Etiological treatment of Chagas disease patients with benznidazole lead to a sustained pro-inflammatory profile counterbalanced by modulatory events.

In the present study, we characterized the phagocytic capacity, cytokine profile along with the FCγ-R and TLR expression in leukocytes from Chagas disease patients (indeterminate-IND and cardiac-CARD) before and one-year after Bz-treatment (INDT and CARDT). A down-regulation of IL-17, IFN-γ and IL-10 synthesis by neutrophils was observed in CARDT. The Bz-treatment did not impact on the expression of phagocytosis-related surface molecules or monocyte-derived cytokine profile in INDT. Although CARDT showed unaltered monocyte-phagocytic capacity, up-regulated expression of Fcγ-RI/III and TLR-4 may be related to their ability to produce IL-10 and TGF-ß. Down-regulation of lymphocyte-derived cytokine was observed in INDT whereas up-regulated cytokine profile was observed for lymphocytes in CARDT. Analysis of cytokine network revealed that IND displayed a multifaceted cytokine response characterized by strong connecting axes involving pro-inflammatory/regulatory phagocytes and lymphocytes. On the other hand, CARD presented a modest cytokine network. The Bz-treatment leads to distinct cytokine network: decreasing the links in INDT, with a pivotal role of IL-10(+) monocytes and expanding the connections in CARDT. Our findings highlighted that the Bz-treatment contributes to an overall immunomodulation in INDT and induces a broad change of immunological response in CARDT, eliciting an intricate phenotypic/functional network compatible with beneficial and protective immunological events.

High sensitivity C-reactive protein distribution in the elderly: the Bambuí Cohort Study, Brazil

The measurement of the serum concentration of the acute-phase reactant C-reactive protein (CRP) provides a useful marker in clinical practice. However, the distribution of CRP is not available for all age and population groups. This study assessed the distribution of high sensitivity-CRP (hs-CRP) by gender and age in 1470 elderly individuals from a Brazilian community that participates in the Bambuí Cohort Study. Blood samples were collected after 12 h of fasting and serum samples were stored at -70°C. Measurements were made with a commercial hs-CRP immunonephelometric instrument. More than 50% of the results were above 3.0 mg/L for both genders. Mean hs-CRP was higher in women (3.62 ± 2.58 mg/L) than in men (3.03 ± 2.50 mg/L). This difference was observed for all ages, except for the over-80 age group. This is the first population-based study to describe hs-CRP values in Latin American elderly subjects. Our results indicate that significant gender differences exist in the distribution of hs-CRP, and suggest that gender-specific cut-off points for hs-CRP would be necessary for the prediction of cardiovascular risks.

High sensitivity C-reactive protein distribution in the elderly: the Bambuí Cohort Study, Brazil.

The measurement of the serum concentration of the acute-phase reactant C-reactive protein (CRP) provides a useful marker in clinical practice. However, the distribution of CRP is not available for all age and population groups. This study assessed the distribution of high sensitivity-CRP (hs-CRP) by gender and age in 1470 elderly individuals from a Brazilian community that participates in the Bambuí Cohort Study. Blood samples were collected after 12 h of fasting and serum samples were stored at -70°C. Measurements were made with a commercial hs-CRP immunonephelometric instrument. More than 50% of the results were above 3.0 mg/L for both genders. Mean hs-CRP was higher in women (3.62 ± 2.58 mg/L) than in men (3.03 ± 2.50 mg/L). This difference was observed for all ages, except for the over-80 age group. This is the first population-based study to describe hs-CRP values in Latin American elderly subjects. Our results indicate that significant gender differences exist in the distribution of hs-CRP, and suggest that gender-specific cut-off points for hs-CRP would be necessary for the prediction of cardiovascular risks.

Impaired phagocytic capacity driven by downregulation of major phagocytosis-related cell surface molecules elicits an overall modulatory cytokine profile in neutrophils and monocytes from the indeterminate clinical form of Chagas disease.

