Pavel Kuklínek

Exon 1 polymorphism of the B2BKR gene does not influence the clinical status of patients with hereditary angioedema.

Polymorphic variants of B2 receptors for bradykinin (B2BKR) have been postulated to influence a clinical manifestation of hereditary angioedema. In this study, exon 1 nonanucleotide deletion polymorphism in the B2BKR gene was examined in 37 patients with hereditary angioedema. The patients were grouped according to disease severity or the age of the first clinical manifestation of disease. No significant differences in allelic frequencies were found between particular subgroups of patients. Therefore, we concluded that this polymorphism does not seem to have any significant effect on the course and severity of hereditary angioedema in Caucasians.

Sequence variants of the TNFRSF13B gene in Czech CVID and IgAD patients in the context of other populations.

Mutations in the TNFRSF13B gene, encoding TACI, have been found in common variable immunodeficiency (CVID) and selective IgA deficient (IgAD) patients, but only the association with CVID seems to be significant. In this study, Czech CVID, IgAD and primary hypo/dysgammaglobulinemic (HG/DG) patients were screened for all TNFRSF13B sequence variants. The TNFRSF13B gene was mutated in 4/70 CVID patients (5.7%), 9/161 IgAD patients (5.6%), 1/17 HG/DG patient (5.9%) and none of 195 controls. Eight different mutations were detected, including the most frequent p.C104R and p.A181E mutations as well as 1 novel missense mutation, p.R189K. A significant association of TNFRSF13B gene mutations was observed in both CVID (p=0.01) and IgAD (p=0.002) Czech patients. However, when combined with all published data, only the association with CVID remained significant compared with the controls (9.9% vs. 3.2%, p<10(-6)), while statistical significance disappeared for IgAD (5.7% vs. 3.2%, p=0.145). The silent mutation p.P97P was shown to be associated significantly with CVID compared with the controls in both Czech patients (allele frequency 4.3% vs. 0.2%, p=0.01) and in connection with the published data (5.1% vs. 1.8%, p=0.003). The relevance of some TNFRSF13B gene variants remains unclear and needs to be elucidated in future studies.

No Evidence for Linkage between the Hereditary Angiooedema Clinical Phenotype and the BDKR1, BDKR2, ACE or MBL2 gene

Hereditary angiooedema (HAE) is a life‐threatening disease with poor clinical phenotype correlation with its causal mutation in the C1 inhibitor (SERPING1) gene. It is characterized by substantial symptom variability even in affected members of the same family. Therefore, it is likely that genetic factors outside the SERPING1 gene have an influence on disease manifestation. In this study, functional polymorphisms in genes with a possible disease‐modifying effect, B1 and B2 bradykinin receptors (BDKR1, BDKR2), angiotensin‐converting enzyme (ACE) and mannose‐binding lectin (MBL2), were analysed in 36 unrelated HAE patients. The same analysis was carried out in 69 HAE patients regardless of their familial relationship. No significant influence of the studied polymorphisms in the BDKR1, BDKR2, ACE and MBL2 genes on overall disease severity, localization and severity of particular attacks, frequency of oedema episodes or age of disease onset was detected in either group of patients. Other genetic and/or environmental factors should be considered to be responsible for HAE clinical variability in Caucasians.

Change in referral diagnoses and diagnostic delay in hypogammaglobulinaemic patients during 28 years in a single referral centre.

Background: The classical clinical manifestation of untreated immunoglobulin deficiency comprises predominantly recurrent and complicated respiratory tract infections. Before the 1980s, little was known about the clinical manifestation of immunodeficiency in the general medical population, and also the availability of serum immunoglobulin laboratory determination was not sufficient, leading to a significant diagnostic delay. Methods: We have analysed the diagnostic delay and referral diagnoses in patients in whom any form of primary hypogammaglobulinaemia had been diagnosed at our department, which was established in 1981. Results: Comparing the diagnostic delay in the 1980s (19 patients, median 5.5 years), the 1990s (37 patients, median 3.5 years) and the years 2001–2008 (33 patients, median 1 year), a significant decrease was observed (p < 0.05, Spearman’s correlation coefficient). Also, the median number of pneumonia episodes during the diagnostic delay decreased from 5 in the 1980s, to 1 in the 1990s and to 0 in the period of 2001–2008 (p < 0.05, Spearman’s correlation coefficient). While in the 1980s 17 of the 19 patients had pneumonia in their past history, in the period of 2001–2008 only 13 of the 33 patients were concerned. Conclusions: Our observation documents improved awareness of immunodeficiencies among physicians. It is supposed that earlier diagnosis will prevent complications, improve the quality of life and even survival of hypogammaglobulinaemic patients.

