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Immunological alterations associated with aging (immunosenescence) do not represent a simple unidirectional decline in all functions but develop as a complex remodeling of the immune system, involving multiple reorganization and developmentally regulated changes. In general, most data available about aging were obtained at particular age intervals and most of them come from Caucasian individuals from either Europe or the United States. Here, we report the frequencies of major lymphocyte subsets in healthy Brazilian individuals from 2 distinct geographic regions (Southeast and South) at several age intervals spanning a lifetime period (0–86 years). Overall, we demonstrated that changes in the frequencies of cells related to both innate and adaptive immunity clearly occur with aging in these individuals. These changes were not progressive and equally steady for all cell populations tested but instead showed an oscillatory or rhythmic behavior that was distinctive of each population at different age intervals. We also observed that abrupt changes in the frequencies of immune cells may occur in healthy individuals over 75 years old, suggesting there is an impaired flexibility of the immune system at late stages of life to sustain homeostasis via immune mechanisms. We presented reference ranges for healthy Brazilian individuals at all ages. The knowledge of these parameters in further detail will allow interventions to optimize immune function in advanced age and to improve the quality of life in the elderly.

Summary There has been no systematic study of the immune response of individuals aged over 60 years living in Schistosomiasis mansoni-endemic areas, although senescence is reportedly associated with susceptibility to infection and progressive decline in immune function. We have shown previously, in two endemic areas in Minas Gerais, Brazil, that the frequency of individuals aged over 60 years with chronic schistosomiasis is no longer negligible. Moreover, several elderly individuals who have always lived in these endemic areas stay protected from infection. An important question for studies of ageing and disease control in developing countries is which differences in the immunological profile of these negatively tested (non-infected) individuals can account for their resistance to either infection or reinfection. We show, in the present study, that non-infected (negative) elderly individuals develop innate immune mechanisms of protection that replace the age-associated decline in T cell function. Non-infected elderly individuals from endemic areas of schistosome infection present an increase in the frequency of the natural killer (NK) CD56low subset of NK cells expressing Toll-like receptors (TLR)-1, -2, -3 and -4 as determined by flow cytometry analysis. In addition, the proportion of dendritic cells expressing TLR-1 is elevated as well as the frequency of monocytes expressing TLR-1 and -4. These results suggest that TLR expression by cells of the innate immune system may be related to the negative status of infection in some elderly individuals who are constantly exposed to S. mansoni. Developing mechanisms of protection from infection may represent a biomarker for healthy ageing in this population.

Introdução: Envelhecimento humano está associado com alterações drástica no sistema imune, a maioria relacionada às células T, que resultam no declíneo progressivo de suas funções. Desde que envelhecimento está associado a deficiências na atividade das células T, não é surpresa o fato de indivíduos idosos estarem mais susceptíveis a infecções. Não existem estudos sistemáticos sobre a resposta imune de indivíduos idosos residentes em áreas endêmicas para S. mansoni. Em um estudo prévio em duas áreas endêmicas de Minas Gerais, Brasil, nosso grupo observou um número considerável de indivíduos com idade acima de 60 anos vivendo nessas áreas, que não pode mais ser ignorado. No entanto, nem todos esses idosos tem alta intensidade de infecção e vários deles que sempre viveram nessas áreas endêmicas permanecem protegidos da infecção e apresentam exame de fezes negativo para ovos de S. mansnoni. Uma questão importante para estudos sobre envelhecimento e controle de doenças em países em desenvolvimento é quais diferenças existem no perfil imunológico dos indivíduos que não se infectam, capazes de conferir proteção contra a infecção ou re-infecção. Indivíduos idosos não infectados devem desenvolver mecanismos para compensar a disfunção imune e gerar resposta protetora contra constante ameaça de infecção nessas áreas. Indivíduos e Métodos: No presente estudo foram incluídos 73 indivíduos (38 não infectados e 35 infectados com S. mansoni), divididos em três grupos etários (7-18, 19-59 e 60-94 anos). Avaliamos por ELISA os níveis das citocinas IL-6 e TNF- e do hormônio do envelhecimento DHEA; e por citometria de fluxo a expressão de receptores envolvidos na imunidade inata TLR-1, 2, 3 e 4 em células NK, células dendríticas, monócitos e linfócitos B. Mecanismos reguladores em células T foram avaliados pela expressão de Foxp3 em linfócitos TCD4+CD25+ em cultura (microgranuloma in vitro) e a expressão de LAP em linfócitos CD4+ no sangue periférico. Resultados: Nós mostramos que indivíduos idosos não infectados