Attila Balog

Polymorphism of the TNF-alpha, HSP70-2, and CD14 genes increases susceptibility to severe acute pancreatitis.

Objectives: Proinflammatory cytokines and heat shock proteins play fundamental roles in the pathogenesis of acute pancreatitis. We studied whether polymorphisms of the tumor necrosis factor α (TNF-α), heat shock protein 70-2 (HSP70-2), and CD14 genes correlate with the severity of acute pancreatitis. Methods: Patients with acute pancreatitis (n = 77) of mixed etiology were grouped according to the severity of the disease on the basis of the Ranson scores. Healthy blood donors (n = 71) served as controls. TNF-α-308 polymorphism was determined by NcoI RFLP, HSP70-2 polymorphism by PstI RFLP, and CD14-159 polymorphism by melting point analysis. Results: There was a moderate increase in the frequency of the TNF1/2 genotype (P = 0.046) among patients with severe acute pancreatitis as compared with those with mild disease. A more significant increase was observed in the frequency of the HSP70-2 G allele between groups of patients with mild or severe pancreatitis (18.9% vs. 53%; P < 0.001). Conversely, the A/A genotype was markedly more frequent among the patients with mild pancreatitis (P < 0.0001). There was no significant correlation between CD14-159 promoter polymorphism and the severity of pancreatitis. Conclusion: High frequencies of the HSP70-2 G and the TNF-α -308 A alleles were associated with risk of severe acute pancreatitis. Genotype assessments may be important prognostic tools to predict disease severity and the course of acute pancreatitis. Therefore, genotype assessments may also be used to guide treatment or to identify risk populations for severe acute pancreatitis.

Investigation of the Prognostic Value of TNF-α Gene Polymorphism among Patients Treated with Infliximab, and the Effects of Infliximab Therapy on TNF-α Production and Apoptosis

Objectives: Infliximab, a chimeric anti-tumor necrosis factor (TNF) antibody, is highly effective for the treatment of Crohn’s disease (CD) and rheumatoid arthritis (RA). Our experiments focused on RA and CD patients receiving infliximab. Since cytokine production is largely determined by genetic factors, the promoter polymorphisms of TNF-α were examined among these patients. Additionally, the changes caused by infliximab in the TNF-α-producing ability and apoptosis of peripheral blood mononuclear cells (PBMCs) were investigated. Methods: The TNF-α genotypes were analyzed by PCR-RFLP. The in vitro TNF-α production of the PBMCs was detected by flow cytometric analysis. The TNF-α concentration in the supernatant was measured by bioassay. Apoptosis was detected by annexin V-fluorescein isothiocyanate labeling. Results and Conclusions: 8 of the 12 nonresponder patients carried the TNF A allele associated with high TNF-α production. We suggest that the determination of TNF polymorphism may help identify more suitable candidates for infliximab treatment. Although in vitro infliximab treatment for 48 h resulted in significant (44.2 ± 1.17%) apoptosis in PBMCs, in ex vivo samples from RA patients who received infliximab, apoptosis was only 13.3 ± 1.6%. Furthermore, infliximab did not result in irreversible inhibition of the TNF-α-producing ability or in the significant apoptosis of PBMCs.

Flow cytometric analysis of procalcitonin expression in human monocytes and granulocytes

Procalcitonin (PCT), a precursor of calcitonin is a useful indicator of severe systemic infection and sepsis. For a better understanding of the pathophysiological background of PCT induction, a study was made of the intracellular expression of PCT in various human white blood cell populations-i.e. monocytes and polymorphonuclear granulocytes (PMNs)-and the role of TNF-alpha in the stimulation of their PCT production. The expression of PCT was investigated by flow cytometric analysis with intracellular staining with antibodies to the PCT components calcitonin (CT) and katacalcin (KC). Both human peripheral monocytes and granulocytes expressed PCT, and increased intracellular amounts of the PCT components were demonstrated after stimulation with Staphylococcus aureus as TNF-alpha inducer. The S. aureus induced stimulation of PCT production was inhibited by anti-TNF-alpha monoclonal antibodies. The monocytic cell line U937 expressed considerable intracellular PCT, but S. aureus failed to induce an increase in PCT expression. The determination of intracellular PCT by flow cytometry is a promising and a sensitive method for further investigation of the effects of various cytokines and cytokine-inducing agents in PCT synthesis of human monocytes and granulocytes.