Lack of dehydroepiandrosterone in type I and II hereditary angioedema and role of danazol in steroid hormone conversion

Background:  Hereditary angioedema (HAE) is successfully treated with danazol, a therapeutic steroid compound. To investigate hormones of the hypothalamic‐pituitary‐adrenal (HPA) and hypothalamic‐pituitary‐gonadal (HPG) axis in patients with HAE with and without danazol.

Paracentesis vs. intraperitoneal reinfusion of concentrated ascites — effect on opsonic activity of ascites

Abstract Background. Spontaneous bacterial peritonitis is a frequent and serious complication of cirrhotic ascites. An important defense mechanism of ascites against infection is opsonic activity, where the most important part is played by specific antibodies and C3 and C4 components of complement. The authors wanted to find out whether reinfusion of concentrated ascites or paracentesis can influence this activity. Methods. Twenty-six patients with cirrhosis of the liver and refractory ascites not corresponding to salt restriction and diuretics were divided into two groups. In 14 patients, a total of 25 therapeutic paracenteses were made; in 12 patients, a total of 19 reinfusions of concentrated ascites were performed intraperitoneally. In all patients, levels of IgG, IgA, IgM, C3 and C4 components of complement were assessed in ascites before and 24 h after the intervention. Results. After paracentesis no change of the investigated parameters was found. Reinfusion of concentrated ascites, on the other hand, led to a significant rise of IgG, IgA, IgM, C3 and C4 at the 0.5% level of significance. Conclusion. Treatment of refractory ascites by ultrafiltration and reinfusion intraperitoneally seems to increase opsonic activity of ascites and could be useful in the prevention of the development of spontaneous bacterial peritonitis.

Recurrent aphthous stomatitis and gene variability in selected interleukins: a case-control study

Genetic factors, especially those related to immune system functioning, have been intensively studied to determine their role in the development of recurrent aphthous stomatitis (RAS). The aim of the present study was to analyze gene variability in interleukin (IL)2, IL4 (and its receptor α, IL4Rα), IL10, and IL13, which were selected based on literature review and/or their functional relevance, in Czech patients with RAS and in healthy controls. In total, 252 subjects (178 controls and 74 patients with RAS) were enrolled in this case-control study, and their detailed anamnestic, clinical, and laboratory data were obtained. Nine polymorphisms in the genes encoding interleukins were determined using PCR techniques. There were no significant differences in allele or genotype frequencies of the IL2, IL4, IL4Rα, IL10, and IL13 polymorphisms rs2069762/rs2069763, rs2243250/rs79071878, rs1801275, rs1800896, and rs1800925, respectively, between controls and patients with RAS. The minority alleles rs1800871 and rs1800872, which encode variants of IL10, were associated with a statistically significantly higher risk of RAS, as confirmed by the results of genotype and haplotype analyses. We suggest that variability in the IL10 gene may play an important role in the development of RAS in the Czech population.

The NOD-like receptor (NLRP3) gene variability in patients with recurrent aphthous stomatitis

