Antigen-stimulated Proliferation Research Articles

CEST MRI and MALDI imaging reveal metabolic alterations in the cervical lymph nodes of EAE mice

BackgroundMultiple sclerosis (MS) is a neurodegenerative disease, wherein aberrant immune cells target myelin-ensheathed nerves. Conventional magnetic resonance imaging (MRI) can be performed to monitor damage to the central nervous system that results from previous inflammation; however, these imaging biomarkers are not necessarily indicative of active, progressive stages of the disease. The immune cells responsible for MS are first activated and sensitized to myelin in lymph nodes (LNs). Here, we present a new strategy for monitoring active disease activity in MS, chemical exchange saturation transfer (CEST) MRI of LNs.Methods and resultsWe studied the potential utility of conventional (T2-weighted) and CEST MRI to monitor changes in these LNs during disease progression in an experimental autoimmune encephalomyelitis (EAE) model. We found CEST signal changes corresponded temporally with disease activity. CEST signals at the 3.2 ppm frequency during the active stage of EAE correlated significantly with the cellular (flow cytometry) and metabolic (mass spectrometry imaging) composition of the LNs, as well as immune cell infiltration into brain and spinal cord tissue. Correlating primary metabolites as identified by matrix-assisted laser desorption/ionization (MALDI) imaging included alanine, lactate, leucine, malate, and phenylalanine.ConclusionsTaken together, we demonstrate the utility of CEST MRI signal changes in superficial cervical LNs as a complementary imaging biomarker for monitoring disease activity in MS. CEST MRI biomarkers corresponded to disease activity, correlated with immune activation (surface markers, antigen-stimulated proliferation), and correlated with LN metabolite levels.

B-cell response to seasonal influenza vaccine in mice is amenable to pharmacological modulation through β-adrenoceptor

AimsGiven that deprivation of noradrenaline acting on lymphocytes through β-adrenoceptor influences antibody response, the effects of propranolol treatment beginning two days before immunization with quadrivalent inactivated influenza vaccine (QIV) on IgG response and underlying cellular molecular mechanism in mice were investigated. Main methodsTwenty-one days post-immunization the total QIV antigen-specific IgG titer and IgG subclass titers in sera were determined using ELISA. Additionally, the total counts of germinal centre (GC) B cells, T follicular helper (Tfh) and T follicular regulatory (Tfr) cells in draining lymph nodes (dLNs) and spleens, in vitro proliferation of interacting B cells and Th cells and IL-21 synthesis in Th cells in response to QIV antigens and/or mitogen were attested using flow cytometry analysis. In QIV antigen-stimulated dLN cell and splenocyte cultures were also measured concentrations of INF-γ and IL-4, cytokines upregulating IgG2a and IgG1 synthesis, respectively. Key findingsPropranolol decreased the total QIV antigen-specific IgG titer. This correlated with lower GC B cell count and the shift in Tfr/Tfh cell and Tfr/GC B cell ratio towards Tfr in propranolol-treated mice compared with controls. Consistently, QIV antigen-stimulated proliferation of B cells and Th cells from propranolol-treated mice in vitro was impaired. This correlated with the lower frequency of QIV antigen-specific IL-21-producing cells among Th cells. Additionally, in propranolol-treated mice, in accordance with the changes in INF-γ/IL-4 ratio in dLN cell/splenocyte cultures, serum IgG2a/IgG1 ratio was shifted towards IgG1 reflecting decreased IgG2a response. SignificanceThe study indicates that chronic propranolol treatment may impair response to QIV.

Activity of Lymphostatin, A Lymphocyte Inhibitory Virulence Factor of Pathogenic Escherichia coli, is Dependent on a Cysteine Protease Motif

