Polymorphic Sites at the 3’ Untranslated Region of the HLA-G Gene Are Associated with Differential hla-g Soluble Levels in the Brazilian and French Population

HLA-G molecule has well-recognized tolerogenic properties, and the encoding gene shows lower frequency of polymorphism at the coding region but higher variability at regulatory 5’ and 3’ untranslated (3’UTR) regions. At least three 3’UTR polymorphic sites have been associated with HLA-G mRNA regulation, including the 14 base pair (14bp) Insertion/Deletion, +3142C-G and +3187A-G. We studied the association of polymorphic sites at 3’UTR (sequencing analysis, encompassing the 14bp Ins-Del/+3003T-C/+3010C-G/+3027C-A/+3035C-T/+3142C-G/+3187A-G/+3196C-G polymorphic sites) with plasma soluble HLA-G levels (sHLA-G, detected by ELISA) in 187 French and 153 Brazilian healthy individuals. Allele and genotype frequencies were closely similar in both populations; however, Brazilians showed a higher HLA-G 3’UTR haplotype diversity. Considering sHLA-G levels in both populations altogether, individuals presenting 14bp Del/Del showed higher levels compared to 14bpIns/Ins genotype (P <0.05); those presenting +3010C/G showed higher levels compared to the +3010C-C genotype (P< 0.05); those presenting +3027C-C showed higher levels than the +3027A-A genotype (P< 0.05); and those bearing +3035C-C showed higher levels compared to the +3035C-T (P < 0.01) and +3035T-T (P < 0.05) genotypes. The analyses of 3’UTR haplotypes showed that UTR-1 (DelTGCCCGC) was associated with higher expression of sHLA-G, whereas UTR-5 (InsTCCTGAC) and UTR-7 (InsTCATGAC) with lower expression and other UTRs (UTR-2/3/4/6) exhibited intermediate levels. Since the differential expression of HLA-G may be beneficial or harmful depending on the underlying condition, the identification of individuals genetically programmed to differentially express HLA-G may help on defining novel strategies to control the immune response against the underlying disorder.     

The Paradox of High Availability and Low Recognition of Soluble HLA-G by LILRB1 Receptor in Rheumatoid Arthritis Patients

HLA-G is a regulatory molecule involved in immunologic tolerance. Growing evidence indicates that HLA-G plays a role in the regulation of inflammatory processes and autoimmune diseases. This study aimed at a systematic evaluation of soluble HLA-G (sHLA-G) in plasma of rheumatoid arthritis (RA) patients with long-lasting chronic inflammation. RA patients (n=68) and healthy controls (n=26) had their plasmatic sHLA-G measured by ELISA whereas the binding capability of sHLA-G to its cognate LILRB1 receptor was measured by a Luminex-based assay. All subjects were PCR-genotyped for HLA-G 14bp polymorphism (rs66554220). Significantly higher sHLA-G levels were observed in patients (p<0.001), however no significant differences were observed in LILRB1 binding capacity between RA patients and controls. Remarkably, the proportion of patients presenting specific binding of sHLA-G to LILRB1 was significantly decreased as compared to controls (56% vs. 81%, p=0.027). Patients without rheumatoid factor (RF-) were significantly overrepresented in the group of patients positive for LILRB1 binding as compared to patients without LILRB1 binding (31% vs 10%, p=0.033). Furthermore, methotrexate treated patients (n=58) revealed significantly lower LILRB1 binding to sHLA-G molecules than non-treated patients (medians: 12.2 vs. 67.7 units/ml, p=0.031). Unlike in controls, no significant differences in sHLA-G levels were observed among patients grouped by 14pb genotype. Thus, in a substantial number of late RA patients, the circulating sHLA-G molecules are impaired regarding LILRB1 recognition, meaning that although increased levels are observed; these molecules are not qualified to exert their protective functions against inflammation. Our findings offer new insights into the immunopathology of RA patients with long-lasting anti-RA-treatment and highlight the importance to also measure the binding capability of sHLA-G to LILRB1.     

