Péter Antal-Szalmás

Evaluation of CD14 in host defence.

An extensive search for the cell membrane targets for lipopolysaccharide (LPS), the major causative agent of Gram‐negative septic shock, resulted in the identification of CD14 as the major endotoxin ‘receptor’. Besides recognition of LPS, several new aspects of its biological functions have been described recently. In this review the different CD14 forms, their most important biological and biochemical features, signalling properties, cellular and subcellular distribution and association with different diseases are discussed in detail, showing that these molecules posses several unique biological functions and further proving their central role in innate immunity.

Quantitation of surface CD14 on human monocytes and neutrophils.

The absolute number of membrane‐expressed CD14, the most important endotoxin receptor, on human monocytes and neutrophils shows remarkable variation in the literature. To quantify these numbers two fluorescence methods using fluorescein isothiocyanate (FITC)‐labeled monoclonal antibodies (mAb) were applied. A commercially available set of standard beads was used in flow cytometry to quantitate CD14 with eight different mAbs. Independent from their isotype the various mAbs showed minor differences and indicated that peripheral blood monocytes expressed 99,500–134,600 (115,400 ± 10,600) and neutrophils 1,900–4,400 (3,300 ± 800) CD14 receptors. There was no significant difference in CD14 expression on leukocytes in unprocessed freshly obtained whole blood and after a Ficoll isolation procedure. However, a short temperature shift resulted in a 1.3‐ to 1.6‐fold upregulation of CD14. The results obtained with the reference beads were verified with fluorescence Scatchard analysis and spectrofluorometry using mAb 26ic‐FITC and showed 109,500 CD14 per monocyte and 6,700 CD14 per neutrophil. For comparison the number of CD14 on the monocytic THP‐1 cells and Fcγ‐receptors on human leukocytes were determined using the reference beads and flow cytometry and gave results comparable to published data. Our data indicate that resting human monocytes express roughly 110,000 CD14 molecules on their surface using a simple fluorometric assay. Correct determination of the number of CD14 and other cell surface receptors is of importance in the monitoring of septic patients. J. Leukoc. Biol. 61: 721–728; 1997.

Elevated rate of Thelper1 (TH1) lymphocytes and serum IFN-γ levels in psoriatic patients

Abstract Several disorders are known to be associated with altered Thelper1/Thelper2 (T H 1/T H 2) cytokine balance. Psoriasis is characterized by increased systemic and local production of T H 1 and pro-inflammatory cytokines. Furthermore recent data indicate the dominant presence of T H 1 lymphocytes in the circulation and T H 1 and Tcytotoxic1 (T C 1) cells in lesional skin of psoriatic patients. In order to assess the systemic T H 1/T H 2 imbalance in psoriasis most of the studies so far tested isolated peripheral mononuclear cells. As a new approach we applied a whole blood flow cytometric assay to determine the rate of circulating T H 1/T H 2 and T C 1/Tcytotoxic2 (T C 2) lymphocytes based on their intracellular IFN-γ, IL-4 and IL-10 expression. Besides, serum levels of these cytokines were determined in healthy controls and psoriatic patients by commercial ELISAs. In psoriatic patients we found significantly ( P + /IFN-γ + lymphocytes (30.3±8.8%) while the percent of CD4 + /IL-4 + cells (0.37±0.31%) were significantly ( P + /IFN-γ + : 20.1±7.3% and CD4 + /IL-4 + : 0.78±0.44%). The IL-10-positive CD4 + and CD8 + cells also had higher rate in psoriasis, but the difference between patients and controls was not significant, similarly to the rate of CD8 + /IFN-γ + and CD8 + /IL-4 + lymphocytes. Beside cellular expression, serum IFN-γ levels were also significantly higher (control: 4.9±6.4 pg/ml; psoriatic patients: 35.9±47.0 pg/ml; P H 1/T H 2 balance in psoriasis measured in non-separated whole blood T cells.

Acute phase proteins in the diagnosis and prediction of cirrhosis associated bacterial infections

Bacterial infections are common cause of morbidity and mortality in patients with cirrhosis. The early diagnosis of these infections is rather difficult.

