Fcγ-RI, Fcγ-RII and IL-10 as predictive biomarkers for post-therapeutic cicatrization time in monocytes from cutaneous leishmaniasis patients.

Cutaneous leishmaniasis (CL) treatment is based on therapy with Glucantime® , yet, there are few laboratory methods to monitor its success. In this study, ex vivo and in vitro evaluations of peripheral blood monocytes were performed in a longitudinal study to characterize the impact of Glucantime® on overall phenotypic/functional features of these cells from CL patients to identify predictive biomarkers for post-therapeutic monitoring by flow cytometry. The ex vivo evaluation from CL patients demonstrated a modulatory profile before treatment, with a decrease in TLR-2, FcγRII, HLA-DR, CD86, IFN-γR, TNF, IL-12, NO, and an increase in FcγRIII and IL-10R. Conversely, treatment changes some of these biomarker expressions by decreasing FcγRIII and IL-10R and increasing IFN-γR, IL-12 and NO. Moreover, an in vitro analysis of these patients showed a reduced phagocytic capacity of Leishmania braziliensis and higher levels of IL-10 and TGF-ß modulating functional profile. Regardless of the compromised L. braziliensis phagocytic capacity, treatment re-established the production of IL-12, IL-10, TGF-ß and NO at the basal level. Notably, monocytes from patients with early cicatrization showed enhanced FcγRI and FcγRII expressions and reduced IL-10, which was further corroborated by a baseline fold change analysis. Finally, the logistic regression model emphasized the performance of FcγRI, FcγRII and IL-10 as robust predictive biomarkers for post-therapeutic cicatrization during cutaneous leishmaniasis.

Lactoferrin increases sperm membrane functionality of frozen equine semen.

During cryopreservation, sperm was submitted to an increase in reactive oxygen species generation. This work aimed to improve the quality of frozen equine sperm after the addition of antioxidants lactoferrin (Lf) and catalase (Cat) to a freezing extender. Semen from six stallions was frozen with the extenders: F1) control, INRA 82 freezing extender, F2) F1 + 500 µg/ml Lf and F3) F1 + 200 IU/ml Cat. After thawing, sperm motility parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome-reacted sperm were evaluated with a computer-assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite, hydroperoxide and iron concentrations of frozen semen were measured with spectrophotometry. The percentage of functional membrane sperm treated with Lf was higher (50.7% ± 11.6%) compared to that of the control (37.6% ± 15.6%), while the iron (61.4 ± 11.6 vs 73.3 ± 13.8 mg/dl) and nitrite concentrations (16.3 ± 7.1 vs 25.9 ± 4.2 µM/µg protein) were lower, respectively (p < .05). Thus, it can be suggested that Lf protect stallion spermatozoon during freezing as it has increased the percentage of sperm with functional membrane and decreased the lipid oxidant agents.

Evaluation of Fertilizing Ability of Frozen Equine Sperm Using a Bovine Zona Pellucida Binding Assay.

BACKGROUND: Frozen equine semen has lower fertility compared to cooled semen. Due to the difficulty to obtain equine oocytes, a heterologous zona pellucida binding assay (ZBA) is an alternative method to predict the fertilizing capability of equine frozen sperm. The rate of capacitated and hyperactivated sperm according to their motility characteristics were analyzed with a Computer Assisted Sperm Analyzer. We believe this report describes for the first time the in vitro hyperactivation induction and the heterologous ZBA to predict the fertilizing ability of frozen equine sperm. OBJECTIVE: This work aimed at developing an assay to evaluate the fertilizing ability of frozen equine sperm using a bovine ZBA with the use of an in vitro capacitation and hyperactivation media with procaine and calcium ionophore A23187, respectively. MATERIALS AND METHODS: The sperm motility characteristics, intact and acrosome reacted sperm rates, and number of stallion sperm bound to the bovine ZP were calculated. RESULTS: The procaine group showed a hyperactivation motility pattern, although it improved ZP sperm binding similarly to the capacitation group. CONCLUSION: The capacitation medium improved the IVF capability of frozen equine sperm, allowing the highest possibility of sperm-oocyte interaction.

