Serum biomarkers in patients with unilateral or bilateral active pulmonary tuberculosis: Immunological networks and promising diagnostic applications.

The present observational study was designed to characterize the integrative profile of serum soluble mediators to describe the immunological networks associated with clinical findings and identify putative biomarkers for diagnosis and prognosis of active tuberculosis. The study population comprises 163 volunteers, including 84 patients with active pulmonary tuberculosis/(TB), and 79 controls/(C). Soluble mediators were measured by multiplexed assay. Data analysis demonstrated that the levels of CCL3, CCL5, CXCL10, IL-1ß, IL-6, IFN-γ, IL-1Ra, IL-4, IL-10, PDGF, VEGF, G-CSF, IL-7 were increased in TB as compared to C. Patients with bilateral pulmonary involvement/(TB-BI) exhibited higher levels of CXCL8, IL-6 and TNF with distinct biomarker signatures (CCL11, CCL2, TNF and IL-10) as compared to patients with unilateral infiltrates/(TB-UNI). Analysis of biomarker networks based in correlation power graph demonstrated small number of strong connections in TB and TB-BI. The search for biomarkers with relevant implications to understand the pathogenetic mechanisms and useful as complementary diagnosis tool of active TB pointed out the excellent performance of single analysis of IL-6 or CXCL10 and the stepwise combination of IL-6 â†’ CXCL10 (Accuracy = 84 %; 80 % and 88 %, respectively). Together, our finding demonstrated that immunological networks of serum soluble biomarkers in TB patients differ according to the unilateral or bilateral pulmonary involvement and may have relevant implications to understand the pathogenetic mechanisms involved in the clinical outcome of Mtb infection.

Identification of Anti-Trypanosoma cruzi Lead Compounds with Putative Immunomodulatory Activity.

In seeking substitutions for the current Chagas disease treatment, which has several relevant side effects, new therapeutic candidates have been extensively investigated. In this context, a balanced interaction between mediators of the host immune response seems to be a key element for therapeutic success, as a proinflammatory microenvironment modulated by interleukin-10 (IL-10) is shown to be relevant to potentiate anti-Trypanosoma cruzi drug activity. This study aimed to identify the potential immunomodulatory activities of the anti-T. cruzi K777, pyronaridine (PYR), and furazolidone (FUR) compounds in peripheral blood mononuclear cells (PBMC) from noninfected (NI) subjects and chronic Chagas disease (CD) patients. Our results showed low cytotoxicity to PBMC populations, with 50% cytotoxic concentrations (CC50) of 71.0 µM (K777), 9.0 µM (PYR), and greater than 20 µM (FUR). In addition, K777 showed no impact on the exposure index (EI) of phytohemagglutinin-stimulated leukocytes (PHA), while PYR and FUR treatments induced increased EI of monocytes and T lymphocytes at late stages of apoptosis in NI subjects. Moreover, K777 induced a more prominent proinflammatory response (tumor necrosis factor alpha-positive [TNF-α+] CD8+/CD4+, gamma interferon-positive [IFN-γ+] CD4+/CD8+ modulated by interleukin-10-positive [IL-10+] CD4+ T/CD8+ T) than did PYR (TNF-α+ CD8+, IL-10+ CD8+) and FUR (TNF-α+ CD8+, IL-10+ CD8+). Signature analysis of intracytoplasmic cytokines corroborated the proinflammatory/modulated (K777) and proinflammatory (PYR and FUR) profiles previously found. In conclusion, the lead compound K777 may induce beneficial changes in the immunological profile of patients presenting the chronic phase of Chagas disease and may contribute to a more effective therapy against the disease.

Lifewide profile of cytokine production by innate and adaptive immune cells from Brazilian individuals.

