Renal Function at Hospital Admission and Mortality Due to Acute Kidney Injury after Myocardial Infarction

Background: The role of an impaired estimated glomerular filtration rate (eGFR) at hospital admission in the outcome of acute kidney injury (AKI) after acute myocardial infarction (AMI) has been underreported. The aim of this study was to assess the influence of an admission eGFR<60 mL/min/1.73 m(2) on the incidence and early and late mortality of AMI-associated AKI. Methods: A prospective study of 828 AMI patients was performed. AKI was defined as a serum creatinine increase of >= 50% from the time of admission (RIFLE criteria) in the first 7 days of hospitalization. Patients were divided into subgroups according to their eGFR upon hospital admission (MDRD formula, mL/min/1.73 m(2)) and the development of AKI: eGFR >= 60 without AKI, eGFR<60 without AKI, eGFR >= 60 with AKI and eGFR<60 with AKI. Results: Overall, 14.6% of the patients in this study developed AKI. The admission eGFR had no impact on the incidence of AKI. However, the admission eGFR was associated with the outcome of AMI-associated AKI. The adjusted hazard ratios (AHR, Cox multivariate analysis) for 30-day mortality were 2.00 (95% CI 1.11-3.61) for eGFR, 60 without AKI, 4.76 (95% CI 2.45-9.26) for eGFR >= 60 with AKI and 6.27 (95% CI 3.20-12.29) for eGFR, 60 with AKI. Only an admission eGFR of <60 with AKI was significantly associated with a 30-day to 1-year mortality hazard (AHR 3.05, 95% CI 1.50-6.19). Conclusions: AKI development was associated with an increased early mortality hazard in AMI patients with either preserved or impaired admission eGFR. Only the association of impaired admission eGFR and AKI was associated with an increased hazard for late mortality among these patients.

Neuroimaging during Trance State: A Contribution to the Study of Dissociation

Despite increasing interest in pathological and non-pathological dissociation, few researchers have focused on the spiritual experiences involving dissociative states such as mediumship, in which an individual (the medium) claims to be in communication with, or under the control of, the mind of a deceased person. Our preliminary study investigated psychography - in which allegedly "the spirit writes through the medium's hand" - for potential associations with specific alterations in cerebral activity. We examined ten healthy psychographers - five less expert mediums and five with substantial experience, ranging from 15 to 47 years of automatic writing and 2 to 18 psychographies per month - using single photon emission computed tomography to scan activity as subjects were writing, in both dissociative trance and non-trance states. The complexity of the original written content they produced was analyzed for each individual and for the sample as a whole. The experienced psychographers showed lower levels of activity in the left culmen, left hippocampus, left inferior occipital gyrus, left anterior cingulate, right superior temporal gyrus and right precentral gyrus during psychography compared to their normal (non-trance) writing. The average complexity scores for psychographed content were higher than those for control writing, for both the whole sample and for experienced mediums. The fact that subjects produced complex content in a trance dissociative state suggests they were not merely relaxed, and relaxation seems an unlikely explanation for the underactivation of brain areas specifically related to the cognitive processing being carried out. This finding deserves further investigation both in terms of replication and explanatory hypotheses.

Dissecting Phaseolus vulgaris Innate Immune System against Colletotrichum lindemuthianum Infection

Background: The genus Colletotrichum is one of the most economically important plant pathogens, causing anthracnose on a wide range of crops including common beans (Phaseolus vulgaris L.). Crop yield can be dramatically decreased depending on the plant cultivar used and the environmental conditions. This study aimed to identify potential genetic components of the bean immune system to provide environmentally friendly control measures against this fungus. Methodology and Principal Findings: As the common bean is not amenable to reverse genetics to explore functionality and its genome is not fully curated, we used putative Arabidopsis orthologs of bean expressed sequence tag (EST) to perform bioinformatic analysis and experimental validation of gene expression to identify common bean genes regulated during the incompatible interaction with C. lindemuthianum. Similar to model pathosystems, Gene Ontology (GO) analysis indicated that hormone biosynthesis and signaling in common beans seem to be modulated by fungus infection. For instance, cytokinin and ethylene responses were up-regulated and jasmonic acid, gibberellin, and abscisic acid responses were down-regulated, indicating that these hormones may play a central role in this pathosystem. Importantly, we have identified putative bean gene orthologs of Arabidopsis genes involved in the plant immune system. Based on experimental validation of gene expression, we propose that hypersensitive reaction as part of effector-triggered immunity may operate, at least in part, by down-regulating genes, such as FLS2-like and MKK5-like, putative orthologs of the Arabidopsis genes involved in pathogen perception and downstream signaling. Conclusions/Significance: We have identified specific bean genes and uncovered metabolic processes and pathways that may be involved in the immune response against pathogens. Our transcriptome database is a rich resource for mining novel defense-related genes, which enabled us to develop a model of the molecular components of the bean innate immune system regulated upon pathogen attack.

