Krisztina Rusai

Increased mucosal expression of toll-like receptor (TLR)2 and TLR4 in coeliac disease

Objectives: The dysregulation of adaptive immunity is extensively investigated in celiac disease (CD). Recent data also suggest, however, the implication of innate immunity in CD. Toll-like receptors (TLRs) play a central role in the initiation or maintenance of innate immune responses. The aim of this study was to characterise the expression of TLR2, TLR3, and TLR4 in duodenal biopsy samples taken from children with CD and from controls. Patients and Methods: Duodenal biopsy specimens were collected from 16 children with untreated CD, 9 children with treated CD, and 10 controls. The mRNA expression of TLR2, TLR3, and TLR4 was determined by semiquantitative reverse transcription-polymerase chain reaction. Protein levels of TLRs were determined by Western blot. Results: We found higher TLR2 and TLR4 mRNA expression and protein levels in the duodenal mucosa of children with treated CD and untreated CD compared with controls. TLR2 and TLR4 mRNA expression and protein levels were even higher in the duodenal mucosa of children with treated CD than in untreated CD. TLR3 mRNA expression was increased in the duodenal mucosa of children with treated CD compared with untreated CD and controls. We were able to detect TLR3 protein only in the biopsy specimens of treated patients with CD. Conclusions: The alteration of TLR2 and TLR4 expression in the duodenal mucosa of patients with CD supports the potential implication of innate immune system in the pathomechanism of this disease.

Hypertension augments cardiac Toll-like receptor 4 expression and activity.

Hypertension causes cardiac hypertrophy characterized by low-grade inflammation. Toll-like receptors (TLRs), members of the innate immune system, contribute to cardiac failure. We hypothesized that hypertension is accompanied by enhanced TLR4 expression and activity. Cardiac TLR4 expression was determined in untreated spontaneously hypertensive rats (SHR) and normotensive Wistar–Kyoto rats (WKY; 4, 8, 16 weeks). Besides, hearts of 8-week-old rats were stimulated with the endogenous TLR4 ligand heparansulfate (HS); the proinflammatory mRNA pattern was assessed (tumor necrosis factor-α (TNF-α), interleukin (IL)-6, monocyte chemotactic protein (MCP)-1). Additionally, we induced hypertension in WKY by L-NAME (Nω-nitro-L-arginine-methylester hydrochloride). In both hypertension models the effect of ramipril on TLR4 density was assessed. Cardiac TLR4 distribution was investigated by fluorescence-activated cell sorting analysis. Blood pressure (BP) and heart weight/body weight ratio (HW/BW) were elevated in SHR. Constitutive TLR4 expression was augmented in adolescent and adult, but not young SHR compared with WKY. TLR4 staining was pronounced in cardiomyocytes. HS entailed an aggravated TNF-α and IL-6 mRNA response in cardiac tissue, which was significantly pronounced in SHR. Ramipril (10 mg kg−1 per day) reduced BP, HW/BW and TLR4 expression in SHR. L-NAME also augmented TLR4 expression in WKY. Ramipril (1 mg kg−1 per day) lowered BP but TLR4 expression remained unaffected. High-dose ramipril (10 mg kg−1 per day) however decreased TLR4 expression. Starting from adolescence SHR demonstrated enhanced cardiac TLR4 expression. TLR4 was also upregulated in L-NAME induced hypertension. Thus, enhanced TLR4 expression might be linked to the development and maintenance of hypertension. Finally, the antihypertensive, anti-inflammatory action of angiotensin-converting-enzyme inhibition had no effect on TLR4 expression in therapeutic doses but in a high-dose model.

Genetic polymorphisms of CD14, toll-like receptor 4, and caspase-recruitment domain 15 are not associated with necrotizing enterocolitis in very low birth weight infants.

Objectives: Inadequate response of the innate immune system to bacterial antigens present in the intestinal flora may play a role in the development of necrotizing enterocolitis (NEC). Pattern recognition receptors such as CD14, toll-like receptor (TLR) 4, and caspase-recruitment domain (CARD) 15 bind bacterial lipopolysaccharide and peptidoglycan, and their activation leads to production of inflammatory cytokines. Our aim was to evaluate whether single nucleotide polymorphisms (SNPs) of CD14, TLR4, and CARD15 are associated with the risk of NEC in very low birth weight (VLBW) infants. Patients and Methods: We determined the CD14 C−260T, TLR4 A + 896G, C + 1196T, and CARD15 G + 2722C, C + 2104T, 3020insC functional SNPs in dried blood samples from 118 VLBW infants (of those, 41 developed NEC) and from 146 healthy term newborns using polymerase chain reaction and restriction fragment length polymorphism methods. We tested the association between genotype and risk of NEC. Results: No significant differences were found in the prevalence of CD14 −260T, TLR4 +896G, +1196T, and CARD15 +2722C, +2104T, 3020insC alleles between VLBW infants and healthy term newborns (P = NS). The frequencies of investigated genotypes were similar in infants with and without NEC (P = NS). Furthermore, we did not find any association between genotype and prematurity or sepsis, which are important risk factors of NEC. Conclusions: Carrier state of the tested CD14, TLR4, and CARD15 SNPs is not associated with NEC risk in VLBW infants.

