Exploring causal correlations between circulating levels of cytokines and colorectal cancer risk: A Mendelian randomization analysis.

Colorectal cancer has the highest mortality rate of all digestive system diseases. Considering the debate about cytokines and biases that exist in traditional observational study designs, we performed a two-sample Mendelian randomization (MR) analysis to explore the association of circulating cytokines with CRC risk. In this study, we used cytokine genetic variants from a recently published genome-wide association study (GWAS) including 14,824 European-ancestry participants. Summary-level data for colorectal cancer were obtained from genome-wide association analyses of the FinnGen consortium. In addition, we conducted independent supplementary analyses using genetic variation data of colorectal cancer and cytokines from a large public GWAS in 2021. Among 91 circulating factors, we only found IL-12B to be significantly associated with CRC risk (odds ratio [OR]: 1.19; 95% confidence interval [CI]: 1.00-1.42; p = .046). We used 2021 data for analysis and found that higher Interleukin-12p70 levels (IL-12p70) were revealed to have a significant positive association with CRC risk (OR: 1.27; 95% CI: 1.13-1.43; p < 1.22 × 10-3). Moreover, CRC was suggestively correlated with an elevated level of vascular endothelial growth factor (VEGF) (OR: 1.17; 95% CI: 1.02-1.35; p = .026), macrophage colony-stimulating factor (M-CSF) (OR: 0.85; 95% CI: 0.76-0.96; p = .005), IL-13 (OR: 1.15; 95% CI: 1.02-1.30; p = .028), IL-10 (OR: 1.23; 95% CI: 1.01-1.49; p = .037), and IL-7 (OR: 1.19; 95% CI: 1.02-1.39; p = .024). Our MR studies support that one cytokine IL-12 is significantly associated with CRC risk and that five cytokines VEGF, M-CSF, IL-13, IL-10, and IL-7 are associated with CRC risk.

Safety, Pharmacokinetics, and Pharmacodynamics of the Oral TLR8 Agonist Selgantolimod in Chronic Hepatitis B.

BACKGROUND AND AIMS: In patients with chronic hepatitis B (CHB) infection, activation of toll-like receptor 8 may induce antiviral immunity and drive functional cure. Selgantolimod, a toll-like receptor 8 agonist, was evaluated in patients with CHB who were virally suppressed on oral antiviral treatment or viremic and not on oral antiviral treatment. APPROACH AND RESULTS: In this phase 1b study, patients were randomized 4:1 to receive either selgantolimod or placebo once weekly. Virally suppressed patients received either 1.5 mg (for 2 weeks) or 3 mg (for 2 weeks or 4 weeks). Viremic patients received 3 mg for 2 weeks. The primary endpoint was safety, as assessed by adverse events (AEs), laboratory abnormalities, and vital sign examination. Pharmacokinetic and pharmacodynamic parameters were assessed by plasma analysis. A total of 38 patients (28 virally suppressed, 10 viremic) were enrolled from six sites in Australia, New Zealand, and South Korea. Twenty patients (53%) experienced an AE and 32 (84%) had laboratory abnormalities, all of which were mild or moderate in severity. The most common AEs were headache (32%), nausea (24%), and dizziness (13%). With a half-life of 5 hours, no accumulation of selgantolimod was observed with multiple dosing. Selgantolimod induced transient dose-dependent increases in serum cytokines, including IL-12p40 and IL-1RA, which are important for the expansion and activity of multiple T- cell subsets and innate immunity. CONCLUSION: Selgantolimod was safe and well-tolerated in virally suppressed and viremic patients with CHB and elicited cytokine responses consistent with target engagement. Further studies with longer durations of selgantolimod treatment are required to evaluate efficacy.

Elevated levels of interleukin-12/23p40 may serve as a potential indicator of dysfunctional heart rate variability in type 2 diabetes.

