Lyuba Miteva

The combined effect of interleukin (IL)-10 and IL-12 polymorphisms on induced cytokine production

Interleukin (IL)-12 and IL-10 are immunoregulatory cytokines with an antagonistic effect of the T-helper (Th)1/Th2 cytokine balance and provide a functional link between innate and adaptive immune responses. The aim of the study was to investigate the combined effect of -1082A*G in IL10 and +16974A*C in IL12B single nucleotide polymorphisms (SNPs) on induced cytokine production by stimulated peripheral blood mononuclear cells (PBMCs) isolated from healthy donors. The presence of the high-producer IL-12p40 genotype led to diminished production of IL-10 as determined by the -1082*G allele of SNP in IL10. Significantly decreased IL-10 production was detected in AA+AG/GG in comparison with the low-producer IL-12p40 (AC/CC+AG/GG) genotype combination after stimulation with C3bgp (2+/-4 vs. 29+/-14.2 pg/ml; p=0.0003) and LPS (33.4+/-13.5 vs. 93.3+/-59.6 pg/ml; p=0.019). IL-12p40 production was independent of IL10 genotype. The present results demonstrated that the production of IL-10 from PBMC depended on both -1082A*G in IL10 and +16974A*C in IL12B polymorphisms.

Association of Polymorphisms in Regulatory Regions of Interleukin-12p40 Gene and Cytokine Serum Level with Colorectal Cancer

ABSTRACT The present study investigates the association between IL12Bpro and +16974A/C in 3′ -untranslated region (UTR) polymorphisms of IL12B and IL-12p40 serum level related to development of colorectal cancer (CRC). Our results showed similar distribution of both the investigated polymorphisms among CRC patients and healthy donors, which suggests that these polymorphisms in IL12B were not associated with the development of CRC. However, we found an increased IL-12p40 level in sera from patients compared to healthy donors and the highest level was observed in stage I compared to more advanced stages of CRC. These findings demonstrated that IL-12p40 serum level has an association with the progression of CRC.

Ile105Val GSTP1 polymorphism and susceptibility to colorectal carcinoma in Bulgarian population.

Background and aimsEtiologically, the sporadic colorectal cancer (CRC) is a complex and multifactorial disease that is linked to both exogenic and endogenic factors. Accumulating evidence indicates that susceptibility to cancers, including CRC, is mediated by genetically determined differences in the effectiveness of detoxification of potential carcinogens. A member of the glutathione-S-transferase (GST) family, GSTP1, is an important candidate for involvement in susceptibility to carcinogen-associated CRC. An A→G transition in exon 5 of the GSTP1 gene resulting in Ile105Val amino acid substitution has been identified. This change leads to alteration in catalytic efficiency of variant enzyme. The aim of the current study was to evaluate the influence of Ile105Val GSTP1 polymorphism on susceptibility to CRC.Materials and methodsThe GSTP1 genotyping was conducted in a case-control study of 80 ethnic Bulgarian CRC patients and 126 unaffected controls using polymerase chain reaction restriction fragment length polymorphism method.ResultsA statistically significant case-control difference in genotype frequencies was observed: 0.69 vs 0.54 for Ile/Ile, 0.22 vs 0.39 for Ile/Val, and 0.09 vs 0.07 for Val/Val (p = 0.049). The odds ratio (OR) for Val/Val was close to 1 (0.96, 95%CI: 0.35–2.66, p = 0.942), whereas the OR for Ile/Val was significantly lower, 0.45 (95%CI: 0.24–0.86, p = 0.016), compared to the referent Ile/Ile genotype. Although a prevalence of the GSTP1 variant allele-containing genotypes (Ile/Val or Val/Val) was found in controls than in patients (OR = 0.53, 95%CI: 0.30–0.96, p = 0.035), the allele frequencies did not show significant difference between cases and controls (p = 0.127).ConclusionsBased on the obtained protective effect of Ile/Val GSTP1 genotype, we could suggest that Ile105Val GSTP1 polymorphism may play some role in susceptibility to CRC.

