Peripheral Blood Mononuclear Cell Production Research Articles

TiO2 nanoparticles and bulk material stimulate human peripheral blood mononuclear cells

Nanomaterials are increasingly produced and used throughout recent years. Consequently the probability of exposure to nanoparticles has risen. Because of their small 1–100nm size, the physicochemical properties of nanomaterials may differ from standard bulk materials and may pose a threat to human health. Only little is known about the effects of nanoparticles on the human immune system. In this study, we investigated the effects of TiO2 nanoparticles and bulk material in the in vitro model of human peripheral blood mononuclear cells (PBMC) and cytokine-induced neopterin formation and tryptophan breakdown was monitored. Both biochemical processes are closely related to the course of diseases like infections, atherogenesis and neurodegeneration. OCTi60 (25nm diameter) TiO2 nanoparticles and bulk material increased neopterin production in unstimulated PBMC and stimulated cells significantly, the effects were stronger for OCTi60 compared to bulk material, while P25 TiO2 (25nm diameter) nanoparticles had only little influence. No effect of TiO2 nanoparticles on tryptophan breakdown was detected in unstimulated cells, whereas in stimulated cells, IDO activity and IFN-γ production were suppressed but only at the highest concentrations tested. Because neopterin was stimulated and tryptophan breakdown was suppressed in parallel, data suggests that the total effect of particles would be strongly pro-inflammatory.

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Introduction: Recent preclinical data in murine models, peripheral blood mononuclear cells (PBMCs), and neutrophils isolated from healthy human subjects demonstrate that triterpenoid analogs such as methyl 2-cyano-3,12-dioxoolean-1,9-dien-28-oate (CDDO-Me), are potent Nrf2 activators. However, the possibility of activating the Nrf2 pathway in critically ill patients to restore redox balance has not been studied. Methods: We prospectively enrolled 18 adult patients (≥18 years old) who were hospitalized in participating ICUs and had onset of septic shock within the previous 24–48 h. Blood samples were collected in cell preparation tubes with sodium citrate. PBMCs and monocytes were isolated and treated with vehicle or CDDO-Me (20 nM) for 20 h. Expression of Nrf2-regulated antioxidant genes (NQO1, HO-1, GCLM, GCLC, and FTL) and inflammatory gene IL-6 was measured with quantitative real-time PCR. Cells pretreated with CDDO-Me were stimulated with LPS (100 ng/ml) for an additional 1 h and ROS measured in culture media. A paired, two-tailed t-test was used to compare Nrf2 targets and inflammatory markers between the groups. Results: CDDO-Me treatment increased the expression of NQO1 (p=0.04) and decreased the expression of HO-1 (p=0.03) in PBMCs from patients with septic shock. Purified monocytes exhibited significant increases in the expression of NQO1 (p=0.01) and GCLM (p=0.003) after CDDO-Me treatment. Levels of other Nrf2 target genes (HO-1 and FTL) remained similar to those of vehicle-treated cells. PBMCs showed a trend toward increased IL-6 expression after CDDO-Me treatment, whereas purified monocytes showed a trend toward decreased IL-6. CDDO-Me treatment significantly increased ROS production in PBMCs (p=0.04). Although CDDO-Me pretreatment significantly attenuated ROS production to subsequent LPS exposure (p=0.03), the change was comparable to the change observed in vehicle-treated PBMCs. CDDO-Me pretreatment followed by LPS exposure had no significant effect on ROS levels in purified monocytes, though levels tended to increase. Conclusions: The Nrf2 pathway is differentially responsive to CDDO-Me activation in PBMCs from critically ill patients. Furthermore, CDDO-Me treatment results in increased ROS production. These findings may suggest a suppressed Nrf2 pathway in patients with septic shock.

Gene Expression Profiles of Peripheral Blood Mononuclear Cells Reveal Transcriptional Signatures as Novel Biomarkers of Cardiac Remodeling in Rats With Aldosteronism and Hypertensive Heart Disease

