Maria Bucova

Immunopathogenesis of bronchial asthma

Bronchial asthma is a common immune-mediated disorder characterized by reversible airway inflammation, mucus production, and variable airflow obstruction with airway hyperresponsiveness. Allergen exposure results in the activation of numerous cells of the immune system, of which dendritic cells (DCs) and Th2 lymphocytes are of paramount importance. Although the epithelium was initially considered to function solely as a physical barrier, it is now evident that it plays a central role in the Th2-cell sensitization process due to its ability to activate DCs. Cytokines are inevitable factors in driving immune responses. To the list of numerous cytokines already known to be involved in the regulation of allergic reactions, new cytokines were added, such as TSLP, IL-25, and IL-33. IgE is also a central player in the allergic response. The activity of IgE is associated with a network of proteins, especially with its high- and low-affinity Fc receptors. Understanding the cellular and molecular mechanisms of allergic reactions helps us not only to understand the mechanisms of current treatments, but is also important for the identification of new targets for biological intervention. An IgE-specific monoclonal antibody, omalizumab, has already reached the clinic and similar biological agents will surely follow.

Polymorphisms in the Genes Encoding TGF-β1, TNF-α, and IL-6 Show Association with Type 1 Diabetes Mellitus in the Slovak Population

Numerous cytokines have been shown to participate in the pathogenesis of type 1 diabetes (T1D). As gene polymorphisms can influence cytokine production or function, they may potentially contribute to genetic predisposition to the disease. The aim of this study was therefore to investigate the role of 22 single nucleotide polymorphisms (SNPs) in 13 cytokine and cytokine receptor genes in genetic susceptibility to T1D. Polymerase chain reaction with sequence-specific primers was used to genotype cytokine SNPs and HLA-DRB1 alleles in 151 diabetics and 140 healthy individuals of Slovak origin. Univariate analysis showed that transforming growth factor (TGF)-β1 codon 10 TT homozygotes were significantly more susceptible to developing T1D than C allele carriers (Pcxa0=xa00.0066, ORxa0=xa02.46). Furthermore, tumor necrosis factor (TNF)-α −308 A allele carriers were also significantly overrepresented among the diabetics (Pcxa0=xa00.0031, ORxa0=xa02.62); however, the association of the −308 A allele with T1D might be due to its strong linkage disequilibrium with the susceptibility allele HLA-DRB1*0301. An association was also found with interleukin (IL)-6 −174 G/C and nt565 G/A SNPs; however, its significance was lost when statistical correction was applied. These data suggest that the TGF-β1 codon 10 SNP is among numerous genetic variations with small individual effects on T1D development. Moreover, a possible role of TNF-α and IL-6 SNPs cannot be ruled out, although their association with T1D was due to strong LD with the HLA class II susceptibility allele or did not withstand statistical correction, respectively.

Genetic variations of interleukin‐8, CXCR1 and CXCR2 genes and risk of acute pyelonephritis in children

Acute pyelonephritis (APN) is the most severe form of urinary tract infection, the etiopathogenesis of which is still not well understood. Previous studies demonstrated that chemotaxis of neutrophils into the tissue and across the infected epithelial layer is a key step in rapid bacterial clearance. Variations within genes encoding the major chemokine interleukin‐8 and its receptors CXCR1 and CXCR2 are therefore attractive candidates for participation in genetic predisposition to APN. The aim of our study was to evaluate the association of single nucleotide polymorphisms (SNPs) −251 T/A, +781 C/T, +1633 C/T and +2767 A/T in the IL‐8 gene, +2608u2003G/C in the CXCR1 gene and +1208 C/T in the CXCR2 gene with susceptibility to APN in the Slovak population. PCR–SSP and PCR–RFLP were used to genotype SNPs in 147 children with APN (62 with recurrent and 85 with episodic form) and 215 healthy individuals. Statistical analysis revealed significantly increased frequency of CXCR1 +2608 C allele (Pu2003=u20030.0238, ORu2003=u20032.452, 95% CIu2003=u20031.147–5.243) and GC genotype (Pu2003=u20030.0201, ORu2003=u20032.627, 95% CIu2003=u20031.188–5.810) and lower frequency of CXCR2 +1208 T allele (Pu2003=u20030.0408, ORu2003=u20030.645, 95% CIu2003=u20030.429–0.972) and TT+TC genotypes (Pu2003=u20030.0497, ORu2003=u20030.5273, 95% CIu2003=u20030.288–0.964) in patients with recurrent APN compared with healthy controls. Furthermore, the A allele of IL‐8 −251 T/A SNP was also significantly overrepresented in patients with recurrent APN when compared with those with only single episode of APN (Pu2003=u20030.0439, ORu2003=u20031.627, 95% CIu2003=u20031.019–2.599). Our results indicate that the minor CXCR1 +2608 C allele is associated with significantly increased susceptibility to APN in childhood, while the CXCR2 +1208 T allele confers protection from recurrent APN. Moreover, allele A of the IL‐8 −251 T/A may also increase the risk of developing recurrent attacks after the first‐time APN.

