PBMC Cultures Research Articles

Molecular characterization of duck (Anas platyrynhos) Toll-like receptors, mRNA expressions profile in day-old duckling’s tissues and cytokine response to in vitro TLR agonsists stimulation

TLR repertoire of duck, profiling of their mRNA expression in a range of duckling tissues and cytokine gene expressions upon TLR agonists stimulation in in vitro assay have been investigated. All ten TLR genes orthologous to chicken TLR repertoire were found in duck. Duck TLR genes showed 77-83% similarity at amino acid level to their chicken counterparts. All ten TLRs-TLR1LA, 1LB, 2A, 2B, 3, 4, 5, 7, 15 and 21 mRNA expressions were significantly higher in bursa than other tissues studied, whereas in muscle all TLRs mRNA expressions were significantly lower except for TLR15 (P>0.01). TLR7 gene expression was significantly higher in spleen, bursa and also in lung tissues (P>0.01). The cytokine gene expression levels in duck PBMCs upon LPS and poly I:C stimulation have been quantified. IL-1g gene expression level in LPS stimulated duck PBMC culture was significantly higher at both 12 h and 24 h time intervals (P>0.05). However, there were no significant changes in IFN-ã gene expression levels in poly I:C stimulated duck PBMC culture at both the intervals. TLR gene expression in young ducklings together with cytokine response upon LPS stimulation demonstrates the innate preparedness of younger birds to encounter pathogens and their functional ability to respond to their ligands.

Autoimmunity to Vimentin Is Associated with Outcomes of Patients with Idiopathic Pulmonary Fibrosis.

Autoimmunity has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF); however, the repertoire of autoantigens involved in this disease and the clinical relevance of these autoimmune responses are still being explored. Our initial discovery assays demonstrated that circulating and intrapulmonary vimentin levels are increased in IPF patients. Subsequent studies showed native vimentin induced HLA-DR-dependent in vitro proliferation of CD4 T cells from IPF patients and enhanced the production of IL-4, IL-17, and TGF-β1 by these lymphocytes in contrast to normal control specimens. Vimentin supplementation of IPF PBMC cultures also resulted in HLA-DR-dependent production of IgG with anti-vimentin specificities. Circulating anti-vimentin IgG autoantibody levels were much greater in IPF subjects from the University of Alabama at Birmingham (n = 102) and the University of Pittsburgh (U. Pitt., n = 70) than in normal controls. Anti-vimentin autoantibody levels in IPF patients were HLA biased and inversely correlated with physiological measurements of lung function (i.e., forced expiratory volumes and diffusing capacities). Despite considerable intergroup differences in transplant-free survival between these two independent IPF cohorts, serious adverse outcomes were most frequent among the patients within each population that had the highest anti-vimentin autoantibody levels (University of Alabama at Birmingham: hazard ratio 2.5, 95% confidence interval 1.2-5.3, p = 0.012; University of Pittsburgh: hazard ratio 2.7, 95% confidence interval 1.3-5.5, p = 0.006). These data show that anti-vimentin autoreactivity is prevalent in IPF patients and is strongly associated with disease manifestations. These findings have implications with regard to the pathogenesis of this enigmatic disease and raise the possibility that therapies specifically directed at these autoimmune processes could have therapeutic efficacy.

Abstract 3673: Preclinical toxicology and pharmacology for the 4-1BB/HER2 bispecific PRS-343: A first-in-class costimulatory T cell engager

