Spleen Cell Cultures Research Articles

Recombinant Toxoplasma gondii phosphoglycerate mutase 2 confers protective immunity against toxoplasmosis in BALB/c mice.

Toxoplasmosis is one of the most widespread zoonoses worldwide. It has a high incidence and can result in severe disease in humans and livestock. Effective vaccines are needed to limit and prevent infection with Toxoplasma gondii. In this study, we evaluated the immuno-protective efficacy of a recombinant Toxoplasma gondii phosphoglycerate mutase 2 (rTgPGAM 2) against T. gondii infection in BALB/c mice. We report that the mice nasally immunised with rTgPGAM 2 displayed significantly higher levels of special IgG antibodies against rTgPGAM 2 (including IgG1, IgG2a and IgAs) and cytokines (including IFN-γ, IL-2 and IL-4) in their blood sera and supernatant of cultured spleen cells compared to those of control animals. In addition, an increased number of spleen lymphocytes and enhanced lymphocyte proliferative responses were observed in the rTgPGAM 2-immunised mice. After chronic infection and lethal challenge with the highly virulent T. gondii RH strain by oral gavage, the survival time of the rTgPGAM 2-immunised mice was longer (P < 0.01) and the survival rate (70%) was higher compared with the control mice (P < 0.01). The reduction rate of brain and liver tachyzoites in rTgPGAM 2-vaccinated mice reached approximately 57% and 69% compared with those of the control mice (P < 0.01). These results suggest that rTgPGAM 2 can generate protective immunity against T. gondii infection in BALB/c mice and may be a promising antigen in the further development of an effective vaccine against T. gondii infection.

γδT cells promoted the Th17 immune responses in mice with Chlamydia muridarum lung infection

Objective To investigate the functions of γδT cells in a mouse model of Chlamydia muridarum (Cm) lung infection and their effects on Chlamydia-specific immunity. Methods Wild type (WT) and TCRδ-/- mice were infected with 1×103 inclusion forming units (IFU) of Cm by nasal inhalation to establish the murine model of Chlamydia respiratory infection. The bodyweight of each mouse was monitored daily. The growth of Chlamydia strains in lung tissues was measured by using enzyme immunoassay (EIA) and represented by IFU. ELISA was used to measure the levels of IFN-γ and IL-17 in the supernatants of spleen cell culture. The levels of IFN-γ and IL-17 secreted by CD3+ CD4+ T cells were determined by using the intracellular cytokine staining assay for evaluating the roles and mechanisms of γδT cells in regulating the Th1 and Th17 immune responses to Chlamydia strains. Results The murine model of Chlamydia pneumonitis was established by nasal inhalation of Cm strains. Compared with the WT mice, the TCR-/- mice showed a significant decrease in bodyweight after Cm infection. No significant difference in the growth of Chlamydia strains in lung tissues was observed between mice from the two groups. Results of the ELISA showed that the levels of IFN-γ in the supernatants of spleen cell culture from WT mice group were similar to those form TCRδ-/- mice groups on days 3 and 7 after Cm infection, but less IL-17 was secreted in TCRδ-/- mice than in WT mice on day 7. The intracellular cytokine staining analysis further indicated that the secretion of IL-17 by CD3+ CD4+ T cells in TCRδ-/- mice was significantly decreased as compared with that in WT mice on days 3 and 7 after Cm infection. Conclusion γδT cells protect mice from Chlamydia respiratory infection by promoting the Chlamydia-specific Th17 immune responses. Key words: Chlamydia muridarum; γδT cell; Th17 immune response

Release from Th1-type immune tolerance in spleen and enhanced production of IL-5 in Peyer’s patch by cholera toxin B induce the glomerular deposition of IgA

We examined the pathogenesis of glomerular damage in Th2 type-dependent GATA-3 transgenic (GATA-3 Tg) mice with IgA nephropathy (IgAN). GATA-3 Tg mice were immunized orally using OVA plus cholera toxin B (CTB), and measurement of the serum IgA antibody level and histopathological examination were performed. Marked increases in the serum levels of OVA-specific IgA antibody, IgA and IgG, C3 deposits analogous to those seen in IgAN, and expansion of the matrix in association with mesangial cell proliferation were observed. Furthermore, glomerular IgA deposits were co-localized with mannan-binding lectin (MBL) deposits, which might actually have been abnormal IgA deposits. In GATA-3/TCR-Tg mice that had been orally sensitized with CTB plus OVA and were re-stimulated with OVA in vitro, cultured Peyer’s patch cells showed the enhanced production of IL-5 and supernatants from cultures of spleen cells showed a reduction of TGF-β production with a simultaneous increase in IL-2 production and the recovery of IFN-γ formation. The amount of TGF-β produced by the spleen cells was found to be correlated with the amount of IFN-γ and IL-IL-2 produced by the cells. Also, the percentage of regulatory T cells (Treg) in the spleens of mice sensitized with OVA plus CTB was lower than that in mice orally sensitized with OVA alone. These results suggest that the increased production of IL-5 from Peyer’s patch cells (PPc) and the restored Th1-type immune response might cause the production of abnormal IgA and might induce the deposition of IgA in glomeruli.