Single nucleotide polymorphisms of cytokine genes in the healthy Slovak population

Cytokines are molecules that control and modulate the activities of numerous target cells via binding to specific receptors. The observed differences in the cytokine production among individuals can be, at least partially, explained by gene polymorphisms. Several cytokine gene polymorphisms have been identified to play a role in susceptibility to various diseases, including autoimmune, infectious, allergic or cardiovascular diseases.

Inflammatory marker sTREM-1 reflects the clinical stage and respiratory tract obstruction in allergic asthma bronchiale patients and correlates with number of neutrophils.

The knowledge that asthma is an inflammatory disorder has prompted us to investigate the plasma levels of a new inflammatory marker sTREM-1 that is released from the surfaces of activated neutrophils and monocytes. The plasma levels of sTREM-1 were analysed by a sandwich ELISA test in the cohort of 76 patients with allergic asthma bronchiale and 39 healthy controls. Our results revealed more than 3.5 times higher levels of sTREM-1 in AB patients (92.3u2009pg/mLu2009±u2009125.6) compared with healthy subjects (25.7u2009pg/mLu2009±u20099.2; P = 0.0001). Higher levels of sTREM-1 were found also in patients with exacerbated AB (170.5u2009pg/mLu2009±u200978.2) compared with nonexacerbated AB patients (59.1u2009±u200978.2; P < 0.0001), patients with respiratory tract obstruction (176.4u2009pg/mLu2009±u2009177.8), than those without obstruction (51.99u2009pg/mLu2009± 64.0; P < 0.0001) and patients with anti-IgE therapy (P < 0.0001). Levels of sTREM-1 correlated with number of leucocytes (P = 0.002), and absolute number of neutrophils (P = 0.001). Elevated plasma levels of sTREM-1 reflect the severity, state of exacerbation, presence of respiratory tract obstruction in AB patients and together with increased number of neutrophils point to the role of neutrophils in inflammation accompanying AB.

Association of MCP-1 -2518 A/G single nucleotide polymorphism with the serum level of CRP in Slovak patients with ischemic heart disease, angina pectoris, and hypertension,.

The aim of our work was to find if MCP-1 -2518 (A/G) single nucleotide polymorphism (SNP) influences somehow the serum concentrations of high-sensitive CRP (hsCRP) both in patients suffering from ischemic heart disease (IHD), myocardial infarction (MI), angina pectoris (AP), and hypertension (HT) and in control group of healthy subjects. Totally, 263 patients with the diagnosis of IHD, out of them 89 with MI, 145 with AP, 205 with HT, and also 67 healthy subjects were included in the study. First, we estimated the serum levels of hsCRP. We found that patients with AP had significantly higher serum level of hsCRP than both control group of healthy subjects (P = .043) and IHD patients without AP (P = .026). The presence of the mutant G allele statistically significantly correlated with the higher serum levels of hsCRP in patients with IHD (P = .016), AP (P = .004), and HT (P = .013). Higher correlations were found in men (AP: P = .019; HT: P = .047). In all cases the highest levels of hsCRP were found both in patients and healthy controls with homozygous GG genotype.

Triggering receptor expressed on myeloid cells‐1 and 2 in bronchoalveolar lavage fluid in pulmonary sarcoidosis

Pulmonary sarcoidosis (PS) is characterized by the formation of granulomas in the lungs and has been associated with infection by microorganisms. Triggering receptor expressed on the surface of myeloid cells (TREM)‐1 is overexpressed in response to infection while TREM‐2 is involved in granuloma formation. We hypothesized that these receptors are overexpressed in PS and might be useful for diagnostic testing.

