Energy deficit does not affect immune responses of experimentally infected pacu (Piaractus mesopotamicus)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Humoral immune responses against the malaria vaccine candidate antigen Plasmodium vivax AMA-1 and IL-4 gene polymorphisms in individuals living in an endemic area of the Brazilian Amazon

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Humoral Immune Responses of White-Tailed Deer (Odocoileus virginianus) to Mycobacterium bovis BCG Vaccination and Experimental Challenge with M. bovis

Monitoring of the kinetics of production of serum antibodies to multiple mycobacterial antigens can be useful as a diagnostic tool for the detection of Mycobacterium bovis infection as well as for the characterization of disease progression and the efficacy of intervention strategies in several species. The humoral immune responses to multiple M. bovis antigens by white-tailed deer vaccinated with BCG orally via a lipid-formulated bait (n = 5), orally in liquid form (n = 5), and subcutaneously (n = 6) were evaluated over time after vaccination and after experimental challenge with virulent M. bovis and were compared to the responses by unvaccinated deer (n = 6). Antibody responses were evaluated by using a rapid test (RT), a multiantigen print immunoassay (MAPIA), a lipoarabinomannan enzyme-linked immunosorbent assay (LAM-ELISA), and immunoblotting to whole-cell sonicate and recombinant antigen MPB83. MAPIA and RT detected minimal to no antibody responses over those at the baseline to multiple M. bovis antigens in vaccinated white-tailed deer after challenge. This was in contrast to the presence of more readily detectable antibody responses in nonvaccinated deer with more advanced disease. The LAM-ELISA results indicated an overall decrease in the level of production of detectable antibodies against lipoarabinomannan-enriched mycobacterial antigen in vaccinated animals compared to that in nonvaccinated animals after challenge. Immunoblot data were inconsistent but did suggest the occurrence of unique antibody responses by certain vaccinated groups to Ag85 and HSP70. These findings support further research toward the improvement and potential use of antibody-based assays, such as MAPIA, RT, and LAM-ELISA, as tools for the antemortem assessment of disease progression in white-tailed deer in both experimental and field vaccine trials.

CD25(+) T cell depletion impairs murine squamous cell carcinoma development via modulation of antitumor immune responses

Squamous cell carcinoma (SCC) constitutes a microenvironment that could modulate the antitumor immune response. Also, tumor-infiltrating lymphocytes are believed to play complex regulatory roles in antitumor immunity against SCC. The presence of regulatory T cells (Tregs) has been associated with the suppression of tumor-reactive T cells. However, the underlying mechanism for this T cell dysfunction is not clear. We used a multistage model of SCC to examine the role of Treg cells during tumor development. 7,12-dimethylbenz[a]-anthracene/phorbol 12-myristate 13-acetate treatment and systemic depletion of Treg cells using an anti-CD25 monoclonal antibody (PC61) resulted in a decrease in the number and incidence of papilloma. Furthermore, CD25 depletion increased the proportion of CD8(+) and CD4(+) T cells that were isolated from tumor lesions. The levels of interleukin (IL)-1 beta, IL-10, IL-12, IL-13, interferon-gamma, transforming growth factor-beta and tumor necrosis factor-alpha, but not IL-17, were increased in the tumor microenvironment after Treg depletion. Therefore, our results indicated involvement of CD25(+) T cells in SCC development and in the suppression of the inflammatory immune response.

MyD88 Signaling Pathway Is Involved in Renal Fibrosis by Favoring a T(H)2 Immune Response and Activating Alternative M2 Macrophages

