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Uncaria tomentosa (UT) is a medicinal plant popularly known as cat's claw belonging to the Rubiaceae family that has been reported to display antiviral and anti-inflammatory activities. The chikungunya virus (CHIKV) outbreaks constitute a Brazilian public health concern. CHIKV infection develops an abrupt onset of fever, usually accompanied by a skin rash, besides incapacitating polyarthralgia. There is no vaccine available or treatment for CHIKV infection. The present study evaluates the hydroalcoholic extract of UT bark as a potential antiviral against CHIKV. The in vitro antiviral activity of the UT extract against the Brazilian CHIKV strain was assessed using quantitative reverse transcription polymerase chain reaction, flow cytometry, and plaque assay. Results obtained demonstrated that UT inhibits CHIKV infection in a dose-dependent manner. At the non-cytotoxic concentration of 100 µg/mL, UT exhibited antiviral activity above 90% as determined by plaque reduction assay, and it reduced the viral cytopathic effect. Similarly, a significant virucidal effect of 100 µg/mL UT was observed after 24 and 48 h post-infection. This is the first report on the antiviral activity of UT against CHIKV infection, and the data presented here suggests UT as a potential antiviral to treat CHIKV infection.

Background: Vaccination schedules, as well as their effectiveness and contraindications, need to be evaluated regularly, especially in specific situations. Congenital Zika Syndrome (CZS) is a severe condition that results in extensive functional and neurological impairment of fetuses and newborns due to Zika virus tropism for fetal neural progenitor cells. Down Syndrome (DS) is the leading genetic cause of intellectual disability. The immune impairment in DS has already been described, but little is known about the immune response of CZS children. Thus, CZS and DS are specific conditions that can be considered for a reassessment of the available immunizations. Here, we carried out serological analyses of attenuated vaccines-induced antibodies for measles, rubella, and yellow fever viruses in children aged 2-7, grouped into asymptomatic controls, DS children, and CZS children., Methods: Plasma samples were taken, and vaccination records were compiled during clinical follow-up. Enzymatic immunoassays for quantifying anti-measles and anti-rubella IgG were performed to assess the response to the Measles, Mumps, and Rubella (MMR) vaccine. Plaque Reduction Neutralization Test (PRNT) was performed to investigate neutralizing antibodies in response to the Brazilian vaccine strain of yellow fever (YF-17DD)., Results: We highlight similar levels of anti-measles IgG and neutralizing antibodies for YF-17DD among CZS, DS, and asymptomatic children, although low positivity of measles data was seen in the three groups. In DS children, the 2-4-year-old group had an increased level of anti-measles IgG compared to the older group of children aged five to seven years. Lower anti-rubella IgG levels were observed in CZS and DS children compared to asymptomatic children. For anti-rubella IgG, the good performance of vaccination in asymptomatic children is due to younger ones rather than older ones., Conclusions: There were no reports of adverse events after the use of the MMR and YF-17DD indicating that CZS and DS could continue to receive these vaccines, but our data draws attention to the necessity of monitoring the vaccination response in CZS and DS children over time and the possible need to adhere to national measles vaccination campaigns. Scientific research needs to continue to help develop appropriate CZS and DS health guidelines., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Familiar-Macedo, Dias, Carvalho, Pauvolid-Corrêa, da Silveira, Oliveira, Gonçalves, Vianna, Cardoso, Boy da Silva, Baumblatt and de-Oliveira-Pinto.)

Immunological and cytotoxic mediators are induced in natural infection and are essential for the effectiveness of vaccination. Vaccination is useful to prevent the spread of SARS-CoV-2 and limit the morbidity/mortality of COVID-19. ChAdOx1 nCoV-19 is one of the most widespread vaccines in the world. We compared the detection of anti-S1 SARS-CoV2 IgG and the profile of inflammatory and cytotoxic responses of patients who developed different clinical outcomes of COVID-19 with individuals previously exposed or not to the virus received the first and booster doses of ChAdOx1 nCoV-19. Plasma from 35 patients with COVID-19 and 11 vaccinated were evaluated by multiplex assay. Here, no vaccinated subjects had serious adverse effects. Those vaccinated with a booster dose had higher anti-S1 IgG than mild/moderate and recovered patients. Critically ill and deceased patients had IgG levels like those immunized. By univariate analysis, IL-2, IL-17, and perforin do not differentiate between patients and vaccinated individuals. Granzyme A increased at dose 1, while patients had their levels reduced. High levels of granulysin, sFas, and IL-6 were detected in the deaths, but after vaccination, all were declined. The multivariate analysis supports the role of IL-6 and granulysin as associated and non-confounding variables related to the worst clinical outcome of COVID-19, but not sFas. Our data confirm the ability of the ChAdOx1 vaccine to produce specific antibody levels up to booster time. Furthermore, our data suggest that the vaccine can regulate both the hyper-production and the kinetics of the production of inflammatory and cytotoxic mediators involved in the cytokine storm, such as granulysin and IL-6., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Tuberculosis (TB) and AIDS are the leading causes of infectious diseases death worldwide. Here, we investigated the relationship between from single nucleotide polymorphisms (SNPs) of the NLRP3, CARD8, AIM2, CASP-1, IFI16, and IL-1β inflammasome genes, as well as the profiles of secreted proinflammatory cytokines (e.g., IL-1β, IL-18, IL-33, and IL-6) with the TB clinical profiles, TB-HIV coinfection, and IRIS onset., Methods: The individuals were divided into four groups: TB-HIV group (n=88; 11 of them with IRIS), HIV-1 group (n=20), TB group (n=24) and healthy volunteers (HC) group (n=10), and were followed up at INI/FIOCRUZ and HGNI (Rio de Janeiro/Brazil) from 2006 to 2016. Real-time PCR was used to determine the genotypes of the Single Nucleotide Polymorphism (SNPs), and ELISA was used to measure the plasma cytokine levels. Unconditional logistic regression models were used to perform risk estimations., Results: A higher risk for extrapulmonary TB was associated with the TT genotype (aOR=6.76; P=0.026) in the NLRP3 rs4612666 Single Nucleotide Polymorphism (SNP) and the C-C-T-G-C haplotype (aOR=4.99; P= 0.017) in the NLRP3 variants. This same Single Nucleotide Polymorphism (SNP) was associated with lower risk against extrapulmonary TB when the carrier allele C (aOR=0.15; P=0.021) was present. Among those with HIV-1 infections, a higher risk for TB onset was associated with the GA genotype (aOR=5.5; P=0.044) in the IL1-β rs1143634 Single Nucleotide Polymorphism (SNP). In contrast, lower risk against TB onset was associated with the A-G haplotype (aOR=0.17; P= 0.026) in the CARD8 variants. Higher IL-6 and IL-33 levels were observed in individuals with TB. A higher risk for IRIS onset was associated with CD8 counts ≤ 500 cells/mm 3 (aOR=12.32; P=0.010), the presence of extrapulmonary TB (aOR=6.6; P=0.038), and the CT genotype (aOR=61.06; P=0.026) or carrier allele T (aOR=61.06; P=0.026) in the AIM2 rs2276405 Single Nucleotide Polymorphism (SNP), whereas lower risk against IRIS onset was associated with the AT genotype (aOR=0.02; P=0.033) or carrier allele T (aOR=0.02; P=0.029) in the CARD8 rs2043211 Single Nucleotide Polymorphism (SNP) and the T-G haplotype (aOR=0.07; P= 0.033) in the CARD8 variants. No other significant associations were observed., Conclusions: Our results depict the involvement of genetic polymorphisms of crucial innate immunity genes and proinflammatory cytokines in the clinical outcomes related to TB-HIV coinfection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 de Sá, de Souza, Neira-Goulart, Ribeiro-Alves, Da Silva, Pilotto, Rolla, Giacoia-Gripp, de Oliveira Pinto, Scott-Algara, Morgado and Teixeira.)

