The role of Vitamin D in immuno-inflammatory responses in Ankylosing Spondylitis patients with and without Acute Anterior Uveitis.

Hypothesis:Abnormal Vitamin D (Vit D) level could have consequences on the immuno-inflammatory processes in Ankylosing Spondylitis (AS). Aim:The purpose of this study was to analyze the role of Vitamin D in the interplay between immune and inflammation effectors in AS associated-Acute Anterior Uveitis (AAU). Methods and Results:25-hydroxyvitamin D (Vit D), LL-37 peptide, IL-8 and Serum Amyloid A (SAA) were identified and quantified in the serum/ plasma of thirty-four AS patients [eleven AS patients presenting AAU (AAU AS patients) and twenty-three AS patients without AAU (wAAU AS patients)] and eighteen healthy individuals (Control) using enzyme-linked immunosorbent assay. Acute-phase SAA level was significantly higher in AS patients compared to Controls. Contrary with wAAU AS patients, significantly elevated levels of IL-8, and diminished levels of Vit D characterized AAU AS patients. Regarding LL-37, its level decreased concomitantly with the level of Vit D. When AS patients were subgrouped based on AAU presence or on Vit D level, important associations between immuno-inflammatory assessed markers and AS features were noticed. Generally, Vit D levels were associated indirectly with leukocytes/ neutrophils number or with ESR, CRP, and Fibrinogen levels. The levels of SAA and IL-8 associated directly with AAU or with AAU relapses, especially in AS patients with Vit D insufficiency, while SAA associated directly with infection/ inflammatory markers and with disease activity indexes or with the degree of functional limitation. Discussion:Altered levels of Vit D affect the balance between LL-37, IL-8 and SAA, suggesting an association with AAU, an extra-articular manifestation of AS. Abbreviations:Vit D = Vitamin D, AS = Ankylosing Spondylitis, AAU = Acute Anterior Uveitis, AAU AS = AS patients with AAU, wAAU AS = AS patients without AAU, SSZ = Sulphasalazine, Leu = Leukocytes, Neu = Neutrophils.

Pycnogenol efficacy in the treatment of systemic lupus erythematosus patients.

A pilot study was performed to evaluate the efficacy of Pycnogenol treatment in systemic lupus erythematosus (SLE) patients. Eleven SLE patients were treated with first line medication according to disease activity and in addition, six of them received Pycnogenol and five a placebo. The SLE disease activity index (SLEDAI), serum anti-dsDNA antibodies, fibrinogen, C-reactive protein levels, erythrocyte sedimentation rate, production of reactive oxygen species (ROS) by neutrophils, spontaneous apoptosis and p56(lck) specific activity in peripheral blood lymphocytes were evaluated. Pycnogenol treatment determined a significant reduction of ROS production, apoptosis, p56(lck) specific activity and erythrocyte sedimentation rate. In addition, the decrease of SLEDAI was significant in the Pycnogenol treated group compared with the placebo group (p = 0.018). The results obtained suggest that Pycnogenol could be useful for second line therapy to reduce the inflammatory feature of SLE.

Dysregulation of p56lck kinase in patients with systemic lupus erythematosus.

In this study we investigated one of the possible mechanisms of p56lck down-regulation in peripheral blood lymphocytes (PBLs) from Systemic Lupus Erythematosus (SLE) patients and we correlated p56lck dysregulation with accelerated apoptosis in SLE PBLs. PBLs from SLE patients and healthy donors were isolated. p56lck protein expression and lck mRNA level were estimated by immunoblotting and RT-PCR, respectively. FACS analysis was used to evaluate the apoptosis and p56lck levels in apoptotic and non-apoptotic PBLs. A non-radioactive Tyrosine Kinase Assay Kit was used to measure p56lck activity. Our results demonstrated that PBLs from SLE patients displayed lower levels of lck mRNA and p56lck protein as compared to healthy donors. The apoptosis of fresh or cultured PBLs was enhanced in SLE patients, especially in anti-DNA negative group. The expression of p56lck was inverse correlated with apoptosis of fresh and cultured SLE PBLs, especially in anti-DNA negative patients. Double staining FACS analysis showed that p56lck expression was lower in apoptotic than in non-apoptotic PBLs. p56lck specific activity was directly correlated to apoptosis in SLE PBLs. While the low expression of p56lck may be the result of lower degree of synthesis, the increased specific activity could directly correlated to the extent of apoptosis in SLE PBLs. Based on our observations, we assume that the p56lck dysregulation could play a role in SLE pathogenesis.

p56lck activity and expression in peripheral blood lymphocytes from patients with systemic lupus erythematosus.