Administration of interleukin‐1 receptor antagonist ameliorates renal ischemia‐reperfusion injury

Interleukin (IL)‐1 is a major contributor to inflammation and apoptosis during ischemia/reperfusion (I/R) injury. Its deleterious effects are primarily mediated by the activation of nuclear factor‐κB (NF‐κB). Receptor‐binding and signaling of IL‐1 can be blocked by the IL‐1 receptor antagonist (IL‐1ra). The aim of our study was to characterize effects and mechanisms of IL‐1ra administration on inflammation, apoptosis, and infiltration in renal I/R injury. Renal ischemia was induced in Lewis rats by clamping of the left renal artery for 45 min. Kidneys were removed for histological and molecular analysis 24 h or 5 days after reperfusion. IL‐1ra ameliorated I/R induced renal injury and inflammation. Furthermore, the number of apoptotic tubular cells was lower in IL‐1ra‐treated animals 24 h after ischemia, which was paralleled by a Bax/Bcl‐2 mRNA ratio towards anti‐apoptotic effects. IL‐1ra reduced the expression of monocyte chemoattractant protein‐1 (MCP‐1) mRNA at 24 h and 5 days and that of intracellular adhesion molecule‐1 (ICAM‐1) expression at 24 h in the ischemic reperfused kidneys. Our results indicate that IL‐1ra treatment ameliorates renal I/R injury and this protective effect might be mediated by reduced induction of NF‐κB mediated MCP‐1, ICAM‐1, and a decreased ratio between Bax and Bcl‐2 mRNA expression.

Association between heat shock protein 70s and Toll‐like receptor polymorphisms with long‐term renal allograft survival

Long‐term renal allograft survival has not improved significantly in recent years and only a minority of grafts survives for more than 15 years. To evaluate the association between HSPA1A G(190)C, HSPA1B A(1267)G and TLR4 A(299)G polymorphisms and allograft survival we analyzed DNA of patients with long‐term renal graft function over 15 years (Tx15), consecutively transplanted recipients (Tx), patients with acute rejection and healthy controls. HSPA1B (1267)AA was less prevalent in Tx versus Tx15 (P = 0.02) and versus controls (P = 0.004). HSPA1B (1267)GG was more frequent in Tx versus Tx15 (P = 0.005) and versus controls (P = 0.002). HSPA1B (1267)G allele occurred more often in Tx versus Tx15 (P = 0.03), and versus controls (P = 0.02). TLR4 (299)AG genotype prevalence was increased in Tx15 versus Tx (P = 0.02), while TLR4 (299)G allele was more frequent in Tx15 versus Tx (P = 0.02). The increased frequency of HSPA1B (1267)AA and TLR4 (299)AG genotypes in Tx15 group indicates that better cytoprotective functions in HSPA1B (1267)AA and reduced proinflammatory response in TLR4 (299)AG carriers might have improved renal allograft survival.

The importance of different immunosuppressive regimens in the development of posttransplant diabetes mellitus

Prokai A, Fekete A, Pasti K, Rusai K, Banki NF, Reusz G, Szabo AJ. The importance of different immunosuppressive regimens in the development of posttransplant diabetes mellitus.

Galectin-9 in allergic airway inflammation and hyper-responsiveness in mice.

Background: Galectin-9 (Gal-9) is a member of the growing family of β-galactoside-binding lectins. Gal-9 is an eosinophil chemoattractant and inducer of Th1 cell apoptosis. These effects suggest its potential role in the pathogenesis of asthma. Our aim was to study the expression of Gal-9 in an ovalbumin (OVA)-induced mouse model of allergic asthma. Methods: To investigate the significance of Gal-9 in allergic inflammation and airway hyperresponsiveness (AHR), a group of BALB/c mice was sensitized and challenged with OVA (GOVA). Another group of animals was allergized with OVA and also treated with dexamethasone (DEX) (GOVA+DEX). The control group (GPBS) received phosphate-buffered saline instead of OVA as placebo. Airway reactivity to intravenous methacholine was assessed. Results: The percentage of Gal-9-positive cells and their intracellular Gal-9 content and Th1/Th2 cytokine levels in the bronchoalveolar lavage (BAL) were determined by flow cytometry. Gal-9 mRNA expression and protein level were measured in the lung tissue by real-time RT-PCR and Western blot. In GOVA mice, airway inflammation and mucus hypersecretion developed. DEX treatment inhibited the main features of experimental asthma. The number of Gal-9-positive lymphocytes, eosinophil and neutrophil granulocytes and the levels of Th2 cytokines were higher in the BAL of GOVA compared to GPBS or GOVA+DEX mice. Moreover, Gal-9 protein level was elevated in the lungs of GOVA mice. Conclusions: These results suggest that Gal-9 plays a role as a mediator contributing to the development of allergic airway inflammation. Gal-9 may serve as a recruiter of eosinophil granulocytes and promoter of Th2 dominance.

