The role of environmental enteric dysfunction in the pathogenesis of Schistosoma mansoni-associated morbidity in school-aged children.

BACKGROUND: Studies have implicated schistosomiasis as a cause of intestinal barrier disruption, a salient feature of environmental enteric dysfunction (EED), as eggs translocate from the sterile bloodstream through the gut wall. We examined the longitudinal impact of praziquantel (PZQ) treatment on a) EED biomarkers and b) Insulin growth factor I (IGF-1), a key driver of childhood linear growth, since EED has been implicated in linear growth stunting. METHODOLOGY: 290 children infected with S. mansoni in Brazil were treated with PZQ at baseline. EED biomarkers lipopolysaccharide (LPS) and intestinal fatty acid binding-protein (I-FABP) were measured, as well as IGF-1 at baseline, 6 and 12-months. Multivariate regression analysis was applied to assess associations between S. mansoni intensity and plasma biomarkers (LPS, I-FABP, and IGF-1), controlling for potential confounding variables. PRINCIPAL FINDINGS: At baseline, S. mansoni infection intensities were 27.2% light, 46.9% moderate, and 25.9% heavy. LPS concentrations were significantly reduced at the 12-month visit compared to baseline (p = 0.0002). No longitudinal changes were observed for I-FABP or IGF-1 in the 6- or 12-month periods following baseline treatment. After 6-months, I-FABP concentration was significantly higher in high vs low intensity (p = 0.0017). IGF-1 concentrations were significantly lower among children with high and moderate vs low intensity infections at each study visit. CONCLUSIONS/SIGNIFICANCE: We report that S. mansoni infection impacts LPS, I-FABP and IGF-1. These findings suggest a mechanistic role for EED in schistosomiasis-related morbidities, particularly linear growth.

Immunotherapy for cancer: effects of iron oxide nanoparticles on polarization of tumor-associated macrophages.

Cancer immunotherapy is the most promising trend in oncology, focusing on helping or activating the patient's immune system to identify and fight against cancer. In the last decade, interest in metabolic reprogramming of tumor-associated macrophages from M2-like phenotype (promoting tumor progression) to M1-like phenotypes (suppressing tumor growth) as a therapeutic strategy against cancer has increased considerably. Iron metabolism has been standing out as a target for the reprogramming of tumor-associated macrophages to M1-like phenotype with therapeutic purposes against cancer. Due to the importance of the iron levels in macrophage polarization states, iron oxide nanoparticles can be used to change the activation state of tumor-associated macrophages for a tumor suppressor phenotype and as an anti-tumor strategy.

Phase I and II Clinical Trial Comparing the LBSap, Leishmune®, and Leish-Tec® Vaccines against Canine Visceral Leishmaniasis.

In this study, we performed a phase I and II clinical trial in dogs to evaluate the toxicity and immunogenicity of LBSap-vaccine prototype, in comparison to Leishmune® and Leish-Tec® vaccines. Twenty-eight dogs were classified in four groups: (i) control group received 1 mL of sterile 0.9% saline solution; (ii) LBSap group received 600 µg of Leishmania braziliensis promastigotes protein and 1 mg of saponin adjuvant; (iii) Leishmune®; and (iv) Leish-Tec®. The safety and toxicity of the vaccines were measured before and after three immunizations by clinical, biochemical, and hematological parameters. The clinical examinations revealed that some dogs of LBSap and Leishmune® groups presented changes at the site of vaccination inoculum, such as nodules, mild edema, and local pain, which were transient and disappeared seventy-two hours after vaccination, but these results indicate that adverse changes caused by the immunizations are tolerable. The immunogenicity results demonstrate an increase of B lymphocytes CD21+ regarding the Leishmune® group and monocytes CD14+ concerning LBSap and Leishmune® groups. In the in vitro analyses, an increase in lymphoproliferative activity in LBSap and Leishmune® groups was observed, with an increase of antigen-specific CD4+ and CD8+ T lymphocytes in the LBSap group. A second approach of in vitro assays aimed at evaluating the percentage of antigen-specific CD4+ and CD8+ T lymphocytes producers of IFN-γ and IL-4, where an increase in both IFN-γ producing subpopulations in the LBSap group was observed, also showed an increase in IFN-γ producers in CD8+ lymphocytes in the Leish-Tec® group. Our data regarding immunogenicity indicate that the vaccination process, especially with the LBSap vaccine, generated a protective immune response compatible with L. infantum parasite control. Based on the foregoing, the LBSap vaccine would be suitable for further studies of phase III clinical trial in endemic areas with high prevalence and incidence of canine visceral leishmaniasis (VL) cases.

