Characterization of tumor antigen specific CD8+ T cells and semi-invariant Vα24/Vβ11 NKT cells in tumor invaded liver

Summary: Detailed knowledge on tumor antigen expression and specific immune cells is required for a rational design of immunotherapy for patients with tumor invaded liver. In this study, we confirmed that Cancer/Testis (CT) tumor-associated antigens are frequently expressed in hepatocellular carcinoma (HCC) and searched for the presence of CD8+ T cells specific for these antigens. In 2/10 HLA-A2+ patients with HCC, we found that MAGE-A10 and/or SSX-2 specific CD8+ T cells naturally responded to the disease, since they were enriched in tumor lesions but not in non-tumoral liver. Isolated T cells specifically and strongly killed tumor cells in vitro, suggesting that these CTL were selected in vivo for high avidity antigen recognition, providing the rational for specific immunotherapy of HCC, based on immunization with CT antigens such as MAGE-Al 0 and SSX-2. Type 1 NKT cells express an invariant TCR α chain (Vα24.1α18, paired with Vβ11 in human) and share a specific reactivity to αGalactosylceramide (αGC) presented by CD1d. These cells can display paradoxical immuno-regulatory properties including strong anti-tumor effects upon αGC administration in murine models. To understand why NKT cells were not sufficiently protective against tumor development in patients with tumor invaded liver, we characterized the diversity of Vα24/Vβ11 NKT cells in healthy donors (HD) and cancer patients: NKT cells from HD and patients were generally diverse in terms of TCR β chain (Vβ11) variability and NKT cells from HD showed a variable recognition of αGC loaded CD 1 d multimers. Vα24/ Vβ11 NKT cells can be divided in 3 populations, the CD4, DN (CD4-/CD8-) and CD8 NKT cell subsets that show distinct ability of cytokine production. In addition, our functional analysis revealed that DN and CD8 subsets displayed a higher cytolytic potential and a weaker IFNγ release than the CD4 NKT cell subset. NKT cell subsets were variably represented in the blood of HD and cancer patients. However, HD with high NKT cell frequencies displayed an enrichment of the DN and CD8 subsets, and few of them were suggestive of an oligoclonal expansion in vivo. Comparable NKT cell frequencies were found between blood, non-tumoral liver and tumor of patients. In contrast, we identified a gradual enrichment of CD4 NKT cells from blood to the liver and to the tumor, together with a decrease of DN and CD8 NKT cell subsets. Most patient derived NKT cells were unresponsive upon αGalactosylceramide stimulation ex vivo; NKT cells from few patients displayed a weak responsiveness with different cytokine polarization. The NKT cell repertoire was thus different in tumor tissue, suggesting that CD4 NKT cells infiltrating tumors may be detrimental for protection against tumors and instead may favour the tumor growth/recurrence as recently reported in mice. Résumé en français scientifique : Afin de développer le traitement des patients porteurs d'une tumeur dans le foie par immunothérapie, de nouvelles connaissances sont requises concernant l'expression d'antigènes par les tumeurs et les cellules immunitaires spécifiques de ces antigènes. Nous avons vérifié que des antigènes associés aux tumeurs, tels que les antigènes « Cancer-Testis » (CT), sont fréquemment exprimés par le carcinome hepatocéllulaire (CHC). La recherche de lymphocytes T CD8+ spécifiques (CTL) de ces antigènes a révélé que des CTL spécifiques de MAGE-A10 et/ou SSX-2 ont répondu naturellement à la tumeur chez 2/10 patients étudiés. Ces cellules étaient présentes dans les lésions tumorales mais pas dans le foie adjacent. De plus, ces CTL ont démontré une activité cytolytique forte et spécifique contre les cellules tumorales in vitro, ce qui suggère que ces CTL ont été sélectionnés pour une haute avidité de reconnaissance de l'antigène in vivo. Ces données fournissent une base pour l'immunothérapie spécifique du CHC, en proposant de cibler les antigènes CT tels que MAGE-A10 ou SSX-2. Les cellules NKT de type 1 ont une chaîne α de TCR qui est invariante (chez l'homme, Vα24Jα18, apparié avec Vβ11) et reconnaissent spécifiquement l'αGalactosylceramide (αGC) présenté par CD1d. Ces cellules ont des propriétés immuno¬régulatrices qui peuvent être parfois contradictoires et leur activation par l'αGC induit une forte protection anti-tumorale chez la souris: Afin de comprendre pourquoi ces cellules ne sont pas assez protectrices contre le développement des tumeurs dans le foie chez l'homme, nous avons étudié la diversité des cellules NKT Vα24/Vβ11 d'individus sains (IS) et de patients cancéreux. Les cellules