BACKGROUND: Immunosenescence is associated with several changes in adaptive and innate immune cells. Altered cytokine production is among the most prominent of these changes. The impact of age-related alterations on cytokine global profiles produced by distinct populations of leukocytes from healthy Brazilian individuals was studied. We analysed frequencies of cytokine-producing lymphocytes and innate immune cells from individuals at several ages spanning a lifetime period (0-85 years). RESULTS: Healthy adult individuals presented a balanced profile suggestive of a mature immune system with equal contributions of both innate and adaptive immunity and of both categories of cytokines (inflammatory and regulatory). In healthy newborns and elderly, innate immune cells, especially neutrophils and NK-cells, contributed the most to a balanced profile of cytokines. CONCLUSIONS: Our results support the hypothesis that ageing is not associated with a progressive pro-inflammatory cytokine production by all leukocytes but rather with distinct fluctuations in the frequency of cytokine-producing cells throughout life.

Signatures of CD4+ T and B cells are associated with distinct stages of chronic chagasic cardiomyopathy.

Introduction: Chagas disease is a neglected parasitic disease caused by Trypanosoma cruzi. While most patients are asymptomatic, around 30% develop Chronic Chagasic Cardiomyopathy (CCC). Methods: Here, we employed high-dimensional flow cytometry to analyze CD4+ T and B cell compartments in patients during the chronic phase of Chagas disease, presenting the asymptomatic and mild or moderate/severe cardiac clinical forms. Results: Effector CD27-CD4+ T cells were expanded in both CCC groups, and only mild CCC patients showed higher frequencies of effector memory and T follicular helper (Tfh) cells than healthy donors (CTL) and asymptomatic patients. Unsupervised analysis confirmed these findings and further revealed the expansion of a specific subpopulation composed of Tfh, transitional, and central memory CD4+ T cells bearing a phenotype associated with strong activation, differentiation, and exhaustion in patients with mild but not moderate/severe CCC. In contrast, patients with mild and moderate/severe CCC had lower frequencies of CD4+ T cells expressing lower levels of activation markers, suggesting resting status, than CTL. Regarding the B cell compartment, no alterations were found in naïve CD21-, memory cells expressing IgM or IgD, marginal zone, and plasma cells in patients with Chagas disease. However, expansion of class-switched activated and atypical memory B cells was observed in all clinical forms, and more substantially in mild CCC patients. Discussion: Taken together, our results showed that T. cruzi infection triggers changes in CD4+ T and B cell compartments that are more pronounced in the mild CCC clinical form, suggesting an orchestrated cellular communication during Chagas disease. Conclusion: Overall, these findings reinforce the heterogeneity and complexity of the immune response in patients with chronic Chagas disease and may provide new insights into disease pathology and potential markers to guide clinical decisions.

Efficacy of three benznidazole dosing strategies for adults living with chronic Chagas disease (MULTIBENZ): an international, randomised, double-blind, phase 2b trial.

BACKGROUND: Treatment with benznidazole for chronic Chagas disease is associated with low cure rates and substantial toxicity. We aimed to compare the parasitological efficacy and safety of 3 different benznidazole regimens in adult patients with chronic Chagas disease. METHODS: The MULTIBENZ trial was an international, randomised, double-blind, phase 2b trial performed in Argentina, Brazil, Colombia, and Spain. We included participants aged 18 years and older diagnosed with Chagas disease with two different serological tests and detectable T cruzi DNA by qPCR in blood. Previously treated people, pregnant women, and people with severe cardiac forms were excluded. Participants were randomly assigned 1:1:1, using a balanced block randomisation scheme stratified by country, to receive benznidazole at three different doses: 300 mg/day for 60 days (control group), 150 mg/day for 60 days (low dose group), or 400 mg/day for 15 days (short treatment group). The primary outcome was the proportion of patients with a sustained parasitological negativity by qPCR during a follow-up period of 12 months. The primary safety outcome was the proportion of people who permanently discontinued the treatment. Both primary efficacy analysis and primary safety analysis were done in the intention-to-treat population. The trial is registered with EudraCT, 2016-003789-21, and ClinicalTrials.gov, NCT03191162, and is completed. FINDINGS: From April 20, 2017, to Sept 20, 2020, 245 people were enrolled, and 234 were randomly assigned: 78 to the control group, 77 to the low dose group, and 79 to the short treatment group. Sustained parasitological negativity was observed in 42 (54%) of 78 participants in the control group, 47 (61%) of 77 in the low dose group, and 46 (58%) of 79 in the short treatment group. Odds ratios were 1·41 (95% CI 0·69-2·88; p=0·34) when comparing the low dose and control groups and 1·23 (0·61-2·50; p=0·55) when comparing short treatment and control groups. 177 participants (76%) had an adverse event: 62 (79%) in the control group, 56 (73%) in the low dose group, and 59 (77%) in the short treatment group. However, discontinuations were less frequent in the short treatment group compared with the control group (2 [2%] vs 11 [14%]; OR 0·20, 95% CI 0·04-0·95; p=0·044). INTERPRETATION: Participants had a similar parasitological responses. However, reducing the usual treatment from 8 weeks to 2 weeks might maintain the same response while facilitating adherence and increasing treatment coverage. These findings should be confirmed in a phase 3 clinical trial. FUNDING: European Community's 7th Framework Programme.

