Aluminum hydroxide associated to Schistosoma mansoni 22.6 kDa protein abrogates partial protection against experimental infection but not alter interleukin-10 production

The need to develop a vaccine against schistosomiasis led several researches and our group to investigate proteins from Schistosoma mansoni as vaccine candidates. Sm22.6 is a protein from S. mansoni that shows high identity with Sj22.6 and Sh22.6 (79 and 91%, respectively). These proteins are associated with high levels of IgE and protection to reinfection. Previously, we have shown that Sm22.6 induced a partial protection of 34.5% when used together with Freund's adjuvant and produced a Th0 type of immune response with interferon-g and interleukin-4. In this work, mice were immunized with Sm22.6 alone or with aluminum hydroxide adjuvant and high levels of IgG, IgG1, and IgG2a were measured. Unfortunately, no protection was detected. Since IL-10 is a modulating cytokine in schistosomiasis, we also observed a high level of this molecule in splenocytes of vaccinated mice. In conclusion, we did not observe the adjuvant effect of aluminum hydroxide associated with rSm22.6 in protective immunity.

Variability of the coat protein gene of Grapevine leafroll-associated virus 3 in Brazil

Leafroll is an economically important disease affecting grapevines (Vitis spp.). Nine serologically distinct viruses, Grapevine leafroll-associated virus-1 through 9, are associated with this disease. The present study describes the coat protein gene sequence of four GLRaV-3 isolates occurring in the São Francisco River basin, Northeastern Brazil. The viral RNA was extracted from GLRaV-3 ELISA-positive plants and the complete coat protein gene was amplified by RT-PCR. Sequences were generated automatically and compared to the complete coat protein sequence from North American (NY1) and Chinese (Dawanhong Nº2 and SL10) GLRaV-3 isolates. The four studied isolates, named Pet-1 through 4, showed deduced amino acid identities of 98-100% (Pet-1 through 3) and 95% (Pet-4) with North American and Chinese isolates. A total of seventeen amino acid substitutions was detected among the four characterized isolates in comparison to the NY1, Dawanhong No.2 and SL10 sequences. The results indicated the existence of natural variation among GLRaV-3 isolates from grapevines, also demonstrating a lack of correlation between sequence data and geographic origin. This variability should be considered when selecting regions of the viral genome targeted for reliable and consistent virus molecular detection.

Echovirus 4 associated to hand, foot and mouth disease

Hand, foot and mouth disease (HFMD) is a contagious enteroviral infection occurring primarily in children and characterized by vesicular palmoplantar eruptions and erosive stomatitis. Echovirus 4 (EV-4) has been commonly associated with aseptic meningitis. The association of HFMD with EV-4 has not been reported previously. Two samples of a 14-month child who presented mild fever, sores in the mouth, rash with blisters on the palm of hands and soles of feet were sent to Enteric Viruses Laboratory of Adolfo Lutz Institute. Clinical samples were inoculated in three different cell lines, and those which presented cytopathic effect (CPE), were submitted to Indirect Immunofluorescence Assay (IFA) and "one step" RT-PCR. Agarose gel electrophoresis from RT-PCR product, showed a product with 437 bp, which is characteristic of Enterovirus group. Echovirus 4 was identified by IFA. Although HFMD is a viral infection associated mainly with Enterovirus 71 (HEV-71) and Coxsackievirus A16 (CV-A16), our results demonstrate a diversity of serotype related to HFMD and stress the importance of epidemiological surveillance to this disease and its complications.

Hemophagocytic lymphohistiocytosis associated with H1N1 virus infection and visceral leishmaniasis in a 4.5-month-old infant

We present a case of a 4.5-month-old boy from Turkey with hemophagocytic lymphohistiocytosis (HLH) associated with H1N1 virus and Leishmania spp. coinfection. Because visceral leishmaniasis can mimic hematologic disorders like HLH, it is important to rule out this clinical condition before starting immunosuppressive therapy. In our case, treatment with liposomal amphotericin B resulted in a dramatic resolution of clinical and laboratory abnormalities.