Chikungunya virus (CHIKV) is an arbovirus currently distributed worldwide, causing a disease that shares clinical signs and symptoms with other illnesses, such as dengue and Zika and leading to a challenging clinical differential diagnosis. In Brazil, CHIKV emerged in 2014 with the simultaneous introduction of both Asian and East/Central/South African (ECSA) genotypes. Laboratorial diagnosis of CHIKV is mainly performed by molecular and serological assays, with the latter more widely used. Although many commercial kits are available, their costs are still high for many underdeveloped and developing countries where the virus circulates. Here we described the development and evaluation of a multi-epitope recombinant protein-based IgG-ELISA (MULTREC IgG-ELISA) test for the specific detection of anti-CHIKV antibodies in clinical samples, as an alternative approach for laboratorial diagnosis. The MULTREC IgG-ELISA showed 86.36% of sensitivity and 100% of specificity, and no cross-reactivity with other exanthematic diseases was observed. The recombinant protein was expressed from the binary system insect cell/baculovirus using the crystal-forming baculoviral protein polyhedrin as a carrier of the target recombinant protein to facilitate recovery. The crystals were at least 10 times smaller in size and had an amorphous shape when compared to the polyhedrin wild-type crystal. The assay uses a multi-epitope antigen, representing two replicates of 18 amino acid sequences from the E2 region and a sequence of 17 amino acids from the nsP3 region of CHIKV. The recombinant protein was highly expressed, easy to purify and has demonstrated its usefulness in confirming chikungunya exposure, indeed showing a good potential tool for epidemiological surveillance.

Introduction: It is a consensus that inflammatory mediators produced by immune cells contribute to changes in endothelial permeability in dengue. We propose to relate inflammatory mediators seen in dengue patients with the in vitro alteration of endothelial cells (ECs) cultured with serum from these patients. Methods: Patients with mild (DF) to moderate and severe dengue (DFWS/Sev) were selected. ELISA quantified inflammatory mediators. Expression of adhesion molecules and CD147 were evaluated in the ECs cultured with the patient's serum by flow cytometry. We assessed endothelial permeability by measuring transendothelial electrical resistance in cocultures of ECs with patient serum. Results: Dengue infection led to an increase in inflammatory mediators-the IL-10 distinguished DF from DFWS/Sev. There were no changes in CD31, CD54, and CD106 but decreased CD147 expression in ECs. DFWS/Sev sera induced a greater difference in endothelial permeability than DF sera. Correlation statistical test indicated that low IL-10 and IFN-γ and high CCL5 maintain the integrity of ECs in DF patients. In contrast, increased TNF, IFN-γ, CXCL8, and CCL2 maintain EC integrity in DFWS/Sev patients. Conclusions: Our preliminary data suggest that a subset of inflammatory mediators may be related to the maintenance or loss of endothelial integrity, reflecting the clinical prognosis.

Chikungunya virus (CHIKV) infection causes intense cytokine/chemokine inflammatory responses and debilitating joint pain. Indoleamine2,3-dioxygenase 1 (IDO-1) is an enzyme that initiates the tryptophan degradation that is important in initial host innate immune defense against infectious pathogens. Besides that, IDO-1 activation acts as a regulatory mechanism to prevent overactive host immune responses. In this study, we evaluated IDO-1 activity and cytokine/chemokine patterns in CHIKV patients. Higher IDO-1 (Kyn/Trp ratio) activation was observed during the early acute phase of CHIKV infection and declined in the chronic phase. Importantly, increased concentrations of Tumor Necrosis Factor-α (TNF-α), Interleukin-6 (IL-6), Interferon γ (IFN-γ), C-C motif chemokine ligand 2/Monocyte Chemoattractant Protein-1 (CCL2/MCP-1) and C-X-C motif chemokine ligand 10/Interferon Protein-10 (CXCL10/IP-10) were found in the acute phase of infection, while C-C motif chemokine ligand 4/Macrophage Inflammatory Protein 1 β (CCL4/MIP-1β) was found at increased concentrations in the chronic phase. Likewise, CHIKV patients with arthritis had significantly higher concentrations of CCL4/MIP-1β compared to patients without arthritis. Taken together, these data demonstrated increased IDO-1 activity, possibly exerting both antiviral effects and regulating exacerbated inflammatory responses. CCL4/MIP-1β may have an important role in the persistent inflammation and arthritic symptoms following chikungunya infection.

Advances in knowledge of the pathophysiology of COVID-19 have been acquired; however, the host factors that could explain the mild and severe forms of the disease are not fully understood. Thus, we proposed to evaluate anti-SARS-CoV-2 antibodies and the inflammatory response of different groups of individuals, including healthcare workers (HCW), sick and dead COVID-19 patients and also recovered patients to contribute to this knowledge gap. Our objective is to relate the clinical evolution of these individuals with the level of detection and functionality of specific antibodies and with the production of inflammatory mediators. As main findings, IgA and IgG anti-SARS-CoV-2 were detected in asymptomatic HCW. IFN-γ and TNF-α levels were higher in symptomatic HCWs than patients with COVID-19 and those who died. Patients who died had higher levels of IL-6, IL-10, and CCL2/MCP-1. We found an imbalance between antiviral and pro-inflammatory mediators in the groups, in which IFN-γ and TNF-α seem to be more associated with protection and IL-6 and CCL2/MCP-1 with pathology. Our work is pioneering the Brazilian population and corroborates data from people from other countries.

The co-circulation of chikungunya virus (CHIKV), dengue virus (DENV) and Zika virus (ZIKV) in Rio de Janeiro (RJ), Brazil, caused a challenging triple epidemic, as they share similar clinical signs and symptoms and geographical distribution. Here, we aimed to investigate the clinical and laboratorial aspects of chikungunya suspected cases assisted in RJ during the 2018 outbreak, focusing on the differential diagnosis with dengue and zika. All suspected cases were submitted to molecular and/or serological differential diagnostic approaches to arboviruses. A total of 242 cases suspected of arbovirus infection were investigated and 73.6% (178/242) were molecular and/or serologically confirmed as chikungunya. In RT-qPCR confirmed cases, cycle threshold (Ct) values ranged from 15.46 to 35.13, with acute cases presenting lower values. Chikungunya cases were mainly in females (64%) and the most frequently affected age group was adults between 46 to 59 years old (27%). Polyarthralgia affected 89% of patients, especially in hands and feet. No dengue virus (DENV) and Zika virus (ZIKV) infections were confirmed by molecular diagnosis, but 9.5% (23/242) had serological evidence of DENV exposure by the detection of specific anti-DENV IgM or NS1, and 42.7% (76/178) of chikungunya positive cases also presented recent DENV exposure reflected by a positive anti-DENV IgM or NS1 result. A significantly higher frequency of arthritis ( p = 0.023) and limb edema ( p < 0.001) was found on patients with CHIKV monoinfection compared to dengue patients and patients exposed to both viruses. Lastly, phylogenetic analysis showed that the chikungunya cases were caused by the ECSA genotype. Despite the triple arboviruses' epidemic in the state of RJ, most patients with fever and arthralgia investigated here were diagnosed as chikungunya cases, and the incidence of CHIKV/DENV co-detection was higher than that reported in other studies.