Blood leukocytes from benznidazole-treated indeterminate chagas disease patients display an overall type-1-modulated cytokine profile upon short-term in vitro stimulation with Trypanosoma cruzi antigens.

BACKGROUND: Benznidazole (Bz)-chemotherapy is recommended to prevent Chagas disease progression, despite its limited efficacy during chronic disease. However, the host mechanisms underlying these benefits still remain to be elucidated. METHODS: In this study, we have used short-term whole blood cultures to describe the cytokine profile of Bz-treated Indeterminate Chagas disease patients-(INDt) as compared to untreated patients-(IND). RESULTS: Our findings showed that IND presented increased levels of IL-10+neutrophils, IL-12⁺ and IL-10⁺ monocytes and IFN-γ⁺NK-cells. Moreover, IND showed slight increase of IL-4⁺CD4⁺T-cells and enhanced levels of IL-10⁺CD8⁺T-cells and B-cells. Additional analysis of cytokine Low and High producers also highlighted the presence of High cytokine producers within IND, including IL-10 from CD4⁺ T-cells and IFN-γ from CD8⁺ T-cells, as compared to NI. The Bz-treatment lead to an overall cytokine down-regulation in the innate and adaptive compartments, including low levels of IL-12⁺ and IL-10⁺ neutrophils and monocytes, IFN-γ⁺NK-cells, IL-12⁺, TNF-α⁺, IFN-γ⁺ and IL-5⁺CD4⁺T-cells and IL-10⁺B-cells, along with basal levels of cytokine-expressing CD8⁺T-cells in INDt as compared to IND. The in vitro antigen stimulation shifted the cytokine profile toward a type 1-modulated profile, with increased levels of IL-12⁺ and IL-10⁺ monocytes, IFN-γ⁺ and IL-4⁺NK-cells along with TNF-α⁺ and IFN-γ⁺CD8⁺T-cells. Analysis of Low and High cytokine producers, upon in vitro antigen stimulation, further confirm these data. CONCLUSION: Together, our findings showed that the Bz treatment of Indeterminate Chagas' disease patients shifts the cytokine patterns of peripheral blood monocytes, NK-cells and CD8⁺ T-cells towards a long-lasting Type-1-modulated profile that could be important to the maintenance of a non-deleterious immunological microenvironment.

Further evidence that the expression of CD38 and HLA-DR(+) in CD8(+) lymphocytes does not correlate to disease progression in HIV-1 vertically infected children.