BACKGROUND: Systemic inflammatory processes plausibly contribute to the development of cardiovascular complications, causing increased morbidity and mortality in type 2 diabetes. Circulating inflammatory markers, i.e., interleukin (IL)-6 and tumour necrosis factor-α, are associated with neurocardiac measures. We examined a broad panel of various inflammatory and inflammation-related serum markers to obtain more detailed insight into the possible neuro-immune interaction between cardiovascular regulation and systemic level of inflammation. METHODS: Serum samples from 100 participants with type 2 diabetes were analysed. Heart rate variability, cardiovascular autonomic reflex tests, and cardiac vagal tone tests were performed based on electrocardiographic readings. Data regarding covariates (demographic-, diabetes-, and cardiovascular risk factors) were registered. RESULTS: Increased serum levels of IL-12/IL-23p40 (p < 0.01) and intercellular adhesion molecule (ICAM)-1 (p < 0.007) were associated with diminished heart rate variability measures. After all adjustments, the associations between IL-12/23p40, SDANN and VLF persisted (p = 0.001). Additionally, serum levels of vascular endothelial growth factor (VEGF)-C were associated with response to standing (p = 0.005). DISCUSSION: The few but robust associations between neurocardiac regulation and serum markers found in this study suggest systemic changes in proinflammatory, endothelial, and lymphatic function, which collectively impacts the systemic cardiovascular function. Our results warrant further exploration of IL-12/IL-23p40, ICAM-1, and VEGF-C as possible cardiovascular biomarkers in T2D that may support future decisions regarding treatment strategies for improved patient care.

Suppression of IL-12/IL-23 p40 subunit in the skin and blood of psoriasis patients by Tofacitinib is dependent on active interferon-γ signaling in dendritic cells: Implications for the treatment of psoriasis and interferon-driven diseases.

Interleukin (IL)-12 and IL-23 are pro-inflammatory cytokines produced by dendritic cells (DCs) and associated with Psoriasis (Pso) and Psoriatic Arthritis (PsA) pathogenesis. Tofacitinib, a Janus kinase inhibitor, effectively suppresses inflammatory cascades downstream the IL-12/IL-23 axis in Pso and PsA patients. Here, we investigated whether Tofacitinib directly regulates IL-12/IL-23 production in DCs, and how this regulation reflects responses to Tofacitinib in Pso patients. We treated monocyte-derived dendritic cells and myeloid dendritic cells with Tofacitinib and stimulated cells with either lipopolysaccharide (LPS) or a combination of LPS and IFN-γ. We assessed gene expression by qPCR, obtained skin microarray and blood Olink data and clinical parameters of Pso patients treated with Tofacitinib from public data sets. Our results indicate that in DCs co-stimulated with LPS and IFN-γ, but not with LPS alone, Tofacitinib leads to the decreased expression of IL-23/IL-12 shared subunit IL12B (p40). In Tofacitinib-treated Pso patients, IL-12 expression and psoriasis area and severity index (PASI) are significantly reduced in patients with higher IFN-γ at baseline. These findings demonstrate for the first time that Tofacitinib suppresses IL-23/IL-12 shared subunit IL12B in DCs upon active IFN-γ signaling, and that Pso patients with higher IFN-γ baseline levels display improved clinical response after Tofacitinib treatment.

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.

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.

Effect of interscalene nerve block on the inflammatory response in shoulder surgery: a randomized trial.

BACKGROUND: Comparing techniques of general anesthesia and regional anesthesia in arthroscopic shoulder surgery, some studies have shown differences in the intensity of immediate postoperative pain and neuroendocrine response, but the inflammatory response when using balanced general anesthesia (BGA) vs. an ultrasound-guided (USG) single-dose interscalene block (SDIB) has not been compared. MATERIALS AND METHODS: In a single-center, prospective, randomized clinical trial, the inflammatory response of 2 groups of 10 patients scheduled to undergo arthroscopic shoulder surgery was evaluated through measurement of a panel of cytokines that act on cells of the adaptive immune response to promote or inhibit inflammation, chemokines involved in chemotaxis, the erythrocyte sedimentation rate (ESR), the high-sensitivity C-reactive protein (CRP) level, and the white blood cell (WBC) count in 3 blood samples (before anesthesia, immediately postoperatively, and 24 hours postoperatively) with 2 types of anesthesia (BGA vs. USG SDIB). Postoperative pain intensity (immediately, at 12 hours, and at 24 hours) was also assessed. RESULTS: The ESR and CRP level increased significantly at 24 hours after surgery; however, the increase in ESR (P < .0001) and CRP level (P < .0001) was lower in the USG SDIB group. Significant increases in the levels of soluble interleukin 2 receptor α (P = .022) and interleukin 12p40 (P = .016) occurred in the immediate postoperative period in the USG SDIB group. Immediate postoperative pain showed a significant increase (P < .001) in the BGA group. CONCLUSIONS: In arthroscopic shoulder surgery, the use of a USG SDIB compared with the use of BGA is possibly associated with improved pain control in the immediate postoperative period and lower immunosuppression, even at 24 hours after surgery.