The distinct ability of phagocytes to present antigens, produce cytokines and provide co-stimulatory signals may contribute to the severity of the outcome of Chagas disease. In this paper, we evaluate the phenotypic features of phagocytes along with the cytokine signature of circulating T-cells from Chagas disease patients with indeterminate (IND) and cardiac (CARD) clinical forms of the disease. Our data demonstrated that neutrophils from IND patients displayed an impaired ability to produce cytokines. A lower Trypanosoma cruzi phagocytic index and higher nitric oxide levels were characteristics of monocytes from IND. The impaired phagocytic capacity did not reflect on the levels of anti-T. cruzi IgG, but was detectable in the downregulation of Fc-γR, TLR and CR1 molecules. The monocyte-derived cytokine signature demonstrated that a down-regulated synthesis of IL-12 and a modulatory state were evidenced by a positive correlation between IL-12 and IL-10 with a lower synthesis of TNF-α. The down-regulation of MHC-II and CD86 in monocytes supports the occurrence of particularities in the APC-activation-arm in IND, and may be involved in the T-cell pro-inflammatory pattern counterbalanced by a potent IL-10 response. Our findings support the hypothesis that differential phenotypic features of monocytes from IND may be committed to the induction of a distinct immune response related to low morbidity in chronic Chagas disease.

Strategy to assess the overall cytokine profile of circulating leukocytes and its association with distinct clinical forms of human Chagas disease.

Herein we have employed an alternative strategy to assess the cytokine patterns of circulating leukocytes and correlate dominant cytokine profiles with indeterminate-IND and cardiac-CARD clinical forms of Chagas disease. We have first calculated median percentages of cytokine-positive leukocytes of our study sample to establish, for each cytokine-positive cell population, the cut-off edge that would segregate 'low' and 'high' cytokine producers to build colour diagrams and draw a panoramic cytokine chart. Using this approach we demonstrated that most IND individuals presented a dominant regulatory cytokine profile, whereas CARD individuals displayed a dominant inflammatory cytokine pattern. In addition, radar chart analysis confirmed the dichotomic cytokine balance between IND and CARD groups and further allowed the identification of the relative contribution of each cell population for the global cytokine pattern. Data analysis demonstrated that CD4+ T cells were the major cell population defining the regulatory profile in IND, whereas monocytes and CD4+ T cells determined the inflammatory cytokine pattern in CARD individuals. Interestingly, in vitro stimulation with trypomastigote Trypanosoma cruzi antigen was able to invert the cytokine balances in IND and CARD groups. Upon antigenic stimulation, changes in the frequencies of IL-10-producing CD4+ T cells and monocytes drove IND individuals towards an inflammatory pattern and CARD towards a regulatory cytokine profile. A similar inversion could be found after in vivo treatment of IND and CARD individuals with benzonidazole. Altogether, these findings shed some light into the complex cytokine network underlying the immunopathogenesis of Chagas disease and provide putative immunological biomarkers of disease severity and therapeutic response.

Activation/modulation of adaptive immunity emerges simultaneously after 17DD yellow fever first-time vaccination: is this the key to prevent severe adverse reactions following immunization?

Over past decades the 17DD yellow fever vaccine has proved to be effective in controlling yellow fever and promises to be a vaccine vector for other diseases, but the cellular and molecular mechanisms by which it elicits such broad-based immunity are still unclear. In this study we describe a detailed phenotypic investigation of major and minor peripheral blood lymphocyte subpopulations aimed at characterizing the kinetics of the adaptive immune response following primary 17DD vaccination. Our major finding is a decreased frequency of circulating CD19+ cells at day 7 followed by emerging activation/modulation phenotypic features (CD19+interleukin(IL)10R+/CD19+CD32+) at day 15. Increased frequency of CD4+human leucocyte antigen D-related(HLA-DR+) at day 7 and CD8+HLA-DR+ at day 30 suggest distinct kinetics of T cell activation, with CD4+ T cells being activated early and CD8+ T cells representing a later event following 17DD vaccination. Up-regulation of modulatory features on CD4+ and CD8+ cells at day 15 seems to be the key event leading to lower frequency of CD38+ T cells at day 30. Taken together, our findings demonstrate the co-existence of phenotypic features associated with activation events and modulatory pathways. Positive correlations between CD4+HLA-DR+ cells and CD4+CD25high regulatory T cells and the association between the type 0 chemokine receptor CCR2 and the activation status of CD4+ and CD8+ cells further support this hypothesis. We hypothesize that this controlled microenviroment seems to be the key to prevent the development of serious adverse events, and even deaths, associated with the 17DD vaccine reported in the literature.