A simple method for identifying bone marrow mesenchymal stromal cells with a high immunosuppressive potential

Background aimsThe beneficial activity of mesenchymal stromal cells (MSC) in allogeneic hematopietic stem cell transplantation requires correct use in terms of cell dose and timing of infusion and the identification of biomarkers for selection. The immunosuppressive bone marrow (BM)-derived MSC (BM-MSC) functions have been associated with the production of soluble HLA-G molecules (sHLA-G) via interleukin (IL)-10. We have established a reliable method for evaluating BM-MSC HLA-G expression without the influence of peripheral blood mononuclear cells (PBMC).MethodsThirteen BM-MSC from donors were activated with recombinant IL-10 or co-cultured with 10 different phytohemagglutinin (PHA)-treated PBMC (PHA-PBMC). Membrane-bound and sHLA-G expression was evaluated by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively; lymphoproliferation was measured by (methyl-³H)thymidine.ResultsThe results demonstrated the ability of IL-10 to stimulate both membrane-bound and sHLA-G production by BM-MSC. The levels of HLA-G expression induced by IL-10 in BM-MSC were associated with the inhibition of PHA-PBMC proliferation (sHLA-G, P = 0.0008, r = 0.9308; membrane HLA-G, P = 0.0005, r = 0.9502).ConclusionsWe propose the evaluation of sHLA-G production in IL-10-treated BM-MSC cultures as a possible marker of immunoregulatory function.     

Antigens HLA-G, sHLA- G and sHLA- class I in reproductive failure

It can be supposed that relation between HLA-G polymorphism and sHLA-G protein expression are associated with successful embryo implantation and pregnancy maintenance. The aim of the study was the estimation specific differences in expression of sHLA-G and sHLA- class I antigens in women with reproductive failure in comparison with fertile women. The study sample enrolled 80 women, divided into 2 groups. The study group (B) enrolled 60 women with reproductive failure including 20 women with 3 recurrent spontaneous abortions in the first trimester of pregnancy (RSA), 20 women with empty sac (ES) and 20 women with 3 consecutive in-vitro fertilization failures (IVFf). The control group (C) enrolled 20 fertile women with at least 2 children. Soluble HLA- class I antigens (sHLA-I) and soluble HLA-G (sHLA-G) were determined using ELISA test kits from IBio Vendor Labolatory Medicine, Inc. HLA-G allele found in individuals in our study were identified by comparing the obtained bp sequences of exon 2., 3. and 4. with bp sequences of HLA-G antigen published at the Nolan Research Institute website. The highest concentration of sHLA-I is noted among women with HLA-G 10401 allele which differed significantly for the mean sHLA-I concentration calculated for all the remaining alleles (p<0.0001). The most prevalent alleles were: HLA-G 10101, 10102 and 10108 with sHLA-I concentrations among women bearing those alleles significantly lower in comparison to the HLA-G 10401 carriers (p<0.001). Allele 10101 and 10102 was related to the lower significantly plasma sHLA-I concentrations than 10108 allele (p<0.02). Lowest mean sHLA-G values were observed in the IVFf group with significant difference from the remaining groups (p<0.05). To conclude, sHLA-G molecules is associated to certain HLA-G alleles and imply that sHLA-G levels are under genetic control. Low concentration sHLA-G seems to be prognostically important in IVF failure.     

Cytokine gene polymorphisms and cytokine levels in pulmonary tuberculosis

Polymorphisms in the cytokine genes are known to influence cytokine levels and may be associated with outcome of infections. We investigated the polymorphisms in the cytokine genes namely IFN-gamma (+874 and +5644), IL-2 (-330 and +160), IL-4 (VNTR), IL-6 (-174), IL-10 (-1082 and -819) and IL-12B (+1188) in 188 normal healthy subjects (NHS) and 166 pulmonary tuberculosis patients (PTB) using polymerase chain reaction-based methods. To study the influence of cytokine gene polymorphisms on cytokine levels, phytohaemagglutinin and culture filtrate antigen of Mycobacterium tuberculosis-induced cytokine levels were measured by ELISA from 72-h-old peripheral blood mononuclear cell culture supernatants. Significantly decreased frequency of TT genotype of IL-2 -330 polymorphism (p=0.024, odds ratio (OR) 0.53, 95% CI 0.31-0.92) was observed in patients compared to NHS. The genotype frequencies of other polymorphisms were not different between patients and NHS. IL-12p40 levels were significantly decreased among NHS with AA genotype of IL-12B gene polymorphism compared to NHS with AC genotype (p<0.05). Increased levels of IL-12p40 were observed among patients with CC genotype of IL-12B gene compared to patients with other genotypes (p<0.01). The present study suggests that the TT genotype of IL-2 -330 polymorphism may be associated with the protection to PTB in south India. Further, +1188 polymorphism of IL-12B gene either alone or in combination with closely linked genes may regulate IL-12p40 production and may play a major role on acquired immunity to tuberculosis.     

