Immunoregulatory mechanisms in Chagas disease: modulation of apoptosis in T-cell mediated immune responses.

BACKGROUND: Chronic Chagas disease presents different clinical manifestations ranging from asymptomatic (namely indeterminate) to severe cardiac and/or digestive. Previous results have shown that the immune response plays an important role, although no all mechanisms are understood. Immunoregulatory mechanisms such as apoptosis are important for the control of Chagas disease, possibly affecting the morbidity in chronic clinical forms. Apoptosis has been suggested to be an important mechanism of cellular response during T. cruzi infection. We aimed to further understand the putative role of apoptosis in Chagas disease and its relation to the clinical forms of the disease. METHODS: Apoptosis of lymphocytes, under antigenic stimuli (soluble T. cruzi antigens - TcAg) where compared to that of non-stimulated cells. Apoptosis was evaluated using the expression of annexin and caspase 3(+) by T cells and the percentage of cells positive evaluated by flow cytometry. In addition activation and T cell markers were used for the identification of TCD4(+) and TCD8(+) subpopulations. The presence of intracellular and plasma cytokines were also evaluated. Analysis of the activation status of the peripheral blood cells showed that patients with Chagas disease presented higher levels of activation determined by the expression of activation markers, after TcAg stimulation. PCR array were used to evaluate the contribution of this mechanism in specific cell populations from patients with different clinical forms of human Chagas disease. RESULTS: Our results showed a reduced proliferative response associated a high expression of T CD4(+)CD62L(-) cells in CARD patients when compared with IND group and NI individuals. We also observed that both groups of patients presented a significant increase of CD4(+) and CD8(+) T cell subsets in undergoing apoptosis after in vitro stimulation with T. cruzi antigens. In CARD patients, both CD4(+) and CD8(+) T cells expressing TNF-α were highly susceptible to undergo apoptosis after in vitro stimulation. Interestingly, the in vitro TcAg stimulation increased considerably the expression of cell death TNF/TNFR superfamily and Caspase family receptors genes in CARD patients. CONCLUSIONS: Taken together, our results suggest that apoptosis may be an important mechanism for the control of morbidity in T. cruzi infection by modulating the expression of apoptosis genes, the cytokine environment and/or killing of effector cells.

Public engagement for the conduct of a controlled human infection study testing vaccines against Necator americanus (hookworm) in areas of active hookworm transmission in Brazil.

Controlled Human Infection Models (CHIS) involve administering human pathogens to healthy participants in controlled medical settings, which can elicit complex bioethical issues. Understanding how the community perceives such studies can significantly increase the participant's sense of cooperation and increases the researcher's and the participant's transparency. The current study describes the development of an educational intervention to achieve these ends as it aims to (1) analyze perceptions of the Controlled Human Infection Studies (CHIS), and (2) evaluate the participants' comprehension of the CHIS. METHODS: This is a qualitative action research that includes the development of an educational intervention with residents of a rural area in Minas Gerais, Brazil, where there is continuous natural transmission of the human pathogen Necator americanus ("hookworm"). In this area, it is intended to carry out a proposed phase 3 vaccine clinical trial in the future to test the efficacy of hookworm vaccines using controlled human infection. Two data collection strategies were used: an educational intervention and a focus group. RESULTS: The participants' perceptions showed distinct perspectives on CHIS. On one side, they recognized that the investigation is essential for the community, but on the other side, they thought that there would be resistance to its conduct by fear of infection. The idea that the study would generate a benefit for the greater good, contributing to the prevention of hookworm infection, was clearly stated. The participants perceived that the study offered concrete risks that could be reduced by constant monitoring by the researchers. They also mentioned the importance of access to information and the positive influence those who express interest in participating in the study can exert in the community. In relation to comprehension the participants memorized the information, mobilized it to explain everyday situations and created strategies to disseminate the study and engage the community in its development. By repeating and making sense of the information, the participant not only assimilates the knowledge transmitted, but also creates new knowledge. CONCLUSION: We concluded that an educational process of discussion and dialogue around participants' perceptions about the CHIS, promotes understanding and allows ways to disseminate information about the research to be collectively created.

Co-expression of human sialyltransferase improves N-glycosylation in Leishmania tarentolae and optimizes the production of humanized therapeutic glycoprotein IFN-beta.