Plasma biomarkers profile of female dogs with mammary carcinoma and its association with clinical and pathological features.

The immunological biomarkers profiles were evaluated using Luminex as putative measures to monitor canine mammary carcinomas (MCs). Forty female dogs were categorized into benign mixed tumour (MC-BMT = 28) and mammary carcinoma (MC=12). The ascendant biomarker signatures were used to compare the groups. For example, a higher frequency of MC-BMT animals producing IL-6, CXCL-8 and CXCL-10 was observed, whereas for the MC group IL-2 and CXCL-8 were detected. MC-BMT animals without metastasis had an increase in the levels of IL-2, CXCL-8, CXCL-10, IL-6, TNF-α, IL-15 and a decrease in IL-10 and CXCL-8. MC-BMT animals with metastasis showed only an increase in CXCL-10 and a decrease in IL-18. After comparing the ascendant signatures following the presence of metastasis in both groups, a higher frequency of dogs exhibiting IL-10 production was observed. Pearson correlation (P = 0.0273) and receiver operating characteristic (ROC) curve analysis revealed that this pattern was associated with worse outcome and lower survival rates in MC animals.

Trophoblast-macrophage crosstalk on human extravillous under Toxoplasma gondii infection.

INTRODUCTION: The interaction between human extravillous trophoblasts and macrophages has an important role in implantation and placentation. However, any dysfunction in this communication system is associated with pregnancy pitfalls, and a Toxoplasma gondii infection can be a potential problem in this crosstalk. Therefore, the aim of this study was to assess the influence of infected macrophages on cytokine production and the incidence of apoptosis in T. gondii-infected extravillous trophoblast cells. METHODS: HTR-8/SVneo cells were treated with supernatant from macrophages infected or not by T. gondii (conditioned medium) in order to analyze apoptosis and cytokine production in comparison to uninfected control conditions. RESULTS: The IL-6 secretion by HTR-8/SVneo cells increased synergistically by treatment with conditioned medium and T. gondii infection. The apoptosis index of HTR-8/SVneo cells was also upregulated by treatment with conditioned medium and infection. In addition, a low expression of Fas/CD95 and a high soluble FasL release were observed during infection, although no significant change was observed in the proliferation of T. gondii. DISCUSSION: The parasite modulates the high apoptosis index in HTR-8/SVneo cells in order to favor its establishment inside its host cells. On the other hand, the conditioned medium from uninfected macrophages restores the apoptosis rates, although the effect of the infection seems to be stronger. In conclusion, our results showed that T. gondii infection in human extravillous trophoblasts is able to modulate the trophoblast-macrophage crosstalk.

Protective Profile Involving CD23/IgE-mediated NO Release is a Hallmark of Cutaneous Leishmaniasis Patients from the Xakriabá Indigenous Community in Minas Gerais, Brazil.

In this study, we described, for the first time, specific aspects of an anti-Leishmania immune response in a Brazilian Xakriabá indigenous community. Induction of an intracellular NO pathway, triggered by the binding of IgE to CD23 receptor in IFN-γ/IL-4 cytokines environment, was evaluated in localized cutaneous leishmaniasis (LCL) carriers and positive Montenegro skin test (MST) individuals without skin lesion (MT(+) SL(-)). Our data demonstrated that the higher frequency of CD23(+) CD14(+) monocytes and the increased serum levels of IgE observed in the LCL group were even higher in LCL carriers with late lesions (LCL≥60). Furthermore, patients with LCL presented increased NO production after Leishmania (Viannia) braziliensis stimulation and this NO profile was independent of the time of the lesion (recent LCL<60 or late LCL≥60). We also showed that the increased frequency of IFN-γ(+) and IL-4(+) CD4(+) T cells is related to the MT(+) SL(-) group. The results of biomarker signature curves demonstrated that in the MT(+) SL(-) group, the index signature was characterized by DAF-2T(+) CD14(+)/IL-4(+) CD8(+)/IFN-γ(+) CD4(+)/IL-4(+) CD4(+). On the other hand, the LCL group presented a higher index of DAF-2T(+) CD14(+)/CD23(+) CD14(+)/IL-4(+) CD8(+), associated with a lower index of IFN-γ(+) CD8(+). Considering the time of lesion, data analysis demonstrated that the main differences observed were highlighted in LCL<60 patients, with a higher index of CD23(+) CD14(+), which was also present in LCL≥60 patients. In conclusion, our data suggest that the protective immune response involving CD23-IgE-mediated NO release is a hallmark of patients with LCL. However, in MT(+) SL(-) individuals, another different leishmanicidal mechanism seems to be involved.