BACKGROUND: In adults, an increase in CD8(+)CD38(+) T cell levels is a strong indicator of disease progression in HIV infection. However, in children, data are conflicting. Slow-progressing children (SPC) provide an exceptional resource for the investigation and clarification of the immunological and virological mechanisms of natural control of HIV infection and can be used to investigate prognostic indicators of disease progression. OBJECTIVES: To investigate the immune activation status and T regulatory (Treg) cell levels in SPC. STUDY DESIGN: A cross-sectional study was carried out on 28 children 8 years old and older who were vertically infected with HIV. The children were stratified into 3 groups according to their clinical outcome: SPC (anti-retroviral-therapy-naïve; ≥8 years-old; CD4 ≥20%; viral load <25,000 copies), IF/VF (anti-retroviral-therapy but with no therapeutic response), and IS/VS (anti-retroviral therapy with good therapeutic response). Uninfected children (NI) were assessed as healthy control group. RESULTS: A higher percentage of activated CD8(+) T cells were found in all HIV infected children, regardless of the evolution of disease. The activation of CD8(+) T cells was not associated with either viral load or the percentage of CD4(+) T cells. In addition, Treg cell levels did not show any correlation with the clinical outcome or the activation status of CD8(+) T cells. CONCLUSIONS: HIV-1-infected children presented an increased percentage of activated CD8(+) T cells and an unaltered percentage of Treg cells, regardless of their clinical evolution. Thus, these immunological parameters should not be used for prognostic evaluation.

Cytokine signatures of innate and adaptive immunity in 17DD yellow fever vaccinated children and its association with the level of neutralizing antibody.

BACKGROUND: The live attenuated yellow fever (YF) vaccines have been available for decades and are considered highly effective and one of the safest vaccines worldwide. METHODS: The impact of YF-17DD-antigens recall on cytokine profiles of YF-17DD-vaccinated children were characterized using short-term cultures of whole blood samples and single-cell flow cytometry. This study enrolled seroconverters and nonseroconverters after primovaccination (PV-PRNT⁺ and PV-PRNT⁻), seroconverters after revaccination (RV-PRNT⁺), and unvaccinated volunteers (UV-PRNT⁻). RESULTS: The analysis demonstrated in the PV-PRNT⁺ group a balanced involvement of pro-inflammatory/regulatory adaptive immunity with a prominent participation of innate immunity pro-inflammatory events (IL-12⁺ and TNF-α⁺ NEU and MON). Using the PV-PRNT⁺ cytokine signature as a reference profile, PV-PRNT⁻ presented a striking lack of innate immunity proinflammatory response along with an increased adaptive regulatory profile (IL-4⁺CD4⁺ T cells and IL-10⁺ and IL-5⁺CD8⁺ T cells). Conversely, the RV-PRNT⁺ shifted the overall cytokine signatures toward an innate immunity pro-inflammatory profile and restored the adaptive regulatory response. CONCLUSIONS: The data demonstrated that the overall cytokine signature was associated with the levels of PRNT antibodies with a balanced innate/adaptive immunity with proinflammatory/regulatory profile as the hallmark of PV-PRNT(MEDIUM⁺), whereas a polarized regulatory response was observed in PV-PRNT⁻ and a prominent proinflammatory signature was the characteristic of PV-PRNT(HIGH⁺).

Trypanosoma cruzi: desferrioxamine decreases mortality and parasitemia in infected mice through a trypanostatic effect.

Desferrioxamine (DFO) is a potent iron chelator that is also known to modulate inflammation and act as an efficient antioxidant under normal conditions and under oxidative stress. Many in vitro and in vivo studies have shown the efficacy of DFO in the treatment of viral, bacterial and protozoan infections. DFO is known to reduce the intensity of Trypanosoma cruzi infections in mice even during a course of therapy that is not effective in maintaining anaemia or low iron levels. To further clarify these findings, we investigated the action of DFO on mouse T. cruzi infection outcomes and the direct impact of DFO on parasites. Infected animals treated with DFO (5 mg/animal/day) for 35 days, beginning 14 days prior to infection, presented lower parasitemia and lower cumulative mortality rate. No significant effect was observed on iron metabolism markers, erythrograms, leukograms or lymphocyte subsets. In the rapid method for testing in vivo T. cruzi susceptibility, DFO also induced lower parasitemia. In regard to its direct impact on parasites, DFO slightly inhibited the growth of amastigotes and trypomastigotes in fibroblast culture. Trypan blue staining showed no effects of DFO on parasite viability, and only minor apoptosis in trypomastigotes was observed. Nevertheless, a clear decrease in parasite mobility was detected. In conclusion, the beneficial actions of DFO on mice T. cruzi infection seem to be independent of host iron metabolism and free of significant haematological side effects. Through direct action on the parasite, DFO has more effective trypanostatic than trypanocidal properties.

