Introduction and Aims:In our previous studies we found that exercise partially prevents chronic kidney disease (CKD)-induced muscle atrophy.Effective therapeutic strategies to treat Chronic Kidney Disease (CKD)-induced muscle atrophy are urgently needed.Low frequency electrical stimulation (LFES) induces muscle contraction which mimics acupuncture and exercise, and may be effective in preventing muscle atrophy.Methods: CKD was induced in 20-25g mice by 5/6th nephrectomy.CKD and control mice were treated with LFES for 15 days.Results: LFES prevented soleus and EDL muscle weight loss in CKD mice ( p<0.05, LFES/CKD vs. CKD) as well as loss of hind-limb muscle grip.LFES countered the CKD-induced decline in the IGF-1 signaling pathway, leading to increased protein synthesis, decreased protein degradation and increased myogenesis markers.There is an acute (immediate) response phase during which inflammation cytokines (IFN and IL-6) increased.M1 macrophage markers ( pro-inflammation: IL-1β) were also increased acutely, but M2 markers (anti-inflammation: arg-1, IL-10 and IL-4) were increased 2-days after initiation of LFES.In addition, microRNA-1 and -206 were decreased in the acute phase after starting LFES.Conclusions: We conclude that LFES ameliorates CKD-induced skeletal muscle atrophy by up-regulation of the IGF-1 signaling pathway which improves protein metabolism and promotes myogenesis.The potential mechanisms leading to upregulation of IGF are 1) decrease of microRNA-1 and -206 in the early phase of LEFS resulting in reversal of the inhibition of IGF-1 production by these microRNAs or 2) direct secretion of IGF-1 by M2 macrophages in the later phase inflammatory response.

In COPD airway obstruction causes hypoventilated areas in the lung leading to alveolar hypoxia. HIF-1 α plays an important role in the response to hypoxia and protection of cells against harmful effects. Role of alveolar oxygen tension and HIF-1α in the development of COPD are unknown. Heathly volunteers (n=7) and 9 patients with COPD were enrolled into clinical examinations including lung function test and blood gas analysis. Mass spectrometry was used to measure exhaled O2 on the level of functional reserve capacity (FRC) and residual volume (RV). HIF1α mRNA and protein expression of alveolar epithelial cells (A549) were analyzed by RT-PCR and flow cytometry using hypoxia (FiO2: 20,9% (control); 13±1%; 6±1%; 1±1%). Exhaled O2 on the level of FRC did not change,(C:15.6±1.2 vs. COPD: 15,8±1.4 %), while on the level of RV the alveolar FiO2 was significantly decreased (C: 13,6±1,8 vs. COPD: 11.6±2.1%, p<0.05) in patients with COPD and the value correlated to pO2 and showed negative correlation to RV. HIF-1α mRNA and protein expression significantly increased in mild hypoxia, extreme hypoxia did not change the mRNA expression. Hypoxia significantly increased the A549 cell count in all treated groups (FiO2 13±1%: 13,33x105; FiO2 6±1%:13,2x105; FiO2 1±1%: 12,86x105 vs. control FiO2 20,9%: 7,73x105; p<0.01). In COPD the exhaled O2 level is decreased in parellel to hyperinflation, demonstrating that there are more severe hypoxic areas in the lungs compare to physiological conditions. Mild hypoxia increases HIF1α mRNA and protein expression, while in severe hypoxia these changes are absent.

Recently, it has been suggested that the gene called Parkinson’s disease 7 (PARK7) might be an upstream activator of hypoxia-inducible factor (HIF)-1α, which plays a major role in sustaining intestinal barrier integrity. Furthermore, PARK7 has been proposed to participate in the Toll-like receptor (TLR)-dependent regulation of the innate immune system. Our aim was to investigate the involvement of PARK7 in the pathogenesis of coeliac disease (CD). Duodenal biopsy specimens were collected from 19 children with untreated CD, five children with treated CD (maintained on gluten-free diet), and ten children with histologically normal duodenal biopsies. PARK7 mRNA expression and protein level were determined by real-time polymerase chain reaction (PCR) and Western blot, respectively. Localization of PARK7 was visualized by immunofluorescence staining. Protein level of PARK7 increased in the duodenal mucosa of children with untreated CD compared to children with treated CD or to control biopsies ( p <0.03). We detected intensive PARK7 staining in the epithelial cells and lamina propria of the duodenal mucosa of children with untreated CD compared with that in control biopsies. Our finding that mucosal expression of PARK7 is increased suggests that PARK7 is involved in the pathogenesis of gastrointestinal diseases, notably CD. Our results suggest that PARK7 may alter processes mediated by HIF-1α and TLR4, which supports a role for PARK7 in the maintenance of epithelial barrier integrity, immune homeostasis, or apoptosis.