Benznidazole treatment during early-indeterminate Chagas' disease shifted the cytokine expression by innate and adaptive immunity cells toward a type 1-modulated immune profile.

Trypanosoma cruzi-infected children was treated with benznidazole (Bz) during the early-indeterminate disease (E-IND) and the cytokine pattern of innate and adaptive immune compartments were evaluated prior to the treatment and 1 year after it. At first, we observed that the ex vivo cytokine profile of circulating leukocytes from E-IND (n = 6) resembled the one observed for healthy schoolchildren (n = 7). Additionally, in vitro stimulation with T. cruzi antigens drove the E-IND cytokine pattern toward a mixed immune profile with higher levels of IFN-gamma+, TNF-alpha+ and IL-4+ NK cells, increased numbers of IFN-gamma+, TNF-alpha+ and IL-10+ CD4+ T cells in addition to enhanced frequency of TNF-alpha+/IL-4+ CD19+ lymphocytes. Interestingly, upon T. cruzi antigen in vitro stimulation, E-IND CD8+ lymphocytes displayed a selective enhancement of IFN-gamma expression, accounting for a global type 1-modulated cytokine microenvironment. A shift toward a type 1-modulated profile was also the hallmark of Bz-treated children (E-IND(T)). In this context, despite the mixed overall ex vivo cytokine profile observed for NK and CD8+ T cells, increased ability of these leukocytes to produce IFN-gamma in response to T. cruzi antigens was reported. Most noteworthy was the IL-10 production evidenced at T lymphocytes, mainly CD4+ cells, as well as B lymphocytes, both ex vivo and upon antigen stimulation. Together, these findings gave evidence that NK cells and CD8+ T lymphocytes are the major sources of IFN-gamma, a pivotal cytokine for successful therapeutic response in human Chagas' disease. Moreover, our data have also brought additional information, pointing out IL-10 production by CD4+ cells and B lymphocytes, as the putative key element for parasite clearance in the absence of deleterious tissue damage.

Are increased frequency of macrophage-like and natural killer (NK) cells, together with high levels of NKT and CD4+CD25high T cells balancing activated CD8+ T cells, the key to control Chagas' disease morbidity?

The immunological response during early human Trypanosoma cruzi infection is not completely understood, despite its role in driving the development of distinct clinical manifestations of chronic infection. Herein we report the results of a descriptive flow cytometric immunophenotyping investigation of major and minor peripheral blood leucocyte subpopulations in T. cruzi-infected children, characterizing the early stages of the indeterminate clinical form of Chagas' disease. Our results indicated significant alterations by comparison with uninfected children, including increased values of pre-natural killer (NK)-cells (CD3- CD16+ CD56-), and higher values of proinflammatory monocytes (CD14+ CD16+ HLA-DR++). The higher values of activated B lymphocytes (CD19+ CD23+) contrasted with impaired T cell activation, indicated by lower values of CD4+ CD38+ and CD4+ HLA-DR+ lymphocytes, a lower frequency of CD8+ CD38+ and CD8+ HLA-DR+ cells; a decreased frequency of CD4+ CD25HIGH regulatory T cells was also observed. These findings reinforce the hypothesis that simultaneous activation of innate and adaptive immunity mechanisms in addition to suppression of adaptive cellular immune response occur during early events of Chagas' disease. Comparative cross-sectional analysis of these immunophenotypes with those exhibited by patients with late chronic indeterminate and cardiac forms of disease suggested that a shift toward high values of macrophage-like cells extended to basal levels of proinflammatory monocytes as well as high values of mature NK cells, NKT and regulatory T cells, may account for limited tissue damage during chronic infection favouring the establishment/maintenance of a lifelong indeterminate clinical form of the disease. On the other hand, development of an adaptive cell-mediated inflammatory immunoprofile characterized by high levels of activated CD8+ cells and basal levels of mature NK cells, NKT and CD4+ CD25HIGH cells might lead to late chronic pathologies associated with chagasic heart disease.

Clinical value of anti-live Leishmania (Viannia) braziliensis immunoglobulin G subclasses, detected by flow cytometry, for diagnosing active localized cutaneous leishmaniasis.