Phenotypic and Functional Signatures of Peripheral Blood and Spleen Compartments of Cynomolgus Macaques Infected With T. cruzi: Associations With Cardiac Histopathological Characteristics.

We performed a detailed analysis of immunophenotypic features of circulating leukocytes and spleen cells from cynomolgus macaques that had been naturally infected with Trypanosoma cruzi, identifying their unique and shared characteristics in relation to cardiac histopathological lesion status. T. cruzi-infected macaques were categorized into three groups: asymptomatic [CCC(-)], with mild chronic chagasic cardiopathy [CCC(+)], or with moderate chronic chagasic cardiopathy [CCC(++)]. Our findings demonstrated significant differences in innate and adaptive immunity cells of the peripheral blood and spleen compartments, by comparison with non-infected controls. CCC(+) and CCC(++) hosts exhibited decreased frequencies of monocytes, NK and NKT-cell subsets in both compartments, and increased frequencies of activated CD8+ T-cells and GranA+/GranB+ cells. While a balanced cytokine profile (TNF/IL-10) was observed in peripheral blood of CCC(-) macaques, a predominant pro-inflammatory profile (increased levels of TNF and IFN/IL-10) was observed in both CCC(+) and CCC(++) subgroups. Our data demonstrated that cardiac histopathological features of T. cruzi-infected cynomolgus macaques are associated with perturbations of the immune system similarly to those observed in chagasic humans. These results provide further support for the validity of the cynomolgus macaque model for pre-clinical research on Chagas disease, and provide insights pertaining to the underlying immunological mechanisms involved in the progression of cardiac Chagas disease.

A novel Trypanosoma cruzi secreted antigen as a potential biomarker of Chagas disease.

Chagas drug discovery has been hampered by a lack of validated assays to establish treatment efficacy in pre-clinical animal models and in patients infected with T. cruzi. Reduced levels of parasite secreted antigens in the blood of infected hosts could be used to demonstrate treatment efficacy. A published proteomic study of parasite secreted antigens identified the hypothetical protein Tc_5171 as a secreted antigen. In this report, we developed Tc_5171 specific antibodies and showed that the native protein was expressed by the three life cycle stages of the parasite. Anti-peptide antibodies were able to detect the parasite antigen in blood of infected mice during the acute and the chronic phase of infection. Benznidazole treatment of infected mice significantly reduced their blood antigen levels. Of clinical significance, patients diagnosed with Chagas disease, either asymptomatic or with cardiac clinical symptoms had significantly higher Tc_5171 antigen levels compared to endemic controls. Pair-wise analysis, before and after Benznidazole treatment, of patients with asymptomatic Chagas disease showed a significant reduction in antigen levels post treatment. Taken together, our results indicate that Tc_5171 could be used as a novel biomarker of Chagas disease for diagnosis and to assess treatment efficacy.

Etiological treatment during early chronic indeterminate Chagas disease incites an activated status on innate and adaptive immunity associated with a type 1-modulated cytokine pattern.

Pro-inflammatory immune response is usually associated with Chagas disease pathogenesis, but is also relevant to treatment effectiveness. Cross-sectional studies have suggested that this activated state may persist for years after therapeutic intervention. However, short-term longitudinal investigation has suggested that the Benznidazole treatment (Bz-treatment) leads to decreased immunological activation. In order to elucidate this issue, we performed a longitudinal study to evaluate the immunological status following Bz-treatment during early indeterminate Chagas disease. Our results demonstrated that Bz-treatment led to higher activation status of circulating monocytes but was negatively associated with the number of IL-12(+)CD14(+) cells. Moreover, Bz-treatment triggered a high frequency of circulating CD3(-)CD16(+)CD56(-) NK cells, in addition to elevated activation status associated with a type 1-modulated cytokine pattern. Bz-treatment induced substantial T and B-cell activation status associated with an overall IL-10 modulated type 1 cytokine profile. In summary, these findings provide new information regarding immune activation status following the etiological treatment of Chagas disease. These results suggest that in addition to the increased number of activated leukocytes in the peripheral blood, Bz-treatment may also involve a qualitative change in their functional capacity that drives their activation state toward a modulated cytokine profile. These changes may account for the benefits of etiological treatment of Chagas disease.

