High interleukin-18 and low FOXP3 mRNAs in peripheral blood of women with severe systemic lupus erythematosus: a cross-sectional study.

Gene expression analysis of peripheral blood cells may provide valuable information about the triggered molecular processes in systemic lupus erythematosus (SLE). The study aimed to quantify the mRNA in peripheral blood of seven target genes, including inflammatory cytokine genes (IL23A, IL12B, TNFA, IL18), and T regulatory-related genes (FOXP3, TGFB1, IL10) in patients with SLE and to correlate expression levels with disease activity and/or clinical manifestations. The relative quantification of target genes was performed using real-time polymerase chain reaction in peripheral blood obtained from 28 adult SLE females and 17 healthy women. The highest up-regulation in the blood of SLE patients was observed for IL23A with a median 9.54 (p < 0.0001), followed by TGFB1 (median: 2.07; p = 0.047) and IL10 (median: 1.84; p = 0.013). IL12B and TNFA were significantly down-regulated in patients compared to controls (median: 0.521; p = 0.0023, and median: 0.519; p = 0.0003, respectively). FOXP3 mRNA was lower among patients with higher degree of disease activity (median: 0.338; p = 0.029) and showed inverse correlation with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). IL18 mRNA correlated positively with the SLEDAI and was highly expressed during severe flares (median: 1.216; p = 0.021). IL18 up-regulation was associated with anti-dsDNA antibody positivity, while FOXP3 down-regulation with lupus nephritis. Our study pointed out the relationship of SLE disease activity and particular clinical manifestations with IL18 and FOXP3 expression, and the significant contribution of IL23A in the SLE immunopathogenesis. Hence, the peripheral blood cytokine mRNAs should be exploited as novel prognostic and diagnostic biomarkers.

Interleukin-10-1082A/G polymorphism is associated with renal parenchymal damage in congenital anomalies of the kidney and urinary tract.

AIM: The aim of the study was to investigate whether the functional IL10-1082A/G polymorphism exert a role in congenital anomalies of the kidney and urinary tract (CAKUT) in children. Also, the serum IL-10 and its association with genotype and renal parenchymal damage in CAKUT were explored. METHODS: In the current case-control study, 134 paediatric cases of CAKUT and 382 unrelated controls were included. The genotyping of IL10-1082A/G polymorphism was performed by amplification refractory mutation system-PCR and IL-10 serum level was determined by ELISA. RESULTS: Although, the genotype and allelic frequencies of IL10-1082 A/G polymorphism in cases and controls were similar (χ2 = 0.459; P = 0.79 and χ2 = 0.426; P = 0.51, respectively), significant different genotype distribution between patients with or without parenchymal damage/reduction was observed (χ2 = 6.9; P = 0.032). The GG-genotype was more frequent in cases with renal parenchymal damage/reduction compared to patients with preserved parenchyma (22% vs. 9%; OR = 2.987; 95% CI = 0.979-9.468; P = 0.031). On the contrary, the heterozygous genotype was less frequent among cases with parenchymal damage/reduction compared to cases with preserved parenchyma (39% vs. 59%; OR = 0.453; 95% CI = 0.214-0.958; P = 0.024). Additionally, the serum IL-10 was significantly higher in CAKUT patients compared to age-sex-matched controls (median 11.98; IQR: 7.14-31.6 vs. 5.92; IQR: 4.68-14.8; P = 0.0057). Among carriers of GG-genotype significantly higher IL-10 level was detected in cases with parenchymal damage/reduction, than cases with preserved parenchyma (P = 0.028). CONCLUSION: Our results suggested that the functional -1082A/G polymorphism in IL10 is associated with risk of renal parenchymal damage/reduction rather than genetic predisposition to CAKUT. Additionally, our study supposes that immunoregulatory cytokine IL-10 might have a significant role in CAKUT.

A Role of Cytokine Gene Polymorphisms in Cognitive Functioning.

