The sequences of IL18, TNF-α and INF-γ genes in Iraqi patients with Visceral Leishmaniasis

IntroductionIn adults and children with RSV infection, a polymorphism in the IL-6 promoter at position −174 predicted illness magnitude. Also, polymorphisms in the IL-10, TNFα and INFγ genes were associated with immune responsiveness and the frequency of complications. Here, the effect of these polymorphisms on illness and seroconversion during infection with rhinovirus type 39 (RV39) was evaluated.MethodsSeventy-two adults were genotyped for the selected polymorphisms, experimentally exposed to RV39 and followed for infection, seroconversion and symptoms/signs of illness. Regression analysis was used to determine if these polymorphisms predicted seroconversion and illness magnitude in 57 infected subjects.ResultsThe low production IL-6 (−174, C/C) phenotype was associated with greater symptom magnitudes and the INFγ (+874) phenotype predicted the frequency of seroconversion. No relationship between the IL-10 or TNFα polymorphisms and any measured outcome was documented. IL-6 protein measured in nasal wash fluids of 51 subjects was positively correlated with symptom magnitude but was independent of the IL-6 (−174) genotypes representing high and low production phenotypes.ConclusionsThese results document significant associations between the IL-6 (−174) and INFγ (+874) gene polymorphisms and specific responses to experimental RV39 infection. For the IL-6 (−174) polymorphism, the results replicate those for RSV infection.

This study aimed to determine whether anti-rK39 antibodies were diagnostic markers for visceral leishmaniasis (kala-azar) and to evaluate the correlation between age and gender in disease occurrence in Iraqi patients. In addition, it aimed to evaluate the correlation between thyroid hormones, i.e., thyroid-stimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4) and anti-rK39 antibodies. Immunochromatographic technique used for anti-rK39 antibodies detection. Enzyme-linked immunosorbent assay was used for determining the serum TSH, T3, and T4 levels. One hundred thirty-eight patients with visceral leishmaniasis were included. The mean age was 27.65±11.60 years. Sixty-one patients (44.2%) were males, and their mean age was 29.65±11.10 years. The mean age of females was 26.12±11.89 years. Anti-rK39 antibodies were detected in 11.59% of patients. Anti-rK39 antibodies were equally detected (5.8%) in both genders without a significant difference (p=0.212) or correlation between gender and anti-rK39 antibodies (p=0.623). There was neither a significant difference (p>0.05) nor correlation between gender; age groups according to gender and anti-rK39 antibodies (p>0.05). Both males and females who were positive for anti-rK39 antibodies had normal TSH, T3, and T4 levels. Only one patient who was positive for anti-rK39 antibodies had an elevated T4 level (>12 μg/dL). Neither a significant difference nor correlation was reported among genders; anti-rK39 antibody positivity (p>0.05); and TSH, T3, and T4 levels. Anti-rK39 antibodies, a daignostic marker for visceral leishmaniasis have no correlation with patients age and gender. Serum TSH and T3 levels were not affected by visceral leishmaniasis. Visceral leishmaniasis causes the increase in serum T4 levels. Thyroid involvement appears to be uncommon in patients who present with visceral leishmaniasis.

Objective To study the relationship between lung ventilation function of workers exposed to rare earth dust and their IL-2,IL-6 and TNF-α gene polymorphism.Methods TNF-α gene polymorphism were identified by RFLP-PCR,IL-2 and IL-6 gene polymorphisms were identified by PCR-CTPP analysis.Lung ventilation function was deteced by instrument of ventilation function.Results Compared with controls,there was no statistic significance in frequencies distribution of TNF-α gene polymorphism(X2=4.03,P＞0.05),IL-2 gene polymorphism(X2=2.21,P＞0.05)and IL-6 gene polymorphism(X2=1.05,P＞0.05).Compared with IL-2 gene wild type,IL-2 homozygote type increased the risk of lung ventilation dysfunction by 4.29 folds(95% CI 1.09～16.9).Conclusions Compared with controls,incidence of ventilation function of workers exposed to rare earth dust is in ascending trend.IL-2(G/G)gene type induces more serious inflammation reaction than the others. Key words: IL2; IL-6; TNF-α; Gene polymorphism; Lung ventilation dysfunction

