Monitoring of human CD4 T cell responses in metastatic melanoma and arthritic patients

SUMMARY : Detailed knowledge of the different components of the immune system is required for the development of new immunotherapeutic strategies. CD4 T lymphocytes represent a highly heterogeneous group of cells characterized by various profiles of cytokine production and effector vs. regulatory functions. They are central players in orchestrating adaptive immune responses: unbalances between the different subtypes can lead either to aggressive autoimmune disorders or can favour the uncontrolled growth of malignancies. In this study we focused on the characterization of human CD4 T cells in advanced stage melanoma patients as well as in patients affected by various forms of autoimmune inflammatory spondyloarthropathies. In melanoma patients we report that a population of FOXP3 CD4 T cells, known as regulatory T cells, is overrepresented in peripheral blood, and even more in tumor-infitrated lymph nodes as well as at tumor sites, as compared to healthy donors. In tumor-infiltrated lymph nodes, but not in normal lymph nodes or in peripheral blood, FOXP3 CD4 T cells feature a highly differentiated phenotype (CD45RA-CCR7+/-), which suggests for a recent encounter with their cognate antigen. FOXP3 CD4 T cells have been described to be an important component of the several known immune escape mechanisms. We demonstrated that FOXP3 CD4 T cells isolated from melanoma patients exert an in vitro suppressive action on autologous CD4 T cells, thus possibly inhibiting an efficient anti-tumor response. Next, we aimed to analyse CD4 T cells at antigen-specific level. In advanced stage melanoma patients, we identified for the first time, using pMHCII multimers, circulating CD4 T cells specific for the melanoma antigen Melan-A, presented by HLA-DQB1 *0602. Interestingly, in a cohort of melanoma patients enrolled in an immunotherapy trails consisting of injection of a Melan-A derived peptide, we did not observe signif cant variations in the ex vivo frequencies of Melan-A specific CD4 T cells, but important differences in the quality of the specific CD4 T cells. In fact, up to 50% of the ex vivo Melan-A/DQ6 specific CD4 T cells displayed a regulatory phenotype and were hypoproliferative before vaccination, while more effector, cytokine-secreting Melan-A/DQ6 specific CD4 T cells were observed after immunization. These observations suggest that peptide vaccination may favourably modify the balance between regulatory and effector tumor-specific CD4 T cells. Finally, we identified another subset of CD4 T cells as possible mediator of pathology in a group of human autoimmune spondyloarthropathies, namely Th17 cells. These cells were recently described to play a critical role in the pathogenesis of some marine models of autommunity. We document an elevated presence of circulating Th17 cells in two members of seronegative spondyloarthropathies, e.g. psoriatic arthritis and ankylosing spondylitis, while we do not observe increased frequencies of Th17 cells in peripheral blood of rheumatoid arthritic patients. In addition, Th17 cells with a more advanced differentiation state (CD45RA-CCR7-CD27-) and polyfunctionality (concomitant secretion of IL-17, IL-2 and TNFα) were observed exclusively in patients with seronegative spondylarthropathies. Together, our observations emphasize the importance of CD4 T cells in various diseases and suggest that immunotherapeutic approaches considering CD4 T cells as targets should be evaluated in the future.

The role of antibody concentration and avidity in antiviral protection.

Neutralizing antibodies are necessary and sufficient for protection against infection with vesicular stomatitis virus (VSV). The in vitro neutralization capacities and in vivo protective capacities of a panel of immunoglobulin G monoclonal antibodies to the glycoprotein of VSV were evaluated. In vitro, neutralizing activity correlated with avidity and with neutralization rate constant, a measure of on-rate. However, in vivo, protection was independent of immunoglobulin subclass, avidity, neutralization rate constant, and in vitro neutralizing activity; above a minimal avidity threshold, protection depended simply on a minimum serum concentration. These two biologically defined thresholds of antibody specificity offer hope for the development of adoptive therapy with neutralizing antibodies.

Alzheimer's disease: the amyloid hypothesis and the Inverse Warburg effect.

Epidemiological and biochemical studies show that the sporadic forms of Alzheimer's disease (AD) are characterized by the following hallmarks: (a) An exponential increase with age; (b) Selective neuronal vulnerability; (c) Inverse cancer comorbidity. The present article appeals to these hallmarks to evaluate and contrast two competing models of AD: the amyloid hypothesis (a neuron-centric mechanism) and the Inverse Warburg hypothesis (a neuron-astrocytic mechanism). We show that these three hallmarks of AD conflict with the amyloid hypothesis, but are consistent with the Inverse Warburg hypothesis, a bioenergetic model which postulates that AD is the result of a cascade of three events-mitochondrial dysregulation, metabolic reprogramming (the Inverse Warburg effect), and natural selection. We also provide an explanation for the failures of the clinical trials based on amyloid immunization, and we propose a new class of therapeutic strategies consistent with the neuroenergetic selection model.