Lymphostatin (LifA) is a 366 kDa protein expressed by attaching & effacing Escherichia coli. It plays an important role in intestinal colonisation and inhibits the mitogen- and antigen-stimulated proliferation of lymphocytes and the synthesis of proinflammatory cytokines. LifA exhibits N-terminal homology with the glycosyltransferase domain of large clostridial toxins (LCTs). A DTD motif within this region is required for lymphostatin activity and binding of the sugar donor uridine diphosphate N-acetylglucosamine. As with LCTs, LifA also contains a cysteine protease motif (C1480, H1581, D1596) that is widely conserved within the YopT-like superfamily of cysteine proteases. By analogy with LCTs, we hypothesised that the CHD motif may be required for intracellular processing of the protein to release the catalytic N-terminal domain after uptake and low pH-stimulated membrane insertion of LifA within endosomes. Here, we created and validated a C1480A substitution mutant in LifA from enteropathogenic E. coli strain E2348/69. The purified protein was structurally near-identical to the wild-type protein. In bovine T lymphocytes treated with wild-type LifA, a putative cleavage product of approximately 140 kDa was detected. Appearance of the putative cleavage product was inhibited in a concentration-dependent manner by bafilomycin A1 and chloroquine, which inhibit endosome acidification. The cleavage product was not observed in cells treated with the C1480A mutant of LifA. Lymphocyte inhibitory activity of the purified C1480A protein was significantly impaired. The data indicate that an intact cysteine protease motif is required for cleavage of lymphostatin and its activity against T cells.

Lymphoproliferative responses to dendritic cell presentation of sensitizing allergens in atopic children with multiple allergies

BackgroundPeripheral blood mononuclear cells (PBMCs) proliferate inconsistently, rendering current lymphoproliferation assays unreliable in diagnosis. ObjectiveTo investigate the utility and nature of proliferation responses in allergy by comparison of the standard lymphoproliferation with a new dendritic cell (DC) stimulated assay. MethodsMonocyte-derived DCs were pulsed with allergens and incubated with autologous T cells for 7 days. DC-stimulated and standard PBMC proliferation responses to 3 common dietary allergens in children with allergy and without atopy were measured by incorporation of tritiated thymidine and reduction of carboxyl fluorescein succinimidyl ester staining. ResultsThe DC presentation of sensitizing allergens induced significantly higher proliferative responses than PBMC stimulation (P = .04) and greater distinction between normal and allergic responses. DC-induced stimulation indices of children without sensitivity and those with allergy were significantly different with all 3 foods (P < .001). All children with allergy presented with peanut allergy and 12 of 14 (86%) β-lactoglobulin–pulsed DC preparations proliferated more than 3.3-fold above un-pulsed cells, but 8 of 18 children (44%) with ovalbumin egg allergy showed proliferation below this level. The stimulation index of DC tritiated thymidine incorporation correlated closely with carboxyl fluorescein succinimidyl ester reduction (P < .001). Sensitivity of detection of peanut, milk, or egg allergy was 100%, 85.7%, or 55.6% and specificity was 60%, 88.9%, or 86.7%, respectively. DC-stimulated T cells expressed increased levels of CD45 RO and CD25 and most produced interferon-γ. DC-stimulated proliferation correlated with total immunoglobulin E and peanut antigen-stimulated proliferation correlated with peanut specific immunoglobulin E (P = .03). ConclusionThe DC-induced lymphoproliferation had higher sensitivity, specificity, and reproducibility than the standard assay and caused increased memory and activated T-cell proliferation in children with food allergy.

Alloimmune Monitoring after Islet Transplantation: A Prospective Multicenter Assessment of 25 Recipients

Islet transplantation is an effective treatment for selected patients with type 1 diabetes. However, an accurate test still lacks for the early detection of graft rejection. Blood samples were prospectively collected in four university centers (Geneva, Grenoble, Montpellier, and Strasbourg). Peripheral blood mononuclear cells were stimulated with donor splenocytes in the presence of interleukin-2. After 24 h of incubation, interferon- (IFN-) ELISpot analysis was performed. After a total of 5 days of incubation, cell proliferation was assessed by fluorescence-activated cell sorting (FACS) analysis for Ki-67. Immunological events were correlated with adverse metabolic events determined by loss of 1 point of -score and/or an increased insulin intake 10%. Twenty-five patients were analyzed; 14 were recipients of islets alone, and 11 combined with kidney. Overall, 76% (19/25) reached insulin independence at one point during a mean follow-up of 30.7 months. IFN- ELISpot showed no detectable correlation with adverse metabolic events [area under the curve (AUC)=0.57]. Similarly, cell proliferation analysis showed no detectable correlation with adverse metabolic events (CD3+/CD4+ AUC=0.54; CD3+/CD8+ AUC=0.55; CD3/CD56+ AUC=0.50). CD3/CD56+ cell proliferation was significantly higher in patients with combined kidney transplantation versus islet alone (6 months, p=0.010; 12 months, p=0.016; and 24 months, p=0.018). Donor antigen-stimulated IFN- production and cell proliferation do not predict adverse metabolic events after islet transplantation. This suggests that the volume of transplanted islets is too small to produce a detectable systemic immune response and/or that alloimmune rejection is not the sole reason for the loss of islet graft function.

