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Melanoma is a type of skin cancer with increasing incidence worldwide and high lethality. Conventional forms of treatment are not effective in advanced cancer stages. Hence, immunotherapeutic approaches have been tested to modulate immune response against tumor cells. Some vaccine models using tumor-associated antigens (TAAs) such as glycoprotein 100 (gp100) have been studied, but their expected effectiveness has not been shown until now. Antigen immunogenicity is a crucial point to improve the immune response, and therefore mutations are inserted in peptide sequences. It is possible to understand the interactions which occur between peptides and immune system molecules through computer simulation, and this is essential in order to guide efficient vaccine models. In this work, we have calculated the interaction binding energies of crystallographic data based on modified gp100 peptides and HLA-A*0201 using density functional theory (DFT) and the molecular fractionation with conjugated caps (MFCC) approach. Our results show the most relevant residue-residue interactions, the impact of three mutations in their binding sites, and the main HLA-A*0201 amino acids for peptide–HLA binding.

Aim: To analyze the effects of exposure to a high-fat diet during the perinatal period and after weaning on white adipose tissue accumulation and gene expression of TNF- α and NF- κB.Method: Wistar female rats were fed with high-fat (H) or control (C) diet during pregnancy and lactation. The offspring were allocated into four groups: Control Control (CC), offspring of mothers GC, fed a control diet after weaning; Control High-fat (CH), offspring of mothers GC, fed a hight-fat diet after weaning; High-fat Control (HC), offspring of mothers GH, fed with control diet after weaning; and High-fat High-fat (HH), offspring of mothers GH, fed a H diet after weaning.Results: HH and HC groups showed increased body weight compared to CC group and increases in caloric intake, larger amount of white adipose tissue and adipocyte size compared to CC and CH groups. The HH and CH groups showed higher NF-kB expression in white adipose tissue compared to the CC and HC groups, and the HH group also showed higher TNF- α expression. In the hypothalamus, the HH and HC groups exhibited higher TNF- α expression compared to the CC and CH groups.Conclusion: Perinatal and post-weaning exposure to the high-fat diet increases the amount of white adipose tissue, adipocyte size, and expression of the inflammatory genes TNF-α and NF-kB.

Multi-walled carbon nanotubes functionalized with naringenin have been developed as new drug carriers to improve the performance of lung cancer treatment. The nanocarrier was characterized by Transmission Electron Microscopy (TEM), Fourier-Transform Infrared Spectroscopy (FTIR), X-ray photoelectron spectroscopy, Raman Spectroscopy, and Differential Scanning Calorimetry (DSC). Drug release rates were determined in vitro by the dialysis method. The cytotoxic profile was evaluated using the MTT assay, against a human skin cell line (hFB) as a model for normal cells, and against an adenocarcinomic human alveolar basal epithelial (A569) cell line as a lung cancer in vitro model. The results demonstrated that the functionalization of carbon nanotubes with naringenin occurred by non-covalent interactions. The release profiles demonstrated a pH-responsive behavior, showing a prolonged release in the tumor pH environment. The naringenin-functionalized carbon nanotubes showed lower cytotoxicity on non-malignant cells (hFB) than free naringenin, with an improved anticancer effect on malignant lung cells (A549) as an in vitro model of lung cancer., This work was supported by the Banco do Nordeste (grant FUNDECI/2016.0015), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Apoio à Pesquisa e à Inovação Tecnológica do Estado de Sergipe (Fapitec) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Eliana B. Souto would like to acknowledge the contributions from the Portuguese Science and Technology Foundation (FCT/MCT) and from European Funds (PRODER/COMPETE) for the projects M-ERA-NET/0004/2015-PAIRED and UIDB/04469/2020 (strategic fund), co-financed by FEDER, under the Partnership Agreement PT2020., info:eu-repo/semantics/publishedVersion

Leishmaniasis corresponds to a group of neglected tropical diseases caused by flagellated protozoa belonging to the Leishmania genus. This widely spread illness is found in over 90 countries in the tropic and sub-tropic regions and also in southern Europe. As parasites exclusively infect the highly phagocytic cells (macrophages), this aspect can be exploited for targeted delivery making use of nanoparticles. Drug-loaded nanoparticles have been proposed to improve the bioavailability of classical drugs commonly in use as standard leishmania therapy, to overcome parasitic resistance and side effects and improve the treatment efficacy. Infected macrophages are expected to recognize drug-loaded nanoparticles which undergo phagocytosis releasing the drug inside macrophages. This approach can further be exploited for the development of nanovaccines. This paper provides an overview of the current disease status worldwide, its classical pharmacological treatments and how these can be improved by the use of featured nanoparticles specific tailored for such a complex disease. Several types of nanoparticles have been proposed while others have already reached clinical trials. This article is protected by copyright., This research was funded by Banco do Nordeste (grant FUNDECI/2016.0015), Coordenação Aperfeiçoamento de Pessoal de Nivel Superior (CAPES) and Fundação de Ámparo à Pesquisa do Estado de Sergipe (FAPITEC) (PROCESSO: 88887.159533/2017–2100 extração, encapsulação e caracterização de bioativos para o interesse biotecnologico), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 301964/2019-0 Chamada 06/2019, and Chamada CNPq no. 01/2019) and the Portuguese Science and Technology Foundation (FCT) project UIDB/04469/2020 (strategic fund)., info:eu-repo/semantics/publishedVersion

IntroductionIn this systematic review, we analysed studies that assessed the brown adipose tissue (BAT) activity in the high-fat/cafeteria diet model of obesity in rats.Material and methodsScopus, PubMed, Embase, and ScienceDirect databases were searched from January 2017 to November 2017. Using specific combinations of medical subject heading (MeSH) descriptors, seven papers remained after the inclusion and exclusion criteria.ResultsMost papers showed an increase in BAT thermogenesis in rodents fed high-fat/cafeteria diet. Some studies did not mention the diet composition or housing temperature, and the most of them investigated the thermogenesis superficially, being limited to the analysis of the UCP 1 expression.ConclusionsDespite the consolidated use of high-fat/cafeteria diets as a model to induce obesity, the identification of the energy expenditure arm has been slow, especially the direct quantitative assessment of the contribution of BAT to the increase in metabolic rate in rats fed a cafeteria/high-fat diet.

This systematic review examined the effects of paternal exposure to a high-fat diet on the likelihood of offspring developing health consequences, including metabolic conditions. While the connection between a mother’s diet and offspring health has been well established, our understanding of whether offspring health is affected by a father’s diet remains limited. This systematic review was performed according to the Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) recommendations. The PubMed, Scopus, and Embase electronic databases were searched using combinations of the MESH terms: obesogenic diet, high-fat diet, cafeteria diet, paternal diet, parental diet, programming, paternal effects, and paternal programming. Sixteen studies were selected after assessing articles for eligibility criteria. The main outcomes concerning offspring health related to metabolic disorders. The offspring of fathers exposed to a high-fat diet displayed elevated gene expression and serum levels of leptin, decreased gene expression and serum levels of adiponectin, insulin resistance, glucose intolerance, hyperglycemia, hyperinsulinemia, changes in the transcriptome of pancreatic islet tissues, increased triglycerides, and increased expression of lipogenic genes. The available evidence suggests that paternal exposure to a high-fat diet may induce harmful effects on the health of offspring.