[Immunological markers of defeat of nervous tissue as an element of predictive model of damage of nervous system at syphilis.]

To develop predictive model of damage of nervous system on the basis of definition of concentration of interleukins-23, 12p40 and also a glial fibrillar acid protein (GFAP) in liquor of patients with various forms of syphilis. Comprehensive laboratory examination of patients with neurosyphilis and syphilis without specific damage of nervous system who were observed in venereologic office of BOUZAS of OO «Clinical Dermatovenerologic Clinic¼ of Omsk is conducted. To all patients were carried out: a serological blood analysis, serological and clinical trial of liquor, and also immunological research of liquor (interleukins - 23, 12p40, and also GFAP). On the basis of the research IL-23, SILT-12p40, GFAP, the level of protein and a pleocytosis in liquor the predictive model of development of neurosyphilis in patients with syphilis without specific damage of nervous system is offered. The analysis of immunological changes in liquor of patients showed that the research of a number of cytokines and markers of damage of nervous tissue to liquor as the most specific and reliable, especially in the absence of clinical symptomatology from central nervous system can be an integral part of diagnostics of neurosyphilis also.

Cholesterol Modification of p40-Specific Small Interfering RNA Enables Therapeutic Targeting of Dendritic Cells.

Small interfering RNA (siRNA)-based therapies allow targeted correction of molecular defects in distinct cell populations. Although efficient in multiple cell populations, dendritic cells (DCs) seem to resist siRNA delivery. Using fluorescence labeling and radiolabeling, we show that cholesterol modification enables siRNA uptake by DCs in vitro and in vivo. Delivery of cholesterol-modified p40 siRNA selectively abolished p40 transcription and suppressed TLR-triggered p40 production by DCs. During immunization with peptide in CFA, cholesterol-modified p40 siRNA generated p40-deficient, IL-10-producing DCs that prevented IL-17/Th17 and IFN-γ/Th1 responses. Only cholesterol-modified p40-siRNA established protective immunity against experimental autoimmune encephalomyelitis and suppressed IFN-γ and IL-17 expression by CNS-infiltrating mononuclear cells without inducing regulatory T cells. Because cholesterol-modified siRNA can thus modify selected DC functions in vivo, it is intriguing for targeted immune therapy of allergic, autoimmune, or neoplastic diseases.

Norovirus in symptomatic and asymptomatic individuals: cytokines and viral shedding.

Noroviruses (NoV) are the most common cause of epidemic gastroenteritis world-wide. NoV infections are often asymptomatic, although individuals still shed large amounts of NoV in their stool. Understanding the differences between asymptomatic and symptomatic individuals would help in elucidating mechanisms of NoV pathogenesis. Our goal was to compare the serum cytokine responses and faecal viral RNA titres of asymptomatic and symptomatic NoV-infected individuals. We tested serum samples from infected subjects (n = 26; 19 symptomatic, seven asymptomatic) from two human challenge studies of GI.1 NoV for 16 cytokines. Samples from prechallenge and days 1-4 post-challenge were tested for these cytokines. Cytokine levels were compared to stool NoV RNA titres quantified previously by reverse transcription-polymerase chain reaction (RT-qPCR). While both symptomatic and asymptomatic groups had similar patterns of cytokine responses, the symptomatic group generally exhibited a greater elevation of T helper type 1 (Th1) and Th2 cytokines and IL-8 post-challenge compared to the asymptomatic group (all P < 0·01). Daily viral RNA titre was associated positively with daily IL-6 concentration and negatively with daily IL-12p40 concentration (all P < 0·05). Symptoms were not associated significantly with daily viral RNA titre, duration of viral shedding or cumulative shedding. Symptomatic individuals, compared to asymptomatic, have greater immune system activation, as measured by serum cytokines, but they do not have greater viral burden, as measured by titre and shedding, suggesting that symptoms may be immune-mediated in NoV infection.