Liver damage in schistosomiasis is reduced by adipose tissue-derived stem cell therapy after praziquantel treatment.

BACKGROUND: In view of the potential immunosuppressive and regenerative properties of mesenchymal stem cells (MSC), we investigated whether transplantation of adipose tissue-derived stem cells (ASC) could be used to control the granulomatous reaction in the liver of mice infected with Schistosoma mansoni after Praziquantel (PZQ) treatment. METHODOLOGY/PRINICPAL FINDINGS: C57BL/6 mice infected with S. mansoni were treated with PZQ and transplanted intravenously with ASC from uninfected mice. Liver morpho-physiological and immunological analyses were performed. The combined PZQ/ASC therapy significantly reduced the volume of hepatic granulomas, as well as liver damage as measured by ALT levels. We also observed that ASC accelerated the progression of the granulomatous inflammation to the advanced/curative phase. The faster healing interfered with the expression of CD28 and CTLA-4 molecules in CD4+ T lymphocytes, and the levels of IL-10 and IL-17 cytokines, mainly in the livers of PZQ/ASC-treated mice. CONCLUSIONS: Our results show that ASC therapy after PZQ treatment results in smaller granulomas with little tissue damage, suggesting the potential of ASC for the development of novel therapeutic approaches to minimize hepatic lesions as well as a granulomatous reaction following S. mansoni infection. Further studies using the chronic model of schistosomiasis are required to corroborate the therapeutic use of ASC for schistosomiasis.

PD1 and PDL1 molecules control suppressor activity of regulatory T cells in chronic Chagas cardiomyopathy patients.

INTRODUCTION: Chagas disease, caused by the protozoan Trypanosoma cruzi (T. cruzi), is the fourth most important tropical disease, which affects approximately 7 million people worldwide. The mechanisms involved in the development of this disease are not completely well understood. An important protective role of regulatory T cells (Treg) in Chagas disease has been observed; however, the specific mechanisms remain unclear. We evaluated apoptosis as a possible mechanism mediated by Treg cells (CD4+CD25HighFOXP3+) to orchestrate the immune response in chronic Chagas disease. METHODS AND RESULTS: Patients with Chagas disease were grouped as the indeterminate (IND; asymptomatic patients with Chagas disease; n = 10) and dilated cardiomyopathy (CARD; n = 10). Healthy T. cruzi-negative individuals (NI; n = 10) were included as a control group. In order to evaluate the apoptotic cell profile, the expression of PD1, PD1L, CD39, CD95, CD95L molecules were investigated. We also evaluated the proportion of CD14+ cells expressing caspase 3. The IND group presented a substantially higher expression of CD39 by Treg cells as compared to the CARD group. On the other hand, the CARD group showed higher expression of PD-1 by Treg cells than both NI and IND groups. Significant positive correlations were observed between Treg CD95L+ cells and CD14 cells expressing caspase 3 as well as between Treg CD39 cells and CD14+ Caspase3+ cells in the IND group. CONCLUSION: Our data indicate that the expressions of different molecules that induce apoptosis are associated with suppressive mechanisms mediated by Treg cells and suggest a possible role for PD1 and PDL1 molecules in the morbidity of chronic Chagas disease.