In searching for a noninvasive surrogate tissue having mimicry with the prooxidant/-proinflammatory hypertensive heart disease (HHD) phenotype, we turned to peripheral blood mononuclear cells (PBMC). We tested whether iterations in [Ca2+]i, [Zn2+]i and oxidative stress in cardiomyocytes and PBMC would complement each other eliciting similar shifts in gene expression profiles in these tissues demonstrable during preclinical (wk 1) and pathologic (wk 4) stages of aldosterone/salt treatment (ALDOST). Inappropriate neurohormonal activation contributes to pathologic remodeling of myocardium in HHD associated with aldosteronism. In rats receiving chronic ALDOST, evidence of reparative fibrosis replacing necrotic cardiomyocytes and coronary vasculopathy appears at wk 4 associated with the induction of oxidative stress by mitochondria that overwhelms endogenous, largely Zn2+-based, antioxidant defenses. Biomarker-guided prediction of risk prior to the appearance of cardiac pathology would prove invaluable. In PBMC and cardiomyocytes, quantitation of cytoplasmic free Ca2+ and Zn2+, H2O2 and 8-iosprostane levels, as well as isolation of RNA and gene expression, together with statistical and clustering analyses, and confirmation of genes by in situ hybridization and RT-PCR, were performed. Compared to controls, at wk 1 and 4 ALDOST, we found comparable: increments in [Ca2+]i, [Zn2+]i and 8-isoprotane coupled to increased H2O2 production in cardiac mitochondria and PBMC, together with the common networks of expression profiles dominated by genes involved in oxidative stress, inflammation and repair. These included three central Ingenuity pathway-linked genes: p38MAPK, a stress-responsive protein; NFκB, a redox-sensitive transcription factor and a proinflammatory cascade it regulates; and TGF-β1, a fibrogenic cytokine involved in tissue repair. Significant overlapping demonstrated in the molecular mimicry of PBMC and cardiomyocytes during preclinical and pathologic stages of ALDOST implicates that transcriptional signatures of PBMC may serve as early noninvasive and novel sentinels predictive of impending pathologic remodeling in HHD.

Inosine-Mediated Modulation of RNA Sensing by Toll-Like Receptor 7 (TLR7) and TLR8

RNA-specific adenosine deaminase (ADAR)-mediated adenosine-to-inosine (A-to-I) editing is a critical arm of the antiviral response. However, mechanistic insights into how A-to-I RNA editing affects viral infection are lacking. We posited that inosine incorporation into RNA facilitates sensing of nonself RNA by innate immune sensors and accordingly investigated the impact of inosine-modified RNA on Toll-like receptor 7 and 8 (TLR7/8) sensing. Inosine incorporation into synthetic single-stranded RNA (ssRNA) potentiated tumor necrosis factor alpha (TNF-α) or alpha interferon (IFN-α) production in human peripheral blood mononuclear cells (PBMCs) in a sequence-dependent manner, indicative of TLR7/8 recruitment. The effect of inosine incorporation on TLR7/8 sensing was restricted to immunostimulatory ssRNAs and was not seen with inosine-containing short double-stranded RNAs or with a deoxy-inosine-modified ssRNA. Inosine-mediated increase of self-secondary structure of an ssRNA resulted in potentiated IFN-α production in human PBMCs through TLR7 recruitment, as established through the use of a TLR7 antagonist and Tlr7-deficient cells. There was a correlation between hyperediting of influenza A viral ssRNA and its ability to stimulate TNF-α, independent of 5'-triphosphate residues, and involving Adar-1. Furthermore, A-to-I editing of viral ssRNA directly enhanced mouse Tlr7 sensing, when present in proportions reproducing biologically relevant levels of RNA editing. Thus, we demonstrate for the first time that inosine incorporation into immunostimulatory ssRNA can potentiate TLR7/8 activation. Our results suggest a novel function of A-to-I RNA editing, which is to facilitate TLR7/8 sensing of phagocytosed viral RNA.

Pro-Inflammatory Action of MIF in Acute Myocardial Infarction via Activation of Peripheral Blood Mononuclear Cells

ObjectivesMacrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, has been implicated in the pathogenesis of multiple inflammatory disorders. We determined changes in circulating MIF levels, explored the cellular source of MIF, and studied the role of MIF in mediating inflammatory responses following acute myocardial infarction (MI).Methods and ResultsWe recruited 15 patients with MI, 10 patients with stable angina and 10 healthy volunteers and measured temporal changes of MIF in plasma. Expression of MIF, matrix metalloproteinase-9 (MMP-9) and interleukin-6 (IL-6) in cultured peripheral blood mononuclear cells (PBMCs) and the media were measured by ELISA or real-time PCR. Compared to controls, plasma levels of MIF and IL-6 were significantly elevated at admission and 72 h post-MI. In contrast, expression of MIF, MMP-9 and IL-6 by PBMCs from MI patients was unchanged at admission, but significantly increased at 72 h. Addition of MIF activated cultured PBMCs by upregulating expression of inflammatory molecules and also synergistically enhanced stimulatory action of IL-1β which were inhibited by anti-MIF interventions. In a mouse MI model we observed similar changes in circulating MIF as seen in patients, with reciprocal significant increases in plasma MIF and reduction of MIF content in the infarct myocardium at 3 h after MI. MIF content in the infarct myocardium was restored at 72 h post-MI and was associated with robust macrophage infiltration. Further, anti-MIF intervention significantly reduced inflammatory cell infiltration and expression of monocyte chemoattractant protein-1 at 24 h and incidence of cardiac rupture in mice post-MI.ConclusionMI leads to a rapid release of MIF from the myocardium into circulation. Subsequently MIF facilitates PBMC production of pro-inflammatory mediators and myocardial inflammatory infiltration. Attenuation of these events, and post-MI cardiac rupture, by anti-MIF interventions suggests that MIF could be a potential therapeutic target following MI.