The production of therapeutic glycoproteins is primarily expensive due to the necessity of culturing mammalian cells. These systems often require complex and costly culture media and typically yield low amounts of protein. Leishmania tarentolae, a non-pathogenic protozoan to mammals, has emerged as a cost-effective alternative system for heterologous glycoprotein expression due to its suitability for large-scale production using low-cost culture media, and its ability to perform mammalian-like post-translational modifications, including glycosylation. Nevertheless, differences in the carbohydrate residues at the end of N-glycan chains are observed in Leishmania compared to mammalian cells due to the absence of biosynthetic enzymes in Leishmania that are required for the incorporation of terminal sialic acid. In this study, a genetically optimized L. tarentolae cell line was engineered for the production of recombinant interferon-ß (IFN-ß) featuring a complete mammalian N-glycosylation profile. Genomic and metabolomic analyses revealed that heterologous expression of the sialyltransferase enzyme and cultivation in a medium containing sialic acid were sufficient to generate mammalian-like protein N-glycosylation. N-glycan mass spectrometry analysis demonstrated a glycosylation pattern compatible with the incorporation of sialic acid into the glycan structure. In vitro IFN-ß activity indicated that the expressed protein exhibited reduced inflammatory effects compared to IFN-beta produced by other platforms, such as bacteria, non-optimized L. tarentolae, and mammalian cells.

Deletion of MIF gene from live attenuated LdCen-/- parasites enhances protective CD4+ T cell immunity.

Vaccination with live attenuated Leishmania parasites such as centrin deleted Leishmania donovani (LdCen-/-) against visceral leishmaniasis has been reported extensively. The protection induced by LdCen-/- parasites was mediated by both CD4+ and CD8+ T cells. While the host immune mediators of protection are known, parasite determinants that affect the CD4+ and CD8+ T cell populations remain unknown. Parasite encoded inflammatory cytokine MIF has been shown to modulate the T cell differentiation characteristics by altering the inflammation induced apoptosis during contraction phase in experimental infections with Leishmania or Plasmodium. Neutralization of parasite encoded MIF either by antibodies or gene deletion conferred protection in Plasmodium and Leishmania studies. We investigated if the immunogenicity and protection induced by LdCen-/- parasites is affected by deleting MIF genes from this vaccine strain. Our results showed that LdCen-/-MIF-/- immunized group presented higher percentage of CD4+ and CD8+ central memory T cells, increased CD8+ T cell proliferation after challenge compared to LdCen-/- immunization. LdCen-/-MIF-/- immunized group presented elevated production of IFN-γ+ and TNF-α+ CD4+ T cells concomitant with a reduced parasite load in spleen and liver compared to LdCen-/-group following challenge with L. infantum. Our results demonstrate the role of parasite induced factors involved in protection and long-term immunity of vaccines against VL.

Retrospective cohort study to evaluate the continuous use of anticholesterolemics and diuretics in patients with COVID-19.

Purpose: The purpose of this study is to evaluate the interference of the continuous use of drug classes in the expression of biomarkers during the first week of hospitalization and in the prognosis of patients with COVID-19. Methods: The patients diagnosed with COVID-19 and confirmed with SARS-CoV-2 by RT-qPCR assay underwent the collection of fasting whole blood samples for further analysis. Other data also extracted for this study included age, sex, clinical symptoms, related comorbidities, smoking status, and classes of continuous use. Routine serum biochemical parameters, including alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, C-reactive protein, N-terminal fragment of B-type natriuretic peptide, and cardiac troponin, were measured. Results: In this cross-sectional study, a total of 176 patients with COVID-19 hospitalizations were included. Among them, 155 patients were discharged (88.5%), and 21 patients died (12%). Among the drug classes evaluated, we verified that the continuous use of diuretic 4.800 (1.853-11.67) (p = 0.0007) and antihypercholesterolemic 3.188 (1.215-7.997) (p = 0.0171) drug classes presented a significant relative risk of death as an outcome when compared to the group of patients who were discharged. We evaluated biomarkers in patients who used continuous antihypercholesterolemic and diuretic drug classes in the first week of hospitalization. We observed significant positive correlations between the levels of CRP with cardiac troponin (r = 0.714), IL-6 (r = 0.600), and IL-10 (r = 0.900) in patients who used continuous anticholesterolemic and diuretic drug classes and were deceased. In these patients, we also evaluated the possible correlations between the biomarkers AST, NT-ProBNP, cardiac troponin, IL-6, IL-8, and IL-10. We observed a significantly negative correlations in AST levels with NT-ProBNP (r = -0.500), cardiac troponin (r = -1.00), IL-6 (r = -1.00), and IL-10 (r = -1.00) and a positive correlation with IL-8 (r = 0.500). We also observed significant negative correlation in the levels of NT-ProBNP with IL-10 (r = -0.800) and a positive correlation with cardiac troponin (r = 0.800). IL-6 levels exhibited positive correlations with cardiac troponin (r = 0.800) and IL-10 (r = 0.700). Conclusion: In this study, we observed that hospitalized COVID-19 patients who continued using anticholesterolemic and diuretic medications showed a higher number of correlations between biomarkers, indicating a poorer clinical prognosis. These correlations suggest an imbalanced immune response to injuries caused by SARS-CoV-2.