Specific antigen serologic tests in leprosy: implications for epidemiological surveillance of leprosy cases and household contacts

BACKGROUND There is a lack of straightforward tests for field application and known biomarkers for predicting leprosy progression in infected individuals. OBJECTIVE The aim was to analyse the response to infection by Mycobacterium leprae based on the reactivity of specific antigens: natural disaccharide linked to human serum albumin via an octyl (NDOHSA), a semisynthetic phenolic glycolipid-I (PGL-I); Leprosy Infectious Disease Research Institute Diagnostic-1 (LID-1) and natural disaccharide octyl - Leprosy Infectious Disease Research Institute Diagnostic-1 (NDOLID). METHODS The study population consisted of 130 leprosy cases diagnosed between 2010 and 2015 and 277 household contacts. An enzyme-linked immunosorbent assay (ELISA) was used to analyse the reactivity of antibodies against NDOHSA, LID-1 and NDOLID. The samples and controls were tested in duplicate, and the antibody titer was expressed as an ELISA index. Data collection was made by home visits with application of questionnaire and dermatological evaluation of all household contacts to identify signs and symptoms of leprosy. FINDINGS Significant differences in the median ELISA results were observed among leprosy cases in treatment, leprosy cases that had completed treatment and household contacts. Higher proportions of seropositivity were observed in leprosy cases in treatment. Seropositivity was also higher in multibacillary in relation to paucibacillary, with the difference reaching statistical significance. Lower titers were observed among cases with a longer treatment time or discharge. For household contacts, the differences according to the clinical characteristics of the leprosy index case were less pronounced than expected. Other factors, such as the endemicity of leprosy, exposure outside the residence and genetic characteristics, appeared to have a greater influence on the seropositivity. MAIN CONCLUSIONS Serologic tests could be used as auxiliary tools for determining the operational classification, in addition to identifying infected individuals and as a strategy for surveillance of household contacts.

A Vaccine Therapy for Canine Visceral Leishmaniasis Promoted Significant Improvement of Clinical and Immune Status with Reduction in Parasite Burden.

Herein, we evaluated the treatment strategy employing a therapeutic heterologous vaccine composed of antigens of Leishmania braziliensis associated with MPL adjuvant (LBMPL vaccine) for visceral leishmaniasis (VL) in symptomatic dogs naturally infected by Leishmania infantum. Sixteen dogs received immunotherapy with MPL adjuvant (n = 6) or with a vaccine composed of antigens of L. braziliensis associated with MPL (LBMPL vaccine therapy, n = 10). Dogs were submitted to an immunotherapeutic scheme consisting of 3 series composed of 10 subcutaneous doses with 10-day interval between each series. The animals were evaluated before (T0) and 90 days after treatment (T90) for their biochemical/hematological, immunological, clinical, and parasitological variables. Our major results showed that the vaccine therapy with LBMPL was able to restore and normalize main biochemical (urea, AST, ALP, and bilirubin) and hematological (erythrocytes, hemoglobin, hematocrit, and platelets) parameters. In addition, in an ex vivo analysis using flow cytometry, dogs treated with LBMPL vaccine showed increased CD3+ T lymphocytes and their subpopulations (TCD4+ and TCD8+), reduction of CD21+ B lymphocytes, increased NK cells (CD5-CD16+) and CD14+ monocytes. Under in vitro conditions, the animals developed a strong antigen-specific lymphoproliferation mainly by TCD4+ and TCD8+ cells; increasing in both TCD4+IFN-γ+ and TCD8+IFN-γ+ as well as reduction of TCD4+IL-4+ and TCD8+IL-4+ lymphocytes with an increased production of TNF-α and reduced levels of IL-10. Concerning the clinical signs of canine visceral leishmaniasis, the animals showed an important reduction in the number and intensity of the disease signs; increase body weight as well as reduction of splenomegaly. In addition, the LBMPL immunotherapy also promoted a reduction in parasite burden assessed by real-time PCR. In the bone marrow, we observed seven times less parasites in LBMPL animals compared with MPL group. The skin tissue showed a reduction in parasite burden in LBMPL dogs 127.5 times higher than MPL. As expected, with skin parasite reduction promoted by immunotherapy, we observed a blocking transmission to sand flies in LBMPL dogs with only three positive dogs after xenodiagnosis. The results obtained in this study highlighted the strong potential for the use of this heterologous vaccine therapy as an important strategy for VL treatment.