Distinct phenotypes of antigen-selected CD8 T cells emerge at different stages of an in vivo immune response.

We have previously described a unique system for identifying Ag-selected CD8 T cells during an in vivo response in normal mice. In this system, lymphocytes isolated from DBA/2 mice injected i.p. with HLA-CW3 transfected syngeneic (H-2d) P815 cells show a remarkable expansion of CD8 cells that utilize TCR expressing the V beta 10 gene segment and additional structural features characteristic of Kd-restricted CW3-specific CTL clones. We have now taken advantage of this system to characterize the surface phenotype of CD8 cells selected by Ag in vivo. We observed several distinct phenotypes at different stages of the response. At the peak of the response, Ag-selected cells were low in CD62L and CD45RB expression but displayed high levels of CD44. In addition, there was a partial down-regulation of CD8 and TCR. Cells of this phenotype were present in lymphoid tissues for several mo after immunization. Much later in the response, Ag-selected cells expressed higher levels of CD8 and TCR. Moreover, a distinct subset of these long-term immune cells emerged that now expressed CD62L and CD45RB. Analysis of CD8 cells from different tissues also revealed certain differences, particularly in TCR and co-receptor levels from liver-derived cells compared with circulating cells at the peak of the response. Our findings suggest that the function of Ag-selected CD8 cells may be regulated over time and according to location by subtle changes in cell-surface phenotype.

Repeated exposure to Lutzomyia intermedia sand fly saliva induces local expression of interferon-inducible genes both at the site of injection in mice and in human blood.

During a blood meal, Lutzomyia intermedia sand flies transmit Leishmania braziliensis, a parasite causing tegumentary leishmaniasis. In experimental leishmaniasis, pre-exposure to saliva of most blood-feeding sand flies results in parasite establishment in absence of any skin damages in mice challenged with dermotropic Leishmania species together with saliva. In contrast, pre-immunization with Lu. intermedia salivary gland sonicate (SGS) results in enhanced skin inflammatory exacerbation upon co-inoculation of Lu. intermedia SGS and L. braziliensis. These data highlight potential unique features of both L. braziliensis and Lu. intermedia. In this study, we investigated the genes modulated by Lu. intermedia SGS immunization to understand their potential impact on the subsequent cutaneous immune response following inoculation of both SGS and L. braziliensis. The cellular recruitment and global gene expression profile was analyzed in mice repeatedly inoculated or not with Lu. intermedia. Microarray gene analysis revealed the upregulation of a distinct set of IFN-inducible genes, an immune signature not seen to the same extent in control animals. Of note this INF-inducible gene set was not induced in SGS pre-immunized mice subsequently co-inoculated with SGS and L. braziliensis. These data suggest the parasite prevented the upregulation of this Lu. intermedia saliva-related immune signature. The presence of these IFN-inducible genes was further analyzed in peripheral blood mononuclear cells (PBMCs) sampled from uninfected human individuals living in a L. braziliensis-endemic region of Brazil thus regularly exposed to Lu. intermedia bites. PBMCs were cultured in presence or absence of Lu. intermedia SGS. Using qRT-PCR we established that the IFN-inducible genes induced in the skin of SGS pre-immunized mice, were also upregulated by SGS in PBMCs from human individuals regularly exposed to Lu. intermedia bites, but not in PBMCs of control subjects. These data demonstrate that repeated exposure to Lu. intermedia SGS induces the expression of potentially host-protective IFN-inducible genes.

La toxicologie forensique, une discipline scientifique en plein essor [Forensic toxicology, a growing scientific discipline]

La recherche de substances ayant pu jouer un rôle direct ou indirect dans la cause de la mort ou ayant pu modifier le comportement d'un individu constitue la mission première de la toxicologie forensique. L'augmentation de la consommation de substances illégales et de médicaments dans les sociétés modernes a fait connaître à la toxicologie forensique un essor très important ces dernières décennies. D'autre part, les développements technologiques analytiques ont permis d'obtenir des outils très sensibles et spécifiques pour la recherche et le dosage d'une multitude de substances dans une grande variété d'échantillons biologiques, parfois présentes à des concentrations très faibles, conséquence d'une dose équivalente à la prise d'une seule unité galénique. Forensic toxicology has to bring evidence of substances that could have been involved directly or indirectly in the cause of death or that could influence the behaviour of somebody. The increase of the consumption of illegal and legal drugs in modern societies during last decades gave a boost to forensic toxicology. Moreover, improvement with analytical technology gave tools with high degrees of sensitivity and specificity for the screening and quantification of a large amount of substances in various biological specimens, even with very low concentration resulting of a single dose of medication.

