Different Profiles of Cytokines, Chemokines and Coagulation Mediators Associated with Severity in Brazilian Patients Infected with Dengue Virus.

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-1ß, 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-1ß 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.

Dengue Virus Induces NK Cell Activation through TRAIL Expression during Infection.

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 IFNα-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 IFNα 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.

Inflammatory early events associated to the role of P2X7 receptor in acute murine toxoplasmosis.

Activation of the purinergic P2X7 receptor by extracellular ATP (eATP) potentiates proinflammatory responses during infections by intracellular pathogens. Extracellular ATP triggers an antimicrobial response in macrophages infected with Toxoplasma gondii in vitro, suggesting that purinergic signaling may stimulate host defense mechanisms against toxoplasmosis. Here, we provide in vivo evidence in support of this hypothesis, by showing that P2X7-/- mice are more susceptible than P2X7+/+ mice to acute infection by the RH strain of T. gondii, and that this phenomenon is associated with a deficient proinflammatory response. Four days post-infection, peritoneal washes from infected P2X7-/- mice had no or little increase in the levels of the proinflammatory cytokines IL-12, IL-1ß, IFN-γ, and TNF-α, whose levels increased markedly in samples from infected P2X7+/+ mice. Infected P2X7-/- mice displayed an increase in organ weight and histological alterations in some of the 'shock organs' in toxoplasmosis - the liver, spleen and mesenteric lymph nodes. The liver of infected P2X7-/- mice had smaller granulomas, but increased parasite load/granuloma. Our results confirm that the P2X7 receptor is involved in containing T. gondii spread in vivo, by stimulating inflammation.

The purinergic receptor P2X7 role in control of Dengue virus-2 infection and cytokine/chemokine production in infected human monocytes.

Purinergic signaling has a crucial role in intracellular pathogen elimination. The P2X7 purinergic receptor (P2X7R), once activated by ATP, leads to pro-inflammatory responses including reactive oxygen species production. ATP can be released by injured cells, as endogenous danger signals. Dengue fever may evolve to a severe disease, leading to hypovolemic shock and coagulation dysfunctions as a result of a cytokine storm. Our aim was to evaluate the role of P2X7R activation during Dengue virus (DENV) infection. Extracellular ATP inhibited viral load in pretreated monocytes, as measured by NS1 secretion and by decrease in DENV(+) P2X7(+) cell frequencies, suggesting that P2X7R is involved in the antiviral response. Nitric oxide (NO) has anti-DENV properties and is decreased after DENV infection. NO production after ATP stimulation is abrogated by KN62 treatment, a specific P2X7R inhibitor, indicating that P2X7R likely is acting in the virus containment process. Additionally, TNF, CXCL8, CCL2 and CXCL10 factors that are associated with dengue severity were modulated by the P2X7R activation. We conclude that P2X7R is directly involved in the modulation of the antiviral and inflammatory process that occurs during DENV infection in vitro, and may have an important role in patient recovery in a first moment.

Pyrimidinergic Receptor Activation Controls Toxoplasma gondii Infection in Macrophages.

Infection by the protozoan parasite Toxoplasma gondii is highly prevalent worldwide and may have serious clinical manifestations in immunocompromised patients. T. gondii is an obligate intracellular parasite that infects almost any cell type in mammalian hosts, including immune cells. The immune cells express purinergic P2 receptors in their membrane--subdivided into P2Y and P2X subfamilies--whose activation is important for infection control. Here, we examined the effect of treatment with UTP and UDP in mouse peritoneal macrophages infected with T. gondii tachyzoites. Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis. On the other hand, UTP and UDP treatments induced early egress of tachyzoites from infected macrophages, in a Ca2+-dependent manner, as shown by scanning electron microscopy analysis, and videomicroscopy. In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole. The use of selective agonists and antagonists of the receptor subtypes P2Y2 and P2Y4 and P2Y6 showed that premature parasite egress may be mediated by the activation of these receptor subtypes. Our results suggest that the activity of P2Y host cell receptors controls T. gondii infection in macrophages, highlighting the importance of pyrimidinergic signaling for innate immune system response against infection. Finally the P2Y receptors should be considered as new target for the development of drugs against T. gondii infection.