Antibody Production, Anaphylactic Signs, and T-Cell Responses Induced by Oral Sensitization With Ovalbumin in BALB/c and C3H/HeOuJ Mice

PurposeTwo mouse strains, BALB/c and C3H/HeOuJ, broadly used in the field of food allergy, were compared for the evaluation of the allergenic potential of ovalbumin (OVA).MethodsSensitization was made by administering 2 different OVA doses (1 and 5 mg), with cholera toxin as Th2-polarizing adjuvant. Antibody levels, severity of anaphylaxis, and Th1 and Th2 responses induced by the allergen were assessed. In addition, because the mice selected had functional toll-like receptor 4, the influence of contamination with lipopolysaccharide (LPS) on the immunostimulating capacity of OVA on spleen cells was also evaluated.ResultsBoth strains exhibited similar susceptibility to OVA sensitization. The 2 protein doses generated similar OVA-specific IgE and IgG1 levels in both strains, whereas C3H/HeOuJ mice produced significantly more IgG2a. Oral challenge provoked more severe manifestations in C3H/HeOuJ mice as indicated by the drop in body temperature and the severity of the anaphylactic scores. Stimulation of splenocytes with OVA led to significantly higher levels of Th2 and Th1 cytokines in BALB/c, and these were less affected by protein contamination with LPS.ConclusionsThe antibody and cytokine levels induced by OVA in BALB/c mice and the observation that BALB/c spleen cell cultures were more resistant than those of C3H/HeOuJ mice to the stimulus of LPS make this strain prone to exhibit Th2-mediated food allergic reactions and very adequate for the study of the features of OVA that make it allergenic.

Evaluation of Trichodysplasia Spinulosa-Associated Polyomavirus Capsid Protein as a New Carrier for Construction of Chimeric Virus-Like Particles Harboring Foreign Epitopes.

Recombinant virus-like particles (VLPs) represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV) viral protein 1 (VP1) was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes.

Bifidobacterium longum CCM 7952 Promotes Epithelial Barrier Function and Prevents Acute DSS-Induced Colitis in Strictly Strain-Specific Manner.

BackgroundReduced microbial diversity has been associated with inflammatory bowel disease (IBD) and probiotic bacteria have been proposed for its prevention and/or treatment. Nevertheless, comparative studies of strains of the same subspecies for specific health benefits are scarce. Here we compared two Bifidobacterium longum ssp. longum strains for their capacity to prevent experimental colitis.MethodsImmunomodulatory properties of nine probiotic bifidobacteria were assessed by stimulation of murine splenocytes. The immune responses to B. longum ssp. longum CCM 7952 (Bl 7952) and CCDM 372 (Bl 372) were further characterized by stimulation of bone marrow-derived dendritic cell, HEK293/TLR2 or HEK293/NOD2 cells. A mouse model of dextran sulphate sodium (DSS)-induced colitis was used to compare their beneficial effects in vivo.ResultsThe nine bifidobacteria exhibited strain-specific abilities to induce cytokine production. Bl 372 induced higher levels of both pro- and anti-inflammatory cytokines in spleen and dendritic cell cultures compared to Bl 7952. Both strains engaged TLR2 and contain ligands for NOD2. In a mouse model of DSS-induced colitis, Bl 7952, but not Bl 372, reduced clinical symptoms and preserved expression of tight junction proteins. Importantly, Bl 7952 improved intestinal barrier function as demonstrated by reduced FITC-dextran levels in serum.ConclusionsWe have shown that Bl 7952, but not Bl 372, protected mice from the development of experimental colitis. Our data suggest that although some immunomodulatory properties might be widespread among the genus Bifidobacterium, others may be rare and characteristic only for a specific strain. Therefore, careful selection might be crucial in providing beneficial outcome in clinical trials with probiotics in IBD.