To investigate whether the TNF-α, IL-2, IL-4 and IL-10 genes contribute to variations in vaccine-induced immune responses after immunization with the inactivated Japanese encephalitis vaccine (IJEV), a total of 369 individuals who received the IJEV were enrolled. Based on Japanese encephalitis virus (JEV) neutralization antibodies (NAbs), the individuals were divided into seropositive (SP) and seronegative (SN) groups. Then, 17 SNPs in the TNF-α, IL-2, IL-4 and IL-10 genes were genotyped using the TaqMan method. Although there was no association of the TNF-α, IL-2, IL-4 and IL-10 genes with JEV seropositivity triggered by JEV vaccination when all the individuals in the SP and SN groups were compared, differences were observed in a subgroup analysis. In the male group, rs2243291 in the IL-4 gene showed a difference between the JEV SP and SN groups with the overdominant model (P = .045), and the C/G genotypes conferred more JEV seropositivity (OR = 1.87; 95% CI: 1.01–3.49); the CT genotype of rs3093726 in the TNF-α gene showed higher JEV NAbs geometric mean titer (GMT) than the TT genotype (P = .018, CT: 1.677 ± 0.144 vs TT: 1.271 ± 0.039). Furthermore, the rs1800629 genotype in the TNF-α gene and the rs1800896 genotype in the IL-10 gene exhibited a trend of association with JEV seropositivity in the female group, but the difference was not significant. The present study suggested that the polymorphisms in the cytokine genes could be associated with sex-specific JEV NAbs seroconversion. However, more samples should be studied, and further functional verification should be performed.

Zebrafish (Danio rerio) is an established model for studying various metabolic diseases. The aim of this study was to evaluate the effect of resveratrol as a natural polyphenol on reducing inflammation caused by hyperglycemia (diabetes) and its effect on digestive tissue as well as TNF-α, IFN-γ, and INL1β genes in zebrafish. Within a 20-day period, the research was performed on 120 adult zebrafish, which were randomly classified into eight groups: two experimental treatments (induced glucose = +G) and (without glucose = -G), where each main group was as follows: CTRL = control and RSV resveratrol with doses 10, 20, and 30μmol/L. At the end of the period, the blood glucose level was measured using glucose test strip, staining of intestinal tissue was done by hematoxylin and eosin (H&E), and expression of INF-γ, IL1-β, and TNF-α genes extracted from the intestinal was measured via internal method RT-PCR. Data analysis in this study was performed using SPSS software version 21. One-way ANOVA and mean comparison of treatments by Duncan test were used for data analysis. All statistical analyses were performed at a significant level (P < 0.5) where the mean data were presented with standard deviation. According to the results, the lowest blood sugar level at the end of the experiment belonged to the group (G-RSV20) where no significant difference was observed between treatments (P > 0.05). The highest expression of IL1-β gene belonged to the (G + CTRL) group (P < 0.05), while the (G + RSV20) group showed the lowest expression of the INF-γ gene and had a significant difference with other groups (P < 0.05). In (G + RSV10) treatment, the lowest expression of TNF-α gene was observed and there was no significant difference with other treatments (P > 0.05). Resveratrol would improve the absorption of nutrients in the intestinal tissue by increasing the number of goblet cells as well as the width and height of the villi. Collectively, this study indicated that treatment with resveratrol could improve metabolic-mediated performances by reducing blood glucose, increasing food absorption in the intestine tissue, and reducing the expression of inflammatory genes in type 2 diabetic zebrafish model.

