Hemolysis-induced lethality involves inflammasome activation by heme.

The increase of extracellular heme is a hallmark of hemolysis or extensive cell damage. Heme has prooxidant, cytotoxic, and inflammatory effects, playing a central role in the pathogenesis of malaria, sepsis, and sickle cell disease. However, the mechanisms by which heme is sensed by innate immune cells contributing to these diseases are not fully characterized. We found that heme, but not porphyrins without iron, activated LPS-primed macrophages promoting the processing of IL-1ß dependent on nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3). The activation of NLRP3 by heme required spleen tyrosine kinase, NADPH oxidase-2, mitochondrial reactive oxygen species, and K(+) efflux, whereas it was independent of heme internalization, lysosomal damage, ATP release, the purinergic receptor P2X7, and cell death. Importantly, our results indicated the participation of macrophages, NLRP3 inflammasome components, and IL-1R in the lethality caused by sterile hemolysis. Thus, understanding the molecular pathways affected by heme in innate immune cells might prove useful to identify new therapeutic targets for diseases that have heme release.

Interferon-γ and granulocyte/monocyte colony-stimulating factor production by natural killer cells involves different signaling pathways and the adaptor stimulator of interferon genes (STING).

Natural killer (NK) cells are important for innate immunity in particular through the production of IFN-γ and GM-CSF. Both cytokines are important in restoration of immune function of tolerized leukocytes under inflammatory events. The expression of TLRs in NK cells has been widely studied by analyzing the mRNA of these receptors, rarely seeking their protein expression. We previously showed that murine spleen NK cells express TLR9 intracellularly and respond to CpG oligodeoxynucleotide (CpG-ODN) by producing IFN-γ and GM-CSF. However, to get such production the presence of accessory cytokines (such as IL-15 and IL-18) was required, whereas CpG-ODN or accessory cytokines alone did not induce IFN-γ or GM-CSF. We show here that TLR9 overlaps with the Golgi apparatus in NK cells. Furthermore, CpG-ODN stimulation in the presence of accessory cytokines induces the phosphorylation of c-Jun, STAT3, and IκBα. IFN-γ and GM-CSF production requires NF-κB and STAT3 activation as well as Erk-dependent mechanisms for IFN-γ and p38 signaling for GM-CSF. Using knock-out-mice, we show that UNC93b1 and IL-12 (produced by NK cells themselves) are also necessary for IFN-γ and GM-CSF production. IFN-γ production was found to be MyD88- and TLR9-dependent, whereas GM-CSF was TLR9-independent but dependent on STING (stimulator of interferon genes), a cytosolic adaptor recently described for DNA sensing. Our study thereby allows us to gain insight into the mechanisms of synergy between accessory cytokines and CpG-ODN in NK cells. It also identifies a new and alternative signaling pathway for CpG-ODN in murine NK cells.

Heme induces programmed necrosis on macrophages through autocrine TNF and ROS production.

Diseases that cause hemolysis or myonecrosis lead to the leakage of large amounts of heme proteins. Free heme has proinflammatory and cytotoxic effects. Heme induces TLR4-dependent production of tumor necrosis factor (TNF), whereas heme cytotoxicity has been attributed to its ability to intercalate into cell membranes and cause oxidative stress. We show that heme caused early macrophage death characterized by the loss of plasma membrane integrity and morphologic features resembling necrosis. Heme-induced cell death required TNFR1 and TLR4/MyD88-dependent TNF production. Addition of TNF to Tlr4(-/-) or to Myd88(-/-) macrophages restored heme-induced cell death. The use of necrostatin-1, a selective inhibitor of receptor-interacting protein 1 (RIP1, also known as RIPK1), or cells deficient in Rip1 or Rip3 revealed a critical role for RIP proteins in heme-induced cell death. Serum, antioxidants, iron chelation, or inhibition of c-Jun N-terminal kinase (JNK) ameliorated heme-induced oxidative burst and blocked macrophage cell death. Macrophages from heme oxygenase-1 deficient mice (Hmox1(-/-)) had increased oxidative stress and were more sensitive to heme. Taken together, these results revealed that heme induces macrophage necrosis through 2 synergistic mechanisms: TLR4/Myd88-dependent expression of TNF and TLR4-independent generation of ROS.