Evaluation of anti-asthmatic and antioxidant potential of Boerhavia procumbens in toluene diisocyanate (TDI) treated rats

Aim of the studyAsthma is an ailment of airways characterized by activation of the T helper (Th) 2 lymphocytes and subsequent movement of inflammatory cells. Boerhavia procumbens of family Nyctaginaceae is locally used for the treatment of asthma, cough, hemorrhoids, dropsy, cardiac, eyes and kidney problems. We have evaluated its methanol extract (BPM) as a therapeutic candidate for asthma against toluene diisocyanate (TDI) allergic model in rat.The BPM extract was obtained from the whole plant of B. procumbens in methanol. Sprague-Dawley male 36 rats (200–250g) were categorized into 6 groups having six rats in each category. The animals were provoked (10%) and sensitized (5%) by TDI. Animals of groups I–III were vehicle control (ethyl acetate), diseased control (TDI) and reference control (TDI+dexamethasone {2.5mg/kg bw}), respectively. Animals of group IV (TDI+200mg/kg bw) and group V (TDI+400mg/kg bw) were administered with BPM whereas group VI was administered with 400mg/kg bw alone of BPM. Protective effects of BPM were determined by counting the number of leucocytes and estimation of interleukines in blood, bronchoalveolar lavage (BAL) and in in vitro culture of spleen cells. Estimation of antioxidant enzymes, lipid peroxides and H2O2 and histopathology of lungs were carried out for antioxidant potential of plant extract used. ResultsMethanol extract of B. procumbens suppressed the asthmatic symptoms and inhibited the infiltration of eosinophils and lymphocytes in lungs of TDI provoked rats. Administration of BPM to TDI provoked rats, dose dependently, inhibited the release of interleukins (IL)-2 in serum and IL-4, IL-6 interferon gamma (IFN-γ) in bronchoalveolar lavage (BAL) and in in vitro culture of spleen cells, and ameliorated the oxidative stress in lung tissues. Quantitative scoring of the lung histopathology exhibited protective effects of BPM and the inflammation, mucus, thickening of peribronchial smooth muscle layer and subepithelial deposition of collagen induced with TDI were ameliorated.The BPM has the anti-inflammatory properties that may be used to treat the asthma and inflammatory related ailments.

Emergence of long-lived autoreactive plasma cells in the spleen of primary warm auto-immune hemolytic anemia patients treated with rituximab

Primary warm autoimmune hemolytic anemia (wAIHA) is a rare autoimmune disease in which red blood cells are eliminated by IgG autoantibodies. We analyzed the antibody-secreting cells in the spleen and the peripheral blood of wAIHA patients in various contexts of treatment. Plasmablasts were observed in peripheral blood of newly diagnosed wAIHA patients and, accordingly, active germinal center reactions were present in the spleen of patients receiving short-term corticosteroid therapy. Long-term corticosteroid regimens markedly reduced this response while splenic plasma cells were able to persist, a fraction of them secreting anti-red blood cell IgG in vitro. In wAIHA patients treated by rituximab and who underwent splenectomy because of treatment failure, plasma cells were still present in the spleen, some of them being autoreactive. By using a set of diagnostic genes that allowed us to assess the plasma cell maturation stage, we observed that these cells displayed a long-lived program, differing from the one of plasma cells from healthy donors or from wAIHA patients with various immunosuppressant treatments, and more similar to the one of normal long-lived bone-marrow plasma cells. Interestingly, an increased level of B-cell activating factor (BAFF) was observed in the supernatant of spleen cell cultures from such rituximab-treated wAIHA patients. These results suggest, in line with our previous report on primary immune thrombocytopenia, that the B-cell depletion induced by rituximab promoted a suitable environment for the maturation and survival of auto-immune long-lived plasma cells in the spleen.

Treatment with Vitamin D/MOG Association Suppresses Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model to study multiple sclerosis (MS). Considering the tolerogenic effects of active vitamin D, we evaluated the therapeutic effect of myelin oligodendrocyte glycoprotein (MOG) associated with active vitamin D in EAE development. EAE was induced in female C57BL/6 mice by immunization with MOG emulsified with Complete Freund’s Adjuvant plus Mycobacterium tuberculosis. Animals also received two intraperitoneal doses of Bordetella pertussis toxin. One day after immunization, mice were treated with 0,1μg of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) every other day during 15 days (on days 1, 3, 5, 7, 9, 11, 13 and 15). MOG (150μg) was co-administered on days 3 and 11. The administration of 1,25(OH) 2D3 or MOG determined significant reduction in EAE incidence and in clinical scores. When MOG was associated with 1,25(OH) 2D3 the animals did not develop EAE. Spleen and central nervous system (CNS) cell cultures from this group produced less IL-6 and IL-17 upon stimulation with MOG in comparison to the EAE control group. In addition, this treatment inhibited dendritic cells maturation in the spleen and reduced inflammatory infiltration in the CNS. The association of MOG with 1,25(OH) 2D3 was able to control EAE development.