residentes em área endêmica para S. mansoni apresentam maior freqüência de NKCD56low expressando TLR-1, 2, 3 e 4; maior freqüência de células dendríticas expressando TLR-1 e maior freqüência de monócitos que expressam TLR-1 e 4. Além disso, também podemos observar que indivíduos idosos infectados possuem maior freqüência de células CD4+CD25+Foxp3+ e CD4+LAP+. As dosagens de citocinas não foram diferentes entre os grupos e a dosagem de DHEA foi menor nos indivíduos idosos infectados quando comparados com adultos infectados, indicando que variações nos níveis desse hormônio em nossos indivíduos pode estar mais associado ao sinergismo dos efeitos do envelhecimento e da infecção crônica por S. mansoni. Conclusões: Nós mostramos no presente estudo que dois mecanismos imunológicos contribuem para distinguir indivíduos idosos infectados de idosos saudáveis (não infectados). Primeiro, indivíduos idosos saudáveis desenvolvem mecanismos de imunidade inata que operam na deficiência das células T durante o envelhecimento, conferindo proteção. Segundo, células T reguladoras cronicamente ativadas, capazes de prejudicar a imunidade protetora, estão presentes com maior freqüência em indivíduos idosos infectados. A partir desses resultados, podemos sugerir que a expressão de TLR em células da imunidade inata pode ser relacionada com a condição de negativo para infecção por S. mansoni em alguns indivíduos idosos expostos constantemente à infecção. O desenvolvimento de mecanismos de proteção contra infecção pode representar um biomarcador de envelhecimento saudável nesta população. Introduction: Several aging-related immunological alterations are already described in medical literature, mostly in the T cell compartment. Senescence is reportedly associated with susceptibility to infection and progressive decline in immune function. We have shown previously, in two endemic areas in Minas Gerais, Brazil, that the frequency of individuals over 60 with chronic schistosomiasis is no longer negligible. There has been no systematic study of the immune response of individuals over 60 living in S. mansoni endemic areas. Since aging is associated with a decline in T cell function, it is not surprising that individuals over 60 would be more susceptible to infection. However, not all aged individuals in endemic areas have high intensity of infection, several old individuals who always lived in these endemic areas stay protected from infection. Non-infected, negative individuals may develop compensatory mechanisms to cope with immune dysfunction and to generate protective responses against the constant threat of infection in these areas. An important question for studies of aging and disease control in developing countries is which differences in the immunological profile of these negatively tested (non-infected) individuals can account for their resistance to either infection or re-infection. Subjects and Methods: We studied from 73 individuals (38 health controls and 35 with chronic schistosomiasis) divided into tree age groups (7-18, 19-59 and 60-94 years), were analyzed. Levels of citokines (IL-6 and TNF-) and dehifroepiandrosterone (DHEA) in the plasma were measured. Were analyzed PBMC from two of these 3 groups of individuals (adult and elderly) by flow cytometry for the expression of TLR-1, 2, 3 e 4 in NK cells, NKCD56low, NKCD56high cells, dendritic cells, monocytes and lymphocytes B. Moreover, we have accessed the frequency of regulatory T cells by the expression of latency-associated peptide (LAP) (PE-labeled anti-LAP) in CD4+ T cells (FITC-labeled anti-CD4) and expression of CD25 and FoxP3 in CD4+ T cells. Results: There was no difference in the levels of cytokines among groups. Levels of DHEA were decreased in infected elderly group when compared with infected adult group and infected young group. This can be effects of the synergism among immunesenescence and schistosome chronic infection. We showed, in the present study, that healthy aged individuals (non-infected) develop innate immune mechanisms of protection that replace the age associated decline in T cell function. Non-infected aged individuals from endemic areas of schistosome infection present increase in the frequency of NKCD56low subset of NK cells expressing TLR 1, 2, 3 and 4 as determined by flow cytometry analysis. In addition, the proportion of dendritic cells expressing TLR1 is elevated as well as the frequency of monocytes expressing TLR1 and 4. Moreover, we have observed that infected elderly individuals showed an increase in the frequency CD4+CD25+Foxp3+ and CD4+LAP+ T cells. Conclusions: We showed that two mechanisms contribute to distinguish between infected and non-infected aged individuals. First, healthy aged individuals (non-infected) develop innate immune responses to replace the decline in T cell function that is observed with aging. Second, chronically activated regulatory T cells, that may impair protective immune responses, are more vigorous in infected aged individuals. These results suggest that TLR expression by cells of the innate immune system may be related to the negative status of infection in some aged individuals that are constantly exposed to S. mansoni. Developing mechanisms of protection from infection may represent a biomarker for healthy aging in this population.