Factors associated with vaccination against Influenza A (H1N1) in the elderly

This study aimed to investigate the sociodemographic, clinical and behavioral factors and receiving information about the vaccine against pandemic influenza A (H1N1) associated with vaccination of elderly people. Study of quantitative and transversal nature, in which 286 elderly residents in Fortaleza, CE, Brazil participated. The association between variables was analyzed by the Pearson chi-square test, considering a 95% confidence interval and significance level (p≤0.05). The results revealed that, unlike the sociodemographic characteristics, many clinical, behavioral and informational aspects correlated significantly with adherence to Influenza A (H1N1) vaccination. It is believed that the findings can be used in strategies to control and prevent infection by viral subtypes within the elderly population, extensible even to other vaccine-preventable diseases, especially in light of possible future pandemics.

A novel Th cell epitope of Candida albicans mediates protection from fungal infection.

Fungal pathogens are a frequent cause of opportunistic infections. They live as commensals in healthy individuals but can cause disease when the immune status of the host is altered. T lymphocytes play a critical role in pathogen control. However, specific Ags determining the activation and function of antifungal T cells remain largely unknown. By using an immunoproteomic approach, we have identified for the first time, to our knowledge, a natural T cell epitope from Candida albicans. Isolation and sequencing of MHC class II-bound ligands from infected dendritic cells revealed a peptide that was recognized by a major population of all Candida-specific Th cells isolated from infected mice. Importantly, human Th cells also responded to stimulation with the peptide in an HLA-dependent manner but without restriction to any particular HLA class II allele. Immunization of mice with the peptide resulted in a population of epitope-specific Th cells that reacted not only with C. albicans but also with other clinically highly relevant species of Candida including the distantly related Candida glabrata. The extent of the reaction to different Candida species correlated with their degree of phylogenetic relationship to C. albicans. Finally, we show that the newly identified peptide acts as an efficient vaccine when used in combination with an adjuvant inducing IL-17A secretion from peptide-specific T cells. Immunized mice were protected from fatal candidiasis. Together, these results uncover a new immune determinant of the host response against Candida ssp. that could be exploited for the development of antifungal vaccines and immunotherapies.

Metodologia de Análise de Investimentos em Sistemas e Tecnologias da Informação, pelas empresas caboverdeanas