High levels of T helper 17 cell (Th17)-related cytokines have been shown in acute Zika virus (ZIKV) infection. We hypothesized that the high levels of Th17-related cytokines, associated with a regulatory environment during pregnancy, create a favorable milieu for the differentiation of CD4+Th17 cells. We present data from a cross-sectional study on mothers who confirmed ZIKV infection by qRT-PCR and their children. We also recruited non-pregnant women infected with ZIKV in the same period. ZIKV infection occurred between 2015 and 2017. We collected samples for this study between 2018 and 2019, years after the initial infection. We highlight that, after in vitro stimulation with ZIKV CD4 megapool (ZIKV MP), we found a lower frequency of IL-17-producing CD4+ T cells (Th17), especially in the mothers, confirmed by the decrease in IL-17 production in the supernatant. However, a higher frequency of CD4+ IL-17+ IFN-γ+ T cells (Th1Th17) responding to the ZIKV MP was observed in the cells of the mothers and children but not in those of the non-pregnant women. Our data indicate that the priming of CD4 T cells of the Th1Th17 phenotype occurred preferentially in the mothers who gave birth to children with CZS and in the children.

The incidence of dengue in Latin America has increased dramatically during the last decade. Understanding the pathogenic mechanisms in dengue is crucial for the identification of biomarkers for the triage of patients. We aimed to characterize the profile of cytokines (IFN-γ, TNF-α, IL-1β, IL-6, IL-18 and IL-10), chemokines (CXCL8/IL-8, CCL2/MCP-1 and CXCL10/IP-10) and coagulation mediators (Fibrinogen, D-dimer, Tissue factor-TF, Tissue factor pathway inhibitor-TFPI and Thrombomodulin) during the dengue-4 epidemic in Brazil. Laboratory-confirmed dengue cases had higher levels of TNF-α ( p < 0.001), IL-6 ( p = 0.005), IL-10 ( p < 0.001), IL-18 ( p = 0.001), CXCL8/IL-8 ( p < 0.001), CCL2/MCP-1 ( p < 0.001), CXCL10/IP-10 ( p = 0.001), fibrinogen ( p = 0.037), D-dimer ( p = 0.01) and TFPI ( p = 0.042) and lower levels of TF ( p = 0.042) compared to healthy controls. A principal component analysis (PCA) distinguished between two profiles of mediators of inflammation and coagulation: protective (TNF-α, IL-1β and CXCL8/IL-8) and pathological (IL-6, TF and TFPI). Lastly, multivariate logistic regression analysis identified high aspartate aminotransferase-to-platelet ratio index (APRI) as independent risk factors associated with severity (adjusted OR: 1.33; 95% CI 1.03-1.71; p = 0.027), the area under the receiver operating characteristics curve (AUC) was 0.775 (95% CI 0.681-0.869) and an optimal cutoff value was 1.4 (sensitivity: 76%; specificity: 79%), so it could be a useful marker for the triage of patients attending primary care centers.

Th17 cells are recognized as indispensable in inducing protective immunity against bacteria and fungi, as they promote the integrity of mucosal epithelial barriers. It is believed that Th17 cells also play a central role in the induction of autoimmune diseases. Recent advances have evaluated Th17 effector functions during viral infections, including their critical role in the production and induction of pro-inflammatory cytokines and in the recruitment and activation of other immune cells. Thus, Th17 is involved in the induction both of pathogenicity and immunoprotective mechanisms seen in the host's immune response against viruses. However, certain Th17 cells can also modulate immune responses, since they can secrete immunosuppressive factors, such as IL-10; these cells are called non-pathogenic Th17 cells. Here, we present a brief review of Th17 cells and highlight their involvement in some virus infections. We cover these notions by highlighting the role of Th17 cells in regulating the protective and pathogenic immune response in the context of viral infections. In addition, we will be describing myocarditis and multiple sclerosis as examples of immune diseases triggered by viral infections, in which we will discuss further the roles of Th17 cells in the induction of tissue damage.

Intrahost genetic diversity is thought to facilitate arbovirus adaptation to changing environments and hosts, and it might also be linked to viral pathogenesis. Dengue virus serotype 2 (DENV-2) has circulated in Brazil since 1990 and is associated with severe disease and explosive outbreaks. Intending to shed light on the viral determinants for severe dengue pathogenesis, we sought to analyze the DENV-2 intrahost genetic diversity in 68 patient cases clinically classified as dengue fever ( n = 31), dengue with warning signs ( n = 19), and severe dengue ( n = 18). Unlike previous DENV intrahost diversity studies whose approaches employed PCR, here we performed viral whole-genome deep sequencing from clinical samples with an amplicon-free approach, representing the real intrahost diversity scenario. Striking differences were detected in the viral population structure between the three clinical categories, which appear to be driven mainly by different infection times and selection pressures, rather than being linked with the clinical outcome itself. Diversity in the NS2B gene, however, showed to be constrained, irrespective of clinical outcome and infection time. Finally, 385 non-synonymous intrahost single-nucleotide variants located along the viral polyprotein, plus variants located in the untranslated regions, were consistently identified among the samples. Of them, 124 were exclusively or highly detected among cases with warning signs and among severe cases. However, there was no variant that by itself appeared to characterize the cases of greater severity, either due to its low intrahost frequency or the conservative effect on amino acid substitution. Although further studies are necessary to determine their real effect on viral proteins, this heightens the possibility of epistatic interactions. The present analysis represents an initial effort to correlate DENV-2 genetic diversity to its pathogenic potential and thus contribute to understanding the virus's dynamics within its human host.

Background: Zika virus (ZIKV) infection causes for mild and self-limiting disease in healthy adults. In newborns, it can occasionally lead to a spectrum of malformations, the congenital Zika syndrome (CZS). Thus, little is known if mothers and babies with a history of ZIKV infection were able to develop long-lasting T-cell immunity. To these issues, we measure the prevalence of ZIKV T-cell immunity in a cohort of mothers infected to the ZIKV during pregnancy in the 2016-2017 Zika outbreak, who gave birth to infants affected by neurological complications or asymptomatic ones. Results: Twenty-one mothers and 18 children were tested for IFN-γ ELISpot and T-cell responses for flow cytometry assays in response to CD4 ZIKV and CD8 ZIKV megapools (CD4 ZIKV MP and CD8 ZIKV MP). IFN-γ ELISpot responses to ZIKV MPs showed an increased CD4 and CD8 T-cell responses in mothers compared to children. The degranulation activity and IFN-γ-producing CD4 T cells were detected in most mothers, and children, while in CD8 T-cells, low responses were detected in these study groups. The total Temra T cell subset is enriched for IFN-γ+ CD4 T cells after stimulation of CD4 ZIKV MP. Conclusion: Donors with a history of ZIKV infection demonstrated long-term CD4 T cell immunity to ZIKV CD4 MP. However, the same was not observed in CD8 T cells with the ZIKV CD8 MP. One possibility is that the cytotoxic and pro-inflammatory activities of CD8 T cells are markedly demonstrated in the early stages of infection, but less detected in the disease resolution phase, when the virus has already been eliminated. The responses of mothers' T cells to ZIKV MPs do not appear to be related to their children's clinical outcome. There was also no marked difference in the T cell responses to ZIKV MP between children affected or not with CZS. These data still need to be investigated, including the evaluation of the response of CD8 T cells to other ZIKV peptides., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Badolato-Corrêa, Carvalho, Paiva, Familiar-Macedo, Dias, Pauvolid-Corrêa, Fernandes-Santos, Lima, Gandini, Silva, Baeta Cavalcanti, de Oliveira, de Oliveira Vianna, de Azeredo, Cardoso, Grifoni, Sette, Weiskopf and de-Oliveira-Pinto.)