To the Editor: In Argentina, 14 autochthonous human cases of visceral leishmaniasis (VL) were reported during 1925–1989. These cases occurred in different localities in Salta, Jujuy, Santiago del Estero, and Chaco Provinces of northwestern Argentina (Figure A1), where cutaneous leishmaniasis (CL) caused principally by Leishmania (Viannia) braziliensis is endemic. It had been postulated that scattered/sporadic VL cases could be caused by visceralization of dermatrophic Leishmania spp. because of 1) absence of already recognized L. (Leishmania) infantum vector species; 2) geographic overlap with the region where CL is endemic; 3) simultaneous symptoms of CL; or 4) lack of detailed parasitic characterization at the molecular level for cases of suspected VL (1). However, during recent decades, urban outbreaks of VL have spread to southern regions of South America (Mato Grosso do Sul, Brazil, and Asuncion, Paraguay) near the northern border with Argentina. In May 2006, an autochthonous human case of VL was reported in Posadas (northeastern Argentina); it was associated with the canine visceral form of the disease. In addition, the presence of Lutzomyia longipalpis sandflies was also reported (2). Currently, 58 human VL cases have been reported in Posadas (3), and >7,000 infected dogs, Lu. longipalpis sandflies, and canine VL have been found 350 km south of Posadas (4). During 2007–2008, new VL cases in 4 children and 7 dogs were reported clustering in time and space in La Banda-Santiago del Estero in the dry Chaco region of Argentina. This focus showed a different pattern from that found in the only urban outbreak of VL reported (nearly the same number of cases in humans and dogs, and the suspected vector was Lu. migonei sandflies instead of Lu. longipalpis sandflies) (5). We report a case of autochthonous human VL in Salta Argentina that was caused by L. (L.) infantum. This parasite was characterized by cytochrome b (cytb) gene sequencing. Sequencing of this gene has been validated for precise characterization of Leishmania spp (6,7). On September 9, 2009, a 44-year-old man from Salta, Argentina (Figure A1), was admitted to the Infectious Disease Service at Hospital Senor del Milagro in Salta. The patient had fever, weight loss, dyspepsia, and splenomegaly that evolved over 3 weeks. Physical examination showed cutaneous and mucosal paleness. His general condition was feverish and rapidly deteriorating. Laboratory tests at the time of final diagnosis showed anemia, leukocytopenia, thrombocytopenia, and increased levels of lactate dehydrogenase. Results of urinalysis and coproculture were negative for parasites. Electrophoresis of serum proteins showed increased levels of gamma globulins. The differential diagnosis was negative for malaria, mycosis, autoimmune hepatitis, and lymphoma. A bone marrow smear showed abundant amastigotes by Giemsa staining (Figure, panel A). The patient was treated with liposomal amphotericin B, 3 mg/day for 7 days, and recovered (8). After a comprehensive interview, we verified that this patient had not been in the VL-endemic area in Argentina. However, he had worked (deforestation activities) during January–February 2009 on a farm in Finca Las Maravillas (22o3′29.30″S, 63o14′28.17″W), where he had been bitten by phlebotomines and acquired the disease. This farm was situated in the dry Chaco region near the border with Bolivia and Paraguay (zones with VL) (9), a region with intensive deforestation and agricultural activities. For species identification, DNA was extracted from a bone marrow aspirate and peripheral blood. We amplified by nested PCR and sequenced the cytb gene (Figure, panel B) (6). The aligned 817-bp sequence obtained showed 100% homology with the cytb gene of the MHOM/TN/80/IPT1 L. (L.) infantum World Health Organization reference strain (Tunisian strain) and 99.3% homology with the MHOM/BR/74/PP75 L. (L.) chagasi strain (Brazilian strain) (7). L. (L.) infantum was identified as the causative agent of this VL case in Salta, Argentina, where VL cases had not been seen for 50 years. Our findings indicate that this case was not caused by visceralization or a dermatropic Leishmania spp. We suggest that the scattered pattern of VL incidence in the dry Chaco region is caused by an enzootic cycle with accidental human transmission (5). There are no reports of Lu. longipalpis sandflies in the study area or surrounding areas (10). Nevertheless, studies on natural infections of vector sandflies and reservoir-host animals (especially dogs) are needed. Therefore, the search for naturally infected sandflies and reservoirs of this infection should be intensified. Epidemiologic surveys of dogs are needed to identify spread of VL foci in areas of deforestation. Deforestation could alter vector and reservoir range and parasite density in the enzootic cycle and increase human exposure to infected vectors. Figure Case of autochthonous human visceral leishmaniasis in a 44-year-old man, identified by parasitologic diagnosis and molecular detection of the causative species, Salta, Argentina. A) Leishmania amastigotes in a bone marrow smear. N, nucleus; K, kinetoplast; ...