Cross-reactivity of anti-human cytokine monoclonal antibodies used as a tool to identify novel immunological biomarkers in domestic ruminants.

Eleven commercially available PE-labeled anti-human (IL-1-α, IL-6, IL-8, TNF-α, IL-17A, IL-5, IL-10, IL-12 and IL-13) and anti-mouse (IL-10, TNF-α) cytokine monoclonal antibodies (mAbs) were tested for cross-reactivity with cattle, goat, and sheep cytokines. Cross-reactivity was assessed by comparative analysis with the standard reactivity of the target species. Our data demonstrated that anti-human IL-1-α, IL-6, IL-8, IL-17A and IL-10 mAbs cross-react with all ruminant species tested. Anti-human IL-5 mAb showed a strong cross-reactivity with cattle and goat IL-5, while anti-human TNF-α mAb showed a selective cross-reactivity with goat TNF-α. No cross-reactivity with the ruminant cytokines was observed for anti-human IL-12 and IL-13 mAbs or for the two anti-mouse cytokine mAbs tested. The present study demonstrated the cross-reactivity of various anti-human cytokine mAbs with cattle, sheep, and goat cytokines, increasing the range of immunological biomarkers for studies in veterinary medicine.

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.

Trypan blue exclusion assay by flow cytometry

Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.

Trypan blue exclusion assay by flow cytometry.

Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.

Differential apoptosis in BeWo cells after infection with highly (RH) or moderately (ME49) virulent strains of Toxoplasma gondii is related to the cytokine profile secreted, the death receptor Fas expression and phosphorylated ERK1/2 expression.

INTRODUCTION: Alterations of apoptosis are commonly associated with pregnancy complications and abortion. Modulation of apoptosis is a relevant feature of Toxoplasma gondii infection and it is related to parasite strain types. The aim of the present study was to evaluate the possible factors that are involved in the differential apoptosis of BeWo cells infected with distinct T. gondii strain types. METHODS: Human trophoblastic cells (BeWo cell line) were infected with RH or ME49 strains, the cytokine production was measured and the phosphorylation of anti-apoptotic ERK1/2 protein was analyzed. Also, cells were treated with different cytokines, infected with RH or ME49 strain, and analyzed for apoptosis index and Fas/CD95 death receptor expression. RESULTS: ME49-infected BeWo cells exhibited a predominantly pro-inflammatory cytokine profile, whereas cells infected with RH strain had a higher production of anti-inflammatory cytokines. Also, the incidence of apoptosis was higher in ME49-infected cells, which have been treated with pro-inflammatory cytokines compared to cells infected with RH and treated with anti-inflammatory cytokines. Moreover, Fas/CD95 expression was higher in cells infected with either ME49 or RH strain and treated with pro-inflammatory cytokines compared to anti-inflammatory cytokine treatment. The phosphorylation of ERK1/2 protein increased after 24 h of infection only with the RH strain. CONCLUSION: These results suggest that opposing mechanisms of interference in apoptosis of BeWo cells after infection with RH or ME49 strains of T. gondii can be associated with the differential cytokine profile secreted, the Fas/CD95 expression and the phosphorylated ERK1/2 expression.