Abstract Background. 4-1BB (CD137) is a key costimulatory immunoreceptor and a highly promising therapeutic target in cancer. To overcome toxicity and efficacy limitations of current 4-1BB targeting antibodies, we have developed PRS-343, a 4-1BB/HER2 bispecific based on Anticalin® technology. We have previously reported on the generation and characterization of PRS-343 with regard to preclinical proof-of-concept and basic drug-like properties.1 Here, we describe the preclinical dataset supporting initiation of a first-in-patient trial. Methods and Results. The pharmacology of PRS-343 was investigated by ex vivo assays based on mixed culture of human PBMC and tumor cell lines. We find that 4-1BB costimulated T cells prominently increase production of IL-2, GM-CSF, TNF-α and IFN-γ. Using a set of immortal cancer cell lines spanning a range of HER2 surface copy numbers, we identified a copy number threshold above which PRS-343 reliably elicited T cell costimulation with a high potency and an EC50 in the subnanomolar range. PRS-343 was well tolerated in a repeat-dose study in cynomolgus monkeys, with no overt toxicity and no significant drug-related toxicological findings. Pharmacokinetic assessment confirmed dose-proportional exposure of the animals during the course of the study. In a mouse model of human PBMC-induced xenograft-vs-host disease (xGvHD), PRS-343 did not show an acceleration of xGvHD development, in contrast to a 4-1BB targeting benchmark. Again utilizing ex vivo assays, we found no PRS-343 induced T cell costimulation in a panel of primary cells, showing that physiological levels of HER2 are insufficient for activation. In a cytokine release assay, proinflammatory cytokine induction by PRS-343 in the absence of a primary TCR stimulation was negligible. Conclusion. The ex vivo experiments described indicate that HER2 expression level is expected to be a reliable marker for patient stratification for PRS-343. The toxicology assessment of PRS-343 indicates that the benign toxicity profile of trastuzumab is retained in PRS-343 with regard to HER2 targeting, and that PRS-343 is expected to elicit its costimulatory effects strictly on T cells also receiving a primary TCR signal and strictly localized to HER2-positive tumors. This is in agreement with in vivo mouse model data showing PRS-343 leads to tumor-localized CD8+ T cell expansion,1 and supports the potential of PRS-343 as an efficacious yet well tolerable 4-1BB costimulating agent. The reported data is the basis for the trial design of a first in patient Phase 1 trial with PRS-343. 1Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B016. Citation Format: Marlon J. Hinner, Rachida-Siham Bel Aiba, Thomas Jaquin, Sven Berger, Manuela Dürr, Corinna Schlosser, Andrea Allersdorfer, Christine Rothe, Louis A. Matis, Shane A. Olwill. Preclinical toxicology and pharmacology for the 4-1BB/HER2 bispecific PRS-343: A first-in-class costimulatory T cell engager [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3673. doi:10.1158/1538-7445.AM2017-3673

Temsirolimus-induced interstitial lung disease as an on-target effect in patients with metastatic renal cell carcinoma.

e16086 Background: An inhibitor for mammalian target of rapamycin (mTORi), temsirolimus, has been used for metastatic renal cell carcinoma (mRCC) pts with poor risk. mTOR is known to regulate proliferation of lymphocytes, which has been rationale of applying mTORi for preventing graft rejection in transplantation. However, nobody knows the immune status of mRCC pts treated with temsirolimus. In addition, accumulation of clinical practices of temsirolimus has revealed a variety of AEs including interstitial lung disease (ILD). The underlying mechanisms of ILD by mTORi remain uncertain. Moreover, the clinical impact of ILD on the clinical response to mTORi remains unknown. So, we have conducted a prospective clinical trial investigating immune parameters in mRCC pts treated with temsirolimus. This project was denominated in Japanese m-TOR Immune Modulation trial (J-TORIM) (UMIN000009662). Methods: J-TORIM was a prospective observational study recruited pts at 25 hospitals in Japan with a targeted sample size of 30. The primary objective was to evaluate whether administration of temsilorimus has consistent effects on immune parameters collected from the peripheral blood before and 3-to-5 weeks after the beginning of temsirolimus therapy. The secondary objectives were to evaluate the associations of immune parameters or ILD with ORR, PFS, and OS, and the associations of immune parameters with ILD. Immune parameters include PBMC culture with con A or lipopolysaccharide (LPS), PBMC flow cytometry with anti- CD4, CD8, CD3, PD-1, PD-L1, CD25, Foxp3 mAbs. Results: Twenty-eight patients were enrolled in the study. ORR of temsirolimus was 10.7% (1 CR and 2 PR). The most commonly reported treatment-related AEs were ILD (28.5%) and stomatitis (28.5%). There were no significant changes after temsirolimus therapy for any immunological parameters. Interestingly, a significant correlation between ILD development and ORR was observed (p < 0.01). In addition, a significant increase of regulatory T cells was also observed after the recovery of ILD (p < 0.05). Conclusions: This is the first prospective study demonstrating that ILD development can be predictive for response to temsirolimus in mRCC pts. Clinical trial information: 000009662.

Immune response after autologous hematopoietic stem cell transplantation in type 1 diabetes mellitus

BackgroundThis study explored the details of the immune response after autologous hematopoietic stem cell transplantation (AHSCT) treatment in type 1 diabetes mellitus.MethodsPeripheral blood mononuclear cells (PBMCs) from 18 patients with type 1 diabetes mellitus were taken at baseline and 12 months after AHSCT or insulin-only therapy. The lymphocyte proliferation, mRNA expression and secretion of pro-inflammatory and anti-inflammatory cytokines belonging to T-helper type 1 (Th1), T-helper type 17 (Th17) and regulatory T (Treg) cells in PBMC culture supernatants were assessed.ResultsCompared with patients receiving insulin-only treatment, the patients receiving AHSCT treatment showed better residual C-peptide secretion, lower anti-GAD titers and less exogenous insulin dosages after 12 months of follow-up. AHSCT treatment was associated with significantly reduced Th1 and Th17 cell proportions as well as decreased IFN-γ, IL-2, IL-12p40 and IL-17A levels in the PBMC culture supernatants (all P < 0.05). Although there was no significant Treg cell expansion after AHSCT treatment, we observed increased IL-10, TGF-β and Foxp3 mRNA expression and increased TGF-β levels. However, we found no significant changes in the T-cell subpopulations after insulin treatment, except for higher IL-12p40 mRNA expression and a lower proportion of Treg cells.ConclusionsAHSCT treatment was associated with decreased expansion and function of Th1 and Th17 cells, which may explain the better therapeutic effect of AHSCT compared with the traditional intensive insulin therapy.Trial registrationClinicaltrials.gov NCT00807651. Registered 18 December 2008.