Induction of a Th1 immune response and suppression of IgE via immunotherapy with a recombinant hybrid molecule encapsulated in liposome-protamine-DNA nanoparticles in a model of experimental allergy.

Liposome-protamine-DNA nanoparticles (LPD) are safe, effective, and non-toxic adjuvants that induce Th1-like immune responses. We hypothesized that encapsulation of allergens into liposomes could be an appropriate option for immunotherapy. The present study evaluated the immunotherapeutic potential of a recombinant hybrid molecule (rHM) encapsulated in LPD nanoparticles in a murine model of Chenopodium album allergy. BALB/c mice were sensitized with the allergen in alum, and the immunotherapy procedure was performed by subcutaneous injections of LPD-rHM, rHM, or empty LPD at weekly intervals. Sensitized mice developed a Th2-biased immune response characterized by strong specific IgG1 and IgE production, IL-4, and the transcription factor GATA3 in spleen cell cultures. Treatment with the LPD-rHM resulted in a reduction in IgE and a marked increase in IgG2a. The LPD-rHM induced allergen-specific responses with relatively high interferon-gamma production, as well as expression of the transcription factor T-bet in stimulated splenocytes. In addition, lymphoproliferative responses were higher in the LPD-rHM-treated mice than in the other groups. Removal of the nanoparticles from the rHM resulted in a decrease in the allergen's immunogenicity. These results indicate that the rHM complexed with LPD nanoparticles has a marked suppressive effect on the allergic response and caused a shift toward a Th1 pathway.

Allergenicity characteristics of germinated soybean proteins in a BALB/c mouse model

Soybean proteins are widely used in many food products. However they also commonly cause food allergy. This study aimed to characterize the allergenicity of germinated soybean proteins in a BALB/c mouse model. Mice were orally sensitized with germinated soybean proteins or soybean proteins using cholera toxin as adjuvant. Anaphylactic shock reactions as well as changes in body temperature, specific antibody levels, mast cell protease-1 (mMCP-1) concentrations, morphological structure of duodenum, and cytokines were determined after the mice were challenged with germinated soybean proteins or soybean proteins. In contrast to soybean proteins, oral sensitization to germinated soybean proteins did not result in anaphylactic shock symptoms or decreased body temperature in mice. However, a minor damage of the intestinal villus existed after the challenge. A tendency toward decreased allergen-specific IgE, IgG and IgG1 levels, and mMCP-1 concentration was observed, accompanied by a repression of IL-4, IL-5, IL-13 and IFN-γ production in spleen cell cultures. Results indicate that germinated soybean proteins did not provoke remarkable allergic reactions compared to soybean proteins. Germinated soybean proteins have the potential to be a safe dietary formula for humans at risk of soybean allergy. However, additional studies on the underlying mechanisms and clinical trials are necessary.

Th1 Cytokine Production Induced by Lactobacillus acidophilus in BALB/c Mice Bearing Transplanted Breast Tumor.

Background:The immunomodulative effects of Lactic Acid Bacteria as probiotics have been already demonstrated.Objectives:The current study aimed to evaluate the effect of oral administration of Lactobacillus acidophilus on the immune responses and patterns of cytokine production in the BALB/c mice bearing breast cancer.Materials and Methods:The current study used thirty inbred BALB/c mice, six- to eight-week-old; they were divided into two groups of 15 each. One group was used as control in each assay. The L. acidophilus (ATCC4356) used in the study was inoculated in MRS broth and cultivated overnight at 37°C under anaerobic conditions, then collected by centrifugation, and re-suspended in Phosphate-buffered Saline (PBS) media. After preparation of the proper amount of the suspension, it was orally administered to the mice via gavage and the control mice received an equal volume of PBS in the same manner.Results:The results showed that oral administration of L. acidophilus as a potent immunostimulator agent could motivate the proliferation of immune cells. Moreover, it could increase the production of IFN-γ and decrease the production of IL-4, known as Th2 cytokines, in the spleen cell culture. The results showed that the survival time of the L. acidophilus administered mice significantly increased in comparison to that of the control mice.Conclusions:The current study findings suggested that L. acidophilus can promote immune responses with Th1 bias and may increase the antitumor response. Further, the consumption of this probiotic strain may help to manage the immune response in tumor condition, but more studies are needed to investigate the other mechanisms of this effect.