Accelerated telomere shortening in peripheral blood lymphocytes after occupational polychlorinated biphenyls exposure

Polychlorinated biphenyls (PCBs) are organochlorine pollutants with a worldwide dissemination. We examined telomere length (TL) in peripheral blood cells of 207 individuals with a high body burden of PCBs due to occupational exposure in a transformer recycling company. Whereas TL in granulocytes was not affected, the age-adjusted TL in lymphocytes (∆TLLymph) of exposed individuals was significantly shorter than expected [−0.77 kb; 95 % confidence interval (CI) −0.9316; −0.6052; p = 0.0001]. PCB exposure did not affect lymphocyte numbers or T cell receptor excision circle (TREC) levels in T cells, suggesting that PCBs cause loss of telomeric DNA in T cells due to their metabolic activation and antigen-stimulated proliferation. In support of this hypothesis, blood plasma levels of PCB-exposed individuals inhibited expression of telomerase, the telomere elongating enzyme in vitro in antigen-specific T cell proliferation assays. 3-OH-CB28, a downstream metabolite of the lower chlorinated PCB-28 in PCB-exposed individuals (mean blood plasma concentration: 0.185 ± 0.68 ng/mL), inhibited telomerase gene expression within 48 h of incubation in lymphoproliferative assays starting at a concentration of 0.27–6.75 µg/mL and accelerated telomere shortening in long-term cell culture experiments. Accelerated telomere shortening due to PCB exposure may lead to limitations of cell renewal and clonal expansion of lymphocyte populations. As PCB-related immune dysfunctions have been linked to increased susceptibility to infectious diseases and increased risk of cancer, our data provide a possible explanation, for how PCBs could promote infections and cancer through limiting immune surveillance.

Regulatory T and B lymphocytes in a spontaneous autoimmune polyneuropathy.

B7-2(-/-) non-obese diabetic (NOD) mice develop a spontaneous autoimmune polyneuropathy (SAP) that mimics the progressive form of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). In this study, we focused on the role of regulatory T cells (Tregs ) and regulatory B cells (Bregs ) in SAP. We found that deletion of B7-2 in female NOD mice led to a lower frequency and number of Tregs and Bregs in spleens and lymph nodes. Tregs but not Bregs suppressed antigen-stimulated splenocyte proliferation, whereas Bregs inhibited the T helper type 1 (Th1) cytokine response. Both Tregs and Bregs induced an increase in CD4(+) interleukin (IL)-10(+) cells, although less effectively in the absence of B7-2. Adoptive transfer studies revealed that Tregs , but not Bregs , suppressed SAP, while Bregs attenuated disease severity when given prior to symptom onset. B cell deficiency in B cell-deficient (muMT)/B7-2(-/-) NOD mice prevented the development of SAP, which would indicate that the pathogenic role of B cells predominates over its regulatory role in this model. We conclude that Bregs and Tregs control the immunopathogenesis and progression of SAP in a non-redundant fashion, and that therapies aimed at expansion of Bregs and Tregs may be an effective approach in autoimmune neuropathies.

Mouse BMDC-dependent T Cell Polarization Assays

In response to exposure to antigen, T cells whose T cell receptor (TCR) are capable of recognizing the self MHC-antigen derived peptide complex, respond to the antigen and differentiate into one of several subsets, namely TH1, TH2, TH17, Treg, etc. characterized by the signature cytokine they secrete, namely IFN-γ, IL-4, IL-17 or IL-10, respectively, referred to as syngeneic polarization as the MHC presenting the foreign antigen/epitope is self-derived. T cell responses following incubation for defined periods, usually 3 days for mouse splenocytes, are routinely measured by assessing the antigen-stimulated proliferation of T cells by measuring the radiolabeled precursor thymidine incorporated into the genomic DNA of the dividing T cell; the direction of polarization is assessed by measuring the cytokine produced by the proliferating or non-proliferating responding T cells using ELISA of culture supernatants or by intracellular cytokine staining followed by flow cytometry. In the protocols detailed below, we describe the use of syngeneic mouse bone marrow-derived primary dendritic cells (BMDC) as APC to stimulate spleen derived T cells. The proliferative response of the T cells is measured by incorporation of radiolabeled precursor thymidine into the genomic DNA and their direction of polarization is assessed by measuring the cytokines they secrete, namely IFN-γ, IL-4 and IL-17 over a 72 h period using ELISA. In addition, we used flow cytometry after intracellular cytokine staining to detect IL-17 positive T cells within the CD3+/CD4+/CD25low population. Prior live infection of BMDC with strains of Mycobacterium bovis- Bacille Calmette Guerin (BCG) was used as antigen to pre-condition the BMDC that presented antigens derived therefrom to T cells. We also measured cytokines secreted within 6 to 8 h of BCG infection by BMDC in order to correlate the BMDC cytokine profile with subsequent direction of T cell polarization.