Co-Stimulation of PAFR and CD36 Is Required for oxLDL-Induced Human Macrophages Activation

The oxidative process of LDL particles generates molecules which are structurally similar to platelet-activating factor (PAF), and some effects of oxidized LDL (oxLDL) have been shown to be dependent on PAF receptor (PAFR) activation. In a previous study, we showed that PAFR is required for upregulation of CD36 and oxLDL uptake. In the present study we analyzed the molecular mechanisms activated by oxLDL in human macrophages and the contribution of PAFR to this response. Human adherent monocytes/macrophages were stimulated with oxLDL. Uptake of oxLDL and CD36 expression were determined by flow cytometry; MAP kinases and Akt phosphorylation by Western blot; IL-8 and MCP-1 concentration by ELISA and mRNA expression by real-time PCR. To investigate the participation of the PI3K/Akt pathway, G alpha i-coupled protein or PAFR, macrophages were treated with LY294002, pertussis toxin or with the PAFR antagonists WEB2170 and CV3988, respectively before addition of oxLDL. It was found that the addition of oxLDL to human monocytes/macrophages activates the PI3K/Akt pathway which in turn activates the MAPK (p38 and JNK). Phosphorylation of Akt requires the engagement of PAFR and a G alpha i-coupled protein. The upregulation of CD36 protein and the uptake of oxLDL as well as the IL-8 production are dependent on PI3K/Akt pathway activation. The increased CD36 protein expression is dependent on PAFR and G alpha i-coupled protein. Transfection studies using HEK 293t cells showed that oxLDL uptake occurs with either PAFR or CD36, but IL-8 production requires the co-transfection of both PAFR and CD36. These findings show that PAFR has a pivotal role in macrophages response to oxLDL and suggest that pharmacological intervention at the level of PAFR activation might be beneficial in atherosclerosis.

Sepsis-Induced Acute Lung Injury (ALI) Is Milder in Diabetic Rats and Correlates with Impaired NFkB Activation

Acute lung injury (ALI) develops in response to a direct insult to the lung or secondarily to a systemic inflammatory response, such as sepsis. There is clinical evidence that the incidence and severity of ALI induced by direct insult are lower in diabetics. In the present study we investigated whether the same occurs in ALI secondarily to sepsis and the molecular mechanisms involved. Diabetes was induced in male Wistar rats by alloxan and sepsis by caecal ligation and puncture surgery (CLP). Six hours later, the lungs were examined for oedema and cell infiltration in bronchoalveolar lavage. Alveolar macrophages (AMs) were cultured in vitro for analysis of I kappa B and p65 subunit of NF kappa B phosphorylation and MyD88 and SOCS-1 mRNA. Diabetic rats were more susceptible to sepsis than non-diabetics. In non-diabetic rats, the lung presented oedema, leukocyte infiltration and increased COX2 expression. In diabetic rats these inflammatory events were significantly less intense. To understand why diabetic rats despite being more susceptible to sepsis develop milder ALI, we examined the NF kappa B activation in AMs of animals with sepsis. Whereas in non-diabetic rats the phosphorylation of I kappa B and p65 subunit occurred after 6 h of sepsis induction, this did not occur in diabetics. Moreover, in AMs from diabetic rats the expression of MyD88 mRNA was lower and that of SOCS-1 mRNA was increased compared with AMs from non-diabetic rats. These results show that ALI secondary to sepsis is milder in diabetic rats and this correlates with impaired activation of NF kappa B, increased SOCS-1 and decreased MyD88 mRNA.

Glutamine Supplementation Stimulates Protein-Synthetic and Inhibits Protein-Degradative Signaling Pathways in Skeletal Muscle of Diabetic Rats