Inflammation contributes to the pathogenesis of chronic kidney disease (CKD). Molecules released by the inflamed injured tissue can activate toll-like receptors (TLRs), thereby modulating macrophage and CD4+ T-cell activity. We propose that in renal fibrogenesis, M2 macrophages are recruited and activated in a T helper subset 2 cell (TH2)-prone inflammatory milieu in a MyD88- dependent manner. Mice submitted to unilateral ureteral ligation (UUO) demonstrated an increase in macrophage infiltration with collagen deposition after 7 d. Conversely, TLR2, TLR4 and MyD88 knockout (KO) mice had an improved renal function together with diminished TH2 cytokine production and decreased fibrosis formation. Moreover, TLR2, TLR4 and MyD88 KO animals exhibited less M2 macrophage infiltration, namely interleukin (IL)-10+ and CD206+ CD11bhigh cells, at 7 d after surgery. We evaluated the role of a TH2 cytokine in this context, and observed that the absence of IL-4 was associated with better renal function, decreased IL-13 and TGF- β levels, reduced arginase activity and a decrease in fibrosis formation when compared with IL-12 KO and wild-type (WT) animals. Indeed, the better renal outcomes and the decreased fibrosis formation were restricted to the deficiency of IL-4 in the hematopoietic compartment. Finally, macrophage depletion, rather than the absence of T cells, led to reduced lesions of the glomerular filtration barrier and decreased collagen deposition. These results provide evidence that future therapeutic strategies against renal fibrosis should be accompanied by the modulation of the M1:M2 and TH1:TH2 balance, as TH2 and M2 cells are predictive of fibrosis toward mechanisms that are sensed by innate immune response and triggered in a MyD88-dependent pathway.

Proteins of Leishmania (Viannia) shawi confer protection associated with Th1 immune response and memory generation

Background: Leishmania (Viannia) shawi parasite was first characterized in 1989. Recently the protective effects of soluble leishmanial antigen (SLA) from L. (V.) shawi promastigotes were demonstrated using BALB/c mice, the susceptibility model for this parasite. In order to identify protective fractions, SLA was fractionated by reverse phase HPLC and five antigenic fractions were obtained. Methods: F1 fraction was purified from L. (V.) shawi parasite extract by reverse phase HPLC. BALB/c mice were immunized once a week for two consecutive weeks by subcutaneous routes in the rump, using 25 mu g of F1. After 1 and 16 weeks of last immunization, groups were challenged in the footpad with L. (V.) shawi promastigotes. After 2 months, those same mice were sacrificed and parasite burden, cellular and humoral immune responses were evaluated. Results: The F1 fraction induced a high degree of protection associated with an increase in IFN-gamma, a decrease in IL-4, increased cell proliferation and activation of CD8(+)T lymphocytes. Long-term protection was acquired in F1-immunized mice, associated with increased CD4(+) central memory T lymphocytes and activation of both CD4+ and CD8(+) T cells. In addition, F1-immunized groups showed an increase in IgG2a levels. Conclusions: The inductor capability of antigens to generate memory lymphocytes that can proliferate and secrete beneficial cytokines upon infection could be an important factor in the development of vaccine candidates against American Tegumentary Leishmaniasis.

Probiotic cheese attenuates exercise-induced immune suppression in Wistar rats

Intense physical activity results in a substantial volume of stress and hence a significant probability of immunosuppression in athletes, with milk proteins being, perhaps, the most recommended protein supplements. Consumption of a probiotic cheese can attenuate immune suppression induced by exhausting exercise in rats. A popular Brazilian fresh cheese (Minas Frescal cheese) containing Lactobacillus acidophilus LA14 and Bifidobacterium longum BL05 was fed for 2 wk to adult Wistar rats, which then were brought to exhaustion on the treadmill. Two hours after exhaustion, the rats were killed and material was collected for the determination of serum uric acid, total and high-density lipoprotein cholesterol fraction, total protein, triacylglycerols, aspartate aminotransferase, alanine aminotransferase, creatine kinase, and blood cell (monocyte, lymphocyte, neutrophil, and leukocyte) counts. Exercise was efficient in reducing lymphocyte counts, irrespective of the type of ingested cheese, but the decrease in the group fed the probiotic cheese was 22% compared with 48% in the animals fed regular cheese. Monocyte counts were unaltered in the rats fed probiotic cheese compared with a significant decrease in the rats fed the regular cheese. Most importantly, ingestion of the probiotic cheese resulted in a >100% increase in serum high-density lipoprotein cholesterol and a 50% decrease in triacylglycerols. We conclude that probiotic Minas Reseal cheese may be a viable alternative to enhance the immune system and could be used to prevent infections, particularly those related to the physical overexertion of athletes.