The effects of Kv1.3 and IKCa1 potassium channel inhibition on calcium influx of human peripheral T lymphocytes in rheumatoid arthritis

OBJECTIVE The transient increase of the cytoplasmic free calcium level plays a key role in the process of lymphocyte activation. Kv1.3 and IKCa1 potassium channels are important regulators of the maintenance of calcium influx during lymphocyte activation and present a possible target for selective immunomodulation. DESIGN Case-control study. SUBJECTS AND METHODS We took peripheral blood samples from 10 healthy individuals and 9 recently diagnosed rheumatoid arthritis (RA) patients receiving no anti-rheumatic treatment. We evaluated calcium influx kinetics following activation in CD4, Th1, Th2 and CD8 cells applying a novel flow cytometry approach. We also assessed the sensitivity of the above subsets to specific inhibition of the Kv1.3 and IKCa1 potassium channels. RESULTS The peak of calcium influx in lymphocytes isolated from RA patients is reached more rapidly, indicating that they respond more quickly to stimulation compared to controls. In healthy individuals, the inhibition of the IKCa1 channel decreased calcium influx in Th2 and CD4 cells to a lower extent than in Th1 and CD8 cells. On the contrary, the inhibition of Kv1.3 channels resulted in a larger decrease of calcium entry in Th2 and CD4 than in Th1 and CD8 cells. No difference was detected between Th1 and Th2 or CD4 and CD8 cells in the sensitivity to IKCa1 channel inhibition among lymphocytes of RA patients. However, specific inhibition of the Kv1.3 channel acts differentially on calcium influx kinetics in RA lymphocyte subsets. Th2 and particularly CD8 cells are inhibited more dominantly than Th1 and CD4 cells. CONCLUSION The inhibition of Kv1.3 channels does not seem to be specific enough in peripheral RA lymphocytes, since anti-inflammatory Th2 cells are also affected to a noteworthy extent.

In vivo confocal microscopic evaluation of corneal Langerhans cell density, and distribution and evaluation of dry eye in rheumatoid arthritis

Corneal Langerhans cells (LCs) offer the opportunity to gain insight into the activity of the innate immunity. We examined the density and the distribution of LCs and compared the results with dry-eye parameters in rheumatoid arthritis (RA). Fifty-two RA patients with various degrees of disease activity and 24 healthy subjects were enrolled. Peripheral and central LC number and morphology were assessed with in vivo laser confocal microscopy. In addition, ocular surface disease index (OSDI), lid parallel conjunctival folds, Schirmer test, and tear break-up time (TBUT) were evaluated. The prevalence of central and peripheral LC, and the central LC morphology values (LCM) were higher than normal in RA. Within the RA group, LC prevalence and morphology were not affected by disease activity. However, patients on anti-TNF or glucocorticosteroid (GCS) therapy exhibited normal LCM, and normal central and peripheral LC density. OSDI was higher and TBUT was lower than normal in RA. The alteration of LC in RA suggests an active inflammatory process in the cornea, which may reflect an increased activation state of the innate immune system—even in inactive stages of RA and without ocular symptoms. The results also indicate ocular effects of GCS therapy in RA.

Soluble urokinase plasminogen activator receptor (suPAR) in the assessment of inflammatory activity of rheumatoid arthritis patients in remission.

Abstract Background: Soluble urokinase plasminogen activator receptor (suPAR) is a biomarker increasingly used for the assessment of systemic inflammation. We aimed to evaluate suPAR for the assessment of inflammatory activity in rheumatoid arthritis (RA) patients in remission. Methods: In our cross-sectional study we measured plasma suPAR and C-reactive protein (CRP) levels as well as erythrocyte sedimentation rate (ESR) in 120 RA patients at various stages of disease activity and 29 healthy age-matched controls. Results: suPAR, CRP and ESR values were higher in RA patients compared to healthy individuals. When suPAR levels were analyzed according to DAS28 scores of RA patients, suPAR level in the subgroup with DAS28≤2.6 was lower than in the subgroup with DAS28>2.6, but still higher than in controls [4.45 (3.33–5.56) ng/mL vs. 3.66 (3.10–4.67) ng/mL vs. 2.80 (2.06–3.42) ng/mL, p<0.0001, median (interquartile range)]. In contrast, CRP and ESR values were comparable in the subgroup with DAS28≤2.6 and in healthy individuals. We further analyzed the correlation between the number of tender and/or swollen joints and suPAR levels in RA patients in remission. suPAR values were significantly higher in patients with four tender and/or swollen joints than in patients with 2–3 or 0–1 tender and/or swollen joints. Conclusions: While CRP and ESR values indicate remission of the chronic inflammatory process in RA, suPAR values are still elevated compared to healthy individuals. suPAR might be particularly valuable in the recognition of inflammatory activity in patients who are in remission according to DAS28 scores but have symptoms of tender and/or swollen joints.

