Population-Based Multicentric Survey of Hepatitis B Infection and Risk Factor Differences among Three Regions in Brazil

This multicentric population-based study in Brazil is the first national effort to estimate the prevalence of hepatitis B (HBV) and risk factors in the capital cities of the Northeast. Central-West, and Federal Districts (2004-2005). Random multistage cluster sampling was used to select persons 13-69 years of age. Markers for HBV were tested by enzyme-linked immunosorbent assay. The HBV genotypes were determined by sequencing hepatitis B surface antigen (HBsAg). Multivariate analyses and simple catalytic model were performed. Overall. 7,881 persons were inculded < 70% were not vaccinated. Positivity for HBsAg was less than 1% among non-vaccinated persons and genotypes A, D, and F co-circulated. The incidence of infection increased with age with similar force of infection in all regions. Males and persons having initiated sexual activity were associated with HBV infection in the two settings: healthcare jobs and prior hospitalization were risk factors in the Federal District. Our survey classified these regions as areas with HBV endemicity and highlighted the risk factors differences among the settings.

Validation of an ELISA method for the serological diagnosis of canine brucellosis due to Brucella canis

In the present study, the validation of an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of canine brucellosis is described. Two different antigenic extracts, obtained by heat or ultrasonic homogenization of microbial antigens from a wild isolate of Brucella canis bacteria, were compared by ELISA and Western blot (WB). A total of 145 canine sera were used to define sensitivity, specificity and accuracy of the ELISA as follows: (1) sera from 34 animals with natural B. canis infection, confirmed by blood culture and PCR, as well as 51 sera samples from healthy dogs with negative results by the agar-gel immunodiffusion (ACID) test for canine brucellosis, were used as the control panel for B. cants infection; and (2) to scrutinize the possibility of cross reactions with other common dog infections in the same geographical area in Brazil, 60 sera samples from dogs harboring known infections by Leptospira sp., Ehrlichia canis, canine distemper virus (CDV), Neospora caninum, Babesia canis and Leishmania chagasi (10 in each group) were included in the study. The ELISA using heat soluble bacterial extract (HE-antigen) as antigen showed the best values of sensitivity (91.18%), specificity (100%) and accuracy (96.47%). In the WB analyses, the HE-antigen showed no cross-reactivity with sera from dogs with different infections, while the B. canis sonicate had various protein bands identified by those sera. The performance of the ELISA standardized with the heat soluble B. canis antigen indicates that this assay can be used as a reliable and practical method to confirm infection by this microorganism, as well as a tool for seroepidemiological studies. (C) 2010 Elsevier Ltd. All rights reserved.

Dipeptidyl peptidase IV in the hypothalamus and hippocampus of monosodium glutamate obese and food-deprived rats

Proline-specific dipeptidyl peptidases are emerging as a protease family with important roles in the regulation of signaling by peptide hormones related to energy balance. The treatment of neonatal rats with monosodium glutamate (MSG) is known to produce a selective damage on the arcuate nucleus with development of obesity. This study investigates the relationship among dipeptidyl peptidase IV (DPPIV) hydrolyzing activity, CD26 protein, fasting, and MSG model of obesity in 2 areas of the central nervous system. Dipeptidyl peptidase IV and CD26 were, respectively, evaluated by fluorometry, and enzyme-linked immunosorbent assay and reverse transcriptase polymerase chain reaction in soluble (SF) and membrane-bound (MF) fractions from the hypothalamus and hippocampus of MSG-treated and normal rats, submitted or not to food deprivation (FD). Dipeptidyl peptidase IV in both areas was distinguished kinetically as insensitive (DI) and sensitive (DS) to diprotin A. Compared with the controls, MSG and/or FD decreased the activity of DPPIV-DI in the SF and MF from the hypothalamus, as well as the activity of DPPIV-DS in the SF from the hypothalamus and in the MF from the hippocampus. Monosodium glutamate and/or FD increased the activity of DPPIV-DI in the MF from the hippocampus. The monoclonal protein expression of membrane CD26 by enzyme-linked immunosorbent assay decreased in the hypothalamus and increased in the hippocampus of MSG and/or FD relative to the controls. The existence of DPPIV-like activity with different sensitivities to diprotin A and the identity of insensitive with CD26 were demonstrated for the first time in the central nervous system. Data also demonstrated the involvement of DPPIV-DI/CD26 hydrolyzing activity in the energy balance probably through the regulation of neuropeptide Y and beta-endorphin levels in the hypothalamus and hippocampus. (C) 2011 Elsevier Inc. All rights reserved.

