Differential requirement for CD4 help in the development of an antigen-specific CD8+ T cell response depending on the route of immunization.

Previous studies in our laboratory have shown that DBA/2 mice injected i.p. with syngeneic P815 tumor cells transfected with the HLA-CW3 gene (P815-CW3) showed a dramatic expansion of activated CD8+CD62L- T cells expressing exclusively the Vbeta10 segment. We have used this model to study the regulatory mechanisms involved in the development of the CW3-specific CD8+ response, with respect to different routes of immunization. Whereas both intradermal (i.d.) and i.p. immunization of DBA/2 mice with P815-CW3 cells led to a strong expansion of CD8+CD62L-Vbeta10+ cells, only the i.d. route allowed this expansion after immunization with P815 cells transfected with a minigene coding for the antigenic epitope CW3 170-179 (P815 miniCW3). Furthermore, depletion of CD4+ T cells in vivo completely abolished the specific response of CD8+CD62L-Vbeta10+ cells and prevented the rejection of P815-CW3 tumor cells injected i.p., whereas it did not affect CD8S+CD62L-Vbeta10+ cell expansion after i.d. immunization with either P815-CW3 or P815 miniCW3. Finally, the CW3-specific CD8+ memory response was identical whether or not CD4+ T cells were depleted during the primary response. Collectively, these results suggest that the CD8+ T cell response to P815-CW3 tumor cells injected i.p. is strictly dependent upon recognition of a helper epitope by CD4+ T cells, whereas no such requirement is observed for i.d. injection.

Intermediate and definitive hosts of Schistosoma mansoni in Corrientes province, Argentina

Corrientes province is located in the humid subtropical region of Argentina northeast on the left riverbank of Paraná River in the border with the South of Brazil. This is a region without schistosomiasis but planorbid and rodents that would serve as host of the life cycle of Schistosoma mansoni inhabit here. The objective of this work is to know the role of rodent as definitive host of schistosomiasis. Biomphalaria tenagophila (4 to 8 mm Ø) from Maloyas, exposed each to 10 miracidia of SJ2 strain of S. mansoni natives from Brazil were susceptible (5%). The degree of compatibility was Class II of Frandsen. Five wild rodents captured in the same ecological niche were exposed transcutaneously to infection with 40 cercariae for animal: two Olygoryzomys flavescens, two Holochilus braziliensis, and one Scapteromys tuncidus. Only one H. braziliensis eliminated eggs in feces. Prepatent period was of 83 days. With these feces, two of six (33.3%) B. tenagophila from Maloyas were infected with miracidium. It was demonstrated, in an area free of schistosomiasis, that life cycle S. mansoni is closed with planorbid and rodents that live in the same ecological niche.

Biomphalaria tenagophila potencial vector of Schistosoma mansoni in the Paraná River basin (Argentina and Paraguay)

Susceptibility and compatibility experiments were carried out with 700 Biomphalaria tenagophila from the Paraná River basin exposed to infection with Schistosoma mansoni. Individual infection was performed with 10 miracidia of SJ2 strain from the Paraiba valley (Brazil) originally infective to B. tenagophila. These snails were laboratory-breed progeny of B. tenagophila collected from six localities of Argentina and one from Paraguay. From Argentina: Rincón de Vences (7%) and Posadas (11%) became infected with S. mansoni and the calculation of Frandsen's index (TCP/100) shows that they were Class II poorly compatible. Those snails from Goya (22%), Maloyas (5%), and Berón de Astrada (3%) were Class III compatible to the S. mansoni. None of the 100 snails exposed from Caá-Catí became infected (Class 0 incompatible). Tested samples from Paraguay (Encarnación) were infected (20%) and compatible (Class III). It was also studied the persistence of the infection in 244 snails of the first generation (F1) of those that were susceptible from three places. It was demonstrated an increment of the susceptibility in the F1 from Maloyas (chi2 = 27.22; p = 0.0001) and Posadas (chi2 = 4.24; p = 0.04). The results point out the possibility that schistosomiasis might be able to spread into the Paraná River basin where B. tenagophila exists.