Phialophora verrucosa is a dematiaceous fungus that causes mainly chromoblastomycosis, but also disseminated infections such as phaeohyphomycosis and mycetoma. These diseases are extremely hard to treat and often refractory to current antifungal therapies. In this work, we have evaluated the effect of 1,10-phenanthroline-5,6-dione (phendione) and its metal-based complexes, [Ag (phendione)2]ClO4 and [Cu(phendione)3](ClO4)2.4H2O, against P. verrucosa, focusing on (i) conidial viability when combined with amphotericin B (AmB); (ii) biofilm formation and disarticulation events; (iii) in vitro interaction with human macrophages; and (iv) in vivo infection of Galleria mellonella larvae. The combination of AmB with each of the test compounds promoted the additive inhibition of P. verrucosa growth, as judged by the checkerboard assay. During the biofilm formation process over polystyrene surface, sub-minimum inhibitory concentrations (MIC) of phendione and its silver(I) and copper(II) complexes were able to reduce biomass and extracellular matrix production. Moreover, a mature biofilm treated with high concentrations of the test compounds diminished biofilm viability in a concentration-dependent manner. Pre-treatment of conidial cells with the test compounds did not alter the percentage of infected THP-1 macrophages; however, [Ag(phendione)2]ClO4 caused a significant reduction in the number of intracellular fungal cells compared to the untreated system. In addition, the killing process was significantly enhanced by post-treatment of infected macrophages with the test compounds. P. verrucosa induced a typically cell density-dependent effect on G. mellonella larvae death after 7 days of infection. Interestingly, exposure to the silver(I) complex protected the larvae from P. verrucosa infection. Collectively, the results corroborate the promising therapeutic potential of phendione-based drugs against fungal infections, including those caused by P. verrucosa.

Exposure to the hight-fat diet may alter the control of food intake promoting hyperphagia and obesity. The objective of this study was to investigate the effects of this diet on dopamine receptors (drd1 and drd2), proopiomelanocortin (pomc), neuropeptideY (npy) genes expression, and preference food in adult rats. Wistar female rats were fed a hight-fat or control diet during pregnancy and lactation. The offspring were allocated into groups: Lactation – Control (C) and High-fat (H). Post-weaning – Control Control (CC), offspring of mothers C, fed a control diet after weaning; Control Hight-fat (CH), offspring of mothers C, fed a hight-fat diet after weaning; Hight-fat Control (HC), offspring of mothers H, fed with control diet after weaning; and Hight-fat Hight-fat (HH), offspring of mothers H, fed a H diet after weaning. The groups CH and HH presented greater expression of drd1 in comparison to the CC. The drd2 of CH and HC presented higher gene expression than did CC. HH presented higher pomc expression in comparison to the other groups. HC also presented greater expression in comparison to CH. The npy of HH presented greater expression in relation to CH and HC. HH and HC have had a higher preference for a high-fat diet at 102º life’s day. The high-fat diet altered the gene expression of the drd1, drd2, pomc and npy, and influencing the food preference for high-fat diet. Resumo A exposição à dieta hiperlipídica pode alterar o controle da ingestão de alimentos, promovendo hiperfagia e obesidade. O objetivo deste estudo foi investigar os efeitos dessa dieta sobre a expressão gênica dos receptores de dopamina (drd1 e drd2), da proopiomelanocortina (pomc) e neuropeptídeo Y (npy), e preferência alimentar em ratos adultos. Ratas Wistar foram alimentadas com uma dieta hiperlipídica ou controle durante a gestação e lactação. Os descendentes foram alocados em grupos: Lactação – Controle (C) e Hiperlipídica (H). Pós-desmame – Controle Controle (CC), descendentes das genitoras do grupo controle e alimentados com dieta controle após o desmame; Controle Hiperlipídica (CH), descendentes das genitoras do grupo controle e alimentados com dieta hiperlipídica após o desmame; Hiperlipídica Controle (HC), descendentes das genitoras do grupo hiperlipídica e alimentados com dieta controle após o desmame; Hiperlipídica Hiperlipídica (HH), descendentes das genitoras do grupo hiperlipídica e alimentados com dieta hiperlipídica após o desmame. Os grupos CH e HH apresentaram maior expressão de drd1 em comparação ao CC. O drd2 de CH e HC apresentou maior expressão gênica que o CC. HH apresentou maior expressão de pomc em comparação com os outros grupos. O HC também apresentou maior expressão de pomc em comparação ao CH. O npy do HH apresentou maior expressão em relação ao CH e HC. HH e HC tiveram uma preferência maior por uma dieta rica em gordura no 102º dia de vida. A dieta hiperlipídica alterou a expressão gênica dos drd1, drd2, pomc e npy e influenciou na preferência alimentar pela dieta hiperlipídica.

BackgroundSevere thrombocytopenia can be a determinant factor in the morbidity ofPlasmodium vivax(Pv), the most widespread human malaria. Although immune mechanisms may drivePv-induced severe thrombocytopenia (PvST), the current data on the cytokine landscape in PvST is scarce, and often conflicting. The analysis of the bidirectional circuit of inflammatory mediators and miRNAs would lead to a better understanding of the mechanisms underlying PvST.MethodsWe combined Luminex proteomics, NanoString miRNA quantification, and machine learning, to evaluate an extensive array of plasma mediators in uncomplicatedPvpatients, whose blood platelet counts varied from reference values to PvST.ResultsUnsupervised clustering analysis identified PvST-linked signatures comprised of both inflammatory (CXCL10, CCL4, and IL-18) and regulatory (IL-10, IL-1Ra, HGF) mediators. As part of PvST signatures, IL-6 and IL-8 were critical to discriminatePvsubgroups, while CCL2 and IFN-γ from healthy controls. Supervised machine learning spotlighted IL-10 inPv-mediated thrombocytopenia, and provided evidence for a potential signaling route involving IL-8 and HGF. Finally, we identified a set of miRNAs capable of modulating these signaling pathways.ConclusionsThe results place IL-10 and IL-8/HGF in the center of PvST and propose investigating these signaling pathways across the spectrum of malaria infections.

Aim This study aimed to verify the correlation between murine measurements and retroperitoneal adipose tissue in rats exposed to the high-fat diet. Material and methods: Wistar male adult rats, descendants of mothers who consumed a high-fat diet during pregnancy and lactation and fed the same diet after weaning were used. At 60 days of life, body weight, longitudinal axis and waist circumference (WC) were measured. The Body Mass Index (BMI) and the Lee Index were calculated for a posterior analysis of the correlation with the amount of retroperitoneal adipose tissue dissected on the same day. For analysis of the data, the Pearson correlation test was used, considering statistical significance for p

Aim Obesity during pregnancy is one of the most established risk factors for negative long-term programming. The aim of the present study was to investigate the effects of maternal consumption of a high-fat diet during pregnancy and lactation on the weight gain, visceral adipose tissue and cholesterolemia in neonatal rats. Methods Wistar rats were divided into two groups according to the mother's diet during pregnancy and lactation: Control group (CG, n = 12) were the offspring of rats fed a standard diet (4% lipid) and the Test group (TG, n = 12) were pups rats fed on a high fat diet (23% lipid). The weight of the animals was measured on alternate days until the 22nd day of life, when collected visceral adipose tissue and blood were collected for biochemical analysis. For statistical analysis the Student t test, Sidak´s teste and two way ANOVA was used, with p