There have been reports of neurological abnormalities associated with the Zika virus (ZIKV), such as congenital Zika syndrome (CZS) in children born to mothers infected during pregnancy. We investigated how the immune response to ZIKV during pregnancy is primed and conduct a thorough evaluation of the inflammatory and cytotoxic profiles as well as the expression of CCR5 and CX3CR1. We compared the reactivity of T cells to ZIKV peptides in convalescent mothers infected during pregnancy. The child's clinical outcome (i.e., born with or without CZS) was taken to be the variable. The cells were stimulated in vitro with ZIKV peptides and evaluated using the ELISPOT and flow cytometry assays. After in vitro stimulation with ZIKV peptides, we observed a tendency toward a higher Interferon gamma (IFN-γ)-producing T cell responses in mothers who had asymptomatic children and a higher CD107a expression in T cells in mothers who had children with CZS. We found a higher frequency of T cells expressing CD107a+ and co-expressing CX3CR1+CCR5+, which is much clearer in the T cells of mothers who had CZS children. We suggest that this differential profile influenced the clinical outcome of babies. These data need to be further investigated, including the evaluation of other ZIKV peptides and markers and functional assays.

Dengue virus (DENV) co-circulation in Brazil represents a challenge for treatment and vaccine development. Despite public health impact, the occurrence of coinfections with other viruses is a common event. Increased T cell activation and altered inflammatory response are found during DENV coinfection with Human Immunodeficiency Virus (HIV) impacting HIV-pathogenesis. Even with Antiretroviral therapy (ART), HIV- treated patients had chronic immune activation and lymphocyte apoptosis. However, apoptotic mechanisms have not been investigated during coinfection with DENV. Our attention was attracted to apoptotic cell markers expressions in PBMCs from DENV and DENV/HIV coinfected patients. We found CD4/CD8 ratio inversion in most coinfected patients. CD4 T and CD8 T-cell subsets from DENV and DENV/HIV groups expressed low levels of anti-apoptotic protein Bcl-2. Furthermore, CD8 CD95 double positive cells frequency expressing low levels of Bcl-2 were significantly higher in these patients. Additionally, the density of Bcl-2 on classical monocytes (CD14 ++ CD16 - ) was significantly lower during DENV infection. Upregulation of pro-apoptotic proteins and anti-apoptotic proteins were found in DENV and DENV/HIV, while catalase, an antioxidant protein, was upregulated mainly in DENV/HIV coinfection. These findings provide evidence of apoptosis triggering during DENV/HIV coinfection, which may contribute to knowledge of immunological response during DENV acute infection in HIV-patients treated with ART.

Background: Arboviruses co-circulating within a population that are transmitted by the same vector have the potential to cause coinfections. Coinfections with dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) have been occurring in Brazil, but it is not well-understood how human responses vary during mono- or coinfections and whether they play different roles in pathogenesis., Methods: We investigated the clinical, virological, and immunological status during patients' acute infections, focusing on the CCL/CXC chemokines, proinflammatory, as well as anti-inflammatory cytokines levels quantified by ELISAs. Viral load was determined by qRT-PCR in serum samples from 116 acute DENV, ZIKV, CHIKV, DENV/ZIKV, and CHIKV/ZIKV-infected adult patients from Brazil., Results: Most of the acute patients displayed fever, headache, prostration, and myalgia, regardless of the type of arbovirus infection. Zika viral load was higher in CHIKV/ZIKV coinfected patients compared with ZIKV or DENV/ZIKV infections. All infected individuals presented increased concentrations of C-X-C motif chemokine ligand 10/interferon protein-10 (CXCL10/IP-10), C-C motif chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1), and tumor necrosis factor alpha (TNF-α) compared to healthy donors. Interestingly, the ZIKV group separated from CHIKV/ZIKV due to higher levels of interleukin-10 (IL-10) and lower levels of TNF-α. While DENV/ZIKV differentiated from CHIKV due to their higher levels of CCL2/MCP-1, in CHIKV- and CHIKV/ZIKV-infected patients, levels of CXC10/IP-10, CCL2/MCP-1, and migration inhibitory factor (MIF) were associated with CHIKV viral load. By contrast, in DENV/ZIKV- and CHIKV/ZIKV-infected patients, levels of CXCL10/IP-10, CCL2/MCP-1, and TNF-α showed a significant inverse correlation with ZIKV viral load., Conclusions: From all the circulating mediators measured, we detected differences of IL-10, TNF-α, and CCL2/MCP-1 between arbovirus groups. We hypothesize that CXC10/IP-10, CCL2/MCP-1, and MIF in the CHIKV-infected group could regulate the CHIKV viral load, while CXC10/IP-10, CCL2/MCP-1, and TNF-α in DENV/ZIKV, and CHIKV/ZIKV groups, could regulate ZIKV viral load., (© 2020 S. Karger AG, Basel.)

Dengue virus (DENV) can cause diseases ranging from dengue fever (DF) to more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Whether antiviral T cells contribute to the protection against or pathogenesis of severe disease is not well defined. Here, we identified antigen-specific IL-10 + IFN-γ + double-positive (DP) CD4 T cells during acute DENV infection. While the transcriptomic signatures of DP cells partially overlapped with those of cytotoxic and type 1 regulatory CD4 T cells, the majority of them were non-cytotoxic/Tr1 and included IL21, IL22, CD109, and CCR1. Although we observed a higher frequency of DP cells in DHF, the transcriptomic profile of DP cells was similar in DF and DHF, suggesting that DHF is not associated with the altered phenotypic or functional attributes of DP cells. Overall, this study revealed a DENV-specific DP cell subset in patients with acute dengue disease and argues against altered DP cells as a determinant of DHF., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

During a pathogenic infection, an inflammatory process is triggered in which several inflammatory mediators, such as cytokines, chemokines, growth factors, complement system components, nitric oxide, and others induce integrity alteration on the endothelial barrier. Chemokines are responsible for regulating leukocyte trafficking under homeostatic conditions as well as activating immune system cells under inflammatory conditions. They are crucial molecules in the early stages of infection, leading to the recruitment of immune cells, namely neutrophils, monocytes, natural killer (NK) cells, natural killer T cells (NKT), dendritic cells (DC), T lymphocytes and all cells expressing chemokine receptors for inflammatory sites. Other functions, such as collagen production, tissue repair, a proliferation of hematopoietic precursors and angiogenesis, are also performed by these molecules. Chemokines, amongst inflammatory mediators, play a key role in dengue immunopathogenesis. Dengue fever is a disease caused by the dengue virus (DENV). It is characterized by a broad spectrum of clinical manifestations ranging from asymptomatic cases to mild and severe symptomatic ones. As for the latter, the appearance of hemorrhagic manifestations and changes in vascular permeability may lead the patient to develop cavitary effusions, organ involvement, and even death. As chemokines exert an influence on various homeostatic and inflammatory processes, acting vigorously on vascular endothelial activation and cell migration, the main purpose of this chapter is to discuss the influence of chemokines on the alteration of endothelial permeability and migration of T lymphocytes in DENV infection., (Copyright © 2019. Published by Elsevier B.V.)

