Thogoto virus infection induces sustained type I interferon responses that depend on RIG-I-like helicase signaling of conventional dendritic cells.

Type I interferon (IFN-α/β) induction upon viral infection contributes to the early antiviral host defense and ensures survival until the onset of adaptive immunity. Many viral infections lead to an acute, transient IFN expression which peaks a few hours after infection and reverts to initial levels after 24 to 36 h. Robust IFN expression often is conferred by specialized plasmacytoid dendritic cells (pDC) and may depend on positive-feedback amplification via the type I IFN receptor (IFNAR). Here, we show that mice infected with Thogoto virus (THOV), which is an influenza virus-like orthomyxovirus transmitted by ticks, mounted sustained IFN responses that persisted up to 72 h after infection. For this purpose, we used a variant of THOV lacking its IFN-antagonistic protein ML, an elongated version of the matrix (M) protein [THOV(ΔML)]. Of note, large amounts of type I IFN were also found in the serum of mice lacking the IFNAR. Early IFN-α expression seemed to depend on Toll-like receptor (TLR) signaling, whereas prolonged IFN-α responses strictly depended on RIG-I-like helicase (RLH) signaling. Unexpectedly, THOV(ΔML)-infected bone marrow-derived pDC (BM-pDC) produced only moderate IFN levels, whereas myeloid DC (BM-mDC) showed massive IFN induction that was IPS-1-dependent, suggesting that BM-mDC are involved in the massive, sustained IFN production in THOV(ΔML)-infected animals. Thus, our data are compatible with the model that THOV(ΔML) infection is sensed in the acute phase via TLR and RLH systems, whereas at later time points only RLH signaling is responsible for the induction of sustained IFN responses.

Complete genetic characterization of a Brazilian dengue virus type 3 strain isolated from a fatal outcome

We have determined the complete nucleotide and the deduced amino acid sequences of Brazilian dengue virus type 3 (DENV-3) from a dengue case with fatal outcome, which occurred during an epidemic in the state of Rio de Janeiro, Brazil, in 2002. This constitutes the first complete genetic characterization of a Brazilian DENV-3 strain since its introduction into the country in 2001. DENV-3 was responsible for the most severe dengue epidemic in the state, based on the highest number of reported cases and on the severity of clinical manifestations and deaths reported.

Seroepidemiological study of herpes simplex virus type 2 in patients with the acquired immunodeficiency syndrome in the city of Niterói, Rio de Janeiro, Brazil

This study was designed to determine the seroprevalence of herpes simplex virus type 2 (HSV-2) and to evaluate its association with age, sex as well as other demographic and behavioural factors in 150 human immunodeficiency virus (HIV) positive adults patients attending the general medical outpatient ward for routine care of Niterói, state of Rio de Janeiro, Brazil. Serum samples were screened for HSV-2 antibodies using an indirect ELISA. Eighty-three patients were men (mean age: 38.8) and 67 were women (mean age: 35.4). The estimated prevalence of HSV-2 was 52% (95% CI: 44-60%) and it was higher among men (53%) than among women (50.7%). Overall, the age of first sexual intercourse and past history of genital herpes were associated with HSV-2 seropositivity. Analysis by gender disclosed significant association of number of lifetime sex partners only among men. Although HSV-2 antibodies were frequent in the study group, genital herpes was reported by 21.8% of the HSV-2 positive subjects, indicating low awareness of the HSV-2 infection. These results may have public health importance for Brazil as the high rate of HSV-2 infection may act as a cofactor of HIV transmission.

Overlapping, additive and counterregulatory effects of type II and I interferons on myeloid dendritic cell functions.

Dendritic cells (DCs) are central player in immunity by bridging the innate and adaptive arms of the immune system (IS). Interferons (IFNs) are one of the most important factors that regulate both innate and adaptive immunity too. Thus, the understanding of how type II and I IFNs modulate the immune-regulatory properties of DCs is a central issue in immunology. In this paper, we will address this point in the light of the most recent literature, also highlighting the controversial data reported in the field. According to the wide literature available, type II as well as type I IFNs appear, at the same time, to collaborate, to induce additive effects or overlapping functions, as well as to counterregulate each one's effects on DC biology and, in general, the immune response. The knowledge of these effects has important therapeutic implications in the treatment of infectious/autoimmune diseases and cancer and indicates strategies for using IFNs as vaccine adjuvants and in DC-based immune therapeutic approaches.