Immunological adjuvant effect of the peptide fraction from the larvae of Musca domestica.

BackgroundThe larvae of Musca domestica (Diptera: Muscidae) have been used traditionally for malnutritional stagnation, decubital necrosis, osteomyelitis, ecthyma and lip scald and also to treat coma and gastric cancer in the traditional Chinese medicine. Its immunomodulatory effects in naïve mice in relation to the traditional uses were also reported. However, the immunological adjuvant potentials of this insect have not yet been studied.MethodsThe peptide fraction from the larvae of Musca domestica L. (MDPF) was evaluated for its adjuvant potentials on the immune responses to ovalbumin (OVA) and avian influenza vaccine (rL–H5) by determining antigen-specific antibody titers, splenocyte proliferation, activity of natural killer (NK) cell, the secretion of cytokines from splenocytes in the immunized mice.ResultsMDPF significantly enhanced not only the concanavalin A (Con A)-, lipopolysaccharide (LPS)- and antigen-stimulated splenocyte proliferation, but serum antigen-specific IgG, IgG1, IgG2a, and IgG2b antibody titers in the mice immunized with OVA and rL–H5. MDPF also remarkably promoted the killing activities of NK cells in splenocytes from the mice immunized with rL–H5. Furthermore, MDPF significantly promoted the production of Th1 (IL-2 and IFN-γ) and Th2 (IL-10) cytokines from splenocytes in the immunized mice.ConclusionsThe results indicated that MDPF had a potential to increase both cellular and humoral immune responses and elicit a balanced Th1/Th2 response, and that MDPF may be a safe and efficacious vaccine adjuvant candidate.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-015-0951-6) contains supplementary material, which is available to authorized users.

Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice.

Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important public health problem and veterinary concern. Although there is no vaccine for human toxoplasmosis, many attempts have been made to develop one. Promising vaccine candidates utilize proteins, or their genes, from microneme organelle of T. gondii that are involved in the initial stages of host cell invasion by the parasite. In the present study, we used different recombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6) or combinations of these proteins (TgMIC1-4 and TgMIC1-4-6) to evaluate the immune response and protection against experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinant TgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated by reduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4 or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% of mice, respectively, survived to the acute phase of infection. In addition, these vaccinated mice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and 68%, respectively. The protective effect was related to the cellular and humoral immune responses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serum antibodies. Our results demonstrate that microneme proteins are potential vaccines against T. gondii, since their inoculation prevents or decreases the deleterious effects of the infection.

Adjuvant-active fraction from Albizia julibrissin saponins improves immune responses by inducing cytokine and chemokine at the site of injection.

The total saponin from the stem bark of Albizia julibrissin (AJSt) has previously showed the adjuvant potentials in mice. In this study, AJSt was subjected to resin column chromatography to afford four fractions (AJS30, AJS50, AJS75 and AJS95), and these fractions were further compared for the hemolytic activities and adjuvant potentials on the immune response to ovalbumin (OVA) and recombinant fowl pox virus vector-based avian influenza vaccine (rFPV). AJSt, AJS50, AJS75 and AJS95 showed a slight hemolytic effect. AJSt, AJS50 and AJS75 significantly enhanced not only the concanavalin A (Con A)-, lipopolysaccharide (LPS)- and antigen-stimulated splenocyte proliferation, but also serum antigen-specific IgG, IgG1, IgG2a and IgG2b antibody titers in the mice immunized with OVA and rFPV. AJSt, AJS50 and AJS75 also significantly promoted the NK cell activity and delayed-type hypersensitivity (DTH) in the OVA-immunized mice. Furthermore, the mechanisms of adjuvant action were explored by determining the effects of AJS75 on cytokines and chemokines at the site of injection using antibody array. AJS75 induced or up-regulated the protein expression of 12 cytokines (IL-12p40, IL-12p40/p70, IFN-γ, IL-13, IL-1β, IL-6, IL-10, TNF-α, sTNFR I, sTNFR III, IL-3 and IL-9) and 10 chemokines (Eotaxin, I-TAC, MIG, MIP-1α, RANTES, TECK, Fracatlkine, FasL, M-CSF and GM-CSF) in the injected muscles. The results suggested that AJS75, the most adjuvant-active fraction of AJSt, could improve antigen-specific both cellular and humoral immune responses and simultaneously elicit a Th1/Th2 response by inducing cytokine and chemokine at the site of injection.