Effect of high pressure-assisted crosslinking of ovalbumin and egg white by transglutaminase on their potential allergenicity

Abstract Transglutaminase (TG) modifies proteins by amine incorporation, crosslinking and deamination. It is used as an important tool in food processing. The aim of this work was to crosslink ovalbumin (OVA) and egg white (EW) with TG, either under high pressure (HP) or under atmospheric pressure on previously HP-treated proteins, to assess the allergenic potential of polymerized allergens. The susceptibility of OVA and EW to TG-mediated crosslinking was enhanced by HP (400 MPa, 40 °C, 30 min). Simultaneous or sequential HP and TG treatments led to the formation of high-molecular weight polymers, but left a substantial amount of monomeric proteins. Results of digestibility experiments, binding to immunoglobulin E (IgE) from egg allergic patients and stimulation of spleen and mesenteric lymph node cell cultures of EW-sensitized BALB/c mice showed that resistance to proteolysis, IgE-binding and immunostimulatory properties of the HP and TG-treated proteins were not substantially different from those of the native ones. Industrial Relevance The use of transglutaminase as a crosslinking agent has been suggested for many industrial applications, as in the case of meat, fish, milk or soy proteins, which are good substrates for this enzyme. This work widens the possible use of transglutaminase, for modifying less suitable substrates for crosslinking, as egg proteins, through the use of high pressure before or during crosslinking. In our study, HP-assisted TG-induced OVA and EW crosslinking enhanced gastroduodenal digestion but did not reduce the allergenicity of egg proteins.

Potential therapeutic effect of Allium cepa L. and quercetin in a murine model of Blomia tropicalis induced asthma.

BackgroundAsthma is an inflammatory condition characterized by airway hyperresponsiveness and chronic inflammation. The resolution of inflammation is an essential process to treat this condition. In this study we investigated the effect of Allium cepa L. extract (AcE) and quercetin (Qt) on cytokine and on smooth muscle contraction in vitro and its therapeutic potential in a murine model of asthma.MethodsAcE was obtained by maceration of Allium cepa L. and it was standardized in terms of quercetin concentration using high performance liquid chromatography (HPLC). In vitro, using AcE 10, 100 or 1000 μg/ml or Qt 3.5, 7.5, 15 μg/ml, we measured the concentration of cytokines in spleen cell culture supernatants, and the ability to relax tracheal smooth muscle from A/J mice. In vivo, Blomia tropicalis (BT)-sensitized A/J mice were treated with AcE 100, 1000 mg/kg or 30 mg/kg Qt. We measured cell influx in bronchoalveolar lavage (BAL), eosinophil peroxidase (EPO) in lungs, serum levels of Bt-specific IgE, cytokines levels in BAL, and lung histology.ResultsWe observed a reduction in the production of inflammatory cytokines, a relaxation of tracheal rings, and a reduction in total number of cells in BAL and EPO in lungs by treatment with AcE or Qt.ConclusionAcE and Qt have potential as antiasthmatic drugs, as they possess both immunomodulatory and bronchodilatory properties.

Development of a walleye spleen stromal cell line sensitive to viral hemorrhagic septicemia virus (VHSV IVb) and to protection by synthetic dsRNA

A cell line, WE-spleen6, has been developed from the stromal layer of primary spleen cell cultures. On conventional plastic, WE-spleen6 cells had a spindle-shaped morphology at low cell density but grew to become epithelial-like at confluency. On the commercial extracellular matrix (ECM), Matrigel, the cells remained spindle-shaped and formed lumen-like structures. WE-spleen6 cells had intermediate filament protein, vimentin and the ECM protein, collagen I, but not smooth muscle α-actin (SMA) and von Willebrand factor (vWF) and lacked alkaline phosphatase and phagocytic activities. WE-spleen6 was more susceptible to infection with VHSV IVb than a fibroblast and epithelial cell lines from the walleye caudal fin, WE-cfin11f and WE-cfin11e, respectively. Viral transcripts and proteins appeared earlier in WE-spleen6 cultures as did cytopathic effect (CPE) and significant virus production. The synthetic double-stranded RNA (dsRNA), polyinosinic: polycytidylic acid (pIC), induced the antiviral protein Mx in both cell lines. Treating WE-spleen6 cultures with pIC prior to infection with VHSV IVb inhibited the early accumulation of viral transcripts and proteins and delayed the appearance of CPE and significant viral production. Of particular note, pIC caused the disappearance of viral P protein 2 days post infection. WE-spleen6 should be useful for investigating the impact of VHSV IVb on hematopoietic organs and the actions of pIC on the rhabdovirus life cycle.