Mapping the molecular characteristics of Brazilian human T-cell lymphotropic virus type 1 Env (gp46) and Pol amino acid sequences for vaccine design

This study was carried out to evaluate the molecular pattern of all available Brazilian human T-cell lymphotropic virus type 1 Env (n = 15) and Pol (n = 43) nucleotide sequences via epitope prediction, physico-chemical analysis, and protein potential sites identification, giving support to the Brazilian AIDS vaccine program. In 12 previously described peptides of the Env sequences we found 12 epitopes, while in 4 peptides of the Pol sequences we found 4 epitopes. The total variation on the amino acid composition was 9 and 17% for human leukocyte antigen (HLA) class I and class II Env epitopes, respectively. After analyzing the Pol sequences, results revealed a total amino acid variation of 0.75% for HLA-I and HLA-II epitopes. In 5 of the 12 Env epitopes the physico-chemical analysis demonstrated that the mutations magnified the antigenicity profile. The potential protein domain analysis of Env sequences showed the loss of a CK-2 phosphorylation site caused by D197N mutation in one epitope, and a N-glycosylation site caused by S246Y and V247I mutations in another epitope. Besides, the analysis of selection pressure have found 8 positive selected sites (w = 9.59) using the codon-based substitution models and maximum-likelihood methods. These studies underscore the importance of this Env region for the virus fitness, for the host immune response and, therefore, for the development of vaccine candidates.

Effect of acute and chronic exercise on fat oxidation and hormonal and plasma metabolite kinetics in obese and severely obese men

Acute exercise increases energy expenditure (EE) during exercise and post-exercise recovery [excess post-exercise oxygen consumption (EPOC)] and therefore may be recommended as part of the multidisciplinary management of obesity. Moreover, chronic exercise (training) effectively promotes an increase in insulin sensitivity, which seems to be associated with increased fat oxidation rates (FORs). The main purpose of this thesis is to investigate 1) FORs and extra-muscular factors (hormones and plasma metabolites) that regulate fat metabolism during acute and chronic exercise; and 2) EPOC during acute post-exercise recovery in obese and severely obese men (class II and III). In the first study, we showed that obese and severely obese men present a lower exercise intensity (Fatmax) eliciting maximal fat oxidation and a lower reliance on fat oxidation at high, but not at low and moderate, exercise intensities compared to lean men. This was most likely related to an impaired muscular capacity to oxidize non-esterified fatty acids (NEFA) rather than decreased plasma NEFA availability or a change in the hormonal milieu during exercise. In the second study, we developed an accurate maximal incremental test to correctly and simultaneously evaluate aerobic fitness and fat oxidation kinetics during exercise in this population. This test may be used for the prescription of an appropriate exercise training intensity. In the third study, we demonstrated that only 2 wk of exercise training [continuous training at Fatmax and adapted high-intensity interval training (HIIT)], matched with respect to mechanical work, may be effective to improve aerobic fitness, FORs during exercise and insulin sensitivity, which suggest that FORs might be rapidly improved and that adapted HIIT is feasible in this population. The increased FORs concomitant with the lack of changes in lipolysis during exercise suggest an improvement in the mismatching between NEFA availability and oxidation, highlighting the importance of muscular (oxidative capacity) rather than extra-muscular (hormones and plasma metabolites) factors in the regulation of fat metabolism after a training program. In the fourth study, we observed a positive correlation between EE during exercise and EPOC, suggesting that a chronic increase in the volume or intensity of exercise may increase EE during exercise and during recovery. This may have an impact in weight management in obesity. In conclusion, these findings might have practical implications for exercise training prescriptions in order to improve the therapeutic approaches in obesity and severe obesity. -- L'exercice aigu augmente la dépense énergétique (DE) pendant l'exercice et la récupération post-exercice [excès de consommation d'oxygène post-exercise (EPOC)] et peut être utilisé dans la gestion multidisciplinaire de l'obésité. Quant à l'exercice chronique (entraînement), il est efficace pour augmenter la sensibilité à l'insuline, ce qui semble être associé à une amélioration du débit d'oxydation lipidique (DOL). Le but de cette thèse est d'étudier 1) le DOL et les facteurs extra-musculaires (hormones et métabolites plasmatiques) qui régulent le métabolisme lipidique pendant l'exercice aigu et chronique et 2) l'EPOC lors de la récupération aiguë post-exercice chez des hommes obèses et sévèrement obèses (classe II et III). Dans la première étude nous avons montré que les hommes obèses et sévèrement obèses présentent une plus basse intensité d'exercice (Fatmax) correspondant au débit d'oxydation lipidique maximale et un plus bas DOL à hautes, mais pas à faibles et modérées, intensités d'exercice comparé aux sujets normo-poids, ce qui est probablement lié à une incapacité musculaire à oxyder les acides gras non-estérifiés (AGNE) plutôt qu'à une diminution de leur disponibilité ou à un changement du milieu hormonal pendant l'exercice. Dans la deuxième étude nous avons développé un test maximal incrémental pour évaluer simultanément l'aptitude physique aérobie et la cinétique d'oxydation des lipides pendant l'exercice chez cette population. Dans la troisième étude nous avons montré que seulement deux semaines d'entraînement (continu à Fatmax et intermittent à haute intensité), appariés par la charge de travail, sont efficaces pour améliorer l'aptitude physique aérobie, le DOL pendant l'exercice et la sensibilité à l'insuline, ce qui suggère que le DOL peut être rapidement amélioré chez cette population. Ceci, en absence de changements de la lipolyse pendant l'exercice, suggère une amélioration de la balance entre la disponibilité et l'oxydation des AGNE, ce qui souligne l'importance des facteurs musculaires (capacité oxydative) plutôt que extra-musculaires (hormones et métabolites plasmatiques) dans la régulation du métabolisme lipidique après un entraînement. Dans la quatrième étude nous avons observé une corrélation positive entre la DE pendant l'exercice et l'EPOC, ce qui suggère qu'une augmentation chronique du volume ou de l'intensité de l'exercice pourrait augmenter la DE lors de l'exercice et lors de la récupération post-exercice. Ceci pourrait avoir un impact sur la gestion du poids chez cette population. En conclusion, ces résultats pourraient avoir des implications pratiques lors de la prescription des entraînements dans le but d'améliorer les approches thérapeutiques de l'obésité et de l'obésité sévère.