O aumento da competitividade das empresas caboverdeanas impõe-se sobretudo por causa do momento histórico que particularmente o país atravessa. A graduação para país de rendimento médio, a adesão à Organização Mundial de Comércio e o estatuto de parceria especial com a União Europeia constituem razões de grande vulto para as empresas nacionais e outras que empreendem no mercado caboverdeano, definirem eficazmente e eficientemente os seus processos organizacionais de forma a atingirem patamares de produtividade e performance aos níveis internacionais. A envolvente externa às empresas caboverdeanas tem sido alvo de alterações profundas com a globalização dos mercados e o novo paradigma de economia baseada no conhecimento. Para que as empresas mantenham posições competitivas são muitos os desafios que terão que superar. Inexoravelmente, terão que reorganizar e redefinir os seus processos de negócio e suas estratégias de forma coerente. Os SI/TI constituem o factor potencial de reorganização e modernização, a alavanca de diferenciação competitiva e da concretização de negócios e resultados adicionais. O objectivo deste trabalho é o desenvolvimento e a validação de uma metodologia de enquadramento, análise e avaliação de investimentos em SI/TI. O procedimento proposto é fundamentado pela literatura e nas práticas das empresas cabo-verdianas e visa apoiar as empresas a atingirem estágios de performance e produtividade elevados, decorrentes da utilização optimizada dos recursos oferecidos pelos SI/TI. Porque elevadas somas de recursos financeiros são investidos em Sistemas e Tecnologias de Informação e nem todos os benefícios associados são quantificáveis e dado que os riscos associados são elevados, propõe-se com este trabalho um procedimento metodológico de apoio às empresas. Esta metodologia simplificada de análise de investimentos em Sistemas e Tecnologias da Informação propõe uma abordagem científica, sólida e objectiva de avaliação de investimentos em SI/TI, visando propiciar incremento do valor acrescentado dos produtos e/ou serviços das empresas. Os resultados obtidos a partir das análises quantitativas e qualitativas elaboradas permitem retratar a prática das empresas caboverdeanas no domínio de análise e avaliação de investimentos em SI/TI e gerar inferências importantes sobre o comportamento dos responsáveis empresariais e de SI/TI. The increase in competition by the Capeverdean companies is taking place mainly because of the historic moment the country is going through. The recent nomination of Cape Verde as a medium development country, the adhesion to the WCO (World Commerce Organization) and the special partnership with the European Union are relevant reasons for the national companies and others that operate in the Capeverdean market to define efficiently their organizational processes in order to reach levels of productivity and performance comparable to the international ones. The external component of the Capeverdean companies has been undergoing deep changes with market globalization and with the new economic paradigma based on kowledge. In order to maintain their competitive position there are many challenges to overcome. Surely and steadily, they will have to reorganize and redefine their business processes and strategies in a coherent way. The IS/IT (information systems / information technologies) constitute the potential factor of reorganization and modernization, the boost-lever that will produce competitive diferentiation and concretization of businesses and additional results. The main goal of this work is development and validation of a methology of insertion, analysis and evaluation of investments in the IS/IT. The proposed procedure is fundamented by the literature and practice of the Capeverdian companies, and aims at supporting these companies so that they can reach high stages of performance and productivity due to the correct use of the resources offered by the IS/IT. As high amounts of financial resources are invested in Information Systems and Tehnologies, and because of the fact that not all associated benefits may be quantified, and since the associated risks are high, with this work we propose a methodological procedure to support these companies. This simplified analysis methodology in Information and Technologies Systems proposes a sound and objective scientific approach to evaluate investments in IS/IT aiming at providing an increase of the added value of the products and/or services supplied by these companies. The incoming results from the quantitative and qualitative analyses made will allow portraying the practice of the capeverdean companies in the domain of analysis and evaluation of investments in IS/IT and generating important inferences on the behaviour of the executives and IS/IT.

Evaluation of prenatal care in unit with family health strategy

We analyzed prenatal care (PN) provided at a unit of the Family Health Strategy Service in São Paulo, according to the indicators of the Program for the Humanization of Prenatal and Birth (PHPB). We compared adequacy of PN in terms of sociodemographic variables, procedures, examinations and maternal and perinatal outcomes. Cross-sectional study with data from records of 308 pregnant women enrolled in 2011. We observed early initiation of PN (82.1%), conducting of a minimum of six consultations (84.1%), puerperal consultation (89.0%); to the extent that there is a sum of the actions, there is a significant drop in the proportion of adequacy. Prenatal care was adequate for 67.9%, with a significant difference between adequacy groups in relation to gestational age and birth weight. Prenatal care deficiencies exist, especially in regards to registration of procedures, exams and immunization. The difference between adequacy groups with respect to perinatal outcomes reinforces the importance of prenatal care that adheres to the parameters of the PHPB.


Time standards of nursing in Primary Health Care: an observational study

Abstract OBJECTIVE To determine time standards for interventions and activities conducted by nursing professionals in Family Health Units (FHU) in Brazil to substantiate the calculation of work force. METHOD This was an observational study carried out in 27 FHU, in 12 municipalities in 10 states, in 2013. In each unit, nursing professionals were observed every 10 minutes, for eight work hours, on five consecutive days via the work sampling technique. RESULTS A total of 32,613 observations were made, involving 47 nurses and 93 nursing technicians/assistants. Appointments were the main intervention carried out by nurses, with a mean time of 25.3 minutes, followed by record-keeping, which corresponded to 9.7%. On average, nursing technicians/assistants spent 6.3% of their time keeping records and 30.6 intervention minutes on immunization/vaccination control. CONCLUSION The study resulted in standard times of interventions carried out by the FHU nursing team, which can underpin the determination of nursing staff size and human resource policies. Furthermore, the study showed the panorama of interventions currently employed, allowing for the work process to be reviewed and optimized.