Platelets play a role in hemostasis, coagulation, angiogenesis, inflammation and immune response is one of the most affected cells in dengue. Here we describe some aspects of platelets by observing their specific circulating mediators, the ability to interact with the virus and morphological consequences of this interaction, activation markers and intraplatelet protein contents in dengue. We conducted this study using dengue-patients as well as healthy donors. Immunoenzymatic assay, flow cytometry, transmission electron microscopy and intraplatelet proteins expression assays were carried out. Briefly, we found an increase in sCD62L, NO or TBX2 ratio in platelet count, mostly in patients with the worse clinical outcome. After in vitro DENV infection or during natural infection, platelets underwent morphological alteration with increased expression of platelet activation markers, particularly in natural infections. Analysis of intraplatelet protein contents revealed different angiogenic and inflammatory profiles, maintaining or not extracellular matrix integrity between DF and DFWS patients. Thus, platelets are frequently affected by dengue, either by altering their own functionality, as "carrier" of the virus, or as an antiviral and mediator-secreting effector cell. Thus, strategies aimed at recovering platelet amounts in dengue seem to be essential for a better clinical outcome of the patients., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction: Zika virus (ZIKV) and dengue virus (DENV) co-circulated during latest outbreaks in Brazil, hence, it is important to evaluate the host cross-reactive immune responses to these viruses. So far, little is known about human T cell responses to ZIKV and no reports detail adaptive immune responses during DENV/ZIKV coinfection., Methods: Here, we studied T cells responses in well-characterized groups of DENV, ZIKV, or DENV/ZIKV infected patients and DENV-exposed healthy donors. We evaluated chemokine receptors expression and single/multifunctional frequencies of IFNγ, TNF, and IL2-producing T cells during these infections. Even without antigenic stimulation, it was possible to detect chemokine receptors and IFNγ, TNF, and IL2-producing T cells from all individuals by flow cytometry. Additionally, PBMCs' IFNγ response to DENV NS1 protein and to polyclonal stimuli was evaluated by ELISPOT., Results: DENV and ZIKV infections and DENV/ZIKV coinfections similarly induced expression of CCR5, CX3CR1, and CXCR3 on CD4 and CD8 T cells. DENV/ZIKV coinfection decreased the ability of CD4 + T cells to produce IFNγ + , TNF + , TNF  +  IFNγ + , and TNF  +  IL2 + , compared to DENV and ZIKV infections. A higher magnitude of IFNγ response to DENV NS1 was found in donors with a history of dengue infection, however, a hyporesponsiveness was found in acute DENV, ZIKV, or DENV/ZIKV infected patients, even previously infected with DENV., Conclusion: Therefore, we emphasize the potential impact of coinfection on the immune response from human hosts, mainly in areas where DENV and ZIKV cocirculate., (© 2017 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.)

Background: The current triple epidemic caused by dengue, zika and chikungunya constitutes a serious health problem in Brazil. The aim of this study was to investigate acute samples (up to the 7 days of symptoms) from patients presenting acute fever syndrome suspected as arboviral infection and characterize the clinical and laboratorial profile during the co-circulation of dengue, zika and chikungunya in Campo Grande, Mato Grosso do Sul (MS), midwest region of Brazil., Methods: All suspected cases (n=134) were tested by using serological and molecular diagnostic tests including DENV, ZIKV and CHIKV RT-PCR, Dengue nonstructural protein 1 (NS1) antigen capture ELISA, anti- DENV IgM ELISA and anti-CHIKV IgM ELISA. In addition, clinical, hematological and biochemical parameters of infected patients were analyzed., Results:  It was observed that 79.1% of the blood samples were confirmed for ZIKV and/or DENV infection Of those, 38.0% patients were DENV monoinfected, 26.8% were ZIKV monoinfected and 13.4% were DENV/ZIKV co-infected. Seven patients presented Chikungunya IgM antibodies indicating a previous CHIKV infection. Common symptoms included fever, rash, arthralgia, myalgia, prostration, headache and conjunctivitis. Statistical analysis showed that pruritus and edema were associated with ZIKV infection while prostration and vomiting were more associated with dengue. Additionally, total protein and ALT levels were significantly different in DENV patients compared to ZIKV ones. Some DENV infected patients as well as co-infected needed hospitalization and venous hydration. Otherwise, most ZIKV infected patients presented mild clinical course. Among the pregnant women studied (n=11), three were ZIKV monoinfected while four were DENV monoinfected and two were DENV-1/ZIKV coinfected. In general, normal birth outcomes were observed except for the death due to respiratory insufficiency of one baby born to a mother coinfected with DENV-1/ZIKV., Conclusions:  Herein, we provide evidence of the co-circulation of DENV, ZIKV and CHIKV infections in the Campo Grande, MS, Brazil, with a high frequency of DENV-1/ZIKV coinfection. Laboratorial diagnosis poses a challenge where those arboviruses are endemic and differential diagnosis proved to imperative for cases characterization. The knowledge about disease severity during arbovirus coinfections is still scarce and there are several issues emphasizing the importance of adequate management of patients at risk areas.

While progress has been made in characterizing humoral immunity to Zika virus (ZIKV) in humans, little is known regarding the corresponding T cell responses to ZIKV. Here, we investigate the kinetics and viral epitopes targeted by T cells responding to ZIKV and address the critical question of whether preexisting dengue virus (DENV) T cell immunity modulates these responses. We find that memory T cell responses elicited by prior infection with DENV or vaccination with tetravalent dengue attenuated vaccines (TDLAV) recognize ZIKV-derived peptides. This cross-reactivity is explained by the sequence similarity of the two viruses, as the ZIKV peptides recognized by DENV-elicited memory T cells are identical or highly conserved in DENV and ZIKV. DENV exposure prior to ZIKV infection also influences the timing and magnitude of the T cell response. ZIKV-reactive T cells in the acute phase of infection are detected earlier and in greater magnitude in DENV-immune patients. Conversely, the frequency of ZIKV-reactive T cells continues to rise in the convalescent phase in DENV-naive donors but declines in DENV-preexposed donors, compatible with more efficient control of ZIKV replication and/or clearance of ZIKV antigen. The quality of responses is also influenced by previous DENV exposure, and ZIKV-specific CD8 T cells from DENV-preexposed donors selectively upregulated granzyme B and PD1, unlike DENV-naive donors. Finally, we discovered that ZIKV structural proteins (E, prM, and C) are major targets of both the CD4 and CD8 T cell responses, whereas DENV T cell epitopes are found primarily in nonstructural proteins. IMPORTANCE The issue of potential ZIKV and DENV cross-reactivity and how preexisting DENV T cell immunity modulates Zika T cell responses is of great relevance, as the two viruses often cocirculate and Zika virus has been spreading in geographical regions where DENV is endemic or hyperendemic. Our data show that memory T cell responses elicited by prior infection with DENV recognize ZIKV-derived peptides and that DENV exposure prior to ZIKV infection influences the timing, magnitude, and quality of the T cell response. Additionally, we show that ZIKV-specific responses target different proteins than DENV-specific responses, pointing toward important implications for vaccine design against this global threat., (Copyright © 2017 American Society for Microbiology.)