The effect of feed supplementation with effective microorganisms (EM) on pro- and anti-inflammatory cytokine concentrations in pigs

Effective microorganisms (EM) are used in numerous fields associated with agriculture. The beneficial effects of EM on the general health of pigs and on production parameters are also determined by the influence of these microbes on immunity. The aim of the study was to assess the effect of feed supplementation with EM on the concentration of pro- and anti-inflammatory cytokines in the serum and in a culture of PBMCs with and without ConA stimulation in pigs. ELISA kits were used to determine the cytokine levels in the porcine serum and the PBMC culture supernatants. Evaluation of the serum concentration of cytokines revealed statistically significantly higher concentrations of IL-2, IL-4, IL-10, IFN-γ and TNF-α in the pigs receiving EM Bokashi. The highest concentration of TGF-β in the experimental group was noted on the final test day. Evaluation of cytokine concentrations in the PBMC cultures revealed statistically significantly higher concentrations of IL-2, IL-4 and IL-10 from the third day of the experiment. Statistically significantly higher concentrations of TNF-α were obtained in the unstimulated PBMC culture on the second test day and in the culture treated with ConA on the second and the third test days. The results of our study indicate that supplementation of pig feed with EM Bokashi activates the cell-mediated and humoral immune response, ensuring that Th1/Th2 balance is maintained and enhancing immune processes protecting the body against infection.

Altered miR-155 Expression in Allergic Asthmatic Airways.

We and others have previously identified microRNAs (miRNAs) with pathological roles in animal models of asthma, where miR-146a and miR-155 have been described to play important roles in inflammatory responses. To date, few studies have investigated miRNA expression in human asthmatics. In the current study, significantly lower levels of miR-155 were detected in cell-free sputum from allergic asthmatics compared to healthy controls. Induced sputum isolated from allergic asthmatics in and out of pollen season revealed that miR-155 expression, but not miR-146a, is reduced in lymphocytes in season compared to post-season. In contrast, miR-155 was found to increase, whereas miR-146a decreased in PBMCs and cell-free PBMC culture media upon T cell receptor stimulation via αCD3/CD28 in both allergic asthmatics and healthy controls. Our findings suggest that miR-155 is differentially expressed exvivo in airways of allergic asthmatics compared to healthy controls, which may have implications in the local immune response in allergic asthma.

THE IMMUNOSTIMULAN POTENTIAL OF TENGGULUN (PROTIUM JAVANICUM) LEAVES TOWARDS T CELL CD4+ AND IFNγ SECRETION ON PBMC CHICKEN

One of the plants with immunostimulant activity is Tenggulun leaves which contain of flavonoid, like terpenoid. The aim of this research is to find the potential of Tenggulun’s leaves extract to have the immunostimulant activities. The potential of immunostimulant activity is identified by the increasing the amount of T-cell CD4+ expression and IFNγ secretion. The research method is conducted through cultured chicken PBMC which is infected by ND virus; it is then treated with Tenggulun’s leaves extract with immunostimulant. The result of immunocitochemistry examination CD4+ secretion on PBMC cultures shows how tenggulun is significantly different from the control in the secretion CD4+. The 10μg of tenggulun extract can modulate the T cell CD4+ secretion 68.8±0.83. It is significantly different from K (control) (p&lt;0,05) and treatment group K+, P0, and P1. The examination of IFNγ level using ELISA from tenggulun leaves extract of 10μg doses were inoculated after being infected by ND virus contained immonostimulant potential in increasing the secretion of IFNγ 120.91±6.44. It is significantly different from K-, K+, and P1, yet not significantly different from P0. The content of terpenoid can increase IFNγ secretion on the macrofag cells culture and limfosit cells.

Effect of thermal processing on T cell reactivity of shellfish allergens - Discordance with IgE reactivity.