NKT peuvent être sous-divisées en 3 populations : Les CD4, DN (CD4- /CD8-) ou CDS, qui ont la capacité de produire des cytokines différentes. Nos analyses fonctionnelles ont aussi révélé que les sous-populations DN et CD8 ont un potentiel cytolytique plus élevé et une production d'IFNγ plus faible que la sous-population CD4. Ces sous-populations sont représentées de manière variable dans le sang des IS ou des patients. Cependant, les IS avec un taux élevé de cellules NKT ont un enrichissement des sous- populations DN ou CDS, et certains suggèrent qu'il s'agit d'une expansion oligo-clonale in vivo. Les patients avaient des fréquences comparables de cellules NKT entre le sang, le foie et la tumeur. Par contre, la sous-population CD4 était progressivement enrichie du sang vers le foie et la tumeur, tandis que les sous-populations DN ou CD8 était perdues. La plupart des cellules NKT des patients ne réagissaient pas lors de stimulation avec l'αGC ex vivo et les cellules NKT de quelques patients répondaient faiblement et avec des polarisations de cytokines différentes. Ces données suggèrent que les cellules NKT CD4, prédominantes dans les tumeurs, sont inefficaces pour la lutte anti-tumorale et pourraient même favoriser la croissance ou la récurrence tumorale. Donc, une mobilisation spécifique des cellules NKT CD4 négatives par immunothérapie pourrait favoriser l'immunité contre des tumeurs chez l'homme. Résumé en français pour un large public Au sein des globules blancs, les lymphocytes T expriment un récepteur (le TCR), qui est propre à chacun d'entre eux et leur permet d'accrocher de manière très spécifique une molécule appelée antigène. Ce TCR est employé par les lymphocytes pour inspecter les antigènes associés avec des molécules présentatrices à la surface des autres cellules. Les lymphocytes T CD8 reconnaissent un fragment de protéine (ou peptide), qui est présenté par une des molécules du Complexe Majeur d'Histocompatibilité de classe I et tuent la cellule qui présente ce peptide. Ils sont ainsi bien adaptés pour éliminer les cellules qui présentent un peptide issu d'un virus quand la cellule est infectée. D'autres cellules T CD8 reconnaissent des peptides comme les antigènes CT, qui sont produits anormalement par les cellules cancéreuses. Nous avons confirmé que les antigènes CT sont fréquemment exprimés par le cancer du foie. Nous avons également identifié des cellules T CD8 spécifiques d'antigènes CT dans la tumeur, mais pas dans le foie normal de 2 patients sur 10. Cela signifie que ces lymphocytes peuvent être naturellement activés contre la tumeur et sont capables de la trouver. De plus les lymphocytes issus d'un patient ont démontré une forte sensibilité pour reconnaître l'antigène et tuent spécifiquement les cellules tumorales. Les antigènes CT représentent donc des cibles intéressantes qui pourront être intégrés dans des vaccins thérapeutiques du cancer du foie. De cette manière, les cellules T CD8 du patient lui-même pourront être induites à détruire de manière spécifique les cellules cancéreuses. Un nouveau type de lymphocytes T a été récemment découvert: les lymphocytes NKT. Quand ils reconnaissent un glycolipide présenté par la molécule CD1d, ils sont capables, de manière encore incomprise, d'initier, d'augmenter, ou à l'inverse d'inhiber la défense immunitaire. Ces cellules NKT ont démontré qu'elles jouent un rôle important dans la défense contre les tumeurs et particulièrement dans le foie des souris. Nous avons étudié les cellules NKT de patients atteints d'une tumeur dans le foie, afin de comprendre pourquoi elles ne sont pas assez protectrice chez l'homme. Les lymphocytes NKT peuvent être sous-divisés en 3 populations: Les CD4, les DN (CD4-/CD8-) et les CD8. Ces 3 classes de NKT peuvent produire différents signaux chimiques appelés cytokines. Contrairement aux cellules NKT DN ou CDS, seules les cellules NKT CD4 sont capables de produire des cytokines qui sont défavorables pour la défense anti-tumorale. Par ailleurs nous avons trouvé que les cellules NKT CD4 tuent moins bien les cellules cancéreuses que les cellules NKT DN ou CD8. L'analyse des cellules NKT, fraîchement extraites du sang, du foie et de la tumeur de patients a révélé que les cellules NKT CD4 sont progressivement enrichies du sang vers le foie et la tumeur. La large prédominance des NKT CD4 à l'intérieur des tumeurs suggère que, chez l'homme, ces cellules sont inappropriées pour la lutte anti-tumorale. Par ailleurs, la plupart des cellules NKT de patients n'étaient pas capables de produire des cytokines après stimulation avec un antigène. Cela explique également pourquoi ces cellules ne protègent pas contre les tumeurs dans le foie.