In this study, we investigated the effect of glutamine (Gln) supplementation on the signaling pathways regulating protein synthesis and protein degradation in the skeletal muscle of rats with streptozotocin (STZ)-induced diabetes. The expression levels of key regulatory proteins in the synthetic pathways (Akt, mTOR, GSK3 and 4E-BP1) and the degradation pathways (MuRF-1 and MAFbx) were determined using real-time PCR and Western blotting in four groups of male Wistar rats; 1) control, non-supplemented with glutamine; 2) control, supplemented with glutamine; 3) diabetic, non-supplemented with glutamine; and 4) diabetic, supplemented with glutamine. Diabetes was induced by the intravenous injection of 65 mg/kg bw STZ in citrate buffer (pH 4.2); the non-diabetic controls received only citrate buffer. After 48 hours, diabetes was confirmed in the STZ-treated animals by the determination of blood glucose levels above 200 mg/dL. Starting on that day, a solution of 1 g/kg bw Gln in phosphate buffered saline (PBS) was administered daily via gavage for 15 days to groups 2 and 4. Groups 1 and 3 received only PBS for the same duration. The rats were euthanized, and the soleus muscles were removed and homogenized in extraction buffer for the subsequent measurement of protein and mRNA levels. The results demonstrated a significant decrease in the muscle Gln content in the diabetic rats, and this level increased toward the control value in the diabetic rats receiving Gln. In addition, the diabetic rats exhibited a reduced mRNA expression of regulatory proteins in the protein synthesis pathway and increased expression of those associated with protein degradation. A reduction in the skeletal muscle mass in the diabetic rats was observed and was alleviated partially with Gln supplementation. The data suggest that glutamine supplementation is potentially useful for slowing the progression of muscle atrophy in patients with diabetes.

Glucocorticoid and Estrogen Receptors Are Reduced in Mitochondria of Lung Epithelial Cells in Asthma

Mitochondrial glucocorticoid (mtGR) and estrogen (mtER) receptors participate in the coordination of the cell's energy requirement and in the mitochondrial oxidative phosphorylation enzyme (OXPHOS) biosynthesis, affecting reactive oxygen species (ROS) generation and induction of apoptosis. Although activation of mtGR and mtER is known to trigger anti-inflammatory signals, little information exists on the presence of these receptors in lung tissue and their role in respiratory physiology and disease. Using a mouse model of allergic airway inflammation disease and applying confocal microscopy, subcellular fractionation, and Western blot analysis we showed mitochondrial localization of GR alpha and ER beta in lung tissue. Allergic airway inflammation caused reduction in mtGR alpha, mtER beta, and OXPHOS enzyme biosynthesis in lung cells mitochondria and particularly in bronchial epithelial cells mitochondria, which was accompanied by decrease in lung mitochondrial mass and induction of apoptosis. Confirmation and validation of the reduction of the mitochondrial receptors in lung epithelial cells in human asthma was achieved by analyzing autopsies from fatal asthma cases. The presence of the mitochondrial GR alpha and ER beta in lung tissue cells and especially their reduction in bronchial epithelial cells during allergic airway inflammation suggests a crucial role of these receptors in the regulation of mitochondrial function in asthma, implicating their involvement in the pathophysiology of the disease.

Kinin B1 Receptor in Adipocytes Regulates Glucose Tolerance and Predisposition to Obesity

Background: Kinins participate in the pathophysiology of obesity and type 2 diabetes by mechanisms which are not fully understood. Kinin B-1 receptor knockout mice (B-1(-/-)) are leaner and exhibit improved insulin sensitivity. Methodology/Principal Findings: Here we show that kinin B-1 receptors in adipocytes play a role in controlling whole body insulin action and glucose homeostasis. Adipocytes isolated from mouse white adipose tissue (WAT) constitutively express kinin B-1 receptors. In these cells, treatment with the B-1 receptor agonist des-Arg(9)-bradykinin improved insulin signaling, GLUT4 translocation, and glucose uptake. Adipocytes from B-1(-/-) mice showed reduced GLUT4 expression and impaired glucose uptake at both basal and insulin-stimulated states. To investigate the consequences of these phenomena to whole body metabolism, we generated mice where the expression of the kinin B-1 receptor was limited to cells of the adipose tissue (aP2-B-1/B-1(-/-)). Similarly to B-1(-/-) mice, aP2-B-1/B-1(-/-) mice were leaner than wild type controls. However, exclusive expression of the kinin B1 receptor in adipose tissue completely rescued the improved systemic insulin sensitivity phenotype of B-1(-/-) mice. Adipose tissue gene expression analysis also revealed that genes involved in insulin signaling were significantly affected by the presence of the kinin B-1 receptor in adipose tissue. In agreement, GLUT4 expression and glucose uptake were increased in fat tissue of aP2-B-1/B-1(-/-) when compared to B-1(-/-) mice. When subjected to high fat diet, aP2-B-1/B-1(-/-) mice gained more weight than B-1(-/-) littermates, becoming as obese as the wild types. Conclusions/Significance: Thus, kinin B-1 receptor participates in the modulation of insulin action in adipocytes, contributing to systemic insulin sensitivity and predisposition to obesity.