Crustacean allergy is a major cause of food-induced anaphylaxis. We showed previously that heating increases IgE reactivity of crustacean allergens. Here we investigate the effects of thermal processing of crustacean extracts on cellular immune reactivity. Raw and cooked black tiger prawn, banana prawn, mud crab and blue swimmer crab extracts were prepared and IgE reactivity assessed by ELISA. Mass spectrometry revealed a mix of several allergens in the raw mud crab extract but predominant heat-stable tropomyosin in the cooked extract. PBMC from crustacean-allergic and non-atopic control subjects were cultured with the crab and prawn extracts and proliferation of lymphocyte subsets was analysed by CFSE labelling and flow cytometry. Effector responses were assessed by intracellular IL-4 and IFN-γ, and regulatory T (CD4+CD25+CD127loFoxp3+) cell proportions in cultures were also compared by flow cytometry. For each crustacean species, the cooked extract had greater IgE reactivity than the raw (mud crab p<0.05, other species p<0.01). In contrast, there was a trend for lower PBMC proliferative responses to cooked compared with raw extracts. In crustacean-stimulated PBMC cultures, dividing CD4+ and CD56+ lymphocytes showed higher IL-4+/IFN-γ+ ratios for crustacean-allergic subjects than for non-atopics (p<0.01), but there was no significant difference between raw and cooked extracts. The percentage IL-4+ of dividing CD4+ cells correlated with total and allergen-specific IgE levels (prawns p<0.01, crabs p<0.05). Regulatory T cell proportions were lower in cultures stimulated with cooked compared with raw extracts (mud crab p<0.001, banana prawn p<0.05). In conclusion, cooking did not substantially alter overall T cell proliferative or cytokine reactivity of crustacean extracts, but decreased induction of Tregs. In contrast, IgE reactivity of cooked extracts was increased markedly. These novel findings have important implications for improved diagnostics, managing crustacean allergy and development of future therapeutics. Assessment of individual allergen T cell reactivity is required.

TNF Blockade Maintains an IL-10+ Phenotype in Human Effector CD4+ and CD8+ T Cells.

CD4+ and CD8+ effector T cell subpopulations can display regulatory potential characterized by expression of the prototypically anti-inflammatory cytokine IL-10. However, the underlying cellular mechanisms that regulate expression of IL-10 in different T cell subpopulations are not yet fully elucidated. We recently showed that TNF inhibitors (TNFi) promote IL-10 expression in human CD4+ T cells, including IL-17+ CD4+ T cells. Here, we further characterized the regulation of IL-10 expression via blockade of TNF signaling or other cytokine/co-stimulatory pathways, in human T cell subpopulations. Addition of the TNFi drug adalimumab to anti-CD3-stimulated human CD4+ T cell/monocyte cocultures led to increased percentages of IL-10+ cells in pro-inflammatory IL-17+, IFNγ+, TNFα+, GM-CSF+, and IL-4+ CD4+ T cell subpopulations. Conversely, exogenous TNFα strongly decreased IL-10+ cell frequencies. TNF blockade also regulated IL-10 expression in CD4+ T cells upon antigenic stimulation. Using time course experiments in whole peripheral blood mononuclear cell (PBMC) cultures, we show that TNF blockade maintained, rather than increased, IL-10+ cell frequencies in both CD4+ and CD8+ T cells following in vitro stimulation in a dose- and time-dependent manner. Blockade of IL-17, IFNγ, IL-6R, or CD80/CD86-mediated co-stimulation did not significantly regulate IL-10 expression within CD4+ or CD8+ T cell subpopulations. We show that TNF blockade acts directly on effector CD4+ T cells, in the absence of monocytes or CD4+ CD25highCD127low regulatory T cells and independently of IL-27, resulting in higher IL-10+ frequencies after 3 days in culture. IL-10/IL-10R blockade reduced the frequency of IL-10-expressing cells both in the presence and absence of TNF blockade. Addition of recombinant IL-10 alone was insufficient to drive an increase in IL-10+ CD4+ T cell frequencies in 3-day CD4+ T cell/monocyte cocultures, but resulted in increased IL-10 expression at later time points in whole PBMC cultures. Together, these data provide additional insights into the regulation of IL-10 expression in human T cells by TNF blockade. The maintenance of an IL-10+ phenotype across a broad range of effector T cell subsets may represent an underappreciated mechanism of action underlying this widely used therapeutic strategy.

Schistosoma antigens downregulate CXCL9 production by PBMC of HTLV-1-infected individuals