Suppression of immunoglobulin production in human peripheral blood mononuclear cells by monocytes via secretion of heavy-chain ferritin

In vitro antigen stimulation of peripheral blood mononuclear cells (PBMCs) does not induce immunoglobulin (Ig) production. However, pretreatment of PBMCs with l-leucyl-l-leucine methyl ester (LLME) prior to in vitro stimulation removes the suppression of Ig production. In the present study, we attempted to identify the target cells of LLME and determine the mechanisms by which Ig production in PBMCs is suppressed. We found that CD14+ monocytes are involved in the suppression of Ig production in PBMCs. Furthermore, we confirmed that heavy-chain ferritin derived from CD14+ monocytes suppresses Ig production in PBMCs, possibly through iron sequestration.

Simplified process for the production of anti–CD19-CAR–engineered T cells

Background aimsAdoptive immunotherapy with the use of chimeric antigen receptor (CAR)-engineered T cells specific for CD19 has shown promising results for the treatment of B-cell lymphomas and leukemia. This therapy involves the transduction of autologous T cells with a viral vector and the subsequent cell expansion. We describe a new, simplified method to produce anti-CD19-CAR T cells. MethodsT cells were isolated from peripheral blood mononuclear cell (PBMC) with anti-CD3/anti-CD28 paramagnetic beads. After 2 days, the T cells were added to culture bags pre-treated with RetroNectin and loaded with the retroviral anti-CD19 CAR vector. The cells, beads and vector were incubated for 24 h, and a second transduction was then performed. No spinoculation was used. Cells were then expanded for an additional 9 days. ResultsThe method was validated through the use of two PBMC products from a patient with B-cell chronic lymphoblastic leukemia and one PBMC product from a healthy subject. The two PBMC products from the patient with B-cell chronic lymphoblastic leukemia contained 11.4% and 12.9% T cells. The manufacturing process led to final products highly enriched in T cells with a mean CD3+ cell content of 98%, a mean expansion of 10.6-fold and a mean transduction efficiency of 68%. Similar results were obtained from the PBMCs of the first four patients with acute lymphoblastic leukemia treated at our institution. ConclusionsWe developed a simplified, semi-closed system for the initial selection, activation, transduction and expansion of T cells with the use of anti-CD3/anti-CD28 beads and bags to produce autologous anti-CD19 CAR–transduced T cells to support an ongoing clinical trial.

Regulation of macrophage immune responses by antipsychotic drugs

Antipsychotic drugs (APDs) have been used to ease clinical psychotic symptoms. APDs have also been recently discovered to induce immune regulation. Our previous studies found that atypical APDs risperidone and clozapine could inhibit INF-γ production of human peripheral blood mononuclear cells (PBMC) and could inhibit Th1 differentiation. This study further investigates APD effects on monocyte-derived macrophages, which are the major antigen-presenting cells in PBMC. Our data suggest that adhesion, phagocytosis and reactive oxygen species production of monocytic cell lines would be inhibited by haloperidol, risperidone or clozapine. Also, that APDs inhibited the production of LPS-stimulated macrophages IL-6 and IL-8 suggests that risperidone and clozapine may inhibit inflammation. We further discovered that risperidone and clozapine could inhibit IL-12 production and increase IL-10 production of LPS-stimulated macrophages. These results indicated that risperidone and clozapine could inhibit Th1 differentiation not only by increasing INF-γ production of PBMC but by inhibiting the release of Th1-inducing cytokines and increasing Th2-inducing cytokines of LPS-stimulated macrophages to modulate and regulate immune responses.

