INTRODUÇÃO: A incidência de pacientes apresentando alergia à proteína do leite de vaca (APLV) após os 5 anos de idade vem crescendo. Definir se estes pacientes tolerariam a ingestão de alimento produzido com leite processado a altas temperaturas (LPAT) proporcionaria melhor qualidade de vida, definiria melhor prognóstico e possibilitaria avaliar a indicação de dessensibilização com muffin. OBJETIVO: (1) identificar quais pacientes com APLV persistente aos quatro anos poderiam tolerar a ingestão de LPAT, (2) descrever as características clínicas e laboratoriais dos grupos reativo e não reativo ao LPAT, e (3) compara-las entre os dois grupos. MÉTODOS: Estudo transversal, utilizando amostra de conveniência, incluindo todos os pacientes acompanhados no ambulatório de alergia alimentar do Instituto da Criança HCFMUSP que preenchiam os critérios de inclusão e que concordaram em realizar o TPO, entre janeiro/2013 e novembro/2014. Os pacientes foram admitidos em hospital-dia sob supervisão médica e submetidos à ingestão de um muffin contendo 2,8 gramas de proteína do leite de vaca. Foram definidos como tolerantes se não apresentassem nenhuma reação alérgica. Estes pacientes foram submetidos na sequência a novo TPO com leite de vaca in natura para excluir a tolerância ao leite de vaca. RESULTADOS: Foram realizados 38 TPO com LPAT, sendo que 30 pacientes (15 masculinos) preencheram todos os critérios de inclusão. A mediana da idade foi de 7 anos e 7 meses (4a10m -14a2m). 14 pacientes (46%) não apresentaram reação após a ingestão do muffin, sendo considerados como não reativos. A análise comparativa entre os grupos reativos e não reativos ao LPAT, não mostrou diferença estatisticamente significante quanto às características clínicas: idade (p=0,8), sexo (p=0,4), história pessoal de rinite (p=0,7), história pessoal de asma (p=0,7), história pessoal de outras alergias (p=0,6), história familiar de rinite (p=0,7), história familiar de asma (p=0,3), história familiar de outras alergias (p=0,1), relato de anafilaxia prévia (p=0,07), relato de ingestão de traços de leite previamente ao TPO (p=0,4), relato de reação alérgica no último ano antes da provocação (p=0,6), relato de anafilaxia no último ano antes do TPO (p=0,6). Não se observou diferença estatisticamente significante entre os dois grupos para IgE total (p=0,1) e eosinófilos (p=0,6). O teste de puntura para leite de vaca e frações mostrou diferença estatisticamente significante para ?-lactoalbumina (p= 0,01) e para a caseína (p = 0,004); em relação ao ImmunoCAP® apenas para a caseína (p= 0,05) essa diferença foi significante. Ao avaliar estes pacientes 1 ano após o TPO, nenhum dos 16 pacientes que foram reativos ao LPAT estava ingerindo leite de vaca, enquanto 28% dos pacientes que foram tolerantes ao LPAT estavam consumindo leite de vaca in natura sem reação (p=0,037). CONCLUSÃO: O estudo mostrou que os pacientes com APLV desta amostra brasileira apresentaram 2 diferentes fenótipos, sendo que aproximadamente metade tolerou o LPAT. Sendo assim, o TPO para LPAT deve ser considerado para pacientes com APLV, sempre sob supervisão médica e estrutura segura e adequada, pois pode contribuir para uma mudança no paradigma do seguimento destes pacientes. Teste de puntura e ImmunoCAP® para caseína podem sugerir quais pacientes estariam tolerantes ao TPO com LPAT, reforçando dados da literatura internacional / INTRODUCTION: The incidence of patients with cow\'s milk allergy (CMA) after the age of 5 has been growing. Defining whether these patients can tolerate the ingestion of food produced with baked milk without allergy reaction could provide a better quality of life, a better prognosis and would make it possible to evaluate indication of desensitization with baked milk. OBJECTIVE: (1) To identify which patients with persistent CMA at the age of four could tolerate the baked milk, (2) to describe the clinical and laboratory characteristics of the baked milk reactive group and the baked milk non-reactive group, and (3) to compare those two groups. METHODS: A cross-sectional study was conducted between January/2013 and November/2014. A convenience sample was applied, including all the patients followed in the Food Allergy Center of the Instituto da Criança HCFMUSP, who met inclusion criteria and agreed to carry out the oral food challenge (OFC). The patients were admitted to a day-hospital under medical supervision. They were submitted to a muffin intake containing 2.8 grams of cow\'s milk protein, and then classified as tolerant if they did not present any allergic reaction. To exclude cow\'s milk tolerance these patients were submitted to a new OFC with cow\'s milk in natura. RESULTS: 38 OFC with baked milk were performed, 30 patients (15 male) met all of the inclusion criteria. The median of age was 7 years and 7 months (4y10m -14y2m). 