Jerko Barbić

Association Studies of Gene Polymorphisms in Toll-Like Receptors 2 and 4 in Croatian Patients with Acute Myocardial Infarction

The aim of the study was to assess the frequency of SNP896A/G in the Toll‐like receptor (TLR) 4 gene and SNP1350T/C in the TLR2 gene in patients with acute myocardial infarction (AMI) and to analyse the association of these SNPs with risk factors for atherosclerosis and clinical aspects of AMI in a sample of the Croatian population. We included 240 participants in the study: 120 AMI patients and 120 sex‐ and age‐matched healthy blood donor controls. The SNP1350T/C variant in the TLR2 gene showed a lower frequency in the AMI patient group than in the control group (P = 0.033). The frequency of SNP896A/G variants in the TLR4 gene between the patients and the controls did not differ (P = 0.286). Significantly, fewer people had SNP1350T/C in the TLR2 gene (P = 0.003) among the participants with arterial hypertension than those without it. The frequency of SNP896A/G in TLR4 was the same in hypertensive patients compared with normotensive subjects (P = 0.088). SNP1350T/C in TLR2 was less frequent in the AMI patients and in those with hypertension. Thus, SNP1350T/C in TLR2 might play a protective role against AMI and arterial hypertension. The frequency of SNP896A/G in the TLR4 gene was not associated with AMI and arterial hypertension. Other risk factors for atherosclerosis and clinical aspects of myocardial infarction were not associated with the genotype distribution of the examined genes.

Human leptospirosis in eastern Croatia, 1969–2003: Epidemiological, clinical, and serological features

This survey presents epidemiological, serological and clinical features of 270 patients (85% males, 18% children) treated for leptospirosis from 1969 to 2003 at the Clinic for Infective Diseases, University Hospital Osijek, Osijek, eastern Croatia. 75% of the admissions were between July and October. The route of transmission was mostly by indirect contact with domestic animals, less frequently by direct contact with urine or tissue of infected animals. Clinical presentation included signs and symptoms with expected and common frequency, with the exception of jaundice (62%) and aseptic meningitis (60%), which occurred with higher incidence than previously reported. Acute renal failure ensued in 53% of patients, 7% of whom required haemodialysis. No deaths were observed. Therapy consisted of antimicrobials (penicillin and doxycycline) and symptomatic measures. Diagnosis was confirmed by microscopic agglutination test (MAT). There were in total 18 serological types of Leptospira detected, and types L. sejroe, L.pomona, L. australis and L. icterohaemorrhagiae prevailed. During the last 10 y some new types were observed. Leptospirosis was not rare in the region of eastern Croatia, and its course could be life-threatening if not recognized and adequately treated.

The relationship between interferon-γ gene polymorphism and acute kidney allograft rejection.

Cytokine gene polymorphisms have been associated with modified gene expression and cytokine production. Gamma interferon (IFN‐γ) plays an important role in the pathogenesis of kidney transplant rejection. This study evaluated the association between IFN‐γ gene polymorphisms and the history of acute allograft rejection in 53 adult first‐transplant recipients receiving cadaveric kidney grafts. They were followed up in a single centre until 2006, for a median time of 4 years after transplantation (1–22 years). IFN‐γ gene polymorphisms +874 T/A (rs2430561) were determined by polymerase chain reaction (PCR). T/T high IFN‐γ genotype was found in 12, intermediate T/A in 29 and low A/A in 12 patients. Twenty‐six acute kidney rejection episodes were evidenced in 20 patients, of which none occurred in the 12 patients with low IFN‐γ genotype A/A. Age, gender, number of HLA (human leukocyte antigen) mismatches, ABO blood groups, HLA, time after transplantation, creatinine clearance and immunosuppressive regimens were excluded as confounding factors associated with IFN‐γ genotype distribution between rejectors and non‐rejectors. IFN‐γ gene polymorphisms could be an important risk factor for acute kidney transplant rejection, whereas the low A/A IFN‐γ genotype could be protective against rejection.