Chronic Low-Grade Inflammation in Childhood Obesity Is Associated with Decreased IL-10 Expression by Monocyte Subsets.

Chronic low-grade inflammation is related to the development of comorbidities and poor prognosis in obesity. Monocytes are main sources of cytokines and play a pivotal role in inflammation. We evaluated monocyte frequency, phenotype and cytokine profile of monocyte subsets, to determine their association with the pathogenesis of childhood obesity. Children with obesity were evaluated for biochemical and anthropometric parameters. Monocyte subsets were characterized by flow cytometry, considering cytokine production and activation/recognition molecules. Correlation analysis between clinical parameters and immunological data delineated the monocytes contribution for low-grade inflammation. We observed a higher frequency of non-classical monocytes in the childhood obesity group (CO) than normal-weight group (NW). All subsets displayed higher TLR4 expression in CO, but their recognition and antigen presentation functions seem to be diminished due to lower expression of CD40, CD80/86 and HLA-DR. All subsets showed a lower expression of IL-10 in CO and correlation analyses showed changes in IL-10 expression profile. The lower expression of IL-10 may be decisive for the maintenance of the low-grade inflammation status in CO, especially for alterations in non-classical monocytes profile. These cells may contribute to supporting inflammation and loss of regulation in the immune response of children with obesity.

Impact of LbSapSal Vaccine in Canine Immunological and Parasitological Features before and after Leishmania chagasi-Challenge.

Dogs represent the most important domestic reservoir of L. chagasi (syn. L. infantum). A vaccine against canine visceral leishmaniasis (CVL) would be an important tool for decreasing the anxiety related to possible L. chagasi infection and for controlling human visceral leishmaniasis (VL). Because the sand fly salivary proteins are potent immunogens obligatorily co-deposited during transmission of Leishmania parasites, their inclusion in an anti-Leishmania vaccine has been investigated in past decades. We investigated the immunogenicity of the "LbSapSal" vaccine (L. braziliensis antigens, saponin as adjuvant, and Lutzomyia longipalpis salivary gland extract) in dogs at baseline (T0), during the post-vaccination protocol (T3rd) and after early (T90) and late (T885) times following L. chagasi-challenge. Our major data indicated that immunization with "LbSapSal" is able to induce biomarkers characterized by enhanced amounts of type I (tumor necrosis factor [TNF]-α, interleukin [IL]-12, interferon [IFN]-γ) cytokines and reduction in type II cytokines (IL-4 and TGF-ß), even after experimental challenge. The establishment of a prominent pro-inflammatory immune response after "LbSapSal" immunization supported the increased levels of nitric oxide production, favoring a reduction in spleen parasitism (78.9%) and indicating long-lasting protection against L. chagasi infection. In conclusion, these results confirmed the hypothesis that the "LbSapSal" vaccination is a potential tool to control the Leishmania chagasi infection.

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.

Integrative literature review of the reported uses of serological tests in leprosy management

Abstract: An integrative literature review was conducted to synthesize available publications regarding the potential use of serological tests in leprosy programs. We searched the databases Literatura Latino-Americana e do Caribe em Ciências da Saúde, Índice Bibliográfico Espanhol em Ciências da Saúde, Acervo da Biblioteca da Organização Pan-Americana da Saúde, Medical Literature Analysis and Retrieval System Online, Hanseníase, National Library of Medicine, Scopus, Ovid, Cinahl, and Web of Science for articles investigating the use of serological tests for antibodies against phenolic glycolipid-I (PGL-I), ML0405, ML2331, leprosy IDRI diagnostic-1 (LID-1), and natural disaccharide octyl-leprosy IDRI diagnostic-1 (NDO-LID). From an initial pool of 3.514 articles, 40 full-length articles fulfilled our inclusion criteria. Based on these papers, we concluded that these antibodies can be used to assist in diagnosing leprosy, detecting neuritis, monitoring therapeutic efficacy, and monitoring household contacts or at-risk populations in leprosy-endemic areas. Thus, available data suggest that serological tests could contribute substantially to leprosy management.