The changes in cognitive functions that occur with aging and in various pathological conditions are a subject of growing interest. Experimental and clinical data justify the hypothesis about the influence the immune system exerts on cognitive processes. The balance between pro-inflammatory and anti-inflammatory cytokines has been established as a necessary factor for normal cognitive functioning. Cytokine production is under strong genetic control and various single nucleotide polymorphisms (SNPs) in cytokine genes have been described. As cytokine SNPs have been demonstrated to affect the gene expression or the functional activity of the immune protein this logically led to the suggestion about the role of these polymorphisms in cognitive functioning. Studies exploring the association between different genetic variants of cytokine gene polymorphisms and cognitive abilities in healthy subjects and in demented patients show divergent results. The review of relevant literature suggests that SNPs implement their effect on cognition in large interactions with each other, as well as with many other factors, some of which still remain to be identified. This article summarizes the contemporary knowledge about the correlations between SNPs in cytokine genes and cognitive status in humans. Further research is needed to determine the precise role and the molecular mechanisms of action of the SNPs in cognitive processes.

Cognitive Impairment in Multiple Sclerosis.

Multiple sclerosis (MS) is a socially significant immune-mediated disease, characterized by demyelination, axonal transection and oligodendropathy in the central nervous system. Inflammatory demyelination and neurodegeneration lead to brain atrophy and cognitive deficit in up to 75% of the patients. Cognitive dysfunctions impact significantly patients' quality of life, independently from the course and phase of the disease. The relationship between pathological brain findings and cognitive impairment is a subject of intensive research. Summarizing recent data about prevalence, clinical specificity and treatment of cognitive disorders in MS, this review aims to motivate the necessity of early diagnosis and complex therapeutic approach to these disturbances in order to reduce the social burden of the disease.

Significance of -1082A/G polymorphism of IL10 gene for progression of colorectal cancer and IL-10 expression.

The role of functional polymorphism within IL10 (rs1800896) in colorectal cancer (CRC) still remains elusive. The aim of present study was to investigate the significance of -1082A/G polymorphism in IL10 on CRC risk, progression, and overall survival in a cohort of Bulgarian patients. Also, a functional role of this polymorphism on systemic and local level of IL10 mRNA quantity and serum IL-10 level was explored. A group of 119 patients with sporadic CRC and 154 age-sex-matched controls were genotyped by allele-specific PCR. The quantification of mRNA and serum IL-10 levels was performed by real-time PCR and ELISA assays, respectively. The genotype and allelic frequency among cases and controls was similar. However, we observed significant elevation of G-allele and GG-genotype frequencies among advanced CRC. G-allele was overrepresented in advanced CRC patients (49 %) compared to early CRC (35 %) with OR = 1.77; 95%CI 1.018 ÷ 3.083; P = 0.031. A significant upregulated expression of IL10 mRNA was observed among AG/GG-genotypes in tumor tissue compared to homozygous AA-genotype (RQ value 68.3 vs. 6.68; P = 0.0062). Also, GG-genotype of -1082A/G polymorphism in IL10 was positively associated with higher serum IL-10 among early CRC patients and controls, in contrast to advanced cases. Although, investigated polymorphism in IL10 has no significant impact of overall survival among Bulgarian CRC patients, we found a significant relationship of high pre-operative serum level of IL-10 with poor survival of CRC (P = 0.023). Our findings indicate a significant impact of -1082A/G polymorphism of IL10 on CRC progression, rather than genetic predisposition and prognosis of CRC.

Monocytes expression of IL-12 related and IL-10 genes in association with development of colorectal cancer.

The main regulator of anti-tumor immune response is the activity of monocytes, suggesting that the produced cytokines may have a prognostic role. This study investigates gene expression of interleukin (IL)-12-related cytokine and IL-10 in stimulated monocytes from colorectal cancer (CRC) patients. Relative quantification of IL-12A, IL-12B, IL-23A and IL-10 mRNA transcripts was performed on the third hours after stimulation by real-time qPCR. We also explored an inhibitor of JNK signaling pathway activation for the observed cytokine gene expression. A strong downregulation of IL-12B mRNA expression in CRC monocytes compared to healthy donors was observed. The rate of transcription of IL-12B in stimulated monocytes was associated with the stage of CRC. The expression of IL-12A gene in stimulated monocytes from patients with advanced was lower than early cancer. Moreover, we observed stage dependent JNK inhibition mediated reduction in IL-12A expression. The hyporesponsiveness was strongly expressed in monocytes from advanced then early stages of CRC. Expression of IL-10 mRNA was almost equally in CRC monocytes from early stages and healthy donors. We demonstrated that altered gene expression profiles of IL-12A, IL-12B, IL-23A at mRNA level in CRC monocytes was associated with tumor development and can be attributed to anticancer immune response.

