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Competing Interests: Conflicts of interest None disclosed.

Using transgenic mice that replicate hepatitis B virus (HBV) at high levels in the liver as recipients of HBV-specific cytotoxic T lymphocytes (CTLs), we showed that the chemokines responsive to gamma-2/IFN-gamma inducible protein ([Crg2]IP-10) and monokine induced by interferon-gamma (Mig) are rapidly and strongly induced in the liver after CTL transfer. The transferred CTLs produce neither chemokine; rather, they activate (via the secretion of IFN-gamma) hepatocytes and nonparenchymal cells of the liver to produce (Crg2)IP-10 and Mig. Importantly, blocking these chemokines in vivo reduces the recruitment of host-derived lymphomononuclear cells into the liver and the severity of the liver disease without affecting the IFN-gamma-dependent antiviral potential of the CTLs. The finding that neutralization of these chemokines is associated with maintenance of antiviral effects but diminished tissue damage may be significant for the development of immunotherapeutic approaches for the treatment of chronic HBV infection.

Using transgenic mice that replicate hepatitis B virus (HBV) at high levels in the liver as recipients of HBV-specific cytotoxic T lymphocytes (CTLs), we showed that the chemokines responsive to gamma-2/IFN-gamma inducible protein ([Crg2]IP-10) and monokine induced by interferon-gamma (Mig) are rapidly and strongly induced in the liver after CTL transfer. The transferred CTLs produce neither chemokine; rather, they activate (via the secretion of IFN-gamma) hepatocytes and nonparenchymal cells of the liver to produce (Crg2)IP-10 and Mig. Importantly, blocking these chemokines in vivo reduces the recruitment of host-derived lymphomononuclear cells into the liver and the severity of the liver disease without affecting the IFN-gamma-dependent antiviral potential of the CTLs. The finding that neutralization of these chemokines is associated with maintenance of antiviral effects but diminished tissue damage may be significant for the development of immunotherapeutic approaches for the treatment of chronic HBV infection.

Diverse conditions for stimulating human mononuclear cells to release thymocyte costimulatory factors were tested for their contribution to the generation of supernatants high titers of these monokines. Activity titers increased with LPS concentration, reaching a plateau between 1 and 10 microng/ml. Indomethacin did not modify the monokine, but the assay for thymocyte costimulatory activity was substantially affected by inhibitory substances produced by the monocytes in the absence of indomethacin. The use of nylon wool columns to trap the cells was shown to be effective in raising cellular densities without decreasing activity titers. As result, the yield per cell could be maintained even in the absence of serum, an important step toward the goal of purifiying bioactive from crude broths.

Direct acting antiviral agents (DAAs) are a group of antiviral drugs that inhibit specific non-structural proteins of the virus and disrupt viral replication and infection. DAAs regimens for hepatitis C virus (HCV) infection provide a particular event to tackle mechanistic intracellular relationships between the innate immunity and HCV, potentially providing perceptions about the rate of the viral replication and complex decay. Interleukin 29 (IL-29) prevents the replication of HCV. IFN-inducible protein 10 (IP-10) plays a significant role in the pathogenesis of HCV infection. MIG/CXCL9 are produced by inflammatory and stromal cells such as hepatocytes following either stimulation by interferon lambda (IFNγ) or viral infection. This study aimed to evaluate the co-expression of IL-29, IP-10 and MIG in peripheral blood mononuclear cells (PBMCs) from untreated and treated chronic HCV patients with DAAs. This study included group of twenty naïve HCV patients, group of twenty sustained viral response (SVR) patients and a control group that consisted of 10 healthy subjects. All subjects were tested for liver enzymes, serum albumin level, total serum bilirubin, platelet count, prothrombin activity and viral load. Relative gene expression of IL-29, IP-10, and MIG in PBMCs from all subjects was determined using real time PCR. The mean value of IL-29, IP-10 and MIG gene expression significantly increased in both naïve HCV and SVR groups of patients as compared to normal subjects. The corresponding value was significantly lower in patients with SVR compared to naïve HCV patients. Infection with HCV significantly trigged the co-expression of IL-29, IP-10, and CXCL9 (MIG) genes in PBMCs of chronic hepatitis C patients and significantly down-regulated in those who achieved SVR after successful DAAs therapy. Keywords: IP10; MIG; IL29; HCV; DAAs; gene expression.