HTLV-1 is the causal agent of Adult T cell Leukemia/lymphoma (ATLL) and HTLV-1-associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). The immune response to HTLV-1-infection is polarized to the Th1-type, and the presence of CXCL9/CXCL10 chemokines may lead to an increase in the recruitment of pro-inflammatory molecules in spinal cord tissue, contributing to the damage observed in the development of HAM/TSP. It has been observed that in chronic helminth-infections, such as schistosomiasis, there is a deviation toward the Th2/regulatory immune response. ObjectiveTo evaluate the ability of Schistosoma spp. proteins to decrease the in vitro CXCL9 and CXCL10 production by PBMC of HTLV-1-infected individuals. MethodsThe Schistosoma proteins rSm29, rSh-TSP-2 and PIII were added to PBMC cultures of HTLV-1-infected individuals and the levels of chemokines in the supernatants were measured using a sandwich ELISA method. ResultsThe addition of rSm29 to the cultures resulted in decreased production of CXCL9 in all the analyzed individuals and HAM/TSP group (18167±9727pg/mL, p=0.044; 20237±6023pg/mL, p=0.028, respectively) compared to the levels in unstimulated cultures (19745±9729pg/mL; 25078±2392pg/mL, respectively). The addition of rSh-TSP-2 decreased the production of CXCL9 in all studied individuals and carriers group (16136±9233pg/mL, p=0.031; 13977±8857pg/mL, p=0.026) vs unstimulated cultures (19745±9729pg/mL; 18121±10508pg/mL, respectively). Addition of PIII did not alter the results. There was no significant change in the levels of CXCL10 by the addition of the studied proteins. ConclusionThe Schistosoma proteins used in this study were able to down modulate the production of CXCL9, a chemokine associated with the inflammatory process in HTLV-1-infection.

ZW38, a Novel Azymetric Bispecific CD19-Directed CD3 T Cell Engager Antibody Drug Conjugate with Controlled T Cell Activation and Improved B Cell Cytotoxicity

Although blinatumomab is an approved treatment for Philadelphia chromosome negative relapsed or refractory (r/r) precursor-B cell ALL and is under development for r/r B cell NHL, blinatumomab has several limitations impacting fuller therapeutic utility. For instance, blinatumomab therapy requires continuous infusion due to its rapid clearance owing to its size and lack of an antibody Fc and has been associated with potentially life-threatening CNS toxicities and cytokine release syndrome (Viardot A et al, Blood 2016; Goebeler ME et al, J Clin Oncol 2016; Topp et al, Lancet Oncol 2015). In addition, blinatumomab treatment is associated with higher incidence of relapse in patients with high disease burden, and its T cell redirected killing is limited by T cell immunosuppression (e.g. PD-1/PD-L1 up-regulation [Köhnke et al, J Hematol & Oncol 2015]; Treg suppression [Duell et al, ASH abstract 2014]). ZW38 is designed to address each of these limitations and represents a best in class CD19-directed CD3 T cell engager and a novel class of bispecific antibody drug conjugate (ADC).ZW38 contains an Azymetric IgG1-like Fc that carries mutations in the CH2 domain preventing FcgR dependent ADCC and ADCP and exhibits typical IgG1-like PK in rodent studies. Transient expression in mammalian CHO cells demonstrate ZW38 can be expressed at a titre of hundreds mg/L and can be purified using conventional IgG antibody methods and resins with typical IgG step purification yields and high heterodimer purity. Additionally, ZW38 has been conjugated to a microtubule inhibitor that lacks bystander killing.ZW38 antibody paratopes have been engineered to favor T cell-B cell functional engagement and selective target B cell cytotoxicity. In vitro studies demonstrated that ZW38 binds to human CD19+ B cells with >30-fold higher affinity than to human CD3+ T cells. Similar to blinatumomab, ZW38 can redirect the killing of target cancer B cells via T cell subtypes from human PBMC and its cytotoxicity is target B cell dependent. At concentrations that result in efficacious B cell depletion, ZW38 does not overly activate T cells. ZW38 is specifically engineered to induce more ’controlled’ T cell activation than blinatumomab while still mediating sufficient T cell redirected target B cell depletion. ZW38 mediates T cell activation, cytokine release, and proliferation at nanomolar potency. By design, the sufficient, lower cytokine levels necessary for B cell killing may reduce the risk of cytokine release syndrome and T cell anergy. ZW38 exhibited potent growth inhibition in a panel of different B cell ALL and NHL cancer lines including but not limited to: G2, Nalm-6, RS4-11, Daudi, SUDHL-4 and SUDHL-6. In comparison to blinatumomab, ZW38 exhibits superior target B cell depletion in in vitro co-cultures of Raji lymphoma cells and human PBMC (ZW38 depletes > 90% B cells; blinatumomab depletes 20-90% B cells depending on the PBMC donor). In addition, ZW38 was effective in killing target B cells in PBMC cultures in which PD-1 has been up-regulated, which were resistant to blinatumomab killing in this assay. The dual mechanisms of action of ZW38, redirected T cell cytotoxicity and ADC cytotoxicity, may prolong and/or boost response rates, lower the incidence of relapse, and reduce the likelihood of acquired resistance while its ’controlled’ T cell activation profile may reduce the risk of life-threatening and potentially fatal neurotoxicity and CRS. DisclosuresNg:Zymeworks: Employment, Patents & Royalties. Spreter:Zymeworks: Employment, Patents & Royalties. Davies:Zymeworks Biopharmaceuticals: Employment. Wickman:Zymeworks: Employment.

