An Elevated IL10 mRNA Combined with Lower TNFA mRNA Level in Active Rheumatoid Arthritis Peripheral Blood.

We aimed to investigate the expression of pro-inflammatory cytokine genes TNFA, IL6, IL12B, IL23, IL18 and immunoregulatory genes FOXP3, TGFB1, and IL10 in the peripheral blood of patients with rheumatoid arthritis (RA) at messenger ribonucleic acid (mRNA) level. The total RNA was isolated from peripheral blood samples. Real-time quantitative PCR was used to perform TaqMan-based assays to quantify mRNAs from 8 target genes. IL23A was upregulated (1.7-fold), whereas IL6 (5-fold), FOXP3 (4-fold), and IL12B (2.56-fold) were downregulated in patients compared to controls. In addition, we found a strong positive correlation between the expression of FOXP3 and TNFA and a moderate correlation between FOXP3 and TGFB1. These data showed the imbalance of the T helper (Th) 1/Th17/ T regulatory (Treg) axis at a systemic level in RA. In cases with active disease, the IL10 gene expression was approximately 2-fold higher; in contrast, the expression of FOXP3 was significantly decreased (3.38-fold). The main part of patients with higher disease activity expressed upregulation of IL10 and downregulation of TNFA. Different disease activity cohorts could be separated based on IL10, TNFA and IL12B expression combinations. In conclusion, our results showed that active disease is associated with an elevated IL10 and lower TNFA mRNA level in peripheral blood cells of RA patients.

Influence of IL10 and TGFB1 Promoter Polymorphisms on Serum Cytokine Levels in Development and Severity of RA.

In our study, we focused on the role of the immunosuppressive cytokines TGF-ß1 and IL-10 in RA and, in particular, the influence of the IL10-1082 A/G (rs1800896) and TGFB1-509C/T (rs1800469) promoter polymorphisms on their levels as a prerequisite for RA and disease activity clinical features. We found significantly higher IL-10 and lower TGF-ß1 serum levels in women with RA than in controls. Patients who carried the -1082AA and AG genotypes had significantly higher levels of lnIL-10 compared to GG in contrast to healthy women carrying the same genotypes. The heterozygous -1082AG genotype was less frequent in RA cases (45.4%) than in healthy women (56.1%) and could be a protective factor for RA development (over-dominant model, OR = 0.66 95% CI 0.38-1.57). In addition, RA patients carrying the heterozygous -1082AG genotype were less likely to be anti-CCP positive than those carrying the homozygous AA/GG genotypes (37.1% vs. 62.9%; OR = 0.495. 95% CI 0.238-1.029, p = 0.058). There was no association between TGFB1 -509C/T SNP and susceptibility to RA and no relation between systemic TGF-ß1 levels and rs1800469 genotypes. In conclusion, the IL10-1082 genotypes affect the serum levels of IL-10 in women with RA in a different way from that in healthy women and appear to play a role in the genetic predisposition and autoantibody production in the Bulgarian population.

Impact of IL12B Polymorphisms on Genetic Susceptibility and IL-12p40 and IL-23 Serum Levels in Rheumatoid Arthritis.

The aim of this study was to evaluate the possible association of IL12B gene polymorphisms with serum levels of IL-12p40, IL-23 and genetic susceptibility to rheumatoid arthritis (RA) in the Bulgarian population. Genotyping for IL12Bpro (rs17860508) and IL12B A/C - 3' UTR (rs3212227) polymorphisms was performed by polymerase chain reaction (PCR)-based methods in 125 RA patients and 239 healthy controls. The IL-23 and IL-12p40 serum levels were measured by enzyme-linked immunosorbent assay (ELISA). An association was established between the rs17860508 polymorphism and RA susceptibility in Bulgarian population with an increased frequency of rs17860508 minor allele-2 and homozygous genotype-22 in RA patients. The rs17860508 risk RA genotype-22 was also significantly correlated to elevated serum IL-23 in RA patients. Although, there was no association between the rs3212227 and genetic predisposition to RA, significantly increased serum levels of both Il-12p40 and IL-23 were observed in RA patients with the rs3212227 AA genotype. Furthermore, the distribution of haplotypes and genotype combination in our cohort indicated increased RA risk in individuals carrying the rs17860508/rs3212227 2/A haplotype or 2.2/AC+CC combination, while 1/A haplotype or 1.1/AA combination may be protective for RA. In conclusion, our study demonstrates a functional effect of IL12B polymorphisms on IL-12p40 and IL-23 cytokine levels in RA patients and suggests a leading role for IL12B rs17860508 in the genetic predisposition to RA, while IL12B rs3212227 significantly modify the RA risk in Bulgarian population.