DIFFERENTIATION OF DENDRITIC CELLS WITH TOLEROGENIC PROFILE FROM HUMAN CD14+ SPLEEN CELLS

Event Abstract Back to Event DIFFERENTIATION OF DENDRITIC CELLS WITH TOLEROGENIC PROFILE FROM HUMAN CD14+ SPLEEN CELLS Karen D. Álvarez1, Mauricio Rojas1, 2 and Cristiam M. Álvarez1* 1 Universidad de Antioquia, Grupo de Inmunología Celular e Inmunogenética, Colombia 2 Unversidad de Antioquia, Sede de Investigación Universitaria, Unidad de Citometría de Flujo, Colombia The long-term allograft survival has been attributed to the development of operational tolerance due different mechanisms as clonal deletion, anergy and immune regulation. Previous studies have demonstrated that tolerogenic dendritic cells play a key role in the induction and maintenance of the peripheral tolerance. Therapies based on tolerogenic DCs transfer, differentiated with pharmacological agents, are a promising strategy for transplantation tolerance induction while reducing dependency on immunosuppressive drugs, however the reduced number of the circulating CD14+ cells represents a limitation for the development of the therapeutic strategies. Recently, studies have suggested that the human spleen may constitute an extramedullary reservoir of monocytes in humans. Based on this considerations, we proposed to evaluate whether the methods described to differentiate dendritic cells whit tolerogenic markers from blood monocytes in vitro are also valid in CD14+ cells derived from human spleen. Dendritic cells were differentiated from blood and spleen CD14+ cells cultured in vitro with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 for 7 days and in the absence or presence of vitamin D3. By flow cytometry, we evaluated the percentage of differentiation of Lin1-HLA-DR+ Cells and surface expression of ILT3, PD-L1 and PD-L2 as markers of tolerogenic dendritic cells. The percentage of differentiated DCs was not significantly different between peripheral blood and spleen CD14+ cells cultures (75.96±16.23% vs 78.45 ±14.26%); and the presence of Vitamin D3 did not interfered with the differentiation process (79.06 ±16.06% vs 77.11±14.21%). Interestingly, when these cells were differentiated in the presence of Vitamin D3, we observed higher percentage of tolerogenic markers as demonstrated by high surface levels of ILT3, PD-L1 and PD-L2 molecules. The increased expression of these molecules were significantly both in spleen and blood cells cultures except PD-L1 which was higher only in spleen differentiated dendritic cells. We could demonstrate that the methods described to differentiate DC from blood monocytes are applicable to CD14+ cells derived from human spleen. These findings open the possibility of using spleen from deceased donors as a source of dendritic cells precursors for the development of new therapeutic treatments that permit the induction and maintenance of transplantation tolerance. Keywords: transplant tolerance, cell therapy, Tolerogenic dendritic cells, Monocytes, Immunotherapy Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015. Presentation Type: Poster Presentation Topic: Transplantation immunology Citation: Álvarez KD, Rojas M and Álvarez CM (2015). DIFFERENTIATION OF DENDRITIC CELLS WITH TOLEROGENIC PROFILE FROM HUMAN CD14+ SPLEEN CELLS. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00096 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: 24 Apr 2015; Published Online: 14 Sep 2015. * Correspondence: PhD. Cristiam M Álvarez, Universidad de Antioquia, Grupo de Inmunología Celular e Inmunogenética, Medellín, Colombia, cristian.alvarez@udea.edu.co 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 Karen D Álvarez Mauricio Rojas Cristiam M Álvarez Google Karen D Álvarez Mauricio Rojas Cristiam M Álvarez Google Scholar Karen D Álvarez Mauricio Rojas Cristiam M Álvarez PubMed Karen D Álvarez Mauricio Rojas Cristiam M Álvarez 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.