Immune Modulation by Different Types of β2→1-Fructans Is Toll-Like Receptor Dependent

Introductionβ2→1-fructans are dietary fibers. Main objectives of this study were 1) to demonstrate direct signalling of β2→1-fructans on immune cells, 2) to study whether this is mediated by the pattern recognition receptors Toll-like receptors (TLRs) and nucleotide-binding oligomerisation domain-containing proteins (NODs), and 3) to relate the observed effects to the chain length differences in β2→1-fructans.MethodsFour different β2→1-fructan formulations were characterised for their chain length profile. Human peripheral blood mononuclear cells (PBMCs) were stimulated in vitro with β2→1-fructans, and production of IL-1Ra, IL-1β, IL-6, IL-10, IL-12p70, and TNF-α was analysed. Reporter cells for TLRs and NODs were incubated with β2→1-fructans and analysed for NF-κB/AP-1 activation.ResultsCytokine production in human PBMCs was dose- and chain length-dependent. Strikingly, short chain enriched β2→1-fructans induced a regulatory cytokine balance compared to long chain enriched β2→1-fructans as measured by IL-10/IL-12 ratios. Activation of reporter cells showed that signalling was highly dependent on TLRs and their adapter, myeloid differentiation primary response protein 88 (MyD88). In human embryonic kidney reporter cells, TLR2 was prominently activated, while TLR4, 5, 7, 8, and NOD2 were mildly activated.Conclusionsβ2→1-fructans possess direct signalling capacity on human immune cells. By activating primarily TLR2, and to a lesser extent TLR4, 5, 7, 8, and NOD2, β2→1-fructan stimulation results in NF-κB/AP-1 activation. Chain length of β2→1-fructans is important for the induced activation pattern and IL-10/IL-12 ratios.

Perifosine inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production via regulation multiple signaling pathways: New implication for Kawasaki disease (KD) treatment

Kawasaki disease (KD) is a multisystem vasculitis of unknown etiology, with coronary artery aneurysms occurring in majority of untreated cases. Tumor necrosis factor (TNF)-α is the pleiotropic inflammatory cytokine elevated during the acute phase of KD, which induces damage to vascular endothelial cells to cause systemic vasculitis. We here investigated the potential role of perifosine, a novel Akt inhibitor, on TNFα expression in LPS-stimulated macrophages and in ex-vivo cultured peripheral blood mononuclear cells (PBMCs) of acute KD patients. Here, we found that perifosine inhibited LPS-induced TNFα expression and production in mouse macrophages (RAW 264.7 cells and bone marrow-derived macrophages (BMDMs)). Meanwhile, perifosine administration down-regulated TNFα production in PBMCs isolated from acute KD patients. For the mechanism study, we found that perifosine significantly inhibited Akt and ERK/mitogen-activated protein kinases (MAPK) signaling, while activating AMP-activated protein kinase (AMPK) signaling in both patients’ PBMCs and LPS-stimulated macrophages. Interestingly, although perifosine is generally known as an Akt inhibitor, our data suggested that ERK inhibition and AMPK activation, but not Akt inactivation were possibly involved in perifosine-mediated inhibition against TNFα production in monocytes. In conclusion, our data suggested that perifosine significantly inhibited TNFα production via regulation multiple signaling pathways. The results of this study should have significant translational relevance in managing this devastating disease.

Pepsin Digest of Wheat Gliadin Fraction Increases Production of IL-1β via TLR4/MyD88/TRIF/MAPK/NF-κB Signaling Pathway and an NLRP3 Inflammasome Activation

Celiac disease (CD) is a gluten-responsive, chronic inflammatory enteropathy. IL-1 cytokine family members IL-1β and IL-18 have been associated with the inflammatory conditions in CD patients. However, the mechanisms of IL-1 molecule activation in CD have not yet been elucidated. We show in this study that peripheral blood mononuclear cells (PBMC) and monocytes from celiac patients responded to pepsin digest of wheat gliadin fraction (PDWGF) by a robust secretion of IL-1β and IL-1α and a slightly elevated production of IL-18. The analysis of the upstream mechanisms underlying PDWGF-induced IL-1β production in celiac PBMC show that PDWGF-induced de novo pro-IL-1β synthesis, followed by a caspase-1 dependent processing and the secretion of mature IL-1β. This was promoted by K+ efflux and oxidative stress, and was independent of P2X7 receptor signaling. The PDWGF-induced IL-1β release was dependent on Nod-like receptor family containing pyrin domain 3 (NLRP3) and apoptosis-associated speck like protein (ASC) as shown by stimulation of bone marrow derived dendritic cells (BMDC) from NLRP3−/− and ASC−/− knockout mice. Moreover, treatment of human PBMC as well as MyD88−/− and Toll-interleukin-1 receptor domain-containing adaptor-inducing interferon-β (TRIF)−/− BMDC illustrated that prior to the activation of caspase-1, the PDWGF-triggered signal constitutes the activation of the MyD88/TRIF/MAPK/NF-κB pathway. Moreover, our results indicate that the combined action of TLR2 and TLR4 may be required for optimal induction of IL-1β in response to PDWGF. Thus, innate immune pathways, such as TLR2/4/MyD88/TRIF/MAPK/NF-κB and an NLRP3 inflammasome activation are involved in wheat proteins signaling and may play an important role in the pathogenesis of CD.