INSULIN REGULATES CYTOKINES AND INTERCELLULAR ADHESION MOLECULE-1 GENE EXPRESSION THROUGH NUCLEAR FACTOR-kappa B ACTIVATION IN LPS-INDUCED ACUTE LUNG INJURY IN RATS

Diabetic patients have increased susceptibility to infection, which may be related to impaired inflammatory response observed in experimental models of diabetes, and restored by insulin treatment. The goal of this study was to investigate whether insulin regulates transcription of cytokines and intercellular adhesion molecule 1 (ICAM-1) via nuclear factor-kappa B (NF-kappa B) signaling pathway in Escherichia coli LIPS-induced lung inflammation. Diabetic male Wistar rats (alloxan, 42 mg/kg, iv., 10 days) and controls were instilled intratracheally with saline containing LPS (750 mu g/0.4 mL) or saline only. Some diabetic rats were given neutral protamine Hagedorn insulin (4 IU, s.c.) 2 h before LIPS. Analyses performed 6 h after LPS included: (a) lung and mesenteric lymph node IL-1 beta, TNF-alpha, IL-10, and ICAM-1 messenger RNA (mRNA) were quantified by real-time reverse transcriptase-polymerase chain reaction; (b) number of neutrophils in the bronchoalveolar lavage (BAL) fluid, and concentrations of IL-1 beta, TNF-alpha, and IL-10 in the BAL were determined by the enzyme-linked immunosorbent assay; and (c) activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were quantified by Western blot analysis. Relative to controls, diabetic rats exhibited a reduction in lung and mesenteric lymph node IL-1 beta (40%), TNF-alpha (similar to 30%), and IL-10 (similar to 40%) mRNA levels and reduced concentrations of IL-1 beta (52%), TNF-alpha (62%), IL-10 (43%), and neutrophil counts (72%) in the BAL. Activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were almost suppressed in diabetic rats. Treatment of diabetic rats with insulin completely restored mRNA and protein levels of these cytokines and potentiated lung ICAM-1 mRNA levels (30%) and number of neutrophils (72%) in the BAL. Activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were partially restored by insulin treatment. In conclusion, data presented suggest that insulin regulates transcription of proinflammatory (IL-1 beta, TNF-alpha) and anti-inflammatory (IL-10) cytokines, and expression of ICAM-1 via the NF-kappa B signaling pathway.

Complement 4 phenotypes and genotypes in Brazilian patients with classical 21-hydroxylase deficiency

The aim of this work was to analyse C4 genotypes, C4 protein levels, phenotypes and genotypes in patients with the classical form of 21-hydroxylase deficiency. Fifty-four patients from 46 families (36 female, 18 male; mean age 10.8 years) with different clinical manifestations (31 salt-wasting; 23 simple-virilizing) were studied. Taq I Southern blotting was used to perform molecular analysis of the C4/CYP21 gene cluster and the genotypes were defined according to gene organization within RCCX modules. Serum C4 isotypes were assayed by enzyme-linked immunosorbent assay. The results revealed 12 different haplotypes of the C4/CYP21 gene cluster. Total functional activity of the classical pathway (CH50) was reduced in individuals carrying different genotypes because of low C4 concentrations (43% of all patients) to complete or partial C4 allotype deficiency. Thirteen of 54 patients presented recurrent infections affecting the respiratory and/or the urinary tracts, none of them with severe infections. Low C4A or C4B correlated well with RCCX monomodular gene organization, but no association between C4 haplotypes and recurrent infections or autoimmunity was observed. Considering this redundant gene cluster, C4 seems to be a well-protected gene segment along the evolutionary process.