Design and production of dengue virus chimeric proteins useful for developing tetravalent vaccines.

Dengue virus (DENV) is a Flavivirus estimated to cause 390 million infections/year. Currently, there is no anti-viral specific treatment for dengue, and efficient DENV vector control is still unfeasible. Here, we designed and produced chimeric proteins containing potential immunogenic epitopes from the four DENV serotypes in an attempt to further compose safer, balanced tetravalent dengue vaccines. For this, South American DENV isolate sequences were downloaded from the NCBI/Virus Variation/Dengue virus databases and intraserotype-aligned to generate four consensuses. Four homologous DENV sequences were retrieved using BLAST and then interserotype-aligned. In parallel, sequences were subjected to linear B epitope prediction analysis. Regions of the envelope and NS1 proteins that are highly homologous among the four DENV serotypes, non-conserved antigenic regions and the most antigenic epitopes found in the C, prM, E and NS1 DENV proteins were used to construct 11 chimeric peptides. Genes encoding the chimeric proteins were commercially synthesized, and proteins were expressed, purified by affinity chromatography and further subjected to ELISA assays using sera from individuals infected with DENVs 1, 2, 3 or 4. As a proof-of-concept, the chimeric EnvEpII protein was selected to immunize BALB/c and C57BL/6 mice strains. The immunization with EnvEpII protein associated with aluminum induced an increased number of T CD4+ and CD8+ cells, high production of IgG1 and IgG2 antibodies, and increased levels of IL-2 and IL-17 cytokines, in both mouse strains. Because the EnvEpII protein associated with aluminum induced an efficient cellular response by stimulating the production of IL-2, IL-4, IL-17 and induced a robust humoral response in mice, we conclude that it resembles an efficient specific response against DENV infection. Although further experiments are required, our results indicate that epitope selection by bioinformatic tools is efficient to create recombinant proteins that can be used as candidates for the development of vaccines against infectious diseases.

HIV aspartyl protease inhibitors modify the percentage of activated leukocytes, as well as serum levels of IL-17A and NO during experimental leishmaniasis.

HIV aspartyl protease inhibitors are able to modulate multiple defense mechanisms. However, their influence on the immune response against Leishmania has rarely been investigated. The aim of our study was to investigate whether in vivo treatment with HIV aspartyl protease inhibitors is able to modulate the immune response during Leishmania infection. Using Leishmania (L.) amazonensis-infected mice, we analyzed the disease evolution and parasite load, immunophenotypic profiles of splenic T and B lymphocytes, numbers of lymphoid aggregates in the spleen, percentages of circulating atypical lymphocytes and reactive monocytes, and serum levels of cytokines and nitric oxide (NO) after 30 days of oral treatment with lopinavir/ritonavir (LPV/RTV) or atazanavir (ATV). We observed that LPV/RTV and ATV did not modify the disease evolution or parasite load. However, the antiretroviral treatment induced an increase in activated lymphocytes in the spleen and blood, as well as a decrease in CD69 expression in T and B lymphocytes in the spleen. The treatment also resulted in an increase in activated monocytes in the blood. In addition, antiretrovirals decreased levels of IL-17A and increased levels of NO in sera from Leishmania-infected mice. Thus, our results demonstrate for the first time that in vivo treatment with HIV aspartyl protease inhibitors modifies innate and adaptative immune responses during Leishmania infection and suggest that these drugs could change the clinical course of leishmaniasis in HIV infected-individuals.