Introduction Understanding associations between food preferences and maternal nutrition during pregnancy and lactation could inform efforts to understanding the obesity mechanisms and provide insight to prevent it. Objective: To identify studies that investigated the effects of nutritional interventions during the pregnancy and lactation on the food preferences of offspring. Method: The review was conducted with search for articles in the databases: Scopus, Pubmed, Medline, LILACS, Scielo and Science Direct. Exclusion criteria were used: reviews, human studies, studies with drugs or other substances not related to food. Results: At the end of the search in the databases, 176 references were found. After use the exclusion criteria, reading the titles, abstracts and full articles, were selected 11 articles to compose the review. Conclusion: The selected studies suggested that unbalanced nutrition in early life alters the food preference and neural components related to the consumption of fatty and sugary foods in offspring rodents. Resumo Introdução O entendimento das associações entre as preferências alimentares e nutrição materna durante a gravidez e lactação poderia colaborar para a compreensão dos mecanismos da obesidade e fornecer informações para prevenir essa infermidade. Objetivo: Identificar estudos que investigaram os efeitos das intervenções nutricionais durante a gravidez e lactação em preferências alimentares dos descendentes. Método: A revisão foi conduzida com busca de artigos nas bases de dados: Scopus, Pubmed, Medline, Lilacs, Scielo e Science Direct. Os critérios de exclusão utilizados: revisões, estudos em humanos, estudos com drogas ou outras substâncias não-alimentares. Resultados: No final da pesquisa nas bases de dados, 176 referências foram encontradas. Depois de usar os critérios de exclusão, lendo os títulos, resumos e artigos completos, 11 artigos foram selecionados para compor a revisão. Conclusão: Os estudos selecionados sugeriram que a nutrição desequilibrada no início da vida altera a preferência alimentar e componentes neurais relacionadas com o consumo de alimentos gordurosos e açucarados em prole de roedores.

The largest ever recorded epidemic of the chikungunya virus (CHIKV) began in 2004 and affected four continents. Acute symptomatic infections are typically associated with the onset of fever and often debilitating polyarthralgia/polyarthritis. In this study, a systems biology approach was used to analyze the blood transcriptomes of adults acutely infected with CHIKV. Gene signatures that were associated with viral RNA amounts and to the onset of symptoms were identified. Among those genes, the putative role of Eukaryotic Initiation Factor (eIF) family genes and apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC3A) in the CHIKV replication process were displayed. We further compared these signatures with those induced by dengue virus infection and rheumatoid arthritis. Finally, we demonstrated that CHIKV infection in mice induced IL-1 beta production in a mechanism highly dependent on the inflammasome NLRP3 activation. The findings provided valuable insights into the virus–host interactions during the acute phase and could be useful in the investigation of new and effective therapeutic interventions.

The largest ever recorded epidemic of the Chikungunya virus (CHIKV) broke out in 2004 and affected four continents. Acute symptomatic infections are typically associated with the onset of fever and often debilitating polyarthralgia/polyarthritis. In this study, a systems biology approach was adopted to analyze the blood transcriptomes of adults acutely infected with the CHIKV. Gene signatures that were associated with viral RNA levels and the onset of symptoms were identified. Among these genes, the putative role of the Eukaryotic Initiation Factor (eIF) family genes and apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC3A) in the CHIKV replication process were displayed. We further compared these signatures with signatures induced by the Dengue virus infection and rheumatoid arthritis. Finally, we demonstrated that the CHIKV in vitro infection of murine bone marrow-derived macrophages induced IL-1 beta production in a mechanism that is significantly dependent on the inflammasome NLRP3 activation. The observations provided valuable insights into virus-host interactions during the acute phase and can be instrumental in the investigation of new and effective therapeutic interventions., Author summary The Chikungunya virus (CHIKV) has infected millions of people worldwide and presents a serious public health issue. Acute symptomatic infections caused by contracting this mosquito-transmitted arbovirus are typically associated with an abrupt onset of fever and often debilitating polyarthralgia/ polyarthritis, as well as prolonged periods of disability in some patients. These dramatic effects call for a careful evaluation of the molecular mechanisms involved in this puzzling infection. By analyzing the blood transcriptome of adults acutely infected with CHIKV, we were able to provide a detailed picture of the early molecular events induced by the infection. Additionally, the systems biology approach revealed genes that can be investigated extensively as probable therapeutic targets for the disease.

Fonsecaea pedrosoi is a dematiaceous fungus and the main causative agent of chromoblastomycosis that is a chronic disease usually affecting the human skin and subcutaneous tissues, which causes deformations and incapacities, being frequently refractory to available therapies. A typical globe-shaped, multiseptated and pigmented cells, known as sclerotic cells, are found in the lesions of infected individuals. In the present work, we have investigated the production of aspartic-type peptidase in F. pedrosoi sclerotic cells as well as the effect of peptidase inhibitors (PIs) on its enzymatic activity and viability. Our data showed that sclerotic cells are able to secrete pepstatin A-sensible aspartic peptidase when grown under chemically defined conditions. In addition, aspartic PIs (ritonavir, nelfinavir, indinavir, and saquinavir), which are clinically used in the HIV chemotherapy, significantly decreased the fungal peptidase activity, varying from 55 to 99%. Moreover, sclerotic cell-derived aspartic peptidase hydrolyzed human albumin, an important serum protein, as well as laminin, an extracellular matrix component, but not immunoglobulin G and fibronectin. It is well-known that aspartic peptidases play important physiological roles in fungal cells. With this task in mind, the effect of pepstatin A, a classical aspartic peptidase inhibitor, on the F. pedrosoi proliferation was evaluated. Pepstatin A inhibited the fungal viability in both cellular density- and drug-concentration manners. Moreover, HIV-PIs at 10 μM powerfully inhibited the viability (>65%) of F. pedrosoi sclerotic cells. The detection of aspartic peptidase produced by sclerotic cells, the parasitic form of F. pedrosoi, may contribute to reveal new virulence markers and potential targets for chromoblastomycosis therapy.

Fonsecaea pedrosoi is the main etiological agent of chromoblastomycosis, a recalcitrant disease that is extremely difficult to treat. Therefore, new chemotherapeutics to combat this fungal infection are urgently needed. Although aspartic peptidase inhibitors (PIs) currently used in the treatment of human immunodeficiency virus (HIV) have shown anti-F. pedrosoi activity their exact mechanisms of action have not been elucidated. In the present study, we have investigated the effects of four HIV-PIs on crucial virulence attributes expressed by F. pedrosoi conidial cells, including surface molecules and secreted enzymes, both of which are directly involved in the disease development. In all the experiments, conidia were treated with indinavir, nelfinavir, ritonavir and saquinavir (100 μM) for 24 h, and then fungal cells were used to evaluate the effects of HIV-PIs on different virulence attributes expressed by F. pedrosoi. In comparison to untreated controls, exposure of F. pedrosoi cells to HIV-PIs caused (i) reduction on the conidial granularity; (ii) irreversible surface ultrastructural alterations, such as shedding of electron dense and amorphous material from the cell wall, undulations/invaginations of the plasma membrane with and withdrawal of this membrane from the cell wall; (iii) a decrease in both mannose-rich glycoconjugates and melanin molecules and an increase in glucosylceramides on the conidial surface; (iv) inhibition of ergosterol and lanosterol production; (v) reduction in the secretion of aspartic peptidase, esterase and phospholipase; (vi) significant reduction in the viability of non-pigmented conidia compared to pigmented ones. In summary, HIV-PIs are efficient drugs with an ability to block crucial biological processes of F. pedrosoi and can be seriously considered as potential compounds for the development of new chromoblastomycosis chemotherapeutics.