Emergence of Long-Lived Autoreactive Plasma Cells in the Spleen of Primary Warm Auto-Immune Hemolytic Anemia Patients Treated with Rituximab

Introduction: We recently proposed that B-cell depletion in immune thrombocytopenia (ITP) promotes the generation of long-lived plasma cells in the spleen, some of them being auto-reactive; but it remained possible that this observation was related to ITP itself rather than to B-cell depletion. Primary warm autoimmune hemolytic anemia (wAIHA) is a rare disease characterized by IgG auto-antibodies directed against antigens at the surface of red blood cells (RBCs) antigens, leading to their accelerated destruction. The use of B-cell depletion in wAIHA leads to 60 % to 70% of overall response at one-year and beyond. Nevertheless, 30-40 % of patients may resist to rituximab and then require a splenectomy. Nothing is known about antibody secreting cells (ASC) in the spleen of wAIHA patients, who have previously been treated or not with rituximab. In this study, we analyzed at the single cell level the splenic ASC from patients with chronic and active wAIHA, previously treated or not with rituximab (RTX), and we compared them with splenic ASC from ITP patients, and with splenic and bone marrow plasma cells from healthy donors (HD).Methods: We took advantage of the different therapeutic outcomes to analyze the splenic B-cell compartment of wAIHA patients, not previously treated with RTX (n=6), or after failure RTX treatment (n=3).Splenic tissues from organ donors and bone marrow from cardiovascular thoracotomy were used as controls. Blood samples from wAIHA (n=19), and (HD) (n=8) enrolled in this study were obtained after giving written informed consent in accordance with the Declaration of Helsinki.Results: We observed by flow cytometry and microscopy that the spleen from wAIHA patients who received less than 3 months of steroid therapy was the site of a B-cell response characterized by the presence of Bcl6+ germinal-center (GC) B-cells. Furthemore, splenic ASC secreted anti-red blood cell IgG in vitro. In line with this observation, we observed in the peripheral blood from patients with a newly diagnosed wAIHA (n=11), that short-lived IgG plasmablasts were increased compared with HD (n=8) (Mean 4.2 ± 0.84 % vs 0.99 ± 0.19% of CD19+ cells, p< 0.01). Moreover, for patients receiving long term steroid therapy (> 6 months) the plasmablast response was suppressed in the peripheral blood (Mean: 0.68 ± 0.2 % of CD19+ cells, n=8) and the splenic GC B-cell reaction was impaired (n=3). We conclude that short-lived IgG ASC result from an over-activity of GC reactions in wAIHA. We then analyzed the spleen of 3 patients who failed to respond to RTX, and observed a residual population of CD19+B-cells (median: 0.9% of lymphoid cells), including non-proliferative memory B-cells and plasma cells (PC). A fraction of these residual PC secreted anti-red blood cells IgG in vitro, thus accounting for the faillure of the B-cell depletion therapy. By using a single cell multiplex quantitative RT-PCR (Fluidigm dynamic arrays), we showed that such RTX-resistant plasma cells display a long-lived transcriptional program, which differs from PC from untreated wAIHA patients or HD, as well as from plasmablasts. Interestingly, the gene expression profile of wAIHA long-lived plasma cells segregated with long-lived PC previously observed in the spleen of ITP patients treated with rituximab. By a principal component analysis, we observed a gradient of maturation from plasmablasts to bone marrow plasma cells in which PC from RTX-treated spleens segregated close to bone marrow PC. We also observed that the cytokine BAFF was increased in the supernatants of spleen cell cultures from wAIHA patients treated with rituximab compared with controls (p< 0.05), suggesting, in keeping with our previous report in ITP, a role for BAFF in the differentiation of short-lived plasma cells into long-lived plasma cells.Conclusion: The presence of splenic long-lived autoreactive PC in wAIHA may explain why some patients cannot achieve a response after RTX. Our results show that, the B-cell depletion induced by rituximab itself, as opposed to the underlying auto-immune condition, promotes a suitable environment for the maturation of auto-immune long-lived plasma cells in the spleen. Targeting specifically some factors such as BAFF right after rituximab injection could be an interesting therapeutic option in the future. DisclosuresNo relevant conflicts of interest to declare.

Antiallergic Effects of Caffeic Acid in Blomia Tropicalis Murine Model of Experimental Asthma

