Elevated Activation of Neutrophil Toll-Like Receptors in Patients with Acute Severe Leptospirosis: An Observational Study.

Leptospirosis is the leading cause of zoonotic morbidity and mortality globally, yet little is known about the immune mechanisms that may contribute to pathogenesis and severe disease. Although neutrophils are a key component of early immune responses to infection, they have been associated with tissue damage and inflammation in some febrile infections. To assess whether neutrophils contribute to the pathogenesis observed in severe leptospirosis, we quantitated levels of neutrophil activation markers in patients with varying disease severities. Hospitalized leptospirosis patients had significantly higher levels of toll-like receptors 2 and 4 (TLR2 and TLR4, respectively) on peripheral neutrophils than healthy controls, with the highest levels detected in patients with organ dysfunction. We observed no significant differences in other neutrophil baseline activation markers (CD62L and CD11b) or activation capacity (CD62L and CD11b levels following stimulation), regardless of disease severity. Our results provide preliminary evidence supporting the hypothesis that higher initial bacterial loads or inadequate or delayed neutrophil responses, rather than TLR-driven inflammation, may drive severe disease outcomes.

Sera from Visceral Leishmaniasis Patients Display Oxidative Activity and Affect the TNF-α Production by Macrophages In Vitro.

Mammalian protection against leishmanial infection depends on the development of an effective immune response. Zoonotic visceral leishmaniasis (ZVL) patients are usually unable to mount an effective immune response against the parasite and indeed appear to be severely immunosuppressed. This suppression has strong nonspecific and specific components mediated by serum factors and leishmanicidal activity of infected macrophages, respectively. The lipid profile has been shown to be altered in ZVL patients' sera. This work aimed at (i) determining the HDL, Apo A1, LDL, and VLDL concentrations in ZVL patients' sera; (ii) investigating the oxidative effect of ZVL patients' sera on the ß-carotene matrix; (iii) measuring IL-10, IL-6, IL-12p40, and tumour necrosis factor-α (TNF-α) concentrations in the macrophage cultures, to which 10% of ZVL patients' serum had been added. Levels of HDL, LDL fraction, and apolipoprotein A1 in ZVL patients' sera were lower than those of healthy individuals' sera, except for the mean level of VLDL. The matrix of ß-carotene and linoleic acid system was oxidized in the presence of ZLV patients' sera. The presence of ZVL patients' sera did not modify the cytokine production of IL-6, IL-12p40, and IL-10 by human macrophages in vitro but TNF-α production was altered, probably due to lack of macrophage stimulation by lipoprotein.

Repeated praziquantel treatments remodel the genetic and spatial landscape of schistosomiasis risk and transmission.

Repeated treatments with praziquantel reduce schistosomiasis prevalence and morbidity, but transmission persists and populations often recover within a few years. To identify factors associated with persistence, we surveyed and treated all identified Schistosoma mansoni infections in two rural Brazilian communities (Jenipapo and Volta do Rio) in 2009, 2012 and 2013. Eggs were collected from all infected individuals and genotyped with 11 microsatellite markers to evaluate parasite differentiation and diversity. After successive rounds of community-wide treatment, prevalence decreased from 45% to 24% then 16%. Intensity of infection decreased by 57% over this period, and the number of eggs transmitted to the environment decreased by 92%. During all time periods the majority of eggs were excreted by those >15years of age. The incidence was 23% in 2012 and 15% in 2013, consistent with a decrease in transmission. There was little immigration or gene flow over a distance of 6km. On reinfection, infrapopulations were moderately differentiated indicating that pretreatment multilocus genotypes were not fully reacquired. The effective population size responded to census population decline more rapidly than differentiation. Reinfection was concentrated in the downstream portion of Jenipapo, consistent with the observed increased human fecal contamination. At this scale and in this area S. mansoni infections exist on a fragmented landscape with a highly focal pattern of transmission that may facilitate future elimination.