Bovine papillomavirus type 2 detection in the urinary bladder of cattle with chronic enzootic haematuria

The bovine papillomavirus type 2 (BPV-2) involvement in the aetiology of chronic enzootic haematuria associated to bracken fern ingestion has been suggested for a long time. However, a few reports have shown the presence of the BPV-2 in urinary bladder tumors of cattle. The aim of this study was to investigate the presence of the BPV-2 infection in the urinary bladder of cattle with chronic enzootic haematuria in Brazilian cattle herds. Sixty-two urinary bladders were collected from adult cattle in beef herds from the north region of the state of Paraná, Brazil. According to clinical and pathological finds the specimens were distributed in three groups: the group A was constituted by 22 urinary bladders with macroscopic lesions collected at necropsy of cattle with clinical signs of chronic enzootic haematuria; the group B by 30 urinary bladders with macroscopic lesions collected in a slaughterhouse of cows coming from bracken fern-endemic geographical region; and the group C (control) by 10 urinary bladders without macroscopic lesions collected from asymptomatic cattle in a bracken fern-free geographical region. By a semi-nested polymerase chain reaction (PCR) assay, with an internal control, a fragment of the BPV-2 L1 gene with 386 bp length was amplified in 36 (58%) urinary bladder. The rate of BPV-2 positive urinary bladders was 50% (11/22) for group A, 80% (24/30) for group B, and 10% (1/10) for group C (control). The rate of the positive results found in groups A and B that included urinary bladder samples with macroscopic lesions was 67% (35/52) and the detection of the BPV-2 in both groups was significantly higher (P < 0.05) than in the control group. RFLP with Rsa I and Hae III enzymes evaluated the specificity of the BPV-2 amplicons. The PCR internal control that amplified a 626 bp fragment of the ND5 gene of the bovine mitochondrial genome was amplified in all analyzed samples and excluded false-negatives or invalid results in the semi-nested PCR. These results suggest the BPV-2 involvement in the chronic enzootic haematuria aetiology and open the perspective of the development of new strategies for the control of this disease that is the major cause of economical losses in beef herds from many Brazilian geographical regions.

Identification and molecular characterization of Van A-type vancomycin-resistant Enterococcus faecalis in Northeast of Brazil

The isolation of vancomycin resistant enterococci (VRE) in Brazil has rapidly increased, following the world wide tendency. We report in the present study the first isolation of vancomycin resistant Enterococcus faecalis (VRE) in the Northeast of Brazil. The four VRE isolates were characterized for antimicrobial susceptibility, genotypic typing by macro restriction of chromosomal DNA followed by pulsed-field gel electrophoresis and for characterization of the Tn1546-like element and plasmid contents. The isolates showed resistance to multiple antibiotics and a single genotype profile, suggesting the dissemination of a single clone among the patients. Tn1546 associated to genetic elements as plasmids shows the importance of infection control measures to avoid the spreading of glycopetide resistance by conjugative transfer of VanA elements.

Type 2 Diabetes for people with a Learning Disability

�� Pictorial information about type 2 diabetes for people with a learning disability.

Primary resistance of human immunodeficiency virus type 1 in a reference center in Recife, Pernambuco, Brazil

To assess the prevalence of primary resistance of human immunodeficiency virus type 1 (HIV-1) to antiretrovirals, 84 patients chronically infected with HIV without prior antiretroviral treatment from Northeast Brazil were studied. Genotyping was performed using the ViroSeqTM Genotyping System. Thimidine analog mutations occurred in 3 (3.6%) patients. Accessory mutations related to NRTI occurred in 6 (7.1%) and related to PI in 67 (79.8%). Subtypes B (72.6%), F (22.6%), B/F 3 (3.6%), and C (1.2%) were detected. A low prevalence of major mutations related to NRTI in patients chronically infected by HIV was observed.

Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli

Strains of enterotoxigenic Escherichia coli (ETEC) are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT) were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.

Human immunodeficiency virus type- 1 subtypes of infected patients in Espírito Santo, Brazil

Genetic variability of human immunodeficiency virus type - 1(HIV-1) is a potential threat for both diagnosis and treatment of HIV/AIDS, as well as the development of effective vaccines. Up to now, HIV subtypes circulating among HIV-positive patients in the state of Espírito Santo were not known. In the present study, blood samples from 100 therapy-naïve HIV-1 infected patients were collected and the HIV subtype was determined through the Heteroduplex Mobility Assay (HMA). Ninety-seven out of 100 studied samples were subtyped by HMA, 73 samples (75.2%) were from subtype B, 9 (9.3%) from subtype F, 3 (3.1%) from subtype C, 6 (6.2%) Benv/Fgag, and another 6 (6.2%) Fenv/Bgag, what suggests that recombinant viruses were present in the studied samples. Twenty-eight percent of the subtype B samples were represented by the Brazilian B" subtype, which were identified by RFLP with Fok I. Data presented here demonstrate that the epidemiological characteristics of the HIV epidemic in the state of Espírito Santo are similar to those from the other Southeastern states and helped to better understand the genetic polymorphism of HIV in Brazil.

Concurrent infection with dengue virus type-2 and DENV-3 in a patient from Ceará, Brazil

Dengue outbreaks have occurred in several regions in Brazil and cocirculating dengue virus type 1 (DENV-1), DENV-2, and DENV-3 have been frequently observed. Dual infection by DENV-2 and DENV-3 was identified by type-specific indirect immunofluorescence assay and confirmed by reverse transcription polymerase chain reaction in a patient in Ceará with a mild disease. This is the first documented case of simultaneous infection with DENV-2 and DENV-3 in Brazil. Sequencing confirmed DENV-2 and DENV-3 (South-East/American) genotype III and (SriLanka/India), genotype III respectively.

N-Terminal Modifications Improve the Receptor Affinity and Pharmacokinetics of Radiolabeled Peptidic Gastrin-Releasing Peptide Receptor Antagonists: Examples of 68Ga- and 64Cu-Labeled Peptides for PET Imaging.

Gastrin-releasing peptide receptors (GRPrs) are overexpressed on a variety of human cancers, providing the opportunity for peptide receptor targeting via radiolabeled bombesin-based peptides. As part of our ongoing investigations into the development of improved GRPr antagonists, this study aimed at verifying whether and how N-terminal modulations improve the affinity and pharmacokinetics of radiolabeled GRPr antagonists. METHODS: The potent GRPr antagonist MJ9, Pip-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (Pip, 4-amino-1-carboxymethyl-piperidine), was conjugated to 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA), and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and radiolabeled with (68)Ga and (64)Cu. The GRPr affinity of the corresponding metalloconjugates was determined using (125)I-Tyr(4)-BN as a radioligand. The labeling efficiency of (68)Ga(3+) was compared between NODAGA-MJ9 and NOTA-MJ9 in acetate buffer, at room temperature and at 95°C. The (68)Ga and (64)Cu conjugates were further evaluated in vivo in PC3 tumor xenografts by biodistribution and PET imaging studies. RESULTS: The half maximum inhibitory concentrations of all the metalloconjugates are in the high picomolar-low nanomolar range, and these are the most affine-radiolabeled GRPr antagonists we have studied so far in our laboratory. NODAGA-MJ9 incorporates (68)Ga(3+) nearly quantitatively (>98%) at room temperature within 10 min and at much lower peptide concentrations (1.4 × 10(-6) M) than NOTA-MJ9, for which the labeling yield was approximately 45% under the same conditions and increased to 75% at 95°C for 5 min. Biodistribution studies showed high and specific tumor uptake, with a maximum of 23.3 ± 2.0 percentage injected activity per gram of tissue (%IA/g) for (68)Ga-NOTA-MJ9 and 16.7 ± 2.0 %IA/g for (68)Ga-NODAGA-MJ9 at 1 h after injection. The acquisition of PET images with the (64)Cu-MJ9 conjugates at later time points clearly showed the efficient clearance of the accumulated activity from the background already at 4 h after injection, whereas tumor uptake still remained high. The high pancreas uptake for all radiotracers at 1 h after injection was rapidly washed out, resulting in an increased tumor-to-pancreas ratio at later time points. CONCLUSION: We have developed 2 GRPr antagonistic radioligands, which are improved in terms of binding affinity and overall biodistribution profile. Their promising in vivo pharmacokinetic performance may contribute to the improvement of the diagnostic imaging of tumors overexpressing GRPr.

