András Treszl

Decreased number of FoxP3+ regulatory T cells in preeclampsia

Systemic inflammation is characteristic for preeclampsia (PE). A hypothesis for immune dysregulation is that the function of regulatory T cells (CD4+FoxP3+, Tregs) inhibiting the activation of lymphocytes is impaired. We investigated the proportion of Tregs and their cellular network in preeclamptic women. Fifteen preeclamptic and 17 healthy pregnant women were enrolled in the 32nd gestational week (median age 29 (range 22–45) and 32 (range 26–38) years, respectively). PE was diagnosed according to international criteria at a median of 30 gestational weeks (range 21–31). Peripheral blood was taken and blood mononuclear cells were isolated. Flow cytometry was used to determine the proportion of regulatory (CD4+FoxP3+) T cells, lymphoid and myeloid dendritic cells, natural killer and natural killer T cells, naive and memory and activated CD4+ and CD8+cells. The proportion of Tregs and that of naive CD4+CD45RA+ cells was lower in preeclamptic than in control women (p = 0.025, p = 0.04, respectively). The proportion of other investigated cell types did not differ. Low Treg numbers may support the notion that PE shares similar features to autoimmune disorders. Low Treg numbers are not reflected in the proportion of activated lymphocytes, at least in this stage of pregnancy. This does not exclude, however, the functional alterations of these cell types.

Reduced CD4+T cell activation in children with type 1 diabetes carrying the PTPN22/Lyp 620Trp variant

The 620Trp variant of the LYP protein, encoded by the lymphoid tyrosine phosphatase 22 gene (PTPN22), is associated with autoimmunity. In this study we aimed at characterising the role of this variant on lymphocyte activation. We analysed cytokine secretion and proliferation of peripheral blood mononuclear cells (PBMCs) and CD4(+)T cells in a cohort of clinically non-diabetic, multiple autoantibody-positive children, healthy controls and in children with type 1 diabetes (T1D). We found a decreased proliferation and IL-2 production of CD4(+)T cells after anti-CD3/anti-CD28 stimulation (p=0.04 for IL-2) among T1D patients. In addition, a profoundly decreased intracellular calcium flux in CD4(+)T cells after PHA stimulus was detected among 620Trp carriers. In contrast, no effect of this polymorphism on tuberculin and tetanus toxoid induced PBMC proliferation and cytokine secretion was observed in autoantibody positive children, healthy controls and children with newly-diagnosed T1D. In conclusion, the LYP 620Trp variant is associated with reduced activation, proliferation and IL-2 production in CD4(+)T cells among T1D patients. In accordance with our previous findings on the key role of this variant on disease progression, this mechanism is likely to contribute to the development of beta-cell specific autoimmunity.

Genetic variants of the interleukin-18 promoter region (-607) influence the course of necrotising enterocolitis in very low birth weight neonates

Interleukin-18-607 polymorphism, as one of the genetic features of host defence, might be associated with the risk for intestinal perforation in very low birth-weight neonates with necrotising enterocolitis. Necrotising enterocolitis (NEC) is a life-threatening disease affecting at least 7% of very low birth weight (VLBW) infants [3]. An immature immune response against a wide range of pathogens is one of the major determinants of this condition. Macrophage-produced immune mediators, including interleukin (IL)-18, play an important role in the immune processes of the intestine [5]. IL-18 plays a key role in the host defence by inducing interferon gamma, amplifying the Th1 cytokine production and stimulating CD8+ and NK cells’ cytotoxicity and IL-8 accumulation [4]. A pathogenic role of altered IL-18 levels in extensive inflammatory processes affecting the intestinal wall has been suggested by data obtained in patients with inflammatory bowel disease [4,5]. Alterations of IL-18 production have been associated with single nucleotide polymorphisms of the IL-18 gene, i.e. C variants at position 607 of the promoter region are associated with somewhat higher levels of IL-18 mRNA [2]. Therefore in our investigation, the association between IL-18 C-607A polymorphism and NEC was studied in VLBW infants. For this purpose, the medical records of 46 VLBW neonates (age 28.9±3.3 weeks, birth weight 1102± 259 g) with NEC were reviewed. Infants were grouped according to the staging criteria of Bell et al. [1]: stage1: presence of abdominal distension and bloody stool (n= 17), stage 2: presence of intestinal pneumatosis (n= 21), and stage 3: presence of perforated bowel (n=8). As controls, 90 gestational age and birth weight matched VLBW infants (age 29.5±2.6 weeks, birth weight 1170±240 g) were enrolled from the same cohort. IL-18 C-607A polymorphism was analysed in dried blood spots by multiplex touch down PCR. Informed parental consent was obtained. After inactivation of the haemoglobin content [6], PCR was performed according to the method of Giedraitis et al. [2] with the following modifications: 20 mM Tris-HCl pH 8.4, 2.5 U Taq, 2.1 mM MgCl2. Each primer was used at a concentration of 0.5 lM and the final volume was 60 ll. Data were analysed with Fisher’s exact test. The genotype frequencies of the investigated VLBW population showed no difference compared to those of a normal population as described by Giedraitis et al. [2]. The prevalence of the A-607 allele was similar in VLBW infants with or without NEC (0.43 versus 0.37). The number of CC, CA and AA carriers was also similar. Analysing the association of genotype with disease severity, the frequency of the AA genotype was higher in infants with stage 3 NEC compared to those with stages 1 and 2 and those without NEC (4/8 versus 4/38 and 7/90, P<0.05 and P<0.05, respectively). Our results suggest that the risk of NEC is not associated with the presence of A-607 alleles. However, a higher prevalence of the AA genotype was found in babies with a severe form of the disease, i.e. perforation. Assuming the hypothesis that IL-18 levels have a defencive activity in intestinal inflammation in NEC and the A-607 polymorphism is associated with slightly lower IL-18 mRNA production [2], our results indicates that the presence of an AA genotype might adversely affect the outcome of NEC in VLBW babies. Supported by OTKA grants F 032024-038151.