In this study we analyzed the activity and the expression of p56lck protein tyrosine kinase in peripheral blood lymphocytes (PBLs) from systemic lupus erythematosus (SLE) patients and from healthy donors. The p56lck activity, determined by a non-radioactive Tyrosine Kinase Assay Kit, was significantly higher in active SLE PBLs and discriminated this group of patients from inactive SLE patients (p = 0.002) and healthy donors (p = 0.009). p56lck level decreased in SLE lymphocytes (especially for inactive SLE lymphocytes, p = 0.005) when compared to healthy donors. These differences were also reflected by the specific activity of p56lck that was clearly elevated in active SLE lymphocytes when compared to inactive SLE (p = 0.022) or healthy donors lymphocytes (p = 0.006). A positive correlation between the activity of p56lck and the tyrosine phosphorylation level in active SLE lymphocytes was found.

Modulation of cell adhesion by tyrosine kinases and phosphatases inhibitors.

Integrin-mediated activation of monocytes is an important aspect involved in the increase of proinflammatory cytokine messages. Tyrosine phosphorylation of proteins is one of the earliest events involved in these processes: Therefore, we selected two inhibitors, one for tyrosine kinases (quercitin) and another for tyrosine phosphatases (sodium orthovanadate) and we studied their capacity to modulate monocyte adhesion to fibronectin. Our results showed that quercitin strongly inhibits both tyrosine phosphorylation and cell adhesion. Sodium orthovanadate induces a modest increase of tyrosine phosphorylation and a weak enhancement of cell adhesion. When a combination of the two inhibitors was used, the tyrosine phosphorylation level displayed a strong enhancement. In contrast, cell adhesion was inhibited, but to the same degree. These observations indicate that tyrosine kinases may be more important than tyrosine phosphatases in the modulation of cell adhesion by flavonoid compounds.

Experimental studies on bacterial product CANTASTIM derived from Pseudomonas aeruginosa with immunomodulatory properties. IV. Tyrosine phosphorylation as an effect of stimulation on different cell populations.

The bacterial product derived from Pseudomonas aeruginosa (trade mark-CANTASTIM) proved immunomodulatory effects in different systems, both in vitro and in vivo experimental animal models, as well as in clinical trials. Among the results obtained regarding CANTASTIM, the following immunomodulatory properties could be mentioned: an increase of the activated T cell subpopulations and humoral-mediated immune processes, facilitation of phagocytic processes, stimulation of cytotoxic activity reflected in the improvement of the capacity of defense in several tumoral and infectious diseases. To better elucidate the intimate mechanisms by which CANTASTIM modulates the cellular functions on different cellular populations, we used tyrosine phosphorylation as an estimate of cell activation on peripheral blood lymphocytes (PBL) and a monocyte cell line (THP-1). In PBL, the treatment with CANTASTIM renders them more susceptible to CD3 stimulation than non-treated cells. In monocytes, CANTASTIM and two phospholipid components of CANTASTIM modulated in a different manner the cellular adhesion on fibronectin and tyrosine phosphorylation leading to the conclusion that these phospholipid components do not fully explain CANTASTIM modulatory properties on cell adhesion processes.

Tyrosine phosphorylation in peripheral lymphocytes from patients with systemic lupus erythematosus.

A comparative study of tyrosine phosphorylation was performed on peripheral blood lymphocytes from systemic lupus erythematosus (SLE) patients and from healthy donors. Freshly isolated SLE lymphocytes presented an elevated tyrosine phosphorylation level when compared to healthy donors lymphocytes (p = 0.005). Among all phosphorylated proteins, those called p120, p110, p80 and p55-p60 were more phosphorylated. The level of tyrosine phosphorylation of p120 and p110 proteins discriminated significantly (p = 0.0048, respectively, p = 0.02) between SLE patients and healthy donors. Lymphocytes form SLE patients and healthy donors were then stimulated by cross-linking T cell antigens (CD3, CD4, CD8) to further distinguish the signal transduction between normal and pathologic lymphocytes. No statistical differences in the tyrosine phosphorylation pattern, following CD4 or CD8 cross-linking, were observed between SLE patients and healthy donors lymphocytes. CD3 cross-linking induced an effect on tyrosine phosphorylation different in SLE patients versus healthy donors lymphocytes. Thus, the lymphocytes of SLE patients were refractile in anti-CD3 stimulation in comparison with the healthy donors lymphocytes. Chi-square analysis demonstrated that a significantly larger number of healthy donors responded to anti-CD3 stimulation compared to SLE patients (p = 0.03). The high frequency of tyrosine phosphorylation of p110 and p80 proteins, following CD3 stimulation, in normal versus SLE lymphocytes, suggested that these proteins could be involved in abnormal signal transduction in SLE cells.