Therapeutical targeting of nucleic acid-sensing Toll-like receptors prevents experimental cerebral malaria.

Excessive release of proinflammatory cytokines by innate immune cells is an important component of the pathogenic basis of malaria. Proinflammatory cytokines are a direct output of Toll-like receptor (TLR) activation during microbial infection. Thus, interference with TLR function is likely to render a better clinical outcome by preventing their aberrant activation and the excessive release of inflammatory mediators. Herein, we describe the protective effect and mechanism of action of E6446, a synthetic antagonist of nucleic acid-sensing TLRs, on experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA. We show that in vitro, low doses of E6446 specifically inhibited the activation of human and mouse TLR9. Tenfold higher concentrations of this compound also inhibited the human TLR8 response to single-stranded RNA. In vivo, therapy with E6446 diminished the activation of TLR9 and prevented the exacerbated cytokine response observed during acute Plasmodium infection. Furthermore, severe signs of ECM, such as limb paralysis, brain vascular leak, and death, were all prevented by oral treatment with E6446. Hence, we provide evidence that supports the involvement of nucleic acid-sensing TLRs in malaria pathogenesis and that interference with the activation of these receptors is a promising strategy to prevent deleterious inflammatory responses that mediate pathogenesis and severity of malaria.

Malaria primes the innate immune response due to interferon-gamma induced enhancement of toll-like receptor expression and function.

Malaria-induced sepsis is associated with an intense proinflammatory cytokinemia for which the underlying mechanisms are poorly understood. It has been demonstrated that experimental infection of humans with Plasmodium falciparum primes Toll-like receptor (TLR)-mediated proinflammatory responses. Nevertheless, the relevance of this phenomenon during natural infection and, more importantly, the mechanisms by which malaria mediates TLR hyperresponsiveness are unclear. Here we show that TLR responses are boosted in febrile patients during natural infection with P. falciparum. Microarray analyses demonstrated that an extraordinary percentage of the up-regulated genes, including genes involving TLR signaling, had sites for IFN-inducible transcription factors. To further define the mechanism involved in malaria-mediated "priming," we infected mice with Plasmodium chabaudi. The human data were remarkably predictive of what we observed in the rodent malaria model. Malaria-induced priming of TLR responses correlated with increased expression of TLR mRNA in a TLR9-, MyD88-, and IFNgamma-dependent manner. Acutely infected WT mice were highly susceptible to LPS-induced lethality while TLR9(-/-), IL12(-/-) and to a greater extent, IFNgamma(-/-) mice were protected. Our data provide unprecedented evidence that TLR9 and MyD88 are essential to initiate IL12 and IFNgamma responses and favor host hyperresponsiveness to TLR agonists resulting in overproduction of proinflammatory cytokines and the sepsis-like symptoms of acute malaria.

[Thoughts regarding researchers utilizing Grounded Theory]. / Reflexões sobre o pesquisador nas trilhas da Teoria Fundamentada nos Dados.

This descriptive-reflexive study was performed with the objective to present the characteristics of researchers who use the Grounded Theory method, and outline the development of aptitudes for the researcher to become a Grounded Theoretician. The theoretical discussion was based on the frameworks of this methodology and supported by the literature. The article presents the main demands of qualitative studies using Grounded Theory, and important behaviors, attitudes and characteristics developed by the researchers. It is concluded that learning about Grounded Theory involves more than operationalizing a group of procedures and techniques. It also involves facing challenges to change one's attitude as a researcher and develop new ways of thinking and researching, gathering knowledge based on data to form a theory.

Detection of hepatitis C virus in platelets: evaluating its relationship to antiviral therapy outcome.