Altered immunoregulatory profile during anti-tumour necrosis factor treatment of patients with inflammatory bowel disease

Inflammatory bowel disease (IBD) can be treated effectively by anti-tumour necrosis factor (TNF) therapy. We set out to investigate the unclear immunoregulatory mechanisms of the treatment. Thirty-four patients with IBD treated with anti-TNF were included. Lymphocytes from peripheral blood and intestinal biopsies were analysed by flow cytometry. Regulation of antigen-stimulated proliferation was analysed by blocking of interleukin (IL)-10, transforming growth factor (TGF)-β or depletion of CD25(+) cells in peripheral blood mononuclear cell cultures. No changes in CD4(+)CD25(+), CD25(+)TNF-RII(+) or CD4(+)CD25(+) forkhead box protein 3 (FoxP3(+)) T cells could be observed in peripheral blood after, in comparison to before, 6 weeks of treatment. The suppressive ability of CD4(+)CD25(+) cells did not change. There was an initial decrease of CD4(+)CD25(+) cells in intestinal mucosa after 2 weeks of treatment, followed by an increase of these cells from weeks 2 to 6 of treatment (P < 0·05). This was accompanied by an increased percentage of CD69(+) cells among these cells after 6 weeks of treatment compared to before treatment (P < 0·01). There was also an increase of mucosal T helper type1 cells from weeks 2 to 6 (P < 0·05). In addition, CD25(+)TNF-RII(+) cells in the mucosa were decreased after 6 weeks of treatment compared to before treatment (P < 0·05). Before treatment, peripheral blood mononuclear cell baseline proliferation was increased when IL-10 was blocked (P < 0·01), but not after. In CD25(+) cell-depleted cultures proliferation increased after treatment (P < 0·05). Our data indicate that anti-TNF treatment leads to an induction of effector T cells. Anti-TNF therapy has no significant impact on regulatory T cells in IBD, although the composition of regulatory T cell subsets may change during treatment.

Evaluation of three recombinant multi-antigenic vaccines composed of surface and secretory antigens of Toxoplasma gondii in murine models of experimental toxoplasmosis

The great clinical and economical impact of Toxoplasma gondii infections makes the development of an effective vaccine for controlling toxoplasmosis an extremely important aim. In the presented study, we evaluate the protective and immunogenic properties of three recombinant subunit vaccines composed of rROP2+rGRA4+rSAG1, rROP2+rROP4+rGRA4 and rROP2+rROP4+rSAG1 proteins of T. gondii in an experimental toxoplasmosis model in the C3H/HeJ and C57BL/6 mouse strains. All three recombinant vaccines induced partial protection as measured by the reduction of brain cyst burden following challenge with five tissue cysts of the low virulence DX T. gondii strain. The level of protection was dependent on the antigen composition of the vaccine and the genetic background of the laboratory animals. The strongest protection against chronic toxoplasmosis was induced in both C3H/HeJ and C57BL/6 mice by the mixture of rhoptry proteins rROP2 and rROP4 combined with tachyzoite major protein rSAG1. The average parasite burden in these groups of mice was reduced by 71% and 90%, respectively, compared to non-vaccinated mice. The observed protective effect was related to the vaccine-induced cellular and humoral immune responses, as measured by the antigen-induced release of the Th1 cytokines IFN-γ and IL-2, the antigen-stimulated proliferation of spleen cells of vaccinated animals in comparison to control animals and the development of systemic antigen-specific IgG1 and IgG2a (C3H/HeJ) or IgG2c (C57BL/6) antibodies. Our studies show that recombinant rROP2, rROP4, rGRA4 and rSAG1 antigens may be promising candidates for a subunit vaccine against toxoplasmosis. Additionally, we demonstrate that the ideal composition of vaccine antigens can be equally effective in mice with different genetic backgrounds and variable levels of innate resistance to toxoplasmosis, resulting in strong protection against T. gondii invasion.