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.

Genetic variations in the IL-12B gene in association with IL-23 and IL-12p40 serum levels in ankylosing spondylitis.

In the present study, we evaluated the implication of IL12Bpro (rs17860508) and IL12B 3' UTR A/C single nucleotide polymorphisms (SNPs) (rs3212227) for the ankylosing spondylitis (AS) development and the impact of IL12B genetic variations on IL-23 and IL-12p40 production and musculoskeletal disease characteristics. 80 patients with AS and 242 healthy controls were studied. Genotyping for the rs3212227 was performed by restriction fragment length polymorphisms-polymerase chain reaction (PCR) and for the rs17860508 by allele-specific PCR. Cytokines were measured by an enzyme-linked immunosorbent assay (ELISA). Clinical status was evaluated by calculation of the Ankylosing Spondylitis Disease Activity Score (ASDAS) using the C-reactive protein (CRP) level, the Bath Ankylosing Spondylitis Functional Index (BASFI) and the Bath Ankylosing Spondylitis Metrology Index (BASMI). An association was found for the rs17860508 polymorphism with AS under the allelic, the dominant, and the co-dominant models. Rs3212227 was not attributable to AS susceptibility by itself, but the carriage of C allele in the genotype amplifies the genetic risk for AS in the carriers of the high-risk IL12Bpro 2-allele, especially in homozygosity. Circulating IL-23 and IL-12p40 were raised among AS patients, as some of the genotypes of both IL12B polymorphisms positively regulate their expression. Carriage of the IL12Bpro genotype 2.2 has been linked to a worsened functional disability, while 3' UTR CC genotype-with severe disease activity. IL12B polymorphisms can impact AS susceptibility and modulate IL-23 and IL-12p40 production levels, and have a contribution to the disease phenotype.

Serum concentration of renin-angiotensin system components in association with ACE I/D polymorphism among hypertensive subjects in response to ACE inhibitor therapy.

Background: Renin-angiotensin system (RAS) is a complex network of enzymes and peptides with the essential role in blood pressure control. The relationships between RAS components, RAS-related genetic polymorphisms and therapy response in essential hypertension (EH) were widely explored but the results were inconclusive. Aim: The aim of this study was to explore the functional role of ACE insertion/deletion (I/D) polymorphism on the systemic quantity of angiotensin-converting enzyme (ACE), its homolog - ACE2, chymase and angiotensin II in EH patients with respect to achieved therapeutic blood pressure control. Results: Genotyping of ACE I/D polymorphism was performed among 140 patients with EH from Bulgaria. The serological analyses reveal the significant elevation of the serum quantity of all investigated enzymes in EH than normotensive controls. In addition, serum ACE2 (183.57 pg/ml; vs. 151.78 pg/ml; p = 0.02) and chymase (68.5 pg/ml; vs. 23.66 pg/ml; p = 0.034) were significantly higher in patients with uncontrolled EH than controlled EH in response to ACE-inhibitory therapy. Also, ACE I/D polymorphism showed a significant impact on the serum ACE and chymase levels. ACE quantity was the highest among carriers of DD-genotype, followed by ID and II-genotype. Contrary, chymase was in the highest quantity in II-genotype compared to ID-genotype (p = 0.025) and DD-genotype (p = 0.044). Conclusions: Our results suggest that insufficient blood pressure control by ACE-inhibitory therapy could be associated with elevation of serum ACE2 and chymase levels. Also, it appears that ACE I/D polymorphism may influence the circulating quantity of chymase in addition to ACE.

n-3 polyunsaturated fatty acids provoke a specific transcriptional profile in rabbit adipose-derived stem cells in vitro.