A Recombinant Protein Based on Trypanosoma cruzi P21 Enhances Phagocytosis

Background: P21 is a secreted protein expressed in all developmental stages of Trypanosoma cruzi. The aim of this study was to determine the effect of the recombinant protein based on P21 (P21-His(6)) on inflammatory macrophages during phagocytosis. Findings: Our results showed that P21-His(6) acts as a phagocytosis inducer by binding to CXCR4 chemokine receptor and activating actin polymerization in a way dependent on the PI3-kinase signaling pathway. Conclusions: Thus, our results shed light on the notion that native P21 is a component related to T. cruzi evasion from the immune response and that CXCR4 may be involved in phagocytosis. P21-His(6) represents an important experimental control tool to study phagocytosis signaling pathways of different intracellular parasites and particles.

On the Evolution of Hexose Transporters in Kinetoplastid Potozoans

Glucose, an almost universally used energy and carbon source, is processed through several well-known metabolic pathways, primarily glycolysis. Glucose uptake is considered to be the first step in glycolysis. In kinetoplastids, a protozoan group that includes relevant human pathogens, the importance of glucose uptake in different phases of the life cycles is well established, and hexose transporters have been proposed as targets for therapeutic drugs. However, little is known about the evolutionary history of these hexose transporters. Hexose transporters contain an intracellular N- and C-termini, and 12 transmembrane spans connected by alternate intracellular and extracellular loops. In the present work we tested the hypothesis that the evolutionary rate of the transmembrane span is different from that of the whole sequence and that it is possible to define evolutionary units inside the sequence. The phylogeny of whole molecules was compared to that of their transmembrane spans and the loops connecting the transmembrane spans. We show that the evolutionary units in these proteins primarily consist of clustered rather than individual transmembrane spans. These analyses demonstrate that there are evolutionary constraints on the organization of these proteins; more specifically, the order of the transmembrane spans along the protein is highly conserved. Finally, we defined a signature sequence for the identification of kinetoplastid hexose transporters.

Cytokine Balance in Human Malaria: Does Plasmodium vivax Elicit More Inflammatory Responses than Plasmodium falciparum?

Background: The mechanisms by which humans regulate pro-and anti-inflammatory responses on exposure to different malaria parasites remains unclear. Although Plasmodium vivax usually causes a relatively benign disease, this parasite has been suggested to elicit more host inflammation per parasitized red blood cell than P. falciparum. Methodology/Principal Findings: We measured plasma concentrations of seven cytokines and two soluble tumor necrosis factor (TNF)-alpha receptors, and evaluated clinical and laboratory outcomes, in Brazilians with acute uncomplicated infections with P. vivax (n = 85), P. falciparum (n = 30), or both species (n = 12), and in 45 asymptomatic carriers of low-density P. vivax infection. Symptomatic vivax malaria patients, compared to those infected with P. falciparum or both species, had more intense paroxysms, but they had no clear association with a pro-inflammatory imbalance. To the contrary, these patients had higher levels of the regulatory cytokine interleukin (IL)-10, which correlated positively with parasite density, and elevated IL-10/TNF-alpha, IL-10/interferon (IFN)-gamma, IL-10/IL-6 and sTNFRII/TNF-alpha ratios, compared to falciparum or mixed-species malaria patient groups. Vivax malaria patients had the highest levels of circulating soluble TNF-alpha receptor sTNFRII. Levels of regulatory cytokines returned to normal values 28 days after P. vivax clearance following chemotherapy. Finally, asymptomatic carriers of low P. vivax parasitemias had substantially lower levels of both inflammatory and regulatory cytokines than did patients with clinical malaria due to either species. Conclusions: Controlling fast-multiplying P. falciparum blood stages requires a strong inflammatory response to prevent fulminant infections, while reducing inflammation-related tissue damage with early regulatory cytokine responses may be a more cost-effective strategy in infections with the less virulent P. vivax parasite. The early induction of regulatory cytokines may be a critical mechanism protecting vivax malaria patients from severe clinical complications.