OBJECTIVE: To evaluate the clinical value of flow cytometry anti-live promastigate antibody (FC-ALPA), for diagnosing active cutaneous leishmaniasis. METHOD: Serum samples from 145 individuals living in endemic areas for localized cutaneous leishmaniasis (population 1) were classified as having the disease or not and then tested for their IgG reactivity by indirect immunofluorescence assay and FC-ALPA-IgG. The results of FC-ALPA-IgG were expressed as percentage of positive fluorescent parasite. Both tests were also evaluated in serum samples of people with visceral leishmaniasis and Chagas disease (population 1A). RESULTS: In population 1, FC-ALPA-IgG performed better than the immunofluorescence assay regarding sensitivity, specificity and predictive values. Analysis of the results according to the likelihood ratios indicated that a percentage of positive fluorescent parasite 60% it reinforces diagnosis of the disease (likelihood ratio = 7.0). Immunofluorescent assay is of little value (likelihood ratio=2.04). In population 1A, both tests performed worse, but FC-ALPA-IgG achieved better statistical indexes than immunofluorescent assay. CONCLUSION: The FC-ALPA-IgG is a valuable method for serological diagnosis of localized cutaneous leishmaniasis. FC-ALPA-IgG1/ALPA-IgG2 combined analysis is an additional serological tool for discriminating localized visceral leishmaniasis, Chagas disease and visceral leishmaniasis in areas where these infections co-exist.

Chagasic patients with indeterminate clinical form of the disease have high frequencies of circulating CD3+CD16-CD56+ natural killer T cells and CD4+CD25High regulatory T lymphocytes.

Several studies have demonstrated that different clinical manifestations of human Chagas' disease are associated with distinct and complex host-parasite relationships directly involving the immune system. In this context, it has been proposed that tissue damage might be more severe in the absence of regulatory mechanisms that involve both innate and adaptive immune responses. Herein, we describe a descriptive phenotypic profile focusing on the frequency of major regulatory T cells [CD4+CD25high and natural killer T (NKT) lymphocytes] in different clinical forms of Chagas' disease. Ex vivo immunophenotyping of whole blood demonstrated that the indeterminate clinical form displays a higher frequency of both CD4+CD25high and NKT regulatory cells (CD3+CD16-CD56+), associated with increased levels of circulating cytotoxic NK cells (CD3-CD16+CD56+ and CD3-CD16+CD56dim NK cells). By contrast, the increased percentage of activated CD8+HLA-DR+ T-cell subset was exclusively associated with severe clinical forms of Chagas' disease. We hypothesize that regulatory T cells may be able to control the deleterious cytotoxic activity in the indeterminate clinical form by inhibiting the activation of CD8+HLA-DR+ T cells. The lack of regulated populations in cardiac and digestive clinical forms could account for impaired immune response that culminates in strong cytotoxic activity and tissue damage.

Phenotypic features of peripheral blood leucocytes during early stages of human infection with Trypanosoma cruzi.

We performed a cross-sectional flow cytometric analysis of peripheral blood mononuclear cells to evaluate human immunologic status during early stages of Trypanosoma cruzi infection in children. We identified major immunological features corresponding to three proposed phases of disease: early acute (EA) phase, late acute (LA) phase and recent chronic (RC) phase. EA phase was accompanied by expansion of conventional B cells, up-regulation of CD54 on monocytes and down-regulation of CD54 on T cells and not associated with monocyte-activation phenotypes or changes of natural killer (NK) population. LA phase was characterized by a selective increase in a distinct lineage of NK cells (CD16+CD56-), as well as a persistent expansion of B cells and down-regulation of CD54 on T cells. RC phase showed persistent low levels of CD54 molecule on T cells and an increase of B cells, mainly triggered by expansion of the B1-cell subset, as well as increased expression of human leucocyte antigen (HLA-DR) by monocytes. These findings reinforce the hypothesis that T. cruzi-derived antigens are able to activate NK cells before the development of T-cell-mediated immunity. Moreover, our data support previous findings of increased levels of B1 lymphocytes during human Chagas' disease and show that this event is already present during initial stages of chronic infection.

In utero sensitization in Chagas' disease leads to altered lymphocyte phenotypic patterns in the newborn cord blood mononuclear cells.