Dry Eye and Corneal Langerhans Cells in Systemic Lupus Erythematosus

Purpose. Investigation of dry eye and corneal Langerhans cells (LCs) in systemic lupus erythematosus (SLE). Methods. Prospective consecutive case series of 27 SLE patients and 27 control subjects. Dry eye was evaluated by lid-parallel conjunctival folds (LIPCOF), Schirmer test, tear break-up time (TBUT), and ocular surface disease index (OSDI) questionnaire. In vivo investigation of corneal LCs density and morphology (LCM) was performed with confocal corneal microscopy (Heidelberg Retina Tomograph with Rostock Cornea Module). Results. Tear production and stability were pathological in SLE subjects compared to control (Schirmer: 8.45 ± 9.82 mm/5 min versus 11.67 ± 3.21 mm/5 min; TBUT: 6.86 ± 3.53 s versus 11.09 ± 3.37 s). OSDI was significantly greater in SLE patients (25.95 ± 17.92) than in controls (11.06 ± 7.18). Central LC density was greater in SLE patients (43.08 ± 48.67 cell/mm2) than in controls (20.57 ± 21.04 cell/mm2). There was no difference in the peripheral LC density (124.78 ± 165.39 versus 78.00 ± 39.51 cell/mm2). LCM was higher in SLE patients in the centre (1.43 ± 0.79) and in the periphery (2.89 ± 0.42) compared to controls (centre: 1.00 ± 0.69, periphery: 2.35 ± 0.54). Conclusions. Significant changes in dry eye parameters and marked increase of central LCs could be demonstrated in SLE patients. SLE alters not only the LC density but also the morphology, modifies corneal homeostasis, and might contribute to the development of dry eye.

Corneal Langerhans cell and dry eye examinations in ankylosing spondylitis.

APCs of the ocular surface, including corneal Langerhans cells (LCs), offer the opportunity to gain insight into the activity of innate immunity. We examined corneal LCs and dry eye parameters in ankylosing spondylitis (AS). Twenty-four AS patients with varying degrees of disease activity and 24 healthy participants were enrolled. Central and peripheral LC numbers, and Langerhans cell morphology (LCM) were assessed with in vivo laser confocal microscopy. In addition, ocular surface disease index, lid parallel conjunctival folds, tear break up time, and Schirmer test were evaluated. LC densities and central LCM were greater in AS patients than in the controls. Moreover, LCM was significantly greater in patients with higher systemic inflammation according to elevated C-reactive protein (CRP). Also, tear production was greatly suppressed in patients with more severe onset of the systemic inflammation according to the Bath Ankylosing Spondylitis Disease Activity Index and elevated CRP. Greater corneal LC density and LCM in AS may reflect an increased activation state of the innate immune system of the cornea in AS, which correlates with the systemic activity of AS even without ocular symptoms. Nonetheless, higher systemic inflammation might impair tear production, and it might partly explain the dry eye mechanism.

Plasma soluble urokinase plasminogen activator receptor (suPAR) levels in systemic lupus erythematosus

Objective: Soluble urokinase plasminogen activator receptor (suPAR) is a biomarker of systemic inflammation. We aimed to characterize plasma suPAR levels in SLE patients. Methods: We measured plasma suPAR, C reactive protein (CRP) and erythrocyte sedimentation rate (ESR) in 89 SLE patients and 29 healthy controls. Results: suPAR and ESR values were higher in SLE than in controls, while CRP levels were comparable. ROC analysis of suPAR levels indicated a cut-off value of 5.70 ng/mL to distinguish patients with high disease activity (SLEDAI >8). Conclusion: suPAR might be an objective marker for identifying SLE patients with active disease.

Successful use of tocilizumab in a patient with rheumatoid arthritis following severe pancytopenia during etanercept therapy.

Severe cytopenia, including neutropenia and anemia, may occasionally occur during anti-tumor necrosis factor &agr; (TNF-&agr;) therapy. However, its mechanism is poorly understood, and little is known concerning the rationale of the choice of biologic therapy after a severe episode of cytopenia. The authors present the case of a 68-year-old rheumatoid arthritis patient in whom severe pancytopenia developed soon after the initiation of etanercept therapy. After resolution, the interleukin 6 receptor-blocking agent tocilizumab was introduced, which resulted in long-lasting complete remission of the rheumatoid arthritis without any adverse effects. The apoptosis-inducing effects of 3 TNF-&agr; blockers and tocilizumab on peripheral blood mononuclear cells of the patient were compared by means of annexin V and propidium iodide labeling and flow cytometry. In concert with the clinical events, the anti-TNF-&agr; agents demonstrated significantly higher apoptotic activities than that of tocilizumab. Tocilizumab appeared safe after anti-TNF-&agr;-induced cytopenia possibly caused by apoptosis induction.