HLA-G UTR Haplotype Conservation in the Malian Population: Association with Soluble HLA-G

The HLA-G molecule plays an important role in immunomodulation. In a previous study carried out on a southern French population our team showed that HLA-G haplotypes, defined by SNPs in the coding region and specific SNPs located in 5′URR and 3′UTR regulatory regions, are associated with differential soluble HLA-G expression (sHLA-G). Furthermore, the structure of these HLA-G haplotypes appears to be conserved in geographically distant populations.The aim of our study is to confirm these expectations in a sub-Saharan African population and to explore additional factors, such as HLA-A alleles, that might influence sHLA-G expression.DNA and plasma samples were collected from 229 Malians; HLA-G and HLA-A genotyping were respectively performed by the Snap Shot® method and by Luminex™ technology. sHLA-G dosage was performed using an ELISA kit. HLA-G and HLA-A allelic and haplotypic frequencies were estimated using an EM algorithm from the Gene[Rate] program. Associations between genetic and non genetic parameters with sHLA-G were performed using a non-parametric test with GRAPH PAD Prism 5.Our results reveal a good conservation of the HLA-G UTR haplotype structure in populations with different origins and demographic histories. These UTR haplotypes appear to be involved in different sHLA-G expression patterns. Specifically, the UTR-2 haplotype was associated with low sHLA-G levels, displaying a dominant negative effect. Furthermore, an allelic effect of both HLA-G and HLA-A, as well as non genetic parameters, such as age and gender possibly linked to osteogenesis and sexual hormones, also seem to be involved in the modulation of sHLA-G.These data suggest that further investigation in larger cohorts and in populations from various ethnical backgrounds is necessary not only to detect new functional polymorphism in HLA-G regulatory regions, but also to reveal the extent of biological phenomena that influence sHLA-G secretion and this might therefore have an impact on transplantation practice.     

A single nucleotide polymorphism in 3'-untranslated region contributes to the regulation of Toll-like receptor 4 translation

We have previously shown that a single nucleotide polymorphism rs11536889 in the 3'-untranslated region (UTR) of TLR4 was associated with periodontitis. In this study the effects of this single nucleotide polymorphism on Toll-like receptor (TLR) 4 expression were investigated. Monocytes from subjects with the C/C genotype expressed higher levels of TLR4 on their surfaces than those from subjects with the other genotypes. Peripheral blood mononuclear cells (PBMCs) from the C/C and G/C subjects secreted higher levels of IL-8 in response to lipopolysaccharide (LPS), a TLR4 ligand, than the cells from the G/G subjects. However, there was no significant difference in TLR4 mRNA levels in PBMCs from the subjects with each genotype. After stimulation with tripalmitoylated CSK(4) (Pam(3)CSK(4)), TLR4 mRNA levels increased in PBMCs from both the C/C and G/G subjects, whereas TLR4 protein levels increased in PBMCs from the C/C but not G/G subjects. Transient transfection of a series of chimeric luciferase constructs revealed that a fragment of 3'-UTR containing rs11536889 G allele, but not C allele, suppressed luciferase activity induced by LPS or IL-6. Two microRNAs, hsa-miR-1236 and hsa-miR-642a, were predicted to bind to rs11536889 G allele. Inhibition of these microRNAs reversed the suppressed luciferase activity. These microRNA inhibitors also up-regulated endogenous TLR4 protein on THP-1 cells (the G/G genotype) after LPS stimulation. Furthermore, mutant microRNAs that bind to the C allele inhibited the luciferase activity of the construct containing the C allele. These results indicate that genetic variation of rs11536889 contributes to translational regulation of TLR4, possibly by binding to microRNAs.     