I diseases continue to be one of the biggest health problems in the world, affecting millions of people annually M. abscessus and other species of rapidly growing mycobacteria (RGM) are naturally resistant to antimicrobial compounds and disinfectants because they have an impermeable cell wall composed by peptideoglycan and mycolic acids. These RGM are responsible for various hospital outbreaks worldwide, causing lung infections in patients with cystic fibrosis, chronic lung disease (bronchiectasis, nodules and cavitations), post-surgical infections and skin and soft tissue infections in immunocompromised patients. The resistance of M. abscessus (Mabs) to the medications used in current therapy challenges the search for new treatment strategies. Previous studies on the search for new natural compounds with antimicrobial action highlighted the potential of Bixa orellana (urucum). The seeds of this plant are already used in folk medicine for treating heart disease, gastrointestinal problems and respiratory infections. In this study, we evaluated potential anti-inflammatory activities of hydroalcoholic (BoEH) and ethyl acetate (BoEA) extracts of B. orellana leaves, using a murine model of peritonitis induced by heat killed Mabs. C57BL/6 mice were orally treated with different concentrations of BoEH or BoEA. After one hour, peritonitis was induced by inoculation of 1x108 CFU of heat killed Mabs. BoEH and BoEA inhibited the migration of total leukocytes (Figure 1A-B), migration of polymorphonuclear cells (Figure 1C-D) and mononuclear cells (Figure 1E-F) into the peritoneum in the periods analyzed 4 and 24 hours after the induction of peritonitis. Our results suggest anti-inflammatory actions of the extracts tested, indicating this plant as natural source of compounds with potential for pharmacological and biotechnological applications.I is a member of the IL-17 family, and is known as CTLA8 in the mouse. It is produced by T lymphocytes and NK cells and has proinflammatory roles, inducing cytokine and chemokine production. However, its role in tumor biology remains controversial. We investigated the effects of locally produced IL-17A by transferring the gene, encoding it into mouse tumor cells including B16 melanoma, and MethA fibrosarcoma, either in a secretory or a membrane-bound form. Expression of the membranebound form on CT26 colon cancer cells dramatically enhanced their proliferation in in vitro. The enhanced growth was shown to be due to an increased rate of cell cycle progression. After synchronizing cells by adding and withdrawing colcemid, the rate of cell cycle progression in the cells expressing the membrane-bound form of IL-17A was much faster than that of the control cells. Both secretory and membrane-bound IL-17A induced the expression of Sca-1 on the cancer cells, which is commonly associated with aggressive phenotype of cancer cells. When tumor clones were grafted into syngeneic BALB/c mice, the tumor clones expressing the membranebound form IL-17A grew rapidly; those expressing the secretory form also grew faster than the wild type CT26 cells, but slower than the clones expressing the membrane-bound form. These results indicate that IL-17A promotes tumorigenicity, in part, by enhancing cell cycle progression. This finding should be considered in treating tumors and immune-related diseases.P which patients are at a higher risk for recurrent chronic rhinosinusitis with nasal polyps (CRSwNP) is one of the most challenging problems in clinical rhinology. A direct association between nasal polyp and eosinophil/neutrophil counts was reported. This study aimed to identify difference of eosinophils and neutrophils for formation of polyp by DNA methylation in CRS. We have previously shown that KRT 19, NR2F2, ADAMTS1, and ZNF222 levels are changed in nasal polyps (NPs) of patients with chronic rhinosinusitis (CRS) in patients. A study was performed from 30 patients with CRS with bilateral NP, examining the prognostic role of eosinophil and neutrophil levels. 30 patients with CRS were classified by the rate of eosinophils and neutrophils in tissue. The methylated genes detected by DNA methylation microarray were validated by methylation-specific polymerase chain reaction (PCR), bisulfite sequencing, and real-time PCR. DNA methylation microarray identified 43,674 CpG islands in 518 genes. Specific genes were found to have a hypermethylated signal, and some genes were significantly hypomethylated in the promoter region in eosinophils compared with neutrophils. Real-time PCR showed that the expression levels of genes were changed in eosinophils, when compared with neutrophils. We clearly demonstrated that the two subgroups of CRSwNP had characteristic differences in DNA methylation, which allows for pathophysiologically meaningful differentiations with likely therapeutic consequences. Further studies are needed to confirm the significance of these epigenetic factors in the mechanisms underlying NP formation.Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease where animal models are used to study its pathogenesis. We have developed a mouse model of an autoimmune disease that resembles human lupus by the injection of liposomes bearing non-bilayer phospholipid arrangements (NPA). We detected the presence of IgG antibodies against NPA in the serum of mice with lupus and patients with SLE. In this work, we determined by citofluorometry the presence of plasmacytoid dendritic cells (pDC) the main producer of type I interferons, and of NKT cells in the secondary lymphoid organs of mice 30 and 60 days after the injection of liposomes with or without NPA induced with 8 mM promazine. In both groups of mice injected with liposomes bearing NPA a significant increase of pDC cells (5-fold) was found which correlates with the high concentration of type I interferon previously detected in this mouse model of lupus and in patients with SLE. A significant increase of NKT (3-fold) was detected at 30 days, specifically in the NKT subpopulation CD4 + that is known to cooperate with B cells in response of lipid antigens; this increase suggests its probable involvement in adaptive immune responses, which lead to the production of anti-NPA IgG antibodies. The increase of pDC and NKT cells found by cytometry in secondary lymphoid organs in this work, suggests their involvement in the formation of anti-NPA IgG antibodies and the development of the disease resembling human lupus.Statement of the Problem: Recurrent aphthous stomatitis (RAS) is a multifactorial disease with an unclear etiopathogenesis, resulting from the interplay between genetic and environmental factors.1 As the dysregulation of the immune system can play a role in the RAS development2, single nucleotide polymorphisms (SNPs) in the genes for immune and inflammatory molecules were studied.3,4 The NOD-like receptor (NLRP3) gene, encoding the component of the inflammasome, has been proposed as one of the candidate genes for RAS.5 The aim of our study was to investigate three SNPs (rs4612666, rs10754558, rs3806265) in NLRP3 gene in patients with RAS and healthy controls in the Caucasian population Methodology: A total of 200 Czech subjects were enrolled in this case-control study. 143 healthy controls, 57 patients with RAS were genotyped by method based on polymerase chain reaction using 5′ nuclease TaqMan® assays. Clinical parameters such as complete blood count, levels of immunoglobulins including allergen-specific immunoglobulin E or presence of antibodies against cytomegalovirus, Epstein-Barr virus were determined in RAS patients. Findings: Although no significant differences in the NLRP3 (rs10754558, rs3806265) allele and genotype frequencies between patients with RAS and controls were observed, statistically significant differences in NLRP3 rs4612666 genotype frequencies between subjects with RAS and controls were found. Carriers of NLRP3 rs4612666 TT genotype had a higher risk of developing RAS in comparison to subjects with CT + CC genotypes (OR = 16.71, 95% CI = 1.96-142.14, P = 0.0024). No association between NLRP3 haplotypes and RAS was detected. Conclusion & Significance: In contrast to the previous study5, associations between NLRP3 (rs10754558, rs3806265) polymorphisms and RAS were not confirmed. However, we suggest that NLRP3 rs4612666 polymorphism can strongly influence the risk of developing RAS in the Czech population.Results: The prevalence of vitamin D insufficiency was 64% in 76 asthmatic and 62.5% in 446 atopic individuals; however there was no significant association between vitamin D and this outcomes. Negative correlation was found between vitamin D and specific IgE levels to Dermatophagoides pteronyssinus on atopic subjects (r=-0.11, p=0.04). Genetic variants in CYP2R1 gene, rs7935792 (C allele) (Beta 1.66; 95% CI 0.20-3.11) and rs7129781 (C allele) (Beta 1.55; 95% CI 0.07-2.96), were associated with vitamin D serum levels. In addition, the same variants had suggestive protection on asthma, but it was not significant (OR 0.74; 95% CI 0.39; 1.39; OR 0.73; 95% CI 0.38; 1.37, respectively). VDR variants rs7965397 (G allele) was positively associated with atopy (OR 1.43; 95% CI, 1.07-1.92); rs4328262 (G allele) (OR 1.44; 95% CI 1.09-1.90) and asthma rs2408876 (C allele) (OR 2.31; 95% CI; 1.18-4.53); rs2238317 (T allele) (OR 2.19; 95% CI 1.02-4.72).