Phenotypic and molecular characterization of proliferating and differentiated GnRH-expressing GnV-3 cells.

GnRH neurons provide the primary driving force upon the neuroendocrine reproductive axis. Here we used GnV-3 cells, a model of conditionally immortalized GnRH-expressing neurons, to perform an analysis of cell cycle and compare the gene expression profile of proliferating cells with differentiated cells. In the proliferation medium, 45 ± 1.5% of GnV-3 cells are in S-phase by FACS analysis. In the differentiation medium, only 9 ± 0.9% of them are in S-phase, and they acquire the characteristic bipolar shape displayed by preoptic GnRH neurons in vivo. In addition, GnV-3 cells in the differentiated state exhibit electrophysiological properties characteristic of neurons. Transcriptomic analysis identified up-regulation of 1931 genes and down-regulation of 1270 genes in cells grown in the differentiation medium compared to cells in the proliferation medium. Subsequent gene ontology study indicated that genes over-expressed in proliferating GnV-3 cells were mainly involved in cell cycle regulations, whereas genes over-expressed in differentiated cells were mainly involved in processes of differentiation, neurogenesis and neuronal morphogenesis. Taken together, these data demonstrate the occurrence of morphological and physiological changes in GnV-3 cells between the proliferating and the differentiated state. Moreover, the genes differentially regulated between these two different states are providing novel pathways potentially important for a better understanding of the physiology of mature GnRH neurons.

A comprehensive characterization of genome-wide copy number aberrations in colorectal cancer reveals novel oncogenes and patterns of alterations.