Growth arrested live-attenuated Leishmania infantum KHARON1 null mutants display cytokinesis defect and protective immunity in mice.

There is no safe and efficacious vaccine against human leishmaniasis available and live attenuated vaccines have been used as a prophylactic alternative against the disease. In order to obtain an attenuated Leishmania parasite for vaccine purposes, we generated L. infantum KHARON1 (KH1) null mutants (ΔLikh1). This gene was previously associated with growth defects in L. mexicana. ΔLikh1 was obtained and confirmed by PCR, qPCR and Southern blot. We also generate a KH1 complemented line with the introduction of episomal copies of KH1. Although ΔLikh1 promastigote forms exhibited a growth pattern similar to the wild-type line, they differ in morphology without affecting parasite viability. L. infantum KH1-deficient amastigotes were unable to sustain experimental infection in macrophages, forming multinucleate cells which was confirmed by in vivo attenuation phenotype. The cell cycle analysis of ΔLikh1 amastigotes showed arrested cells at G2/M phase. ΔLikh1-immunized mice presented reduced parasite burden upon challenging with virulent L. infantum, when compared to naïve mice. An effect associated with increased Li SLA-specific IgG serum levels and IL-17 production. Thus, ΔLikh1 parasites present an infective-attenuated phenotype due to a cytokinesis defect, whereas it induces immunity against visceral leishmaniasis in mouse model, being a candidate for antileishmanial vaccine purposes.

Intradermal Immunization of Leishmania donovani Centrin Knock-Out Parasites in Combination with Salivary Protein LJM19 from Sand Fly Vector Induces a Durable Protective Immune Response in Hamsters.

BACKGROUND: Visceral leishmaniasis (VL) is a neglected tropical disease and is fatal if untreated. There is no vaccine available against leishmaniasis. The majority of patients with cutaneous leishmaniasis (CL) or VL develop a long-term protective immunity after cure from infection, which indicates that development of an effective vaccine against leishmaniasis is possible. Such protection may also be achieved by immunization with live attenuated parasites that do not cause disease. We have previously reported a protective response in mice, hamsters and dogs with Leishmania donovani centrin gene knock-out parasites (LdCen-/-), a live attenuated parasite with a cell division specific centrin1 gene deletion. In this study we have explored the effects of salivary protein LJM19 as an adjuvant and intradermal (ID) route of immunization on the efficacy of LdCen-/- parasites as a vaccine against virulent L. donovani. METHODOLOGY/PRINCIPAL FINDINGS: To explore the potential of a combination of LdCen-/- parasites and salivary protein LJM19 as vaccine antigens, LdCen-/- ID immunization followed by ID challenge with virulent L. donovani were performed in hamsters in a 9-month follow up study. We determined parasite burden (serial dilution), antibody production (ELISA) and cytokine expression (qPCR) in these animals. Compared to controls, animals immunized with LdCen-/- + LJM19 induced a strong antibody response, a reduction in spleen and liver parasite burden and a higher expression of pro-inflammatory cytokines after immunization and one month post-challenge. Additionally, a low parasite load in lymph nodes, spleen and liver, and a non-inflamed spleen was observed in immunized animals 9 months after the challenge infection. CONCLUSIONS: Our results demonstrate that an ID vaccination using LdCen-/-parasites in combination with sand fly salivary protein LJM19 has the capability to confer long lasting protection against visceral leishmaniasis that is comparable to intravenous or intracardial immunization.

Application of rapid in vitro co-culture system of macrophages and T-cell subsets to assess the immunogenicity of dogs vaccinated with live attenuated Leishmania donovani centrin deleted parasites (LdCen-/-).