Functional genetic polymorphisms in interleukin-12B gene in association with systemic lupus erythematosus.

Genetic polymorphisms in cytokine genes, which influence gene expression, may have an important impact on SLE susceptibility and severity. The aim of this study was to examine the possible influence of two functional polymorphisms in cis-regulatory regions of IL12B gene in the susceptibility and clinical symptoms of SLE in Bulgarian patients. Female SLE patients (n = 141) and 124 healthy women were included in the study. Genotyping for the IL12B A/C polymorphism in 3'UTR was performed by restriction fragment length polymorphisms PCR assay and for the IL12Bpro polymorphism by allele-specific PCR. Genotype-22 of IL12Bpro polymorphism was overrepresented among women with SLE (28 vs. 17%; OR = 1.875; 95% CI: 1.037 ÷ 3.390; P = 0.037). Respectively, a higher frequency of allele-2 was found in patients than in controls (51% vs. 40%; OR = 1.566; 95% CI: 1.110 ÷ 2.210; P = 0.011). Also, we found significantly elevated frequency of genotype-22 of IL12Bpro polymorphism among SLE patients who were simultaneously carrier of genotype-AA of SNP in 3'UTR. Women with both homozygous genotypes, genotype-AA of SNP in 3'UTR and genotype-22 of IL12Bpro polymorphism, had a 2.1-fold significantly elevated risk for SLE development. The carrying of genotype-11 of IL12Bpro polymorphism was positively associated with hematological and neuropsychiatric manifestations of SLE. We have provided evidence that the IL12Bpro polymorphism was associated with SLE development among Bulgarian women. Although a strong individual effect of SNP in 3'UTR of IL12B was not detected, we found that genotype-22 of IL12Bpro was predominantly combined with genotype-AA of SNP in 3'UTR among SLE patients and this combination elevates the risk of SLE.

The Role of Transforming Growth Factor-ß1 Gene Polymorphism and Its Serum Levels in Hashimoto's Thyroiditis.

BACKGROUND: TGF-ß1 gene (TGFB1) is one of the target genes involved in genetic predisposition to autoimmune diseases, particularly Hashimoto's thyroiditis (HT). OBJECTIVE: In the present study, we attempted to investigate whether -509C/T SNP (rs1800469) in the promoter of TGFB1 is associated with the genetic susceptibility and clinical characteristics of Bulgarian patients with HT. We also analyzed serum TGF-ß1 levels in different stages of the disease and its association with the -509C/T polymorphism in the TGFB1 promoter. METHODS: The study recruited 121 female out-patients with autoimmune thyroiditis and 250 agematched healthy women (HC). Genotyping of the rs1800469 was performed by restriction fragment length polymorphism (RFLP)-PCR assay. The serum concentrations of latent acid-activated TGF-ß1 protein were determined by the quantitative sandwich ELISA method. RESULTS: Upon testing different types of inheritance, a significant risk was found for heterozygotes (CT) with OR=1.640; p=0.05 under the codominant model. The significantly higher risk for developing Hypothyroidism was calculated again for CT-genotype patients with OR=1.789. According to the hormone reference values, a significant association of CT genotype with decreased TSH (75.4%) simultaneously with increased free T4 hormone (94%) levels was also calculated. When patients were stratified by genotype and compared to the same genotype in HC, we observed that the decreased levels in serum TGF-b1 were significant for patients who carried the C-allele in their genotype. CONCLUSION: We suggest that heterozygous genotype CT is a genetic risk factor for developing more severe HT due to enhanced free T4 serum level at the onset of the disease, before developing the hypothyroid stage.

Circulating levels of interleukin-17A, tumor necrosis factor-alpha, interleukin-18, interleukin-10, and cognitive performance of patients with relapsing-remitting multiple sclerosis.

