Putative regulatory functions of SNPs associated with bronchial asthma, arterial hypertension and their comorbid phenotype

Linkage disequilibrium (LD) of single nucleotide polymorphisms (SNPs) of TLR4/AL160272.2 (rs1927914, rs1928298, rs7038716, rs7026297, rs7025144) was estimated in the Slavs of West Siberia. We further investigated an association of SNPs in TLR4/AL160272.2 (rs1927914, rs7038716, rs7025144), SERPINA1 (rs1980616), ATXN2/BRAP (rs11065987), IL2RB (rs2284033), NT5C2 (rs11191582), CARD8 (rs11669386), ANG/RNASE4 (rs1010461), and ABTB2/ САТ (rs2022318) genes with bronchial asthma (BA), arterial hypertension (AH) and their comorbidity. Then, the disease-associated SNPs were annotated in silico in relation to their potential regulatory functions. Strong LD was detected between rs1928298 and rs1927914, as well as rs7026297 and rs7038716 in the Slavs of West Siberia. It was found that the rs1927914 G allele of the TLR4 gene and the rs1980616 C allele of the SERPINA1 gene are associated with the predisposition to BA. These SNPs can affect binding affinity of transcription factors of the Pou and Klf4 families, as well as the expression levels of the TLR4 and SERPINA1 genes. The rs11065987 allele A of the ATXN2/BRAP genes, the rs11669386 A allele of the CARD8 gene, the rs2284033 allele G of the IL2RB gene, and the rs11191582 allele G of the NT5C2 gene were associated with the risk of AH. These variants can alter binding affinity of the Hoxa9, Irf, RORalpha1 and HMG-IY transcription factors, as well as the expression levels of the ALDH2, CARD8, NT5C2, ARL3, and SFXN2 genes in blood cells/vessels/heart, respectively. The risk of developing a comorbid phenotype of AD and AH is associated with the A allele of rs7038716 and the T allele of rs7025144 of the TLR4/AL160272.2 genes, the A allele of rs1010461 of the ANG gene and the C allele of rs2022318 of the ABTB2/CAT genes. Variants rs7038716 and rs7025144 can change the expression levels of the TLR4 gene in blood cells, while rs1010461 and rs2022318 influence the expression levels of the ANG and RNASE4 genes as well as the CAT and ABTB2 genes in blood cells, lungs/vessels/heart.

The effect of Borago officinalis on the signaling pathway of the NLRP3 inflammasome complex, TLR4 and some inflammatory cytokines in type II diabetic patients with acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a life-threatening condition in which the lungs become severely inflamed, causing the alveoli to constrict or fill with fluid, which prevents the lungs from functioning properly. This disease becomes more dangerous when it occurs in patients with diabetes. Because of the clinical condition of these patients, it is not possible to treat them with usual medicines. One of the best options for treating these people is to use herbs. Borage (Borago officinalis) is a medicinal herb that, in addition to its anti-inflammatory properties, is also able to control blood sugar. Therefore, in the current study, the effect of borage oil was considered on the signaling pathway of the NLRP3 inflammasome complex, TLR4, and serum levels of inflammatory cytokines (IL-1? and IL-18) in type II diabetic patients with ARDS. For this purpose, 25 diabetic type II patients with ARDS were divided into three groups by ARDS Berlin Definition. Then, after providing the demographic and clinical characteristics of the patients, they were treated with 30 mg/day borage oil for seven days. The expression of NLRP3 and TLR4 genes (by Real-time PCR technique) and serum levels of IL-1? and IL-18 (by ELISA test) were evaluated before and after treatment with borage oil through blood samples taken from patients. The results showed that serum levels of inflammatory cytokines (IL-1? and IL-18), NLRP3 gene, and TLR4 gene were significantly decreased in diabetic type II patients with mild ARDS by treating with borage oil. IL-1? serum level and TLR4 were significantly decreased in diabetic type II patients with moderate ARDS. But there was not any significant decrease or increase in IL-1?, IL-18, NLRP3 gene, and TLR4 gene in diabetic type II patients with severe ARDS after 7 days of treatment with borage oil. According to the obtained results, borage oil can act as a double-edged blade. Thus, in the early and middle stages of ARDS, borage oil can be effective in reducing the inflammasome pathway of inflammation and also reduce blood sugar levels in these diabetic patients. But in the severe stage of ARDS, it not only does not help to treat the ARDS; it also increases systolic and diastolic blood pressure in diabetic patients.