Polymorphisms in IL-10 and INF-γ genes are associated with early atherosclerosis in coronary but not in carotid arteries: A study of 122 autopsy cases of young adults

Human endogenous retroviruses (HERVs) constitute about 8% of the human genome. The overexpression of HERVs has been detected in various inflammatory disorders like neuro-inflammation disorders and cancer. Interestingly, it has been reported that stress conditions facilitate HERV expression. Space travel exposes astronauts to microgravity environments (a stress condition), which may result in the activation of HERVs and might influence pathogenic outcomes during and after space flight. This study aimed to elucidate the transcriptional activity of three HERV families (W, K, and H) and cytokine genes (IL-1, IL-6, and TNF-α) in different cell lines under microgravity (μg) conditions and compare them with the results obtained under normal gravity (ng; 1g). We evaluated the expression of HERVs (HERV-K env, HERV-K gag, HERV-W env, and HERV-H env) and cytokine gene expression (IL-1, IL-6, and TNF-α) in neuroblastoma (SH-SY5Y), HEp-2, and Caco-2 cell lines under simulated μg and 1g conditions. In SH-SY5Y cells, the expression level of the IL-1, IL-6, HERV-H env, HERV-K env, HERV-K gag, and HERV-W env genes was significantly increased when exposed to short-term μg (3 and 6 h). The expression of TNF-α remained unchanged throughout all time points. Additionally, in Caco-2 cells, the expression of the HERV-K env, HERV-K gag, and IL-1 genes was significantly higher after 6 h of incubation in μg conditions compared to 1g. There was no statistically significant difference in the expression levels of the HERV-W env, HERV-H env, IL6, and TNF-α genes between the μg and 1g conditions. Moreover, in HEp-2 cells, the expression of the IL-1, IL6, TNF-α, HERV-H env, HERV-K env, HERV-K gag, and HERV-W env genes significantly increased following short-term incubation in μg (3 h, 6 h) and then decreased to levels comparable to those observed in the 1g condition. Taken together, the dysregulation of cytokine and HERV gene expression was observed under the simulated μg condition. The patterns of these dysregulations varied throughout cell lines, which demands further investigation for human health protection in space.

This study was carried out to investigate some cytokines (INF-g, IL-10, TNF-a) on blood samples of Iraqi children with Visceral leishmaniasis (VL) before and during treatment with stibogluconate and healthy people serving as control, using ELISA kits. Samples were confirmed parasitologically by bone marrow smears. The results of cytokine showed that there were significant increases in the levels of all the investigated cytokines (INF-g, IL-10, TNF-a) in the sera of patients with VL during active disease in comparison with the control group. On the other hand, patients after treatment showed a dramatic and progressive decline in serum levels of all the tested cytokine during the whole period-21 days- of therapy. These declines were started from day-7 to go back to the normal or comparable to the level of healthy controls after 21-days. Key words: Visceral leishmaniasis, cytokines, ELISA, INF-g, IL-10, TNF-a, stibogluconate.

For the best of our knowledge, there is no information about molecular characterization of Iraqiisolates of visceral leishmaniasis, the present work aimed to characterize three different Iraqiisolates of Leishmania infantum by polymerase chain reaction (PCR), restriction fragmentlength polymorphism (RFLP) and sequencing methods.Three isolates from bone marrow of Iraqi patients infected with kala azar were used in thisstudy. The isolates were already diagnosed by isoenzyme as Leishmania infantum. Patients wereinhabiting different parts of Baghdad. The samples after microscopic examination were culturedon modified NNN media. Then DNA was extracted for amplifying ITS1 (internal transcribedspacer 1) gene by PCR. Identification of samples was studied using RFLP (digestion with Apo1restriction enzyme) and sequencing of PCR products.The PCR of all samples showed a band under about 500 bp. The results by using PCR-RFLPmethod showed no restriction with digestion with Apo1 restriction enzyme. The results ofsequencing showed differences with all separated gene from Leishmania infantum in the genebank.In this study we found that the sequences of ITS1 gene of Leishmania infantum separated fromIraqi patients are different from other samples, as there is no similarity with Leishmaniainfantum (MHOM/TN/80/IPI1). The more similarity is with the Iranian isolate of Leishmaniainfantum (MCAN/IR/97/LON) with 41%, whereas the similarity with Crithidia luciliae internaltranscribed spacer 1, ITS1 is 96%.