Differential expression of the inflammation marker IL12p40 in the at-risk mental state for psychosis: a predictor of transition to psychotic disorder?

BACKGROUND: The identification of biomarkers of transition from the at-risk mental state (ARMS) to psychotic disorder is important because early treatment of psychosis is associated with improved outcome. Increasing evidence points to an inflammatory contribution to psychosis. We questioned whether raised levels of plasma inflammatory markers predict transition from ARMS to psychotic disorder and whether any such predictors could be reduced by omega-3 (ω-3) polyunsaturated fatty acids (PUFAs). METHODS: We measured the levels of 40 neuroinflammation biomarkers using a commercially available immunoassay kit. Firstly, we compared inflammatory markers in subjects in the ARMS who transitioned to psychotic disorder (n = 11) compared to subjects who did not (n = 28). Then we compared inflammatory markers in all subjects before and after ω-3 PUFA treatment (n = 40). RESULTS: Our data provides preliminary evidence that elevations in the baseline plasma levels of the inflammatory marker IL12/IL23p40 are associated with transition from ARMS to psychotic disorder. IL12/IL23p40 levels did not change following 12 weeks administration of ω-3 PUFAs. These findings provide evidence that elevated plasma IL12/IL23p40 is a potential biomarker of increased risk for transition to psychotic disorder. CONCLUSION: Further studies are required to confirm and extend this finding. Our results do not provide support for the possibility that administration of ω-3 PUFAs act to reduced transition to psychotic disorder by reducing blood levels of IL12/IL23p40. TRIAL REGISTRATION: ClinicalTrials.gov, a service of the U.S. National Institutes of Health, Identifier: NCT00396643 , last updated December 20, 2007. Retrospectively registered.

Effects of Caffeine Supplementation on Plasma and Blood Mononuclear Cell Interleukin-10 Levels After Exercise.

This study compared the response of interleukin (IL)-10, and also of IL-6 and IL-12 p40, to exercise and caffeine supplementation between plasma and blood mononuclear cells (BMNCs). Participants in the study (n = 28) were randomly allocated in a double-blind fashion to either caffeine (n = 14) or placebo (n = 14) treatments. One hour before completing a 15-km run competition, athletes took 6 mg/kg body mass of caffeine or a placebo. Plasma and BMNCs were purified from blood samples taken before and after competition. Concentrations of interleukins (IL-10, IL-6, and IL-12 p40), cyclic adenosine monophosphate (cAMP), caffeine, adrenaline, and cortisol were measured in plasma. IL-10, IL-6, and IL-12 p40 and cAMP levels were also determined in BMNCs. Exercise induced significant increases in IL-6 and IL-10 plasma levels, with higher increases in the caffeine-supplemented group. After 2-hr recovery, these levels returned to almost preexercise values. However, no effect of caffeine on BMNC cytokines was observed. IL-10, IL-6, and IL-12 p40 levels in BMNCs increased mainly at 2 hr postexercise. cAMP levels increased postexercise in plasma and after recovery in BMNCs, but no effects of caffeine were observed. In conclusion, caffeine did not modify cytokine levels in BMNCs in response to exercise. However, higher increases of IL-10 were observed in plasma after exercise in the supplemented participants, which could suppose an enhancement of the anti-inflammatory properties of exercise.

Staphylococcus epidermidis Bacteremia Induces Brain Injury in Neonatal Mice via Toll-like Receptor 2-Dependent and -Independent Pathways.