Multiple sclerosis (MS) is associated with cytokine imbalance and high rate (40-70%) of cognitive impairment. The objective of this study is to investigate the relationship between serum concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-17A, IL-18, IL-10, and cognitive performance in patients with relapsing-remitting MS (RRMS). Methods The study comprised 159 patients with RRMS (mean age 40.08 ± 8.48 years) in remission phase and 86 age-, gender-, and education-matched healthy controls. Paced Auditory Serial Addition Test (PASAT), Symbol Digit Modalities test (SDMT), and Isaacs test were used for assessment of working memory, attention, visuo-perceptual abilities, information processing speed, and executive functions. Serum cytokine concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Results Patients had significantly increased serum concentrations of TNF-alpha and IL-17A and decreased levels of IL-10 compared to the controls (p < 0.05). Negative correlation was found between serum TNF-alpha and SDMT score in patients with disease evolution longer than 10 years (rxy = -0.258 p = 0.033); PASAT and SDMT scores were in negative correlation with concentration of IL-17A (rxy = -0.229 p = 0.004; rxy = -0.166 p = 0.041). Cognitive impairment was established in 46.5% (n = 74) of the patients. Cognitively impaired patients had significantly higher serum IL-17A than cognitively preserved individuals (p = 0.007). Multiple linear regression analysis revealed IL-17A as a significant predictor of cognitive performance in RRMS patients. Conclusion The results from this study suggest that pro-inflammatory cytokines IL-17A and TNF-alpha simultaneously with decreased IL-10 are involved in cognitive deterioration in RRMS.

Interleukin-10-1082 promoter polymorphism in association with cytokine production and sepsis susceptibility.

OBJECTIVE: To investigate the -1082 (A/G) polymorphism in the promoter of the IL-10 gene in terms of IL-10 production from stimulated peripheral blood mononuclear cells (PBMC) and to evaluate the relationship of this polymorphism with susceptibility to severe sepsis and the outcome of the disease. DESIGN: Case-control study. SETTING: Research laboratory of Molecular Biology and Immunology and University Hospital ICU, Faculty of Medicine, Trakia University. PATIENTS: A total of 53 healthy volunteers and 33 patients in ICU meeting the criteria for severe sepsis were included. MEASUREMENTS AND RESULTS: The amplification refractory mutation system PCR was used for IL-10-1082 polymorphism detection. Isolated PBMC were stimulated with either C3-binding glycoprotein (C3bgp), lipopolysaccharide (LPS), phytohemagglutinin (PHA),or pokeweed mitogen (PWM). IL-10 production was measured in culture supernatants. The AA genotype was associated with lower IL-10 production in LPS-, PHA- or PWM-stimulated healthy PBMC. Patients with severe sepsis had significant elevation of A allele, compared with healthy controls (74.2% vs 52.8%; p=0.0062). Carriage of at least one copy of IL-10-1082 G allele in sepsis patients and in healthy controls resulted in a statistically significant increase in IL-10 production from stimulated PBMC. Surviving sepsis patients had a significant decrease of IL-10-1082 allele G frequency, compared with controls (17% vs 47.2%; p=0.012). An association between increased IL-10 production and poor outcome from sepsis was observed. CONCLUSION: The A allele of the -1082 polymorphism in the interleukin-10 gene promoter is associated with sepsis susceptibility, whereas G allele is associated with higher stimulated interleukin-10 production and increased mortality in severe sepsis.

High interleukin 12 and low interleukin 10 production after in vitro stimulation detected in sepsis survivors.