Human Leukocyte Antigen-G (HLA-G) Polymorphism and Expression in Breast Cancer Patients

Human leukocyte antigen-G (HLA-G) is known to be implicated in a tumor-driven immune escape mechanism in malignancies. The purpose of this study was to investigate HLA-G polymorphism and expression in breast cancer. HLA-G alleles were determined by direct DNA sequencing procedures from blood samples of 80 breast cancer patients and 80 healthy controls. Soluble HLA-G (sHLA-G) was measured by enzyme-linked immunosorbent assay (ELISA) from serum specimens. HLA-G expression in breast cancer lesions was also analyzed by immunohistochemistry staining. The presence of HLA-G 3′ untranslated region (UTR) 14-bp sequence was analyzed and found to be associated with reduced risk of breast cancer susceptibility based on HLA-G expression in tissues (P = 0.0407). Levels of sHLA-G were higher in the breast cancer group (median 117.2 U/mL) compared to the control group (median 10.1 U/mL, P<0.001). The area under the receiver operating characteristic curve (AU-ROC) values of sHLA-G for differentiating breast cancer from normal controls and for detecting metastasis from other stages of breast cancer were 0.89 and 0.79, respectively. HLA-G polymorphism and expression may be involved in breast carcinogenesis and sHLA-G concentrations could be used as a diagnostic marker for detecting breast cancer.     

Peripheral mononuclear cell IL-10 and IL-12 production is not impaired in patients with advanced cancer and severe infection

We assessed whether interleukin (IL)-10 and IL-12 production of blood mononuclear cells (PBMC) after lipopolysaccharide (LPS 0.1 microgram/ml, 1.0 microgram/ml) or phytohemagglutinin (PHA 5 microgram/ml) stimulation in advanced cancer patients with severe infections (IA, n=10) differs from those without infection (AC,n =10) or healthy controls HC (n=10). PBMCs in IA produced statistically higher median IL-12 levels than those in HC; LPS 0.1 (P=0.05), LPS 1.0 (P=0.019) and PHA (P=0.024). IL-10 production was always higher in AC than in HC. Both the IL-10 (LPS 1.0; P=0.018) and IL-12 (LPS 0.1;P =0.046) production of PBMCs were significantly higher in IA than one month afterwards. In conclusion, the PBMC production of IL-10 and IL-12 in advanced cancer patients with or without infection was not impaired but rather enhanced compared to that of HC. One month after infection, however, PBMC production clearly decreased, suggesting a kind of hyporeactivity.     

Hepatitis C virus genotype and HIV coinfection affect cytokine mRNA levels in unstimulated PBMC but do not shift the T1/T2 balance

Rapid progression of hepatitis C virus (HCV) disease in patients with HIV/HCV may reflect different cytokine responses and be influenced by HCV genotype. This is addressed by a study of patients with HIV/HCV coinfection and infection with HCV genotype 2 or 3 (2/3). They are compared with coinfected patients infected with genotype 1 and HCV monoinfected patients matched for HCV genotype. IFN-gamma, IL-10, IL-4 and IL-4delta2 mRNA were quantified by real-time PCR in unstimulated PBMC and after in vitro stimulation with HCV core or nonstructural 3/4A antigen. In unstimulated PBMC, levels of IFN-gamma and IL-4 mRNA were lowest in HIV/HCV genotype 1 patients, intermediate in HIV/HCV genotype 2/3 patients and highest in HCV genotype 2/3 patients. Neither HCV genotype nor HIV affected levels of IL-10 mRNA in unstimulated PBMC or IFN-gamma, IL-4 and IL-10 mRNA in PBMC stimulated with HCV antigens. Levels of IL-4 and IL-4delta2 mRNA correlated in mitogen-stimulated PBMC from all patient groups but both were low in HIV/HCV genotype 1 patients. Serum soluble CD30 levels (a putative marker of a T2 cytokine environment) did not differ between patient groups. The data do not suggest a shift in the T1/T2 balance driven by HIV coinfection or HCV genotype but either may affect IL-4 bioavailability.     

Interleukin-18 single nucleotide polymorphisms contribute to the susceptibility to brucellosis in Iranian patients

It seems that IL-18 has a crucial role in immunity against Brucella infection. Since the expression of IL-18 can be affected by polymorphisms in its gene, we decided to investigate any probable relationship between the six different IL-18 gene polymorphisms and brucellosis. A total of 193 patients with brucellosis and 83 healthy farmers who consumed contaminated raw milk and dairy products from the animals with brucellosis, were included in this study. All the individuals were genotyped for six IL-18 polymorphisms at positions -656, -607, -137, +113, +127 and codon 35/3, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The distributions of alleles for IL-18 polymorphisms at positions -137G/+113T/+127C/codon 35/3A (correlated with higher production of IL-18) were significantly higher in healthy controls than in patients (P=0.012, 0.012, 0.012 and 0.0018, respectively). It could be suggested that individuals who inherited the aforementioned genotypes/alleles are able to produce higher levels of IL-18 at the onset of infection, and it leads to more IFN-gamma production and control Brucella infection before the emerging brucellosis.     