BACKGROUND: Staphylococcus epidermidis causes late-onset sepsis in preterm infants. Staphylococcus epidermidis activates host responses in part via Toll-like receptor 2 (TLR2). Epidemiologic studies link bacteremia and neonatal brain injury, but direct evidence is lacking. METHODS: Wild-type and TLR2-deficient (TLR2-/-) mice were injected intravenously with S. epidermidis at postnatal day 1 prior to measuring plasma and brain cytokine and chemokine levels, bacterial clearance, brain caspase-3 activation, white/gray matter volume, and innate transcriptome. RESULTS: Staphylococcus epidermidis bacteremia spontaneously resolved over 24 hours without detectable bacteria in the cerebrospinal fluid (CSF). TLR2-/- mice demonstrated delayed S. epidermidis clearance from blood, spleen, and liver. Staphylococcus epidermidis increased the white blood cell count in the CSF, increased interleukin 6, interleukin 12p40, CCL2, and CXCL1 concentrations in plasma; increased the CCL2 concentration in the brain; and caused rapid (within 6 hours) TLR2-dependent brain activation of caspase-3 and TLR2-independent white matter injury. CONCLUSIONS: Staphylococcus epidermidis bacteremia, in the absence of bacterial entry into the CSF, impairs neonatal brain development. Staphylococcus epidermidis bacteremia induced both TLR2-dependent and -independent brain injury, with the latter occurring in the absence of TLR2, a condition associated with an increased bacterial burden. Our study indicates that the consequences of transient bacteremia in early life may be more severe than commonly appreciated, and our findings may inform novel approaches to reduce bacteremia-associated brain injury.

The relationship between blood IL-12p40 level and melanoma progression.

Cytokines such as Interleukin (IL)-12p70 ("IL-12") and IL-23 can influence tumor progression. We tested the hypothesis that blood levels of IL-12p40, the common subunit of both cytokines, are associated with melanoma progression. Blood from 2,048 white melanoma patients were collected at a single institution between March 1998 and March 2011. Plasma levels of IL-12p40 were determined for 573 patients (discovery), 249 patients (Validation 1) and 244 patients (Validation 2). Per 10-unit change of IL-12p40 level was used to investigate associations with melanoma patient outcome among all patients or among patients with early or advanced stage. Among stage I/II melanoma patients in the pooled data set, after adjustment for sex, age, stage and blood draw time from diagnosis, elevated IL-12p40 was associated with melanoma recurrence [hazard ratio (HR) = 1.04 per 10-unit increase in IL-12p40, 95% CI 1.02-1.06, p = 8.48 × 10(-5) ]; Elevated IL-12p40 was also associated with a poorer melanoma specific survival (HR = 1.06, 95% CI 1.03-1.09, p = 3.35 × 10(-5) ) and overall survival (HR = 1.05, 95% CI 1.03-1.08, p = 8.78×10(-7) ) in multivariate analysis. Among stage III/IV melanoma patients in the pooled data set, no significant association was detected between elevated IL-12p40 and overall survival, or with melanoma specific survival, with or without adjustment for the above covariates. Early stage melanoma patients with elevated IL-12p40 levels are more likely to develop disease recurrence and have a poorer survival. Further investigation with a larger sample size will be needed to determine the role of IL-12p40 in advanced stage melanoma patients.

Anti-IFN-γ autoantibodies in adults with disseminated nontuberculous mycobacterial infections are associated with HLA-DRB1*16:02 and HLA-DQB1*05:02 and the reactivation of latent varicella-zoster virus infection.

Adult patients with disseminated nontuberculous mycobacterial (dNTM) infections usually have severe immune system defects. Recently, several studies have shown that anti-IFN-γ autoantibodies may play an important role in the pathogenicity of dNTM infections. A considerable proportion of reported cases of anti-IFN-γ autoantibodies show either clinical or laboratory evidence of autoimmune disease. In the present study, we identified 19 formerly healthy adults who later developed dNTM infections, of whom 17 were further investigated immunologically. High-titer anti-IFN-γ autoantibodies capable of inhibiting IL-12 production in vitro were found in the plasma of all of these patients. In addition to dNTM infection, 35% and 71% of our patients also suffered from salmonellosis and herpes zoster, respectively. This observation suggests that IFN-γ may be crucial in controlling salmonella infection and reactivating latent varicella-zoster virus infection in humans. 2 HLA alleles, DRB1*16:02 DQB1*05:02 (odds ratio 8.68; 95% confidence interval, 3.47-21.90; P = 1.1 × 10(-6); Pc = 3.08 × 10(-5) and odds ratio 7.16; 95% confidence interval, 3.02-17.05; P = 1 × 10(-7); Pc = 1.4 × 10(-6), respectively), were found in 82% (14 of 17) of our patients. In conclusion, our data suggest that anti-IFN-γ autoantibodies may play a critical role in the pathogenesis of dNTM infections and reactivation of latent varicella-zoster virus infection and are associated with HLA-DRB1*16:02 and HLA-DQB1*05:02.