Severe Jaccoud's arthropathy in systemic lupus erythematosus.

Jaccoud's arthropathy (JA) is a clinical situation nowadays present mostly in systemic lupus erythematosus (SLE). It is characterized by the presence of joint deformities such as "swan neck," ulnar deviation and "Z-thumb" resembling rheumatoid arthritis (RA) but that are passively correctable and without bone erosion on plain radiographs. From our cohort of SLE patients with JA, we selected a subgroup with a more severe form of this arthropathy and looked at their clinical and laboratory profile as well as studied the magnetic resonance imaging (MRI) findings or ultrasound (US) obtained from the hand with most evident deformities. Seven SLE patients with a severe form of JA were identified. All seven patients have "swan neck," ulnar deviation and "Z-thumb" deformities. Two out of seven had "mutilans-type JA" and four had fixed deformities in the metacarpophalangeal (MCP) joints. The MRI of the hand with more evident deformity clinically performed in six cases and US performed in one case showed mild synovitis in five and moderate synovitis in two patients, mild flexor tenosynovitis in six and severe tenosynovitis in one. Only two small bone erosions were observed in the second and third MCP joints of one patient with moderate synovitis. Severe JA compromises the functional capacity of the joints and imposes the risk of misdiagnosis of RA. With the improvement of the survival rate of SLE and the lack of specific prophylactic or therapeutical measures for JA, it is reasonable to assume that more and more cases of severe JA are going to be identified.

The relative contribution of immigration or local increase for persistence of urban schistosomiasis in Salvador, Bahia, Brazil.

Urbanization is increasing across the globe, and diseases once considered rural can now be found in urban areas due to the migration of populations from rural endemic areas, local transmission within the city, or a combination of factors. We investigated the epidemiologic characteristics of urban immigrants and natives living in a neighborhood of Salvador, Brazil where there is a focus of transmission of Schistosoma mansoni. In a cross-sectional study, all inhabitants from 3 sections of the community were interviewed and examined. In order to determine the degree of parasite differentiation between immigrants and the native born, S. mansoni eggs from stools were genotyped for 15 microsatellite markers. The area received migrants from all over the state, but most infected children had never been outside of the city, and infected snails were present at water contact sites. Other epidemiologic features suggested immigration contributed little to the presence of infection. The intensity and prevalence of infection were the same for immigrants and natives when adjusted for age, and length of immigrant residence in the community was positively associated with prevalence of infection. The population structure of the parasites also supported that the contribution from immigration was small, since the host-to-host differentiation was no greater in the urban parasite population than a rural population with little distant immigration, and there had been little differentiation in the urban population over the past 7 years. Public health efforts should focus on eliminating local transmission, and once eliminated, reintroduction from distant migration is unlikely.

Oxidative stress markers correlate with renal dysfunction and thrombocytopenia in severe leptospirosis.

Leptospirosis is a zoonotic disease that causes severe manifestations such as Weil's disease and pulmonary hemorrhage syndrome. The aim of this study was to evaluate whether reactive oxygen species (ROS) production and antioxidant reduced glutathione (GSH) levels are related to complications in patients hospitalized with leptospirosis. The ROS production and GSH levels were measured in blood samples of 12 patients and nine healthy controls using chemiluminescence and absorbance assays. We found that ROS production was higher and GSH levels were lower in leptospirosis patients compared with healthy individuals. Among patients, GSH depletion was correlated with thrombocytopenia and elevated serum creatinine, whereas a strong positive correlation was observed between ROS production and elevated serum potassium. Additional investigation of the biological significance of ROS production and GSH levels is warranted as they may guide the development of novel adjuvant therapies for leptospirosis targeting oxidative stress.

Characteristics of the human host have little influence on which local Schistosoma mansoni populations are acquired.