Na+,K+‐ATPase is modulated by angiotensin II in diabetic rat kidney – another reason for diabetic nephropathy?

Angiotensin II (ANGII) plays a central role in the enhanced sodium reabsorption in early type 1 diabetes in man and in streptozotocin‐induced (STZ) diabetic rats. This study investigates the effect of untreated STZ‐diabetes leading to diabetic nephropathy in combination with ANGII treatment, on the abundance and localization of the renal Na+,K+‐ATPase (NKA), a major contributor of renal sodium handling. After 7 weeks of STZ‐diabetes (i.v. 65 mg kg−1) a subgroup of control (C) and diabetic (D7) Wistar rats were treated with ANGII (s.c. minipump 33 μg kg−1 h−1 for 24 h; CA and D7A). We measured renal function and mRNA expression, protein level, Serin23 phosphorylation, subcellular distribution, and enzyme activity of NKA α‐1 subunit in the kidney cortex. Diabetes increased serum creatinine and urea nitrogen levels (C versus D7), as did ANGII (C versus CA, D7 versus D7A). Both diabetes (C versus D7) and ANGII increased NKA α‐1 protein level and enzyme activity (C versus CA, D7 versus D7A). Furthermore, the combination led to an additive increase (D7 versus D7A, CA versus D7A). NKA α‐1 Ser23 phosphorylation was higher both in D7 and ANGII‐treated rats in the non‐cytoskeletal fraction, while no signal was detected in the cytoskeletal fraction. Control kidneys showed NKA α‐1 immunopositivity on the basolateral membrane of proximal tubular cells, while both D7 and ANGII broadened NKA immunopositivity towards the cytoplasm. Our study demonstrates that diabetes mellitus (DM) increases the mRNA expression, protein level, Ser23 phosphorylation and enzyme activity of renal NKA, which is further elevated by ANGII. Despite an increase in total NKA quantity in diabetic nephropathy, the redistribution to the cystosol suggests the Na+ pump is no longer functional. ANGII also caused translocation from the basolateral membrane, thus in diabetic states where ANGII level is acutely elevated, the loss of NKA will be exacerbated. This provides another mechanism by which ANGII blockade is likely to be protective.

The serum and glucocorticoid-regulated kinase 1 in hypoxic renal injury.

The serum- and glucocorticoid-inducible kinase 1 (SGK1) is a serine threonine protein kinase activated through the phosphatidylinositol 3-kinase (PI3-kinase) pathway and counteracting apoptosis. Protein expression and activation of SGK1 are increased in various models of cell stress. The present study explored the role of SGK1 in renal hypoxia/ischemia induced apoptosis. HEK 293 cells were exposed in vitro to hypoxia/reoxygenation (H/R), which increased SGK1 transcript levels, SGK1 protein abundance and SGK1 phosphorylation. H/R injury further enhanced the percentage of apoptotic cells, an effect significantly blunted by prior SGK1 overexpression. In vivo renal ischemia/reperfusion (I/R) injury increased SGK1 transcript levels and SGK1 protein abundance. I/R enhanced apoptosis, an effect significantly more pronounced in gene targeted mice lacking SGK1. In conclusion, SGK1 is up-regulated and counteracts apoptosis following H/R in vitro and ischemia in vivo.

Role of serum and glucocorticoid-regulated kinase-1 in the protective effects of erythropoietin during renal ischemia/reperfusion injury

Erythropoietin (EPO) protects the kidneys from ischemia/reperfusion (I/R) injury; however, the exact signalling mechanisms are not fully understood. The serum and glucocorticoid-regulated kinase 1 (SGK1) is an anti-apoptotic protein kinase regulated through the phosphatidylinositol 3-kinase (PI3-kinase) pathway by cellular stimuli, hormones and growth factors. The objective of the present study was to examine the role of SGK1 in the renoprotective effects of EPO in renal I/R injury. In vitro, cultures of HEK293 cells were exposed to 16h hypoxia. Incubation with EPO at a doses of 400U/ml exerted a protective effect on cell death assessed by LDH release and Annexin V FACS analysis. This was paralleled by up-regulation of SGK1 expression, as well as phosphorylation. Downregulation of SGK1 expression by small interfering RNA technique ameliorated the anti-apoptotic effect of EPO treatment. In an in vivo rat model of unilateral renal I/R injury, rats were treated with 500U/kg EPO 24h prior to ischemia. EPO resulted in less severe tissue injury and ameliorated the elevation in creatinine and urea nitrogen levels 24h after reperfusion. Furthermore, SGK1 expression and phosphorylation were higher in EPO compared to vehicle-treated rats as demonstrated by real-time PCR, Western blot and immunofluorescence technique. We conclude that EPO protects from renal I/R injury and SGK1 might contribute to the mediation of EPO effects under ischemic conditions.