Boosting of Replication Competent NYVAC Primed HIV-1-Specific T-Cell Responses by HIV Synthetic Long Peptides (SLP)

Background: To enhance the induction of insert specific immune responses, a new generation of replication competent poxvirus vectors was designed and evaluated against non-replicating poxvirus vectors in a HIV vaccine study in non human primates.Methods: Rhesus macaques were immunized with either the non-replicating variant NYVAC-GagPolNef HIV-1 clade C or the replicating NYVAC-GagPolNef-C-KC, boosted with HIVGag- PolEnv-SLP and immune responses were monitored.Results: Gag-specific T-cell responses were only detected in animals immunized with the replicating NYVAC-GagPolNef-C-KC variant. Further enhancement and broadening of the immune response was studied by boosting the animals with novel T-cell immunogens HIVconsv synthetic long peptides (SLP), which direct vaccine-induced responses to the most conserved regions of HIV and contain both CD4 T-helper and CD8 CTL epitopes. The adjuvanted (Montanide ISA-720) SLP divided into subpools and delivered into anatomically separate sites enhanced the Gag-specific T-cell responses in 4 out of 6 animals, to more than 1000 SFC/106 PBMC in some animals. Furthermore, the SLP immunization broadened the immune response in 4 out of 6 animals to multiple Pol epitopes. Even Env-specific responses, to which the animals had not been primed, were induced by SLP in 2 out of 6 animals.Conclusion: This new immunization strategy of priming with replicating competent poxvirus NYVAC-HIVGagPolNef and boosting with HIVGagPolEnv-SLP, induced strong and broad Tcell responses and provides a promising new HIV vaccine approach. This study was performed within the Poxvirus T-cell Vaccine Discovery Consortium (PTVDC) which is part of the CAVD program.

Welcome To Medicare Physical Exam, September 2007

Medicare will cover a one-time preventive physical exam within the first six months that you have Part B. This benefit is for all Medicare beneficiaries including those under age 65. How much does the exam cost? You pay 20% of the Medicare approved amount after you meet the yearly Part B deductible ($131 for 2007). Since this exam may be your first Medicare-covered service, you could meet your entire Part B deductible for the year. Medicare will cover the exam if performed by a physician, physician assistant, nurse practitioner, or clinical nurse specialist. What should I expect during the exam? The “Welcome to Medicare Physical” will include the following: 1. A review of your medical and social history. 2. A review of your potential risk factors for depression. 3. A review of your functional ability and level of safety. 4. Blood pressure, height, weight and vision test 5. An electrocardiogram (EKG) 6. Education and counseling on the above five items. 7. A written plan explaining screenings and other recommended preventive services. All seven elements must be documented in order for the physical to be covered by Medicare. The exam does not include clinical laboratory tests. Medicare will pay for a one-time ultrasound screening for abdominal aortic aneurysms for beneficiaries who are at risk (has a family history or a man age 65 to 75 who has smoked at least 100 cigarettes in his lifetime.) Only Medicare beneficiaries who receive a referral from the Welcome to Medicare physical exam will be covered for this benefit. There is no Part B deductible, but you or your supplemental insurance will be responsible for the coinsurance. What should I take to the exam? You should bring the following when you go to your “Welcome to Medicare” physical exam: • Medical records, including immunization records (if you are seeing a doctor for the first time) • Family health history • A list of current prescription drugs, how often you take them, and why.

Activation of human melanoma reactive CD8+ T cells by vaccination with an immunogenic peptide analog derived from Melan-A/melanoma antigen recognized by T cells-1.

PURPOSE: As compared with natural tumor peptide sequences, carefully selected analog peptides may be more immunogenic and thus better suited for vaccination. However, T cells in vivo activated by such altered analog peptides may not necessarily be tumor specific because sequence and structure of peptide analogs differ from corresponding natural peptides. EXPERIMENTAL DESIGN: Three melanoma patients were immunized with a Melan-A peptide analog that binds more strongly to HLA-A*0201 and is more immunogenic than the natural sequence. This peptide was injected together with a saponin-based adjuvant, followed by surgical removal of lymph node(s) draining the site of vaccination. RESULTS: Ex vivo analysis of vaccine site draining lymph nodes revealed antigen-specific CD8+ T cells, which had differentiated to memory cells. In vitro, these cells showed accelerated proliferation upon peptide stimulation. Nearly all (16 of 17) of Melan-A-specific CD8+ T-cell clones generated from these lymph nodes efficiently killed melanoma cells. CONCLUSIONS: Patient immunization with the analog peptide leads to in vivo activation of T cells that were specific for the natural tumor antigen, demonstrating the usefulness of the analog peptide for melanoma immunotherapy.