BACKGROUND/AIMS: Detection of HCV has been documented in extrahepatic sites such as platelets. However, its influence on antiviral therapy outcome is unknown. In this study, we investigated the relationship between the detection of HCV in platelets from a cohort of 48 chronically HCV-infected patients and response to antiviral therapy. METHODOLOGY: This study comprised of 19 males and 29 females, mean age 54.9 +/- 8.72 years, followed-up in Rio de Janeiro, Brazil, between August 2004 and October 2006. HCV-RNA was detected in serum and platelets (pre-treatment, end-of-treatment and 24 weeks after completion of therapy) by reverse transcription-nested polymerase chain reaction. Patients with genotype 1 or 4 were treated with peginterferon-alfa/ribavirin for 48 weeks, and patients with genotype 3 received interferon-alfa/ribavirin for 24 weeks. RESULTS: Baseline detection of HCV in platelets was found not to be related to therapy outcome. However, significant associations between detection rates of HCV in platelets and serum at the end-of-treatment (p = 0.0203), and 24 weeks after completion of therapy (p = 0.0016) were observed. Interestingly, HCV was detected in platelets from two patients with normal ALT who lost detectable serum HCV at the end-of-treatment and, after 24 weeks of followup, relapsed virologically in serum. CONCLUSIONS: Our data suggest that patients with HCV persistence in platelets by the end-of-treatment appear to be at an increased risk of recurrent HCV infection.

Molecular analysis and patterns of ALT and hepatitis C virus seroconversion in haemodialysis patients with acute hepatitis.

BACKGROUND: Haemodialysis (HD) continues to carry the risk of hepatitis C virus (HCV) transmission, with delayed seroconversion and often normal alanine aminotransferase (ALT) values increasing the likelihood of undetected infection and thus uninterrupted spread of HCV. The aim of this study was to identify the characteristic patterns of ALT changes and seroconversion during an outbreak of HCV in a HD unit. We also wanted to establish the relationship between infecting viruses using molecular analysis. METHODS: All patients (n = 72) and staff (n = 23) of the HD unit were prospectively followed for 14 months. Serial measurements for ALT, HCV antibody and HCV-RNA were performed besides HCV sequence analysis. RESULTS: The initial screening for anti-HCV and HCV-RNA confirmed chronic infection in 16/72 (22%) subjects and identified three subjects with recent seroconversion. In addition, five cases were reverse transcription-polymerase chain reaction positive alone for a total of eight recent cases. The interval between the initial observation of ALT changes and seroconversion varied from 1 to 8 months, and in several individuals ALT fluctuations only below the upper limit of normal were detected. However, relating each subject's ALT values to ALT at baseline, ALT levels increased between 1.6- and 4.7-fold. Molecular analysis provided evidence for transmission from two chronically infected source patients, probably because of inappropriate infection control measures. CONCLUSION: Our data highlight the importance of well-implemented safety precautions and regular HCV-RNA testing to prevent the further spread of HCV in this population, and suggest the use of ALT baseline values to identify infections that may remain unnoticed otherwise.

Ninjurin 1 asp110ala single nucleotide polymorphism is associated with protection in leprosy nerve damage.

Leprosy is the major cause of non-traumatic neuropathy. Herein, we investigated the role of ninjurin 1, an adhesion molecule involved in nerve regeneration in leprosy. Our results demonstrated that M. leprae stimulates in vitro up-regulation of ninjurin mRNA in cultured Schwann and blood cells as well as in vivo mRNA and protein expression in leprosy nerve biopsies. A polymorphism (asp110ala) was investigated in a case-control study (1123 individuals) and no association was found with leprosy per se or with disseminated forms. Nevertheless, ala110 was associated with functional nerve impairment (OR=2.42; p=0.02 for ala/ala) and with lower mRNA levels. Our data suggests that asp110ala could be a valuable genetic marker of nerve damage in leprosy.

Detection of hepatitis C virus in platelets: evaluating its relationship to viral and host factors.

BACKGROUND/AIMS: Interaction of platelets with HCV is presumed to be one of the pathogenic mechanisms implicated in HCV-associated thrombocytopenia. Nevertheless, analysis of factors influencing the detection of HCV in platelets is not well understood. In this study, we investigated the relationship between the detection of HCV in platelets from a cohort of 39 chronically HCV-infected patients and several viral and host factors. METHODOLOGY: This study comprised of 14 males and 25 females with a median age of 53 years, followed-up in Rio de Janeiro, Brazil, between August 2003 and December 2004. HCV-RNA was detected in serum and platelet samples by reverse transcription-nested polymerase chain reaction. Genotypes were determined by using direct nucleotide sequencing of the PCR products and plasma viral loads by using HCV-Amplicor Monitor 2.0. RESULTS: When compared on the basis of the results of the detection of HCV-RNA in platelets, patients did not differ significantly in relation to viral load and genotype, platelet count, aminotransferases and degree of hepatic fibrosis. CONCLUSIONS: Our data suggest that HCV can be detected in platelets of chronically HCV-infected patients independent of these cofactors, including circulating HCV load. Studies on HCV dynamics are needed to provide new insights into HCV binding to platelets.