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.

Plasma cytokine expression is associated with cardiac morbidity in chagas disease.

The expression of immune response appears to be associated with morbidity in Chagas disease. However, the studies in this field have usually employed small samples of patients and statistical analyses that do not consider the wide dispersion of cytokine production observed in these patients. The aim of this study was to evaluate the plasma cytokine levels in well-defined clinical polar groups of chagasic patients divided into categories that better reflect the wide cytokine profile and its relationship with morbidity. Patients infected with Trypanosoma cruzi (T. cruzi) were grouped as indeterminate (IND) and cardiac (CARD) forms ranging from 23 to 69 years of age (mean of 45.6±11.25). The IND group included 82 individuals, ranging from 24 to 66 years of age (mean of 39.6±10.3). The CARD group included 94 patients ranging from 23 to 69 years of age (mean of 48±12.52) presenting dilated cardiomyopathy. None of the patients have undergone chemotherapeutic treatment, nor had been previously treated for T. cruzi infection. Healthy non-chagasic individuals, ranging from 29 to 55 years of age (mean of 42.6±8.8) were included as a control group (NI). IND patients have a higher intensity of interleukin 10 (IL-10) expression when compared with individuals in the other groups. By contrast, inflammatory cytokine expression, such as interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interleukin 1 beta (IL-1ß), proved to be the highest in the CARD group. Correlation analysis showed that higher IL-10 expression was associated with better cardiac function, as determined by left ventricular ejection fraction and left ventricular diastolic diameter values. Altogether, these findings reinforce the concept that a fine balance between regulatory and inflammatory cytokines represents a key element in the establishment of distinct forms of chronic Chagas disease.

Profile of natural killer cells after a previous natural Vaccinia virus infection in an in vitro viral re-exposure.

The present study compares the profile of NK cells in an in vitro re-exposure by Vaccinia virus (VACV), in groups that have had a previous vaccination or natural infection. Our data suggests that stimulation with VACV triggers a cytotoxic response by NK cells marked by an increase of NCRs: NKp30, NKp44, and NKp46 in infected (vaccinated and unvaccinated) subjects and in non-infected vaccinated patients, when compared with non-infected unvaccinated individuals. However, the degranulation and secretion processes are inhibited in infected (vaccinated and unvaccinated) subjects and in the non-infected vaccinated patients, when compared with non-infected unvaccinated individuals. We demonstrated that stimulation with VACV downregulates the percentage of expression of Perforin, Granzyme A, and CD107a, but upregulate CD94 in infected (vaccinated and unvaccinated) subjects and in non-infected vaccinated patients, when compared with non-infected unvaccinated individuals. Furthermore, the percentage of IFN-γ(+) NK cells was significantly lower in non-infected unvaccinated subjects, when compared with infected (vaccinated and unvaccinated) and non-infected vaccinated individuals. Our results also show that the percentage of TNF-α(+) NK cells was significantly higher in infected (vaccinated and unvaccinated) subjects and in non-infected vaccinated patients, when compared with non-infected unvaccinated individuals, after in vitro stimulation with UV-inactivated VACV. Our data suggest that the expression of NCRs NKp30, NKp44, NKp46 and cytokines by NK cells are important in the innate response against VACV.

Cytokine and nitric oxide patterns in dogs immunized with LBSap vaccine, before and after experimental challenge with Leishmania chagasi plus saliva of Lutzomyia longipalpis.