To develop a comprehensive overview of copy number aberrations (CNAs) in stage-II/III colorectal cancer (CRC), we characterized 302 tumors from the PETACC-3 clinical trial. Microsatellite-stable (MSS) samples (n = 269) had 66 minimal common CNA regions, with frequent gains on 20 q (72.5%), 7 (41.8%), 8 q (33.1%) and 13 q (51.0%) and losses on 18 (58.6%), 4 q (26%) and 21 q (21.6%). MSS tumors have significantly more CNAs than microsatellite-instable (MSI) tumors: within the MSI tumors a novel deletion of the tumor suppressor WWOX at 16 q23.1 was identified (p<0.01). Focal aberrations identified by the GISTIC method confirmed amplifications of oncogenes including EGFR, ERBB2, CCND1, MET, and MYC, and deletions of tumor suppressors including TP53, APC, and SMAD4, and gene expression was highly concordant with copy number aberration for these genes. Novel amplicons included putative oncogenes such as WNK1 and HNF4A, which also showed high concordance between copy number and expression. Survival analysis associated a specific patient segment featured by chromosome 20 q gains to an improved overall survival, which might be due to higher expression of genes such as EEF1B2 and PTK6. The CNA clustering also grouped tumors characterized by a poor prognosis BRAF-mutant-like signature derived from mRNA data from this cohort. We further revealed non-random correlation between CNAs among unlinked loci, including positive correlation between 20 q gain and 8 q gain, and 20 q gain and chromosome 18 loss, consistent with co-selection of these CNAs. These results reinforce the non-random nature of somatic CNAs in stage-II/III CRC and highlight loci and genes that may play an important role in driving the development and outcome of this disease.

In vitro characterization of sunitinib eluting beads.

Purpose: To load embolization particles (DC-Beads, Biocompatibles, UK) with an anti-angiogenic agent (sunitinib) and to characterize the in vitro properties of the Beads-drug association.Materials: DC Beads of 100-300µm were loaded using a specially designed 10mg/ml sunitinib solution. Loading profile was studied by spectrophotometry of the supernatant solution at 430nm at different time points. Release experiment was performed using the USP method 4 (flow-through cell). Spectrophotometric determination at 430nm was used to measure drug concentration in the eluting solution.Results: We were able to load >98% of the drug in the DC-Beads in 2 hours. The maximum concentration was 20mg sunitinib/ml DC Beads. Loaded Beads gradually released 59% of the loaded drug in the eluting solution, by an ionic exchange mechanism,over 6 hours.Conclusions: DC Beads could be loaded with the multi tyrosine kinase inhibitor sunitinib using a specially designed solution. High drug payload can be achieved. The loaded DC Beads released the drug in an ionic eluting solution with an interesting release profile.

Biophysical characterization of the interaction of endotoxins with hemoglobins.

Bacterial endotoxin (lipopolysaccharide, LPS) is the major component of the outer leaflet of the outer membrane in gram-negative bacteria. During severe infections, bacteria may reach the blood circuit of humans, and endotoxins may be released from the bacteria due to cell division or cell death. In particular enterobacterial forms of LPS represent extremely strong activator molecules of the human immune system causing a rapid induction of cytokine production in monocytes and macrophages. Various mammalian blood proteins have been documented to display LPS binding activities mediating normally decreasing effects in the biological activity of LPS. In more recent studies, the essential systemic oxygen transportation protein hemoglobin (Hb) has been shown to amplify LPS-induced cytokine production on immune cells. The mechanism responsible for this effect is poorly understood. Here, we characterize the interaction of hemoglobin with LPS by using biophysical methods. The data presented, revealing the changes of the type and size of supramolecular aggregates of LPS in the presence of Hb, allow a better understanding of the hemoglobin-induced increase in bioactivity of LPS.

Genomic characterization of a Brazilian TT Virus isolate closely related to SEN virus-F

SEN virus (SENV) is a circular, single stranded DNA virus that has been first characterized in the serum of a human immunodeficiency virus type 1 (HIV-1)-infected patient. Eight genotypes of SENV (A-H) have been identified and further recognized as variants of TT virus (TTV) in the family Circoviridae. Here we describe the first genomic characterization of a SENV isolate (5-A) from South America. Using 'universal' primers, able to amplify most, if not all, TTV/SENV genotypes, a segment of > 3 kb was amplified by polymerase chain reaction from the serum of an HIV-1 infected patient. The amplicon was cloned and a 3087-nucleotide sequence was determined, that showed a high (85%) homology with the sequence of the Italian isolate SENV-F. Proteins encoded by open reading frames (ORFs) 1 to 4 consisted of 758, 129, 276, and 267 amino acids, respectively. By phylogenetic analysis, isolate 5-A was classified into TTV genotype 19 (phylogenetic group 3), together with SENV-F and TTV isolate SAa-38.