Circulating cytokines and chemokines associated with plasma leakage and hepatic dysfunction in Brazilian children with dengue fever.

Dengue fever is usually a benign acute viral infection transmitted by arthropods but may evolve to severe clinical manifestations such as coagulation and/or hemodynamic disorders, caused mainly by an increase of vascular permeability. Deregulated circulating immunological factors have been associated with severity. In Brazil severe cases appeared in children only recently and we evaluated the profile of cytokine/chemokine kinetics in 134 hospitalized young patients during the epidemic in Rio de Janeiro in 2008. Inflammatory cytokines TNF and IFNγ were found elevated during the acute phase in children as well as the anti-inflammatory IL10 and chemokines MIF and CXCL10/IP10, all last three persisting longer during the recovery phase. Severe disease fitting the dengue hemorrhagic fever pattern (WHO, 1997) was associated with higher IL10 and CXCL10/IP10 circulating levels (peak levels at seven days with P<0.01 and P<0.001 respectively as compared to DF). These factors were higher in patients pulmonary effusion or ascites (P<0.05 for IL10 and P<0.01 for CXCL10/IP10). Both factors were also associated with liver changes such as AST increase correlated with CXCL10/IP10 (r=0.4300 with P<0.0001) and patients presenting painful hepatomegaly showed higher circulating levels of IL10 (P<0.01, at 7-9 days) and of CXCL10/IP10 (P<0.05, 4-6 days and P<0.001, 7-9 days) when compared to patients without apparent liver alterations. Most cases presented a history of prior infection (93%). This is the first study demonstrating cytokine and chemokine association with severity during dengue fever in Brazilian children. IL10 and CXCL10/IP10 play a role in the disease severity associated with induction of vascular leakage and a novel association with changes in liver dysfunction.

Characterizing the presence and sensitivity of the P2X7 receptor in different compartments of the gut.

Purinergic signaling has been established as an important feature of inflammation and homeostasis. The expression of a number of P2 receptor subtypes in the gut has been reported. In this study, using a well-known permeabilization method that is assessed by flow cytometry, we show that lymphocytes and macrophages from the mesenteric lymph nodes (MLN) and the peritoneal cavity exhibit different sensitivities to extracellular ATP. Compared with the macrophages, the lymphocytes are more sensitive to ATP in the MLN compartment, whereas in the peritoneal cavity the macrophages are more sensitive to ATP than the lymphocytes. In addition, we have shown that the epithelial cells from the small bowel are more resistant to the ATP effects than the cells from the colon. These cells, however, become susceptible after exposure to IFN-γ. Furthermore, by examining parameters such as pH manipulation, the exposure to divalent cations and the P2X7 antagonist Brilliant Blue G, and the use of cells from P2X7(-/-) mice, we have shown that the P2X7 receptors are the ATP-activated receptors responsible for the permeabilization phenomenon. In addition, using Western blot analysis, we have demonstrated the changes in the P2X7 receptor expression in immune cells isolated from different sites in the gut and in the gut-associated lymphoid tissues. Our findings suggest the existence of the site-specific modulation of P2X7 receptors on epithelial and immune cells, and we define purinergic signaling as a new regulatory element in the control of inflammation and cell fate in the gut and in the gut-associated lymphoid tissues.

Host-cell lipid rafts: a safe door for micro-organisms?

The lipid raft hypothesis proposed that these microdomains are small (10-200 nM), highly dynamic and enriched in cholesterol, glycosphingolipids and signalling phospholipids, which compartmentalize cellular processes. These membrane regions play crucial roles in signal transduction, phagocytosis and secretion, as well as pathogen adhesion/interaction. Throughout evolution, many pathogens have developed mechanisms to escape from the host immune system, some of which are based on the host membrane microdomain machinery. Thus lipid rafts might be exploited by pathogens as signalling and entry platforms. In this review, we summarize the role of lipid rafts as players in the overall invasion process used by different pathogens to escape from the host immune system.