Cytokine profile and FVIII inhibitors development in haemophilia A.

Haemophilia A is a hereditary bleeding disorder linked to the X chromosome characterized by a deficiency or defect in the coagulation factor VIII (FVIII). Individuals with this coagulopathy require constant infusions of FVIII to maintain their physical integrity and haemostasis. During treatment, some patients develop an immune response that produces antibodies to FVIII, also called inhibitors, affecting the pro-coagulant activity of this protein. Despite the clinical relevance of FVIII inhibitors, the immune mechanisms that lead to their production are not known. This study investigated the immunological cytokine profile using plasma from HA patients which were either positive or negative for FVIII inhibitors and from healthy individuals. The results showed that healthy individuals and HA patients that do not develop FVIII inhibitors have a mixed immune response profile with high secretion of IFN-γ, TNF-α IL-2 and IL-5. In contrast, HA patients with FVIII inhibitors exhibited an anti-inflammatory/regulatory immune response characterized by low levels of all measured cytokines except for IL-4 and IL-10. This profile may be related to the development and maintenance of the FVIII inhibitors. By comparing the cytokine profiles of the three different groups we have established a model explaining the immune activation resulting in the production of FVIII inhibitors in haemophilia A patients.

Trophoblast cells are able to regulate monocyte activity to control Toxoplasma gondii infection.

INTRODUCTION: Toxoplasma gondii is an intracellular parasite that causes severe disease when the infection occurs during pregnancy. Trophoblast cells constitute an important maternal-fetal barrier, with monocytes concentrating around them. Thus, interactions between trophoblasts and monocytes are important for maintaining a successful pregnancy, especially in cases of infection. This study aimed to evaluate the role of trophoblast cells (BeWo line) on monocyte (THP-1 line) activity in the presence or absence of T. gondii infection. METHODS: THP-1 cells were stimulated with supernatants of BeWo cells, previously infected or not with T. gondii, and then infected with parasites. The supernatant of both cells were collected and analyzed for cytokine production and T. gondii proliferation in THP-1 cells was determined. RESULTS: The results showed that after infection, the pattern of cytokines secreted by THP-1 and BeWo cells was characterized as a pro-inflammatory profile. Furthermore, supernatant of BeWo cells infected or not, was able to change the cytokine profile secreted by infected THP-1 cells, and this supernatant became THP-1 cells more able to control T. gondii proliferation than those that had not been stimulated. DISCUSSION: This effect was associated with secretion of interleukin (IL)-6 by the THP-1 cells and soluble factors secreted by BeWo cells, such as IL-6 and MIF. CONCLUSION: Together, these results suggest that trophoblast cells are able to modulate monocyte activity, resulting in the control of T. gondii infection and subsequent maintenance of pregnancy.

Comparative phenotypic profile of subpopulations of peripheral blood leukocytes in European (Bos taurus taurus) and Zebu cattle (Bos taurus indicus).

Differences in cellular and humoral immunity in Zebu (Bos taurus indicus) and European (B. taurus taurus) cattle breeds, which may be related to differences in resistance or susceptibility to infectious or parasitic diseases, are largely unknown. This study aimed to perform a comparative analysis of innate and adaptive immunity of European (including Holstein, Brown Swiss, and Hereford) and Zebu (including Gir, Nelore, and Guzera) breeds, by assessing their peripheral blood leukocyte profiles (i.e., monocytes, eosinophils, and lymphocytes, including CD4(+) and CD8(+) T cells, and CD21(+) B cells). Higher frequencies of cells involved in innate immunity were observed in Zebu breeds, particularly monocytes and non-T and non-B cells (13.37 ± 0.9058 and 37.67 ± 1.55, respectively). This finding may contribute to the increased resistance of B. taurus indicus to certain infectious and parasitic diseases. Considering other leukocyte populations in the peripheral blood, among-breed variation was greater than differences between the two subspecies. This study will serve as a basis for further investigations regarding comparative immunology and resistance to infectious and parasitic diseases of cattle.