Increased frequency of intracellular interleukin (IL)-13 and IL-10, but not IL-4, expressing CD4+ and CD8+ peripheral T cells of patients with atopic dermatitis.

Summary Background  A number of studies exist demonstrating the increased expression of type 2 cytokines and decreased capacity to produce interferon‐γ (IFN‐γ) in peripheral blood mononuclear cells (PBMCs) of patients with atopic dermatitis (AD).

Elevated human epididymis protein 4 concentrations in chronic kidney disease

Background Human epididymis protein 4 (HE4) has recently become an available tumour biomarker for detecting ovarian cancer along with the standard cancer antigen 125 (CA125). However, it is unknown if the levels of HE4 and CA125 may be altered in subjects who have impaired renal function with no ovarian disorders. Methods In 113 female patients at different stages of chronic kidney disease (CKD) with no ovarian and lung cancer and 68 subjects with normal renal and ovarian function, HE4 and CA125 concentrations were analysed by using chemiluminescent microparticle immunoassay (Architect®, Abbott) and electrochemiluminescent immunoassay (Modular E170®, Roche), respectively. Renal function was evaluated by measuring serum creatinine and urea concentrations (Cobas Integra-800®, Roche). Estimated glomerular filtration rate (eGFR in mL/min/1.73 m2) was calculated by the 4v-MDRD formula. Results Significantly increased HE4 concentrations (P < 0.0001) were found in individuals with differently decreased eGFR values (<90 mL/min/1.73 m2) compared with clinical controls. CA125 serum concentration was higher than normal in subjects with CKD3 (eGFR = 30–59 mL/min/1.73 m2), but significant elevation (P = 0.006) in CA125 concentrations was seen only in those who had severe renal failure (CKD4-5; eGFR < 30 mL/min/1.73 m2). These tendencies were independent of age in our study cohort, and seemed to be more evident among women in premenopausal status. Conclusions HE4 concentrations may be elevated in CKD patients with no ovarian and lung cancer. Thus, HE4 results should be interpreted cautiously in women with renal disorders.

Immune dysfunction in cirrhosis.

Innate and adaptive immune dysfunction, also referred to as cirrhosis-associated immune dysfunction syndrome, is a major component of cirrhosis, and plays a pivotal role in the pathogenesis of both the acute and chronic worsening of liver function. During the evolution of the disease, acute decompensation events associated with organ failure(s), so-called acute-on chronic liver failure, and chronic decompensation with progression of liver fibrosis and also development of disease specific complications, comprise distinct clinical entities with different immunopathology mechanisms. Enhanced bacterial translocation associated with systemic endotoxemia and increased occurrence of systemic bacterial infections have substantial impacts on both clinical situations. Acute and chronic exposure to bacteria and/or their products, however, can result in variable clinical consequences. The immune status of patients is not constant during the illness; consequently, alterations of the balance between pro- and anti-inflammatory processes result in very different dynamic courses. In this review we give a detailed overview of acquired immune dysfunction and its consequences for cirrhosis. We demonstrate the substantial influence of inherited innate immune dysfunction on acute and chronic inflammatory processes in cirrhosis caused by the pre-existing acquired immune dysfunction with limited compensatory mechanisms. Moreover, we highlight the current facts and future perspectives of how the assessment of immune dysfunction can assist clinicians in everyday practical decision-making when establishing treatment and care strategies for the patients with end-stage liver disease. Early and efficient recognition of inappropriate performance of the immune system is essential for overcoming complications, delaying progression and reducing mortality.