There has been no systematic study of the immune response of individuals over 60 residing in areas endemic for Schistosoma mansoni infection although senescence is reportedly associated with susceptibility to infection and progressive decline in immune function. We have shown previously, in two endemic areas in Minas Gerais, Brazil, that the frequency of individuals over 60 with chronic schistosomiasis is no longer negligible. Several aging-related immunological alterations are already described in medical literature, mostly in the T-cell compartment. Since aging is associated with a decline in T-cell function, it is not surprising that individuals over 60 would be more susceptible to infection. However, not all aged individuals in endemic areas have high intensity of infection; some of them display a negative stool-screening test for the presence of schistosome eggs and low levels of serum antibodies reactive with S. mansoni antigens indicating they are not infected. Non-infected, negative individuals may develop compensatory mechanisms to cope with immune dysfunction and to generate protective responses against the constant threat of infection in these areas. Herein, we reviewed previous reports from our group showing that two mechanisms contribute to distinguish between infected and egg negative aged individuals. First, egg negative aged individuals develop innate immune responses to replace the decline in T-cell function that is observed with aging. Second, chronically activated regulatory T cells, that may impair protective immune responses, are more vigorous in infected aged individuals. We propose that egg negative individuals may be considered as an example of healthy aging in areas endemic for infectious disease.

The determination of the rabies neutralizing antibody (VNA) response after immunization against rabies is an acceptable index of the efficacy of a vaccine and a successful treatment. Several tests have been developed in attempt to improve the assessment of VNA, from mice inoculation to cell-culture fluorescence inhibition tests. All of them, however, present special difficulties in terms of reading or accuracy. The present study describes a neutralization test performed in cell-culture appraised by flow cytometry (FC). Serial dilutions of the serum samples were mixed in vitro with rabies virus before the addition of BHK-21 cells. After 24h-incubation, cells were released by trypsin treatment, fixed and permeabilized with a p-formaldehyde solution and stained with a rabies virus nucleocapsid protein-specific antibody conjugate. The percentage of virus infection inhibition caused by specific antibodies present in the serum were evaluated in a Beckton & Dickinson FACSCalibur® flow cytometer. A correlation curve between the IU/ml content and the percentage of infective inhibition was built with a reference serum and the VNA titers of serum samples were obtained by extrapolation. Titers obtained by FC and standard test showed an effective pairing results (p < 0.01), with a correlation coefficient (r) = 0.7. These results permit to envisage the FC as a suitable technique to evaluate VNA in sera from immunized animals and likely in human serum samples. Nevertheless, new studies comparing FC to gold-standard techniques are required for determining the FC values of Sensibility and Specificity . A titulação de anticorpos neutralizantes contra o vírus rábico (AcN) pós-imunização é um parâmetro aceito como indicador de eficácia da vacina e do sucesso do processo de imunização. Este estudo descreve um teste de neutralização realizado em cultura de células, analisado através da técnica de citometria de fluxo. Diluições seriadas de amostras de soro foram misturadas in vitro com vírus rábico e adicionados a células BHK-21. Após incubação de 24 h, as células foram individualizadas por tratamento com tripsina, fixadas e permeabilizadas com p-formaldeído e coradas com conjugado específico. A porcentagem de inibição da infecção viral causada pelos anticorpos específicos presentes nas amostras de soro foi determinada em citômetro de fluxo Beckton & Dickinson FACSCalibur® e comparada com um soro de referência internacional. Os títulos de AcN foram determinados por extrapolação. Os níveis de correlação (r) entre os títulos obtidos com CF e SFIMT foi de 0.7712 (p < 0,001), e entre CF e ELISA de 0,6702 (p < 0,001). Os resultados indicam que a CF pode ser utilizada na análise de títulos de anticorpos neutralizantes em amostras de soro, com a vantagem de ser um método automatizado. No entanto, devem ser realizados novos estudos, comparando a CF com as técnicas consideradas como padrão ouro, para que os valores reais de Sensibilidade e Especificidade sejam determinados.

Despite the absence of current official reports showing the number of cattle infected by rabies, it is estimated that nearly 30,000 bovines are lost each year in Brazil. In order to minimize the important economic losses, control of the disease is achieved by eliminating bat colonies and by herd vaccination. In this study, we compare the antibody response in cattle elicited by vaccination with an attenuated ERA vaccine (AEvac) and an inactivated-adjuvanted PV (IPVvac) vaccine. The antibody titers were appraised by cell-culture neutralization test and ELISA, and the percentage of seropositivity was ascertained for a period of 180 days. IPVvac elicited complete seropositivity rates from day 30 to day 150, and even on day 180, 87% of the sera showed virus-neutralizing antibody titers (VNA) higher than 0.5IU/ml. There were no significant differences between the VNA titers and seropositivity rates obtained with IPVvac in the two methods tested. AEvac, however, elicited significantly lower titers than those observed in the group receiving inactivated vaccine. In addition, the profiles of antirabies IgG antibodies, evaluated by ELISA, and VNA, appraised by cell-culture neutralization test, were slightly different, when both vaccines were compared.