Multiple sclerosis risk variant HLA-DRB1*1501 associates with high expression of DRB1 gene in different human populations.

The human leukocyte antigen (HLA) DRB1*1501 has been consistently associated with multiple sclerosis (MS) in nearly all populations tested. This points to a specific antigen presentation as the pathogenic mechanism though this does not fully explain the disease association. The identification of expression quantitative trait loci (eQTL) for genes in the HLA locus poses the question of the role of gene expression in MS susceptibility. We analyzed the eQTLs in the HLA region with respect to MS-associated HLA-variants obtained from genome-wide association studies (GWAS). We found that the Tag of DRB1*1501, rs3135388 A allele, correlated with high expression of DRB1, DRB5 and DQB1 genes in a Caucasian population. In quantitative terms, the MS-risk AA genotype carriers of rs3135388 were associated with 15.7-, 5.2- and 8.3-fold higher expression of DQB1, DRB5 and DRB1, respectively, than the non-risk GG carriers. The haplotype analysis of expression-associated variants in a Spanish MS cohort revealed that high expression of DRB1 and DQB1 alone did not contribute to the disease. However, in Caucasian, Asian and African American populations, the DRB1*1501 allele was always highly expressed. In other immune related diseases such as type 1 diabetes, inflammatory bowel disease, ulcerative colitis, asthma and IgA deficiency, the best GWAS-associated HLA SNPs were also eQTLs for different HLA Class II genes. Our data suggest that the DR/DQ expression levels, together with specific structural properties of alleles, seem to be the causal effect in MS and in other immunopathologies rather than specific antigen presentation alone.