Altered cytokine expression of peripheral blood lymphocytes in polymyositis and dermatomyositis

Objective: To investigate the intracellular and soluble cytokine levels and T cell subsets in peripheral blood of patients with active and inactive polymyositis and dermatomyositis. Methods: The frequencies of T and B lymphocytes, T helper (Th), and T cytotoxic (Tc) cells and of interferon γ (IFNγ), interleukin (IL)4, and IL10 expression of CD4+ or CD8+ cells were determined by flow cytometry. The concentrations of soluble cytokines were measured with commercial enzyme linked immunosorbent assays. Results: In active dermatomyositis there was a decreased percentage of T (CD3+) lymphocytes and Tc (CD8+) lymphocytes, decreased IFNγ expression of CD4+ and CD8+ cells, but an increase in B and IL4 producing CD4+ lymphocyte frequencies. These prominent changes disappeared in the inactive stage of the disease. In polymyositis no significant change in these lymphocyte subsets or in intracellular cytokine expression could be detected in either the active or the inactive form. The frequency of IL4+/IFNγ+ Th cells was calculated and a significantly increased Th2/Th1 frequency was found in active dermatomyositis, and a decreased frequency in inactive dermatomyositis, compared with the control population. Conclusions: There appears to be a difference between polymyositis and dermatomyositis in the level of peripheral blood lymphocytes and their intracellular cytokine content. These findings provide further evidence for a difference in the pathogenesis of polymyositis and dermatomyositis.

Ferritin Prevents Calcification and Osteoblastic Differentiation of Vascular Smooth Muscle Cells

Vascular calcification plays a role in the pathogenesis of atherosclerosis, diabetes, and chronic kidney disease. Human aortic smooth muscle cells (HSMCs) undergo mineralization in response to elevated levels of inorganic phosphate (Pi) in an active and well-regulated process. This process involves increased activity of alkaline phosphatase and increased expression of core binding factor alpha-1, a bone-specific transcription factor, with the subsequent induction of osteocalcin. Mounting evidence suggests an essential role for the heme oxygenase 1 (HO-1)/ferritin system to maintain homeostasis of vascular function. We examined whether induction of HO-1 and ferritin alters mineralization of HSMCs provoked by high Pi. Upregulation of the HO-1/ferritin system inhibited HSMC calcification and osteoblastic differentiation. Of the products of the system, only ferritin and, to a lesser extent, biliverdin were responsible for the inhibition. Ferritin heavy chain and ceruloplasmin, which both possess ferroxidase activity, inhibited calcification; a site-directed mutant of ferritin heavy chain, which lacked ferroxidase activity, failed to inhibit calcification. In addition, osteoblastic transformation of HSMCs provoked by elevated Pi (assessed by upregulation of core binding factor alpha-1, osteocalcin, and alkaline phosphatase activity) was diminished by ferritin/ferroxidase activity. We conclude that induction of the HO-1/ferritin system prevents Pi-mediated calcification and osteoblastic differentiation of human smooth muscle cells mainly via the ferroxidase activity of ferritin.

Measurement of intracellular interferon-gamma and interleukin-4 in whole blood T lymphocytes from patients with systemic lupus erythematosus

Contradictory data are available about the dominance of T-helper 1 (T(H)1), or T-helper 2 (T(H)2) cytokines in systemic lupus erythematosus (SLE). Therefore, intracellular interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) production of T lymphocytes was measured in whole blood of healthy donors and active and inactive SLE patients by flow cytometry. The percentage of IFN-gamma and IL-4 positive cells was low (<1%) in unstimulated samples of the healthy controls, while that of IFN-gamma and IL-4 positive cells in the stimulated cells was 25.2+/-10.6% and 0.6+/-1.5%, respectively. No significant difference was found between SLE patients and healthy controls and between active and inactive patients in these parameters either in the unstimulated or in the stimulated samples. One patient with severe disease had as high as 11.8% IL-4 positive cells and 12.5% IFN-gamma positive cells in the stimulated samples, but after the initiation of intensive corticosteroid and cytostatic therapy, the percentage of IL-4 positive T cells decreased (4.76%) while that of IFN-gamma positive T cells increased (47.91%). We conclude that the intracellular IL-4 and IFN-gamma expression of T lymphocytes does not differ markedly between SLE patients and healthy controls, with the possible exception of severe disease, when marked IL-4 overproduction may exist beside low IFN-gamma production. Furthermore, corticosteroid and cytostatic therapy might normalize this altered IFN-gamma/IL-4 ratio.