Healthcare-Associated Infective Endocarditis: a Case Series in a Referral Hospital from 2006 to 2011

Background: Healthcare-associated infective endocarditis (HCA-IE), a severe complication of medical care, shows a growing incidence in literature. Objective: To evaluate epidemiology, etiology, risk factors for acquisition, complications, surgical treatment, and outcome of HCA-IE. Methods: Observational prospective case series study (2006-2011) in a public hospital in Rio de Janeiro. Results: Fifty-three patients with HCA-IE from a total of 151 cases of infective endocarditis (IE) were included. There were 26 (49%) males (mean age of 47 ± 18.7 years), 27 (51%) females (mean age of 42 ± 20.1 years). IE was acute in 37 (70%) cases and subacute in 16 (30%) cases. The mitral valve was affected in 19 (36%) patients and the aortic valve in 12 (36%); prosthetic valves were affected in 23 (43%) patients and native valves in 30 (57%). Deep intravenous access was used in 43 (81%) cases. Negative blood cultures were observed in 11 (21%) patients, Enterococcus faecalis in 10 (19%), Staphylococcus aureus in 9 (17%), and Candida sp. in 7 (13%). Fever was present in 49 (92%) patients, splenomegaly in 12 (23%), new regurgitation murmur in 31 (58%), and elevated C-reactive protein in 44/53 (83%). Echocardiograms showed major criteria in 46 (87%) patients, and 34 (64%) patients were submitted to cardiac surgery. Overall mortality was 17/53 (32%). Conclusion: In Brazil HCA-IE affected young subjects. Patients with prosthetic and native valves were affected in a similar proportion, and non-cardiac surgery was an infrequent predisposing factor, whereas intravenous access was a common one. S. aureus was significantly frequent in native valve HCA-IE, and overall mortality was high.

Sex Differences in High Sensitivity C-Reactive Protein in Subjects with Risk Factors of Metabolic Syndrome

Abstract Background: Metabolic syndrome (MetS) is associated with a higher risk of all-cause mortality. High-sensitivity C-reactive protein (hsCRP) is a prototypic marker of inflammation usually increased in MetS. Women with MetS-related diseases present higher hsCRP levels than men with MetS-related diseases, suggesting sex differences in inflammatory markers. However, it is unclear whether serum hsCRP levels are already increased in men and/or women with MetS risk factors and without overt diseases or under pharmacological treatment. Objective: To determine the impact of the number of MetS risk factors on serum hsCRP levels in women and men. Methods One hundred and eighteen subjects (70 men and 48 women; 36 ± 1 years) were divided into four groups according to the number of MetS risk factors: healthy group (CT; no risk factors), MetS Ͱ4; 2, MetS = 3, and MetS ≥ 4. Blood was drawn after 12 hours of fasting for measurement of biochemical variables and hsCRP levels, which were determined by immunoturbidimetric assay. Results: The groups with MetS risk factors presented higher serum hsCRP levels when compared with the CT group (p < 0.02). There were no differences in hsCRP levels among groups with MetS risk factors (p > 0.05). The best linear regression model to explain the association between MetS risk factors and hsCRP levels included waist circumference and HDL cholesterol (r = 0.40, p < 0.01). Women with MetS risk factors presented higher hsCRP levels when compared with men (psex < 0.01). Conclusions: Despite the absence of overt diseases and pharmacological treatment, subjects with MetS risk factors already presented increased hsCRP levels, which were significantly higher in women than men at similar conditions.

Activated peripheral lymphocytes with increased expression of cell adhesion molecules and cytotoxic markers are associated with dengue fever disease

The immune mechanisms involved in dengue fever and dengue hemorrhagic/dengue shock syndrome are not well understood. The ex vivo activation status of immune cells during the dengue disease in patients was examined. CD4and CD8 T cells were reduced during the acute phase. Interestingly, CD8 T cells co-expressing activation marker HLA-DR, Q, P, and cytolytic granule protein-Tia-1 were significantly higher in dengue patients than in controls. Detection of adhesion molecules indicated that in dengue patients the majority of T cells (CD4 and CD8) express the activation/memory phenotype, characterized as CD44HIGH and lack the expression of the naïve cell marker, CD62L LOW. Also, the levels of T cells co-expressing ICAM-1 (CD54), VLA-4, and LFA-1 (CD11a) were significantly increased. CD8 T lymphocytes expressed predominantly low levels of anti-apoptotic molecule Bcl-2 in the acute phase, possibly leading to the exhibition of a phenotype of activated/effector cells. Circulating levels of IL-18, TGF-b1 and sICAM-1 were significantly elevated in dengue patients. Early activation events occur during acute dengue infection which might contribute to viral clearance. Differences in expression of adhesion molecules among CD4 and CD8 T cells might underlie the selective extravasation of these subsets from blood circulation into lymphoid organs and/or tissues. In addition, activated CD8 T cells would be more susceptible to apoptosis as shown by the alteration in Bcl-2 expression. Cytokines such as IL-18, TGF-b1, and sICAM-1 may be contributing by either stimulating or suppressing the adaptative immune response, during dengue infection, thereby perhaps establishing a relationship with disease severity.