The increase in severe dengue (SD) cases has caused great impact on public health and has concerned authorities of countries where the disease is endemic and epidemics reach high proportions. The recognition of progression signs of this severe disease during the initial febrile phase can be difficult, since the symptoms are often indistinguishable from other febrile diseases. The aim of this study was to evaluate the clinical manifestations and laboratory findings in patients from two dengue outbreaks and their association with the disease. The study was conducted in patients ( n = 153) with signs and symptoms consistent with dengue occurred during two distinct epidemics, 2010 and 2013, in the city of Campos dos Goytacazes, Rio de Janeiro, Brazil. According to the 2009 World Health Organization criteria, patients were classified as dengue without warning signs ([DwoWS] 60.6%, 57/94), dengue with warning signs ([DwWS] 30.9%, 29/94), and SD (4.25%, 4/94). Patients with DwWS/SD presented lower platelet and leukocyte counts and higher transaminase levels when compared with the DwoWS ones. Interestingly, patients from the epidemic of 2010 caused by dengue virus 2 (DENV-2) had lower platelet counts than patients of the 2013 epidemic caused by DENV-4. Furthermore, plasma leakage, gastrointestinal bleeding, and pleural effusion, hallmarks for a more severe disease, were also more frequently observed in those cases. Although previous studies may have extensively reported the wide range of the clinical aspects of dengue, the characterization of DENV-4 is desirable considering the burden of the disease during epidemics, especially for the health units and hospitals performing patient's management.

Tissue Factor (TF) is the initiator of coagulation and Tissue Factor Inhibitor (TFPI) is the physiological inhibitor of the TF/FVIIa complex. Circulating levels of TF and TFPI were quantified in dengue patients and the relationships with disease severity and infecting serotype analysed. A significant decrease in TF and TPFI plasma levels was observed in mild DF patients compared with severe dengue. Furthermore, both factors were associated with haemorrhagic manifestations. Finally, TF levels were significantly increased in DENV-1/2 infected patients as compared with DENV-4. These findings suggest that activation of TF-pathway is an important component of DENV -related coagulation disorders., (Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Dengue is an acute febrile illness with a wide spectrum of signs and symptoms ranging from mild to severe forms characterized by plasma leakage that can be fatal. NK cells are one of the main effectors in early infection and may play an important role in dengue pathogenesis. We investigated NK cell involvement during dengue infection. A higher frequency of NK cell subsets and TRAIL+NK cells was found in mild DF cases when compared to that in severe cases or healthy donors. NK activation markers such as CD107a and TLR3 were upregulated in patients' cells compared to those in healthy donors. In addition, IL12 related to NK cell activation were upregulated in mild DF cases. In vitro PBMC culture models show that DENV-stimulated and IFN α -stimulated NK cells were able to express TRAIL, suggesting an indirect activation of cells, regarding TRAIL expression. Type I IFN receptor blockage on DENV-stimulated PBMCs showed TRAIL expression on NK cells is partially IFN α dependent. In addition, during PBMC stimulation, TRAIL expression on NK cells was inversely correlated with DENV-positive monocytes. Therefore, we observed DENV-induced activation of NK cell populations. A higher activation of NK cells would promote limited viral spread, resulting in decreased inflammatory response, contributing to protection against dengue severity.

Background: Dengue is a major problem in Brazil. Epidemiological and clinical aspects were characterized in patients from two epidemics which occurred in Mato Grosso do Sul, Brazil., Methods: Dengue cases were classified according to the 2009 WHO criteria, tested by serological and molecular biology tests and analysed for nonstructural protein 1 (NS1) antigenemia., Results: Dengue was confirmed in 78.7% (48/61) and 75.6% (118/156) of the cases studied in 2010 and 2013, respectively. DENV-1 and DENV-2 were the serotypes involved in the 2010 epidemic and DENV-4 in the 2013 one. Most of the cases were classified as dengue without warning; however, severe dengue was observed in 18.7% (9/48) of the cases in 2010 and less observed in DENV-4 cases. NS1 levels were higher in patients with dengue with warning signs and severe dengue in 2010. Circulating aspartate aminotransferase (AST) and alanine transferase (ALT) were altered in all groups, independently of the infecting serotype or epidemic. Patients with DENV-1 and DENV-2 presented significant lower monocyte counts when compared to patients with DENV-4. An inverse correlation was found between platelet count, leucocytes, monocytes and NS1 levels., Conclusions: Epidemics caused by the prevalence of distinct DENV serotypes had different impacts and clinical characteristics in a same scenario and, despite the occurrence of secondary infections, the DENV-4 emergence was not associated with severe cases., (© The Author 2016. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Dengue is an infectious disease caused by dengue virus (DENV). In general, dengue is a self-limiting acute febrile illness followed by a phase of critical defervescence, in which patients may improve or progress to a severe form. Severe illness is characterized by hemodynamic disturbances, increased vascular permeability, hypovolemia, hypotension, and shock. Thrombocytopenia and platelet dysfunction are common in both cases and are related to the clinical outcome. Different mechanisms have been hypothesized to explain DENV-associated thrombocytopenia, including the suppression of bone marrow and the peripheral destruction of platelets. Studies have shown DENV-infected hematopoietic progenitors or bone marrow stromal cells. Moreover, anti-platelet antibodies would be involved in peripheral platelet destruction as platelets interact with endothelial cells, immune cells, and/or DENV. It is not yet clear whether platelets play a role in the viral spread. Here, we focus on the mechanisms of thrombocytopenia and platelet dysfunction in DENV infection. Because platelets participate in the inflammatory and immune response by promoting cytokine, chemokine, and inflammatory mediator secretion, their relevance as "immune-like effector cells" will be discussed. Finally, an implication for platelets in plasma leakage will be also regarded, as thrombocytopenia is associated with clinical outcome and higher mortality.

Despite being the most significant arboviral disease worldwide, dengue has no antiviral treatment or reliable severity predictors. It has been shown that apoptotic cells from blood and tissues may be involved in the complex pathogenesis of dengue. However, very little is known about the interplay between proapoptotic and antiapoptotic mediators in this disease. Therefore, plasma levels of the three proapoptotic mediators Fas ligand (FasL), tumor necrosis factor-α (TNF-α), and TNF-related apoptosis-inducing ligand (TRAIL) were measured in dengue patients. Patients were classified according to the World Health Organization classification of dengue revised in 2009. Additionally, inhibitors of apoptosis protein (IAPs) were determined in plasma (Survivin) and peripheral blood mononuclear cells (PBMCs) lysates (cIAP-1, cIAP-2, XIAP). Levels of apoptotic proteins in plasma were correlated with counts of blood cells. FasL and TRAIL levels were elevated in dengue patients without warning signs when compared to patients with severe dengue and controls. Dengue patients with warning signs showed decreased levels of Survivin compared to patients with severe dengue and controls. TRAIL was inversely correlated with counts of lymphocyte subsets. In contrast, Survivin was positively correlated with leukocyte counts. There was a trend of elevated IAPs levels in PBMCs of patients with severe dengue. The results suggest a likely antiviral effect of TRAIL in dengue. It appears that TRAIL might be involved with apoptosis induction of lymphocytes, whereas IAPs might participate in protecting leukocytes from apoptosis. Further research is needed to explore the interactions between pro and antiapoptotic molecules and their implications in dengue pathogenesis., (© 2013 Wiley Periodicals, Inc.)