Variation rhythms of lymphocyte subsets during healthy aging.

Immunological alterations associated with aging (immunosenescence) do not represent a simple unidirectional decline in all functions but develop as a complex remodeling of the immune system, involving multiple reorganization and developmentally regulated changes. In general, most data available about aging were obtained at particular age intervals and most of them come from Caucasian individuals from either Europe or the United States. Here, we report the frequencies of major lymphocyte subsets in healthy Brazilian individuals from 2 distinct geographic regions (Southeast and South) at several age intervals spanning a lifetime period (0-86 years). Overall, we demonstrated that changes in the frequencies of cells related to both innate and adaptive immunity clearly occur with aging in these individuals. These changes were not progressive and equally steady for all cell populations tested but instead showed an oscillatory or rhythmic behavior that was distinctive of each population at different age intervals. We also observed that abrupt changes in the frequencies of immune cells may occur in healthy individuals over 75 years old, suggesting there is an impaired flexibility of the immune system at late stages of life to sustain homeostasis via immune mechanisms. We presented reference ranges for healthy Brazilian individuals at all ages. The knowledge of these parameters in further detail will allow interventions to optimize immune function in advanced age and to improve the quality of life in the elderly.

Non-conventional flow cytometry approaches to detect anti-Trypanosoma cruzi immunoglobulin G in the clinical laboratory.

We have recently developed a flow cytometric approach to detect anti-live trypomastigote and anti-fixed epimastigote IgG antibodies (FC-ALTA and FC-AFEA) in sera from individuals infected by Trypanosoma cruzi. Here, we present the first evaluation of the applicability of FC-AFEA-IgG as a diagnostic tool for Chagas disease. Performance analysis demonstrated that FC-AFEA-IgG has a sensitivity of 82% and a specificity of 100%. The assessment for prognosis performed by FC-ALTA-IgG1 and FC-AFEA-IgG, after classification of chagasic patients as belonging to indeterminate (IND), cardiac (CARD) or digestive (DIG) clinical forms, showed that most of IND have higher amounts of IgG than individuals' carrying CARD or DIG Chagas disease. FC-AFEA-IgG was also evaluated as a method to monitor chemotherapy efficacy in individuals classified into three distinct categories: not treated (NT), treated but not cured (TNC), and treated and cured (TC). Performance analysis demonstrated that FC-AFEA-IgG has an extraordinary capacity as a serological criterion to assess cure after therapeutic intervention in Chagas disease. These results represent a great advance in the application of serological techniques for clinical investigations on Chagas disease, and they clearly define new directions and perspectives. We intend to continue this field research focusing our attention on the influence of the degree of clinical damage on the FC-ALTA-IgG1 and FC-AFEA-IgG reactivity.

Mycobacterial Hsp65 antigen upregulates the cellular immune response of healthy individuals compared with tuberculosis patients.

Previously we showed that 65-kDa Mycobacterium leprae heat shock protein (Hsp65) is a target for the development of a tuberculosis vaccine. Here we evaluated peripheral blood mononuclear cells (PBMC) from healthy individuals or tuberculosis patients stimulated with two forms of Hsp65 antigen, recombinant DNA that encodes Hsp65 (DNA-HSP65) or recombinant Hsp65 protein (rHsp65) in attempting to mimic a prophylactic or therapeutic study in vitro, respectively. Proliferation and cytokine-producing CD4+ or CD8+ cell were assessed by flow cytometry. The CD4+ cell proliferation from healthy individuals was stimulated by DNA-HSP65 and rHsp65, while CD8+ cell proliferation from healthy individuals or tuberculosis patients was stimulated by rHSP65. DNA-HSP65 did not improve the frequency of IFN-gamma+ cells from healthy individuals or tuberculosis patients. Furthermore, we found an increase in the frequency of IL-10-producing cells in both groups. These findings show that Hsp65 antigen activates human lymphocytes and plays an immune regulatory role that should be addressed as an additional antigen for the development of antigen-combined therapies.

