetylases such as trichostatin A (TSA) and sodium butyrate (NaBut) potentiated TNF-induced expression of several natural NF-kappa B-driven promoters. This transcriptional synergism observed between TNF and TSA (or NaBut) required intact kappa B sites in all promoters tested and was biologically relevant as demonstrated by RNase protection on two instances of endogenous NF-kappa B-regulated gene transcription. Importantly, TSA prolonged both TNF-induced DNA-binding activity and the presence of NF-kappa B in the nucleus. We showed that the p65 subunit of NF-kappa B was acetylated in vivo. However, this acetylation was weak, suggesting that other mechanisms could be implicated in the potentiated binding and transactivation activities of NF-kappa B after TNF plus TSA versus TNF treatment. Western blot and immunofluorescence confocal microscopy experiments revealed a delay in the cytoplasmic reappearance of the I kappa B alpha inhibitor that correlated temporally with the prolonged intranuclear binding and presence of NF-kappa B. This delay was due neither to a defect in I kappa B alpha mRNA production nor to a nuclear retention of I kappa B alpha but was rather due to a persistent proteasome-mediated degradation of I kappa B alpha. A prolongation of I kappa B kinase activity could explain, at least partially, the delayed I kappa B alpha cytoplasmic reappearance observed in presence of TNF plus TSA.

Impact of ageing on proteasome structure and function in human lymphocytes.

Key actors of the immune response, lymphocytes exhibit functional deficits with advancing age. For instance, the age-related decline in lymphocyte proliferation may be related to alteration in the degradation of crucial proteins such as cell-cycle regulators. Degradation of these proteins is mediated by the ubiquitin-26S proteasome system. The proteasome is also the major "housekeeping" proteolytic complex responsible for eliminating intracellular damaged proteins. To investigate the occurrence of proteasome structural and functional age-related alterations, 26S proteasome was purified from peripheral blood lymphocytes of 20-63-year-old donors. Changes in peptidase activity were measured and modifications in the proteasome particle structure were analysed using bi-dimensional electrophoresis. We found the age-related decline of 26S proteasome-specific activity to be associated with an increased yield of post-translational modifications of proteasome subunits, while proteasome content and subunit composition were unchanged. In particular, some catalytic and assembly subunits of the 20S proteasome were preferentially modified with age. Western blotting of proteasome subunits resolved by bi-dimensional electrophoresis showed some of these modified subunits to be glycated, conjugated with a lipid peroxidation product and/or ubiquitinated. In conclusion, it is suggested that structural alterations of proteasome subunits may contribute to the observed decline of proteasome activity with age and could play a major role in immune senescence.

Impairment of proteasome structure and function in aging.

Damage to macromolecules, and in particular protein, implicated in the cellular degeneration that occurs during the aging process, is corroborated by the accumulation of oxidative end-products over time. Oxidized protein build up is commonly seen as a hallmark of cellular aging. Protein turnover is essential to preserve cell function and the main proteolytic system in charge of cytosolic protein degradation is the proteasome. The proteasome is a multi-catalytic proteolytic complex, which recognizes and selectively degrades oxidatively damaged and ubiquitinated proteins. One of the hypothesis put forward to explain the accumulation of altered proteins is the decrease of proteasome activity with age. Indeed, accumulation of altered protein can be explained by increased protein alteration, decreased protein degradation or the combination of both. A short description of proteasome structure and of its role in cellular functions is first given. Then, accumulation of damaged protein is presented with emphasis on the pathways implicated in the formation of altered proteins. Finally, evidence for an age-related impairment of proteasome structure and function that has been reported by different groups is provided in the light of proteasomal dysfunction induced upon oxidative stress. It is now clear that proteasome activity is declining with age and that the loss in proteasome activity during aging is dependent of at least three different mechanisms: decreased proteasome expression; alterations and/or replacement of proteasome subunits and formation of inhibitory cross-linked proteins. However, it is also clear that events leading to the age- and disease-related loss of proteasome function have not yet been fully characterized.