The inhibition of JNK and p38 MAPKs downregulates IL-10 and differentially affects c-Jun gene expression in human monocytes

Interleukin-10 is the most important anti-inflammatory cytokine that controls the progress of the immune response. The molecular mechanisms driving the IL10 gene regulation are not well understood. To gain insight into this process we studied the IL-10 expression on mRNA and protein levels, together with c-Jun, FOXP3 and RelA transcription factors gene expression in human monocytes. We investigated also, the involvement of JNK and p38 transduction pathways in IL-10, c-Jun, FOXP3 and RelA gene expression. The quantity determination of IL-10 was performed by ELISA. qRT-PCR was performed for the detection of mRNA transcripts. The pharmacological inhibitors SP600125 and SB202190 were used to explore JNK and p38 MAPKs involvement in IL10, c-Jun, FOXP3 and RelA gene expression. The measurement of IL-10 mRNA synthesis, triggered by lipopolysaccharide (LPS) or C3 binding glycoprotein (C3bgp) showed that stimulation with both inducers led to similar high level of IL-10 mRNA synthesis, whereas C3bgp was the stronger inducer of IL-10 production than LPS. JNK and p38 inhibition significantly decreased IL-10 expression in stimulated cells. C3bgp and LPS induced comparatively low expression of FOXP3, RelA and c-Jun mRNA in monocytes. The inhibition of p38 MAPK in stimulated monocytes resulted in significant enhancement of c-Jun mRNA synthesis suggesting the functional relation between p38 MAPK and c-Jun gene expression. We concluded that the IL10 gene transcription did not associate with enhancement of c-Jun, RelA and FOXP3 gene expression and strictly depended on the JNK and p38 MAPKs activation in stimulated human monocytes.

Advanced Colorectal Cancer Is Associated With Enhanced Il-23 and IL-10 Serum Levels

Objective: Cytokines play a pivotal role in the induction of host immune responses against tumor growth and are involved in the development and progression of colorectal cancer in humans. Methods: A group of 48 patients and 27 healthy volunteers were included in the study. The quantitative determination of IL-23, IL-17, IL-10, and IFN-a serum levels were performed by ELISA. Results: We observed significantly enhanced IL-23 in CRC patients compared to the controls. The serum level of IL-10 was significantly higher in CRC patients than healthy donors. The highest level of IL-10 was detected in patients with stage IV, which was significantly greater than those in stages I, II, and III. Conclusions: We found that IL-23 and IL-10 levels were significantly elevated in patients’ sera, in contrast to IL-17 and IFN-a, and this serum cytokine profile may play a role in tumor development. In addition, an increased level of IL-10 was strongly associated with the progression of colorectal carcinoma (CRC).

High expression of Foxp3, IL-23p19 and survivin mRNA in colorectal carcinoma

IntroductionCytokines have been suggested to both modulate anti-tumor responses and promote tumor growth.Materials and methodsWe analyzed the expression of pro-inflammatory IL-12p35, IL-12p40, IL-23p19, anti-inflammatory IL-10, antiapoptotic factor survivin, and transcription factors—RelA, c-Jun, and Foxp3 mRNA in patients’ blood, colon carcinoma tissue, and in normal mucosal tissue by real-time polymerase chain reaction. The quantity determination of serum IL-12p40, IL-23, and IL-10 was performed by enzyme-linked immunosorbent assay.ResultsWe observed significantly higher levels in patients for all three analyzed cytokines, with IL-23 concentration change being the highest. We detected the greatest upregulation of IL-23p19, Foxp3 and survivin mRNA in colorectal carcinomas than normal mucosa. A statistically significant upregulation of IL-12p40, IL-10, and c-Jun mRNA but not for IL-12p35 and RelA mRNA in tumor tissue comparing to normal tissue was also established.ConclusionsIn conclusion, we show a characteristic gene expression profile combining markers associated with inhibition of anti-tumor immune response (Foxp3, IL-10), inhibition of apoptosis (survivin), and induction of the cytokines with protumoral activity as IL-12p40 and IL-23p19 (IL-23) in the colorectal tumor tissue but not in peripheral blood of patients.

Induction of immunostimulatory cytokine genes expression in human PBMCs by a novel semiquinone glucoside derivative (SQGD) isolated from a Bacillus sp. INM-1.