Single nucleotide polymorphism of toll-like receptor 4 (TLR4) is associated with juvenile spondyloarthritis in Croatian population

Single nucleotide polymorphisms (SNP) of toll-like and NOD-like receptors have been associated with altered receptor activity and modified production of proinflammatory cytokines leading to a number of diseases. Our aim was to determine whether SNP of TLR2 (Arg753Gln), TLR4 (Asp299Gly, Thr399Ile), and NLRP3 (Q705K) influence susceptibility to juvenile spondyloarthrtis (jSpA) and juvenile idiopathic arthritis (JIA). After the DNA extraction, 26 patients with jSpA, 11 with oligoarticular, polyarticular, or systemic JIA, and 40 healthy controls were genotyped for Arg753Gln, Asp299Gly, Thr399Ile, and Q705K SNP using real-time PCR–SNP analysis. Statistically significant difference in genotype frequency for Thr399Ile SNP of TLR4 was observed in the jSpA (χ2 = 6.705, p = 0.035) and not in the JIA group (χ2 = 3005, p = 0.223). Regarding Asp299Gly SNP, no significant difference in genotype frequency was found; however, allele frequency was significant in both jSpA and JIA patients. No significant difference in genotype or allele frequency was observed for Arg735Gln and Q705K SNP. The399Ile polymorphism of TLR4 may be responsible for altered immune response to microbial infection in variant carriers and represent a mechanism of triggering overproduction of proinflammatory cytokines and long-term inflammation in jSpA. SNP of TLR2, NLRP3, and TLR4 (Asp299Gly) were not associated with jSpA or JIA.

Most common HCV genotypes in patients from north-eastern Croatia.

OBJECTIVE The aims of this study were to determine the HCV-RNA viral load, genotype distribution, risk factors and symptoms of HCVRNA positive viral load in HCV antibody-positive patients from north-eastern Croatia. MATERIALS AND METHODS From January 2009 to December 2011, 203 HCV antibody- positive patients (130 men and 73 women; median age 44.5 years) were analyzed for HCV-RNA by the COBAS TaqMan HCV test and genotyped by the Linear Array HCV Genotyping test (both from Roche). All patients completed a structured questionnaire about risk factors and symptoms. RESULTS The HCV-RNA percentage was 61.1% and was similar for men and women. The HCV-RNA viral load increased with age: while 55% of 20-50 year old patients were HCV-RNA positive, 73% of patients >50 years were positive (p=0.021). Genotype 1 was the most prevalent genotype (79.8%), followed by 3 (12.9%), 4 (6.5%), and 2 (0.8%); genotypes 5 and 6 were not determined. Patients with genotype 1 (median, 50 years) were older than patients with 3 (median, 33.5 years) or 4 (median, 38 years). The blood transfusions performed in Croatian hospitals before 1993 was significantly associated with HCV-RNA positive viral load (p<0.05). CONCLUSION These data indicated an elevated prevalence of genotype 1 in elderly HCV-RNA positive patients and it may continue to rise. Using RNA-based detection in HCV positive-antibody patients would allow early detection of HCV in the acute stage of HCV disease and the increased risk of HCV genotyperelated treatment failure.

Postoperative immunosuppression markers and the occurrence of sepsis in patients with benign and malignant disease