Cynomolgus macaques naturally infected with Trypanosoma cruzi-I exhibit an overall mixed pro-inflammatory/modulated cytokine signature characteristic of human Chagas disease.

BACKGROUND: Non-human primates have been shown to be useful models for Chagas disease. We previously reported that natural T. cruzi infection of cynomolgus macaques triggers clinical features and immunophenotypic changes of peripheral blood leukocytes resembling those observed in human Chagas disease. In the present study, we further characterize the cytokine-mediated microenvironment to provide supportive evidence of the utility of cynomolgus macaques as a model for drug development for human Chagas disease. METHODS AND FINDINGS: In this cross-sectional study design, flow cytometry and systems biology approaches were used to characterize the ex vivo and in vitro T. cruzi-specific functional cytokine signature of circulating leukocytes from TcI-T. cruzi naturally infected cynomolgus macaques (CH). Results showed that CH presented an overall CD4+-derived IFN-γ pattern regulated by IL-10-derived from CD4+ T-cells and B-cells, contrasting with the baseline profile observed in non-infected hosts (NI). Homologous TcI-T. cruzi-antigen recall in vitro induced a broad pro-inflammatory cytokine response in CH, mediated by TNF from innate/adaptive cells, counterbalanced by monocyte/B-cell-derived IL-10. TcIV-antigen triggered a more selective cytokine signature mediated by NK and T-cell-derived IFN-γ with modest regulation by IL-10 from T-cells. While NI presented a cytokine network comprised of small number of neighborhood connections, CH displayed a complex cross-talk amongst network elements. Noteworthy, was the ability of TcI-antigen to drive a complex global pro-inflammatory network mediated by TNF and IFN-γ from NK-cells, CD4+ and CD8+ T-cells, regulated by IL-10+CD8+ T-cells, in contrast to the TcIV-antigens that trigger a modest network, with moderate connecting edges. CONCLUSIONS: Altogether, our findings demonstrated that CH present a pro-inflammatory/regulatory cytokine signature similar to that observed in human Chagas disease. These data bring additional insights that further validate these non-human primates as experimental models for Chagas disease.

Distinct patterns of cellular immune response elicited by influenza non-adjuvanted and AS03-adjuvanted monovalent H1N1(pdm09) vaccine.

The study aimed at identifying biomarkers of immune response elicited by non-adjuvanted-(NAV) and adjuvanted-(AV) H1N1(pdm09) vaccines. The results showed that despite both vaccines elicited similar levels of anti-H1N1 antibodies at day30 after vaccination, higher reactivity was observed in AV at day180. While AV induced early changes in cell-surface molecules on monocytes, CD4+, CD8+ T-cells and B-cells, NAV triggered minor changes, starting later on at day3. Furthermore, AV induced a late and persistent increase in TLR gene expression after day3, except for tlr4, while NAV displayed earlier but transient tlr3/4/7/9 up-regulation. Contrasting with NAV, prominent chemokine gene expression (cxcl8,cxcl9,ccl5) and a broad spectrum up-regulation of plasmatic biomarkers (CXCL8,IL-6,IL-1ß,IL-12,IL-10) was evident in AV, which showed a major involvement of TNF and IL-10. Similarly, AV induced a robust IL-10-modulated proinflammatory storm, with early and persistent involvement of TNF-α/IL-12/IFN-γ axis derived from NK-cells, CD4+ and CD8+ T-cells along with promiscuous production of IL-4/IL-5/IL-13. Conversely, NAV promotes a concise and restricted intracytoplasmic chemokine/cytokine response, essentially mediated by TNF-α and IL-4, with late IL-10 production by CD8+ T-cells. Systems biology approach underscored that AV guided the formation of an imbricate network characterized by a progressive increase in the number of neighborhood connections amongst innate and adaptive immunity. In AV, the early cross-talk between innate and adaptive immunity, followed by the triad NK/CD4+/CD8+ T-cells at day3, sponsored a later/robust biomarker network. These findings indicate the relevance of adjuvanted vaccination to orchestrate broad, balanced and multifactorial cellular immune events that lead ultimately to a stronger H1N1 humoral immunity.