BACKGROUND: Brazil remains the country in the Americas with the highest prevalence of schistosomiasis. A combination of control efforts and development, however, has sharply reduced its intensity and distribution. The acquisition of specific schistosome populations may be dependent on host characteristics such as sex, age, geography, work, habits and culture. How these and other host characteristics align with parasite subpopulations may guide approaches to improve control. METHODOLOGY: A cohort of more than 90% of the residents in two rural communities in Brazil participated in an epidemiologic survey of demographic, socio-economic and behavioral characteristics. The variables sex, age, intensity of infection, socio-economic index, % lifetime spent on site, previous infection, and trips outside the district were used to group parasites infecting individuals. Schistosoma mansoni infection status was determined by examination of stools submitted on 3 different days. The aggregate of eggs collected from the whole stool was used to determine degree of population differentiation from allele frequencies for 15 microsatellites. CONCLUSIONS/SIGNIFICANCE: Infection prevalence was 41% for these communities, and the epidemiologic characteristics were similar to many of the endemic areas of Brazil and the world. Parasite population structuring was observed between the two communities (Jost's D 0.046, CI95% 0.042-0.051), although separated by only 8 km and connected by a highway. No structuring was observed when infected individuals were stratified by host's biologic, demographic or epidemiologic characteristics. Those most heavily infected best reflected the communities' overall parasite diversity. The lack of differentiation within villages suggests that individuals are likely to get infected at the same sites or that the same parasite multilocus genotypes can be found at most sites. The geographic structuring between villages and the lack of structuring by age of the host further supports the impression of a population little affected by migration or drift.

Cytokine response signatures in disease progression and development of severe clinical outcomes for leptospirosis.

BACKGROUND: The role of the immune response in influencing leptospirosis clinical outcomes is not yet well understood. We hypothesized that acute-phase serum cytokine responses may play a role in disease progression, risk for death, and severe pulmonary hemorrhage syndrome (SPHS). METHODOLOGY/PRINCIPAL FINDINGS: We performed a case-control study design to compare cytokine profiles in patients with mild and severe forms of leptospirosis. Among patients hospitalized with severe disease, we compared those with fatal and nonfatal outcomes. During active outpatient and hospital-based surveillance we prospectively enrolled 172 patients, 23 with mild disease (outpatient) and 149 with severe leptospirosis (hospitalized). Circulating concentrations of pro- and anti-inflammatory cytokines at the time of patient presentation were measured using a multiplex bead array assay. Concentrations of IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-17A, and TNF-α were significantly higher (P<0.05) in severe disease compared to mild disease. Among severe patients, levels of IL-6 (P<0.001), IL-8 (P = 0.0049) and IL-10 (P<0.001), were higher in fatal compared to non-fatal cases. High levels of IL-6 and IL-10 were independently associated (P<0.05) with case fatality after adjustment for age and days of symptoms. IL-6 levels were higher (P = 0.0519) among fatal cases who developed SPHS than among who did not. CONCLUSION/SIGNIFICANCE: This study shows that severe cases of leptospirosis are differentiated from mild disease by a "cytokine storm" process, and that IL-6 and IL-10 may play an immunopathogenic role in the development of life-threatening outcomes in human leptospirosis.

Schistosoma mansoni population structure and persistence after praziquantel treatment in two villages of Bahia, Brazil.

Praziquantel has been used to treat schistosome infections since 1979 and currently is the only chemotherapeutic agent in production for this purpose, raising concerns about the potential for the emergence of drug resistance. In practice, 10-20% of infected patients will continue to excrete eggs after treatment. It is not understood to what degree this represents selection of a resistant population or incomplete elimination due to the presence of immature worms at the time of treatment. We used a population genetics approach to test whether or not persistent Schistosomamansoni parasites were drawn from the same population as susceptible parasites. In this study, stool samples were collected from 96% of individuals in two small Brazilian communities (populations 482 and 367) and examined for S.mansoni eggs. The combined prevalence of S.mansoni infections in the villages was 41%. Total egg DNA was extracted from each sample and was genotyped at 15 microsatellite markers. Day-to-day variation of the infrapopulation from an individual human host was low (median differentiation using Jost's D=0.010), so that a single stool was representative of the genotypes present in stool eggs, at least in the short term. Average pairwise analysis of D among all pre-treatment infrapopulations suggested moderate differentiation (mean D=0.082 and 0.122 for the two villages), whereas the pre-treatment component population differentiation between the two communities was 0.047. The differentiation of the component population remaining after treatment from the fully susceptible component population was low (mean D=0.007 and 0.020 for the two villages), suggesting that the persistent parasites were not selected by praziquantel treatment. We will continue to follow these communities for evidence of selection or changes in population structure.