CD147 modulates the differentiation of T-helper 17 cells in patients with rheumatoid arthritis.

The role of CD147 in regulation of rheumatoid arthritis (RA) is not fully elucidated. The aim of this study was to investigate the effect of cell-to-cell contact of activated CD14+ monocytes with CD4+ T cells, and the modulatory role of CD147 on T-helper 17 (Th17) cells differentiation in patients with RA. Twenty confirmed active RA patients and twenty normal controls were enrolled. CD4+ T cells and CD14+ monocytes were purified by magnetic beads cell sorting. Cells were cultured under different conditions in CD4+ T cells alone, direct cell-to-cell contact co-culture of CD4+ and CD14+ cells, or indirect transwell co-culture of CD4+ /CD14+ cells in response to LPS and anti-CD3 stimulation with or without anti-CD147 antibody pretreatments. The proportion of IL-17-producing CD4+ T cells (defined as Th17 cells) was determined by flow cytometry. The levels of interleukin (IL)-17, IL-6, and IL-1β in the supernatants of cultured cells were measured by ELISA. The optimal condition for in vitro induction of Th17 cells differentiation was co-stimulation with 0.1 μg/mL of LPS and 100 ng/mL of anti-CD3 for 3 days under direct cell-to-cell contact co-culture of CD4+ and CD14+ cells. Anti-CD147 antibody reduced the proportion of Th17 cells, and also inhibited the productions of IL-17, IL-6, and IL-1β in PBMC culture from RA patients. The current results revealed that Th17 differentiation required cell-to-cell contact with activated monocytes. CD147 promoted the differentiation of Th17 cells by regulation of cytokine production, which provided the evidence for pathogenesis and potential therapeutic targets for RA.

PROTEIN REKOMBINAN 38 KDA MYCOBACTERIUM TUBERCULOSIS DALAM INTERLEUKIN-2 DAN INTERLEUKIN-4 SERTA LIMFOSIT T CD3+

Tuberculosis remains a serious global health problem despite the widespread use of the vaccine against tuberculosis (TB). Up to now, the only available TB vaccine, Mycobacterium bovis BCG has a very wide efficacy range from 0 until 80 percent protection so the development of a new vaccine is needed. The new protein as a candidate vaccine should be assessed for their immunogenicity. The purpose of this study was to examine whether Mycobacterium tuberculosis 38 kDa recombinant protein could stimulate a cellular immune response especially CD3+T lymphocytes to express IL-2 and IL-4 in PBMC cultures. An experimental laboratory research on cultured PBMC of 3 groups consisting of TB patients, contacts of TB positive and healthy subjects, each group consisted of 8 subjects. All PBMC cultures were induced by Mycobacterium tuberculosis 38 kDa recombinant protein, Purified Protein Derivative (PPD) and without antigen as a control. Expression of IL-2 and IL-4 CD3+ T lymphocytes was measured with flowcytometry. In healthy volunteers and TB contacts there was a significant difference in the expression of IL-2 and IL-4 CD3+ T lymphocytes compared with no any treatment. The highest IL-2 expression was in healthy subjects [8.13 (0.622)] while the highest expression of IL-4 was in TB patients [6.436 (4.586)]. Mycobacterium tuberculosis 38 kDa recombinant protein could induce the expression of IL-2 and IL-4 of CD3+ T lymphocytes in healthy subjects, TB contacts and TB patients and there were a significance differences in the expression of all groups

Glucose-dependent insulinotropic polypeptide (GIP) dose-dependently reduces osteoclast differentiation and resorption

A role for glucose-dependent insulinotropic polypeptide (GIP) in controlling bone resorption has been suspected. However uncertainty remains to identify whether GIP act directly on osteoclasts. The aim of the present study were (i) to identify in different osteoclast differentiation models (human peripheral blood mononuclear cells-PBMC, murine bone marrow macrophage-BMM and murine Raw 264.7 cells) whether GIP was capable of reducing osteoclast formation and resorption; (ii) ascertain whether the highly potent GIP analogue N-AcGIP was capable of inducing a response at lower concentrations and (iii) to decipher the molecular mechanisms responsible for such effects. [d-Ala2]-GIP dose-dependently reduced osteoclast formation at concentration as low as 1nM in human PBMC and 10nM in murine BMM cultures. Furthermore, [d-Ala2]-GIP also reduced the extent of osteoclast resorption at concentration as low as 1nM in human PBMC and murine BMM cultures. The mechanism of action of [d-Ala2]-GIP appeared to be mediated by reduction in intracellular calcium concentration and oscillation that subsequently inhibited calcineurin activity and NFATc1 nuclear translocation. The potency of the highly potent N-AcGIP was determined and highlighted an effect on osteoclast formation and resorption at concentration ten times lower than observed with [d-Ala2]-GIP in vitro. Furthermore, N-AcGIP was also capable of reducing the number of osteoclast in ovariectomized mice as well as the circulating level of type I collagen C-telopeptide. Pharmacological concentrations required for reducing osteoclast formation and resorption provide the impetus to design and exploit enzymatically stable GIP analogues for the treatment of bone resorption disorders in humans.