Packed Red Blood Cells Suppress T-Cell Proliferation Through a Process Involving Cell–Cell Contact

Packed red blood cell (PRBC) transfusion suppresses immunity and increases morbidity and mortality. Leukocyte reduction has failed to abrogate these effects, thus implicating red blood cells themselves or their components. PRBC impair proliferation of immortal (Jurkat) T cells by depleting arginine from the extracellular environment. The effect of PRBC on isolated ex vivo T-cell proliferation has not been reported. We hypothesize that PRBCs depress mitogen-stimulated proliferation in isolated human and mouse T cells. Human peripheral T cells were isolated by Ficoll-Hypaque gradient, purified by magnetic separation, and stimulated with anti-CD3 or anti-CD28. DO11.10 transgenic mouse splenic T cells were stimulated with ovalbumin. Cells were cultured at 1 x 10(6)/mL in 96-well plates or in 24-transwell plates in the presence of PRBC (0.015-5% by volume, stored for 4-6 weeks). In culture media, arginine and citrulline were varied. Proliferation was measured at 72 hours by thymidine incorporation. T-cell viability, apoptosis, and receptor zeta chain were measured by flow cytometry. PRBC significantly depressed human peripheral and mouse splenic T-cell proliferation in a dose-dependent manner. PRBC arginase blockade by N-omega-hydroxy-nor-l-arginine only partly restored proliferation. Cell contact was required in both cell types for maximal effect. Depressed zeta chain in human peripheral T cells was partly restored by arginase blockade. Salvage by high-dose arginine and citrulline was unsuccessful. Decreased proliferation was not related to cell death. PRBC suppresses mitogen-stimulated human and antigen-stimulated mouse T-cell proliferation by mechanisms independent of arginine depletion. This is a novel mechanism for transfusion-associated immune suppression.

Neonatal Tolerization with Bacterial Antigens Stimulates an Expansion of CD11b+ Leukocytes with Inhibitory Activity in IL-10 Gene-Deficient Mice (39.38)

Abstract Tolerance to antigens of the endogenous flora can be induced by intra-peritoneal injection of 100 microliter of a sterile lysate solution (2mg/ml protein) prepared from fecal material of conventionally-raised mice with enteric micro flora into neonates. In IL-10 gene-deficient mice this treatment results in delayed onset of intestinal inflammation and reduced levels of pro-inflammatory cytokine release with a concomitant increase in CD11b+ leukocytes in spleen. To investigate whether these CD11b+ leukocytes play an active role in the reduction of bacteria-induced immune responses we isolated CD11b+ leukocytes from spleens of 20 week old mice that were tolerized to bacterial antigens as neonates and added these cells in limited dilutions to spleen cell cultures stimulated with bacterial antigens. We found that in bacterial antigen-tolerized, Il-10 gene-deficient mice the amount of CD11b+ leukocytes in spleen doubled compared to the amount found in untreated mice and mice injected with germ-free lysates. Sorted CD11b+ spleen cells from tolerized mice were able to inhibit antigen-stimulated spleen cell proliferation and release of pro-inflammatory cytokines in a concentration-dependent manner suggesting that CD11b+ cells play a regulatory role in the response to bacterial antigens in this IBD model. This work is supported by the Crohn's and Colitis Foundation of Canada.

Di-(2-ethylhexyl) phthalate affects immune cells from atopic prone mice in vitro

Phthalate esters as plasticizers have been widespread in the environment and may be associated with development of allergic diseases such as asthma and atopic dermatitis. However, the underlying mechanisms have not been fully elucidated. The present study investigated the effects of di-(2-ethylhexyl) phthalate (DEHP) on immune cells from atopic prone NC/Nga mice in vitro. Bone marrow-derived dendritic cells (BMDC) as a professional antigen-presenting cell and splenocytes as mixture of immune cells were used. BMDC were differentiated by culture with granulocyte macrophage-colony stimulating factor (GM-CSF) in the presence of DEHP (0.1–10 μM) for 6 days. In another experiments, BMDC were differentiated by culture with GM-CSF for 8 days then these BMDC were exposed to DEHP (0.1–100 μM) for 24 h. Splenocytes were exposed to DEHP for 24 h (0.1–100 μM) or 72 h (0.1–1000 nM). After the culture, the phenotypic markers and the function of BMDC and splenocytes were evaluated. BMDC differentiated in the presence of DEHP showed enhancement in the expression of MHC class II, CD86, CD11c and DEC205, and in their antigen-presenting activity. On the other hand, the function of the differentiated BMDC was not activated by DEHP although DEHP partly enhanced their expression of DEC205. DEHP-exposed splenocytes showed increases in their TCR and CD3 expression, interleukin-4 production, and antigen-stimulated proliferation. These results demonstrate that DEHP enhances BMDC differentiation but not activation and also enhances Th2 response in splenocytes from atopic prone mice. The enhancement might contribute to the aggravating effect of DEHP on allergic disorders.