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.

Cytometry‐acquired calcium‐flux data analysis in activated lymphocytes

Flow cytometry enables the sequential determination of calcium levels in millions of stimulated lymphocytes over a short period of time. Current algorithms available are not suitable for the statistical analysis of this large amount of data. The authors aimed to develop a robust algorithm that fits a function to median values of measured data and provides an opportunity for statistical comparison between different calcium‐flux measurements. The alteration of calcium signal was monitored in CD4+ cells loaded with calcium binding fluorescent dyes and stimulated with phytohemagglutinin; the alteration of calcium signal was monitored for 10 minutes. The authors also reanalyzed published calcium‐flux data of CD3+ cells and Jurkat cells stimulated with different concentrations of anti‐CD3 and thapsigargin. The authors fitted different functions to the medians of data per time unit and identified hormesis function as the best fitting one. On the basis of the optimally fitting function, the authors calculated the most relevant biological descriptors such as starting value, peak, time to reach the maximum, and time to reach 50% of maximum before and after the peak. Statistically significant differences in cell activation kinetics at different stimulatory concentrations were also demonstrated. This approach enables us to characterize the kinetics and distribution of calcium‐flux data derived by flow cytometry and may be a reliable tool for the characterization of lymphocyte activation (for details see: http://calciumflux.intralab.eu).

Genetic variants of the tumour necrosis factor-alpha promoter gene do not influence the development of necrotizing enterocolitis.

Previous studies indicated that elevated tumour necrosis factor‐alpha (TNF‐α) levels may play a role in the development of necrotizing enterocolitis (NEC). The A–238 and A–238 variants of the promoter region of the TNF‐α gene are reportedly associated with altered TNF‐α production. The aim of our study was to determine the impact of these gene polymorphisms on the development and course of NEC in very‐low‐birthweight (VLBW) infants. Dried blood samples from 46 VLBW neonates with NEC were analysed using the method of restriction fragment length polymorphism. Samples from 90 VLBW neonates without NEC were used as controls. The prevalence of alleles with guanine‐adenine transition in the –308 and –238 positions was the same in NEC and control subjects (12% vs 10% and 3% vs 4%, respectively).

Variance of ACE and AT1 receptor gene does not influence the risk of neonatal acute renal failure

Abstract. High neonatal activity of the renin-angiotensin system (RAS) is crucial for the maintenance of glomerular filtration of the newborn. The aim of the present study was to investigate whether genetic polymorphisms leading to lower angiotensin converting enzyme activity (ACE) or impaired functionality of angiotensin II (AII) type 1 receptor (AT1R) might predispose very low birth weight newborns (VLBWs) to the development of acute renal failure (ARF). The medical records of 110 VLBW infants were analyzed. ARF developed in 42 of them during the first postnatal week, while 68 neonates exhibited normal renal function. The ACE I/D polymorphism and the A1166C variants of AT1R were determined from dried blood samples. The frequency of the ACE I allele did not differ in ARF and non-ARF groups (0.307 and 0.284); the frequency of the AT1R C1166 variant was also the same in ARF and non-ARF groups (0.250 and 0.227). Although low activity of RAS has been implicated in the development of neonatal ARF and data indicated that the functionality of RAS is influenced by the I/D variants of the ACE gene and the A1166C variant of the AT1R gene, we could not demonstrate any effect of these polymorphisms on the development of ARF in VLBW infants.