SummaryAimTo investigate associations between the postoperative immune response and the levels of extracellular circulating DNA (cDNA), C-reactive protein (CRP), neutrophil/lymphocyte (N/L) ratio, and regulatory T (Treg) cells in the peripheral blood and their role as potential predictors of postoperative septic complications.MethodsThis was a prospective observational study involving 115 adult patients who underwent elective surgery. Patients were divided into three groups: with benign disease, with malignant disease, and with malignant disease and administration of dexamethasone. Serum CRP levels, N/L ratio, monocyte human leukocyte antigen-DR (HLA-DR) expression, proportion of Treg cells, and cDNA levels were measured at different time points before and after surgery.ResultsAll patients had increased CRP levels after surgery. Septic patients had higher serum CRP levels at baseline. Compared with the other groups, the dexamethasone group had significantly higher CRP levels before and after surgery, a significantly higher N/L ratio before surgery, a significantly lower rise in the N/L ratio after surgery, and a significantly lower HLA-DR expression at baseline, which remained stable after surgery. In the malignant-disease group, we observed a significant postoperative decrease in the HLA-DR expression.ConclusionsOur results suggest that the immunosuppressive effect of surgery and the presence of a malignant disease may contribute to a higher risk of postoperative sepsis. Preoperative CRP levels may be a reliable predictor of sepsis in oncological patients.ZusammenfassungZielder Studie war es, Zusammenhänge zwischen der postoperativen Immunantwort und den Konzentrationen der zirkulierenden extrazellulären DNA (cDNA), des CRPs, des Quotienten Neutrophile/Lymphozyten (N/L) und der regulatorischen T (Treg) Zellen im peripheren Blut sowie deren Rolle als mögliche Vorhersager von postoperativen septischen Komplikationen zu untersuchen.MethodenIn diese prospektive Beobachtungsstudie haben wir 115 erwachsene Patienten, die einer elektiven Operation unterzogen wurden, eingeschlossen. Die Patienten wurden in 3 Gruppen eingeteilt: eine mit benigner Erkrankung, eine mit maligner Erkrankung und eine mit maligner Erkrankung und Gabe von Dexamethason. Die Serum-Konzentrationen des CRPs, der N/L Quotienten, der HLA-DR Expression der Monozyten, des Anteils der Treg Zellen und die Konzentrationen der cDNA wurden zu verschiedenen Zeitpunkten vor und nach der Operation gemessen.ErgebnisseAlle Patienten hatten nach der Operation erhöhte CRP Konzentrationen. Bei den Patienten mit postoperativer Sepsis waren die CRP Ausgangswerte höher. Im Vergleich mit den anderen Gruppen hatte die Gruppe der Patienten mit Dexamethason signifikant höhere CRP Werte vor und nach der Operation. Außerdem waren die N/L Quotienten präoperativ vergleichsweise signifikant erhöht, während postoperativ bei dieser Gruppe ein signifikant niedrigerer Anstieg der N/L Quotienten beobachtet wurde. Die Ausgangswerte der HLA-DR Expression waren bei diesen Patienten signifikant erniedrigt und blieben postoperativ stabil. Bei der Gruppe der Patienten mit maligner Erkrankung beobachteten wir postoperativ einen signifikanten Abfall der HLA-DR Expression.SchlussfolgerungenUnsere Ergebnisse lassen vermuten, dass der immunsuppressive Effekt einer Operation und das Vorliegen einer malignen Erkrankung zu einem erhöhten Risiko für das Auftreten einer postoperativen Sepsis beitragen können. Präoperative CRP Werte scheinen einen verlässlichen Vorhersagewert bezüglich Sepsis bei onkologischen Patienten zu haben.

Antibody combination therapy targeting CD25, CD70 and CD8 reduces islet inflammation and improves glycaemia in diabetic mice.

Destruction of pancreatic islets in type 1 diabetes is caused by infiltrating, primed and activated T cells. In a clinical setting this autoimmune process is already in an advanced stage before intervention therapy can be administered. Therefore, an effective intervention needs to reduce islet inflammation and preserve any remaining islet function. In this study we have investigated the role of targeting activated T cells in reversing autoimmune diabetes. A combination therapy consisting of CD25‐, CD70‐ and CD8‐specific monoclonal antibodies was administered to non‐obese diabetic (NOD) mice with either new‐onset diabetes or with advanced diabetes. In NOD mice with new‐onset diabetes antibody combination treatment reversed hyperglycaemia and achieved long‐term protection from diabetes (blood glucose <13·9 mmol/l) in >50% of mice. In contrast, in the control, untreated group blood glucose levels continued to increase and none of the mice were protected from diabetes (P < 0·0001). Starting therapy early when hyperglycaemia was relatively mild proved critical, as the mice with advanced diabetes showed less efficient control of blood glucose and shorter life span. Histological analysis (insulitis score) showed islet preservation and reduced immune infiltration in all treated groups, compared to their controls. In conclusion, antibody combination therapy that targets CD25, CD70 and CD8 results in decreased islet infiltration and improved blood glucose levels in NOD mice with established diabetes.