ROLE OF TLR2, TLR4 GENE POLYMORPHISM IN DEVELOPING MICROVASCULAR COMPLICATIONS IN ADOLESCENTS WITH TYPE 1 DIABETES MELLITUS

Long-term complications of type 1 diabetes mellitus (T1DM) in children and adolescents are an important problem in modern medicine. Recently, the role of immune mechanisms, in particular, chronic inflammation, in the development of both T1DM and its microvascular complications has been actively discussed. Activation of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) leads to hyperproduction of proinflammatory cytokines, chemokines, adhesion molecules involved in the formation of diabetic microvascular complications. At the same time, TLR2 and TLR4 gene polymorphism alters the immune susceptibility to the endogenous ligands, which may increase the risk of diabetic microangiopathies. The aim of this study is to evaluate the frequency of genotypes and alleles of TLR2 and TLR4 genes distribution and to determine the content of TNFα, IL-1, VCAM-1, fractalkine, endothelin-1 in adolescents with T1DM with microvascular complications. We examined 139 adolescents with T1DM from 14 to 18 years old and 56 healthy teenagers. Patients with T1DM were divided into two groups: Group I – patients with poor glycemic control (HbA1C > 9.0%), (n = 64); Group II – patients with satisfactory glycemic control of T1DM (HbA1C ≤ 9.0%), (n = 75), including adolescents with optimal (HbA1C < 7.5%) and suboptimal glycemic control (7.5% ≤ HbA1C ≤ 9.0%) (ISPAD clinical practice consensus guidelines 2014). According to the presence of microvascular complications, the groups were subdivided into subgroups: Iа (n = 49), IIа (n = 38) – adolescents with verified microvascular disorders: diabetic retinopathy, nephropathy and neuropathy; Ib (n = 15), IIb (n = 37) – without microvascular complications. Allelic variants of TLR genes were determined using test systems GosNII genetics (Moscow). The content of cytokines in blood serum was carried out by the method of enzyme-linked immunosorbent assay “BIOSCIENCE”. Data were analyzed using software packages Statistica version 6.0. The assessment of TLR2 (Arg753Gln) and TLR4 (Thr399Ile) polymorphism distribution did not reveal significant differences between the observed subgroups and the control. In Ia and IIa subgroups (with complications) Asp299Gly variant was noted to be significantly less common when compared to subgroups Ib, IIb and controls. The presence of Gly allele in TLR4 gene was found to disrupt the expression of TNFα and VCAM-1 and can be considered protective for the development of microvascular complications.

TLR4 gene polymorphisms in Egyptian vitiligo patients: insights into emerging association with clinical activity, family history, and response to therapy

Background Vitiligo is a common pigmentary disorder in which autoimmunity has been suggested to play an important role. Toll-like receptor (TLR) family are recognized different molecular structures expressed on immune cells and have been implicated in a number of autoimmune diseases (AIDs) such as vitiligo. The purpose of this study was to investigate the possible association between TLR4 gene polymorphisms: rs11536858, rs1927911, rs1927914 in Egyptian vitiligo patients and their clinical data, their response to therapy. Using PCR-RFLP for TLR4 gene polymorphisms (rs11536858, rs1927911, and rs1927914), both alleles and genotypes were determined after extraction of DNA in a case-control study of 100 vitiligo Egyptian patients and 100 matched age and sex controls. Results The distribution of the protective CT genotype of rs1927914 was higher in the control group. After dividing both patients and controls into 2 age groups (below 18 and above 18 years), no significant associations between the genotypes of the selected TLR4 SNPs and the demographic and clinical data of the vitiligo patients in group 1 (below 18 years) were observed. For group 2 (above 18 years), also no significant associations were found except for the association between the CC genotype of rs1927914 and psychiatric trauma, from one side, and between the CT genotype of rs1927911 and alopecia, from the other side. The association between combined genotypes and the risk of vitiligo showed either higher frequency in patients (risky), or controls (protective), and some equal frequencies (non-significant). The association between haplotypes and risk of vitiligo in patients’ group revealed the highest frequency for the risky ATT and the least frequency for ATC haplotypes. In control group, the protective GCT haplotype showed the highest frequency while the GTC and GCC showed the least frequency. No significant correlations of haplotypes with clinical and demographic data of selected patients’ group were observed apart from that between ACC haplotype and family history of AIDs and between ATT haplotype and remission after phototherapy. Conclusions The significant relationship between TLR4 gene polymorphisms and vitiligo patients charcteristics clarify the role of innate immunity in pathogensis of vitiligo and its effect on the used therapies.