TLR4, IL10RA, and NOD2 mutation in paediatric Crohn’s disease patients: an association with Mycobacterium avium subspecies paratuberculosis and TLR4 and IL10RA expression

Mycobacterium avium subspecies paratuberculosis (MAP) has been implicated in the pathogenesis of Crohn's disease (CD). The role of CD susceptibility genes in association with these microbes is not known. Sixty-two early onset paediatric CD patients and 46 controls with known MAP status were analysed for an association with 34 single nucleotide polymorphisms (SNPs) from 18 CD susceptibility genes. Functional studies on peripheral blood mononuclear cells (PBMCs) were conducted on 17 CD patients with known CD mutations to assess IL-6, IL-10, and TNF-α expression upon stimulation with MAP precipitated protein derivative (PPD) and lipopolysaccharide (LPS). In addition, surface expression of IL10R and TLR4 on resting B cells, NK cells, T cells, and monocytes was assessed. A mutation in TLR4 (rs4986790) and IL10RA (rs22291130) was significantly associated with MAP-positive CD patients compared to MAP-negative CD patients (27.6 vs. 6.1%, p=0.021, and 62.1 vs. 33.3%, p=0.024, respectively). PPD and LPS significantly increased IL-6, IL-10, and TNF-α production in PBMCs. IL-10 and TNF-α production were significantly lower in a subgroup of CD patients (5/12) with a known NOD2 mutation. Receptor for IL-10 was significantly higher expressed on NK cells (CD56low) and on NK T cells harbouring a NOD2 mutations compared to wildtype cells (p=0.031 and 0.005, respectively). TLR4 was significantly higher expressed on NK cells (CD56high) harbouring a NOD2 mutations compared to wildtype cells (p=0.038).

Bisphenol A suppresses interferon-gamma production and related biochemical pathways in human PBMC

Event Abstract Back to Event Bisphenol A suppresses interferon-gamma production and related biochemical pathways in human PBMC Emanuel Raggl1, Simon Geisler1, Johanna M. Gostner1, Dietmar Fuchs1* and Harald Schennach1 1 Innsbruck Medical University, Austria Bisphenol A (BPA) is one of the most widely distributed chemicals. Humans are exposed to it mainly via polycarbonate plastics that are used for food packaging and plastic bottles and contain BPA as a softener. There is accumulating evidence of a carcinogenic and allergy-promoting effect of BPA. In vitro we investigated the impact of BPA on freshly isolated human peripheral blood mononuclear cells (PBMC) from healthy donors. PBMC were incubated with increasing doses of BPA, and after 30 min either left unstimulated or stimulated with the mitogen phytohemagglutinin (PHA) and cell viability was controlled. After 48 h, formation of interferon-gamma (IFN-gamma), macrophage product neopterin as well as tryptophan breakdown by indoleamine 2,3-dioxygenase (IDO), expressed as the kynurenine to tryptophan ratio (Kyn/Trp), were monitored. All these immunobiochemical pathways are characteristic for cell-mediated (= Th1-type) immune response. Upon PHA stimulation, neopterin production and tryptophan breakdown increased significantly compared to unstimulated cells. BPA exerted a dose-dependent suppressive effect in unstimulated and in PHA-stimulated PBMC at 12.5 - 200 µM concentrations. Due to the cross-regulation of Th1- and Th2-type immunity, the suppressive effects of BPA on Th1-type immunity could relate to the allergy-promoting properties of the compound. Moreover, the inhibition of IFN-gamma production and its related antiproliferative effects could be of relevance for the tumor-promoting effect of this chemical. On the other side, the antioxidant compound could also have beneficial effects, possibly delaying or even preventing Th1-type immunopathologies. References Zaknun D, Schroecksnadel S, Kurz K, Fuchs D. Potential role of antioxidant food supplements, preservatives and colorants in the pathogenesis of allergy and asthma. Int Arch Allergy Immunol 2012;157:113-124 Gostner JM, Becker K, Fuchs D, Sucher R. Redox regulation of the immune response. Redox Report 2013;18: 88-94 Gostner J, Ciardi C, Becker K, Fuchs D, Sucher R. Immunoregulatory impact of food antioxidants. Curr Pharm Des (in press) Keywords: bisphenol A, Interferon-gamma, Neopterin, indoleaminedioxygenase, PBMC Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Raggl E, Geisler S, Gostner JM, Fuchs D and Schennach H (2013). Bisphenol A suppresses interferon-gamma production and related biochemical pathways in human PBMC. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00665 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Dietmar Fuchs, Innsbruck Medical University, Innsbruck, Austria, dietmar.Fuchs@i-med.ac.at Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Emanuel Raggl Simon Geisler Johanna M Gostner Dietmar Fuchs Harald Schennach Google Emanuel Raggl Simon Geisler Johanna M Gostner Dietmar Fuchs Harald Schennach Google Scholar Emanuel Raggl Simon Geisler Johanna M Gostner Dietmar Fuchs Harald Schennach PubMed Emanuel Raggl Simon Geisler Johanna M Gostner Dietmar Fuchs Harald Schennach Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Effect of Recombinant α1-Antitrypsin Fc-Fused (AAT-Fc) Protein on the Inhibition of Inflammatory Cytokine Production and Streptozotocin-Induced Diabetes