In the present study, a semiquinone glucoside derivative (SQGD) isolated from a radioresistant bacterium Bacillus sp. INM-1 was evaluated for its immunostimulatory activities. Human peripheral blood mononuclear cells (PBMCs) were stimulated by different doses (30-90 microg/ml) of SQGD for different time (3-12h) intervals at 37°C, and IL-12p40, IL-23p19, IL-10, RelA and c-Jun gene expression analysis was carried out by qRT-PCR method. SQGD dose dependent cytokines protein expression kinetic analysis was carried out using western blotting. As the results of SQGD (30μg/ml) stimulation for 3h at 37°C, significant induction in IL-12p40, IL-23p19 and RelA gene expression was observed in PBMCs compared to unstimulated control cells. However, no such induction in IL-10 and c-Jun gene expression was observed. Time dependent protein expression study indicated significant increase in IL-12p40, IL-12p35, IL-23p19 and RelA protein expression at 3-6h, which was found decrease at 12h upon SQGD treatment. In contrast, IL-10 protein expression was found to enhance significantly at 12h after SQGD treatment to the PBMCs. SQGD dose dependent study showed approximately similar level of induction in IL-12p40, IL-12p35, IL-23p19 and RelA proteins expression at all tested concentration (30-90 microg/ml) compared to control. However, no significant change in the IL-10 and c-Jun protein expression was observed at any SQGD concentration. SQGD treatment (0.25mg/kgbwt.) was also found to enhance anti-keyhole Limpet Hemocynin (KLH) IgM antibodies significantly in the mice immunized by KLH. Thus, SQGD fraction stimulates cellular immunity by inducing immunostimulatory cytokines and humoral immunity by enhancing IgM antibodies and could be a promising immunostimulant. Further studies related to molecular mechanisms offering immunostimulation is underway, will certainly helpful to unravel its mode of action in the biological system.

Colorectal cancer severity and survival in correlation with tumour necrosis factor-alpha

Colorectal cancer (CRC) development is strongly associated with innate immune mechanisms and intestinal inflammation. The aim of the study was to investigate the pre-operative serum levels of TNF-α and its correlation with cancer progression and survival in CRC patients taking into account the genotype of –308G/A promoter polymorphism in TNF-α gene (rs1800629). TNF-α –308G/A genotypes of 119 CRC cases and 177 no CRC controls were determined by restriction fragment length polymorphism assay (RFLP-PCR). TNF-α serum levels were measured by enzyme-linked immunosorbent assay (ELISA). Although no significant differences in allele and genotype frequencies between CRC and controls were observed, it should be noted that the minor allele-A and its homozygous genotype were overrepresented among CRC. In addition, allele-A was more frequent in early CRC patients compared to advanced cases. TNF-α serum level was significantly higher in CRC patients than in controls (36.1 ± 8.4 pg/mL vs. 18.66 ± 11 pg/mL; p = 0.0000001). In the subgroup analysis by tumour–node–metastasis stages, the highest TNF-α level was found in stage IV (42.7 ± 12.5 pg/mL) and was significantly elevated compared to earlier stages of CRC and controls. The survival rate of CRC patients with low TNF-α serum level, estimated as median survival, was significantly higher than that of patients with high levels of TNF-α (38.4 vs. 7.761 months; log rank test p = 0.00015) In conclusion, we can affirm that TNF-α affects tumour development along with disease progression which has an impact on the survival 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.

Increased transforming growth factor β and interleukin 10 transcripts in peripheral blood mononuclear cells of colorectal cancer patients

Aim of the study The ability of immune cells in peripheral blood to produce certain cytokines affects tumour-elicited inflammation. The aim of this study was to investigate the gene expression of interleukin 12A (IL-12A), IL-12B, IL-23A, IL-10, IL-6, transforming growth factor β (TGF-β), HDAC3, and iNOS in peripheral blood mononuclear cells (PBMC) from colorectal cancer (CRC) patients. Material and methods The venous blood for PBMC isolation was collected preoperatively and 10 days after surgery, from CRC patients. After isolation of total RNA and synthesis of cDNA, quantitative real-time PCR assays were performed. Results Our results demonstrated that among investigated cytokine genes IL-10 and TGF-β were significantly upregulated in patients with CRC compared to the control group, while the expression of IL-23 mRNA was significantly decreased in CRC patients. We observed significantly increased mRNA levels in CRC patients’ PBMC before surgery for IL-10 and TGF-β compared to both postoperative and control groups. We also found a significant upregulation of iNOS in early compared to advanced CRC. Conclusions Based on the results we can assume that PBMC gene expression programming in CRC patients drives local differentiation of Th cells towards Treg instead of the Th1 anti-tumour subpopulation.