Coeliac disease is a complex inflammatory disorder of the small intestine with autoimmune features in genetically predisposed individuals and triggered by chronic exposure to gluten of wheat, barley and/or rye (Trynka et al., 2010). The aim of the recent chapter is to introduce and characterize a family of proteins, called heat shock proteins (HSPs), which are known to be key molecules during stress responses (Polla & Cossarizza, 1996). We will discuss the potential involvement of HSPs in the pathomechanism of coeliac disease based on recent scientific results. We will also refer to some future directions and potential therapeutic intervention.

Tacrolimus (Tac) and Cyclosporin A (CyA) are two effective immunosuppressant which are essential therapeutic solutions of the prevention of the allograft rejection. However, it is well known that both of them posses nephrotoxic potential. The precise effect how these drugs act as nephrotoxic agents are still not fully cleared. In this study we investigated in-vivo and real time the effect of calcineurin-inhibitory drugs on renal renin system. Three week old, male C57 black 6 mice (n=15) were divided into three groups: control mice (C), treated with 0.075 mg/kg/day of Tac twice a day (Tac) or 2mg/kg/day of CyA (CyA). After three weeks of administratration serum creatinin was measured, then the renin contant in the collecting duct (CD) and juxtaglomerular apparatus (JGA) were evaluated applying FACS and multi-foton microscopy. The contraction of the vessels was assessed and the consequent fibrosis was determined by Masson staining. Serum creatinin was significantly elevated in the Tac and CyA groups. Both the JGA and CD renin content increased four time higher following the administration with calcineurin inhibitors, which was further supported by multi-foton microscopy, the renin granulation increased remarkably in both localizations. As a result of the local activation vasoconstriction was in present in both treated groups and as early as the third week of the treatment with immunosuppressants fibrotic islands were found in the kidney. In summary, our studies revealed first that calcineurin inhibitors possess nephrotoxic effect on the kidney parenchyma due to the enhanced renin activity not only in JGA but in the collecting duct segment as well. Therefore, the inhibition of renin-angiotensin system could be beneficial in prevention of nephrotoxic effect of the calcineurin inhibitors. However, further studies are needed to reveal what kind of inhibitors and in which combination could provide the most efficient treatment? REG_KM_09-1-2009-0016 BAROSS, REG_KM_09-1-2009-0016, TÁMOP-4.2.1.B-09/1/KMR, TÁMOP-4.2.2/B-10/1-2010-0013, SE-MTA Lendulet LP2001-008/2011

There are about quarter of million patients on chronic renal replacement therapy in Europe, and the estimated number of patients with chronic kidney disease, stages 1-4 is about tenfold higher. Interestingly, regardless of the initiating cause (infection, autoimmune response, chemical insult, radiation or tissue injury etc.), the mechanism of fibrosis is similar in the different chronic kidney diseases and characterized by inflammation. In general, the damaged glomerular or tubular cells release danger signals (Anders, 2010; McDonald et al., 2010) and produce chemotactic stimuli, which trigger the rapid recruitment of leukocytes. The infiltrating immune and the damaged renal cells then produce high levels of proinflammatory cytokines, growth factors, chemokines and adhesion molecules which contribute to glomerular/tubular injury, accumulation of further leukocytes and myofibroblasts, which are the effector cells of renal fibrosis. However the origin of the myofibroblasts is still controversial recent hypotheses suggest that myofibroblasts can originate from different renal cells, such as epithelial and endothelial cells, pericytes or the bone marrow derived fibrocytes. The thus generated myofibroblasts then serves as the key cellular mediator of renal fibrosis. Myofibroblasts have migratory capacity, are resistant to apoptosis, produce several growth factors and cytokines and according to our present knowledge these cells are the main source of the collagen-I and collagen-III rich extracellular matrix in the fibrous tissue. Organ fibrosis is characterized by excessive deposit of extracellular matrix (ECM) leading to glomerular sclerosis and renal tubule-interstitium fibrosis. The excessive deposition of fibrous tissue replaces healthy kidney tissue; the nephrons disappear and the kidney function gradually declines. In this chapter we will summarize our knowledge about the role of immune cells and molecular changes leading to generation of renal myofibroblasts.