Species specialization in cytokine biology: Is interleukin-4 central to the T H1–T H2 paradigm in swine?

The T H1–T H2 paradigm provides an elegant model of directed response to infectious pathogens. Developed in the mouse, the model has provided a framework for systematic and mechanistic studies of immune regulation, protective immunity, and vaccine development in swine. Interleukin-4 (IL-4) plays a central role in the paradigm as a regulatory molecule directing development of the T H2 phenotype, as a developmental cytokine essential for antibody production, and as a soluble diagnostic marker of the T H2 cell type. In contrast, while characterizing the biological properties of porcine IL-4, we discovered that it was not a stimulatory factor for porcine B cells. Rather, it blocked antibody and IL-6 secretion and suppressed antigen-stimulated proliferation of B cells. Inhibition was not reversed by treatment with IL-2 and IL-6 treatment. IL-4 did not stimulate T lymphocyte proliferation, but induced cell growth in lymphoblasts in a dose-dependent fashion. These results suggest that IL-4 plays a different role in pigs than in mice and humans, in which it stimulates B cells and is essential for antibody production. Furthermore, the functions of IL-4 in swine cannot be inferred from results in model systems such as the mouse. General models of disease resistance show substantial variation between pigs and mice at the cellular and molecular level. Advances in somatic cell technologies and animal engineering to enable gene knockouts in pigs, in combination with a continuously expanding immunological toolkit, promise an exciting future for pig immunology, detailed mechanistic elucidation of the T H1–T H2 paradigm, and an improved understanding of the role of IL-4 in porcine immunity to infectious disease.

Immunogenicity of a recombinant envelope domain III protein of dengue virus type-4 with various adjuvants in mice

Dengue fever, a mosquito borne viral disease, has become a major public health problem with dramatic expansion in recent decades. Several dengue vaccines are at developing stage, none are yet available for humans. There is no vaccine or antiviral therapy available for dengue fever till date. Domain III of envelope protein is involved in binding to host receptors and it contains type and subtype-specific epitopes that elicit virus neutralizing antibodies. Hence domain III is an attractive vaccine candidate. In the present study we report the immunomodulatory potential of refolded D4EIII protein in combination with various adjuvants (Freunds Complete adjuvant, Montanide ISA720, Alum). Mice were tested for humoral immune responses by ELISA, immunofluorescence assay and plaque reduction neutralization test. Cell mediated immune response was tested by lymphocyte proliferation assay and cytokine profiling. All the formulations resulted in high antibody titers that neutralized the virus entry in vitro. D4EIII in combination with montanide ISA720 and Feuds complete adjuvant gave highest antibody endpoint titers followed by alum. The level of antigen-stimulated splenocyte proliferation and cytokine production was comparable to that obtained from Con A stimulation and cytokine profiling of stimulated splenocyte culture supernatants indicated that all the adjuvant formulations have induced cell mediated immune response as well. These findings suggest that D4EIII in combination with compatible adjuvants is highly immunogenic and can elicit high titer neutralizing antibodies and cell mediated immune response which plays an important role in intracellular infections, which proves that refolded D4EIII can be a potential vaccine candidate.

Preparation and immunogenic properties of a recombinant West Nile subunit vaccine

While several West Nile vaccines are being developed, none are yet available for humans. In this study aimed at developing a vaccine for humans, West Nile virus (WNV) envelope protein (E) and non-structural protein 1 (NS1) were produced in the Drosophila S2 cell expression system. The C-terminal 20% of the E protein, which contains the membrane anchor portion, was deleted, thus allowing for efficient secretion of the truncated protein (80E) into the cell culture medium. The proteins were purified by immunoaffinity chromatography (IAC) using monoclonal antibodies that were flavivirus envelope protein group specific (for the 80E) or flavivirus NS1 group specific (for NS1). The purified proteins were produced in high yield and used in conjunction with adjuvant formulations to vaccinate mice. The mice were tested for both humoral and cellular immune responses by a plaque reduction neutralization test and ELISA, and by lymphocyte proliferation and cytokine production assays, respectively. The results revealed that the 80E and the NS1 proteins induced both high-titered ELISA and neutralizing antibodies in mice. Splenocytes from immunized mice, cultured in vitro with the vaccine antigens as stimulants, showed excellent proliferation and production of cytokines (IFN-γ, IL-4, IL-5, and IL-10). The level of antigen-stimulated lymphocyte proliferation and cytokine production was comparable to the level obtained from mitogen (phytohemagglutinin or pokeweed) stimulation, indicating a robust cellular response as well. These findings are encouraging and warrant further in vivo studies to determine the protective efficacy of the WNV vaccine candidate.