Interleukin-1beta and receptor antagonist (IL-1Ra) gene polymorphisms and the prediction of the risk of end-stage renal disease.

Cytokines play an important role in the pathogenesis of kidney disease and its progression to end-stage renal disease (ESRD). Inflammation is regulated by the genes of the interleukin 1 (IL-1) gene cluster. Therefore, it was hypothesized that a polymorphism in this gene cluster may be associated with the risk of ESRD. Polymorphisms in the IL-1 gene cluster were examined in a cohort of 222 ESRD patients and 206 controls of similar ethnicity. These individuals were genotyped for IL-1 beta (promoter -511 and exon-5 +3953) genes and a variable number of tandem repeats (VNTR) in the IL-1 receptor antagonist gene (IL-1Ra). There was significant difference in genotype frequencies between ESRD patients and control group for IL-1beta (promoter region and exon-5) and IL-1Ra gene polymorphism (p < 0.001, 0.006 and < 0.001, respectively). A significant difference was observed in IL-1Ra for 1/1 (410/410) and 1/2 (410/240) genotypes, and the risk for ESRD was higher in those carrying the 1/1 genotype (p = 0.014, OR = 1.692, and p < 0.001, OR = 0.163). Also identified was a novel, rare allele of a single copy of 86 bp in ESRD patients as compared with the controls. The haplotype 'T-E2-1' frequency distribution between patients and controls revealed greater than threefold risk (p = 0.001, OR = 3.572, 95% CI = 1.589-8.032). Genetic linkage between the IL-1beta promoter region and exon-5 and between the IL-1beta promoter and IL-1Ra of IL-1 gene demonstrated a strong association among the variants in controls (D' = 0.42, p < 0.001, and D' = 0.39, p=0.001). Thus, the three polymorphisms within the IL-1 cluster are associated with ESRD. This finding is perhaps one of the strongest associations between genotype and ESRD reported, and it suggests that the IL-1 gene cluster affects the risk of development of ESRD.     

EB病毒感染及XRCC1、IL-10基因多态性与甲状腺癌的关联性分析

摘要 目的：探究Epstein-Barr病毒（EB病毒）感染及X射线交错互补修复因子1（XRCC1）、白介素-10（IL-10）基因多态性与甲状腺癌的关联性。方法：选取2020年1月~2022年12月132例甲状腺癌患者为研究组以及同期132例甲状腺良性腺瘤患者为对照组,采用原位杂交技术检测肿瘤标本EB病毒感染情况,聚合酶链反应-限制性内切酶片段长度多态性法检测XRCC1-399G/A位点、IL-10-592C/A位点基因多态性。结果：研究组EB病毒感染阳性率55.3%,高于对照组的33.3%（P<0.05）。研究组XRCC1-399G/A位点GA、AA基因型及A等位基因频率均高于对照组（P<0.05）;两组IL-10-592C/A各基因型频率比较差异无统计学意义,但研究组A等位基因频率高于对照组（P<0.05）。EB病毒感染阳性者较阴性者甲状腺癌风险增加3.337倍（95%CI：1.272~8.752）,携带XRCC1-399位点（GA+AA）型者较GG型风险增加2.438倍（95%CI：1.223~4.859）,携带IL-10-592位点（CA+AA）型者较CC型未增加甲状腺癌风险。不同病理类型甲状腺癌患者EB病毒感染情况及XRCC1-399位点、IL-10-592位点基因型分布比较差异均无统计学意义。结论：EB病毒感染阳性、XRCC1-399G/A位点突变基因型可能是甲状腺癌发病的易感因素,但二者与甲状腺癌病理类型无明显关系,而IL-10-592C/A基因多态性可能与甲状腺癌无关。     

Frequency of HLA-G exon 8 polymorphisms and kidney allograft outcome in Iranian population

The 14-bp polymorphism in exon 8 of the HLA-G gene is associated with HLA-G mRNA stability and the patterns of alternative isoform splicing and may influence the functionality of the HLA-G molecule. HLA-G expression was related to allograft acceptance and fewer episodes of acute rejection during heart, kidney and liver–kidney transplantation. In order to determine a possible correlation between the 14-bp insertion/deletion polymorphism and kidney allograft outcome in our population, genomic DNA was isolated from 144 patients who had received isolated kidney allografts. The recipients was divided into two groups, grafts presenting features of rejection group and a non-rejection group, and compared them with a control group of 100 healthy subjects. There was no significant difference in allelic frequencies of 14-bp insertion/deletion polymorphism between normal controls and kidney transplant patients. No significant difference was found between the RG and the NRG regarding the 14-bp genotypes and alleles. Therefore, additional studies with more sample size from other populations with analysis of other HLA-G polymorphisms are necessary to define this polymorphism as a valuable clinical marker.     