Immune response to cytolethal distending toxin of Aggregatibacter actinomycetemcomitans in periodontitis patients

Background and Objective: Cytolethal distending toxin (CDT) is a genotoxin produced by Aggregatibacter actinomycetemcomitans. In spite of its association with pathogenesis, little is known about the humoral immune response against the CDT. This study aimed to test whether subgingival colonization and humoral response to A. actinomycetemcomitans would lead to a response against CDT. Material and Methods: Sera from periodontally healthy, localized and generalized aggressive periodontitis and chronic periodontitis subjects (n = 80) were assessed for immunoglobulin G titers to A. actinomycetemcomitans serotypes a/b/c and to each CDT subunit (CdtA, CdtB and CdtC) by ELISA. A. actinomycetemcomitans subgingival levels and neutralization of CDT activity were also analyzed. Results: Sera from 75.0% localized and 81.8% generalized aggressive periodontitis patients reacted to A. actinomycetemcomitans. A response to serotype b was detected in localized (66.7%) and generalized aggressive periodontitis (54.5%). Reactivity to A. actinomycetemcomitans correlated with subgingival colonization (R = 0.75, p < 0.05). There was no correlation between A. actinomycetemcomitans colonization or response to serotypes and the immunoglobulin G response to CDT subunits. Titers of immunoglobulin G to CdtA and CdtB did not differ among groups; however, sera of all generalized aggressive periodontitis patients reacted to CdtC. Neutralization of CDT was not correlated with levels of antibodies to CDT subunits. Conclusion: Response to CdtA and CdtB did not correlate with the periodontal status of the subject in the context of an A. actinomycetemcomitans infection. However, a response to CdtC was found in sera of generalized but not of localized aggressive periodontitis subjects. Differences in response to CdtC between generalized and localized aggressive periodontitis subjects indicate that CDT could be expressed differently by the infecting strains. Alternatively, the antibody response to CdtC could require the colonization of multiple sites.

Inhibitory Signals Mediated by Programmed Death-1 Are Involved With T-Cell Function in Chronic Periodontitis

Background: Inhibitory signals mediated via molecules such as programmed death-1 (PD-1) play a critical role in downmodulating immune responses and maintaining peripheral tolerance. We investigated the involvement of cytokines and PD-1 engagement in mediating the T-cell unresponsiveness to bacterial and ubiquitous antigens in periodontal diseases. Methods: Gingival and peripheral blood samples from healthy individuals and patients with chronic periodontitis were collected and used for the subsequent assays. Leukocytes in the lesion site and blood were evaluated using flow cytometry. The production of interferon-gamma, interleukin-10, and transforming growth factor-P proteins was evaluated by enzyme-linked immunosorbent assay (ELISA), and the presence of PD-1+cells in the inflamed gingiva was confirmed by immunofluorescence confocal microscopy for CD4 and PD-1 colocalization. Results: T cells from patients with chronic periodontitis proliferated poorly in response to Aggregatibacter actinomycetem comitans (previously Actinobacillus actinomycetemcomitans) antigen. T-cell unresponsiveness was not associated with imbalanced cytokine production. However, T cells from patients with chronic periodontitis expressed significantly higher levels of PD-1 either upon isolation or after culture with antigens. Moreover, PD-1 blocking did not result in significant T-cell proliferation in cells cultured with phytohemagglutinin or bacterial antigens. The blockade of PD-1 resulted in the increased production of IFN-gamma. In addition, CD4+ and CD8+ T cells expressing PD-1 accumulated in lesions with chronic periodontitis. Conclusion: These data show that PD-1 engagement could be involved in the modulation of IFN-gamma production by T cells in patients with chronic periodontitis. J Periodontol 2009,80:1833-1844.