Molecular survival strategies of Echinococcus multilocularis in the murine host

Larval infection with Echinococcus multilocularis starts with the intrahepatic postoncospheral development of a metacestode that—at its mature stage—consists of an inner germinal and an outer laminated layer (GL & LL). In certain cases, an appropriate host immune response may inhibit parasite proliferation. Several lines of evidence obtained in vivo and in vitro indicate the important bio-protective role of the LL. For instance, the LL has been proposed to protect the GL from nitric oxide produced by periparasitic macrophages and dendritic cells, and also to prevent immune recognition by surrounding T cells. On the other hand, the high periparasitic NO production by peritoneal exsudate cells contributes to periparasitic immunosuppression, explaining why iNOS deficienct mice exhibit a significantly lower susceptibility towards experimental infection. The intense periparasitic granulomatous infiltration indicates a strong host–parasite interaction, and the involvement of cellular immunity in control of the metacestode growth kinetics is strongly suggested by experiments carried out in T cell deficient mouse strains. Carbohydrate components of the LL, such as Em2(G11) and Em492, as well as other parasite metabolites yield immunomodulatory effects that allow the parasite to survive in the host. I.e., the IgG response to the Em2(G11)-antigen takes place independently of alpha-beta + CD4 + T cells, and in the absence of interactions between CD40 and CD40 ligand. Such parasite molecules also interfere with antigen presentation and cell activation, leading to a mixed Th1/Th2-type response at the later stage of infection. Furthermore, Em492 and other (not yet published) purified parasite metabolites suppress ConA and antigen-stimulated splenocyte proliferation. Infected mouse macrophages (AE-MØ) as antigen presenting cells (APC) exhibited a reduced ability to present a conventional antigen (chicken ovalbumin, C-Ova) to specific responder lymph node T cells when compared to normal MØ. As AE-MØ fully maintain their capacity to appropriately process antigens, a failure in T cell receptor occupancy by antigen-Ia complex or/and altered co-stimulatory signals can be excluded. Studying the status of accessory molecules implicated in T cell stimulation by MØ, it could be shown that B7-1 (CD80) and B7-2 (CD86) remained unchanged, whereas CD40 was down-regulated and CD54 (=ICAM-1) slightly up-regulated. FACS analysis of peritoneal cells revealed a decrease in the percentage of CD4+ and CD8 + T cells in AE-infected mice. Taken together the obstructed presenting-activity of AE-MØ appeared to trigger an unresponsiveness of T cells leading to the suppression of their clonal expansion during the chronic phase of AE infection. Interesting information on the parasite survival strategy and potential can be obtained upon in vitro and in vivo treatment. Hence, we provided very innovative results by showing that nitazoxanide, and now also, respectively, new modified compounds may represent a useful alternative to albendazole. In the context of chemotherapeutical repression of parasite growth, we searched also for parasite molecules, whose expression levels correlate with the viability and growth activity of E. multilocularis metacestode. Expression levels of 14-3-3 and II/3-10, relatively quantified by realtime reverse transcription-PCR using a housekeeping gene beta-actin, were studied in permissive nu/nu and in low-permissive wild type BALB/c mice. At 2 months p.i., the transcription level of 14-3-3 was significantly higher in parasites actively proliferating in nu/nu mice compared to parasites moderately growing in wild type mice. Immunoblotting experiments confirmed at the protein level that 14-3-3 was over-expressed in parasites derived from nu/nu mice at 2 months p.i. In vitro-treatment of E. multilocularis with an anti-echinococcal drug nitazoxanide for a period of 8 days resulted in a significant decrease of both 14-3-3 and II/3-10 transcription levels, which correlated with the kinetics of a housekeeping gene, beta-actin. This indicates that 14-3-3-exhibits a good potential as a molecular marker to assess viability and growth activity of the parasite.