Background: Atopic asthma is a chronic airway inflammatory disorder, which is characterized by reversible airway obstruction, airway hyperresponsiveness and production of Th2-dominated response with the production of cytokines IL-4, IL-5 and IL-13. The most common drugs used to treat allergic respiratory diseases, control their symptoms, but also induce long-term side effects. Therefore, in order to mitigate these effects, new treatments with alternative drugs must be pursued to improve asthma control. Among several compounds investigated by the scientific community, caffeic acid (CA), a phenolic acid has shown important biological properties, such as antioxidant and anti-inflammatory. In this study we investigated the antiallergic effects of caffeic acid on inflammatory cytokines, in vitro, and in a murine model of experimental asthma induced by the Blomia tropicalis extract (Bt). Methods: We measured the concentration of cytokines (IL-4, IL-5 and IL-13) on spleen cell culture supernatants in vitro, using 25, 50 or 100 ?M of CA. For in vivo experiments, AJ mice were sensitized (100 ?g/ animal s.c) and challenged (10?g/ animal i.n) with Bt. Sensitized A/J mice were treated or not with CA (10, 100 or 200mg/Kg) or with 3mg/kg of Dexametazone (Dex) and the following parameters were analyzed: number of total cells in bronchoalveolar lavage (BAL); eosinophil peroxidase activity (EPO) in BAL; serum level of specific IgE, IgG1 and IgG2a; and histopathological changes in the lungs. Results: The CA oral treatment caused a reduction in total number of cells in BAL, EPO in lungs, serum level of specific IgG1 and histopathological changes showed attenuate allergic inflammation in the lungs. Additionally, we observed a significant decrease of Th2-type cytokines (IL-4, IL-5 and IL-13) on spleen cell culture supernatants. Conclusion: These results suggest that oral treatment with caffeic acid could be a promising alternative treatment for bronchial asthma.

Genetically engineered Lactococcus lactis protect against house dust mite allergy in a BALB/c mouse model.

BackgroundMucosal vaccine based on lactic acid bacteria is an attractive concept for the prevention and treatment of allergic diseases, but their mechanisms of action in vivo are poorly understood. Therefore, we sought to investigate how recombinant major dust mite allergen Der p2-expressing Lactococcus lactis as a mucosal vaccine induced the immune tolerance against house dust mite allergy in a mouse model.MethodsThree strains of recombinant L. lactis producing Der p2 in different cell components (extracellular, intracellular and cell wall) were firstly constructed. Their prophylactic potential was evaluated in a Der p2-sensitised mouse model, and immunomodulation properties at the cellular level were determined by measuring cytokine production in vitro.ResultsDer p2 expressed in the different recombinant L. lactis strains was recognized by a polyclonal anti-Der p2 antibody. Oral treatment with the recombinant L. lactis prior sensitization significantly prevented the development of airway inflammation in the Der p2-sensitized mice, as determined by the attenuation of inflammatory cells infiltration in the lung tissues and decrease of Th2 cytokines IL-4 and IL-5 levels in bronchoalveolar lavage. In addition, the serum allergen-specific IgE levels were significantly reduced, and the levels of IL-4 in the spleen and mesenteric lymph nodes cell cultures were also markedly decreased upon allergen stimulation in the mice fed with the recombinant L. lactis strains. These protective effects correlated with a significant up-regulation of regulatory T cells in the mesenteric lymph nodes.ConclusionOral pretreatment with live recombinant L. lactis prevented the development of allergen-induced airway inflammation primarily by the induction of specific mucosal immune tolerance.

B cell signatures of BCWD-resistant and susceptible lines of rainbow trout: A shift towards more EBF-expressing progenitors and fewer mature B cells in resistant animals

Bacterial cold water disease (BCWD) is a chronic disease of rainbow trout, and is caused by the Gram-negative bacterium Flavobacterium psychrophilum (Fp), a common aquaculture pathogen. The National Center for Cool and Cold Water Aquaculture has bred two genetic lines of rainbow trout: a line of Fp-resistant trout (ARS-Fp-R or R-line trout) and a line of susceptible trout (ARS-Fp-S, or S-line). Little is known about how phenotypic selection alters immune response parameters or how such changes relate to genetic disease resistance. Herein, we quantify interindividual variation in the distribution and abundance of B cell populations (B cell signatures) and examine differences between genetic lines of naive animals. There are limited trout-specific cell surface markers currently available to resolve B cell subpopulations and thus we developed an alternative approach based on detection of differentially expressed transcription factors and intracellular cytokines. B cell signatures were compared between R-line and S-line trout by flow cytometry using antibodies against transcription factors early B cell factor-1 (EBF1) and paired domain box protein Pax5, the pro-inflammatory cytokine IL-1β, and the immunoglobulin heavy chain mu. R-line trout had higher percentages of EBF+ B myeloid/ progenitor and pre-B cells in PBL, anterior and posterior kidney tissues compared to S-line trout. The opposite pattern was detected in more mature B cell populations: R-line trout had lower percentages of both IgM+ mature B cells and IgM-secreting cells in anterior kidney and PBL compared to S-line trout. In vitro LPS-activation studies of PBL and spleen cell cultures revealed no significant induction differences between R-line and S-line trout. Together, our findings suggest that selective resistance to BCWD may be associated with shifts in naive animal developmental lineage commitment that result in decreased B lymphopoiesis and increased myelopoiesis in BCWD resistant trout relative to susceptible trout.