Denture stomatitis, also known as atrophic candidiasis, remains the most frequent form of oral candidiasis, being detected in approximately 25%-65% of denture-wearing patients. Denture stomatitis is characterized by an erythematous inflammation of mucosal areas covered by prosthesis with preferential localization in the palatal mucosa. Although the clinical entity of this infectious disease is multifactorial, Candida albicans is the major etiological agent. The treatment of this oral infection is difficult because failures and recurrences are extremely common. In the present opinion article, we have presented a brief review on this oral pathology, summarizing the main predisposing factors, clinical diagnosis and current effective options to prevent and treat the affected population.

Nucleotide sequence analyses of the SH gene of 18 mumps virus isolates collected in the 2006-2007 parotitis epidemic in the state of São Paulo identified a new genotype, designated genotype M. This new designation fulfills all the parameters required to define a new mumps virus genotype. The parameters were established by an expert panel in collaboration with the World Health Organization (WHO) in 2005. This information will enhance the mumps virus surveillance program both at the national and global levels.

Recent reports showed that, in mice, symptomatic Plasmodium infection triggers NLRP3/NLRP12-dependent inflammasome formation and caspase-1 activation in monocytes. In humans, few works demonstrated that inflammasome is activated in malaria. As Plasmodiumvivax is a potent inducer of inflammatory response we hypothesised that inflammasome genetics might affect P. vivax malaria clinical presentation. For this purpose, selected SNPs in inflammasome genes were analysed among patients with symptomatic P. vivax malaria. 157 Brazilian Amazon patients with P. vivax malaria were genotyped for 10 single nucleotide polymorphisms (SNPs) in inflammasome genes NLRP1, NLRP3, AIM2, CARD8, IL1B, IL18 and MEFV. Effect of SNPs on hematologic and clinical parameters was analysed by multivariate analysis. Our data suggested an important role of NLRP1 inflammasome receptor in shaping the clinical presentation of P. vivax malaria, in term of presence of fever, anaemia and thrombocytopenia. Moreover IL1B rs1143634 resulted significantly associated to patients' parasitaemia, while IL18 rs5744256 plays a protective role against the development of anaemia. Polymorphisms in inflammasome genes could affect one or other aspects of malaria pathogenesis. Moreover, these data reveal novel aspects of P.vivax/host interaction that involved NLRP1-inflammasome.

Background Neurological involvement during influenza infection has been described during epidemics and is often consistent with serious sequelae or death. Objective To investigate the etiologic agent involved in myelopathy post influenza-like syndrome. Study design This investigation focuses on virus isolation from the cerebrospinal fluid (CSF) collected from a 19-year-old male student presenting with clinical diagnosis of myelopathy post influenza-like syndrome. To achieve this goal, different cell cultures and molecular methodologies were carried out. Results Influenza virus A(H3N2) strain was isolated in MDCK cell culture; virus particles were observed under electron microscopy. Phylogenetics analyses showed that the Brazilian influenza A(H3N2) strains were closely related to the A/Perth/16/2009-like. Conclusion This study demonstrates that influenza virus A(H3N2) strain was the cause of illness of the students. According to the Brazilian influenza virus sentinel surveillance data A/Perth/16/2009-LIKE (H3N2) strain has predominated during the 2010 influenza virus season in Brasilia – DF.

Mutations of the thyroperoxidase (TPO) gene have been reported as being the most severe and frequent abnormality in thyroid iodide organification defect (IOD) causing goitrous congenital hypothyroidism. The objective of this study was to screen and subsequently identify TPO gene mutations in patients with congenital hypothyroidism with evidence of total iodine organification defects (TIOD) or partial iodine organification defect (PIOD) as defined by the perchlorate discharge test. Seven goitrous patients with TIOD and seven patients with PIOD, from three and five unrelated families, respectively, were studied. We were able to detect different TPO genes mutations in patients with TIOD and PIOD. In TIOD families the results were as follows: (1) a homozygous GGCC insertion at exon 8, position 1277 (family 1); (2) compound heterozygosity with a GGCC insertion at exon 8 (1277) and a nucleotide substitution in exon 11 (2068GC) (family 2); (3) compound heterozygosity with the mutation 2068GC in exon 11 and a C insertion in exon 14 between positions 2505-2511 (family 3). In patients with PIOD we have detected: (1) only one heterozygous mutation in two families (4 and 5), in exons 11 and 10 (2084GA and 1780CA); (2) a compound heterozygous condition in one family (family 6), with mutations, respectively in exons 8 and 10 (1242GT and 1780CA); (3) only polymorphisms (family VII) and (4) a heterozygous mutation in the first base of the border exon/intron 9 +1GT (family VIII). We did not detect inactivating mutations in exons 11, 16, and 21 of the THOX2 gene where mutations have been previously described. We concluded that homozygous and compound heterozygous mutations found in TIOD characterized the autosomal recessive mode of inheritance and will translate a nonfunctional protein or a protein that may not reach the apical membrane. As for PIOD, the majority of the studied kindreds had only heterozygous mutations and/or polymorphisms. It is conceivable that these TPO gene sequence alterations may partially affect the functional state of the translated protein or affect its transport to the apical membrane.

Background: The prevalence of anti-hepatitis C virus (HCV) positive test is higher among patients in dialysis and in kidney recipients than in general population. Hepatitis C virus infection is the main cause of chronic liver disease in renal transplant patients. Liver biopsy and virological analysis were performed to clarify the grade of liver damage in kidney recipients. Methods: Renal recipients patients with at least 5 yr under immunosuppression were submitted to clinical and laboratory analysis. Patients who tested anti-HCV positive were candidates to liver biopsy with no regard to transaminase levels. Results: Forty-five patients tested anti-HCV positive and 42 anti-HCV negative. Twenty-six anti-HCV and RNA-HCV positive patients were submitted to liver biopsy. Seventy-three percentage of these patients presented chronic active hepatitis, from these only one patient presented cirrhosis. Only 29% of the anti-HCV positive group presented elevated alanine aminotransferase levels. Anti-HCV positive patients presented longer previous time on dialysis and less rejection episodes than the group anti-HCV negative (p

In 2001, an autochthonous case of dual viremia, resulting from naturally acquired dengue virus DEN-1 and DEN-2 infections was detected during the dengue outbreak that occurred in Barretos, a city with about 105,000 inhabitants in the North region of São Paulo State. Serotype identification was based on virus isolation to C6/36 mosquito cells culture and immunofluorescence assays using type-specific monoclonal antibodies. The double infection was also confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Comparative analysis of the 240-nucleotide sequences of E/NS1 gene junction region between the genome of DEN-1 and DEN-2 isolates of the corresponding reference Nauru and PR 159S1 strains, respectively, showed some nucleotide differences, mainly silent mutations in the third codon position. Results of maximum likelihood phylogenetic analysis of E/NS1 gene sequences indicated that both genotypes of DEN-1 and DEN-2 viruses recovered from double infection in Barretos belonged to genotypes I and III, respectively.