BACKGROUND: Live attenuated Leishmania donovani parasites as LdCen(-/-) were shown to confer protective immunity against Leishmania infection in mice, hamsters, and dogs. Strong immunogenicity in dogs vaccinated with LdCen(-/-) has been previously reported, including increased antibody response favoring Th1 response lymphoproliferative responses, CD4(+) and CD8(+) T-cells activation, increased levels of Th1 and reduction of Th2 cytokines, in addition to a significant reduction in parasite burden after 18 and 24 months post virulent parasite challenge. METHODS: Aimed at validating a new method using in vitro co-culture systems with macrophages and purified CD4(+) or CD8(+) or CD4(+):CD8(+) T-cells of immunized dogs with both LdCen(-/-) and Leishmune® to assess microbicide capacity of macrophages and the immune response profile as the production of IFN-γ, TNF-α, IL-12, IL-4 and IL-10 cytokines. RESULTS AND DISCUSSION: Our data showed co-cultures of macrophages and purified T-cells from dogs immunized with LdCen(-/-) and challenged with L. infantum were able to identify high microbicidal activity, especially in the co-culture using CD4(+) T-cells, as compared to the Leishmune® group. Similarly, co-cultures with CD8(+) T-cells or CD4(+):CD8(+) T-cells in both experimental groups were able to detect a reduction in the parasite burden in L. infantum infected macrophages. Moreover, co-cultures using CD4(+) or CD8(+) or CD4(+):CD8(+) T-cells from immunized dogs with both LdCen(-/-) and Leishmune® were able to identify higher levels of IFN-γ and IL-12 cytokines, reduced levels of IL-4 and IL-10, and a higher IFN-γ/IL-10 ratio. While the highest IFN-γ levels and IFN-γ/IL-10 ratio were the hallmarks of LdCen(-/-) group in the co-culture using CD4(+) T-cells, resulting in strong reduction of parasitism, the Leishmune® immunization presented a differential production of TNF-α in the co-culture using CD4(+):CD8(+) T-cells. CONCLUSION: The distinct conditions of co-culture systems were validated and able to detect the induction of immune protection. The method described in this study applied a new, more accurate approach and was able to yield laboratory parameters useful to test and monitor the immunogenicity and efficacy of Leishmania vaccines in dogs.

Arginine Supplementation Induces Arginase Activity and Inhibits TNF-α Synthesis in Mice Spleen Macrophages After Intestinal Obstruction.

BACKGROUND: The purpose of this study was to assess the effect of arginine supplementation on arginase activity, tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) synthesis in cultured splenic macrophages from a murine model of intestinal obstruction (IO). The effects of nitric oxide synthase (iNOS) inhibition were also studied using iNOS knockout animals. MATERIAL AND METHODS: Male C57BL6/J wild-type (WT) and iNOS knockout (iNOS-/-) mice were randomized into 6 groups: Sham and Sham-/- (standard chow), IO and IO-/- (standard chow + IO), and Arg and Arg-/- (standard chow supplemented with arginine + IO). After 7 days of treatment with standard or supplemented chow, IO was induced. Arginase activity as well as TNF-α and IL-10 levels were analyzed in splenic macrophage cultures. RESULTS: Arginine supplementation and the absence of iNOS increased arginase activity in splenic macrophages (Arg, IO-/-, and Arg-/- groups vs the Sham group; P < .05). Arginine was also related to a decrease in TNF-α levels (Arg vs IO group, P < .05) and maintenance of IL-10 levels (Arg vs other groups, P > .05). The inhibition of iNOS did not result in effects on the concentration of cytokines (Sham-/-, IO-/-, and Arg-/- vs other, P < .05). CONCLUSIONS: Arginine supplementation and iNOS inhibition led to increased arginase activity. Arginine availability decreased plasma TNF-α levels, which may be directly related to nitric oxide derived from arginine.

Vaccination using live attenuated Leishmania donovani centrin deleted parasites induces protection in dogs against Leishmania infantum.