IL-8 is associated with non-viremic state and IFN-γ with biochemical activity in HCV-seropositive blood donors.

OBJECTIVE: The aim of this study was to explore a possible association between the pattern of serum cytokines with the virological and biochemical status of hepatitis C virus (HCV)-seropositive blood donors. METHODS: 23 non-viremic and 33 viremic HCV-seropositive blood donors based on HCV-RNA tests, and 29 healthy individuals were included. Cytometric bead array assays were performed to detect cytokines. RESULTS: The subjects were classified as low, medium or high cytokine producers based on the tertile distribution. The absence of detectable viremia was associated with high IL-1ß and IL-8 producers. Conversely, elevated levels of IL-6, IL-10 and IL-12 were associated with detectable viremia. An increased frequency of high IL-1ß producers was observed frequently in the non-viremic recombinant immunoblot assay (RIBA)-indeterminate subjects, while the high IL-4, IL-6, IL-8, IL-10 and IL-12 producers were more frequent in the non-viremic RIBA-positive subjects. Furthermore, the levels of IL-1ß and IL-8 were higher in viremic subjects with a low level of alanine-aminotransferase (ALT), whereas the level of IFN-γ was increased among viremic subjects with a high ALT level. CONCLUSION: IL-1ß and IL-8 were more likely to be associated with a non-viremic or less severe HCV infection, whereas IL-2 and IFN-γ levels correlated with a high ALT level.

Dengue hemorrhagic fever is associated with polymorphisms in JAK1.

To identify genes associated with the clinical presentation of dengue, 50 cases of probable or possible dengue hemorrhagic fever (DHF), 236 dengue fever (DF), and 236 asymptomatic infections were genotyped for 593 single-nucleotide polymorphisms (SNPs) in 56 genes across the type 1 interferon (IFN) response pathway as well as other important candidate genes. By single locus analysis comparing DHF with DF, 11 of the 51 markers with P<0.05 were in the JAK1 gene. Five markers were significantly associated by false discovery rate criteria (q<0.20 when P<6 × 10(-4)). The JAK1 SNPs showed differential distribution by ethnicity and ancestry consistent with epidemiologic observations in the Americas. The association remained significant after controlling for ancestry and income. No association was observed with markers in the gene encoding CD209 (DC-SIGN). An association between DHF and JAK1 polymorphisms is in agreement with expression profiles showing generalized decreased type 1 IFN-stimulated gene expression in these patients.

Analysis of Schistosoma mansoni population structure using total fecal egg sampling.

Many parasite populations are difficult to sample because they are not uniformly distributed between several host species and are often not easily collected from the living host, thereby limiting sample size and possibly distorting the representation of the population. For the parasite Schistosoma mansoni, we investigated the use of eggs, in aggregate, from the stools of infected individuals as a simple and representative sample. Previously, we demonstrated that microsatellite allele frequencies can be accurately estimated from pooled DNA of cloned S. mansoni adults. Here, we show that genotyping of parasite populations from reproductively isolated laboratory strains can be used to identify these specific populations based on characteristic patterns of allele frequencies, as observed by polyacrylamide gel electrophoresis and automated sequencer analysis of fluorescently labeled PCR products. Microsatellites used to genotype aggregates of eggs collected from stools of infected individuals produced results consistent with the geographic distribution of the samples. Preferential amplification of smaller alleles, and stutter PCR products, had negligible effect on measurement of genetic differentiation. Direct analysis of total stool eggs can be an important approach to questions of population genetics for this parasite by increasing the sample size to thousands per infected individual and by reducing bias.