Gonococcal lipooligosaccharide sialylation: virulence factor and target for novel immunotherapeutics.

Gonorrhea has become resistant to most conventional antimicrobials used in clinical practice. The global spread of multidrug-resistant isolates of Neisseria gonorrhoeae could lead to an era of untreatable gonorrhea. New therapeutic modalities with novel mechanisms of action that do not lend themselves to the development of resistance are urgently needed. Gonococcal lipooligosaccharide (LOS) sialylation is critical for complement resistance and for establishing infection in humans and experimental mouse models. Here we describe two immunotherapeutic approaches that target LOS sialic acid: (i) a fusion protein that comprises the region in the complement inhibitor factor H (FH) that binds to sialylated gonococci and IgG Fc (FH/Fc fusion protein) and (ii) analogs of sialic acid that are incorporated into LOS but fail to protect the bacterium against killing. Both molecules showed efficacy in the mouse vaginal colonization model of gonorrhea and may represent promising immunotherapeutic approaches to target multidrug-resistant isolates. Disabling key gonococcal virulence mechanisms is an effective therapeutic strategy because the reduction of virulence is likely to be accompanied by a loss of fitness, rapid elimination by host immunity and consequently, decreased transmission.

Body language in health care: a contribution to nursing communication.

OBJECTIVE: to classify body language used in nursing care, and propose "Body language in nursing care" as an analytical category for nursing communication. METHOD: quantitative research with the systematic observation of 21:43 care situations, with 21 members representing the nursing teams of two hospitals. Empirical categories: sound, facial, eye and body expressions. RESULTS: sound expressions emphasized laughter. Facial expressions communicated satisfaction and happiness. Eye contact with members stood out in visual expressions. The most frequent body expressions were head movements and indistinct touches. CONCLUSION: nursing care team members use body language to establish rapport with patients, clarify their needs and plan care. The study classified body language characteristics of humanized care, which involves, in addition to technical, non-technical issues arising from nursing communication.

DNA-Containing Immunocomplexes Promote Inflammasome Assembly and Release of Pyrogenic Cytokines by CD14+ CD16+ CD64high CD32low Inflammatory Monocytes from Malaria Patients.

UNLABELLED: High levels of circulating immunocomplexes (ICs) are found in patients with either infectious or sterile inflammation. We report that patients with either Plasmodium falciparum or Plasmodium vivax malaria have increased levels of circulating anti-DNA antibodies and ICs containing parasite DNA. Upon stimulation with malaria-induced ICs, monocytes express an NF-κB transcriptional signature. The main source of IC-induced proinflammatory cytokines (i.e., tumor necrosis factor alpha [TNF-α] and interleukin-1ß [IL-1ß])in peripheral blood mononuclear cells from acute malaria patients was found to be a CD14(+) CD16 (FcγRIIIA)(+) CD64 (FcγRI)(high) CD32 (FcγRIIB)(low) monocyte subset. Monocytes from convalescent patients were predominantly of the classical phenotype (CD14(+) CD16(-)) that produces high levels of IL-10 and lower levels of TNF-α and IL-1ß in response to ICs. Finally, we report a novel role for the proinflammatory activity of ICs by demonstrating their ability to induce inflammasome assembly and caspase-1 activation in human monocytes. These findings illuminate our understanding of the pathogenic role of ICs and monocyte subsets and may be relevant for future development of immunity-based interventions with broad applications to systemic inflammatory diseases. IMPORTANCE: Every year, there are approximately 200 million cases of Plasmodium falciparum and P. vivax malaria, resulting in nearly 1 million deaths, most of which are children. Decades of research on malaria pathogenesis have established that the clinical manifestations are often a consequence of the systemic inflammation elicited by the parasite. Recent studies indicate that parasite DNA is a main proinflammatory component during infection with different Plasmodium species. This finding resembles the mechanism of disease in systemic lupus erythematosus, where host DNA plays a central role in stimulating an inflammatory process and self-damaging reactions. In this study, we disclose the mechanism by which ICs containing Plasmodium DNA activate innate immune cells and consequently stimulate systemic inflammation during acute episodes of malaria. Our results further suggest that Toll-like receptors and inflammasomes have a central role in malaria pathogenesis and provide new insights toward developing novel therapeutic interventions for this devastating disease.