Experimental analysis of allergic airway inflammation (AAI) in animals and humans is associated with coordinate gene induction. Using DNA microarray analysis, we have identified a large panel of AAI signature genes. Unexpectedly, the allergen-challenged lung (a T helper 2 microenvironment) was found to be associated with the expression of T helper 1-associated CXCR3 ligands, monokine induced by IFN-[gamma] (Mig), and IFN-[gamma]-inducible protein of 10 kDa (IP-10). Here we report that Mig functions as a negative regulator of murine eosinophils. Whereas Mig was not able to induce chemotaxis of eosinophils, pretreatment with Mig induced a dose-dependent inhibition of chemoattractant-induced eosinophil transmigration in vitro. Moreover, i.v. administration of low doses of Mig ([approximately equal to] 10-30 /[micro]g/kg) induced strong and specific dose-dependent inhibition of chemokine-, IL-13-, and allergen-induced eosinophil recruitment and, conversely, neutralization of Mig before allergen challenge increased airway eosinophilia. Importantly, Mig also inhibited a CCR3-mediated functional response in eosinophils. These results indicate that the ultimate distribution and function of inflammatory cells within the allergic lung is dictated by a balance between positively and negatively regulatory chemokines. The identification of a naturally occurring eosinophil inhibitory chemokine pathway in vivo provides a strategic basis for future therapeutic consideration.

A cloned subline of the human monocytic leukemia cell line, THP-1, was induced to produce high levels of cytotoxic activity following an 18-hour phorbol myristate acetate (CAS: 16561-29-8) stimulation in vitro. This activity, termed monocyte cell line cytotoxin(s) (MCCT), was tested in vitro on different human continuous cell lines (Chang, ESH-7, GM3104A, HeLa, HT-1080, K562, Mel, T-24) and on primary human fibroblasts (GM3468, Manz). The continuous cell lines exhibited a spectrum of sensitivity to MCCT-containing supernatants whereas the primary fibroblasts were resistant to lysis. Enzymatic degradation and heat denaturation studies indicate that MCCT is a protein. Its bioactivity can be resolved into three lytic peaks after molecular sieving on Ultrogel AcA 44. The major peak, designated alpha MCCT, eluted with a molecular weight of 100,000-140,000 daltons. A minor peak, beta MCCT, was seen at 60,000-80,000 daltons, and a third, unstable minor peak, gamma MCCT, eluted at less than 10,000 daltons. The alpha-lytic peak was examined further and was found to migrate as a single peak in 7% native polyacrylamide gel electrolysis tube gels with an rf of 0.25-0.30. None of the MCCT forms were immunologically cross-reactive with human alpha-lymphotoxin. Various protease inhibitors known to inhibit monokine- and macrophage-mediated direct cell lysis in vitro were tested for their inhibitory effects on alpha MCCT activity. The irreversible binding inhibitor N alpha-p-tosyl-L-lysyl chloromethyl ketone inhibited the biologic activity of alpha MCCT. The reversible binding inhibitors N alpha-p-tosyl-L-arginine methyl ester and soybean trypsin inhibitor were able to block in vitro lytic activity when added to alpha MCCT in the presence of the target cell. In contrast, the inhibitors phenylmethylsulfonyl fluoride, L-1-tosylamide, 2-phenylethyl chloromethyl ketone, and N alpha-acetyl-L-lysine methyl ester were not effective in blocking cytolysis. Finally, the hydrogen peroxide scavenger catalase inhibited alpha MCCT lytic activity in vitro; however, the hypochlorous acid scavengers alanine, serine, and valine were without effect.