Exosomes in human semen impair antigen-presenting cell function and decrease antigen-specific T cell responses

Abstract Exposure to semen is the primary route of transmission for many sexually transmitted infections. Accumulating evidence suggests that components in semen directly impair leukocytes, which could compromise the protective efficacy of vaccine-induced immune responses in the mucosa. Exosomes, small microvesicles with immunomodulatory functions, are present at an average concentration of 2.2 × 1013 particles per ejaculate (n = 18). These seminal exosomes (SE) efficiently and rapidly entered peripheral and vaginal dendritic cells (DCs), whereas T cell uptake was poor. In PBMC cultures, SE impaired memory T cell function, reducing the production of TNF-alpha, IL-2 and/or IFN-gamma in response to virus-derived peptides an average of 60% for CD4+ T cells and 48% for CD8+ T cells, in a dose-responsive manner (n = 6). Reduction of cytokine production by memory CD8+ T cells was reduced to a greater extent for protein antigens as compared to peptide antigens, implying that antigen-presenting cell function was impaired by the presence of SE. Exposing only DCs, as opposed to bulk PBMCs, to SE also blocked subsequent CD8+ memory T cell activation, reducing the proportion of cells making cytokines by 22% (n = 5 donors). Expression of CD107a, a marker of cytotoxic T cell degranulation, was decreased by 42% in PBMC cultures exposed to SE and superantigen (n=4). SE also impaired vaginal T cell cytokine and degranulation responses to superantigen. Thus, SE likely inhibit antigen-specific T cell responses by impairing antigen-presenting cell function. Understanding how programmed immune responses are altered by the presence of semen is important to developing the next generation of vaccine and preventative treatments against sexually transmitted disease

IgG1 and Th1 responses to DosR, Rpf and active growth phase antigens of Mycobacterium tuberculosis in Brazilian tuberculosis patients.

Abstract Tuberculosis (TB) is serious problem worldwide. New antigens from Mycobacterium tuberculosis are being investigated in order to improve the diagnosis of latent TB infection and TB disease. In this study, serum levels of IgG1 antibodies against active growth phase antigens (ESAT-6/CFP-10, Rv0717 and Rv3353), dormancy-related (DosR) antigens (Rv1733, Rv1737, Rv2029 and Rv2628), and resuscitation promoting factors (Rpfs Rv0867 and Rv2389) were evaluated in patients with TB before and after chemotherapy using ELISA. Antigen-induced IFN-γ, TNF-α, IL-17, IL-10 and IL-4 were evaluated in supernatants of PBMC cultures. Our results indicate that patients with pulmonary TB express high levels of IgG1 antibodies against ESAT-6/CFP-10, Rv0717, Rv3353, Rv1733, Rv2029 and Rv2628 Rv0867 in the active phase of the disease in comparison to healthy controls (p&amp;lt;0.001). These levels declined to control levels after the completion of six months treatment. ROC analysis confirmed the good performance of Rv0717, Rv1733, Rv3353, Rv0867, Rv2029 and Rv2628 antigens. Interestingly, Rv0717 and Rv1733 antigens induced an IgG1 peak response after 1–3 months of chemotherapy (P&amp;lt;0.01). Similarly, IFN-γ and TNF-α peaked early during treatment in response to ESAT-6/CFP-10, Rv1733 and Rv2029, and declined to control levels after 6 months of chemotherapy. The study groups did not differ in respect to IL-17, IL-10 and IL-4. Taken together, these data further reinforce a possible correlation of IgG1 production with Th1 responses in TB. Moreover, detecting IgG1 antibodies against M. tuberculosis antigens, including DosR and Rpf proteins, may represent an additional tool in the diagnosis of tuberculosis. Financial support: FAPEMIG, CNPq and CAPES, EU and NWO-TOP.