Neonates are sensitized in utero to maternal circulating antigens and idiotypes that eventually cross the placental barrier. We believe that children born of mothers under long lasting antigenic stimulation, as in a chronic infection, would be affected by these maternal influences and show differences in the phenotypic repertoire of lymphocytes. To test this hypothesis, we evaluated flow cytometry studies in cord blood mononuclear cells (CBMC) from children born of chagasic mothers without congenital disease, with special attention to T and B cells and expression of activation markers. We have also evaluated the peripheral blood mononuclear cells (PBMC) of these children 6 months after delivery. We show that CBMC of children born of infected mothers have high proliferative responses to antigenic stimulation, significantly lower mean percentages of CD3+ T cells, CD4+ T cells and diminished expression of the costimulatory molecule CD28 in the CD8+ T cell subset. Interestingly, this subpopulation has an increased expression of the MHC class II gene product as evidenced by the expression of HLADR. It is noteworthy that the patterns observed in CBMC T lymphocyte populations of these children closely resemble earlier findings on lymphocytic profiles of chronic chagasic adult patients and those of their mothers. We also show that, 6 months after delivery, some alterations observed at birth are reversed to levels observed in noninfected individuals.

Neonatal idiotypic exposure alters subsequent cytokine, pathology, and survival patterns in experimental Schistosoma mansoni infections.

Exposure to maternal idiotypes (Ids) or antigens might predispose a child to develop an immunoregulated, asymptomatic clinical presentation of schistosomiasis. We have used an experimental murine system to address the role of Ids in this immunoregulation. Sera from mice with 8-wk Schistosoma mansoni infection, chronic (20-wk infection) moderate splenomegaly syndrome (MSS), or chronic hypersplenomegaly syndrome (HSS) were passed over an S. mansoni soluble egg antigen (SEA) immunoaffinity column to prepare Ids (8WkId, MSS Id, HSS Id). Newborn mice were injected with 8WkId, MSS Id, HSS Id, or normal mouse immunoglobulin (NoMoIgG) and infected with S. mansoni 8 wk later. Mice exposed to 8WkId or MSS Id as newborns had prolonged survival and decreased morbidity compared with mice that received HSS Id or NoMoIgG. When stimulated with SEA, 8WkId, or MSS Id, spleen cells from mice neonatally injected with 8WkId or MSS Id produced more interferon gamma than spleen cells from mice neonatally injected with HSS Id or NoMoIgG. Furthermore, neonatal exposure to 8WkId or MSS Id, but not NoMoIgG or HSS Id, led to significantly smaller granuloma size and lower hepatic fibrosis levels in infected mice. Together, these results indicate that perinatal exposure to appropriate anti-SEA Ids induces long-term effects on survival, pathology, and immune response patterns in mice subsequently infected with S. mansoni.

Schistosoma mansoni: susceptibility differences between male and female mice can be mediated by testosterone during early infection.

In murine Schistosoma mansoni infections, fewer adult worms develop in male than in female mice infected with the same number of cercariae. To evaluate a potential role for testosterone in this phenomenon, testosterone levels were manipulated in groups of CBA/J mice that were then infected and monitored for survival rates, worm burdens, organomegaly, and egg production. By 16 weeks of infection, more than 80% of mice in groups with low levels of testosterone (untreated females, castrated males, or carrier-treated castrates) were dead, while less than 40% of those in groups with high levels of testosterone (sham-castrated males, testosterone-treated castrates, or testosterone-treated female mice) succumbed to infection. The mean number of worms recovered from mice in the low testosterone level groups was comparable among groups, and significantly greater than that from those in high-testosterone-level groups. The degree of organomegaly observed correlated strongly with worm burden, but the number of hepatic eggs per female worm did not differ significantly between groups. When male mice were castrated or sham-castrated 5 weeks after S. mansoni infection, no significant differences in host survival occurred. Furthermore, female mice treated with testosterone demonstrated reduced worm burdens if the testosterone was given 10 days prior to infection but not if the testosterone was given 10 days or 5 weeks after infection. Thus, the host sex bias observed in parallel-infected male and female mice appears to be related to the presence of male gonadal tissue or testosterone early in infection, during the development of immature schistosomules.

Interferon-gamma production by peripheral blood mononuclear cells from residents of an area endemic for Schistosoma mansoni.