Hereditary angio-oedema with C1 inhibitor deficiency: Characteristics and diagnostic delay of Czech patients from one centre.

BACKGROUND Hereditary angio-oedema (HAE) is manifested by repeated episodes of localised subcutaneous or sub-mucosal oedema. Symptoms are extremely variable in frequency, localisation, and severity. Atypical or mild clinical symptoms of the disease may lead to erroneous diagnosis, causing diagnostic delay. The goal of this study was to assess how diagnostic delay has changed over 33 years at a single referral centre. METHODS We analysed diagnostic delay and first symptoms of HAE in patients who were diagnosed at an immunology department between 1980 and 2013. Patients records were analysed. RESULTS The median diagnostic delay in 77 HAE type 1 and 2 patients was seven (range, 0-42) years. The difference observed in diagnostic delay between probands (18 [0-42] years) and others (1 [0-37] year) was significant (p<0.001). Our data show a significant negative correlation between the length of diagnostic delay and the year of diagnosis in our group of patients (p=0.024). The median age of first symptoms among all HAE patients (N=64) was 17 (1-40) years. The first symptoms of HAE in 64 patients were analysed. Twenty-six patients had abdominal, seventeen peripheral, five facial, two urogenital, and three had laryngeal oedema as the first manifestation of the disease. The last death that was attributed to HAE was in 1977. CONCLUSIONS Our observations demonstrate improved awareness of HAE among physicians, as documented by the significant decrease in diagnostic delay. It is believed that earlier treatment will improve patient quality of life and life expectancy.