OBJECTIVE To screen and subsequently sequence the TPO gene for mutations in patients with congenital goitre, hypothyroidism and evidence for an organification defect (positive perchlorate discharge test). PATIENTS We have studied seven hypothyroid and congenitally goitrous patients from three unrelated families. DESIGN AND MEASUREMENTS We have measured serum thyroid hormone levels, 131I uptake, serum TSH and serum Tg concentrations. Denaturing gradient gel electrophoresis (DGGE) of PCR amplified genomic DNA was used to screen for mutations in the TPO gene. RESULTS DGGE identified the presence of two frameshift mutations: a GGCC duplication in exon 8 (homozygous in one family and heterozygous in the other family) and a heterozygous insertion of a single nucleotide (C) at position 2505-2511 in exon 14. In addition, we have detected an alteration in exon 11, not yet described in the literature, derived from a single nucleotide substitution of a C to G at position 2008, altering the well-conserved amino acid domain among the peroxidases superfamily. This mutation in exon 11 was present in two families that showed heterozygous mutation for exon 8 or for exon 14. CONCLUSIONS These results could support the hypothesis for a putative compound heterozygosity pattern in the affected patients. The altered phenotype (goitre and hypothyroidism since birth) seems justifiable in view of the possible inactivating character of this novel mutation in exon 11.

Pendred's syndrome is an autosomal recessive disease characterized by goiter, impaired iodide organification, and congenital sensorineural deafness. The gene mutated in Pendred's syndrome, PDS (Pendred's syndrome gene), was cloned very recently and encodes the putative sulfate transporter pendrin. Pendred's syndrome may account for up to 10% of the cases with hereditary hearing loss, and pendrin mutations have also been found in a kindred with non-syndromic deafness. In this study, 41 individuals from a large, highly inbred pedigree from Northeastern Brazil were examined for features of Pendred's syndrome. Linkage studies and sequence analysis of the coding region of the PDS gene were performed with DNA from 36 individuals. The index patient, with the classical triad of deafness, positive perchlorate test, and goiter, was found to be homozygous for a deletion of thymidine 279 in exon 3, resulting in a frameshift and a premature stop codon at amino acid 96. This alteration resulted in truncation of the protein in the first transmembrane domain. Two other patients with deafness were found to be homozygous for this mutation; 19 were heterozygous and 14 were homozygous for the wild type allele. Surprisingly, 6 deaf individuals in this kindred were not homozygous for the PDS gene mutation; 3 were heterozygous and 3 were homozygous for the wild type allele, suggesting a probable distinct genetic cause for their deafness. All 3 homozygous individuals for the PDS mutation had goiters. However, goiters were also found in 10 heterozygous individuals and in 6 individuals without the PDS mutation and are most likely caused by iodine deficiency. In conclusion, we identified a novel mutation in the PDS gene causing Pendred's syndrome. The comparison of phenotype and genotype reveals, however, that phenocopies generated by distinct environmental and/or genetic causes are present in this kindred and that the diagnosis of Pendred's syndrome may be difficult without molecular analysis.

A very large cervical tumor that extended to the upper mediastinum was seen in a newborn after an uneventful pregnancy. The computed axial tomography scan confirmed the presence of a solid mass with precise limits and scattered foci of calcifications situated in the anterolateral region of the neck. The infant underwent thyroidectomy on the seventh day after birth. Pathological examination revealed a follicular carcinoma of the thyroid and probable dyshormonogenetic hyperplastic goiter. At 5 months of age, whole body scans indicated the presence of lung and bone metastases, which were treated with therapeutic doses of radioiodine. Genomic DNA was obtained from the newborn, her parents, her paternal aunt, and her paternal grandparents. Denaturing gradient gel electrophoresis analysis of PCR fragments corresponding to exon 14 of the thyroid peroxidase (TPO) gene indicated the presence of a mutant TPO allele present in the propositus, her father, and her paternal grandmother. Sequencing of the TPO gene demonstrated a mutation resulting from an insertion of a single extra cytosine in a stretch of seven cytosines at positions 2505-2511. The insertion caused a frame shift and a stop signal in exon 16. This sequence would translate into a structurally modified and probably inactive TPO protein. We conclude that the aggressive thyroid metastatic carcinoma arose from a dyshormonogenetic goiter caused by a defective TPO protein.

Preface.- Biology of Human Pathogenic Trypanosomatids: Epidemiology, Lifecycle and Ultrastructure.- Selection of Molecular Targets for Drug Development against Trypanosomatids.- A2 and other visceralizing proteins of Leishmania: role in pathogenesis and application for vaccine development.- Arginase in Leishmania.- The heat shock proteins of Trypanosoma cruzi.- The Gp82 Surface Molecule of Trypanosoma cruzi Metacyclic Forms.- The Gp85 Surface Glycoproteins from Trypanosoma cruzi.- Trypanosoma cruzi Trans-Sialidase: Structural Features and Biological Implications.- Surface Topology Evolution of Trypanosoma Trans-Sialidase.- Ecto-Nucleotidases and Ecto-Phosphatases from Leishmania and Trypanosoma Parasites.- Gp63 Function in the Interaction of Trypanosomatids with the Invertebrate Host - Facts and Prospects.- Highlights on Trypanosomatid Aminoacyl-tRNA Synthesis.- The Expected Outcome of the Trypanosoma cruzi Proteomic Map: a Review of its Potential Biological Applications for Drug Target Discovery.- Proteomics advances in the study of Leishmania parasites and leishmaniasis.- Towards the Phosphoproteome of Trypanosomatids. Index.

Fundação Oswaldo Cruz. Centro de Pesquisa René. Laboratório de Malária. Belo Horizonte, MG, Brazil Fundação Oswaldo Cruz. Centro de Pesquisa René. Laboratório de Malária. Belo Horizonte, MG, Brazil Fundação de Medicina Tropical Dr. Heitor Vieira Dourado. Gerência de Malária. Manaus, AM, Brazil Fundação Oswaldo Cruz. Centro de Pesquisa René. Laboratório de Malária. Belo Horizonte, MG, Brazil Fundação Oswaldo Cruz. Centro de Pesquisa René. Laboratório de Malária. Belo Horizonte, MG, Brazil Fundação Oswaldo Cruz. Centro de Pesquisa René. Laboratório de Malária. Belo Horizonte, MG, Brazil Fundação Oswaldo Cruz. Centro de Pesquisa René. Laboratório de Malária. Belo Horizonte, MG, Brazil Universidade Federal de Mato Grosso. Departamento de Clínica Médica. Cuiabá, MT, Brazil Fundação de Medicina Tropical Dr. Heitor Vieira Dourado. Gerência de Malária. Manaus, AM, Brazil Fundação Oswaldo Cruz. Centro de Pesquisa René. Laboratório de Malária. Belo Horizonte, MG, Brazil Background: Given the increasing evidence of Plasmodium vivax infections associated with severe and fatal disease, the identification of sensitive and reliable markers for vivax severity is crucial to improve patient care. Circulating nucleic acids (CNAs) have been increasingly recognized as powerful diagnostic and prognostic tools for various inflammatory diseases and tumors as their plasma concentrations increase according to malignancy. Given the marked inflammatory status of P. vivax infection, we investigated here the usefulness of CNAs as biomarkers for malaria morbidity. Methods and Findings: CNAs levels in plasma from twenty-one acute P. vivax malaria patients from the Brazilian Amazon and 14 malaria non-exposed healthy donors were quantified by two different methodologies: amplification of the human telomerase reverse transcriptase (hTERT) genomic sequence by quantitative real time PCR (qPCR), and the fluorometric dsDNA quantification by Pico Green. CNAs levels were significantly increased in plasma from P. vivax patients as compared to healthy donors (p