α1-Antitrypsin (AAT) is a member of the serine proteinase inhibitor family that impedes the enzymatic activity of serine proteinases, including human neutrophil elastase, cathepsin G and neutrophil proteinase 3. Here, we expressed recombinant AAT by fusing the intact AAT gene to the constant region of IgG1 to generate soluble recombinant AAT-Fc protein. The recombinant AAT-Fc protein was produced in Chinese hamster ovary (CHO) cells and purified using mini-protein A affinity chromatography. Recombinant AAT-Fc protein was tested for antiinflammatory function and AAT-Fc sufficiently suppressed tumor necrosis factor (TNF)-α-induced interleukin (IL)-6 in human peripheral blood mononuclear cells (PBMCs) and inhibited cytokine-induced TNFα by different cytokines in mouse macrophage Raw 264.7 cells. However, AAT-Fc failed to suppress lipopolysaccharide-induced cytokine production in both PBMCs and macrophages. In addition, our data showed that AAT-Fc blocks the development of hyperglycemia in a streptozotocin-induced mouse model of diabetes. Interestingly, we also found that plasma-derived AAT specifically inhibited the enzymatic activity of elastase but that AAT-Fc had no inhibitory effect on elastase activity.

Cytokine response of human mononuclear cells induced by intestinal Clostridium species

Altered composition of intestinal microbiota has been associated with various immunological disorders such as inflammatory bowel disease. Although Clostridium species are major inhabitants of the intestinal tract, their interaction with the host immunological system is yet poorly characterized. In this study, cytokine responses of human monocytic cell line THP-1 and peripheral blood mononuclear cells (PBMC) to six type strains representing common intestinal clostridial species were determined. The strains induced diverse cytokine responses in both THP-1 cells and PBMC. Clostridium perfringens was the most potent inducer of both tumour necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10), as compared to Clostridium histolyticum, Clostridium clostridioforme, Clostridium leptum, Clostridium sporosphaeroides and Blautia coccoides. Interleukin-8 (IL-8) production in PBMC was most efficiently stimulated by C. sporosphaeroides. The same PBMC preparations that responded strongly to Escherichia coli lipopolysaccharide (LPS) also responded strongly to bacterial stimulation. This indicates that the level of responsiveness is an individual feature of mononuclear cell preparations, and that the overall cytokine response is composed by a combination of host factors and microbial structures affecting them. This work supports the idea that the composition of the intestinal clostridial population influences immune responses and is likely to play an important role in intestinal homeostasis.

