Gene expression of catalytic proteasome subunits and resistance toward proteasome inhibition of B lymphocytes from patients with primary sjogren syndrome.

OBJECTIVE: Dysregulation of proteasome subunit ß1i expression has been shown in total blood mononuclear cells (PBMC) from patients with primary Sjögren syndrome (pSS), a B cell-driven systemic autoimmune disorder. METHODS: Proteasome activation was investigated in sorted blood cells from patients with pSS and controls by measuring transcript levels of constitutive (ß1/ß2/ß5) and corresponding immunoproteasome catalytic subunits (ß1i/ß2i/ß5i) using real-time PCR. At protein level, ß1i protein expression was analyzed by immunoblotting. Functional effects of proteasome inhibition on proteolytic activity and induction of apoptosis were also evaluated in cellular subsets. RESULTS: The proteasome was found to be activated in pSS, with upregulation of gene expression of catalytic proteasome subunits. Western blot analysis revealed decreased ß1i protein expression in pSS B lymphocytes, with decreased protein despite increased messenger RNA (mRNA) levels. After proteasome inhibition in vitro, proteolytic activity was less reduced and resistance to apoptosis was increased in B lymphocytes compared to other cells. CONCLUSION: In pSS, catalytic subunits of the proteasome are upregulated at the mRNA level, while dysregulation of subunit ß1i is attributed to B lymphocytes. B cell resistance after proteasome inhibition differs from the classical concept of increased susceptibility toward inhibition in activated cells, supporting the novel notion that susceptibility depends on cellular intrinsic factors and on proteasome activation.

Rimexolone inhibits proliferation, cytokine expression and signal transduction of human CD4+ T-cells.

Rimexolone is a lipophilic glucocorticoid drug used for local application. Only few data are available describing its effects on immune cell functions. In this study we investigated the effects of rimexolone on the proliferation of human CD4+ T-cells using dexamethasone as standard reference. Isolated CD4+ T-cells were pre-incubated with rimexolone or dexamethasone at different concentrations for 10 min (10(-11)/10(-8)/10(-5)M) and stimulated with anti-CD3/anti-CD28 for 96 h. Proliferation was determined by flow cytometry. The percentage of dividing cells was significantly reduced by 10(-5)M rimexolone and dexamethasone; however, the average number of cell divisions was unchanged. In addition, production of IL-2 and other cytokines was reduced by both glucocorticoids at 10(-5)M. Interestingly, we observed a rimexolone-induced down-regulation of CD4 expression in unstimulated and non-dividing cells. The inhibitory effects on proliferation and CD4 expression could be blocked by the glucocorticoid-antagonist RU486 and were not due to glucocorticoid-induced apoptosis. Rimexolone and dexamethasone showed a similar potential to induce IkappaBalpha gene expression. We demonstrate rimexolone and dexamethasone to impair T-cell signalling pathways by rapid non-genomic suppression of the phosphorylation of Akt, p38 and ERK. We conclude that rimexolone and dexamethasone inhibit T-cell proliferation as well as cytokine production of activated CD4+ T-cells in a similar manner. As these inhibitory effects predominantly occur at high concentrations, a relatively high occupation-rate of cytosolic glucocorticoid receptors is needed, but receptor-mediated non-genomic effects may also be involved. It is implied that these effects contribute to the well-known beneficial anti-inflammatory and immunomodulatory effects of glucocorticoid therapy.

Is musculoskeletal ultrasonography an operator-dependent method or a fast and reliably teachable diagnostic tool? Interreader agreements of three ultrasonographers with different training levels.

Objectives. To assess interreader agreements and a learning curve between three (senior, junior, and beginner) different experienced musculoskeletal ultrasonographers. Senior served as the imaging "gold standard". Methods. Clinically dominant joints (finger, shoulder, knee, tibiotalar, and talonavicular) of 15 rheumatoid arthritis (RA) patients were examined by three different experienced ultrasonographers (senior 10 years, junior 10 months, and beginner one month). Each patient's ultrasonographic findings were reported unaware of the other investigators' results. κ coefficients, percentage agreements, sensitivities, and specificities were calculated. Results. 120 joints of 15 RA patients were evaluated. Comparing junior's and beginner's results each to the senior's findings, the overall κ for all examined joints was 0.83 (93%) for junior and 0.43 (76%) for beginner. Regarding the different joints, junior's findings correlate very well with the senior's findings (finger joints: κ = 0.82; shoulder: κ = 0.9; knee: κ = 0.74; tibiotalar joint: κ = 0.84; talonavicular joint: κ = 0.84) while beginner's findings just showed fair to moderate agreements (finger joints: κ = 0.4; shoulder: κ = 0.42; knee: κ = 0.4; tibiotalar joint: κ = 0.59; talonavicular joint: κ = 0.35). In total, beginner's results clearly improved from κ = 0.34 (agreement of 67%) at baseline to κ = 0.78 (agreement of 89%) at the end of the evaluation period. Conclusions. Ultrasonographic evaluation of a ten-month-experienced investigator in comparison to a senior ultrasonographer was of substantial agreement. Agreements between a beginner and a highly experienced ultrasonographer were only fair at the beginning, but during the study including ultrasonographical sessions of 15 RA patients, the beginner clearly improved in musculoskeletal ultrasonography.

Potencies of topical glucocorticoids to mediate genomic and nongenomic effects on human peripheral blood mononuclear cells.

Several different genomic and nongenomic mechanisms are known to mediate the important anti-inflammatory and immunomodulatory effects of glucocorticoids (GC). Genomic effects are the most important while the clinical relevance of nongenomic actions is still a matter of debate. We therefore investigated whether beclometasone and clobetasol are particularly suitable for topical application because of their specific spectrum of genomic and nongenomic actions. For these purposes we compared effects on oxygen consumption as measured with a Clark electrode (nonspecific nongenomic glucocorticoid effects), on interleukin-6 synthesis by means of ELISA (genomic effects) and on apoptosis using flow cytometry (nongenomic and genomic effects) in quiescent and mitogen-stimulated PBMC. Beclometasone and clobetasol indeed had stronger effects on the oxygen consumption of quiescent and stimulated cells at lower concentrations (10(-10) and 10(-8) M) but were less potent at higher concentrations (10(-5) and 10(-4) M) in comparison with dexamethasone. Also in terms of genomic potency, topical GC were more effective than dexamethasone at 10(-10) and 10(-8) M but gave similar results at higher concentrations. The ability of all three GC to induce apoptosis was found to be concentration-dependent and similar at concentrations between 10(-8) and 10(-5) M. But, compared with 10(-4) M dexamethasone, topical GC at 10(-4) M were significantly more effective at inducing apoptosis in both PBMC and Jurkat T-cells. These results show that topical GC have different concentration--(genomic/nongenomic) effect--ratios compared with dexamethasone: besides to the well-known genomic effects there are also significant nongenomic effects of topical glucocorticoids that already at low concentrations might be more therapeutically relevant in certain clinical conditions than currently assumed.