In dengue virus (DENV) infection, complement system (CS) activation appears to have protective and pathogenic effects. In severe dengue fever (DF), the levels of DENV non-structural-1 protein and of the products of complement activation, including C3a, C5a and SC5b-9, are higher before vascular leakage occurs, supporting the hypothesis that complement activation contributes to unfavourable outcomes. The clinical manifestations of DF range from asymptomatic to severe and even fatal. Here, we aimed to characterise CS by their receptors or activation product, in vivo in DF patients and in vitro by DENV-2 stimulation on monocytes. In comparison with healthy controls, DF patients showed lower expression of CR3 (CD11b), CR4 (CD11c) and, CD59 on monocytes. The DF patients who were high producers of SC5b-9 were also those that showed more pronounced bleeding or vascular leakage. Those findings encouraged us to investigate the role of CS in vitro, using monocytes isolated from healthy subjects. Prior blocking with CR3 alone (CD11b) or CR3 (CD11b/CD18) reduced viral infection, as quantified by the levels of intracellular viral antigen expression and soluble DENV non-structural viral protein. However, we found that CR3 alone (CD11b) or CR3 (CD11b/CD18) blocking did not influence major histocompatibility complex presentation neither active caspase-1 on monocytes, thus probably ruling out inflammasome-related mechanisms. Although it did impair the secretion of tumour necrosis factor alpha and interferon alpha. Our data provide strategies of blocking CR3 (CD11b) pathways could have implications for the treatment of viral infection by antiviral-related mechanisms.

Dengue fever, a public health problem in Brazil, may present severe clinical manifestations as result of an increased vascular permeability and coagulation disorders. T cell activation is a critical event for an effective immune response against infection, including the production of cytokines. We aim to reveal mechanisms that modulate the virus-cell interaction, with an emphasis on cell death. Apoptosis is involved in lymphocyte homeostasis, contributes to the clearance of virus-infected cells but also may play a role in the pathogenesis. Phosphatidylserine exposure on CD8T lymphocytes from dengue patients support early apoptotic processes and loss of genomic integrity, observed by DNA fragmentation in T lymphocytes and indicating late apoptosis. These T cells express activation and cytotoxic phenotypes as revealed by CD29 and CD107a upregulation. Higher frequencies of CD95 were detected in T lymphocytes mainly in those with the cytotoxic profile (CD107a+) and lower levels of anti-apoptotic molecule Bcl-2, suggesting that both CD4+ and CD8+ T cell subsets are more susceptible to apoptosis during acute dengue. The analysis of apoptosis-related protein expression profile showed that not only molecules with pro- but also those with anti-apoptotic functions are overexpressed, indicating that survival mechanisms could be possibly protecting cells against apoptosis caused by viral, immune, oxidative and/or genotoxic stresses. These observations led us to propose that in dengue patients there is an association between T cell susceptibility to apoptosis and the activation state. The mechanisms for understanding the immunopathogenesis during dengue infection are discussed., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Dengue virus (DENV) and parvovirus B19 (B19V) infections are acute exanthematic febrile illnesses that are not easily differentiated on clinical grounds and affect the paediatric population. Patients with these acute exanthematic diseases were studied. Fever was more frequent in DENV than in B19V-infected patients. Arthritis/arthralgias with DENV infection were shown to be significantly more frequent in adults than in children. The circulating levels of interleukin (IL)-1 receptor antagonist (Ra), CXCL10/inducible protein-10 (IP-10), CCL4/macrophage inflammatory protein-1 beta and CCL2/monocyte chemotactic protein-1 (MCP-1) were determined by multiplex immunoassay in serum samples obtained from B19V (37) and DENV-infected (36) patients and from healthy individuals (7). Forward stepwise logistic regression analysis revealed that circulating CXCL10/IP-10 tends to be associated with DENV infection and that IL-1Ra was significantly associated with DENV infection. Similar analysis showed that circulating CCL2/MCP-1 tends to be associated with B19V infection. In dengue fever, increased circulating IL-1Ra may exert antipyretic actions in an effort to counteract the already increased concentrations of IL-1β, while CXCL10/IP-10 was confirmed as a strong pro-inflammatory marker. Recruitment of monocytes/macrophages and upregulation of the humoral immune response by CCL2/MCP-1 by B19V may be involved in the persistence of the infection. Children with B19V or DENV infections had levels of these cytokines similar to those of adult patients.

Little is known about the role of chemokines/chemokines receptors on T cells in natural DENV infection. Patients from DENV-2 and -3- outbreaks were studied prospectively during the acute or convalescent phases. Expression of chemokine receptor and activation markers on lymphocyte subpopulations were determined by flow cytometry analysis, plasma chemokine ligands concentrations were measured by ELISA and quantification of CCL5/RANTES(+) cells in liver tissues from fatal dengue cases was performed by immunochemistry. In the acute DENV-infection, T-helper/T-cytotoxic type-1 cell (Th1/Tc1)-related CCR5 is significantly higher expressed on both CD4 and CD8 T cells. The Th1-related CXCR3 is up-regulated among CD4 T cells and Tc2-related CCR4 is up-regulated among CD8 T cells. In the convalescent phase, all chemokine receptor or chemokine ligand expression tends to reestablish control healthy levels. Increased CCL2/MCP-1 and CCL4/MIP-1β but decreased CCL5/RANTES levels were observed in DENV-patients during acute infection. Moreover, we showed an increased CD107a expression on CCR5 or CXCR3-expressing T cells and higher expression of CD29, CD44(HIGH) and CD127(LOW) markers on CCR4-expressing CD8 T cells in DENV-patients when compared to controls. Finally, liver from dengue fatal patients showed increased number of cells expressing CCL5/RANTES in three out of four cases compared to three death from a non-dengue patient. In conclusion, both Th1-related CCR5 and CXCR3 among CD4 T cells have a potential ability to exert cytotoxicity function. Moreover, Tc1-related CCR5 and Tc2-related CCR4 among CD8 T cells have a potential ability to exert effector function and migration based on cell markers evaluated. The CCR5 expression would be promoting an enhanced T cell recruitment into liver, a hypothesis that is corroborated by the CCL5/RANTES increase detected in hepatic tissue from dengue fatal cases. The balance between protective and pathogenic immune response mediated by chemokines during dengue fever will be discussed.

Understanding the dual participation of the immune response in controlling the invader and at the same time causing tissue damage might contribute to the design of effective new vaccines and therapies for Chagas disease. Perforin, a cytolytic protein product of killer cells, is involved in resistance to acute Trypanosoma cruzi infection. However, the contribution of perforin in parasite control and chronic chagasic cardiomyopathy is unclear. Perforin-positive cells were detected in the heart tissue during the acute and chronic phases of infection of C57BL/6 mice inoculated with low dose (10(2) parasites) of the Colombian T. cruzi strain. This protocol led to acute phase survival in both wild-type and perforin null (pfp(-/-)) mice lineages. During the chronic infection, parasitism and inducible nitric oxide synthase (iNOS) as well as interleukin (IL)-4+ and, mainly, interferon (IFN)-gamma+ cells were more elevated in the heart tissue of pfp(-/-) mice. Higher levels of circulating NO and anti-parasite immunoglobulin (Ig)G2c and IgG3, paralleled by a prominent frequency of IFN-gamma+ and IL-10+ splenocytes, were present in pfp(-/-)-infected mice. Therefore, although the perforin-dependent pathway plays a role, it is not crucial for anti-T. cruzi immunity and acute phase survival of mice infected with a low inoculum. Further, perforin deficiency resulted in lower activity of creatine kinase-muscle brain isoform (CK-MB) isoenzyme in serum and a more restricted connexin 43 loss, both of which are markers of the cardiomyocyte lesion. Moreover, perforin deficiency hampered the development of severe electrocardiographic abnormalities. Hence, our results corroborate that perforin-bearing cytotoxic cells might contribute to cardiomyocyte lesion and heart dysfunction during chronic T. cruzi infection, shedding light on immunopathogenesis of chronic chagasic cardiomyopathy.