NK and NKT cell dynamics after rituximab therapy for systemic lupus erythematosus and rheumatoid arthritis.

Biomarkers of clinical response to rituximab (RTX) therapy and early predictors of outcome are still under investigation. We report a flow cytometric immunophenotyping analysis from peripheral blood leukocyte subpopulations of two patients with systemic lupus erythematosus (SLE) associated thrombocytopenia and one patient with rheumatoid arthritis (RA), before and after 6 weeks of treatment with RTX. Our results show a reduced population of CD19(+) expressing cells (B cells) after RTX treatment in all three patients. Increased frequency of peripheral regulatory CD4(+)CD25(high) T cell subset and the CD3(-)CD16(-)CD56(bright) NK cell subset after RTX therapy were also observed in all patients, the latter being more pronounced in the SLE patient with sustained clinical response. In addition, an increased population of NKT cell subsets was observed in the patients with clinical response. This is the first evaluation of NK and NKT cells as biomarkers of clinical response after rituximab therapy in rheumatic diseases.

Potential immunomodulatory effects of plant lectins in Schistosoma mansoni infection.

Lectins are sugar-binding glycoproteins that can stimulate, in a non-antigen-specific fashion, lymphocytes, leading to proliferation and cytokine production. Some lectins are utilized as in vitro mitogenic lymphocyte stimulators and their use as immunomodulators against infectious diseases has been evaluated experimentally. In the experimental murine model, the immune response to schistosomiasis is Th1-like during the initial stage of infection, with a shift towards a Th2-like response after oviposition. We report the response of schistosomiasis patients' (n=37) peripheral blood mononuclear cells (PBMC) to stimulation by lectins, including newly isolated lectins from Brazilian flora, and by Schistosomamansoni soluble egg antigens (SEA). Cytokine production upon lectin stimulation ex vivo was assessed in PBMC supernatants, collected at 24 and 72 h, by sandwich ELISA to IL-5, IL-10, TNF-alpha and IFN-gamma. In PBMC from infected patients all but one of the lectins induced a Th2-like cytokine response, characterized by elevated IL-5 production that was higher than that induced by SEA stimulation alone. Our results show that the Th2 environment present during schistosomiasis is not affected and that it may be further stimulated by the presence of lectins.

Influence of the HLA-DQB1*0201 allele on the immune response in a Schistosoma mansoni infection.

We previously reported the association of the major histocompatibility complex class II HLA-DQB1*0201 allele with hepatosplenic schistosomiasis. The aim of this study was to evaluate the cytokine responses of peripheral blood mononuclear cells (PBMCs) and the serum levels of immunoglobulin isotypes. The study population was selected from a schistosomiasis endemic area. No significant differences in cytokine profiles were detected in PBMCs stimulated with Schistosoma mansoni soluble egg antigen (SEA), regardless of the subjects DQB1*0201 genotype or infection status. However, previously infected DQB1*0201 positive individuals had significantly lower levels of IgG4 compared to DQB1*0201 negative individuals (P<0.05).

Genetic ancestry and income are associated with dengue hemorrhagic fever in a highly admixed population.

To test whether African ancestry is protective for severe dengue, we genotyped 49 hospitalized cases of dengue hemorrhagic fever (DHF) as well as 293 neighborhood cases of dengue fever and 294 asymptomatic controls in Salvador, Bahia, Brazil. Ancestry-informative markers and 282 unlinked SNPs not associated with the clinical presentation of dengue were used to estimate ancestry. After controlling for income, both self-defined Afro-Brazilian ethnicity and African ancestry were protective for DHF (P=0.02, OR=0.28 and P=0.02, OR=0.13, respectively). Income or an index of income indicators, however, was also independently associated with the diagnosis of DHF.