Influence of HLA DRB1 alleles in the susceptibility of rheumatoid arthritis and the regulation of antibodies against citrullinated proteins and rheumatoid factor.

INTRODUCTION The purpose of this study was to investigate the association between HLA-DRB1 alleles with susceptibility to rheumatoid arthritis (RA) and production of antibodies against citrullinated proteins (ACPA) and rheumatoid factor (RF). METHODS We studied 408 patients (235 with RA, 173 non-RA) and 269 controls. ACPA, RF and HLA-DR typing were determined. RESULTS We found an increased frequency of HLA DRB1 alleles with the shared epitope (SE) in ACPA-positive RA. Inversely, HLA DRB1 alleles encoding DERAA sequences were more frequent in controls than in ACPA-positive RA, and a similar trend was found for HLA DR3. However, these results could not be confirmed after stratification for the presence of the SE, probably due to the relatively low number of patients. These data may suggest that the presence of these alleles may confer a protective role for ACPA-positive RA. In RA patients we observed association between SE alleles and ACPA titers in a dose-dependent effect. The presence of HLA DR3 or DERAA-encoding alleles was associated with markedly reduced ACPA levels. No association between RF titers and HLA DR3 or DERAA-encoding alleles was found. CONCLUSIONS HLA DRB1 alleles with the SE are associated with production of ACPA. DERAA-encoding HLA-DR alleles and HLA DR3 may be protective for ACPA-positive RA.

CD209 in inflammatory bowel disease: a case-control study in the Spanish population

BACKGROUND The etiology of Ulcerative Colitis (UC) and Crohn's Disease (CD), considered together as Inflammatory Bowel Diseases (IBD), involves environmental and genetic factors. Although some genes are already known, the genetics underlying these diseases is complex and new candidates are continuously emerging. The CD209 gene is located in a region linked previously to IBD and a CD209 functional polymorphism (rs4804803) has been associated to other inflammatory conditions. Our aim was to study the potential involvement of this CD209 variant in IBD susceptibility. METHODS We performed a case-control study with 515 CD patients, 497 UC patients and 731 healthy controls, all of them white Spaniards. Samples were typed for the CD209 single nucleotide polymorphism (SNP) rs4804803 by TaqMan technology. Frequency comparisons were performed using chi2 tests. RESULTS No association between CD209 and UC or CD was observed initially. However, stratification of UC patients by HLA-DR3 status, a strong protective allele, showed that carriage of the CD209_G allele could increase susceptibility in the subgroup of HLA-DR3-positive individuals (p = 0.03 OR = 1.77 95% CI 1.04-3.02, vs. controls). CONCLUSION A functional variant in the CD209 gene, rs4804803, does not seem to be influencing Crohn's disease susceptibility. However, it could be involved in the etiology or pathology of Ulcerative Colitis in HLA-DR3-positive individuals but further studies are necessary.

Susceptibility and compatibility of Biomphalaria tenagophila from the Río de la Plata basin with Schistosoma mansoni from Brazil

Schistosomiasis has expanded to southern parts of Brazil. Between 2005-2007 the dispersion and the proliferation of Biomphalaria tenagophila was verified in the province of Corrientes near the Brazilian border. In order to study the possibility that schistosomiasis might spread into the basins of the Paraná and Uruguay Rivers, 440 B. tenagophila collected from 10 populations groups were experimentally exposed to infection with Schistosoma mansoni of the SJ2 strain. Snails from five localities were susceptible. Frandsen's index (TCP/100) shows that those snails from Mirungá (11%), Aguacerito (2%) and Curupicay (2%) were Class I and not very compatible. Meanwhile, snails from Copra (6%) and Pay-Ubre (22%), in the Paraná River basin, were Class II and poorly compatible.

The NK cell receptor repertoire: formation, adaptation and exploitation.