Live attenuated Leishmania donovani parasites such as LdCen(-/-) have been shown elicit protective immunity against leishmanial infection in mice and hamster models. Previously, we have reported on the induction of strong immunogenicity in dogs upon vaccination with LdCen(-/-) including an increase in immunoglobulin isotypes, higher lymphoproliferative response, higher frequencies of activated CD4(+) and CD8(+) T cells, IFN-γ production by CD8(+) T cells, increased secretion of TNF-α and IL-12/IL-23p40 and, finally, decreased secretion of IL-4. To further explore the potential of LdCen(-/-) parasites as vaccine candidates, we performed a 24-month follow up of LdCen(-/-) immunized dogs after challenge with virulent Leishmania infantum, aiming determination of parasite burden by qPCR, antibody production (ELISA) and cellular responses (T cell activation and cytokine production) by flow cytometry and sandwich ELISA. Our data demonstrated that vaccination with a single dose of LdCen(-/-) (without any adjuvant) resulted in the reduction of up to 87.3% of parasite burden after 18 months of virulent challenge. These results are comparable to those obtained with commercially available vaccine in Brazil (Leishmune(®)). The protection was associated with antibody production and CD4(+) and CD8(+) proliferative responses, as well as T cell activation and significantly higher production of IFN-γ, IL-12/IL-23p40 and TNF-α, which was comparable to responses induced by immunization with Leishmune(®), with significant differences when compared to control animals (Placebo). Moreover, only animals immunized with LdCen(-/-) expressed lower levels of IL-4 when compared to animals vaccinated either with Leishmune(®) or PBS. Our results support further studies aiming to demonstrate the potential of genetically modified live attenuated L. donovani vaccine to control L. infantum transmission in endemic areas for CVL.

Inflammatory mediators from monocytes down-regulate cellular proliferation and enhance cytokines production in patients with polar clinical forms of Chagas disease.

Exposure to Trypanosoma cruzi parasites induces monocytes and macrophages to produce various endogenous mediators, including prostaglandins and cytokines. To clarify the involvement of monocytes as an important source of inflammatory mediators in Chagas disease patients, we evaluated PBMC before and after depletion of adherent cells (monocytes) from patients with indeterminate (IND) and cardiac (CARD) clinical forms and from non-infected individuals (NI). We demonstrated that after the partial depletion of adherent cells, production of PGE2 was slightly decreased in patients with Chagas disease. Inhibition of the cells by indomethacin increased the proliferation in PBMC cells from patients after antigen stimulation. Pro-inflammatory cytokines as IL-2 and IFN-γ also had a greater decrease after partial depletion of adherent cells in both clinical forms of Chagas disease. IL-10 and IL-5 levels were also reduced after partial depletion of adherent cells both in IND and CARD patients. In addition, we evaluated the APC potential of B cells and observed that the MHCII and CD80 molecules had an increased expression after partial depletion of most monocytes in all groups. Thus, inflammatory mediators produced by monocytes seem to be important to modulate immune responses in Chagas disease by regulating the processes of inflammation and antigen presentation.

Induction of immunogenicity by live attenuated Leishmania donovani centrin deleted parasites in dogs.

Zoonotic visceral leishmaniasis, caused by the intracellular protozoan parasite Leishmania infantum, is a neglected tropical disease that is often fatal when untreated. Dogs are considered the main reservoir of L. infantum in zoonotic VL as the presence of infected dogs may increase the risk for human infection. Canine visceral leishmaniasis (CVL) is a major veterinary and public health problem in Southern Europe, Middle East and South America. Control of animal reservoirs relies on elimination of seropositive dogs in endemic areas. However, treatment of infected dogs is not considered a favorable approach as this can lead to emergence of drug resistance since the same drugs are used to treat human infections. Therefore, vaccination against CVL remains the best alternative in control of the animal reservoirs. In this study, we present data on the immunogenicity profile of a live attenuated parasite LdCen(-/-) in a canine infection model and compared it to that of Leishmune(®), a commercially available recombinant vaccine. The immunogenicity of the LdCen(-/-) parasites was evaluated by antibody secretion, production of intracytoplasmic and secreted cytokines, activation and proliferation of T cells. Vaccination with LdCen(-/-) resulted in high immunogenicity as revealed by the higher IgGTotal, IgG1, and IgG2 production and higher lymphoproliferative response. Further, LdCen(-/-) vaccinated dogs showed higher frequencies of activated CD4+ and CD8+ T cells, IFN-γ production by CD8+ T cells, increased secretion of TNF-α and IL-12/IL-23p40 and decreased secretion of IL-4. These results contribute to the understanding of immunogenicity elicited by live attenuated L. donovani parasites and, consequently, to the development of effective vaccines against visceral leishmaniasis.