Current perspectives in avian salmonellosis: Vaccines and immune mechanisms of protection

Salmonellosis is one of the most prevalent foodborne diseases worldwide. Food animals have been identified as reservoirs for nontyphoid Salmonella infections. in poultry, host-specific Salmonella infections cause fowl typhoid and pullorum diseases that produce economic losses in different parts of the world. Several measures have been used to prevent and control Salmonella infections in poultry, and vaccination is the most practical measure because it avoids contamination of poultry products and by-products and prevents disease in humans. Salmonella vaccines can decrease public health risk by reducing colonization and organ invasion, including invasion of reproductive tissues, and by diminishing fecal shedding and environmental contamination. We review available information on the host-specific and non-host-specific Salmonella serotypes found in poultry and the improved understanding of the pathogenesis of and immune responses to infection. We also include some approaches based on updated publications regarding killed and live attenuated vaccines and their immune mechanisms of protection.

The impact of rising sea temperature on innate immune parameters in the tropical subtidal sea urchin Lytechinus variegatus and the intertidal sea urchin Echinometra lucunter

Ocean temperatures are rising throughout the world, making it necessary to evaluate the impact of these temperature changes on sea urchins, which are well-known bioindicators. This study evaluated the effect of an increase in temperature on the immune response of the subtidal Lytechinus variegatus and the intertidal Echinometra lucunter sea urchins. Both species were exposed to 20 (control), 25 and 30 °C temperatures for 24 h, 2, 7 and 14 days. Counting of coelomocytes and assays on the phagocytic response, adhesion and spreading of coelomocytes were performed. Red and colorless sphere cells were considered biomarkers for heat stress. Moreover, a significant decrease in the phagocytic indices and a decrease in both cell adhesion and cell spreading were observed at 25 and 30 °C for L. variegatus. For E. lucunter, the only alteration observed was for the cell proportions. This report shows how different species of sea urchins respond immunologically to rising temperatures

An interferon regulatory factor element controls the major immune modulator of murine cytomegalovirus

Immune modulation by herpesviruses, such as cytomegalovirus, is critical for the establishment of acute and persistent infection confronting a vigorous antiviral immune response of the host. Therefore, the action of immune-modulatory proteins has long been the subject of research, with the final goal to identify new strategies for antiviral therapy.rnIn the case of murine cytomegalovirus (mCMV), the viral m152 protein has been identified to play a major role in targeting components of both the innate and the adaptive immune system in terms of infected host-cell recognition in the effector phase of the antiviral immune response. On the one hand, it inhibits cell surface expression of RAE-1 and thereby prevents ligation of the activating natural killer (NK)-cell receptor NKG2D. On the other hand, it decreases cell surface expression of peptide-loaded MHC class I molecules thereby preventing antigen presentation to CD8 T cells. Ultimately, the outcome of CMV infection is determined by the interplay between viral and cellular factors.rnIn this context, the work presented here has revealed a novel and intriguing connection between viral m152 and cellular interferon (IFN), a key cytokine of the immune system: rnthe m152 promoter region contains an interferon regulatory factor element (IRFE) perfectly matching the consensus sequence of cellular IRFEs.rnThe biological relevance of this regulatory element was first suggested by sequence comparisons revealing its evolutionary conservation among various established laboratory strains of mCMV and more recent low-passage wild-derived virus isolates. Moreover, search of the mCMV genome revealed only three IRFE sites in the complete sequence. Importantly, the functionality of the IRFE in the m152 promoter was confirmed with the use of a mutant virus, representing a functional deletion of the IRFE, and its corresponding revertant virus. In particular, m152 gene expression was found to be inhibited in an IRFE-dependent manner in infected cells. Essentially, this inhibition proved to have a severe impact on the immune-modulatory function of m152, first demonstrated by a restored direct antigen presentation on infected cells for CD8 T-cell activation. Even more importantly, this effect of IRFE-mediated IFN signaling was validated in vivo by showing that the protective antiviral capacity of adoptively-transferred, antigen-specific CD8 T cells is also significantly restored by the IRFE-dependent inhibition of m152. Somewhat curious and surprising, the decrease in m152 protein simultaneously prevented an enhanced activation of NK cells in acute-infected mice, apparently independent of the RAE-1/NKG2D ligand/receptor interaction but rather due to reduced ‘missing-self’ recognition.rnTaken together, this work presents a so far unknown mechanism of IFN signaling to control mCMV immune modulation in acute infection.rnrn