Inflammatory cytokines in vitro production are associated with Ala16Val superoxide dismutase gene polymorphism of peripheral blood mononuclear cells

Obesity is considered a chronic low-grade inflammatory state associated with a chronic oxidative stress caused by superoxide production (O(2)(-)). The superoxide dismutase manganese dependent (SOD2) catalyzes O(2)(-) in H(2)O(2) into mitochondria and is encoded by a single gene that presents a common polymorphism that results in the replacement of alanine (A) with a valine (V) in the 16 codon. This polymorphism has been implicated in a decreased efficiency of SOD2 transport into targeted mitochondria in V allele carriers. Previous studies described an association between VV genotype and metabolic diseases, including obesity and diabetes. However, the causal mechanisms to explain this association need to be more elucidated. We postulated that the polymorphism could influence the inflammatory response. To test our hypothesis, we evaluated the in vitro cytokines production by human peripheral blood mononuclear cells (PBMCs) carrier's different Ala16Val-SOD2 genotypes (IL-1, IL-6, IL-10, TNF-α, IFN-γ). Additionally, we evaluated if the culture medium glucose, enriched insulin, could influence the cytokine production. Higher levels of proinflammatory cytokines were observed in VV-PBMCs when compared to AA-PBMCs. However, the culture medium glucose and enriched insulin did not affect cytokine production. The results suggest that Ala16Val-SOD2 gene polymorphism could trigger the PBMCs proinflammatory cytokines level. However, discerning if a similar mechanism occurs in fat cells is an open question.     

The combination of the interleukin-1alpha (IL-1alpha-889) genotype and the interleukin-10 (IL-10 ATA) haplotype is associated with increased interleukin-10 (IL-10) plasma levels in healthy individuals

Family studies have demonstrated striking differences between individuals in their ability to produce IL-10 following lipopolysaccharide (LPS) stimulation of whole blood cultures in vitro, suggesting that differences in IL-10 production involve a considerable hereditary component. The first aim of this study was to analyse the possible effect of IL-10 genotypes and haplotypes on IL-10 plasma levels in a healthy Finnish population. As previous reports have demonstrated that endogenously produced IL-1 induces LPS-stimulated IL-10 production and that IL-10 inhibits synthesis of IL-1 in human monocytes, it is apparent that these two cytokines form an autoregulatory feedback loop. Secondly, we were interested whether any relationship could be found between IL-10 and IL-1beta in vivo. To examine this, the influence of IL-1alpha -889, IL-1beta -511 and IL-1Ra VNTR genotypes and IL-10 genotypes/haplotypes (ACC, GCC and ATA) on IL-10 plasma levels, and a putative correlation between IL-10 and IL-1alpha plasma levels were analysed. Four hundred adult blood samples were obtained from the Finnish Red Cross Blood Transfusion Centre, Tampere. The IL-10, IL-1alpha, IL-1beta and IL-1Ra gene polymorphisms were analysed using PCR. IL-1beta and IL-10 plasma levels were measured using an ELISA method. Our results indicated that increased IL-10 plasma levels were associated with the ATA haplotype (p = 0.03) and, surprisingly, with the IL-1alpha allele 2 carrier status (p = 0.02) in healthy individuals. This IL-1alpha 2+/ATA+ combination was found in 93 subjects out of 400 analysed (23%) and was associated with significantly high IL-10 plasma levels (p = 0.002). When individuals were classified into three groups, with no detectable IL-10 plasma levels (n = 145), with moderate levels (n = 152) and with high levels (n = 100) of IL-10, the IL-1alpha2+/ATA+ combination was more likely present among those with high levels than among those with undetectable levels of IL-10 (OR = 3.3, 95% CI 1.8 - 6.0, p < 0.001) or those with moderate levels of IL-10 (OR = 2.0, 95% CI 1.2 - 3.6, p = 0.012). Besides the observed association between IL-1alpha genotype and IL-10 levels, a moderate correlation was found between IL-10 and IL-1beta levels (r = 0.6, p = 0.01) among IL-10 producers (n = 252). The present findings suggest that the genotype combination of IL-1alpha 2+/ATA+ has a regulatory effect on basal IL-10 levels and that among individuals with measurable IL-10 plasma levels, IL-1beta and IL-10 basal levels correlate. Until now, data on the feedback loop between IL-1 and IL-10 cytokines have been based on studies in vitro, but now our results suggest that this relationship may also exist in vivo.     