A Genetic and Pathologic Study of a DENV2 Clinical Isolate Capable of Inducing Encephalitis and Hematological Disturbances in Immunocompetent Mice

Dengue virus (DENV) is the causative agent of dengue fever (DF), a mosquito-borne illness endemic to tropical and subtropical regions. There is currently no effective drug or vaccine formulation for the prevention of DF and its more severe forms, i.e., dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). There are two generally available experimental models for the study of DENV pathogenicity as well as the evaluation of potential vaccine candidates. The first model consists of non-human primates, which do not develop symptoms but rather a transient viremia. Second, mouse-adapted virus strains or immunocompromised mouse lineages are utilized, which display some of the pathological features of the infection observed in humans but may not be relevant to the results with regard to the wild-type original virus strains or mouse lineages. In this study, we describe a genetic and pathological study of a DENV2 clinical isolate, named JHA1, which is naturally capable of infecting and killing Balb/c mice and reproduces some of the symptoms observed in DENV-infected subjects. Sequence analyses demonstrated that the JHA1 isolate belongs to the American genotype group and carries genetic markers previously associated with neurovirulence in mouse-adapted virus strains. The JHA1 strain was lethal to immunocompetent mice following intracranial (i.c.) inoculation with a LD50 of approximately 50 PFU. Mice infected with the JHA1 strain lost weight and exhibited general tissue damage and hematological disturbances, with similarity to those symptoms observed in infected humans. In addition, it was demonstrated that the JHA1 strain shares immunological determinants with the DENV2 NGC reference strain, as evaluated by cross-reactivity of anti-envelope glycoprotein (domain III) antibodies. The present results indicate that the JHA1 isolate may be a useful tool in the study of DENV pathogenicity and will help in the evaluation of anti-DENV vaccine formulations as well as potential therapeutic approaches.

Echinococcus granulosus Antigen B Structure: Subunit Composition and Oligomeric States

Background: Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states. Methodology/Principal Findings: The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3 >rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability. Conclusions/Significance: For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the current knowledge on AgB expression and structure, highlighting issues that may help to understand the parasite adaptive response during chronic infection.

MRKAd5 HIV-1 Gag/Pol/Nef Vaccine-Induced T-Cell Responses Inadequately Predict Distance of Breakthrough HIV-1 Sequences to the Vaccine or Viral Load

Background: The sieve analysis for the Step trial found evidence that breakthrough HIV-1 sequences for MRKAd5/HIV-1 Gag/Pol/Nef vaccine recipients were more divergent from the vaccine insert than placebo sequences in regions with predicted epitopes. We linked the viral sequence data with immune response and acute viral load data to explore mechanisms for and consequences of the observed sieve effect. Methods: Ninety-one male participants (37 placebo and 54 vaccine recipients) were included; viral sequences were obtained at the time of HIV-1 diagnosis. T-cell responses were measured 4 weeks post-second vaccination and at the first or second week post-diagnosis. Acute viral load was obtained at RNA-positive and antibody-negative visits. Findings: Vaccine recipients had a greater magnitude of post-infection CD8+ T cell response than placebo recipients (median 1.68% vs 1.18%; p = 0.04) and greater breadth of post-infection response (median 4.5 vs 2; p = 0.06). Viral sequences for vaccine recipients were marginally more divergent from the insert than placebo sequences in regions of Nef targeted by pre-infection immune responses (p = 0.04; Pol p = 0.13; Gag p = 0.89). Magnitude and breadth of pre-infection responses did not correlate with distance of the viral sequence to the insert (p. 0.50). Acute log viral load trended lower in vaccine versus placebo recipients (estimated mean 4.7 vs 5.1) but the difference was not significant (p = 0.27). Neither was acute viral load associated with distance of the viral sequence to the insert (p>0.30). Interpretation: Despite evidence of anamnestic responses, the sieve effect was not well explained by available measures of T-cell immunogenicity. Sequence divergence from the vaccine was not significantly associated with acute viral load. While point estimates suggested weak vaccine suppression of viral load, the result was not significant and more viral load data would be needed to detect suppression.

Fighting Misconceptions to Improve Compliance with Influenza Vaccination among Health Care Workers: An Educational Project

The compliance with influenza vaccination is poor among health care workers (HCWs) due to misconceptions about safety and effectiveness of influenza vaccine. We proposed an educational prospective study to demonstrate to HCWs that influenza vaccine is safe and that other respiratory viruses (RV) are the cause of respiratory symptoms in the months following influenza vaccination. 398 HCWs were surveyed for adverse events (AE) occurring within 48 h of vaccination. AE were reported by 30% of the HCWs. No severe AE was observed. A subset of 337 HCWs was followed up during four months, twice a week, for the detection of respiratory symptoms. RV was diagnosed by direct immunofluorescent assay (DFA) and real time PCR in symptomatic HCWs. Influenza A was detected in five episodes of respiratory symptoms (5.3%) and other RV in 26 (27.9%) episodes. The incidence density of influenza and other RV was 4.3 and 10.8 episodes per 100 HCW-month, respectively. The educational nature of the present study may persuade HCWs to develop a more positive attitude to influenza vaccination.