Naturally Acquired Antibodies to Plasmodium vivax Duffy Binding Protein (DBP) in Rural Brazilian Amazon

Duffy binding protein (DBP), a leading malaria vaccine candidate, plays a critical role ill Plasmodium vivax erythrocyte invasion. Sixty-eight of 366 (18.6%) subjects had IgG anti-DBP antibodies by enzyme-linked immunosorbent assay (ELISA) in a community-based cross-sectional survey ill the Brazilian Amazon Basin. Despite Continuous exposure to low-level malaria transmission, the overall seroprevalence decreased to 9.0% when the Population was reexamined 12 months later. Antibodies from 16 of 50 (360%) Subjects who were ELISA-positive at the baseline were able to inhibit erythrocyte binding to at least one of two DBP variants tested. Most (13 of 16) of these subjects still had inhibitory antibodies when reevaluated 12 months later. Cumulative exposure to malaria was the strongest predictor of DBP seropositivity identified by Multiple logistic regression models in this population. The poor antibody recognition of DBP elicited by natural exposure to P. vivax in Amazonian populations represents a challenge to be addressed by vaccine development strategies.

Early Increase in Autoantibodies Against Human Oxidized Low-Density Lipoprotein in Hypertensive Patients After Blood Pressure Control

BACKGROUND Oxidized lipoproteins and antioxidized low-density lipoprotein (anti-oxLDL) antibodies (Abs) have been detected in plasma in response to blood pressure (BP) elevation, suggesting the participation of the adaptive immune system. Therefore, treatment of hypertension may act on the immune response by decreasing oxidation stimuli. However, this issue has not been addressed. Thus, we have here analyzed anti-oxLDL Abs in untreated (naive) hypertensive patients shortly after initiation of anti hypertensive therapeutic regimens. METHODS Titers of anti-oxLDL Abs were measured in subjects with recently diagnosed hypertension on stage 1 (n = 94), in primary prevention of coronary disease, with no other risk factors, and naive of anti hypertensive medication at entry. Subjects were randomly assigned to receive perindopril, hydrochlorothiazide (HCTZ), or indapamide (INDA) for 12 weeks, with additional perindopril if necessary to achieve BP control. Abs against copper-oxidized LDL were measured by enzyme-linked immunosorbent assay. RESULTS Twelve-week antihypertensive treatment reduced both office-based and 24-h ambulatory BP measurements (P < 0.0005). The decrease in BP was accompanied by reduction in thiobarbituric acid-reactive substances (TBARS) (P < 0.05), increase in anti-oxLDL Ab titers (P < 0.005), and improvement in flow-mediated dilation (FMD) (P < 0.0005), independently of treatment. Although BP was reduced, we observed favorable changes in anti-oxLDL titers and FMD. CONCLUSIONS We observed that anti-oxLDL Ab titers increase after antihypertensive therapy in primary prevention when achieving BP targets. Our results are in agreement with the concept that propensity to oxidation is increased by essential hypertension and anti-oxLDL Abs may be protective and potential biomarkers for the follow-up of hypertension treatment.

Naturally-acquired humoral immune responses against the N- and C-termini of the Plasmodium vivax MSP1 protein in endemic regions of Brazil and Papua New Guinea using a multiplex assay