The identification of NK cell receptors specific for MHC class I molecules has greatly improved our knowledge of NK cell reactivity and specificity. Inhibitory receptors prevent NK cell activation directed against cells expressing self-MHC class I molecules. Consequently, diseased cells that do not express self-MHC class I molecules become susceptible to NK cell-mediated attack. Because of the specificity and distribution of inhibitory NK cell receptors, cells that express non-self (allogeneic) MHC class I molecules are also susceptible to NK cell reactions. This feature has been exploited in a clinical setting to treat leukemia patients.

Role of gene methylation in antitumor immune response: Implication for tumor progression

Cancer immunosurveillance theory has emphasized the role of escape mechanisms in tumor growth. In this respect, a very important factor is the molecular characterization of the mechanisms by which tumor cells evade immune recognition and destruction. Among the many escape mechanisms identified, alterations in classical and non-classical HLA (Human Leucocyte Antigens) class I and class II expression by tumor cells are of particular interest. In addition to the importance of HLA molecules, tumor-associated antigens and accessory/co-stimulatory molecules are also involved in immune recognition. The loss of HLA class I antigen expression and of co-stimulatory molecules can occur at genetic, transcriptional and post-transcriptional levels. Epigenetic defects are involved in at least some mechanisms that preclude mounting a successful host-antitumor response involving the HLA system, tumor-associated antigens, and accessory/co-stimulatory molecules. This review summarizes our current understanding of the role of methylation in the regulation of molecules involved in the tumor immune response.

Probing the interactions of NK cell receptors with ligand expressed in trans and cis.

Certain receptors on natural killer (NK) cells, which are specific for MHC class I (MHC-I) molecules, do not only interact with ligand expressed on opposing cell membranes (in trans) but also interact with those on the same cell membrane (in cis). Cis interactions have been demonstrated for only a small number of cell surface receptors. However, this has not been tested systematically, raising the possibility that additional receptors may be able to bind ligand expressed in cis. Here we describe a number of approaches to evaluate trans and cis binding of the Ly49A NK cell receptor to its H-2D(d) ligand. These procedures should facilitate the investigation of cis/trans interactions of other receptor-ligand pairs and simplify the analysis of NK cell receptor variants.

Mycobacterium leprae virulence-associated peptides are indicators of exposure to M. leprae in Brazil, Ethiopia and Nepal

Silent transmission of Mycobacterium leprae, as evidenced by stable leprosy incidence rates in various countries, remains a health challenge despite the implementation of multidrug therapy worldwide. Therefore, the development of tools for the early diagnosis of M. leprae infection should be emphasised in leprosy research. As part of the continuing effort to identify antigens that have diagnostic potential, unique M. leprae peptides derived from predicted virulence-associated proteins (group IV.A) were identified using advanced genome pattern programs and bioinformatics. Based on human leukocyte antigen (HLA)-binding motifs, we selected 21 peptides that were predicted to be promiscuous HLA-class I T-cell epitopes and eight peptides that were predicted to be HLA-class II restricted T-cell epitopes for field-testing in Brazil, Ethiopia and Nepal. High levels of interferon (IFN)-γ were induced when peripheral blood mononuclear cells (PBMCs) from tuberculoid/borderline tuberculoid leprosy patients located in Brazil and Ethiopia were stimulated with the ML2055 p35 peptide. PBMCs that were isolated from healthy endemic controls living in areas with high leprosy prevalence (EChigh) in Ethiopia also responded to the ML2055 p35 peptide. The Brazilian EChigh group recognised the ML1358 p20 and ML1358 p24 peptides. None of the peptides were recognised by PBMCs from healthy controls living in non-endemic region. In Nepal, mixtures of these peptides induced the production of IFN-γ by the PBMCs of leprosy patients and EChigh. Therefore, the M. leprae virulence-associated peptides identified in this study may be useful for identifying exposure to M. leprae in population with differing HLA polymorphisms.