During human schistosomiasis host responses to antigens of various parasite life-cycle stages may contribute to whether the severe, hepatosplenic state develops or the patient remains relatively asymptomatic throughout infection, and may play a role in resistance. This study evaluated production of interferon gamma (IFN-gamma) in vitro by schistosome antigen-stimulated peripheral blood mononuclear cells (PBMCs) from asymptomatic patients, and by PBMCs from apparently uninfected, untreated persons living in areas endemic for Schistosoma mansoni ('endemic normals'). IFN-gamma production parallels PBMC proliferation in that schistosomal egg antigens stimulate patent patients' cells poorly, but strongly stimulate PBMCs from 'endemic normals'. This is proportionally true for antigens from adult worms and cercariae. Although asymptomatic patent patients' cells produced little or no IFN-gamma in response to the 3 schistosomal antigenic extracts, their PBMCs, and PBMCs from 'endemic normals', produced expected amounts of IFN-gamma when exposed to phytohaemagglutinin. This implies that persons with patent infections have schistosome antigen-specific defects in their ability to respond to IFN-gamma production that are not exhibited by putatively resistant 'endemic normals'.

Schistosoma mansoni: mortality, pathophysiology, and susceptibility differences in male and female mice.

In parallel studies of Schistosoma mansoni infections in male and female CBA/J mice, major sex-related differences are seen in the development of infection and disease. Upon equal subcutaneous exposures to 45 cercariae female mice present a more severe clinical course with consequent higher mortality than male mice. By 12 weeks of infection, more than 80% of female mice die, while less than 20% of infected males succumb to infection. This greater index of mortality is apparently due to the higher susceptibility of female mice to the development of adult worms. Exposed to 45 cercariae, virtually all females develop patent infections, but 8-34% of male mice do not do so. Also, the recovery rate of adult worms per cercariae from female mice is much higher than that from males, indicating that schistosomula are more successful in developing into adult worms in female mice. Additional studies indicate that this dichotomy of schistosomiasis in the sexes is not restricted to mice of the CBA/J strain, but also occurs in C57BL/6 and outbred CF1 strain mice.

Differential cellular reactivity to adult worm antigens of patients with different clinical forms of schistosomiasis mansoni.

Analysis of the proliferation in vitro of peripheral blood mononuclear cells and the production of interferon-gamma (IFN-gamma) in individuals infected with Schistosoma mansoni and showing different clinical forms of the disease, as well as normal putatively immune individuals from an endemic area, was undertaken using total and fractionated soluble adult worm antigens (SWAP). A higher frequency of detectable response to fractionated antigens in T cell Western blot assays was observed in individuals with the more severe forms of the disease. Analysis of variance showed that, in the Western blot assays, there was a statistically significant difference in the level of cellular proliferation to antigens with low molecular weight (less than 21 kDa) between hepatosplenic patients and those with intestinal and hepatointestinal forms of the disease. No correlation between cellular proliferation and IFN-gamma production was observed. Most of the normal individuals from an endemic area failed to show significant proliferative responses to SWAP T cell Western blot assays or to antigen immobilized on nitrocellulose; they did show significant proliferative responses to whole soluble SWAP with positive IFN-gamma production. The results are consistent with the hypothesis that variations in the cellular immune responses to SWAP influence both the development of pathology and resistance to infection in schistosomiasis mansoni.

Idiotypic sensitization in utero of children born to mothers with schistosomiasis or Chagas' disease.

Cord blood mononuclear cells (CBMC) of neonates born of mothers with Chagas' disease or schistosomiasis exhibited strong proliferative responses against idiotypes expressed on antibodies with specificity for Trypanosoma cruzi or Schistosoma mansoni antigens, respectively. These immunoaffinity-purified preparations were stimulatory if they were prepared from pools of patients' sera or from the mother's own serum, taken early during her pregnancy. These CBMC did not respond to normal immunoglobulin, and CBMC of neonates born of uninfected mothers did not respond to antibodies against either T. cruzi or S. mansoni, or normal immunoglobulin preparations. We propose that in utero exposure of a fetus to some idiotypes expressed on placentally transferred antibodies induces anti-Id T lymphocyte sensitization, which we detect as a proliferative response by CBMC exposed to immunoaffinity-purified antibodies expressing the relevant idiotypes. This is the first experimental evidence that children born of mothers with chronic infections undergo natural in utero idiotypic manipulations and are born possessing cellular anti-Id reactivity.