PREVALENCE AND CHARACTERISTICS OF TUBERCULOSIS IN HIV-INFECTED PATIENTS CONSIDERING THE 299GLY ALLELE TLR4 GENE CARRIAGE

Human immunodeficiency virus (HIV) infection remains one of the most acute problems of modern medicine. Tuberculosis is known as the leading cause of death among the opportunistic infections in HIV-positive people; moreover, TB is also known as resulting in one of three deaths associated with acquired immunodeficiency syndrome. It should be stressed the TB course against the background of HIV infection demonstrates the atypical characteristics, nonspecific clinical symptoms with increasing frequency of extrapulmonary lesions, minimal radiological manifestations, low frequency of the pathogen excretion, and rapid course of the disease. In recent years, researchers around the world have paid considerable attention to studying the effects of genetic variation of genes on the course of infectious diseases in humans, including HIV and tuberculosis, and in particular, to investigating Tool-receptors, innate immune system receptors, which interact with pathogens and stimulate effector mechanisms of innate immunity. The objective of this study is to determine the prevalence and evaluate the features of the TB course before and during the antiretroviral therapy, considering the carriage of the 299Gly allele of the TLR4 gene. To assess the manifestations, clarify the clinical characteristics of the disease in the dynamics before and during the antiretroviral therapy, a retrospective cohort examination of 181 HIV-positive patients before and after the therapy was carried out. The study has demonstrated that, despite the virological and immunological effectiveness of the treatment, the TB detection in HIV-infected patients taking antiretroviral therapy remained almost constant compared to the period before antiretroviral therapy (17.0% vs. 14.9%, > 0.05). Analysis of genotypes of the TLR4 gene showed that during the observation period before antiretroviral therapy in patients with the 299Gly allele there was a 6.3-fold higher risk of developing of disseminated TB forms (OR = 6.29 [95% 1.20-32.99], p = 0.044), compared with carriers of Asp299Asp genotype. In HIV-infected patients with the 299Gly allele of the TLR4 gene on the background of antiretroviral therapy, the risk of TB development is 3.4 times higher (p = 0.008) than in carriers of its homozygous genotype.

TLR4 and TLR9 polymorphism: Probable role in susceptibility among the population of Bihar for Indian visceral leishmaniasis

Genetic variations in the host TLRs genes play an important role in susceptibility and/or resistance to visceral leishmaniasis by altering the host-pathogen interaction. In this study, we investigated the association between polymorphisms of TLR4 (Asp299Gly, Thr399Ile) and TLR-9 (T-1237C), with susceptibility to visceral leishmaniasis. A bi-directional PCR amplification of specific alleles technique was used to characterize the distribution of TLR4 (Asp299Gly and Thr399Ile) and TLR9 (T-1237C) polymorphisms. A total of 60 samples were randomly selected from confirmed visceral leishmaniasis patients and 24 endemic healthy volunteers. The samples were genotyped and allele frequencies were determined. We observed that TLR4 Asp299Gly and Thr399Ile genotypes were more frequent in visceral leishmaniasis patients (10% and 15% respectively) compared to controls (4.2% and 8.3% respectively). However, the differences were not significant in TLR4 Asp299Gly and Thr399Ile alleles and genotypes. In the case of TLR9, we observed the frequency of T1237C genotype was higher in visceral leishmaniasis patients (43.3%) than in healthy controls (33.3%). Statistically significant differences were observed in TLR9 T1237C alleles and genotypes. We concluded that TLR9 T1237C, but not TLR4, gene polymorphisms can be regarded as contributors to visceral leishmaniasis susceptibility among the Indian population of Bihar state.