Background: Progress towards the development of a malaria vaccine against Plasmodium vivax, the most widely distributed human malaria parasite, will require a better understanding of the immune responses that confer clinical protection to patients in regions where malaria is endemic. Methods: Glutathione S-transferase (GST) and GST-fusion proteins representing the N-terminus of the merozoite surface protein 1 of P. vivax, PvMSP1-N, and the C-terminus, PvMSP1-C, were covalently coupled to BioPlex carboxylated beads. Recombinant proteins and coupled beads were used, respectively, in ELISA and Bioplex assays using immune sera of P. vivax patients from Brazil and PNG to determine IgG and subclass responses. Concordances between the two methods in the seropositivity responses were evaluated using the Kappa statistic and the Spearman's rank correlation. Results: The results using this methodology were compared with the classical microtitre enzyme-linked immnosorbent assay ( ELISA), showing that the assay was sensitive, reproducible and had good concordance with ELISA; yet, further research into different statistical analyses seems desirable before claiming conclusive results exclusively based on multiplex assays. As expected, results demonstrated that PvMSP1 was immunogenic in natural infections of patients from different endemic regions of Brazil and Papua New Guinea ( PNG), and that age correlated only with antibodies against the C-terminus part of the molecule. Furthermore, the IgG subclass profiles were different in these endemic regions having IgG3 predominantly recognizing PvMSP1 in Brazil and IgG1 predominantly recognizing PvMSP1 in PNG. Conclusions: This study validates the use of the multiplex assay to measure naturally-acquired IgG antibodies against the merozoite surface protein 1 of P. vivax.

Comparative Study of the Standard Fluorescent Antibody to Membrane Antigen (FAMA) Assay and a Flow Cytometry-Adapted FAMA Assay To Assess Immunity to Varicella-Zoster Virus

A flow cytometry-adapted fluorescent antibody to membrane antigen (FAMA) assay to detect IgG antibodies against varicella-zoster virus (VZV) was developed and tested in 62 serum samples, showing 90.32% accuracy obtained from a receiver operating characteristic (ROC) curve with a 0.9125 (95% confidence interval [CI], 0.829 to 1.00) area below the curve compared to the result with standard FAMA.

Dengue and dengue hemorrhagic fever among adults: Clinical outcomes related to viremia, serotypes, and antibody response

Background. Clinical manifestations of dengue vary in different areas of endemicity and between specific age groups, whereas predictors of outcome have remained controversial. In Brazil, the disease burden predominantly affects adults, with an increasing trend toward progression to dengue hemorrhagic fever (DHF) noted. Methods. A cohort of adults with confirmed cases of dengue was recruited in central Brazil in 2005. Patients were classified according to the severity of their disease. Associations of antibody responses, viremia levels (as determined by real-time polymerase chain reaction [PCR]), and serotypes (as determined by multiplex PCR) with disease severity were evaluated. Results. Of the 185 symptomatic patients > 14 years of age who had a confirmed case of dengue, 26.5% and 23.2% were classified as having intermediate dengue fever (DF)/ DHF (defined as internal hemorrhage, plasma leakage, manifested signs of shock, and/ or thrombocytopenia [platelet count, <= 50,000 platelets/mm(3)]) and DHF, respectively. The onset of intermediate DF/ DHF and DHF occurred at a late stage of disease, around the period of defervescence. Patients with DHF had abnormal liver enzyme levels, with a > 3-fold increase in aspartate aminotransferase level, compared with the range of values considered to be normal. Overall, 65% of patients presented with secondary infections with dengue virus, with such infection occurring in similar proportions of patients in each of the 3 disease category groups. Dengue virus serotype 3 (DV3) was the predominant serotype, and viremia was detected during and after defervescence among patients with DHF or intermediate DF/ DHF. Conclusions. Viremia was detected after defervescence in adult patients classified as having DHF or intermediate DF/ DHF. Secondary infection was not a predictor of severe clinical manifestation in adults with infected with the DV3 serotype.