Adipose-derived stem cells (ADSCs) possess multipotent properties, and their proper functionality is essential for further development of metabolic disorders. In the current study, we explored the impact of two n-3 LC-PUFAs (long-chain polyunsaturated fatty acids, DHA-docosahexaenoic; C22:6, and EPA-eicosapentaenoic; C20:5) on a specific profile of lipolytic-related gene expressions in the in vitro-differentiated subcutaneous and visceral ADSCs from rabbits. The subcutaneous and visceral ADSCs were obtained from 28-day-old New Zealand rabbits. The primary cells were cultured up to passage 4 and were induced for adipogenic differentiation. Thereafter, the differentiated cells were treated with 100 µg EPA or DHA for 48 hr. The total mRNA was isolated and target genes expression evaluated by real-time RCR. The results demonstrated that treatment of rabbit ADSCs with n-3 PUFAs significantly enhanced mRNA expression of Perilipin A, while the upregulation of leptin and Rab18 genes was seen mainly in ADSCs from visceral adipose tissue. Moreover, the EPA significantly enhanced PEDF (Pigment Derived Epithelium Factor) mRNA expression only in visceral cells. Collectively, the results suggest activation of an additional lipolysis pathway most evident in visceral cells. The data obtained in our study indicate that in vitro EPA up-regulates the mRNA expression of the studied lipolysis-associated genes stronger than DHA mainly in visceral rabbit ADSCs.

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.

Alterations in serum levels of IL-17 in contrast to TNF-alpha correspond to disease-modifying treatment in relapsing-remitting multiple sclerosis.

Cytokines of different types play an important role in multiple sclerosis (MS) pathogenesis as mediators and regulators of the immune processes in the central nervous system. The aim of the study was to determine the effect of interferon-beta and glatiramer acetate on serum concentrations of TNF-alpha and IL-17 A and their correlation with the degree of disability in clinically stable patients with relapsing-remitting MS. A cross-sectional, case-control study of 220 patients (68 treatment naïve; 152 treated with interferon-beta or glatiramer acetate) and 99 clinically healthy age-gender-body mass index-matched subjects were performed. Serum cytokine concentrations were measured during remission of the disease by means of ELISA. Treatment naïve patients showed significantly higher levels of IL-17 A than the treated individuals (p = .000109) and controls (p = .000044). Within the treated group, only patients with interferon-beta had significantly higher serum IL-17 than the controls (p = .023). TNF-alpha concentrations were significantly higher in the treated patients compared to the healthy controls (p = .000013), regardless of the type of the therapy. Treatment naïve individuals did not differ from the controls according to their serum TNF-alpha (p = .922). No correlation was found between the serum cytokine concentrations and Expanded Disability Status Scale (EDSS) score (p > .05). Serum concentrations of these cytokines could not be regarded as reliable predictors for the severity of the residual neurological deficit during disease-modifying treatment. Our data suggest that suppression of IL-17 A production as one of the mechanisms underlying the beneficial effect of first-line disease-modifying treatments is stronger in glatiramer acetate than in interferon-beta.

ACE serum level and I/D gene polymorphism in children with obstructive uropathies and other congenital anomalies of the kidney and urinary tract.