14 patients (46.6%) were considered as non-reactive because they did not present any reaction after the muffin intake. The comparative analysis between baked milk reactive group and baked milk non-reactive group did not show statistically significant difference in the clinical characteristics: age (p=0.8), gender (p=0.4), personal history of rhinitis (p=0.7), personal history of asthma (p=0.7), personal history of others allergies (p=0.6), family history of rhinitis (p=0.7), family history of asthma (p=0.3) family history of others allergies (p=0.1), previous anaphylaxis report (p=0.07), report of milk traits intake prior to OFC (p=0.4), allergic reaction in the last year before the OFC (p=0.6), anaphylaxis in the last year before the OFC (p=0.6). There was no statistically significant difference between the two groups for total IgE (p=0.1) and eosinophils (p=0.6). The Prick test for cow\'s milk and fractions showed statistically significant difference for ?-lactalbumin (p = 0.01) and for casein (p =0.004); in relation to the ImmunoCAP® only for casein (p=0.05) this difference was significant. After 1 year of the OFC, none of the patients which have been reactive to the baked milk were ingesting cow\'s milk, while 28% of the baked milk tolerant patients were consuming cow\'s milk in natura without reaction (p=0.037). CONCLUSION: The present study showed that patients with CMA of this brazilian sample presented 2 different phenotypes. Approximately half of them tolerated baked milk at age four. In conclusion, OFC for baked milk should be considered for patients with CMA, always under medical supervision and appropriate structure, so it could contribute for a change in these patients follow-up. Prick test and ImmunoCAP® for casein can suggest which patients would tolerate the OFC with baked milk, strengthening data of international literature

This study was conducted to investigate the role of natural killer cell surface receptors, KIRs and their cognate HLA ligands in preventing HIV-1 acquisition and disease progression in HIV-1 exposed infants. Using DBS stored for 8 years from 21 pregnant South African women we evaluated 3 methods of gDNA extraction with and without whole genome amplification (WGA) to characterize immune-related genes: IL-10, KIR and HLA class I. However, IL-10 SNP typing was only for testing the quality of gDNA. QIAamp DNA mini kit yielded the highest gDNA quality (p<0.05; Wilcoxon Signed Rank Test) with sufficient yield for subsequent analyses. In contrast, WGA was not reliable for SSP-PCR analysis of KIR2DL1, KIR2DS1, KIR2DL5, and KIR2DL3 or high resolution HLA genotyping using a sequence-based approach. A cohort of 370 infants; 124 HIV-1 perinatally infected, 120 exposed uninfected and 126 unexposed healthy infants was used for KIR and HLA genotyping. After adjustment for viral load and multiple comparisons, the frequency of HLA-Cw*04:01 allele was likely to be associated with susceptibility to mother-to-child acquisition of HIV-1 in exposed infected (EI) infants (p=0.05; Logistic Regression analysis). HLA-A*23:01 was likely to be associated with decreased CD4 T lymphocyte count in HIV-1 infected infants (p=0.01; ANOVA), whereas HLA-B*81 tended to be associated with higher CD4 T lymphocyte count (p=0.04, ANOVA). We speculate that HLA-Cw*04:01 interacts with KIR2DL1 and inhibit NK cell responses which predispose the infants to HIV-1 infection. KIR2DS1 and KIR2DL5 were both associated with faster HIV-1 disease progression. Identified protective HLA-class I alleles could be used to present viral epitopes to either NK cells via KIRs or CTLs and enhance immune activation which may promote resistance to HIV-1 infection. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2012.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010.