Activation of the P2X(7) receptor triggers the elimination of Toxoplasma gondii tachyzoites from infected macrophages.

Toxoplasmosis is caused by the protozoan parasite Toxoplasma gondii, which is widespread throughout the world. After active penetration, the parasite is enclosed within a parasitophorous vacuole and survives in the host cell by avoiding, among other mechanisms, lysosomal degradation. A large number of studies have demonstrated the importance of ATP signalling via the P2X(7) receptor, as a component of the inflammatory response against intracellular pathogens. Here we evaluate the effects of extracellular ATP on T. gondii infection of macrophages. ATP treatment inhibits the parasite load and the appearance of large vacuoles in the cytoplasm of intracellular parasites. ROS and NO assays showed that only ROS production is involved with the ATP effects. Immunofluorescence showed colocalization of Lamp1 and SAG1 only after ATP treatment, suggesting the formation of phagolysosomes. The involvement of P2X(7) receptors in T. gondii clearance was confirmed by the use of P2X(7) agonists and antagonists, and by infecting macrophages from P2X(7) receptor-deficient mice. We conclude that parasite elimination might occur following P2X(7) signalling and that novel therapies against intracellular pathogens could take advantage of activation of purinergic signalling.

Cell death induction in Giardia lamblia: effect of beta-lapachone and starvation.

Giardia lamblia is a protozoan that parasitizes the small intestine of vertebrates. It is a cause of intestinal infection and diarrhea and infects millions of people worldwide. This protozoan presents many characteristics common to eukaryotic cells but it lacks organelles found in most eukaryotes (e.g., peroxisomes, typical Golgi complex and mitochondria). Also it presents mitosomes, a relic organelle that appears to be a mitochondrial remnant. Cell death in Giardia was induced by the drug beta-Lapachone and by starvation. Giardia behavior was followed by scanning, transmission and fluorescence microscopy, quantification of cell metabolism using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide), changes in lipid rafts, using DiIC(16) and cholera toxin. Cell shrinkage, chromatin condensation, membrane blebbing and vacuolization provided ultrastructural evidence of apoptosis, whereas the myelinic figures in large vacuoles and LC-3 staining suggested an autophagic process. Lipids rafts were altered by drug treatment and co-localized with regions containing membrane blebbing. The treatment with beta-Lap induced encystation. A search for sequence similarities in databases and protein alignments was carried out. Although Giardia is an amitochondrial organism, it presented some autophagic-like cell death characteristics and several, but not all, apoptotic characteristics, induced by beta-Lapachone and starvation.

The P2X(7) receptor and intracellular pathogens: a continuing struggle.

The purinergic receptor, P2X(7), has recently emerged as an important component of the innate immune response against microbial infections. Ligation of P2X(7) by ATP can stimulate inflammasome activation and secretion of proinflammatory cytokines, but it can also lead directly to killing of intracellular pathogens in infected macrophages and epithelial cells. Thus, while some intracellular pathogens evade host defense responses by modulating with membrane trafficking or cell signaling in the infected cells, the host cells have also developed mechanisms for inhibiting infection. This review will focus on the effects of P2X(7) on control of infection by intracellular pathogens, microbial virulence factors that interfere with P2X(7) activity, and recent evidence linking polymorphisms in human P2X(7) with susceptibility to infection.