Role of c-Myc in homeostasis of memory CD8 T cells

RESUME La mémoire immunologique est essentielle durant la vie et permet aux lymphocytes de répondre plus rapidement et efficacement lors d'une deuxième rencontre avec un antigène connu. Les facteurs contrôlant l'homéostasie des cellules T CD8 mémoires in vivo ne sont pas encore bien définis. Cependant, la prolifération homéostatique de ces cellules dans un hôte déplété en cellules hématopoietiques nécessite l'intéraction du TCR avec les molecules du MHC de class I du soi. De plus, le rôle de cytokines, telles que 1'IL-15 et l'IL-7, est essentiel dans ce mécanisme, aussi bien que dans la maintenance des cellules T CD8 mémoires. Puisque la protéine c-Myc - impliquée dans des mécanismes tells que la division, la prolifération, l'apoptose et la differentiation - a été définie comme étant impliquée dans la réponse à différentes cytokines, nous nous sommes intéressés à l'analyse de l'homéostasie des lymphocytes T CD8 mémoires dans des souris déficientes en c-Myc (c_rnycΔORF/+), qui expriment un niveau réduit de cette protéine. Bien que le développement des cellules T dans le thymus soit normal dans les souris c_rnycΔORF/+, nous avons observé une réduction de 2 à 3 fois dans la population des cellules T CD8 de phenotype mémoire (CD44+) dans les organes lymphoïdes de la périphérie de ces souris. Cette différence ne correspond pas à une réduction de prolifération ou d'expression de protéines de survie telles que Bel-2. Cependant, la prolifération homéostatique de cellules T CD8 c_rnycΔORF/+, mais pas T CD4 c_rnycΔORF/+, est reduite de manière dramatique lorsqu'elles sont transférées dans un hôte irradié. De plus, le transfert adoptif de lymphocytes T dans des souris irradiées déficientes en l'IL-15 nous a permis de montrer que la prolifération homéostatique dépendante de l'IL-15 des cellules T CD8 nécessite l'expression de c-Myc. De plus, contrairement aux cellules T CD8 CD44+ de type sauvage, nous avons observé que l'expansion induite par l'IL-15 des cellules T CD8 CD44+ c_rnycΔORF/+ est altérée aussi bien in vivo (en réponse à une injection de polyI:C) et in vitro. Par conséquent, nos résultats identifient c-Myc comme une nouvelle protéine régulatrice de la signalisation par l'IL-15 impliquée dans l'homéostasie des cellules T CD8 CD44+. SUMMARY Immunological memory is essential throughout life and allows memory lymphocytes to respond faster and more efficiently upon re-encounter of a known antigen. Factors controlling homeostasis of memory CD8 T cells under steady-state conditions in vivo are currently not well defined. However, the homeostatic proliferation of memory CD8 T cells in lymphopenic hosts requires the interaction of the TCR with self MHC class I molecules. In addition, cytokines, such as IL-15 and to a lesser extent IL-7, are essential for both homeostatic proliferation and maintenance of memory CD8 T cells. Since c-Myc, a proto-oncogene involved in cell division, proliferation, apoptosis and differentiation, has been widely implicated in responsiveness to cytokines, we were interested in analyzing homeostasis of memory CD8 T cells in c-myc hypomorph (c_rnycΔORF/+) mice, which express reduced levels of c-Myc. Although T cell development in the thymus was normal in c_rnycΔORF/+ mice, we found a selective 2- to 3-fold reduction in the memory-phenotype CD44high CD8 T cell population in the periphery. Reduced numbers of CD44high CD8 T cells did not correlate with decreased steady-state turnover rate or low expression of survival factors such as Bcl- 2. However, homeostatic proliferation of c_rnycΔORF/+ CD8 T cells, but not c_rnycΔORF/+ CD4 T cells, was dramatically reduced upon transfer into sublethally irradiated wild-type recipients. In addition, upon transfer of c_rnycΔORF/+ and c-myc WT cells into IL-15-/- mice, we observed that IL-15-induced homeostatic proliferation of CD8 T cells requires c-Myc. Moreover, in contrast to c-myc WT CD44high CD8 T cells, IL-15-induced expansion of c_rnycΔORF/+ CD44high CD8 T cells was strongly impaired both in vivo (in response to polyI:C injection) and in vitro. Collectively, our data identify c-Myc as a novel downstream regulator of IL-15 signaling involved in homeostasis of memory CD8 T cells.