Short communication: Effect of heat stress during the dry period on gene expression in mammary tissue and peripheral blood mononuclear cells

Heat stress (HT) during the dry period compromises mammary gland development, decreases future milk production, and impairs the immune status of dairy cows. Our objective was to evaluate the effect of cooling HT cows during the dry period on gene expression of the mammary gland and peripheral blood mononuclear cells (PBMC). Cows were dried off 46d before their expected calving and assigned to 2 treatments, HT or cooling (CL). Cows in the CL group were cooled with sprinklers and fans whereas HT cows were not. After parturition, all cows were housed in a freestall barn with cooling. The PBMC were isolated at dry-off and at −20, 2, and 20d relative to calving from a subset of cows (HT, n=9; CL, n=10), and mammary biopsies were taken at the same intervals (HT, n=7; CL, n=6) for RNA extraction. Gene expression was assessed using a custom multiplex gene expression assay based on traditional reverse transcription-PCR. Genes involved in prolactin (PRL) signaling [PRL receptor long form, PRL receptor short form, suppressor of cytokine signaling (SOCS)2, SOCS3, IGF2, IGF binding protein 5, and cyclin D1], fatty acid metabolism (acetyl-CoA carboxylase α (ACACA) and lipoprotein lipase (LPL)], and IGF1 were evaluated in mammary tissue, and genes related to fatty acid metabolism [ACACA, fatty acid synthase (FASN), and LPL], cytokine production [IL6, IL8, and tumor necrosis factor (TNF)], and IGF1 were evaluated in PBMC. No differences were observed in PRL signaling or fatty acid metabolism gene expression in the mammary gland. In PBMC, HT cows had greater mRNA expression of IGF1 and TNF during the transition period relative to CL and upregulated IL8 and downregulated FASN mRNA expression at 2d relative to calving. We conclude that cooling HT cows during the dry period alters expression of genes involved in cytokine production and lipid metabolism in PBMC.

P006 Cytokine and chemokine responses as predictive markers for progressive, stable or cured Echinococcus multilocularis infection in man

Introduction In man the tissue infiltrative growth of Echinococcus multilocularis metacestodes will cause alveolar echinococcosis (AE) with organ malfunction and finally organ failure. In some AE patients, however, metacestode proliferation may regress, but little is known about the mechanism and determinants which may control or even restrain parasite survival and its growth. Methods In this study, chemokine and cytokine responses were investigated in E. multilocularis patients at different states of infection, i.e. with progressive, stable and cured alveolar echinococcosis. We analyzed the serum levels and the ex vivo cytokine and chemokine responsiveness of peripheral blood mononuclear cells (PBMC) from AE patients, reflecting their innate and memory immune responses to E. multilocularis antigens. First, plasma concentrations of pro-inflammatory cytokines and chemokine were investigated, and then the capacity of PBMC from AE patients and infection-free controls was studied in order to distinguish between progressive, stable and cured E. multilocularis infection. Results The plasma levels of the pro-inflammatory cytokines IL-17B and its soluble receptor (sIL-17RB), and MIP-3 α were highly elevated in AE patients as compared to controls while highest concentrations of IL-17F and its common soluble receptor (sIL-17RA), regulatory IL 27, pro-inflammatory IL 31 and IL 33 were detected in controls. Spontaneous release of several cytokines and chemokine was clearly different between AE patients in different stages of infection and controls. Furthermore, a distinct cellular responsiveness was observed in AE patients when their PBMC were activated with antigens from helminthes, bacteria or mitogens. Pro-inflammatory cytokine production of IL17F and IL 17RA, as well as regulatory IL27, was reduced above baseline level in AE patients when their PBMC were co-culture with Em Antigens. Chemokine production of PBMC in AE patients was not inducible by E.multilocularis antigens for pro-inflammatory monocyte inflammatory proteins (MIP-1 α /CCL3//MIP-1 β /CCL4//MIP-3 α /CCL20). Conclusion These observations disclose a distant dynamic of the cytokine and chemokine profile at different stages of AE. Furthermore, cestode and nematode antigens will differ in their capacity to stimulate cellular cytokine and chemokine production, notably for the activation of pro-inflammatory immune mediators. In summary, the analyses of constitutive and inducible chemokine and cytokine profiles may provide a better understanding of their importance during progression, regression and also for the staging of alveolar echinococcosis.