The role of L-arginine and inducible nitric oxide synthase in intestinal permeability and bacterial translocation.

BACKGROUND: Arginine has been shown to have several immunological and trophic properties in stressful diseases. Its metabolites, nitric oxide (NO) and polyamines, are related to arginine's effects. Thus, the aim of this study was to determine the effects of the NO donor L-arginine and the role of inducible NO synthase (iNOS) on intestinal permeability and bacterial translocation in a model of intestinal obstruction (IO) induced by a simple knot in the terminal ileum. MATERIAL AND METHODS: Male C57BL6/J wild-type (WT) and iNOS knockout (iNOS-/-) mice were randomized into 6 groups: Sham and Sham-/- (standard chow), IO and IO-/- (standard chow +IO), and Arg and Arg-/- (standard chow supplemented with arginine + IO). After 7 days of treatment with standard or supplemented chows, IO was induced and intestinal permeability and bacterial translocation were evaluated. The small intestine and its contents were harvested for histopathological and morphometric analysis and the determination of polyamine concentration. RESULTS: Pretreatment with arginine maintained intestinal permeability (P > .05; Arg and Arg-/- groups vs Sham and Sham-/- groups), increased polyamine concentration in intestinal content (P < .05; Arg vs IO group), and decreased bacterial translocation in WT animals (Arg group vs IO and IO-/- groups). Absence of iNOS also presented a protective effect on permeability but not on bacterial translocation. CONCLUSION: Arginine supplementation and synthesis of NO by iNOS are important factors in decreasing bacterial translocation. However, when intestinal permeability was considered, NO had a detrimental role.

Cell apoptosis induced by hookworm antigens: a strategy of immunomodulation.

While several mechanisms of immunoregulation have been demonstrated for hookworm and other neglected tropical infections, the influence of apoptosis in the immunomodulation of hookworm infection is still poorly understood. In this study, we demonstrate the cytotoxic and pro-apoptotic activity of hookworm antigens in Jurkat T cells, mesenteric lymph nodes lymphocytes of healthy and hookworm-infected hamsters and during human natural infection. Our results showed that in vitrostimulation of Jurkat T cells with antigens induces a significant decrease of cell viability leading to a relevant increase of apoptotic cells. Similar results were also observed in experimental conditions, for both healthy and hookworm-infected hamsters` lymphocytes. Flow cytometric analysis demonstrated that hookworm-infected patients presented a significant increase of CD4+, CD8+, and CD19+lymphocytes in early and/or late apoptosis when compared with non-infected individuals. The downmodulation of TNF receptors, as well as the up-regulation of the pro-apoptotic genes belonging to the BCL-2 and P53 families, suggest that hookworm antigens induced apoptosis by an intrinsic mitochondrial pathway, acting as a sophisticated strategy to safeguard parasite long-term survival in their hosts.

Induction of CD4(+)CD25(+)FOXP3(+) regulatory T cells during human hookworm infection modulates antigen-mediated lymphocyte proliferation.

Hookworm infection is considered one of the most important poverty-promoting neglected tropical diseases, infecting 576 to 740 million people worldwide, especially in the tropics and subtropics. These blood-feeding nematodes have a remarkable ability to downmodulate the host immune response, protecting themselves from elimination and minimizing severe host pathology. While several mechanisms may be involved in the immunomodulation by parasitic infection, experimental evidences have pointed toward the possible involvement of regulatory T cells (Tregs) in downregulating effector T-cell responses upon chronic infection. However, the role of Tregs cells in human hookworm infection is still poorly understood and has not been addressed yet. In the current study we observed an augmentation of circulating CD4(+)CD25(+)FOXP3(+) regulatory T cells in hookworm-infected individuals compared with healthy non-infected donors. We have also demonstrated that infected individuals present higher levels of circulating Treg cells expressing CTLA-4, GITR, IL-10, TGF-ß and IL-17. Moreover, we showed that hookworm crude antigen stimulation reduces the number of CD4(+)CD25(+)FOXP3(+) T regulatory cells co-expressing IL-17 in infected individuals. Finally, PBMCs from infected individuals pulsed with excreted/secreted products or hookworm crude antigens presented an impaired cellular proliferation, which was partially augmented by the depletion of Treg cells. Our results suggest that Treg cells may play an important role in hookworm-induced immunosuppression, contributing to the longevity of hookworm survival in infected people.