Delayed graft function (DGF), a frequent complication after kidney transplantation, occurs among about 60% of recipients of kidneys from deceased donors. DGF has a multifactorial etiology. It is characterized by acute tubular necrosis (ATN) upon biopsy. In this study we sought to identify among a group of recipients of kidneys from deceased donors, the incidence, risk factors, and impacts on patient and graft survivals of DGF.We retrospectively analyzed medical records from renal transplant recipients aged18 years who received a deceased donor kidney graft between January 2003 and December 2006. Kidneys lost during the first week posttransplantation were excluded from this series.Among 165 transplants, 111 (67%) displayed DGF, defined as the need for dialysis during the first week posttransplantation. The incidence of DGF was higher among patients with a cold ischemia time (CIT)24 hours: 85% vs 60%, DGF vs no DGF (P.05), as well as for grafts from older donors. After 1-year follow-up, the DGF group showed worse graft function (serum creatinine 1.6 +/- 0.7 vs 1.3 +/- 0.4 mg/dL; P.05) as well as a greater incidence of graft loss.Prolonged cold ischemia and older donor age were associated with a greater incidence of DGF in this series, leading to prolonged hospitalization, increased risk for an acute rejection episode, and reduced graft function and survival after 1 year.

Rocio virus (ROCV) was responsible for an explosive encephalitis epidemic in the 1970s affecting about 1,000 residents of 20 coastland counties in São Paulo State, Brazil. ROCV was first isolated in 1975 from the cerebellum of a fatal human case of encephalitis. Clinical manifestations of the illness are similar to those described for St. Louis encephalitis. ROCV shows intense antigenic cross-reactivity with Japanese encephalitis complex (JEC) viruses, particularly with Ilheus (ILHV), St. Louis encephalitis, Murray Valley and West Nile viruses. In this study, we report a specific RT-PCR assay for ROCV diagnosis and the molecular characterization of the SPAn37630 and SPH37623 strains. Partial nucleotide sequences of NS5 and E genes determined from both strains were used in phylogenetic analysis. The results indicated that these strains are closely related to JEC viruses, but forming a distinct subclade together with ILHV, in accordance with results recently reported by Medeiros et al. (2007). O vírus Rocio (ROCV) foi responsável por uma explosiva epidemia de encefalite que ocorreu nos anos 70 afetando cerca de 1.000 habitantes de 20 municípios litorâneos do Estado de São Paulo, Brasil. ROCV foi isolado em 1975 de cerebelo de caso humano fatal de encefalite. As manifestações clínicas da doença são semelhantes àquelas descritas para encefalite St. Louis. ROCV apresenta intensa reatividade cruzada com os vírus do Complexo da Encefalite Japonesa (JEV), particularmente com o vírus Ilhéus (ILHV) e com os vírus das encefalites St. Louis, Murray Valley e West Nile. Neste estudo, relatamos o desenvolvimento de um teste de RT-PCR específico para diagnóstico de ROCV e a caracterização molecular das cepas SPAn37630 e SPH37623. Foi realizada a análise filogenética das seqüências parciais dos genes NS5 e E, de ambas as cepas. Os resultados indicaram que essas cepas são intimamente relacionadas ao complexo JEV, mas formando um subgrupo com o ILHV, de acordo com os resultados recentemente publicados por MEDEIROS et al. (2007).

Lentine, Krista/0000-0002-9423-4849; Axelrod, David/0000-0001-5684-0613; Abbott, Kevin/0000-0003-2111-7112 WOS: 000246576700017 PubMed: 17511681 We investigated graft and patient survival implications of simultaneous pancreas kidney (SPK) transplant from old donors. Data describing patients with type 1 diabetes mellitus listed for an SPK transplant from 1994 to 2005 were drawn from Organ Procurement and Transplant Network registries. Allograft survival, patient survival and long-term survival expectations among SPK recipients from young (age < 45 years) and old (age >= 45 years) donors were modeled by multivariate regression. We also examined predictors of reduced early access to young donor transplants. Of 16 496 eligible SPK candidates, 8850 patients (53.6%) received an SPK transplant and 776 (8.8%) of these transplants were from old donors. Reasonable 5-year, death-censored kidney (77.8 %) and pancreas (71.3%) survivals were achieved with old donors. SPK transplantation from both young and old donors predicted lower mortality compared to continued waiting. An additional expected wait of 1.5 years for a young donor equalized long-term survival expectations to that achieved with use of old donors. Early allocation of young donor transplants declined in the more recent era and varied by region, candidate age, blood type and sensitization. We conclude that old SPK donors should be considered for patients with decreased access to young donor transplants. Prospective evaluation of this practice is needed. NIDDK NIH HHSUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK) [K24-DK002886]; PHS HHSUnited States Public Health Service [K08-0730306]

The molecular characterization of SPH253157, a new strain of St. Louis encephalitis virus (SLEV), isolated in 2004 from the first case of human infection recognized in the state of São Paulo, Brazil, is reported. The patient, presenting a febrile illness without neurological involvement, was hospitalized as a probable case of dengue fever. Genomic RNA was isolated from the supernatant of C6/36 cells infected with acute phase-serum specimen of the patient and the envelope gene was amplified by reverse-transcription-polymerase chain reaction. The complete nucleotide sequence of the envelope gene of this isolate was directly sequenced from the amplified products and compared with other Brazilian and American SLEV strains. Phylogenetic analyses were carried out under maximum likelihood criterion with outgroups both included and excluded. Outgroups comprised four flavivirus of the Japanese encephalitis group. Phylogeny also included Bayesian analysis. The results indicated that the new SLEV isolate belongs to lineage III, being closely related to an Argentinean strain recovered from Culex sp. in 1979. It is concluded that there are at least 3 lineages of SLEV in Brazil.

O presente estudo relata o isolamento do vírus da encefalite São Luis (SLEV) de um caso febril humano suspeito de dengue, em São Pedro, Estado de São Paulo. MAC-ELISA realizado com soros das fases aguda e convalescente foi inconclusivo e anticorpos IgG foram detectados por inibição da hemaglutinação para flavivirus. Imunofluorescência indireta com cultura de células C6/36 inoculadas com soro da fase aguda foi positivo para flavivirus mas negativo quando testado com anticorpos monoclonais para dengue. O RNA extraído de cultura de células infectadas foi amplificado na presença de primers universais para o gênero Flavivirus, deduzidos de uma região da proteína não estrutural 5 e diretamente sequenciado. Os resultados da pesquisa no BLAST indicaram que a seqüência apresenta 93% de similaridade de nucleotídeos com a seqüência de SLEV (cepa MS1.7), confirmado por RT-PCR, realizado com primers específicos para SLEV. O fato de SLEV ter sido identificado como a causa de doença humana indica a necessidade de aprimorar a vigilância a fim de detectar precocemente esse agente no Estado de São Paulo e no Brasil. Esse caso é também um alerta para os profissionais de saúde sobre a necessidade de investigações clínicas e epidemiológicas mais completas sobre doenças febris como no caso relatado. Infecções por SLEV podem não ser reconhecidas ou confundidas com outras causadas por arbovírus como a dengue. This paper reports the isolation of St. Louis encephalitis virus (SLEV) from a febrile human case suspected to be dengue, in São Pedro, São Paulo State. A MAC-ELISA done on the patient's acute and convalescent sera was inconclusive and hemagglutination inhibition test detected IgG antibody for flaviviruses. An indirect immunofluorescent assay done on the C6/36 cell culture inoculated with the acute serum was positive for flaviviruses but negative when tested with dengue monoclonal antibodies. RNA extracted from the infected cell culture supernatant was amplified by RT-PCR in the presence of NS5 universal flavivirus primers and directly sequenced. Results of BLAST search indicated that this sequence shares 93% nucleotide similarity with the sequence of SLEV (strain-MSI.7), confirmed by RT-PCR performed with SLEV specific primers. Since SLEV was identified as the cause of human disease, it is necessary to improve surveillance in order to achieve early detection of this agent in the state of São Paulo and in Brazil. This finding is also an alert to health professionals about the need for more complete clinical and epidemiological investigations of febrile illnesses as in the reported case. SLEV infections can be unrecognized or confused with other ones caused by an arbovirus, such as dengue.