Increased survival in B-cell-deficient mice during experimental cerebral malaria suggests a role for circulating immune complexes.

The pathogenesis of malaria, an insect-borne disease that takes millions of lives every year, is still not fully understood. Complement receptor 1 (CR1) has been described as a receptor for Plasmodium falciparum, which causes cerebral malaria in humans. We investigated the role of CR1 in an experimental model of cerebral malaria. Transgenic mice expressing human CR1 (hCR1(+)) on erythrocytes were infected with Plasmodium berghei ANKA and developed cerebral malaria. No difference in survival was observed in hCR1(+) mice compared to wild-type mice following infection with P. berghei ANKA; however, hCR1 detection was significantly diminished on erythrocytes between days 7 and 10 postinfection. hCR1 levels returned to baseline by day 17 postinfection in surviving animals. Immunoblot assays revealed that total erythrocyte hCR1 levels were diminished, confirming that immune complexes in association with erythrocyte hCR1 were likely removed from erythrocytes in vivo by clearance following immune adherence. Decreases in hCR1 were completely dependent on C3 expression, as mice treated with cobra venom factor (which consumes and depletes C3) retained hCR1 on erythrocytes during C3 depletion through day 7; erythrocyte hCR1 decreases were observed only when C3 levels recovered on day 9. B-cell-deficient mice exhibit a marked increase in survival following infection with P. berghei ANKA, which suggests that immune complexes play a central role in the pathogenesis of experimental cerebral malaria. Together, our findings highlight the importance of complement and immune complexes in experimental cerebral malaria. IMPORTANCE Cerebral malaria is a deadly complication of infection with Plasmodium falciparum. Despite its high prevalence, relatively little is understood about its pathogenesis. We have determined that immune complexes are generated and deposited on erythrocytes specifically expressing human complement receptor 1 in a mouse model of cerebral malaria. We also provide evidence demonstrating the importance of immunoglobulins in the pathogenesis of cerebral malaria in mice. These findings may have important implications in human cerebral malaria.

Enfermarias mistas em psiquiatria: memória da enfermagem sobre um novo espaço assistencial no Brasil (1996-2002) / Enfermerías mixtas en psiquiatría: memoria de la enfermería sobre un nuevo espacio asistencial en Brasil (1996-2002) / Mixed wards in psychiatry: records of nursing care on a new assistance space in Brazil (1996-2002)

RESUMO Objetivo: Analisar as implicações da implantação das "Enfermarias Mistas" na configuração do espaço assis tencial do Instituto de Psiquiatria da Universidade Federal do Rio de Janeiro (IPUB), na visão da equipe de enfermagem. Metodo: Estudo sócio-histórico, cujas fontes primárias foram documentos escritos e documentos orais produzidos com 5 profissionais da equipe de enfermagem. Resultados: Nas Enfermarias Mistas a distri buição de doentes por sexo deu lugar a uma distribuição por quadro clínico, propiciando o surgimento de um espaço terapêutico diferenciado e facilitador da reabilitação psicossocial, denominado "enfermaria de portas abertas". A equipe de enfermagem ainda permaneceu com papéis de vigilância e controle, porém com maior liberdade para o planejamento de ações de reabilitação psicossocial. Conclusõa: As Enfermarias Mistas tiveram a equipe de enfermagem como principal agente transformador. Funcionou como estratégia de convivência sem segregação sexual, propiciando a existência de um novo espaço terapêutico, rompendo, dentro dopossível,com o modelo manicomial.