Profound lymphopenia has been observed during many acute viral infections, and our laboratory has previously documented a type 1 IFN-dependent loss of most memory (CD44hi) and some naïve (CD44lo) CD8 T cells immediately preceding the development of the antiviral T cell response at days 2-4 following lymphocytic choriomeningitis virus (LCMV) infection. In this thesis, I will examine additional mechanisms involved in the early attrition of CD8 T cells and evaluate whether antigen-specific and non-specific CD8 T cells are equally susceptible. Lastly, I will examine whether the early attrition of CD8 T cells contributes to the generation of an effective immune response. Poly(I:C), a potent inducer of type 1 IFN, was previously shown to cause the attrition and apoptosis of CD8α+CD44hi cells in normal mice, but not in type 1 IFN receptor–deficient mice (IFN1-R KO). I questioned whether additional molecule(s) might contribute to the type 1 IFN-induced apoptosis of CD8α+CD44hi cells. I used a PCR array to determine the expression of 84 apoptosis-related genes at 6 hours post-poly(I:C) treatment, relative to an untreated control. There was an 11-fold increase in CD40 RNA expression in CD8α+CD44hi cells isolated from poly(I:C)-treated mice. CD40 protein expression was also increased on CD8α+CD44hi cells, peaking between 9 and 12 hours following poly(I:C) treatment, before declining thereafter. This increase in CD40 protein expression directly correlated with an increase in Annexin V reactivity, an indicator of early apoptosis. Nevertheless, CD40 was not required for the loss of CD8α+CD44hi cells, as both wildtype and CD40-deficient mice were equally susceptible to the poly(I:C)-induced attrition. Upon further characterization, I found this population of CD40+CD8α+CD44hi cells to be CD11c+B220-Thy1.2- MHCIIhi, which is consistent with a “lymphoid” CD8α+ DC phenotype. Kinetic analysis revealed a type 1 IFN-dependent increase in this CD8α+ DC population at 12 hours post-poly(I:C) treatment. This increase was only observed in the spleen, as no increase in percentage was observed in the peritoneal cavity (PEC), lungs, inguinal lymph nodes (iLN), or peripheral blood. Collectively, these results suggest that the type 1 IFN-dependent increase in splenic CD8α+DCs accounts for the observed increase in Annexin V reactive cells following poly(I:C) treatment. These findings required a re-evaluation of the type 1 IFN-induced attrition of CD8+CD44hi T cells with an anti-CD8β antibody, which is a more exclusive marker for T cells than the anti-CD8α antibody. Kinetic analysis revealed a significant decrease in splenic CD8β+CD44hi T cells at 12 hours post-poly(I:C) treatment. This reduction in splenic CD8β+CD44hi T cells was not due to trafficking to other organs, as the PECs, lungs, iLN, lungs, and peripheral blood all exhibited significant, although varying, decreases in the percentage of CD8β+CD44hi T cells at 12 hour following poly(I:C) treatment. These data support the notion that the type 1 IFN-induced attrition of CD8β+CD44hiT cells was a “global” phenomenon and could not be completely due to migration out of the spleen. The attrition of CD8β+CD44hi T cells was also dependent upon type 1 IFN at 3 days post-LCMV infection, as there was no significant reduction of this population in IFN1-R KO mice. The loss of wildtype CD8β+CD44hi T cells correlated with an increased activation of caspases 3 and 8, which are enzymes that play essential roles in apoptosis and inflammation. A significant loss of CD4+CD44hi T cells, which also correlated with an increased activation of caspases 3 and 8, was observed at 3 days post-LCMV infection. Collectively, these results suggest that attrition of both CD4+CD44hi and CD8β+CD44hiT cell populations is type 1 IFN-dependent and associated with the activation of caspases following LCMV infection. At 