In the studies presented here, dogs were vaccinated against Leishmania (Leishmania) chagasi challenge infection using a preparation of Leishmania braziliensis promastigote proteins and saponin as adjuvant (LBSap). Vaccination with LBSap induced a prominent type 1 immune response that was characterized by increased levels of interleukin (IL-) 12 and interferon gamma (IFN-γ) production by peripheral blood mononuclear cells (PBMC) upon stimulation with soluble vaccine antigen. Importantly, results showed that this type of responsiveness was sustained after challenge infection; at day 90 and 885 after L. chagasi challenge infection, PBMCs from LBSap vaccinated dogs produced more IL-12, IFN-γ and concomitant nitric oxide (NO) when stimulated with Leishmania antigens as compared to PBMCs from respective control groups (saponin, LB- treated, or non-treated control dogs). Moreover, transforming growth factor (TGF)-ß decreased in the supernatant of SLcA-stimulated PBMCs in the LBSap group at 90 days. Bone marrow parasitological analysis revealed decreased frequency of parasitism in the presence of vaccine antigen. It is concluded that vaccination of dogs with LBSap vaccine induced a long-lasting type 1 immune response against L. chagasi challenge infection.

Analysis using canine peripheral blood for establishing in vitro conditions for monocyte differentiation into macrophages for Leishmania chagasi infection and T-cell subset purification.

Canine visceral leishmaniasis (CVL) is a parasitic disease endemic in many countries, and dogs present as the major natural reservoir of the parasite, Leishmania chagasi (syn. L. infantum). Biomarkers in the canine immune system is an important technique in the course of developing vaccines and treatment strategies against CVL. New methodologies for studying the immune response of dogs during Leishmania infection and after receiving vaccines and treatments against CVL would be useful. In this context, we used peripheral blood mononuclear cells (PBMCs) from healthy dogs to evaluate procedures related to (i) establishment of in vitro conditions of monocytes differentiated into macrophages infected with L. chagasi and (ii) purification procedures of T-cell subsets (CD4(+) and CD8(+)) using microbeads. Our data demonstrated that after 5 days of differentiation, macrophages were able to induce significant phagocytic and microbicidal activity after L. chagasi infection and also showed increased frequency of parasitism and a higher parasite load. Although N-acetyl-ß-d-glucosaminidase (NAG) levels presented similar levels of macrophage culture and L. chagasi infection, a progressive decrease in myeloperoxidase (MPO) levels was a hallmark over 5 days of culture. High purity levels (>90%) of CD4 and CD8 T cells were obtained on a magnetic separation column. We concluded that monocytes differentiated into macrophages at 5 days and displayed an intermediate frequency of parasitism and parasite load 72 h after L. chagasi infection. Furthermore, the purification system using canine T-lymphocyte subsets obtained after 5 days of monocyte differentiation proved efficient for CD4 or CD8 T-cell purification (≥90%). The in vitro analysis using L. chagasi-infected macrophages and purified T cells presented a prospective methodology that could be incorporated in CVL vaccine and treatment studies that aim to analyze the microbicidal potential induced by specific CD4(+) and/or CD8(+) T cells.

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.

Cytokine profile, proliferation and phosphorylation of ERK1/2 and Akt in circulating mononuclear cells from individuals during the chronic intestinal phase of Schistosomiasis mansoni infection.