Antigenic extracts of Leishmania braziliensis and Leishmania amazonensis associated with saponin partially protects BALB/c mice against Leishmania chagasi infection by suppressing IL-10 and IL-4 production

This study evaluated two vaccine candidates for their effectiveness in protecting BALB/c mice against Leishmania chagasiinfection. These immunogenic preparations were composed of Leishmania amazonensisor Leishmania braziliensisantigenic extracts in association with saponin adjuvant. Mice were given three subcutaneous doses of one of these vaccine candidates weekly for three weeks and four weeks later challenged with promastigotes of L. chagasiby intravenous injection. We observed that both vaccine candidates induced a significant reduction in the parasite load of the liver, while the L. amazonensisantigenic extract also stimulated a reduction in spleen parasite load. This protection was associated with a suppression of both interleukin (IL)-10 and IL-4 cytokines by spleen cells in response to L. chagasiantigen. No change was detected in the production of IFN- 47;. Our data show that these immunogenic preparations reduce the type 2 immune response leading to the control of parasite replication.

Trypanosoma rangeli protein tyrosine phosphatase is associated with the parasite's flagellum

Protein tyrosine phosphatases (PTPs) play an essential role in the regulation of cell differentiation in pathogenic trypanosomatids. In this study, we describe a PTP expressed by the non-pathogenic protozoan Trypanosoma rangeli (TrPTP2). The gene for this PTP is orthologous to the T. brucei TbPTP1 and Trypanosoma cruzi (TcPTP2) genes. Cloning and expression of the TrPTP2 and TcPTP2 proteins allowed anti-PTP2 monoclonal antibodies to be generated in BALB/c mice. When expressed by T. rangeli epimastigotes and trypomastigotes, native TrPTP2 is detected as a ~65 kDa protein associated with the parasite's flagellum. Given that the flagellum is an important structure for cell differentiation in trypanosomatids, the presence of a protein responsible for tyrosine dephosphorylation in the T. rangeli flagellum could represent an interesting mechanism of regulation in this structure.

Recombinant hepatitis C virus-envelope protein 2 interactions with low-density lipoprotein/CD81 receptors

Hepatitis C virus (HCV) envelope protein 2 (E2) is involved in viral binding to host cells. The aim of this work was to produce recombinant E2B and E2Y HCV proteins in Escherichia coli and Pichia pastoris, respectively, and to study their interactions with low-density lipoprotein receptor (LDLr) and CD81 in human umbilical vein endothelial cells (HUVEC) and the ECV304 bladder carcinoma cell line. To investigate the effects of human LDL and differences in protein structure (glycosylated or not) on binding efficiency, the recombinant proteins were either associated or not associated with lipoproteins before being assayed. The immunoreactivity of the recombinant proteins was analysed using pooled serum samples that were either positive or negative for hepatitis C. The cells were immunophenotyped by LDLr and CD81 using flow cytometry. Binding and binding inhibition assays were performed in the presence of LDL, foetal bovine serum (FCS) and specific antibodies. The results revealed that binding was reduced in the absence of FCS, but that the addition of human LDL rescued and increased binding capacity. In HUVEC cells, the use of antibodies to block LDLr led to a significant reduction in the binding of E2B and E2Y. CD81 antibodies did not affect E2B and E2Y binding. In ECV304 cells, blocking LDLr and CD81 produced similar effects, but they were not as marked as those that were observed in HUVEC cells. In conclusion, recombinant HCV E2 is dependent on LDL for its ability to bind to LDLr in HUVEC and ECV304 cells. These findings are relevant because E2 acts to anchor HCV to host cells; therefore, high blood levels of LDL could enhance viral infectivity in chronic hepatitis C patients.

High interleukin-4 expression and interleukin-4 gene polymorphisms are associated with susceptibility to human paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is caused by dimorphic fungi from theParacoccidioides brasiliensis complex. Previous studies have demonstrated that the severity of disease is associated with a T-helper 2 immune response characterised by high interleukin (IL)-4 production. In the present study we analysed two polymorphisms in the IL-4gene (-590 C/T and intron-3 microsatellite) in 76 patients with PCM and 73 control subjects from an endemic area. The production of IL-4 by peripheral blood mononuclear cells after antigen or phytohaemagglutinin stimulation was determined by ELISA. A significant correlation was observed between the RP2/RP2 intron-3 genotype and infection with Paracoccidioides sp.(p = 0.011), whereas the RP1/RP1 genotype was correlated with resistance. No significant correlation was observed for the IL-4promoter polymorphism. Furthermore, the low IL-4 expression observed in the control group compared with patients was associated with the RP1/RP1 genotype. These results suggest that IL-4polymorphisms might be associated with the ability of the host to control Paracoccidioides sp.infection. The relevance of this polymorphism is supported by the observation that patients with disease produce high levels of IL-4 following mitogen or antigen stimulation. The IL-4gene is located in the cytokine cluster region of chromosome 5 where other polymorphisms have also been described.