The immune mechanisms involved in dengue fever and dengue hemorrhagic/dengue shock syndrome are not well understood. The ex vivo activation status of immune cells during the dengue disease in patients was examined. CD4 and CD8 T cells were reduced during the acute phase. Interestingly, CD8 T cells co-expressing activation marker HLA-DR, Q, P, and cytolytic granule protein-Tia-1 were significantly higher in dengue patients than in controls. Detection of adhesion molecules indicated that in dengue patients the majority of T cells (CD4 and CD8) express the activation/memory phenotype, characterized as CD44HIGH and lack the expression of the naïve cell marker, CD62L LOW. Also, the levels of T cells co-expressing ICAM-1 (CD54), VLA-4, and LFA-1 (CD11a) were significantly increased. CD8 T lymphocytes expressed predominantly low levels of anti-apoptotic molecule Bcl-2 in the acute phase, possibly leading to the exhibition of a phenotype of activated/effector cells. Circulating levels of IL-18, TGF-b1 and sICAM-1 were significantly elevated in dengue patients. Early activation events occur during acute dengue infection which might contribute to viral clearance. Differences in expression of adhesion molecules among CD4 and CD8 T cells might underlie the selective extravasation of these subsets from blood circulation into lymphoid organs and/or tissues. In addition, activated CD8 T cells would be more susceptible to apoptosis as shown by the alteration in Bcl-2 expression. Cytokines such as IL-18, TGF-b1, and sICAM-1 may be contributing by either stimulating or suppressing the adaptative immune response, during dengue infection, thereby perhaps establishing a relationship with disease severity.

During the innate immune response against infections, Natural Killer (NK) cells are as important effector cells as are Cytotoxic T lymphocytes (CTL) generated after antigenic stimulation in the adaptative response. NK cells increase in numbers, after viral infection or vaccination. We investigated the NK cell and CD8 T lymphocyte status in 55 dengue infected patients. The NK (CD56+CD3-) and CD56+ T cell (CD56+CD3+) rates rise during the acute phase of disease. The majority of NK cells from dengue patients display early markers for activation (CD69, HLA-DR, and CD38) and cell adhesion molecules (CD44, CD11a) during the acute phase of disease. The intracellular cytotoxic granule, TIA-1, is also up-regulated early in NK cells. Most of these markers appear also on CD8+ T lymphocytes but during the late acute phase. Circulating IL-15 is elevated in a significant number of patients during early acute infection and its values were statistically correlated with NK frequencies and cytotoxic markers on NKs. We have therefore shown that dengue virus infection is very likely stimulating a cytotoxic response that may be efficient in controlling the virus in synergism with CD8+ T lymphocytes. Interestingly, the heightened CD56+CD3-, CD56+CD3+, CD56+TIA-1+ and CD56+CD11a+ cell rates are associated with mild dengue clinical manifestations and might indicate a good prognosis of the disease.

Apoptosis and primary necrosis are the two modes of cell death induced by a lethal injury. The majority of structural and biochemical events occurring during cell death can be analysed by flow cytometry. The 7-aminoactinomycin D (7-AAD) assay can be used to detect the loss of membrane integrity during apoptosis of murine thymocytes and human peripheral lymphocytes. We describe here new applications of the 7-AAD assay. It can be applied to a variety of cell lines of different origins, including adherent cell lines, and it allows the co-detection of lipidic antigens such as phosphatidylserine (PS) residues, and biochemical processes linked to apoptosis, such as the loss of mitochondrial transmembrane potential, cardiolipin peroxidation, the expression of the 7A6 mitochondrial antigen and DNA fragmentation. Thus, this assay is a noninvasive method particularly adapted to the analysis of biochemical events associated with cell death. Finally, we show that this assay is not specific for apoptosis since it detects oncosis, the early stage of primary necrosis.

The destruction of CD4 T cells in human immunodeficiency virus (HIV) infection is associated with activation of apoptotic programs, partly mediated by death receptors. The role of CD95L/CD95 in depletion of patients' CD4 T cells is well documented, but the possible contribution of the tumor necrosis factor/tumor necrosis factor receptor (TNF/TNFR) pathway has not been examined. In this study, we found that both TNFR1 and TNFR2 induced marked apoptosis in peripheral T cells from HIV-infected persons, involving both CD4 and CD8 T cells. Longitudinal follow-up of HIV(+) patients suggests an association between the in vivo evolution of CD4 T-cell numbers and variations in susceptibility to TNFR-induced apoptosis. Analysis of molecular mechanisms involved showed that it was not related to altered ex vivo expression of TNFR1-associated death domain, receptor interacting protein, or TNFR-associated factor 2. Susceptibility to TNFR-mediated apoptosis was rather related to Bcl-2 expression, because patients' T cells expressing high levels of Bcl-2 were completely protected from TNFR1- and TNFR2-induced cell death, whereas T cells expressing normal levels of Bcl-2 were not protected in patients in contrast to controls. Early recruitment of caspase-8 and caspase-3 is needed to transduce the apoptotic signals, and expression of both caspases in their active form was detected in blood T cells from HIV(+) patients, whereas it was hardly detected in controls. Moreover, ligation of TNFRs induced increased activation of both caspases in patients' T cells. Together these data demonstrate that exacerbated TNFR-mediated cell death of T cells from HIV-infected individuals is associated with both alteration of Bcl-2 expression and activation of caspase-8 and caspase-3 and may contribute to the pathogenesis of acquired immunodeficiency syndrome.

Background: Increased and premature T cell apoptosis is recognized as a feature of HIV infection, and its normalization during highly active antiretroviral therapy (HAART) is thought to contribute to quantitative CD4 T cell restoration., Design: Cross-sectional study of spontaneous, CD3- and CD95-mediated apoptosis in lymphocytes from 53 HIV-infected individuals taking HAART., Methods: Overnight stimulation of peripheral blood mononuclear cells (PBMC) with coated anti-CD3 or anti-CD95 monoclonal antibodies or incubation overnight in medium. Apoptosis in CD4 and CD8 T cells was measured by flow cytometry. For in vitro assay of antiretroviral drugs, normal PBMC were prestimulated with anti-CD3 monoclonal antibodies and apoptosis was induced by ligation of CD95. The expression of active caspase-8 and caspase-3 was examined by flow cytometry., Results: We report for the first time that important levels of T cell apoptosis may persist under HAART, in spite of a rise in CD4 T cells from baseline and a sustained suppression of plasmatic viral load. Spontaneous CD3- or CD95-induced apoptosis levels were inversely correlated with the in vivo number of CD4 T cells and the CD4/CD8 ratio, but not with the viral load or duration of antiretroviral therapy. Regimens including lamivudine are associated with persistent T cell apoptosis, particularly following CD95 ligation. Lamivudine was also found to stimulate in vitro CD95-induced apoptosis and caspase activation in pre-activated T lymphocytes from healthy donors., Conclusion: The immunomodulatory effect of lamivudine may be one of the contributing factor to increased levels of T cell apoptosis under HAART. The data suggest that there is a requirement for physiological apoptosis during HAART.