ABSTRACT Objective: To analyze the implications of implementing "mixed wards" in the design of assistance space of the Psychiatry Institute of the Federal University of Rio de Janeiro (IPUB), from the perspective of the nursing staff. Method: Sociohistorical study, whose primary sources were written and oral documents produced by 5 professional nursing staff members. Results: The distribution of patients by gender in mixed wards gave rise to a distribution by clinical presentation, which led to the emergence of a differentiated therapeutic space facilitating psychosocial rehabilitation called "open-door ward". The nursing staff still took roles of surveillance and control, but had greater freedom for the planning of psychosocial rehabilitation. Conclusion: Mixed wards worked as a strategy to cohabit without sexual segregation, allowing for the existence of a new therapeutic area, which ended, where possible, with the asylum model.

RESUMEN Objetivo: Analizar las implicancias de la implementación de las "Enfermerías Mixtas" en la configuración del espacio asistencial del Instituto de Psiquiatría de la Universidad Federal de Río de Janeiro (IPUB), en la perspec tiva del personal de enfermería. Método: Estudio socio-histórico, cuyas fuentes primarias fueron documentos escritos y orales producidos con 5 miembros del personal profesional de enfermería. Resultados: La distribu ción de pacientes por género en las Enfermerías Mixtas generó una distribución por cuadro clínico, lo que llevó a la aparición de un espacio diferenciado terapéutico y facilitador de la rehabilitación psicosocial, llamado "enfermería de puertas abiertas". El personal de enfermeira, permaneció con funciones de vigilancia y control, pero con una mayor libertad para la planificación de la rehabilitación psicosocial. Conclusión: Las salas mixtas de hospitalización tuvieron al personal de enfermería como el principal agente de cambio. Funcionó como estrategia de convivencia sin segregación sexual, lo que permitió la existencia de un nuevo espacio terapéutico, rompiendo, posiblemente, con el modelo de aislamiento.

Mosquitoes in degraded and preserved areas of the Atlantic Forest and potential for vector-borne disease risk in the municipality of São Paulo, Brazil.

In order to assess the epidemiological potential of the Culicidae species in remaining areas of the Brazilian Atlantic Forest, specimens of this family were collected in wild and anthropic environments. A total of 9,403 adult mosquitoes was collected from May, 2009 to June, 2010. The most prevalent among species collected in the wild environment were Anopheles (Kerteszia) cruzii, the Melanoconion section of Culex (Melanoconion), and Aedes serratus, while the most common in the anthropic site were Coquillettidia chrysonotum/albifera, Culex (Culex) Coronator group, and An. (Ker.) cruzii. Mosquito richness was similar between environments, although the abundance of individuals from different species varied. When comparing diversity patterns between environments, anthropic sites exhibited higher richness and evenness, suggesting that environmental stress increased the number of favorable niches for culicids, promoting diversity. Increased abundance of opportunistic species in the anthropic environment enhances contact with culicids that transmit vector-borne diseases.

Hepatitis C virus-associated thrombocytopenia: a controlled prospective, virological study.

Chronic hepatitis C virus (HCV) infection has also been associated with the development of several extrahepatic alterations, including thrombocytopenia, and a variety of pathogenic mechanisms are reported to be implicated in this hematological abnormality. Different studies have succeeded in detecting HCV in platelets with discrepant results. Moreover, most of the studies on HCV-associated thrombocytopenia have failed to provide data concerning the infecting genotype, a factor with prognostic implication in chronically HCV-infected patients. To determine whether thrombocytopenia is an extrahepatic alteration dependent on particular HCV genotypes, and to assess the relationship between thrombocytopenia and detection of HCV-RNA (positive strand) in platelets from patients with chronic HCV infection, 106 anti-HCV+/HCV-RNA+ patients (57 thrombocytopenic and 49 non-thrombocytopenic) were prospectively studied. The infecting genotype was analyzed from sera by using direct nucleotide sequencing of the polymerase chain reaction (PCR) products from core region. Genotypes 1a, 1b, and 3a were more prevalent in our patients, and no association between these genotypes and thrombocytopenia was observed ( p=0.891). HCV-RNA was detected in platelets by reverse transcriptase (RT)-nested PCR in the 5' non-coding region with a higher frequency (60%) in thrombocytopenic patients than in non-thrombocytopenic subjects (35%, p=0.017), suggesting that HCV is directly involved in the process that, at least in part, leads to thrombocytopenia.