Genotype and allele frequencies of divalent metal transporter 1 polymorphism in Turkish population

Divalent metal transporter 1 (DMT1 also known as DCT1, NRAMP2 or SCL11A2) is a membrane-bound divalent metal transporter which is conserved from prokaryotes to higher eukaryotes. It has been postulated to play important roles in intestinal iron absorption at the brush border of duodenal enterocytes, erythroid iron utilization, hepatic iron accumulation, placental iron transfer, and other processes. DMT1 gene which contains at least four isoforms (1A/+IRE, 1A/-IRE, 2/+IRE and 2/-IRE) is located on chromosome 12q13 in human. DMT1 mediates the transport of a wide range of metals, including the essential metals Fe2+, Zn2+, Mn2+, Cu2+, Co2+, Ni2+ and toxic metals such as Cd2+ and Pb2+. The intention of this study is to determine that IVS4+44C/A single nucleotide polymorphism in DMT1 gene of Turkish population. For this purpose blood samples from 192 female and 192 male volunteers were analyzed. DMT1 gene was amplified with the polymerase chain reaction-restriction fragment length polymorphism technique and 351 bp oligonucleotide was produced. The amplified oligonucleotides were cut with MnlI restriction enzyme according to their polymorphic characteristics. Digested and undigested products were separated on a 2% agarose gel electrophoresis, visualized by ethidium bromide staining under an ultraviolet illuminator. The genotype frequencies of DMT1 IVS4+44C/A polymorphism were determined as 47.9% for CC, 40.1% for AC and 12.0% for AA genotypes. The frequency of the C allele was found to as 68.0% and of the A allele as 32.0%. The genotype frequencies were consistent with Hardy-Weinberg equilibrium (χ2=2.394; Exact P=0.128).     

Regulation of Bovine Leukemia Virus tax and pol mRNA Levels by Interleukin-2 and -10

Recently, particular cytokines have been identified to affect progression of a variety of diseases and retrovirus infections. Previously, we demonstrated that interleukin-2 (IL-2), IL-12, and gamma interferon increased in peripheral blood mononuclear cells (PBMCs) from animals with early disease and decreased in PBMCs from animals with late disease stages of bovine leukemia virus (BLV) infection. In contrast, IL-10 increased with disease progression. To examine the effects of these cytokines on BLV expression, BLV tax and pol mRNA and p24 protein were quantified by competitive PCR and immunoblotting, respectively. IL-10 inhibited BLV tax and pol mRNA levels in BLV-infected PBMCs; however, the inhibitory effect of IL-10 was prevented in PBMCs depleted of monocytes and/or macrophages (monocyte/macrophages). To determine whether these factors were secreted or monocyte/macrophage associated, monocyte/macrophage-depleted PBMCs were cultured with isolated monocyte/macrophages in transwells where contact between monocyte/macrophages and nonadherent PBMCs was blocked. BLV tax and pol mRNA levels increased in transwell cultures similar to cultures containing nonseparated cells, and IL-10 addition inhibited the increase of BLV tax and pol mRNA. These results suggest that monocyte/macrophages secrete soluble factor(s) that increases BLV mRNA levels and that secretion of these soluble factor(s) could be inhibited by IL-10. In contrast, IL-2 increased BLV tax and pol mRNA and p24 protein production. Thus, IL-10 production by BLV-infected animals with late stage disease may serve to control BLV mRNA levels, while IL-2 may increase BLV mRNA in the early disease stage. To determine a correlation between cell proliferation and BLV expression, the effect of IL-2 and IL-10 on PBMC proliferation was tested. As anticipated, IL-2 stimulated while IL-10 suppressed antigen-specific PBMC proliferation. The present study, combined with our previous findings, suggests that increased IL-10 production in late disease stages suppresses BLV mRNA levels, while IL-2-activated immune responses stimulate BLV expression by BLV-infected B cells.