MCP-1 −2518 A/G Single Nucleotide Polymorphism in Slovak Patients with Systemic Sclerosis

Recent study in a group of German patients with SSc has implicated the SNP in the MCP-1 gene (−2518 A to G) as a factor of susceptibility to SSc. Reflecting the need for replication of genetic association studies, we investigated if this SNP is associated with SSc in another Caucasian population. MCP-1 −2518 A/G genotypes were determined using PCR-SSP in 46 SSc patients and in 449 healthy subjects, all unrelated and of Slovak (Slavonic) origin. The distribution of MCP-1 −2518 A/G genotypes complied with the Hardy-Weinberg equilibrium both in patient and healthy control groups. There was no difference in MCP-1 −2518*G allele frequency between SSc patients and healthy subjects (patients: 0.23; controls: 0.24; P > .05). Furthermore, MCP-1 −2518 GG homozygotes were similarly represented among SSc patients and healthy subjects (P > .05). The association of MCP-1 −2518 A/G SNP with SSc observed originally in German population was not replicated in the Slovak population.

Characteristics of responders to autologous bone marrow cell therapy for no-option critical limb ischemia

BackgroundThe present study investigated factors associated with therapeutic benefits after autologous bone marrow cell (BMC) therapy in patients with “no-option” critical limb ischemia (CLI).Methods and resultsSixty-two patients with advanced CLI (Rutherford category 5 or 6) not eligible for revascularization were randomized to treatment with 40xa0ml of autologous BMCs (SmartPreP2) by local intramuscular (nu2009=u200932) or intra-arterial (nu2009=u200930) application. The primary endpoint was limb salvage and wound healing at 12xa0months. Seven patients (11xa0%) died during the follow-up from reasons unrelated to stem cell therapy. The BMC product of patients with limb salvage and wound healing (33/55) was characterized by a higher CD34+ cell count (pu2009=u20090.001), as well as a higher number of total bone marrow mononuclear cells (BM-MNCs) (pu2009=u20090.032), than that of nonresponders (22/55). Patients with limb salvage and wound healing were younger (pu2009=u20090.028), had lower C-reactive protein levels (pu2009=u20090.038), and had higher transcutaneous oxygen pressure (tcpO2) (pu2009=u20090.003) before cell application than nonresponders. All patients with major tissue loss at baseline (Rutherford 6 stage of CLI, nu2009=u20095) showed progression of limb ischemia and required major limb amputation. In the multiple binary logistic regression model, the number of applied CD34+ cells (pu2009=u20090.046) and baseline tcpO2 (pu2009=u20090.031) were independent predictors of limb salvage and wound healing. The number of administrated BM-MNCs strongly correlated with decreased peripheral leukocyte count after 6xa0months in surviving patients with limb salvage (pu2009=u20090.0008).ConclusionPatients who benefited from autologous BMC therapy for “no-option” CLI were treated with high doses of CD34+ cells. The absolute number of applied BM-MNCs correlated with the improvement of inflammation. We hypothesize that the therapeutic benefit of cell therapy for peripheral artery disease is the result of synergistic effects mediated by a mixture of active cells with regenerative potential. Patients at the most advanced stage of CLI do not appear to be suitable candidates for cell therapy.Trial registrationThe study was approved and registered by the ISRCTN registry. Trial registration: ISRCTN16096154. Registered: 26 July 2016.

Increased Antifungal Antibodies in Bronchoalveolar Lavage Fluid and Serum in Pulmonary Sarcoidosis

The recent studies suggest a role of fungi in development of sarcoidosis. Moreover, the immune response in sarcoidosis and fungal infection shows a striking similarity. We formulated a hypothesis of the possible increase in antifungal antibodies in bronchoalveolar lavage fluid (BALF) and serum in pulmonary sarcoidosis. BALF and serum levels of IgG‐, IgM‐ and IgA‐specific antibodies against the cell wall β‐D‐glucan and mannan of Candida albicans and Saccharomyces cerevisiae were tested in 47 patients (29 pulmonary sarcoidosis patients and 18 patients with other interstitial lung diseases (ILD – control group)) and 170 healthy controls. Our results proved: (1) an increase in IgG‐, IgM‐ and IgA‐specific antifungal antibodies in BALF in pulmonary sarcoidosis compared with the control group (C. albicans: IgG: P = 0.0329, IgM: P = 0.0076, IgA: P = 0.0156; S. cerevisiae: IgG: P = 0.0062, IgM: P = 0.0367, IgA: P = 0.0095) and (2) elevated levels of serum antifungal antibodies in pulmonary sarcoidosis compared with healthy controls (C. albicans: IgG: P = 0.0329, IgM: P = 0.0076, IgA: P = 0.0156; S. cerevisiae: IgG: P > 0.05, IgM: P < 0.05, IgA: P < 0.001). The study showed increased serum and BALF levels of antifungal antibodies in pulmonary sarcoidosis. The hypothesis that fungal infection is one of the possible aetiologic agents of sarcoidosis is interesting and deserves further attention.