A imuno-senescência é o resultado cumulativo do processo de declínio progressivo da atividade do sistema imune devido ao processo de envelhecimento. Ela contribui para o aumento da susceptibilidade a doenças infecciosas, câncer e doenças autoimunes. As alterações relacionadas ao envelhecimento incluem a involução tímica, alterações nos fenótipos celulares, perfil de citocinas e no repertório de linfócitos. De um modo geral, animais e humanos idosos apresentam uma redução no número de linfócitos T virgens com o aumento paralelo dos linfócitos T de memória, uma reatividade imunológica comprometida e uma diminuição na sua capacidade de reagira antígenos novos. Vários trabalhos relatam modificações no desenvolvimento de doenças parasitárias ao longo do processo de envelhecimento no que diz respeito a sua prevalência e intensidade.Aponta-se uma forte associação entre idade e resistência à infecções e reinfecções por parasitos, tais como o Schistosoma mansoni, causador de doença endêmica debilitante e que afeta mais de duzentos milhões de pessoas em países em desenvolvimento.É possível que a menor intensidade observada em adultos resulte da aquisição de imunidade protetora devido ao estímulo antigênico crônico. O objetivo desse trabalho foi avaliar a influência do envelhecimento na reatividade imunológica à infecção pelo Schistosoma mansoniem modelo experimental murino. Verificamos que camundongos C57BL/6 velhos apresentam uma resposta inflamatória diminuída aos antígenos do parasito, resultando em títulos mais baixos de anticorpos específicos. Apesar da resposta imune humoral diminuída, os animais velhos são igualmente susceptíveis à infecção por S. mansoni, e também desenvolvem imunidade protetora frente a uma reinfecção. Em animais velhos infectados, a atividade de eosinófilos parece estar preservada e pode ter contribuído para a eliminaçãode vermes na reinfecção. Possivelmente, a menor polarização na produção decitocinas durante a evolução da patologia altera a dinâmica da formação/involução do granuloma no velho. Camundongos velhos formam granulomas menores durante a fase aguda da infecção, mas não é observado o processo de imunomodulação na fase crônica, como bem descrito nos jovens. Observamos um aumento na frequência de células T com perfil de ativação em animais jovens infectados, entretanto, em camundongos velhos, que já possuem maior proporção de linfócitos ativados, a infecção não altera a frequência dessas células. Devido a esse estado constitutivo de ativação dos linfócitos durante a senescência, é provável que respostas inflamatórias a novos antígenos sejam inibidas por encontrarem o sistema imune altamente povoado por células efetoras. The immunosenescence is defined as a cumulative result of decline in the activity of the immune system activity due to progressive aging, and contributes to increased susceptibility to infectious diseases, cancer and autoimmunediseases. Such aging-related changes include thymic involution, changes in frequencies of activated lymphocytes, in cytokine profile and in lymphocyte repertoire. Generally, aged animals and humans show a reduction in the frequency of naïve T lymphocytes, increased proportion of memory T cells and a diminished immune reactivity to novel antigens. Many studies report changes in the prevalence and intensity of parasitic diseasesduring aging. There is a strong association between age and resistance to infectious diseases, such as schistosomiasis, an endemic disease that affects more than two hundred million people in developing countries. It is possible that the lower infection intensity observed in adults results from the acquisition of protective immunity due to chronic antigenic stimulation. The aim of this work was to evaluate the influence of aging in immune reactivity to infection by Schistosoma mansoni in the mouse. We observed that aged C57BL/6 mice have a reduced inflammatoryresponse to parasite antigens, resulting in lower specific antibody titles. Despite the reduced humoral immune response, old animals were as susceptible to infection as young animals, and they also develop protective immunity against reinfection. Eosinophils activity seemed to be conserved in aged infected animals and may have contributed to worm elimination during reinfection. It is plausible that the altered cytokine balance during disease development changes the dynamics of the granuloma formation/involution in aged mice. Aged mice formed smaller granulomas at acute phase of infection, but they did not develop the process of immunomodulation inchronic phase, as described in young animals. After infection, young mice showed a typical increase in the frequency of activated T cells. On the other hand, we found increased frequencies of activated T cells in non-infected aged mice regardless of their infection status. Lymphocytes in non-infected aged animals were constitutively activated and it is likely that inflammatory responses to novel antigens may be impaired when facing an immune system already highly populated by effector cells.