Interleukin 17A and Toll-like Receptor 4 in Patients with Arterial Hypertension

Background/Aims: Immune responses are involved in arterial hypertension. An observational cross-sectional case control study was conducted to estimate the association between Toll-like receptor 4 (TLR4) expression and interleukin (IL)-17A serum levels in patients with controlled and non-controlled hypertension. Methods: We have enrolled 105 non-complicated otherwise healthy hypertensive patients: 53 with well-controlled blood pressure and 52 non-controlled. TLR4 peripheral monocytes expression and serum IL-17A levels were determined by flow cytometry and ELISA, respectively. Results: Non-controlled patients exhibited higher TLR4 expression than well-controlled (25.60 vs. 21.99, P=0.011). TLR4 expression was lower in well-controlled patients who were prescribed beta blockers (18.9 vs. 22.6, P=0.005) and IL-17A concentration was higher in patients using diuretics in either group (1.41 vs. 2.01 pg/ml, P<0.001; well-controlled 1.3 vs. 1.8 pg/ml, P= 0.023; non-controlled 1.6 vs. 2.3 pg/ml, P=0.001). Correlation between IL-17A concentration and hypertension duration was observed in non-controlled patients (Spearman correlation coefficient . ρ=0.566, P<0.001) whereas in well-controlled patients a correlation was found between hypertension duration and TLR4 expression (ρ=0.322, P=0.020). Conclusions: Arterial hypertension stimulates the immune response regardless of blood pressure regulation status. Prolonged hypertension influences peripheral monocyte TLR4 expression and IL-17A serum levels. Anti-hypertensive drugs have different immunomodulatory effects: diuretics are associated with higher IL-17A concentration and beta-blockers with lower TLR4 expression.

PReS-FINAL-2078: Single nucleotide polymorphisms of toll like receptors 2 and 4 in enthesitis related arthritis and oligo and polyarticular juvenile idiopathic arthritis.

Introduction Single nucleotide polymorphisms of Toll like receptors modify cellular immune response and induce pro-inflammatory cytokine production and therefore could be associated with enthesitis related arthritis (ERA) and/or oligoarticular and polyarticular juvenile idiopathic arthritis (JIA). Objectives To determine whether polymorphisms of TLR2 and TLR4 influence susceptibility to ERA or JIA. Methods DNA was extracted from blood samples of 19 ERA patients, 10 patients with oligoarticular or polyarticular JIA and 40 healthy controls, all diagnosed according to ILAR criteria. Polymorphisms of the TLR2 (Arg753Gln) and TLR4 (Asp299Gly, Thr399Ile) were determined using real time and multiplex PCR. Results All JIA patients were carriers of wild type allele for all three polymorphisms. Regarding Arg753Gln polymorphism of TLR2, only one patient with ERA (5.56%) and 2 healthy controls (5%) were carriers of heterozygous allele. There were no homozygous mutants. All ERA patients had wild type allele for Asp299Gly polymorphism of TLR4. For Thr399Ile polymorphism of TLR4, 21.05% ERA patients were heterozygous (CT variant), and none of the ERA patients was homozygous (TT variant), (CC vs CT variant in ERA OR 3.7500, 95% CI 1.0498-13.3952, p 0.0419, CC vs TT variant OR 31.0000, 95% CI 1.7309-555.1867, p 0.0197). In group of healthy controls, TLR4 polymorphisms Asp299Gly and Thr399Ile were in linkage disequilibrium ; 2 controls were hetrozygous and 6 homozygous variant carriers for both polymorphisms, whereas linkage disequilibrium was not found among patient groups (CC vs CT in controls OR 16.0000, 95% CI 3.5905- 71.2994, p 0.0003, CC vs TT OR 5.3333, 95% CI 2.0099-14.1524, p 0.1944). Conclusion Polymorphisms of TLR2 and TLR4 are not associated with oligoarticular/polyarticular JIA. There was also no evidence that variants of TLR are major risk factors for ERA, however, lack of susceptibility should be confirmed on larger group of patients, since four out of 19 patients (21, 05%) were heterozygous. A study on larger cohort is currently ongoing.