Hipertensión y su relación con el sodio, el potasio, el calcio y el magnesio / Relationship between: Hypertension and sodium, potassium, calcium and magnesium

La presión arterial (PA) elevada es uno de los principales facto-res de riesgo de la enfermedad, cardiaca, cerebrovascular y renal. Su aumento se debe entre otros a factores dietéticos, genéticos y a sus interacciones, entre otros. Un respaldo amplio de evidencia científica, apoya fuertemente el concepto de que los facto-res dietarios afectan la PA. Las modificaciones dietarias y de estilo de vida que disminuyen la PA son: la reducción del consumo de sal, deficiencia en calorías que induce a pérdida de peso, actividad física regular, moderación en la ingesta de alcohol, aumento en la ingesta de potasio y consumo de una dieta total saludable, como la dieta DASH (Dietary Approaches to Prevent and Treat Hypertension). Específicamente las personas de raza negra son más sensibles a la disminución de la PA, cuando se les reduce la ingesta de sal, se aumenta la de potasio y se les ofrece la dieta estilo DASH. Las personas de mayor edad, con alto riesgo de sufrir enfermedades secundarias a la PA elevada, de tipo cardíaco, cerebrovascular y renal, pueden mejorar sus cifras tensionales y prevenir la aparición de complicaciones cuando realizan modificaciones en su alimentación. De interés especial son los avances en estudios de experimentación animal, epidemiológicos y de ensayos clínicos, sobre la relación entre el incremento en el contenido de calcio en la dieta y el efecto protector contra la hipertensión arterial (HTA), no obstante aún continúan las investigaciones para validar dichos hallazgos. Con respecto al magnesio y su asociación con la disminución de la PA, los estudios no son concluyentes y falta aun más evidencia para reconocer su papel en la HTA.

Hypertension is a major risk factor for many cardiovascular diseases includingstroke, coronary heart disease, cardiac failure, and end stage renal disease.Elevated blood pressure results from interactions between environmental,nutritional and genetic factors. A wide scientific evidence strongly supportsthe concept, that nutritional components can affect the blood pressure.There are some comprehensive lifestyle and dietary modifications that cansubstantially lower blood pressure and improve blood pressure control. African-Americans particularly are more sensible to reduce blood pressure whenthey decrease salt consumption, reduced intake of alcohol, increase potassiumintake and follow the DASH Diet. Elderly people, with high blood pressureare at risk for chronic disease and they can also get beneficial effects of healthful lifestyle modifications in the prevention and management of hypertension. Some epidemiologic and clinical trials with animal models have assessed the relationship between increased dietary calcium intake and its protective role for the control of hypertension, therefore scientific studies to prove these facts are still going on and more scientific evidence is necessary.

Giardia lamblia behavior under cytochalasins treatment.

Giardia lamblia, a flagellated protist, is the parasite most commonly found in the intestinal tract of humans and other mammals causing a disease known as giardiasis. This parasite presents several cytoskeletal structures whose major components are microtubules, namely: the ventral adhesive disk, eight flagella axonemes, the median body, and funis. However, the cytoskeletal filamentous structures are poorly understood, and therefore, less studied. In the present work, we used actin-interacting drugs such as cytochalasin B and D to investigate their effects on Giardia ultrastructure. Axenically grown G. lamblia trophozoites were treated with these drugs and analyzed by fluorescence microscopy and scanning and transmission electron microscopy. It was observed that trophozoites became completely misshapen, detached from the glass surface, and failed to complete cell division. The main alterations observed included: (1) disk fragmentation, (2) presence of large vacuoles, (3) alterations in flagella number and flagella internalization, (4) blocked cytokinesis but not the karyokinesis, and (5) presence of membrane undulations and blebs. These findings are discussed.

Centrin in Giardia lamblia - ultrastructural localization.

Giardia lamblia is a multiflagellar parasite and one of the earliest diverging eukaryotic cells. It possesses a complex cytoskeleton based on different groups of microtubular structures - a ventral adhesive disc, four pairs of flagella, a median body and funis. Centrin is an important member of the EF-hand family of calcium-binding proteins, and it is known to show calcium-sensitive contractile behaviour. In the present study, we performed an ultrastructural localization of centrin in G. lamblia using several monoclonal antibodies to centrin. Microtubular structures such as the basal bodies, all the flagella axonemes, the adhesive disc, funis, and the median bodies presented positive labelling to centrin. In addition, the dense rods also demonstrated positive labelling. These results show that centrin is located in key positions related to microtubules. The role of centrin in these dynamic regions is discussed.