Strategy for immunological analysis of pro-inflammatory cytokine marker studies with chronic hepatitis B virus in Southwestern region of Saudi Arabia

Cytokine gene variants of TNF-α and IL-10 in the propensity of type 2 diabetes in south Indian population

Susceptibility to severe influenza A/H1N1pdm09 virus is multifactorial. The present study was carried out in 246 patients infected with A/H1N1pdm09 virus to find out whether single nucleotide polymorphisms (SNPs) in the genes coding for proinflammatory and anti-inflammatory cytokines are associated with disease severity. Among the cases, 129 had mild disease, whereas 117 had severe disease. There were 27 fatal cases. TNFA rs1800629, IFNG rs2430561, IL10 rs1800872, IL10 rs1800896, and CCL2 rs1024611 SNPs were genotyped by polymerase chain reaction-based methods. A significantly higher frequency of TNFA rs1800629 "G/A" genotype was observed in severe and fatal cases compared with mild and survived cases, respectively. In a dominant mode, IL10 rs1800896 "G" allele was significantly negatively associated with disease severity. IL10 rs1800896 "C/A" genotype was significantly associated with fatality in influenza A/H1N1pdm09 infections. The results suggest that SNPs in the IL10 and TNFA genes might be associated with disease severity in influenza A/H1N1pdm09-infected patients.

Osteoarthritis (OA) is one of the major joint diseases, and the synovial inflammation is involved in the pathogenesis and progression of OA. Glucosamine (GlcN) is widely used as a dietary supplement for OA, and is expected to exert the antiinflammatory action in OA. However, the detailed mechanism for the antiinflammatory action of GlcN remains poorly understood. In this study, to elucidate the molecular mechanism involved in the GlcN-medicated regulation of synovial cell activation, we comprehensively analyzed the effect of GlcN on the gene expression using a human synovial cell line MH7A by DNA microarray. The results indicated that GlcN significantly downregulates the expression of 187 genes (≤1/1.5-fold) and upregulates the expression of 194 genes (≥1.5-fold) in IL-1β-stimulated MH7A cells. Interestingly, pathway analysis indicated that among the 10 pathways into which the GlcN-regulated genes are categorized, the 4 pathways are immune-related. Furthermore, GlcN suppressed the expression of proinflammatory cytokine genes (such as IL-6, IL-8, IL-24 and TNF-α genes). In addition, GlcN-mediated O-GlcNAc modification was involved in the downregulation of TNF-α and IL-8 genes but not IL-6 and IL-24 genes, based on the effects of alloxan, an O-GlcNAc transferase inhibitor. Thus, GlcN likely exerts an antiinflammatroy action in OA by suppressing the expression of proinflammatory cytokine genes in synovial MH7A cells by O-GlcNAc modification-dependent and -independent mechanisms.

Associations between IL1B (rs1143634), IL1RN (VNTR, intron 2), IL4 (VNTR, intron 3), TNFA (rs1800629, rs361525), IL6 (rs1800795), and IL10 (rs1800896) genetic polymorphisms in women with recurrent miscarriage (RM) were analyzed. We studied DNA samples of 112 women with RM and 267 women with physiological pregnancy. The IL1RN, IL4 genotypes were identified by PCR techniques, the IL1B, IL6 gene polymorphisms were defined by means of RFLP approach. To detect TNFA and IL10 gene polymorphisms, TaqMan real-time PCR was used. The results have shown that polymorphic loci of IL1B, IL1RN, IL10, TNFA genes were not associated with RM, and early spontaneous abortion risk. The 2R allele of IL4 gene was found to be associated with higher RM risk (OR = 1.52; 95% CI = [1.08-2.14]; P-value (cor) = 0.05), and G allele of IL6 gene was associated with a risk for > 3 early spontaneous abortions (OR = 2.10; 95% CI = [1.24-3.56]; P-value (cor) = 0.05), in an additive inheritance model. Upon evaluation of the data obtained, one may conclude that the IL4 (VNTR intron 3) and IL6 (rs1800795) gene polymorphisms could influence the RM development. These results may be useful for assessment of molecular mechanisms underlying early spontaneous abortion.