The use of the polymerase chain reaction and restriction fragment length polymorphism technique associated with the classical morphology for characterization of Lymnaea columella, L. viatrix, and L. diaphana (Mollusca: Lymnaeidae)

The specific identification of Lymnaeid snails is based on a comparison of morphological characters of the shell, radula, renal and reproductive organs. However, the identification is complicated by dissection process, intra and interspecific similarity and variability of morphological characters. In the present study, polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) techniques targeted to the first and second internal transcribed spacers (ITS1 and ITS2) rDNA and to the mitochondrial 16S ribosomal gene (16S rDNAmt) were used to differentiate the species Lymnaea columella, L. viatrix, and L. diaphana from some localities of Brazil, Argentina, and Uruguay as well as to verify whether the molecular results corroborates the classical morphological method.PCR-RFLP analysis of the ITS1, ITS2, and 16S using 12 restriction enzymes revealed characteristic patterns for L. columella and L. diaphana which were concordant with the classical morphology. On the other hand, for L. viatrix populations a number of 1 to 6 profiles were generated while morphology provided the species pattern results.

Quantifying network properties in multi-electrode recordings: spatiotemporal characterization and inter-trial variation of evoked gamma oscillations in mouse somatosensory cortex in vitro.

Linking the structural connectivity of brain circuits to their cooperative dynamics and emergent functions is a central aim of neuroscience research. Graph theory has recently been applied to study the structure-function relationship of networks, where dynamical similarity of different nodes has been turned into a "static" functional connection. However, the capability of the brain to adapt, learn and process external stimuli requires a constant dynamical functional rewiring between circuitries and cell assemblies. Hence, we must capture the changes of network functional connectivity over time. Multi-electrode array data present a unique challenge within this framework. We study the dynamics of gamma oscillations in acute slices of the somatosensory cortex from juvenile mice recorded by planar multi-electrode arrays. Bursts of gamma oscillatory activity lasting a few hundred milliseconds could be initiated only by brief trains of electrical stimulations applied at the deepest cortical layers and simultaneously delivered at multiple locations. Local field potentials were used to study the spatio-temporal properties and the instantaneous synchronization profile of the gamma oscillatory activity, combined with current source density (CSD) analysis. Pair-wise differences in the oscillation phase were used to determine the presence of instantaneous synchronization between the different sites of the circuitry during the oscillatory period. Despite variation in the duration of the oscillatory response over successive trials, they showed a constant average power, suggesting that the rate of expenditure of energy during the gamma bursts is consistent across repeated stimulations. Within each gamma burst, the functional connectivity map reflected the columnar organization of the neocortex. Over successive trials, an apparently random rearrangement of the functional connectivity was observed, with a more stable columnar than horizontal organization. This work reveals new features of evoked gamma oscillations in developing cortex.

Molecular characterization of Mycobacterium kansasii isolates in the State of São Paulo between 1995-1998

Mycobacterium kansasii is the most common cause of pulmonary nontuberculous mycobacteria infection and classical identification of this pathogen needs a time consuming phenotypic tests. Polymerase chain reaction-restriction fragment lenght polymorphism analysis (PRA) of the gene enconding for the 65kDa heat shock (hsp65) protein offers an easy, rapid, and inexpensive procedure to identify and subtype M. kansasii isolates. In the present study, we performed a retrospective analysis of patients who had mycobacteria identified on the basis of phenotypic tests by means of a review of database at Mycobacteria Laboratory of the Instituto Adolfo Lutz in the period 1995-1998. A total of 9381 clinical isolates were analyzed of which 7777 (82.9%) were identified as M. tuberculosis complex and 1604 (17.1%) as nontuberculous mycobacteria. Of the 296 M. kansasii isolates, 189 (63.8%) isolates obtained from 119 patients were viable and were analyzed by PRA-hsp65. Hundred eight two (98.9%) were classified as M. kansasii type I. Two isolates were classified as type II and III and five isolates were characterized as other Mycobacterium species. Clinical isolates of M. kansasii in the state of São Paulo was almost exclusively subtype I regardless of HIV status.