Biochemical and immunologic predictors of efficacy of treatment or reinfection risk for Schistosoma mansoni.

Most Schistosoma mansoni infections are egg-negative after a single dose of oxamniquine. A cohort of 661 infected children was treated at 6-month intervals and assessed for nutritional and parasitological status. Initial biochemical and immunologic markers were measured in a subset of 84 children. All were treated at the start of therapy and at 6 months. Immunoglobulins only served as markers for active infection. No markers were predictive of cure or reinfection, except initial infection intensity and serum low-density lipoprotein. Ten percent were persistently infected and had no change in infection intensity at any time-point. Several factors suggest that this group was biologically different. In addition to failing to reduce their worm burden, they had significantly higher initial intensity of infection (100 versus 65 eggs/g, P = 0.001) and significantly lower initial serum low-density lipoprotein (72 versus 104 mg/dL, P = 0.045). The biologic plausibility of this observation is discussed.

Schizodeme analysis of Trypanosoma cruzi Colombian strain clones isolated from the acute phase of murine infection.

Colombian strain of Trypanosoma cruzi, biodeme Type III (T. cruzi I), has been cloned by micromanipulation at two phases of the acute infection: early (10 days ) and advanced (30 days). Twelve clones were obtained therefrom. Characterization by their biological and biochemical behavior showed an identity among the several clones and their parental strain, albeit with different degrees of virulence. Molecular characterization of the kinetoplast DNA (kDNA) after amplification by polymerase chain reaction revealed identical profiles of the bands from the kDNA minicircle by the analysis of restriction fragment length polymorphism for the isolated clones, their parental strain, and to the clones isolated at two different phases of the infection. Results suggest the predominance of a "principal clone", in the composition of the Colombian strain, responsible for the biological and biochemical behavior. However, no relationship was detected between the molecular profile of kDNA and the degree of virulence presented by the several clones.

Schizodeme analysis of Trypanosoma cruzi Colombian strain clones isolated from the acute phase of murine infection

Colombian strain of Trypanosoma cruzi, biodeme Type III (T. cruzi I), has been cloned by micromanipulation at two phases of the acute infection: early (10 days ) and advanced (30 days). Twelve clones were obtained therefrom. Characterization by their biological and biochemical behavior showed an identity among the several clones and their parental strain, albeit with different degrees of virulence. Molecular characterization of the kinetoplast DNA (kDNA) after amplification by polymerase chain reaction revealed identical profiles of the bands from the kDNA minicircle by the analysis of restriction fragment lenght polymorphism for the isolated clones, their parental strain, and to the clones isolated at two different phases of the infection. Results suggest the predominance of a "principal clone", in the composition of the Colombian strain, responsible for the biological and biochemical behavior. However, no relationship was detected between the molecular profile of kDNA and the degree of virulence presented by the several clones.

Uso do rituximabe para o tratamento da plaquetopenia do lúpus eritematoso sistêmico: eficácia clínica e efeito nas moléculas de co-estimulação / Use of rituximab for the treatment of systemic lupus erythematosus' thrombocytopenia: clinical efficacy and costimulatory molecules effect

Plaquetopenia é uma complicação não raramente vista no contexto do lúpus eritematoso sistêmico (LES). O seu tratamento inclui o uso de corticosteróide, imunossupressores, imunoglobulina humana e, eventualmente, esplenectomia. Descreve-se um caso de LES com plaquetopenia persistente a despeito do uso de prednisona e azatioprina, que respondeu satisfatoriamente ao uso de rituximabe (RTX). O estudo de citometria de fluxo mostrou depleção dos linfócitos B do sangue periférico, assim como uma queda das moléculas de co-estimulação após o uso da medicação. Não foi observado efeito colateral relacionado à infusão da substância. Dessa forma, o RTX parece estar indicado para casos selecionados de LES com plaquetopenia de difícil controle.