Hookworm products ameliorate dextran sodium sulfate-induced colitis in BALB/c mice.

BACKGROUND: Several lines of evidence have shown that helminthiasis can significantly reduce disease severity in animal models of intestinal inflammation, airway inflammation/hyperreactivity, diabetes, and multiple sclerosis. Identification and characterization of helminth-derived immunomodulatory molecules that contribute to anticolitis effects could lead to new therapeutic approaches in inflammatory bowel diseases (IBDs) without the need for helminth infection. We evaluated the therapeutic potential of adult human hookworm, Ancylostoma ceylanicum, crude (Aw) and excreted/secreted (ES) products on dextran sulfate sodium (DSS)-induced colitis in BALB/c mice. METHODS: Colitis was induced by 5% DSS oral administration for 7 days. Clinical disease severity was monitored daily during concomitant intraperitoneal treatment with helminth-derived products. Additionally, several pathways of immunological modulation induced by A. ceylanicum products (MPO, EPO, Th1, Th2, and Th17 cytokine responses) in the inflamed intestinal microenvironment were assessed. Finally, the histopathological profile of the colon was characterized. RESULTS: Hookworm products are able to modulate the potent proinflammatory response induced by DSS, mainly through the downregulation of Th1 and Th17 cytokines. These proteins also reduce clinical and colonic microscopic inflammation scores as well as EPO and MPO activity. CONCLUSIONS: Ancylostoma ceylanicum Aw and ES mediators have an important therapeutic potential in experimental colitis in mice, which may provide a more socially acceptable form of therapy for patients with IBDs as opposed to using living worms. Our results support the urgency of further isolation and recombinant expression of active hookworm products responsible for the beneficial effects on colitis.

Profile of central and effector memory T cells in the progression of chronic human chagas disease.

BACKGROUND: Chronic Chagas disease presents several different clinical manifestations ranging from asymptomatic to severe cardiac and/or digestive clinical forms. Several studies have demonstrated that immunoregulatory mechanisms are important processes for the control of the intense immune activity observed in the chronic phase. T cells play a critical role in parasite specific and non-specific immune response elicited by the host against Trypanosoma cruzi. Specifically, memory T cells, which are basically classified as central and effector memory cells, might have a distinct migratory activity, role and function during the human Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: Based on the hypothesis that the disease severity in humans is correlated to the quality of immune responses against T. cruzi, we evaluated the memory profile of peripheral CD4(+) and CD8(+) T lymphocytes as well as its cytokine secretion before and after in vitro antigenic stimulation. We evaluated cellular response from non-infected individuals (NI), patients with indeterminate (IND) or cardiac (CARD) clinical forms of Chagas disease. The expression of CD45RA, CD45RO and CCR7 surface molecules was determined on CD4(+) and CD8(+) T lymphocytes; the pattern of intracellular cytokines (IFN-gamma, IL-10) synthesized by naive and memory cells was determined by flow cytometry. Our results revealed that IND and CARD patients have relatively lower percentages of naive (CD45RA(high)) CD4(+) and CD8(+) T cells. However, statistical analysis of ex-vivo profiles of CD4(+) T cells showed that IND have lower percentage of CD45RA(high) in relation to non-infected individuals, but not in relation to CARD. Elevated percentages of memory (CD45RO(high)) CD4(+) T cells were also demonstrated in infected individuals, although statistically significant differences were only observed between IND and NI groups. Furthermore, when we analyzed the profile of secreted cytokines, we observed that CARD patients presented a significantly higher percentage of CD8(+)CD45RA(high) IFN-gamma-producing cells in control cultures and after antigen pulsing with soluble epimastigote antigens. CONCLUSIONS: Based on a correlation between the frequency of IFN-gamma producing CD8+ T cells in the T cell memory compartment and the chronic chagasic myocarditis, we propose that memory T cells can be involved in the induction of the development of the severe clinical forms of the Chagas disease by mechanisms modulated by IFN-gamma. Furthermore, we showed that individuals from IND group presented more T(CM) CD4(+) T cells, which may induce a regulatory mechanism to protect the host against the exacerbated inflammatory response elicited by the infection.