Functional Characterization of a n NTPase Activity of the Hepatitis C Virus Nonstructural Protein 4B

Background: Nonstructural p rotein 4 B (NS4B) i s the m asterorganizer of hepatitis C virus (HCV) replication complexformation. It is a multispanning membrane protein that has beenreported to p ossess NTPase activity. This enzymatic functionhas been poorly studied so far and its role in the HCV life cycleis u nknown. T he present w ork-in-progress a ims at validatingand functionally c haracterizing this a ctivity a nd its r ole in t heviral life cycle.Methods: B ioinformatic analyses were performed to i dentifykey residues for site-directed mutagenesis, both in t he contextof s ubgenomic r eplicons a s well as recombinant v iruses.Mutants were investigated with respect to R NA replication andinfectious particle p roduction. In p arallel, expression andpurification of recombinant wild-type and mutant NS4B proteinsare being pursued to characterize enzymatic activity in vitro.Results: B ioinformatic a nalyses revealed t hat p redictedNTPase features are conserved only in H CV NS4B b ut n ot i nNS4B from other Flaviviridae f amily m embers. A laninesubstitutions were designed to target predicted key Walker A, Band C motifs. These substitutions affected RNA replication andinfectious virus production to v arying degrees. Optimization ofrecombinant protein production is i n progress both in b acterialas well as mammalian expression systems.Conclusions: These studies should yield new insights into thefunctions of this hitherto poorly characterized viral nonstructuralprotein and may reveal novel targets for antiviral intervention inthe future.

Isolation and characterization of the full-length cDNA encoding a member of a novel cytochrome p450 family (CYP320A1) from the tropical freshwater snail, Biomphalaria glabrata, intermediate host for Schistosoma mansoni

Cytochrome p450s (cyp450s) are a family of structurally related proteins, with diverse functions, including steroid synthesis and breakdown of toxins. This paper reports the full-length sequence of a novel cyp450 gene, the first to be isolated from the tropical freshwater snail Biomphalaria glabrata, an important intermediate host of Schistosoma mansoni. The nucleotide sequence is 2291 bp with a predicted amino acid sequence of 584aa. The sequence demonstrates conserved cyp450 structural motifs, but is sufficiently different from previously reported cyp450 sequences to be given a new classification, CYP320A1. Initially identified as down-regulated in partially resistant snails in response to S. mansoni infection, amplification of this gene using RT-PCR in both totally resistant or susceptible snail lines when exposed to infection, and all tissues examined, suggests ubiquitous expression. Characterization of the first cyp450 from B. glabrata is significant in understanding the evolution of these metabolically important proteins.

Genetic characterization of Trypanosoma cruzi natural clones from the state of Paraíba, Brazil

Eighteen Trypanosoma cruzi stocks from the state of Paraíba, Brazil, isolated from man, wild mammals, and triatomine bugs were studied by multilocus enzyme electrophoresis and random primed amplified polymorphic DNA. Despite the low number of stocks, a notable genetic, genotypic, and phylogenetic diversity was recorded. The presence of the two main phylogenetic subdivisions, T. cruzi I and II, was recorded. The strong linkage disequilibrium observed in the population under survey suggests that T. cruzi undergoes predominant clonal evolution in this area too, although this result should be confirmed by a broader sample. The pattern of clonal variation does not suggests a recent origin by founder effect with a limited number of different genotypes.

Characterization of the hcnABC gene cluster encoding hydrogen cyanide synthase and anaerobic regulation by ANR in the strictly aerobic biocontrol agent Pseudomonas fluorescens CHA0.