Design and evaluation of a unique SYBR Green real-time RT-PCR assay for quantification of five major cytokines in cattle, sheep and goats.

BACKGROUND: Today, when more than 60% of animal diseases are zoonotic, understanding their origin and development and identifying protective immune responses in ruminants are major challenges. Robust, efficient and cost-effective tools are preconditions to solve these challenges. Cytokines play a key role in the main mechanisms by which the immune system is balanced in response to infectious pathogens. The cytokine balance has thus become the focus of research to characterize immune response in ruminants. Currently, SYBR Green reverse transcriptase quantitative PCR (RT-qPCR) is the most widely method used to investigate cytokine gene expression in ruminants, but the conditions in which the many assays are carried out vary considerably and need to be properly evaluated. Accordingly, the quantification of gene expression by RT-qPCR requires normalization by multiple reference genes. The objective of the present study was thus to develop an RT-qPCR assay to simultaneously quantify the expression of several cytokines and reference genes in three ruminant species. In this paper, we detail each stage of the experimental protocol, check validation parameters and report assay performances, following MIQE guidelines. RESULTS: Ten novel primer sets were designed to quantify five cytokine genes (IL-4, IL-10, IL-12B, IFN-γ and TNF-α) and five reference genes (ACTB, GAPDH, H3F3A, PPIA and YWHAZ) in cattle, sheep, and goats. All the primer sets were designed to span exon-exon boundaries and use the same hybridization temperature. Each stage of the RT-qPCR method was detailed; their specificity and efficiency checked, proved and are reported here, demonstrating the reproducibility of our method, which is capable of detecting low levels of cytokine mRNA up to one copy whatever the species. Finally, we checked the stability of candidate reference gene expression, performed absolute quantification of cytokine and reference gene mRNA in whole blood samples and relative expression of cytokine mRNA in stimulated PBMC samples. CONCLUSIONS: We have developed a novel RT-qPCR assay for the simultaneous relative quantification of five major cytokines in cattle, sheep and goats, and their accurate normalization by five reference genes. This accurate and easily reproducible tool can be used to investigate ruminant immune responses and is widely accessible to the veterinary research community.

Impaired in-vitro responses to IL-12 and IFN-γ in Iranian patients with Mendelian susceptibility to mycobacterial disease.

INTRODUCTION: Diagnosis of specific molecular defects of Mendelian susceptibility to mycobacterial diseases (MSMD) patients is important with respect to their clinical outcomes and their response to therapy. The aim of this study was to perform functional tests on blood samples of a group of patients who were suspected of having MSMD. METHODS: This study was performed on 11 cases who had mycobacterial infections and suspected MSMD. Whole blood cell culture was performed in presence of different stimulators. The supernatants were assayed for IFN-γ, IL-12p40 by ELISA method. RESULTS: All patients presented with complications of BCG vaccine in the form of localised lymphadenitis or disseminated BCG infection and chronic mycobacterial osteomyelitis. Infections with Salmonella species occurred in two patients. In-vitro studies showed that 10 cases had impaired response to IL-12. However, the baseline levels of IL-12p40 were normal, while one of our patients may have a potential IFN-γ signalling defect or an IL-12p40 defect. CONCLUSIONS: Early detection of MSMD and commencing of appropriate combination therapy could prevent severe or even fatal complications of uncontrolled mycobacterial infections.

Proinflammatory and anti-inflammatory cytokines in adolescents from Southeast Bulgarian cities with different levels of air pollution.