3 days post-LCMV infection, both wildtype CD8β+CD44hi and CD4+CD44hi T cell populations had a higher frequency of cells with fragmented DNA, a hallmark characteristic of the late stages of apoptosis, as revealed by terminal transferase dUTP nick end labeling (TUNEL), relative to uninfected controls. This suggests that the loss of both populations was due to apoptosis. Therefore, I questioned whether the LCMV-induced apoptosis of both CD4+CD44hi and CD8β+CD44hi T cell populations occurred through a mitochondrial-induced pathway involving the pro-apoptotic molecule Bim. The attrition of both CD4+CD44hi and CD8β+CD44hi T cells was significantly higher in wildtype mice compared to Bim KO mice at 3 days post-LCMV infection. Moreover, both wildtype CD8β+CD44hi and CD4+CD44hi T cell populations had higher frequency of TUNEL+ cells, relative to Bim KO populations. These results suggest that the apoptosis of CD8β+CD44hi and CD4+CD44hiT cells, following LCMV infection, might occur through a mitochondrial-induced pathway involving Bim. Studies have shown “lymphoid” CD8α+ DCs to be involved in the phagocytosis of apoptotic lymphocytes. Therefore, I evaluated whether host CD8α+ DCs are capable of phagocytosing apoptotic lymphocytes by adoptively transferring CFSE-labeled wildtype donor splenocytes (Ly5.1) into congenic wildtype hosts (Ly5.2), followed by inoculation with poly(I:C). There was an increased frequency of donor cells (Ly5.1, CFSE+) within the host CD8α+CD11c+ gate at 9 and 12 hours post-poly(I:C) treatment. The results suggest that type 1 IFN-activated CD8α+DCs might aid in the rapid clearance of apoptotic cells during the type 1 IFN-induced attrition associated with viral infections. I next questioned whether TCR engagement by antigen would render CD8 T cells resistant to attrition. I tested whether a high concentration of antigen (GP33 peptide) would protect LCMV-specific naïve TCR transgenic P14 cells specific for the GP33 epitope of LCMV and GP33-specific LCMV-immune cells from depletion. Both naïve P14 and memory GP33-specific donor CD8 T cells decreased substantially 16 hours after inoculation poly(I:C), regardless of whether a high concentration of GP33 peptide was administered to host mice beforehand. The increased activation status of naïve antigen-specific cells via peptide inoculation did not confer resistance to type 1 IFN-induced depletion. Donor naïve P14 and LCMV-specific memory cells were also depleted from day 2 LCMV-infected (Clone 13) hosts by 16 hours post-transfer. These results indicate that antigen engagement does not protect CD8 T cells from the type 1 IFN-induced attrition associated with viral infections. Computer models indicated that early depletion of memory T cells may allow for the generation for a more diverse T cell response to infection by reducing the immunodomination caused by cross-reactive T cells. To test this in a biological system, I questioned whether the reduced apoptosis of the crossreactive memory CD8 population (NP205), in aged LCMV-immune mice (18-22 months), following heterologous virus challenge (PV), would allow it to dominate the immune response. At day 8 post-PV infection, the cross-reactive memory CD8 T cell response (NP205) was more immunodominating in aged LCMV-immune mice relative to younger LCMV-immune mice. This was indicated by the increased ratio of the cross-reactive NP205 response to the newly arising noncross-reactive, PV-specific NP38 response in older LCMV-mice relative to younger LCMV immune-mice, at day 8 post-PV infection. These data suggest that the early attrition of T cells allows for the generation of a more diverse T cell response to infection by reducing the immunodomination caused by crossreactive T cells. Collectively, these findings offer further insight into the early attrition of T cells associated with viral infections.