Genetic diversity of begomoviruses infecting soybean, bean and associated weeds in Northwestern Argentina

The subtropical Northwestern region of Argentina (provinces of Tucumán, Salta, Jujuy, Santiago del Estero and Catamarca) suffers from a high incidence of the whitefly Bemisia tabaci, and the detection of begomoviruses is also common. The Northwest is the main bean-growing region of the country, and approximately 10% of Argentina's soybean crop is grown in this area. We have used a PCR-based assay to establish the identity and genetic diversity of begomoviruses associated with bean and soybean crops in Northwestern Argentina. Universal begomovirus primers were used to direct the amplification of a fragment encompassing the 5' portion of the capsid protein gene. Amplified fragments were cloned, sequenced and subjected to phylogenetic analysis to determine the sequence identity to known begomoviruses. The data indicated the presence of four distinct begomoviruses, all related to other New World begomoviruses. The prevalent virus, which was present in 94% of bean and soybean samples and also in two weed species, is closely related to Sida mottle virus (SiMoV). A virus with high sequence identity with Bean golden mosaic virus (BGMV) was found in beans. The two remaining viruses displayed less than 89% identity with other known begomoviruses, indicating that they may constitute novel species. One of these putative novel viruses was detected in bean, soybean and tomato samples.

Expression of Grapevine leafroll-associated virus 3 coat protein gene in Escherichia coli and production of polyclonal antibodies

Grapevine leafroll-associated virus 3 (GLRaV-3), the main viral species of the grapevine leafroll complex, causes yield and quality reduction in grapes (Vitis spp.). The coat protein gene was RT-PCR-amplified from total RNA extracted from infected grapevine leaves and the amplified fragment was cloned and completely sequenced. The fragment was subsequently subcloned into the pRSET-C expression vector. The recombinant plasmid was used to transform Escherichia coli BL21:DE3 and express the capsid protein. The coat protein, fused to a 6 His-tag, was purified by affinity chromatography using an Ni-NTA resin. The identity of the purified protein was confirmed by SDS-PAGE and Western blot. The in vitro-expressed protein was quantified and used for rabbit immunizations. The antiserum was shown to be sensitive and specific for the detection of GLRaV-3 in grapevine extracts in Western blot and DAS-ELISA assays, with no unspecific or heterologous reactions against other non-serologically related viruses being observed.

Sequence diversity in the coat protein gene of Lettuce big-vein associated virus and Mirafiori lettuce big-vein virus infecting lettuce in Brazil

Lettuce big vein associated virus (LBVaV) and Mirafiori lettuce big vein virus (MLBVV) have been found in mixed infection in Brazil causing the lettuce big vein disease. Analysis of part of the coat protein (CP) gene of Brazilian isolates of LBVaV collected from lettuce, showed at least 93% amino acid sequence identity with other LBVaV isolates. Genetic diversity among MLBVV CP sequences was higher when compared to LBVaV CP sequences, with amino acid sequence identity ranging between 91% to 100%. Brazilian isolates of MLBVV belong to subgroup A, with one RsaI restriction site on the coat protein gene. There is no indication for a possible geografical origin for the Brazilian isolates of LBVaV and MLBVV.

Single nucleotide polymorphisms at 15 codons of the prion protein gene from a scrapie-affected herd of Suffolk sheep in Brazil

Scrapie is a transmissible spongiform encephalopathy of sheeps and goats, associated with the deposition of a isoform of the prion protein (PrPsc). This isoform presents an altered conformation that leads to aggregation in the host's central nervous and lymphoreticular systems. Predisposition to the prion agent infection can be influenced by specific genotypes related to mutations in amino acids of the PrPsc gene. The most characterized mutations occur at codons 136, 154 and 171, with genotypes VRQ being the most susceptible and ARR the most resistant. In this study we have analyzed polymorphisms in 15 different codons of the PrPsc gene in sheeps from a Suffolk herd from Brazil affected by an outbreak of classical scrapie. Amplicons from the PrPsc gene, encompassing the most relevant altered codons in the protein, were sequenced in order to determine each animal's genotype. We have found polymorphisms at 3 of the 15 analyzed codons (136, 143 and 171). The most variable codon was 171, where all described alleles were identified. A rare polymorphism was found at the 143 codon in 4% of the samples analyzed, which has been described as increasing scrapie resistance in otherwise susceptible animals. No other polymorphisms were detected in the remaining 12 analyzed codons, all of them corresponding to the wild-type prion protein. Regarding the risk degree of developing scrapie, most of the animals (96%) had genotypes corresponding to risk groups 1 to 3 (very low to moderate), with only 4% in the higher risks group. Our data is discussed in relation to preventive measures involving genotyping and positive selection to control the disease.