Effect of heat and moisture exchangers on the prevention of ventilator-associated pneumonia in critically ill patients

Ventilator-associated pneumonia (VAP) remains one of the major causes of infection in the intensive care unit (ICU) and is associated with the length of hospital stay, duration of mechanical ventilation, and use of broad-spectrum antibiotics. We compared the frequency of VAP 10 months prior to (pre-intervention group) and 13 months after (post-intervention group) initiation of the use of a heat and moisture exchanger (HME) filter. This is a study with prospective before-and-after design performed in the ICU in a tertiary university hospital. Three hundred and fourteen patients were admitted to the ICU under mechanical ventilation, 168 of whom were included in group HH (heated humidifier) and 146 in group HME. The frequency of VAP per 1000 ventilator-days was similar for both the HH and HME groups (18.7 vs 17.4, respectively; P = 0.97). Duration of mechanical ventilation (11 vs 12 days, respectively; P = 0.48) and length of ICU stay (11 vs 12 days, respectively; P = 0.39) did not differ between the HH and HME groups. The chance of developing VAP was higher in patients with a longer ICU stay and longer duration of mechanical ventilation. This finding was similar when adjusted for the use of HME. The use of HME in intensive care did not reduce the incidence of VAP, the duration of mechanical ventilation, or the length of stay in the ICU in the study population.

Effect of heat and moisture exchangers on the prevention of ventilator-associated pneumonia in critically ill patients.

Ventilator-associated pneumonia (VAP) remains one of the major causes of infection in the intensive care unit (ICU) and is associated with the length of hospital stay, duration of mechanical ventilation, and use of broad-spectrum antibiotics. We compared the frequency of VAP 10 months prior to (pre-intervention group) and 13 months after (post-intervention group) initiation of the use of a heat and moisture exchanger (HME) filter. This is a study with prospective before-and-after design performed in the ICU in a tertiary university hospital. Three hundred and fourteen patients were admitted to the ICU under mechanical ventilation, 168 of whom were included in group HH (heated humidifier) and 146 in group HME. The frequency of VAP per 1000 ventilator-days was similar for both the HH and HME groups (18.7 vs 17.4, respectively; P = 0.97). Duration of mechanical ventilation (11 vs 12 days, respectively; P = 0.48) and length of ICU stay (11 vs 12 days, respectively; P = 0.39) did not differ between the HH and HME groups. The chance of developing VAP was higher in patients with a longer ICU stay and longer duration of mechanical ventilation. This finding was similar when adjusted for the use of HME. The use of HME in intensive care did not reduce the incidence of VAP, the duration of mechanical ventilation, or the length of stay in the ICU in the study population.

Distinct pattern of immunophenotypic features of innate and adaptive immunity as a putative signature of clinical and laboratorial status of patients with localized cutaneous leishmaniasis.

In this study, we have analysed the phenotypic features of innate/adaptive immunity of patients with localized cutaneous leishmaniasis (LCL), categorized according to their clinical/laboratorial status, including number of lesion (L1; L2­4), days of illness duration (≤60;>60) and positivity in the Montenegro skin test (MT−;MT+). Our findings highlighted a range of phenotypic features observed in patients with LCL (↑%HLA-DR+ neutrophils; ↑CD8+ HLA-DR+/CD4+ HLA-DR+ T cell ratio; ↑HLA-DR in B lymphocytes, ↑%CD23+ neutrophils, monocytes and B cells; ↑α-Leishmania IgG and ↑serum NO2⁻ + NO3⁻). Selective changes were observed in L1 (↑%HLA-DR+ neutrophils, ↑CD8+ HLA-DR+/CD4+ HLA-DR+ T cell ratio and ↑serum NO2⁻ + NO3⁻) as compared to L2­4 (↑%CD5− B cells; ↑CD23+ B cells and ↑α-Leishmania IgG). Whilst ≤60 presented a mixed profile of innate/adaptive immunity (↓%CD28+ neutrophils and ↑%CD4+ T cells), >60 showed a well-known leishmanicidal events (↑CD8+ T cells; ↑serum NO2⁻ + NO3⁻ and ↑α-Leishmania IgG). MT+ patients showed increased putative leishmanicidal capacity (↑%HLA-DR+ neutrophils; ↑%CD23+ monocytes; ↑CD8+ HLA-DR+/CD4+ HLA-DR+ T cell ratio and ↑ serum NO2⁻ + NO3⁻). Overall, a range of immunological biomarkers illustrates the complex immunological network associated with distinct clinical/laboratorial features of LCL with applicability in clinical studies.