Chlamydiae host cell interaction and immune evasion strategies

Chlamydiae are obligate intracellular bacteria with a strong global prevalence. They cause infections of the eye, lung and the genital tract and can either replicate in inclusion compartments or persist inside their host cell. In this thesis we focused on two aspects of chlamydiae infection. We hypothesize that transcription factor AP-1 is crucial for a replicative chlamydiae infection in epithelial cells. In addition we suggest that chlamydiae hide inside apoptotic blebs for a silent uptake by macrophages as immune evasion strategy.rnFocusing on AP-1, we could demonstrate that during Chlamydia pneumoniae infection, protein expression and phosphorylation of the AP-1 family member c-Jun significantly increased in a time and dose dependent manner. A siRNA knockdown of c-Jun in HEp-2 cells reduced chlamydial load, resulting in smaller inclusions and a significant lower chlamydial recovery. Furthermore, inhibition of the c-Jun containing AP-1 complexes, using Tanshinone IIA, changed the replicative infection into a persistent phenotype, characterized by (i) smaller, aberrant inclusions, (ii) a strong decrease in chlamydial load, as well as by (iii) its reversibility after removal of Tanshinone IIA. As chlamydiae are energy parasites, we investigated whether Tanshinone IIA interferes with energy/metabolism related processes. rnA role for autophagy or gene expression of glut-1 and c-jun in persistence could not be determined. However we could demonstrate Tanshinone IIA treatment to be accompanied by a significant decrease of ATP levels, probably causing a chlamydiae persistent phenotype.rnRegarding the chlamydial interaction with human primary cells we characterized infection of different chlamydiae species in either pro-inflammatory (type I) or anti-inflammatory (type II) human monocyte derived macrophages (hMDM). We found both phenotypes to be susceptible to chlamydiae infection. Furthermore, we observed that upon Chlamydia trachomatis and GFP-expressing Chlamydia trachomatis infection more hMDM type II were infected. However the chlamydial load was higher in hMDM type I and correspondingly, more replicative-like inclusions were found in this phenotype. Next, we focused on the chlamydial transfer using a combination of high speed live cell imaging and GFP-expressing Chlamydia trachomatis for optimal visualization. Thereby, we could successfully visualize the formation of apoptotic, chlamydiae-containing blebs and the interaction of hMDM with these blebs. Moreover, we observed the development of a replicative infection in hMDM. rnIn conclusion, we demonstrated a crucial role of AP-1 for C. pneumoniae development and preliminary time lapse data suggest that chlamydiae can be transferred to hMDMs via apoptotic blebs. In all, these data may contribute to a better understanding of chlamydial infection processes in humans.rn

Gerbu adjuvant modulates the immune response and thus the course of infection in C56BL/6 mice immunised with Echinococcus multilocularis rec14-3-3 protein

Vaccination with Echinococcus multilocularis 14-3-3 protein can protect mice against primary E. multilocularis infection. The present study investigated the efficacy and efficiency of the adjuvant muramyl dipeptide Gerbu, alone or together with recombinant 14-3-3 protein, to modulate the course of secondary E. multilocularis infection in C56BL/6 mice. The application of Gerbu alone already resulted in a parasite weight reduction when compared with infected control mice, while rec14-3-3 did not add to this effect. Immunological parameters were concurrently assessed with a mixed cell reaction including bone marrow-derived dendritic cells (BMDCs) together with lymph node cells from mice with or without immunisation and/or infection. While mice having received Gerbu adjuvant were found to highly proliferate in response to co-cultivation with 14-3-3-stimulated bone marrow dendritic cells, a sensitisation of BMDCs with vesicle fluid (VF) antigen lead to a striking decrease of the lymphoproliferative response in comparison to that of control mice, raising the hypothesis that immunosuppressive components may be part of this VF-antigen. Anti-14-3-3 antibody production was only found in those mice that had been previously 14-3-3-immunised, whereas all other only-infected mice failed to produce such antibodies. Conclusively, Gerbu adjuvant appears to directly generate a non-specific immune response that contributes to the control of the metacestode growth, putatively in association with a BMDC activity suppressed by components of the VF-antigen.