T helper lymphocytes become polarized upon antigen and cytokine stimuli received after their maturation in the thymus. Since the balance of Th1 and Th2 responses is critical in healthy and pathological immune responses, understanding the molecular base of T cell polarization still remained an important question. Using our Th0/Th1/Th2 hybridoma model system, we performed a comparative study on polarized Th1 and Th2 cells in terms of their membrane raft expression/composition, their TCR mediated activation signaling, and sensitivity to activation‐induced cell death (AICD) using flow and image cytometric methods. We show here that the TCR stimulation induced more intense and sustained Ca2+‐response in Th1 cells compared to Th2 ones correlates well with a shorter nuclear residence time of the Ca2+‐dependent NFAT transcription factor in Th2 cells. In addition, NFAT translocation directly depended on lipid raft integrity/membrane cholesterol level. Expression pattern of raftophilic accessory proteins (CD4, CD59, and CD48) and lipids (GM1, cholesterol) were also different in the Th1 and Th2 hybridomas, similarly to differentiated spleen Th cells. The activation‐induced, remarkably clustered and polarized membrane distribution of TCR/CD3 complex in Th1, but not in Th2 cells, together with an increased raft localization of Kv1.3 ion channels regulating the Ca2+‐response, are consistent with the above properties of NFAT. Finally, the polarized Th cells, especially Th1, were more sensitive to AICD than their unpolarized Th0 precursor. These results suggest that the membrane microdomain organization—Ca2+‐signaling—NFAT activation axis is an important determinant of polarized Th cell effector function and fate. © 2012 International Society for Advancement of Cytometry

Microglia are activated by pathogen-associated molecular patterns and produce proinflammatory cytokines, such as TNF-α, IL-6, and IL-12, and the anti-inflammatory cytokine IL-10. Adenosine is an endogenous purine nucleoside and a ligand of four G protein-coupled adenosine receptors (ARs), which are the A(1)AR, A(2A)AR, A(2B)AR, and A(3)AR. ARs have been shown to suppress TNF-α production by microglia, but their role in regulating IL-10 production has not been studied. In this study, we demonstrate that adenosine augments IL-10 production by activated murine microglia while suppressing the production of proinflammatory cytokines. Because the order of potency of selective AR agonists in inducing IL-10 production was NECA > IB-MECA > CCPA ≥ CGS21680, and the A(2B)AR antagonist MRS1754 prevented the effect of NECA, we conclude that the stimulatory effect of adenosine on IL-10 production is mediated by the A(2B)AR. Mechanistically, adenosine augmented IL-10 mRNA accumulation by a transcriptional process. Using mutant IL-10 promoter constructs we showed that a CREB-binding region in the promoter mediated the augmenting effect of adenosine on IL-10 transcription. Chromatin immunoprecipitation analysis demonstrated that adenosine induced CREB phosphorylation at the IL-10 promoter. Silencing CREB using lentivirally delivered short hairpin RNA blocked the enhancing effect of adenosine on IL-10 production, confirming a role for CREB in mediating the stimulatory effect of adenosine on IL-10 production. In addition, adenosine augmented IL-10 production by stimulating p38 MAPK. Collectively, our results establish that A(2B)ARs augment IL-10 production by activated murine microglia.

Abstract Microglia, the intrinsic macrophages of the central nervous system produce the anti-inflammatory cytokine IL-10 following activation with the bacterial cell wall product peptidoglycan (PGN), which is recognized by Toll-like receptor 2 (TLR2). Adenosine is an endogenous purine nucleoside that binds to specific G protein-coupled receptors (A1, A2A, A2B, and A3), and is a well known modulator of the immune system. In this study we investigated the effect of adenosine on IL-10 production by microglia. Cells were treated with adenosine, or selective adenosine receptor agonists and antagonists, in conjunction with 20 μg/ml PGN for 6 or 24 hours. Adenosine treatment augmented IL-10 production by microglia activated with PGN. The non-selective adenosine receptor agonist NECA was the most potent IL-10 inducer, and its effect was prevented by pretreatment with the A2B antagonist MRS-1754. Adenosine receptor activation augmented IL-10 mRNA levels, and this effect was prevented by blocking transcription with actinomycin D. The stimulatory effect of adenosine on IL-10 production was mediated by p38 because it was reversed with a p38 pathway inhibitor. IL-10 promoter analysis and chromatin immunoprecipitation (CHIP) experiments suggested that CREB activation is necessary for the effect of adenosine. These results demonstrate that A2B adenosine receptor activation augments IL-10 production by PGN-activated microglial cells through a p38- and CREB-mediated pre-transcriptional mechanism.

Th17 cells are the newly described subset of the CD4(+) T lymphocytes. Activated Th17 cells are characterized by their ability to produce IL-17A and other pro-inflammatory cytokines. IL-17A regulates immune function through its cell-surface receptor expressed on epithelial-and endothelial cells, fibroblasts and leukocytes by promoting neutrophil recruitment and releasing further pro-inflammatory mediators. Failures of the susceptible balance of the immunoregulation may lead to unchecked immune response and autoimmune diseases. The central role of Th17 cells and cytokines produced by Th17 cells were confirmed in a wide variety of human autoimmune diseases, including rheumatoid arthritis. Recently Th17 cells and its cytokines come into the focus of immunological research as potential therapeutic targets.