Abstract Background The ability to measure T-cell responses to antigens is proving critical in the field of vaccine development and for understanding immunity to pathogens, allergens and self-antigens. Although a variety of technologies exist for this purpose IFNγ-ELISpot assays are widely used because of their sensitivity and simplicity. However, ELISpot assays cannot be performed on whole blood, and require relatively large volumes of blood to yield sufficient numbers of peripheral blood mononuclear cells. To address these deficiencies, we describe an assay that measures antigen-specific T cell responses through changes in monokine gene transcription. The biological amplification of the IFNγ signal generated by this assay provides sensitivity comparable to ELISpot, but with the advantage that responses can be quantified using small volumes of whole blood. Methods Whole blood or peripheral blood mononuclear cells (PBMCs) from healthy controls and immunosuppressed recipients of solid organ transplants were incubated with peptide pools covering viral and control antigens or mitogen for 20 hours. Total RNA was extracted and reverse transcribed before amplification in a TaqMan qPCR reaction using primers and probes specific for MIG (CXCL9), IP-10 (CXCL10) and HPRT. The induction of MIG and IP-10 in response to stimuli was analysed and the results were compared with those obtained by ELISpot. Results Antigen-specific T cell responses can be measured through the induction of MIG or IP-10 gene expression in PBMCs or whole blood with results comparable to those achieved in ELISpot assays. The biological amplification generated by IFNγ-R signaling allows responses to be detected in as little as 25 μL of whole blood and enables the assay to retain sensitivity despite storage of samples for up to 48 hours prior to processing. Conclusions A monokine-based reporter assay provides a sensitive measure of antigen-specific T cell activation. Assays can be performed on small volumes of whole blood and remain accurate despite delays in processing. This assay may be a useful tool for studying T cell responses, particularly when samples are limited in quantity or when storage or transportation is required before processing.

BackgroundPrior epidemiological studies have established a correlation between inflammatory cytokines and inflammatory bowel disease (IBD). However, the nature of this relationship remains uncertain. Mendelian randomization (MR) study has the advantages of avoiding confounding and reverse causality compared with traditional observational research.ObjectiveWe aimed to evaluate whether genetically determined circulating levels of cytokines are associated with the risk of IBD by using the MR approach.Materials and methodsWe selected genetic variants associated with circulating levels of 28 cytokines at the genome-wide significance level from a genome-wide association study (GWAS) including 8,293 individuals. Summary-level data for IBD (including Crohn’s disease and ulcerative colitis) were obtained from the International Inflammatory Bowel Disease Genetics Consortium and UK Biobank. We performed the primary analysis using the inverse-variance weighted method, as well as sensitivity analyses to test the stability of our results. We subsequently replicated the results of IBD in the UK Biobank dataset. A reverse MR analysis was also conducted to evaluate the possibility of reverse causation.ResultsGenetically predicted elevated levels of interleukin-17 (IL-17) and monokine induced by interferon-gamma (MIG) were associated with an increased risk of IBD[odds ratio (OR): 1.52, 95% confidence interval (CI):1.10-2.08, P =0.010 for IL-17 and OR: 1.58, 95% CI: 1.24-2.00, P = 1.60×10-4 for MIG]. Moreover, we observed suggestive associations between β-NGF and MIP-1β with the risk of Crohn’s disease (OR: 0.71, 95% CI: 0.52-0.98, P = 0.039) and ulcerative colitis (OR: 1.08, 95% CI: 1.01-1.15, P= 0.019). In the reverse MR study, there was no evidence of causal effects of IBD and these cytokines.ConclusionOur study suggests the potential causal associations of IL-17 and MIG with IBD. Further studies are needed to determine whether IL-17 and MIG or their downstream effectors could be useful in the management of IBD.