Polyoma virus nephropathy (PVN) occurs in 3% to 4% of renal transplants, causing graft loss in about 50% of cases. The presence of viral cytopathic changes in graft epithelial cells is the only diagnostic tool for PVN. However, identification of cells with viral inclusions (decoy cells) in urine can be used as a screening tool for viral replication of or for active infection with PV. The aim of the present study was to identify the occurrence of PV active infection in renal transplant recipients. Two hundred forty urine cytology samples, collected from 80 transplant patients with stable renal function, were collected on a monthly basis and stained with the Pap smear for decoy cells. Active infection with polyoma virus was confirmed by urine immunostaining. All samples were analyzed blindly and classified as negative or positive (>1 decoy cell/sample). Among 240 urine cytologies collected from 48 men and 32 women, decoy cells were identified in 37.5%. No differences were observed in serum creatinine or immunosuppressive regimen between patients with positive versus negative cytology. No graft losses occurred secondary to PVN in the present study setting. The incidence of decoy cells in this series (37.5%) was consistent with previous reports (20% to 40%), suggesting that active infection may be confirmed by PV immunohistochemistry. The absence of PVN in this group may be attributed to the low doses of immunosuppressive drugs in the late posttransplant transplant period, but also to the unknown incidence of polyoma virus infection in Brazil.

The authors report the isolation of dengue 3 virus for the first time in Brazil. The patient, resident in Limeira-SP, traveled to Nicaragua on May 16th, 1998, where he stayed for two months. Starting on August 14th he had fever, headache, myalgia, arthralgia, retro-orbital pain and diarrhea. He returned to Brazil on August 16th and was hospitalized in the next day. The patient had full recovery and was discharged on August 20th. The virus was isolated in C6/36 cell culture inoculated with serum collected on the 6th day after the onset of the symptoms. The serotype 3 was identified by indirect immunofluorescence assays performed with type-specific monoclonal antibodies. This serotype was further confirmed by polymerase chain reaction analysis. The introduction of a new dengue serotype in a susceptible population is a real threat for the occurrence of severe forms of the disease. The isolation and identification of dengue virus are important in order to monitoring the serotypes circulating in Brazil and to take the measures necessary to prevent and control an epidemic. Os autores relatam o isolamento do vírus dengue 3 pela primeira vez no Brasil. O paciente, residente em Limeira-SP, viajou em 16 de maio de 1998 para a Nicarágua, onde permaneceu por três meses. A partir de 14 de agosto, ele apresentou febre, dor de cabeça, mialgia, artralgia, dor retro-orbital e diarréia. Ele retornou ao Brasil em 16 de agosto e foi hospitalizado no dia seguinte. O paciente teve completa recuperação, recebendo alta em 20 de agosto. O vírus foi isolado em cultura de células C6/36 inoculadas com soro colhido no 6(0) dia após o início dos sintomas. O sorotipo 3 foi identificado por imunofluorescência indireta com anticorpos monoclonais tipo-específico. O sorotipo foi posteriormente confirmado por PCR. A introdução de um novo sorotipo de dengue em uma população susceptível é uma ameaça para a ocorrência de formas severas da doença. O isolamento e a identificação do vírus dengue são de grande importância a fim de monitorar a introdução de novos sorotipos e para que medidas sejam tomadas para prevenir e controlar novas epidemias.

Pan American Health Organization, Funda??o Nacional de Sa?de (Ministry of Health, Brazil), and CNPq (Brazil) Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brazil. Acambis. Cambridge, Massachusetts. Instituto Adolpho Lutz. S?o Paulo, SP, Brazil. Acambis. Cambridge, Massachusetts. Funda??o Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brazil. Minist?rio da Sa?de. Funda??o Nacional de Sa?de. Instituto Evandro Chagas. Bel?m, PA, Brasil. Funda??o Oswaldo Cruz. Instituto de Tecnologia em Imunobiol?gicos. Rio de Janeiro, RJ, Brazil. Funda??o Oswaldo Cruz. Instituto de Tecnologia em Imunobiol?gicos. Rio de Janeiro, RJ, Brazil. Funda??o Oswaldo Cruz. Instituto de Tecnologia em Imunobiol?gicos. Rio de Janeiro, RJ, Brazil. Minist?rio da Sa?de. Funda??o Nacional de Sa?de. Instituto Evandro Chagas. Bel?m, PA, Brasil. Funda??o Oswaldo Cruz. Instituto de Tecnologia em Imunobiol?gicos. Rio de Janeiro, RJ, Brazil. Instituto Adolpho Lutz. S?o Paulo, SP, Brazil. Minist?rio da Sa?de. Funda??o Nacional de Sa?de. Instituto Evandro Chagas. Bel?m, PA, Brasil. Instituto Adolpho Lutz. S?o Paulo, SP, Brazil. Minist?rio da Sa?de. Funda??o Nacional de Sa?de. Instituto Evandro Chagas. Bel?m, PA, Brasil. Acambis. Cambridge, Massachusetts. Acambis. Cambridge, Massachusetts. The yellow fever (YF) 17D virus is one of the most successful vaccines developed to data. Its use has been estimated to be over 400 million doses with an excellent record of safety. In the past 3 years, yellow fever vaccination was intensified in Brazil in response to higher risk of urban outbreaks of the disease. Two fatal adverse events temporally associated with YF vaccination were reported. Both cases had features similar to yellow fever disease, including hepatitis and multiorgan failure. Two different lots of YF 17DD virus vaccine were administered to the affected patients and also to hundreds of thousands of other individuals without any other reported serious adverse events. The lots were prepared from the secondary seed, which has been in continuous use since 1984. Nucleotide sequencing revealed minor variations at some nucleotide positions between the secondary seed lot virus and the virus isolates from patients; these differences were not consistent across the isolates, represented differences in the relative amount of each nucleotide in a heterogeneous position, and did not result in amino acid substitutions. Inoculation of rhesus monkeys with the viruses isolated from the two patients by the intracerebral (ic) or intrahepatic (ih) route caused minimal viremia and no clinical signs of infection or alterations in laboratory markers. Central nervous system histological scores of rhesus monkeys inoculated ic were within the expected range, and there were no histopathological lesions in animals inoculated ih. Altogether, these results demonstrated the genetic stability and attenuated phenotype of the viruses that caused fatal illness in the two patients. Therefore, the fatal adverse events experienced by the vaccinees are related to individual, genetically determined host factors that regulate cellular susceptibility to yellow fever virus. Such increased susceptibility, resulting in clinically overt disease expression, appears to be extremely rare.