Revisiting the IFN-γ release assay: Whole blood or PBMC cultures? — And other factors of influence

The interferon-γ release assay (IGRA) is a widely used test for the presence of a cell-mediated immune (CMI) response in vitro. This measure is used to test for infection with intracellular pathogens or for validating vaccine efficacy, and it is a widely used test for both human as well as cattle. However, there is no consensus whether to use whole blood cultures or purified PBMCs for the assay, and both cell populations are being used and results compared. Therefore the aim of this study was to compare different culture settings using immune cells from previously vaccinated calves, and to shed light on external factors that could influence the read out in terms of IFN-γ levels. It was found that optimal culture conditions varied between individual animals; when polyclonal activated, cells from whole blood cultures were most responsive, but when activated specifically, the optimal cell concentration/population varied with whole blood, 10×106cells/ml PBMC and 5×106cells/ml PBMC being the highest performing conditions. A further investigation of the distribution of cell populations in PBMCs compared to whole blood was conducted, and a significant (p<0.001) decrease in the percentage of CD3+ T lymphocytes within the PBMCs was found. More specifically, this reduction was due to a significant (p<0.01) decrease in the percentage of γδ+ T lymphocytes. Thus measuring immune responses on purified PBMCs might not give a physiologically relevant output. Additionally, it was tested if the choice of incubation plate would interfere with the level of secreted IFN-γ in whole blood cultures from five calves. Six plates (a–f) were tested and no significant difference in absolute levels of IFN-γ was detected in the six plates when cells were polyclonal and specifically activated. However, we observed a significant (p<0.05) higher background level in a flat-bottom plate from Corning® (cat# 3595) (plate d) compared to two different flat-bottom plates from Corning® (cat# 3596) (plate b) and Nunc™ (cat# 167008) (plate a). Furthermore 4 out of 5 calves had maximum specific IFN-γ expression on plate b, and the relative-to-maximum level on this plate was significant (p<0.05) compared to plate a. Altogether these findings highlight the potential weaknesses of the IFN-γ release assay in terms of the many variables that can influence the results, including the cell culture population, the concentration of cells being cultured, and the plastic ware used for the in vitro culture. These findings stress the importance of documenting the precise assay conditions when publishing results of in vitro IFN-γ release assays.

Leprosy Reactions Show Increased Th17 Cell Activity and Reduced FOXP3+ Tregs with Concomitant Decrease in TGF-β and Increase in IL-6.

Background50% of leprosy patients suffer from episodes of Type 1/ reversal reactions (RR) and Type 2/ Erythema Nodosum Leprosum (ENL) reactions which lead to morbidity and nerve damage. CD4+ subsets of Th17 cells and CD25+FOXP3+ regulatory T cells (Tregs) have been shown to play a major role in disease associated immunopathology and in stable leprosy as reported by us and others. The aim of our study was to analyze their role in leprosy reactions.Methodology and Principle FindingsQuantitative reverse transcribed PCR (qPCR), flowcytometry and ELISA were used to respectively investigate gene expression, cell phenotypes and supernatant levels of cytokines in antigen stimulated PBMC cultures in patients with stable disease and those undergoing leprosy reactions. Both types of reactions are associated with significant increase of Th17 cells and associated cytokines IL-17A, IL-17F, IL-21, IL-23 and chemokines CCL20, CCL22 as compared to matching stable forms of leprosy. Concurrently patients in reactions show reduction in FOXP3+ Treg cells as well as reduction in TGF-β and increase in IL-6. Moreover, expression of many T cell markers, cytokines, chemokines and signaling factors were observed to be increased in RR as compared to ENL reaction patients.ConclusionsPatients with leprosy reactions show an imbalance in Th17 and Treg populations. The reduction in Treg suppressor activity is associated withhigherTh17cell activity. The combined effect of reduced TGF-β and enhanced IL-6, IL-21 cytokines influence the balance between Th17 or Treg cells in leprosy reactions as reported in the murine models and autoimmune diseases. The increase in Th17 cell associated cytokines may contribute to lesional inflammation.

Tumour Necrosis Factor-alpha (TNF-α) and its soluble receptor type1 (sTNFR I) in human active and healed leishmaniases.

The role of tumour necrosis factor-alpha (TNF-α) is not fully understood in human leishmaniasis. We analysed the alterations in the levels of TNF-α, soluble TNF receptor type 1 (sTNFR I), IL-17 and IL-22 productions in active and healed leishmaniases. Blood samples were collected from volunteers with active cutaneous leishmaniasis (ACL), the same subjects after lesion healing (healed CL=HCL), volunteers with active visceral leishmaniasis (AVL), healed VL (HVL) and healthy controls. Levels of cytokines were titrated on Leishmania Ag-stimulated PBMC culture. The mean level of TNF-α production from stimulated cells was significantly higher in ACL than controls (P<0·001) and significantly reduced after treatment in HCL volunteers (P<0·05). The mean level of sTNFR I production was significantly higher in ACL than controls (P<0·001) and significantly reduced after treatment in HCL volunteers (P<0·05). The mean level of IL-22 production in AVL was significantly higher than controls (P<0·05) and was significantly lower in HVL compared with AVL (P<0·001) and controls (P<0·05). The levels of TNF-α (P=0·0025) and sTNFR I (P<0·01) productions from PBMCs showed significant decreasing trend after treatment in each CL volunteer. Reduction in TNF-α is associated with clinical response to treatment and healing of CL lesions due to L.major.