Naturally occurring anti-idiotypic antibodies to anti-phosphotyrosine in systemic lupus erythematosus interact with SRC-homology 2 domains.

We have recently identified in SLE sera naturally occurring anti-idiotypic antibodies against anti-phosphotyrosine antibodies. Analysis of immunochemical properties of these anti-idiotypic antibodies suggest that they are of beta/gamma type mimicking the antigen. The interaction between these anti-idiotypes and SH2 domains of various fusion proteins was analysed by immunoprecipitation and immunoblotting. Our data demonstrate that these anti-idiotypic antibodies specifically bind SH2 domains, with the highest affinity for SH2 domain of lck protein tyrosine kinase. The significance of this interaction is discussed.

Signal transduction in T lymphocytes of SLE patients: lectin-activated phosphorylation on tyrosine.

A comparative study of signal transduction through tyrosine phosphorylation process in peripheral blood lymphocytes from SLE patients and healthy subjects reveal some modifications in the phosphorylation pattern of SLE T lymphocytes. Thus, the level of constitutive tyrosine phosphorylation in resting SLE T lymphocytes is higher than in lymphocytes from healthy subjects. In SLE T lymphocytes, a cellular proteic substrate with an apparent molecular weight of about 37 kDa is constitutively phosphorylated. Some differences in the pattern of phosphorylation are obvious in lectin (Con A, PHA)-activated T lymphocytes. Thus, Con A activation enhances the phosphorylation of cellular substrates with molecular weight in the range of 55-80 kDa from SLE T lymphocytes. Moreover, the 21 kDa substrate is also hyperphosphorylated after PHA activation of SLE lymphocytes.

Identification of anti-idiotypic antibodies to anti-phosphotyrosine antibodies in human sera.

We have recently identified in SLE sera antibodies against phosphotyrosine. They were also detected in normal sera and gammaglobulin preparations, suggesting that they belong to natural autoantibodies. In this paper, the occurrence of anti-idiotypic antibodies against anti-phosphotyrosine antibodies, in the above mentioned samples, is investigated. In order to identify these anti-idiotypic antibodies ELISA, immunoprecipitation and immunoblotting are performed. Our data demonstrate the presence of anti-idiotypic antibodies specific to anti-phosphotyrosine antibodies in SLE sera as well as in normal sera, suggesting that these anti-idiotypic antibodies are also auto-anti-idiotypic antibodies. The densitometry of immunoblots reveals significantly higher levels of anti-idiotypic antibodies in SLE sera. Based on the competition inhibition studies we conclude that some of these anti-idiotypic antibodies belong to beta/gamma type.

Presence and significance of some antibodies/autoantibodies in patients with acute myocardial infarction and idiopathic dilated cardiomyopathy.

By using ELISA technique we determined the anti-myosin, actin, collagen type I and IV, cholesterol and phosphotyrosine proteins antibodies in sera from patients with acute myocardial infarction and idiopathic dilated cardiomyopathy. In idiopathic dilated cardiomyopathy we found a significant increase in five out of six types of antibodies tested. The patients with acute myocardial infarction reveal high levels of anti-myosin antibodies only. Our results suggest that some of the autoantibodies studied by us have a prognostic value and could be involved in maintaining cardiac dysfunctions.

Anti-phosphotyrosine and anti-phosphoserine antibodies in SLE sera.

By using an ELISA method, we identified antiPTyr and antiPSer antibodies in the sera of some SLE patients. Afterwards, antiPTyr and antiPSer antibodies were purified by affinity chromatography on phosphotyramine-CNBr-Sepharose column and on phosphoethanolamine-CNBr-Sepharose, respectively, and the specificity of the purified antibodies was demonstrated by inhibition assays. The study pointed out a higher incidence of antiPTyr than anti PSer antibodies in the sera of these patients, which suggests that some "autoantigens" from membrane might be involved.

Anti-phosphotyrosine antibodies--obtaining and purification.

Anti-phosphotyrosine antibodies were obtained by rabbits immunization using bovine serum albumin-phosphotyrosine complex. Hyperimmune rabbit serum was purified on affinity chromatography column obtained by phosphotyramine binding on Sepharose 4 B, CNBr-activated. The antibodies obtained after purification were tested by ELISA and immunoblotting.