Administration of cannabidiol and imipramine induces antidepressant-like effects in the forced swimming test and increases brain-derived neurotrophic factor levels in the rat amygdala

Objective: Cannabidiol is a chemical constituent from Cannabis sativa and it has multiple mechanisms of action, including antidepressant effects. The main objective of the present study was to evaluate behavioural and molecular effects induced by administration of cannabidiol and imipramine in rats. Methods: In the present study, rats were acutely or chronically treated for 14 days once a day with saline, cannabidiol (15, 30 and 60 mg/kg) or imipramine (30 mg/kg) and the animals behaviour was assessed in forced swimming and open-field tests. Afterwards, the prefrontal cortex, hippocampus and amygdala brain-derived neurotrophic factor (BDNF) levels were assessed by enzyme-linked immunosorbent sandwich assay. Results: We observed that both acute and chronic treatments with imipramine at the dose of 30 mg/kg and cannabidiol at the dose of 30 mg/kg reduced immobility time and increased swimming time; climbing time was increased only with imipramine at the dose of 30 mg/kg, without affecting locomotor activity. In addition, chronic treatment with cannabidiol at the dose of 15 mg/kg and imipramine at the dose of 30 mg/kg increased BDNF levels in the rat amygdala. Conclusion: In conclusion, our results indicate that cannabidiol has an antidepressant-like profile and could be a new pharmacological target for the treatment of major depression.

Detection of Hepatitis B virus subgenotype A1 in a Quilombo community from Maranhao, Brazil

Background: The Brazilian population is mainly descendant from European colonizers, Africans and Native Americans. Some Afro-descendants lived in small isolated communities since the slavery period. The epidemiological status of HBV infection in Quilombos communities from northeast of Brazil remains unknown. The aim of this study was to characterize the HBV genotypes circulating inside a Quilombo isolated community from Maranhao State, Brazil. Methods: Seventy-two samples from Frechal Quilombo community at Maranhao were collected. All serum samples were screened by enzyme-linked immunosorbent assays for the presence of hepatitis B surface antigen ( HBsAg). HBsAg positive samples were submitted to DNA extraction and a fragment of 1306 bp partially comprising HBsAg and polymerase coding regions (S/POL) was amplified by nested PCR and its nucleotide sequence was determined. Viral isolates were genotyped by phylogenetic analysis using reference sequences from each genotype obtained from GenBank (n = 320). Sequences were aligned using Muscle software and edited in the SE-AL software. Bayesian phylogenetic analyses were conducted using Markov Chain Monte Carlo (MCMC) method to obtain the MCC tree using BEAST v.1.5.3. Results: Of the 72 individuals, 9 (12.5%) were HBsAg-positive and 4 of them were successfully sequenced for the 1306 bp fragment. All these samples were genotype A1 and grouped together with other sequences reported from Brazil. Conclusions: The present study represents the first report on the HBV genotypes characterization of this community in the Maranhao state in Brazil where a high HBsAg frequency was found. In this study, we reported a high frequency of HBV infection and the exclusive presence of subgenotype A1 in an Afro-descendent community in the Maranhao State, Brazil.

TESA-blot for the diagnosis of Chagas disease in dogs from co-endemic regions for Trypanosoma cruzi, Trypanosoma evansi and Leishmania chagasi

We standardized serodiagnosis of dogs infected with Trypanosoma cruzi using TESA (trypomastigote excreted-secreted antigen)-blot developed for human Chagas disease. TESA-blot showed 100% sensitivity and specificity. In contrast, ELISA using TESA (TESA-ELISA) or epimastigotes (epi-ELISA) as antigen yielded 100% sensitivity but specificity of 94.1% and 49.4%, respectively. When used in field studies in an endemic region for Chagas disease, visceral leishmaniasis and Trypanosoma evansi (Mato Grosso do Sul state, Central Brazil), positivities were 9.3% for TESA-blot, 10.7% for TESA-ELISA and 32% for epi-ELISA. Dogs from a non-endemic region for these infections (Rondonia state, western Amazonia) where T cruzi is enzootic showed positivity of 4.5% for TESA-blot and epi-ELISA and 6.8% for TESA-ELISA. Sera from urban dogs from Santos, Sao Paulo, where these diseases are absent, yielded negative results. TESA-blot was the only method that distinguished dogs infected with T cruzi from those infected with Leishmania chagasi and/or Trypanosoma evansi. (C) 2009 Published by Elsevier B.V.