Impaired thyroglobulin (Tg) synthesis is one of the putative causes for dyshormonogenesis of the thyroid gland. This type of hypothyroidism is characterized by intact iodide trapping, normal organification of iodide, and usually low serum Tg levels in relation to high TSH, and when untreated the patients develop goiter. In thyroid tissue from a 13-yr-old patient suspected of a thyroglobulin synthesis defect, the Tg mRNA was studied. The complete coding region of 8307 bp was directly sequenced and revealed a homozygous point mutation: a C886T transition in exon 7. Upon translation this mutation would result in a stopcodon at amino acid position 277, replacing the arginine residue. A Tg cDNA construct containing the mutation was expressed in rabbit reticulocyte lysate resulting in a truncated protein of 30 kDa. Expression in the presence of microsomal membranes resulted in a gel shift of this Tg molecule, indicating glycosylation ability. Two other siblings had a clinical presentation like the index patient, while their parents were unaffected. Additional restriction fragment length polymorphism analysis of the pedigree verified that the homozygous nonsense mutation cosegregated with the clinical phenotype. Clinically, hypothyroidism was not severe in the affected siblings because the truncated Tg glycoprotein was still capable of thyroid hormonogenesis.

Two unrelated families (CA and NA) in which an iodide organification defect (lOD) was present in two siblings of each family were studied. These patients had congenital goiters with hypothyroidism and a positive perchlorate discharge test. Examination of the thyroid tissue revealed no thyroid peroxidase (TPO) activity. Histologic findings were consistent with a microfollicular pattern of hyperplasia. Moderate cellular atypia was present, characterized by nuclear pleomorphism and hyperchromatism. Full length thyroglobulin was purified by gel filtration, but was not iodinated. Immunohistochemical studies using a polyclonal anti-human thyroid peroxidase (hTPO) antibody confirmed the presence of immunoreactive TPO protein in the thyroid tissues. Samples of normal and affected individuals were studied with respect to the presence of various fragments using TPO probes of varying sizes. The two affected siblings from family CA were homozygous for fragments 3.9, 4.6, and 7.0 kb (8g111) and 2.3 and 2.9 kb (Ta ql), whereas the parents were heterozygous. In the other family (NA), the Bg/ll digestion and TPO-31 hybridization revealed an interesting and informative polymorphism. The parents showed two different polymorphic patterns: the father had a 5.0/4.6 kb pattern and the mother a 4.7/4.5 kb pattern. However, the two affected siblings showed the same heterozygotic allelic pattern at 4.5/4.6 kb. The restriction fragment length polymorphism detected in these two families suggests an association between the TPO gene and an lOD. Results suggest that in these dyshormonogenetic tissues an altered TPO protein molecule is being synthesized, without detectable in vitro activity, but visible by immunostaining techniques in the goitrous tissue. Mutations in the TPC gene sequence are most likely associated with these changes.

Dengue Fever and Dengue Hemorrhagic Fever are diseases affecting approximately 100 million people/year and are a major concern in developing countries. In the present study, the phylogenetic relationship of six strains of the first autochthonous cases of DENV-4 infection occurred in Sao Paulo State, Parana State and Rio Grande do Sul State, Brazil, 2011 were studied. Nucleotide sequences of the envelope gene were determined and compared with sequences representative of the genotypes I, II, III and Sylvatic for DEN4 retrieved from GenBank. We employed a Bayesian phylogenetic approach to reconstruct the phylogenetic relationships of Brazilian DENV-4 and we estimated evolutionary rates and dates of divergence for DENV-4 found in Brazil in 2011. All samples sequenced in this study were located in Genotype II. The studied strains are monophyletic and our data suggest that they have been evolving separately for at least 4 to 6 years. Our data suggest that the virus might have been present in the region for some time, without being noticed by Health Surveillance Services due to a low level of circulation and a higher prevalence of DENV-1 and DENV- 2., Author Summary Dengue virus infections are a major concern in developing countries, affecting approximately 100 million people/year. The virus has four immunologically related serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) associated with human disease. The virus is widespread in tropical and Sub-Tropical areas of Asia, Africa and Americas. The virus is transmitted by mosquito bites, and is primarily associated with Aedes aegypti as its main vector. To understand the reemergence of DENV-4 in Brazil in 2010–2011 we carried out a Bayesian phylogenetic analysis of the envelope gene sequences sampled in Brazil in 2011. Our results indicate that the studied samples are close related to strains circulating since 1981, when DENV-4 was first introduced in South America, but have gone trough recent evolution for at least 4 to 6 years. Our results also suggests that the virus may have penetrated Brazilian population earlier than 2010, indicating that the virus could have been present but not detected due a higher prevalence of DENV-1 and DENV- 2 and the failure of the surveillance system to locate the milder disease commonly associated with DENV-4.

Severe thrombocytopenia can be a determinant factor in the morbidity of Plasmodium vivax, the most widespread human malaria parasite. Although immune mechanisms may drive P. vivax-induced severe thrombocytopenia (PvST), the current data on the cytokine landscape in PvST is scarce and often conflicting. Here, we hypothesized that the analysis of the bidirectional circuit of inflammatory mediators and their regulatory miRNAs would lead to a better understanding of the mechanisms underlying PvST. For that, we combined Luminex proteomics, NanoString miRNA quantification, and machine learning to evaluate an extensive array of plasma mediators in uncomplicated P. vivax patients with different degrees of thrombocytopenia. Unsupervised clustering analysis identified a set of PvST-linked inflammatory (CXCL10, CCL4, and IL-18) and regulatory (IL-10, IL-1Ra, HGF) mediators. Among the mediators associated with PvST, IL-6 and IL-8 were critical to discriminate P. vivax subgroups, while CCL2 and IFN-γ from healthy controls. Supervised machine learning spotlighted IL-10 in P. vivax-mediated thrombocytopenia and provided evidence for a potential signaling route involving IL-8 and HGF. Finally, we identified a set of miRNAs capable of modulating these signaling pathways. In conclusion, the results place IL-10 and IL-8/HGF in the center of PvST and propose investigating these signaling pathways across the spectrum of malaria infections.

Apoptosis is a well-known specific process of cell death that normally occurs in physiological situations such as tissue or organ development and involution. During tumor growth there is a balance between proliferation and cell death which involves apoptotic mechanisms. In the present study genomic DNAs from 120 breast tumor biopsies were analyzed by agarose gel electrophoresis and none of them presented the fragmentation pattern characteristic of the apoptosis process. However, 33% of the 105 breast cancer patients clearly showed the apoptotic pattern when DNA from blood cells was analyzed. None of the DNAs from healthy volunteer blood cells showed any trace of apoptosis. Since the breast cancer patients were not receiving chemo- or hormone therapy, the possible relationship between blood cortisol levels and the apoptotic pattern found in patient blood cells was investigated. Using a chemoluminescence immunodetection assay, similar cortisol levels were observed in breast cancer patient sera presenting or not apoptotic blood cells and in healthy volunteer sera. Analysis of the clinical data obtained from 60 of these patients showed that patients bearing tumors of smaller size (under 20 mm) were more susceptible to the apoptotic effect in blood cells. According to the Elston grade, it was observed that 7 of 12 patients with grade III tumors (58%) presented apoptotic peripheral blood cells, in contrast to 10 of 48 patients with grade I and grade II tumors. These observations may reflect the immunosuppression characteristic of some breast cancer patients, which may contribute to tumor growth. Therefore, further studies are necessary to elucidate the factor(s) involved in such massive blood cell death.