Epidemiological studies demonstrated that the exposure of different air pollutants including particulate matter (PM) has been related to adverse effect on immune system. Current study was designed to investigate cytokines in blood plasma of adolescent persons continuously exposed to different degrees of ambient air pollutions. Tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), IL-12p40, and IL-10 were chosen as cytokines of proinflammatory and anti-inflammatory immune response. The peripheral venous blood was taken from adolescents living in the cities of Stara Zagora region, Southeast Bulgaria, that is, in Stara Zagora, Kazanlak, and Chirpan. The quantity of cytokines in plasma samples was determined by enzyme-linked immunosorbent assay. Results demonstrated that youths living in Stara Zagora showed significantly smaller quantity of TNF-α, compared with adolescents from Kazanlak and Chirpan. Moreover, adolescents living in Stara Zagora showed significantly higher quantity of IL-10 than students from Kazanlak and Chirpan. Analysis of the data of air quality gives reason to assert that PM10 and PM2.5 have been the main atmospheric pollutants around the monitoring points. The complex air quality assessment based on these criteria determined that the highest air pollution was in the city of Stara Zagora, followed by Chirpan and the relatively unpolluted town was Kazanlak. We concluded that air pollutants, mostly PM2.5, can modulate cytokine production and can change the balance between proinflammatory TNF-α and anti-inflammatory IL-10 production. Increased levels of IL-10 combined with decreased level of TNF-α in adolescents living in Stara Zagora can serve as a biomarker for suppression of T helper 1 (Th1) cell-mediated immunity and exacerbation of Th2 humoral immune response and could be a prerequisite for the development of allergic and autoimmune diseases.

Genetic susceptibility to increased bacterial translocation influences the response to biological therapy in patients with Crohn's disease.

OBJECTIVE: The aetiology of Crohn's disease (CD) has been related to nucleotide-binding oligomerisation domain containing 2 (NOD2) and ATG16L1 gene variants. The observation of bacterial DNA translocation in patients with CD led us to hypothesise that this process may be facilitated in patients with NOD2/ATG16L1-variant genotypes, affecting the efficacy of anti-tumour necrosis factor (TNF) therapies. DESIGN: 179 patients with Crohn's disease were included. CD-related NOD2 and ATG16L1 variants were genotyped. Phagocytic and bactericidal activities were evaluated in blood neutrophils. Bacterial DNA, TNFα, IFNγ, IL-12p40, free serum infliximab/adalimumab levels and antidrug antibodies were measured. RESULTS: Bacterial DNA was found in 44% of patients with active disease versus 23% of patients with remitting disease (p=0.01). A NOD2-variant or ATG16L1-variant genotype was associated with bacterial DNA presence (OR 4.8; 95% CI 1.1 to 13.2; p=0.001; and OR 2.4; 95% CI 1.4 to 4.7; p=0.01, respectively). This OR was 12.6 (95% CI 4.2 to 37.8; p=0.001) for patients with a double-variant genotype. Bacterial DNA was associated with disease activity (OR 2.6; 95% CI 1.3 to 5.4; p=0.005). Single and double-gene variants were not associated with disease activity (p=0.19). Patients with a NOD2-variant genotype showed decreased phagocytic and bactericidal activities in blood neutrophils, increased TNFα levels in response to bacterial DNA and decreased trough levels of free anti-TNFα. The proportion of patients on an intensified biological therapy was significantly higher in the NOD2-variant groups. CONCLUSIONS: Our results characterise a subgroup of patients with CD who may require a more aggressive therapy to reduce the extent of inflammation and the risk of relapse.

Efficiency of double layered microencapsulated probiotic to modulate proinflammatory molecular markers for the management of alcoholic liver disease.

Alcohol-related disorders are one of the challenging current health problems with medical, social, and economic consequences. Endotoxemia, oxidative stress, and release of a variety of inflammatory molecules are established mediators in alcoholic liver injury (ALD). Probiotics like L. plantarum though were reported to attenuate ALD, their in vivo health benefits are limited by their survival and sustenance in the adverse gut conditions. Therefore, to enhance their in vivo performance, chitosan coated alginate beads entrapping L. plantarum were prepared, characterized, and evaluated for their efficacy against ALD in rats. Following chronic alcohol exposure, rats developed endotoxemia, showed enhanced levels of liver enzyme markers, NF-κB levels, and increased cytokines such as TNF- α and IL12/p40 subunit, and reflected significant histological changes in the intestine and liver. However, cosupplementation with double layered microencapsulated probiotic significantly (P < 0.05) reduced the levels of endotoxemia, serum transaminases, NF-κB, and cytokines complemented with restoration of normal histoarchitecture of the intestine and liver. It is being documented here for the first time that the probiotics have the potential to inhibit IL-12/p40 subunit which is a recently explored potential marker for developing novel therapeutic agents. This study reveals that microencapsulation of probiotics may offer a biopharmacological basis for effective management of ALD.