BACKGROUND: The immune response to Schistosoma mansoni is characterized by a granulomatous reaction around the parasite eggs that are trapped in the host liver, and this reaction modulates the immune response during the chronic phase of the disease. The typical peripheral blood mononuclear cell (PBMC) response of patients during the chronic intestinal phase of infection is characterized by a decreased response to an S. mansoni soluble egg antigen. To obtain a greater understanding of Schistosoma infections, this study investigated the effects of the soluble egg antigen (SEA) and soluble adult worm antigen (SWAP) of S. mansoni on cellular proliferation, cytokine production, and ERK1/2 and Akt phosphorylation in PBMCs from infected (XTO) and egg-negative (NI) individuals living in the same endemic area. METHODS: The activation status was evaluated by cell immunophenotypic staining (cytometry). The cell proliferation assay was by CFSE method. Cytokine detection assay (Th1 and Th2) was by Cytometric Bead and Array phosphorylation status was by ELISA. RESULTS: The XTO, NI and BD (blood donor) individuals from an area not endemic for schistosomiasis were compared. The CD4(+) T lymphocyte proliferation rate was lower in the XTO group, but not the NI group, after SEA stimulation compared to the BD group. The CD8(+) T cell proliferation rate was lower in the XTO group in the unstimulated cultures and after both SEA and SWAP stimulation compared to the BD group. Cytokine analysis after either SEA or SWAP stimulation showed a balanced cytokine pattern in the XTO and NI groups. ERK1/2 and Akt phosphorylation were only marginally detected in all groups; however, a decrease in ERK 1/2 phosphorylation was observed in the SWAP-stimulated XTO group compared to both the NI and BD groups. CONCLUSIONS: The data indicate that SEA-stimulated CD4(+) T cells from infected patients have a lower proliferation rate than the same cells from the NI group. Furthermore, we observed that SWAP stimulation influences ERK1/2 phosphorylation in the XTO group.

Distinct cytokine profiles of circulating mononuclear cells stimulated with Staphylococcus aureus enterotoxin A in vitro during early and late episodes of chronic osteomyelitis

We investigated the cytokine profile of peripheral mononuclear cells from chronic osteomyelitis (OST) patients following in vitro stimulation with staphylococcal enterotoxin A (SEA). We demonstrate that stimulation with SEA induced prominent lymphocyte proliferation and high levels of tumour necrosis factor (TNF)-α, interleukin (IL)-4 and IL-10 secretion in both OST and non-infected individuals (NI). Even though stimulation with SEA had no impact on IL-6 production in either patient group, the baseline level of IL-6 production by cells from OST patients was always significantly less than that produced by cells from NI. After classifying the osteomyelitic episodes based on the time after the last reactivation event as "early" (1-4 months) or "late" osteomyelitis (5-12 months), we found that increased levels of TNF-α and IL-4 in combination with decreased levels of IL-6 were observed in the early episodes. By contrast, increased levels of IL-10, IL-2 and IL-6 were hallmarks of late episodes. Our data demonstrate that early osteomyelitic episodes are accompanied by an increased frequency of "high producers" of TNF-α and IL-4, whereas late events are characterised by increased frequencies of "high producers" of IL-10, IL-6 and IL-2. These findings demonstrate the distinct cytokine profiles in chronic osteomyelitis, with a distinct regulation of IL-6 production during early and late episodes.

Infiltrating CD57+ inflammatory cells in head and neck squamous cell carcinoma: clinicopathological analysis and prognostic significance.

This study investigated the immunodetection of CD57+ inflammatory cells in patients with head and neck squamous cell carcinoma (HNSCC) and its association with clinicopathological parameters and overall survival. Data collected from the morphological analysis and immunohistochemical reaction testing of archived HNSCC specimens (n=70) were statistically analyzed by bivariate and multivariate statistical testing at a significance level of P<0.05. The results indicate that CD57+ inflammatory cells predominate within the peritumoral stroma of HNSCC lesions and the existence of two significant relationships: between high CD57+ cell density and the development of a tumor of a large size [odds ratio (OR)=5.610, 95% confidence interval (CI)=1.516-20.763) and between high CD57+ cell density and the development of locoregional metastatic disease (OR=3.401, 95% CI=1.162-9.951). A significant difference in the rate of survival was detected only in HNSCC patients that presented large size tumors (OR=4.747, 95% CI=1.281-17.594). Together, these results suggest that although high CD57+ inflammatory cell density is associated with HNSCC lesions of greater clinical severity, the variable of cell density is not an independent predictor of HNSCC patient survival. Our findings also suggest that the relatively aggressive infiltration of CD57+ inflammatory cells in the peritumoral stroma of head and neck carcinomas may contribute to an ineffective locoregional antitumoral response.