Low plasma lactate concentration as a biomarker of an incompetent brain-pull: a risk factor for weight gain in type 2 diabetes patients.

OBJECTIVE: Body weight development is closely regulated by central nervous mechanisms. As has been demonstrated recently, the capability of the brain to actively demand energy from the body (brain-pull) is indispensable for the maintenance of systemic homeostasis. A deficit in this brain-pull may result in compensatory ingestive behavior followed by weight gain in the medium or long term. The aim of this study was to establish a biomarker of such an incompetent brain-pull. Since lactate is an alternative cerebral energy substrate to glucose, we investigated whether low fasting plasma lactate concentrations are associated with weight gain and increased feelings of hunger in patients with type 2 diabetes over a 3-year period. METHODS: In a population based cohort study 134 type 2 diabetes patients were examined at baseline and 3-year follow-up. Plasma lactate concentrations and additional hormones associated with food intake such as e.g. insulin, or leptin, as well as psychological variables like hunger feelings before and after a standardized breakfast were measured. The relation between fasting plasma lactate concentrations and postprandial hunger as well as follow-up weight was analyzed. RESULTS: Low fasting plasma lactate concentrations predicted a higher 3-year follow-up weight (B=-1.268, SE=0.625, p=0.04). Moreover, low fasting plasma lactate concentrations were associated with more pronounced feelings of postprandial hunger (B=-0.406, SE=0.137, p<0.01). CONCLUSIONS: We conclude that low plasma lactate concentrations may represent a biomarker of an incompetent brain-pull, which is associated with weight gain and increased postprandial hunger in patients with type 2 diabetes mellitus. These results are in line with the view that plasma lactate can be used by the brain as an alternative energy substrate and thereby to some extent prevent overeating and obesity.

The influence of cationic lipid type on in-vitro release kinetic profiles of antisense oligonucleotide from cationic nanoemulsions.

Novel formulations of cationic nanoemulsions based on three different lipids were developed to strengthen the attraction of the polyanionic oligonucleotide (ODN) macromolecules to the cationic moieties on the oil nanodroplets. These formulations were developed to prolong the release of the ODN from the nanoemulsion under appropriate physiological dilutions as encountered in the eye following topical application. Increasing the concentration of the new cationic lipid exhibiting two cationic amine groups (AOA) in the emulsion from 0.05% to 0.4% did not alter markedly the particle size or zeta potential value of the blank cationic nanoemulsion. The extent of ODN association did not vary significantly when the initial concentration of ODN remained constant at 10 microM irrespective of the cationic lipid nature. However, the zeta potential value dropped consistently with the low concentrations of 0.05% and 0.1% of AOA in the emulsions suggesting that an electrostatic attraction occurred between the cationic lipids and the polyanionic ODN molecules at the o/w interface. Only the nanoemulsion prepared with N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salts (DOTAP) remained physically stable over time. DOTAP cationic lipid nanoemulsion was the most efficient formulation capable of retaining the ODN despite the high dilution of 1:100 with simulated tear solution (STS). Less than 10% of the ODN was exchanged in contrast to 40-50% with the other cationic nanoemulsions. The in-vitro release kinetic behavior of ODN exchange with physiological anions present in the STS appears to be complex and difficult to characterize using mathematical fitting model equations. Further pharmacokinetic studies are needed to verify our kinetic assumptions and confirm the in-vitro ODN release profile from DOTAP cationic nanoemulsions.