Posttherapeutic cure criteria in Chagas' disease: conventional serology followed by supplementary serological, parasitological, and molecular tests.

We performed a critical study of conventional serology, followed by supplementary serological, parasitological, and molecular tests, to assess the response to etiologic treatment of Chagas' disease. A group of 94 Chagas' disease patients treated with benznidazole at least 10 years earlier were evaluated from the laboratory and clinical points of view. When conventional serology (enzyme-linked immunosorbent assay [ELISA], indirect immunofluorescence [IIF], and indirect hemagglutination [IHA]) and classic criteria (consistent results with any two of the three tests) or more rigorous criteria (consistent results from the three tests) were used, 10.6% and 8.5% of patients were considered treated and cured (TC) by classic and rigorous criteria, respectively. Patients were then evaluated using supplementary (recombinant ELISA and Trypanosoma cruzi excreted-secreted antigen blotting [TESA-blot]), parasitological (hemoculture), and molecular (PCR) tests. The results of recombinant ELISA were similar to those with the rigorous criterion (three consistent test results). The TESA-blot group showed a higher percentage (21.3%) of negative results than the groups defined by either cure criterion. Hemoculture and PCR gave negative results for all treated and cured (TC) patients, regardless of the criterion used. Recombinant ELISA and TESA-blot tests showed negative results for 70% and 87.5% of the patients categorized as TC by the classic and three-test criteria, respectively. For patients with discordant conventional serology, the supplementary serological and molecular tests were the decisive factor in determining therapeutic failure. Clinical evaluation showed that 62.5% of TC patients presented with the indeterminate form of the disease. Additionally, treated patients with negative TESA-blot results should be reevaluated later with all methodologies used here to verify whether TESA-blot is a reliable way to determine early parasitological cure of Chagas' disease.

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.

Expression of the chemokine receptor CXCR4 on lymphocytes of leprosy patients

Leprosy is caused by Mycobacterium leprae, which induces chronic granulomatous infection of the skin and peripheral nerves. The disease ranges from the tuberculoid to the lepromatous forms, depending on the cellular immune response of the host. Chemokines are thought to be involved in the immunopathogenesis of leprosy, but few studies have investigated the expression of chemokine receptors on leukocytes of leprosy patients. In the present study, we evaluated 21 leprosy patients (M/F: 16/5) with a new diagnosis from the Dermatology Outpatient Clinic of the University Hospital, Federal University of Minas Gerais. The control group was composed of 20 healthy members (M/F: 15/5) of the community recruited by means of announcements. The expression of CCR2, CCR3, CCR5, and CXCR4 was investigated by flow cytometry on the surface of peripheral blood lymphocytes. There was a decrease in percentage of CD3+CXCR4+ and CD4+CXCR4+ lymphocytes in the peripheral blood of leprosy patients (median [range], 17.6 [2.7-41.9] and 65.3 [3.9-91.9], respectively) compared to the control group (median [range], 43.0 [3.7-61.3] and 77.2 [43.6-93.5], respectively). The percentage of CD4+CXCR4+ was significantly lower in patients with the tuberculoid form (median [range], 45.7 [0.0-83.1]) of the disease, but not in lepromatous patients (median [range], 81.5 [44.9-91.9]). The CXCR4 chemokine receptor may play a role in leprosy immunopathogenesis, probably directing cell migration to tissue lesions in tuberculoid leprosy patients.