Effect of ethanolic extract of seeds of Linum usitatissimum (Linn.) in hyperglycaemia associated ROS production in PBMNCs and pancreatic tissue of alloxan induced diabetic rats

ObjectiveTo evaluate the effect of ethanolic extract of seeds of Linum usitatissimum (EELU) in hyperglycemia associated reactive oxygen species (ROS) production in peripheral blood mononuclear cells (PBMNCs) and pancreatic antioxidant enzymes in alloxan induced diabetic rat. MethodsDiabetes was induced in male Wistar rats by alloxan (120 mg/kg, i.p.). After acute and subacute treatment serum glucose was determined. Oral glucose tolerance test (OGTT) was performed in EELU pretreated animals. ROS production in PBMNCs and pancreatic antioxidant enzymes were measured in alloxan induced diabetic rat. ResultsOur results showed that, treatment of EELU (200 and 400 mg/kg) significantly reduced serum glucose level in acute and subacute study. The antihyperglycaemic effects of EELU showed onset at 4th h (P<0.001) and peak effect at 6th h (P<0.001). The effect was sustained until 24th h with 400 mg/kg. In subacute study, significant antihyperglycaemic effect was observed from 14th day (P<0.001) onwards. In EELU treated rat the body weight was significantly (P<0.001) increased as compared to diabetic group on 21st day onwards. In OGTT, increased glucose utilization was observed. Treatment of EELU 400 mg/kg showed significant reversal in pancreatic GSH (P<0.01) and SOD (P<0.05) indicating antioxidant nature of EELU. Flow cytometric estimation of total ROS production in PBMNCs in diabetic rats was significantly increased (P<0.001), whereas EELU treatment showed significant (P<0.001) decrease in PBMNCs ROS. ConclusionsIt is concluded from the investigation that EELU showed antihyperglycaemic effect mediated through inhibition of ROS level in PBMNCs and preservation of endogenous antioxidant enzymes in pancreatic tissue in alloxan induced diabetic rat.

Restoration of corticosteroid sensitivity by p38 mitogen activated protein kinase inhibition in peripheral blood mononuclear cells from severe asthma.

BackgroundSevere asthma accounts for a small number of asthmatics but represents a disproportionate cost to health care systems. The underlying mechanism in severe asthma remains unknown but several mechanisms are likely to be involved because of a very heterogeneous profile. We investigated the effects of a p38MAPK inhibitor in corticosteroid sensitivity in peripheral blood mononuclear cells (PBMCs) from severe asthmatics and the profile of its responders.Methodology/Principal FindingsCorticosteroid sensitivity was determined by measuring dexamethasone inhibition of CD3/28 and TNF-α induced IL-8 production in PBMCs by using ELISA. PBMCs from severe asthmatics were relatively less sensitive to dexamethasone (Dex) as compared to those of non-severe asthmatics and healthy volunteers. The IC50 values of Dex negatively correlated with decreased glucocorticoid receptor (GR) nuclear translocation assessed using immunocytochemistry (r = −0.65; p<0.0005) and with decreased FEV1 (% predicted) (r = 0.6; p<0.0005). A p38α/β inhibitor (SB203580) restored Dex-sensitivity in a subpopulation of severe asthma that was characterized by a defective GR nuclear translocation, clinically by lower FEV1 and higher use of oral prednisolone. We also found that SB203580 partially inhibited GR phosphorylation at serine 226, resulting in increased GR nuclear translocation in IL-2/IL-4 treated corticosteroid insensitive U937s.Conclusions/Significancep38MAPKα/β is involved in defective GR nuclear translocation due to phosphorylation at Ser226 and this will be a useful biomarker to identify responders to p38MAPKα/β inhibitor in the future.

Anti-angiogenesis and immunomodulatory activities of an anti-tumor sesquiterpene bigelovin

Bigelovin is a sesquiterpene lactone isolated from the plant Inula helianthus-aquatica which was traditionally used in cancer treatment in Yunnan, China. We previously demonstrated the potent apoptotic activities of bigelovin. The objective of this study was to investigate the anti-angiogenic and immunomodulatory effects of bigelovin using transgenic zebrafish Tg(fli1a:EGFP)y1 with fluorescent blood vessels and human peripheral blood mononuclear cells (PBMCs), respectively. The growth of subintestinal vessels and gene expression in angiogenesis signaling pathways of bigelovin-treated zebrafish embryos were examined. The proliferation and cytokines production of bigelovin-treated PBMCs were also evaluated. Our results showed that bigelovin could significantly inhibit the growth of subintestinal vessels and down-regulate the expression of Ang2 and Tie2 of zebrafish embryos. Besides, bigelovin could significantly suppress the proliferation and production of Th1 cytokines (IFNγ, IL-2 and IL-12) in human PBMCs. This is the first report of the anti-angiogenic and immunomodulatory activities of bigelovin in zebrafish and human lymphocytes. Our findings suggest that bigelovin may exert multi-target function against tumor in animal model.