AIM: The aim of this study was to investigate the association of an insertion/deletion (I/D) polymorphism in angiotensin-converting enzyme (ACE) gene with serum ACE level in relation to the type and severity of malformations from congenital anomalies of the kidney and urinary tract (CAKUT) spectrum. METHODS: A group of 134 Bulgarian children with CAKUT divided into four subgroups according to the leading malformation and 109 controls were genotyped by classical polymerase chain reaction. The quantitative determination of serum ACE was performed by ELISA method. RESULTS: A significant elevation of DD-genotype was observed in high-grade hydronephrosis compared to low-grade (43% vs. 9%). The carrying of DD-genotype was associated with higher risk for severe hydronephrosis with OR = 7.5 (95% CI: 1.242÷45.278; P = 0.028). Also, elevated serum ACE concentrations in patients with high-grade compared to low-grade hydronephrosis (237.4 ± 45 ng/mL vs 180.5 ± 64 ng/mL; P = 0.0065) were found. ACE level was significantly lower in patients with unilateral renal agenesis; hypo/dysplasia and multicystic dysplastic kidney (156.6 ± 54 ng/mL) than controls (200.6 ± 56.7 ng/mL; P = 0.005) and the remaining CAKUT subgroups. CONCLUSION: The DD genotype of I/D ACE polymorphism encodes the highest serum ACE level may be an additional genetic risk factor contributing to the severe hydronephrosis in Bulgarian patients with obstructive uropathies in contrast to other investigated categories of CAKUT malformations.

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.

Increased transforming growth factor ß 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 ß (TGF-ß), 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-ß 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-ß 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.

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.

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.

Anaplasma and Ehrlichia Species in Ixodidae Ticks Collected from Two Regions of Bulgaria.

The aim of the study was to determine prevalence of Anaplasmataceae-infected ticks in the Black Sea Coast and the Pleven regions of Bulgaria. A total of 350 ticks from different tick species were collected. Two hundred fifty-five ticks were removed from dogs in the Black Sea Coast region, and 95 Ixodes ricinus ticks were collected by flagging vegetation with a white flannel cloth in two areas in the region of Pleven. After the DNA isolation of the ticks, a genus-specific polymerase chain reaction (PCR) was performed to identify Anaplasmataceae. Second PCRs were performed with species-specific primers to identify Ehrlichia canis (E. canis) and Anaplasma phagocytophilum (A. phagocytophilum). The results showed that 26.9% of the Ixodes ricinus ticks were infected with Anaplasmataceae in the Black Sea Coast region and 36.8% in the Pleven region. The infection with E. canis was detected in 35.7% and A. phagocytophilum in 25.0% of positive ticks from the Black Sea Coast region. In the Pleven region, 22.9% of ticks were positive for E. canis, while 42.9% were positive for A. phagocytophilum. The molecular identification of E. canis in ticks collected from Bulgaria was performed for the first time. In conclusion, the present study revealed a higher prevalence of ticks infected with Anaplasmataceae, particularly A. phagocytophilum, in the Pleven region than in the Black Sea Coast region.

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.

Prevalence of Borrelia burgdorferi Sensu Lato in Ixodes ricinus Ticks Collected from Kaylaka Park in Pleven, Bulgaria.

We aimed to determine the presence and distribution of Borrelia burgdorferi sensu lato (s.l.) in Ixodes ricinus ticks collected from urbanized and wild areas in Kaylaka Park (Bulgaria). A total of 546 ticks were collected over three years (2017-2019). The presence of Borrelia in 334 of the collected I. ricinus was detected by dark-field microscopy (DFM) and two nested PCRs (nPCR) targeting the borrelial 5S-23S rRNA intergenic spacer and Flagellin B (FlaB) gene. DFM was performed on a total of 215 ticks, of which 86 (40%) were positive. PCR was performed on 153 of the ticks. In total, 42.5% of the 5S-23S rRNA intergenic spacer and 49% of FlaB were positive. Considering as positive any single tick in which Borrelia sp. was detected regardless of the used method, the infection rate reached 37% (10/27) in the nymphs and 48.5% (149/307) in the adults (48.7% (77/158) females, 48.3% (72/149) males). The incidence of B. burgdorferi infection in I. ricinus did not differ statistically significantly between female, male, and nymph. This study provides evidence that Lyme disease spirochetes are present in various regions of Kaylaka Park with extremely high prevalence in their vectors.

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.