Lymphocyte activation in type 1 diabetes mellitus: the increased significance of Kv1.3 potassium channels.

Kv1.3 and IKCa1 potassium channels participate in the maintenance of calcium-influx during lymphocyte activation. Kv1.3 channels have a prominent role in specific T cell subsets, presenting a possible target for selective immunomodulation. We investigated the impact of Kv1.3 and IKCa1 channel inhibitors on calcium-influx characteristics in human T cells in type 1 diabetes mellitus. We isolated lymphocytes from 9 healthy and 9 type 1 diabetic individuals and measured the alteration of calcium-influx with flow cytometry in the Th1, Th2, CD4 and CD8 subsets after treatment of samples with specific channel inhibitors. Our results indicate an increased reactivity of type 1 diabetes lymphocytes, which is correlated to their increased sensitivity to Kv1.3 channel inhibition. However, the contribution of Kv1.3 channels to calcium flux is not exclusive for a specific lymphocyte subset as previous reports suggest, but is characteristic for each subset investigated. Therefore, the proposed inhibition of Kv1.3 channels as a novel therapeutic approach for the treatment of type 1 diabetes mellitus may have a major effect on overall lymphocyte function in this disease.

Immune Phenotype of Children with Newly Diagnosed and Gluten-Free Diet-Treated Celiac Disease

BackgroundRecent data suggest the involvement of both the adaptive and the innate immune system in celiac disease (CD). However, little is known about the immune phenotype of children with CD and its alteration upon dietary intervention.AimsWe characterized the prevalence of major interacting members of the adaptive and innate immune system in peripheral blood of newly diagnosed children with CD and tested its alteration with the improvement of clinical signs after the introduction of gluten-free diet (GFD).MethodsPeripheral blood was taken from ten children with biopsy-proven CD at the time of diagnosis and after the resolution of clinical symptoms following GFD. As controls, 15 children with functional abdominal pain were enrolled. The prevalence of the cells of adaptive and innate immunity was measured with labeled antibodies against surface markers and intracellular FoxP3 using a flow cytometer.ResultsPatients with CD were found to have lower T helper, Th1 and natural killer (NK), NKT and invariant NKT cell prevalence and with higher prevalence of activated CD4+ cells, myeloid dendritic cells (DC) and Toll-like receptor (TLR) 2 and TLR-4 positive DCs and monocytes compared to controls. After resolution of symptoms on GFD, the majority of these changes normalized, although the prevalence of NK and NKT cell, DC and TLR-2 expressing DCs and monocytes remained abnormal.ConclusionsThe immune phenotype in childhood CD indicates the implication of both adaptive and innate immune system. The normalization of immune abnormalities occurs on GFD, but the kinetics of this process probably differs among different cell types.

The extent to which genotype information may add to the prediction of disturbed perinatal adaptation: None, minor, or major?

Studies have been performed to describe the significance of genetic polymorphisms in complications associated with disturbed perinatal adaptation. Due to the large number of interacting factors, the results of classic statistical methods are often inconsistent. The random forest technique (RFT) is a robust nonparametric statistical approach that overcomes this problem through the calculation of the importance of each factor. We used RFT to reanalyze the importance of 24 genetic polymorphisms in the classification of preterm infants (birth weight, 680–1460 g, n = 100) to affected and unaffected groups according to the presence of acute perinatal complications. The accuracy of classification was between 0.5 and 0.8 for each complication when only birth data were considered. However, when genetic polymorphisms with the highest importance scores (ISs) were included in the analysis, the accuracy of classification according overall morbidity, necrotizing enterocolitis (NEC), acute renal failure (ARF), infant respiratory distress syndrome (IRDS), cardiac failure (CF), and patent ductus arteriosus (PDA) improved from 0.69, 0.60, 0.70, 0.72, 0.68, and 0.57 to 0.77, 0.70, 0.76, 0.77, 0.76, and 0.64, respectively. Our findings suggest that genetic polymorphisms identified by RFT as predictors may improve the risk assessment of preterm infants. RFT is a suitable tool to develop risk factor patterns in this population.