Expression of toll-like receptor 4 gene polymorphism and inflammatory factors in peripheral blood on patients with sepsis

Objective: To observe the presence of toll-like receptor 4 (TLR4) gene polymorphism in the venous blood in patients with sepsis, meanwhile, to observe the expression and the diagnostic value of TLR4 mRNA, interferon-γ (IFN-γ), interleukin-23 (IL-23), procalcitonin (PCT) and C-reactive protein (CRP) in patients with different degrees of sepsis.Methods: The peripheral blood samples from the subjects were collected to extract genomic DNA, gene sequencing and enzyme digestion method were applied to the detection of TLR4 Asp299Gly loci polymorphism. The expression of TLR4 mRNA in the peripheral blood was detected by reverse transcriptase polymerase chain reaction (RT-PCR). The expression levels of IFN-γ, IL-23, PCT and CRP were measured by enzyme linked immunosorbent assay (ELISA).Results: (1) The gene polymorphism was not present in TLR4 Asp299Gly loci; (2) The expression of TLR4 mRNA in the peripheral blood in patients with sepsis: on d1, there were statistically significant differences between the normal group and the group (APACHE II ≤ 20), between the normal group and the group (APACHE II > 20), between the group (APACHE II ≤ 20) and the group (AAPACHE II > 20) (t = 5.741, 14.780 and 10.500, all p < .01). APACHE II stands for Acute Physiology and Chronic Health Evaluation II. On d7, there were statistically significant differences between the normal group and the group (APACHE II ≤ 20), between the normal group and the group (APACHE II > 20), between the group (APACHE II ≤ 20) and the group (APACHE II > 20) (t = 4.186, 13.830 and 9.645, all p < .01); (3) The expression levels of IFN-γ, IL-23, PCT and CRP were obviously upregulated (all p < .01), and TLR4 was positively correlated with IFN-γ and IL-23 (all p < .01); (4) The best cutoff value of TLR4 mRNA at baseline was 891.6 μg/L, with a sensitivity of 100% and a diagnostic specificity of 57%. The best cutoff value of IFN-γ at baseline was 84.5 μg/L, with a sensitivity of 100% and a diagnostic specificity of 57%. The best cutoff value of IL-23 at baseline was 861 μg/L, with a sensitivity of 100% and a diagnostic specificity of 97%.Conclusions: (1) The Asp299Gly polymorphism is not present in TLR4 gene; (2) The expression levels of TLR4 mRNA, IFN-γ and IL-23 are relatively high in patients with sepsis, and will be higher with the increased severity of sepsis; (3) TLR4 mRNA, IFN-γ and IL-23 can be used as molecular biomarkers for the early diagnosis of sepsis.

Minimal role of interleukin 6 and toll-like receptor 2 and 4 in murine models of immune-mediated bone marrow failure

Immune aplastic anemia (AA) results from T cell attack on hematopoietic cells, resulting in bone marrow hypocellularity and pancytopenia. Animal models have been successfully developed to study pathophysiological mechanisms in AA. While we have systemically defined the critical components of the adaptive immune response in the pathogenesis of immune marrow failure using this model, the role of innate immunity has not been fully investigated. Here, we demonstrate that lymph node (LN) cells from B6-based donor mice carrying IL-6, TLR2, or TLR4 gene deletions were fully functional in inducing severe pancytopenia and bone marrow failure (BMF) when infused into MHC-mismatched CByB6F1 recipients. Conversely, B6-based recipient mice with IL-6, TLR2, and TLR4 deletion backgrounds were all susceptible to immune-mediated BMF relative to wild-type B6 recipients following infusion of MHC-mismatched LN cells from FVB donors, but the disease appeared more severe in IL-6 deficient mice. We conclude that IL-6, TLR2, and TLR4, molecular elements important in maintenance of normal innate immunity, have limited roles in a murine model of immune-mediated BMF. Rather, adaptive immunity appears to be the major contributor to the animal disease.