The secondary metabolite hydrogen cyanide (HCN) is produced by Pseudomonas fluorescens from glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis in P. fluorescens CHA0 was investigated. The contiguous structural genes hcnABC encoding HCN synthase were expressed from the T7 promoter in Escherichia coli, resulting in HCN production in this bacterium. Analysis of the nucleotide sequence of the hcnABC genes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC). These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase in the reaction leading from glycine to HCN and CO2. The hcnA promoter was mapped by primer extension; the -40 sequence (TTGGC ... ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT ... ATCAA). The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned from P. fluorescens CHA0 and sequenced. ANR of strain CHA0 was most similar to ANR of P. aeruginosa and CydR of Azotobacter vinelandii. An anr mutant of P. fluorescens (CHA21) produced little HCN and was unable to express an hcnA-lacZ translational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of the hcnA-lacZ fusion. Mutant CHA21 as well as an hcn deletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused by Thielaviopsis basicola, under gnotobiotic conditions. This effect was most pronounced in water-saturated artificial soil, where the anr mutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0. These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot.

Molecular characterization of human T-cell lymphotropic virus coinfecting human immunodeficiency virus 1 infected patients in the Amazon region of Brazil

The present work evaluated the epidemiology of human immunodeficiency virus 1/human T-cell lymphotropic virus (HIV-1/HTLV) coinfection in patients living in Belém (state of Pará) and Macapá (state of Amapá), two cities located in the Amazon region of Brazil. A total of 169 blood samples were collected. The sera were tested by enzyme-linked immunosorbent assay to determine the presence of antibodies anti-HTLV-1/2. Confirmation of infection and discrimination of HTLV types and subtypes was performed using a nested polymerase chain reaction targeting the pX and 5' LTR regions, followed by restriction fragment length polymorphism and sequencing analysis. The presence of anti-HTLV1/2 was detected in six patients from Belém. The amplification of the pX region followed by RFLP analysis, demonstrated the presence of HTLV-1 and HTLV-2 infections among two and four patients, respectively. Sequencing HTLV-1 5' LTR indicated that the virus is a member of the Cosmopolitan Group, Transcontinental subgroup. HTLV-2 strains isolated revealed a molecular profile of subtype HTLV-2c. These results are a reflex of the epidemiological features of HIV-1/HTLV-1/2 coinfection in the North region of Brazil, which is distinct from other Brazilian regions, as reported by previous studies.

Neuroendocrine characterization and anorexigenic effects of telmisartan in diet- and glitazone-induced weight gain.

Telmisartan is an angiotensin II receptor blocker with peroxisome proliferator-activated receptor-gamma agonistic properties. Telmisartan prevents weight gain and decreases food intake in models of obesity and in glitazone-treated rodents. This study further investigates the influence of telmisartan and pioglitazone and their association on weight gain and body composition by examining their influence on neuroendocrine mediators involved in food intake. Male C57/Black 6 mice were fed a high-fat diet, weight matched, and randomized in 4 treatment groups: vehicle, pioglitazone, telmisartan, and pioglitazone-telmisartan. Weight gain, food and water intake, body composition, plasma leptin levels, and the hypothalamic expression of neuroendocrine mediators were analyzed. Additional studies were performed with irbesartan and in angiotensin II 1(A) receptor-knockout mice. Telmisartan abolished weight and fat gain in vehicle- and pioglitazone-treated mice while decreasing food intake, the hypothalamic expression of the agouti-related protein, and plasma leptin levels. Modifications in neuropeptide Y and proopiomelanocortin were not consistent with changes in food intake. The effects on weight gain and expression of the agouti-related protein were intermediate with irbesartan. The effects of telmisartan on weight gain were even more pronounced in angiotensin II 1(A) receptor-knockout mice. This study confirms the anorexigenic effects of telmisartan in mice fed a high-fat diet and suggests for the first time a functional role of telmisartan on hypothalamic orexigenic agouti-related protein regulation. These anorexigenic properties abolish both weight gain and body composition modifications in fat-fed and glitazone-treated mice. The anorexigenic properties are independent from the angiotensin II 1(A) receptor.