Early and nonreversible decrease of CD161++ /MAIT cells in HIV infection

HIV infection is associated with immune dysfunction, perturbation of immune-cell subsets and opportunistic infections. CD161++ CD8+ T cells are a tissue-infiltrating population that produce IL17A, IL22, IFN, and TNFα, cytokines important in mucosal immunity. In adults they dominantly express the semi-invariant TCR Vα7.2, the canonical feature of mucosal associated invariant T (MAIT) cells and have been recently implicated in host defense against pathogens. We analyzed the frequency and function of CD161++ /MAIT cells in peripheral blood and tissue from patients with early stage or chronic-stage HIV infection. We show that the CD161++ /MAIT cell population is significantly decreased in early HIV infection and fails to recover despite otherwise successful treatment. We provide evidence that CD161++ /MAIT cells are not preferentially infected but may be depleted through diverse mechanisms including accumulation in tissues and activation-induced cell death. This loss may impact mucosal defense and could be important in susceptibility to specific opportunistic infections in HIV.

Effects of tributyltin on the immune system of Japanese flounder (Paralichthys olivaceus)

Effects of tributyltin (TBT) which has been used for antifouling paint of ship's hulls and fishing nets on the immune system in Japanese flounder (Paralichthys olivaceus) were investigated. After short-term exposure to a high level of TBT, leucocytes in the head kidney from 1-year-old flounder were examined for the proportion of neutrophils in total leucocytes. Also examined were their respiratory burst activities using flow cytometry, the reduction of nitroblue tetrazolium (NBT) and lysozyme activities. Furthermore, long-term exposures to a relatively low level of TBT using young flounder were also carried out. The proportion of neutrophils in total leucocytes prepared from head kidney in each fish exposed to TBT at 20 microg/L for 5 days and the reduction of NBT by leucocytes prepared from the same experimental conditions increase compared to the control group. The contents were 42.0+/-6.8 and 52.5+/-6.3%, respectively. Significant differences of the NBT reduction were observed between 0 and 20 microg/L TBT exposure groups. On the other hand, the respiratory burst activity of cells in the exposure group clearly showed a tendency to decrease compared to the control group. Furthermore, high level of TBT also inhibited lysozyme activity which plays an important role for the bacteriocidal procedures. However, similar results were not obtained in the exposure group with a relatively low level of TBT. To determine the immunotoxic effects of TBT, infection experiments using pathogens which are naturally occurring should be further investigated.

Persistence of circulating ADAMTS13-specific immune complexes in patients with acquired thrombotic thrombocytopenic purpura

Anti-ADAMTS13 autoantibodies are the main cause of acquired thrombotic thrombocytopenic purpura. Binding of these antibodies to ADAMTS13 eventually results in the formation of antigen-antibody immune complexes. Circulating ADAMTS13-specific immune complexes have been described in acquired thrombotic thrombocytopenic purpura patients, however, the prevalence and persistence of these immune complexes over time has hitherto remained elusive. Here, we analyzed a large cohort of patients with acquired thrombotic thrombocytopenic purpura for the presence of free and complexed anti-ADAMTS13 antibodies. In the acute phase (n=68), 100% of patients had free IgG antibodies and 97% had ADAMTS13-specific immune complexes. In remission (n=28), 75% of patients had free antibodies (mainly IgG) and 93% had ADAMTS13-specific immune complexes. Free antibodies were mainly of subclasses IgG1 and IgG4, whereas IgG4 was by far the most prevalent in ADAMTS13-specific immune complexes. Comparison of ADAMTS13 inhibitor and anti-ADAMTS13 IgG (total and subclasses) antibody titers in acute phase and in remission samples showed a statistically significant decrease in all parameters in remission. Although non-significant, a trend towards reduced or undetectable titers in remission was also observed for ADAMTS13-specific immune complexes of subclasses IgG1, IgG2 and IgG3. For IgG4, no such trend was discernible; IgG4 immune complexes persisted over years, even in patients who had been treated with rituximab and who showed no features suggesting relapse.