OBJECTIVE: To evaluate viability of isolated peripheral blood mononuclear cells (PBMC) and production of cytokines in vitro after stimulation as prognostic factors for survival in sepsis patients. DESIGN: Prospective study of the biological response of PBMC in the onset of severe sepsis. SETTING: Research laboratory of molecular biology and immunology and university hospital ICU, Faculty of Medicine, Trakia University. PATIENTS: Twenty-three patients meeting the criteria for severe sepsis, and 14 control subjects. INTERVENTIONS: Isolated PBMC were stimulated in vitro with: C3-binding glycoprotein (C3bgp; 30 microg), lipopolysaccharide (30 microg), phytohemagglutinin (20 microg), pokeweed mitogen (30 microg), and dexamethasone (500 microg). MEASUREMENTS AND RESULTS: We measured the levels of interleukins (IL) 6, 10, and 12 in culture supernatants. Stimulation with C3bgp and phytohemagglutinin led to significantly lower PBMC secretion of IL-6 in nonsurvivors than in survivors and healthy donors. Stimulation with C3bgp, lipopolysaccharide, and pokeweed mitogen considerably reduced IL-12 production in nonsurvivors. Stimulation with lipopolysaccharide and pokeweed mitogen caused immune cells in nonsurvivors to produce higher levels of IL-10 than in survivors. Survival of PBMC reduced viability for nonsurvivors' PBMC, both spontaneously and as induced by lipopolysaccharide or pokeweed mitogen. CONCLUSIONS: The viability of PBMC at the onset of sepsis and enhanced production of IL-12 and diminished production of IL-10 after stimulation with all stimuli used may be a favorable prognostic factor in sepsis.

C3 binding glycoprotein from Cuscuta europea induced different cytokine profiles from human PBMC compared to other plant and bacterial immunomodulators.

The immunomodulatory properties of bioactive agents include the ability to induce cytokine production by the activated target cell. The effect of immunomodulatory C3 binding glycoprotein isolated from Cuscuta europea on the induction of human PBMC cytokine synthesis and the cell viability was investigated. Isolated PBMC from healthy donors were cultured for 24 h with C3bgp. We also studied the influence of C3bgp on the cytokine production in LPS, PHA, PWM and Dex treated PBMC. The quantitative determination of TNF-alpha, IL-12, IL-6 and IL-10 was performed in culture supernatant by ELISA. Results obtained demonstrated that C3bgp induced proinflammatory and immunoregulatory cytokine production, in the highest degree IL-12, followed by IL-6 and in lower degree TNF-alpha. IL-12 quantity was significantly increased in C3bgp stimulated cultures in comparison with LPS, PHA and PWM stimulated PBMC. C3bgp also increased IL-12 in PHA or PWM stimulated cultures, but not in LPS stimulated culture. C3bgp significantly increased IL-6 production compared to the PHA and PWM but not to LPS stimulation. On the other side, C3bgp inhibited IL-10 production after LPS, PHA and PWM stimulation. Cell viability in C3bgp stimulated cultures retained on the same level from 72 to 120 h of culturing, in contrast to LPS and PHA stimulated cultures. Based on the results presented, we conclude that the C3bgp exhibited immunomodulatory properties on the human PBMC. The ability of PDTC and Dex to down-regulate the effect of C3bgp on the proinflammatory cytokine production suggests that a part of the mechanism of action of C3bgp is mediated through NF-kB signal transduction pathway.

Immunomodulatory effects of C3bgp on the antibody response to hemocyanin in outbred rabbits and the F1 generation of breeding with siblings.

Dynamics and quantitative analyses of monospecific antibody during the primary and secondary humoral responses were determined in outbred rabbits and in the F1 generation of breeding with siblings. The antibody response in rabbits immunized with Keyhole Limpet Hemocyanin (KLH) was studied during a 4-month immunization period. ELISA determination of anti-KLH Ig and anti-KLH IgG alone, in preimmune and immune rabbit sera, was performed. Antibody response in both groups of rabbits was similar when assessed by anti-rabbit Ig but displayed differences when assessed by anti-rabbit IgG. A statistically significant increase in anti-KLH IgG was observed in the F1 inbred rabbits compared to the control group after primary immunization from days 14 to 35. Immunomodulation also elicited differences in the antibody response in the two groups of animals. C3-binding glycoprotein isolated from Cuscuta europea (C3bgp), applied simultaneously with antigen (KLH), produced a much stronger secondary immune response than the antigen alone, in both experimental groups. The enhancement of anti-KLH Ig in C3bgp-treated inbred rabbits was statistically significant in comparison with nontreated inbred rabbits. A significant increase in anti-KLH IgG was observed only for the inbred group after treatment with C3bgp. The results demonstrate that the F1 generation of breeding with sibling leads to significant differences in antibody responses to immunization compared with outbred rabbits, as well as to immunomodulation with C3bgp.