Phenotypic Features of Circulating Leukocytes from Non-human Primates Naturally Infected with Trypanosoma cruzi Resemble the Major Immunological Findings Observed in Human Chagas Disease.

BACKGROUND: Cynomolgus macaques (Macaca fascicularis) represent a feasible model for research on Chagas disease since natural T. cruzi infection in these primates leads to clinical outcomes similar to those observed in humans. However, it is still unknown whether these clinical similarities are accompanied by equivalent immunological characteristics in the two species. We have performed a detailed immunophenotypic analysis of circulating leukocytes together with systems biology approaches from 15 cynomolgus macaques naturally infected with T. cruzi (CH) presenting the chronic phase of Chagas disease to identify biomarkers that might be useful for clinical investigations. METHODS AND FINDINGS: Our data established that CH displayed increased expression of CD32+ and CD56+ in monocytes and enhanced frequency of NK Granzyme A+ cells as compared to non-infected controls (NI). Moreover, higher expression of CD54 and HLA-DR by T-cells, especially within the CD8+ subset, was the hallmark of CH. A high level of expression of Granzyme A and Perforin underscored the enhanced cytotoxicity-linked pattern of CD8+ T-lymphocytes from CH. Increased frequency of B-cells with up-regulated expression of Fc-γRII was also observed in CH. Complex and imbricate biomarker networks demonstrated that CH showed a shift towards cross-talk among cells of the adaptive immune system. Systems biology analysis further established monocytes and NK-cell phenotypes and the T-cell activation status, along with the Granzyme A expression by CD8+ T-cells, as the most reliable biomarkers of potential use for clinical applications. CONCLUSIONS: Altogether, these findings demonstrated that the similarities in phenotypic features of circulating leukocytes observed in cynomolgus macaques and humans infected with T. cruzi further supports the use of these monkeys in preclinical toxicology and pharmacology studies applied to development and testing of new drugs for Chagas disease.

Comparison between qualitative and real-time polymerase chain reaction to evaluate minimal residual disease in children with acute lymphoblastic leukemia.

INTRODUCTION: Minimal residual disease is an important independent prognostic factor that can identify poor responders among patients with acute lymphoblastic leukemia. OBJECTIVE: The aim of this study was to analyze minimal residual disease using immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements by conventional polymerase chain reaction followed by homo-heteroduplex analysis and to compare this with real-time polymerase chain reaction at the end of the induction period in children with acute lymphoblastic leukemia. METHODS: Seventy-four patients diagnosed with acute lymphoblastic leukemia were enrolled. Minimal residual disease was evaluated by qualitative polymerase chain reaction in 57 and by both tests in 44. The Kaplan-Meier and multivariate Cox methods and the log-rank test were used for statistical analysis. RESULTS: Nine patients (15.8%) were positive for minimal residual disease by qualitative polymerase chain reaction and 11 (25%) by real-time polymerase chain reaction considering a cut-off point of 1×10(-3) for precursor B-cell acute lymphoblastic leukemia and 1×10(-2) for T-cell acute lymphoblastic leukemia. Using the qualitative method, the 3.5-year leukemia-free survival was significantly higher in children negative for minimal residual disease compared to those with positive results (84.1%±5.6% versus 41.7%±17.3%, respectively; p-value=0.004). There was no significant association between leukemia-free survival and minimal residual disease by real-time polymerase chain reaction. Minimal residual disease by qualitative polymerase chain reaction was the only variable significantly correlated to leukemia-free survival. CONCLUSION: Given the difficulties in the implementation of minimal residual disease monitoring by real-time polymerase chain reaction in most treatment centers in Brazil, the qualitative polymerase chain reaction strategy may be a cost-effective alternative.