Prior immunity to a related or unrelated pathogen greatly influences the host’s immune response to a subsequent infection and can cause a dramatic difference in disease course, a phenomenon known as heterologous immunity. Heterologous immunity can influence protective immunity, immunopathology and/or immune deviation of cytokine-producing T cell subsets. Examples of heterologous immunity have been well documented in mouse models, as well as during human infections. For example, prior immunity to lymphocytic choriomeningitis virus (LCMV) provides partial protection against vaccinia virus (VV), as LCMV-immune mice show reduced VV titers and increased survival upon lethal dose VV infection. Heterologous protection against VV challenge, as a result of LCMV immunity, is mediated by LCMV-specific CD4 and CD8 T cells, as transfer of LCMV-specific memory T cells can mediate this protective effect in naïve mice. The recognition of a single TCR with more than one MHC-peptide complex is referred to as T cell cross-reactivity. A VV Kb-restricted epitope a11r198 was identified to be able to induce cross-reactive responses from LCMV-specific CD8 T cells. During VV infection, LCMV-specific memory T cells that are cross-reactive to VV epitopes produce IFN-γ early in VV infection. IFN-γ is essential for mediating the protection against VV in LCMV-immune mice, as this heterologous protection is absent in IFN-γR-/-and IFN-γ blocking antibody-treated LCMV-immune mice. In addition to protective immunity, cross-reactive LCMV-specific memory T cells and IFN-γ also induce an altered immunopathology during heterologous VV challenge. LCMV-immune mice show moderate to severe levels of inflammation of the fat tissue, known as panniculitis, in the visceral fat pads upon VV challenge. In humans, panniculitis is a painful condition, most commonly presenting as erythema nodosum. Erythema nodosum is a disease of unknown etiology with no known treatment. It may occur following intracellular bacterial and viral infections, and occasionally happens after vaccination with VV for smallpox. During infections there can be a delicate balance between the ability of immune responses to provide protective immunity, and the tendency to induce immunopathology. By using the mouse model of heterologous immunity between LCMV and VV, we tried to understand how the immunity to LCMV biased the balance between the protective immunity and immunopathology, and what effector molecules were responsible for the pathogenesis of panniculitis in this system. TNF is a pleiotropic cytokine, which is required for normal innate and adaptive immune responses. Its functions range from inducing proliferative responses including cell survival, to destructive responses such as promoting apoptosis and programmed necrosis. In response to inflammatory stimuli, activated macrophages/ monocytes produce large amounts of TNF, and upon activation, T cells, B cells and NK cells also produce TNF. In vitro and in vivo studies have shown that TNF in synergy with IFN-γ plays an important role in mediating host defense against pathogens, such as Listeria monocytogenesand poxviruses in mice and hepatitis B virus and human immunodeficiency virus in humans. However, inappropriate expression of TNF often results in tissue damage. Considering the important role TNF plays in both host defense and mediating autoimmune diseases, we hypothesized that TNF was required for mediating both protective and pathogenic effects in the heterologous immunity between LCMV and VV. We first examined whether TNF was involved in mediating protective heterologous immunity. LCMV-immune mice, that were TNF-deficient as a consequence of genetic deletion (TNF-/-) or receptor blockade by treatment with etanercept (TNFR2: Fc fusion protein), were challenged with VV. These TNF-deficient mice showed normal recruitment and selective expansion of cross-reactive LCMV-specific memory CD8 T cells. They also exhibited efficient clearance of VV similar to LCMV-immune mice with normal TNF function. Thus, we concluded that neither TNF nor lymphotoxin (LT), which uses the same receptors as TNF, was required in mediating protective heterologous immunity against VV. Indeed, prior immunity to LCMV could completely compensate for the role of TNF in protection of naïve mice against VV infection, even under conditions of lethal dose inoculum. Thus, heterologous immunity may help explain why treatment of humans with etanercept is reasonably well tolerated with relatively few infectious complications. One of the histological characteristics of panniculitis is necrosis of adipose tissue. It is known that three members in the TNF superfamily, i.e. TNF/LT, FasL and TRAIL are able to induce necrosis of a target cell. It is also known that TNF is able to induce VV-infected cells to go through necrosis, when apoptosis is blocked in these cells by VV protein. Furthermore, TNF and FasL have already been shown to be associated with some skin and fat pathology. Thus, we hypothesized that TNF, FasL and TRAIL were involved in the pathogenesis of panniculitis in VV infected LCMV-immune mice. By using blocking antibodies or genetically deficient mice, we demonstrated that both TNF/LT and FasL were crucial for inducing panniculitis. Although TNFR1 has been reported to induce programmed necrosis, our data indicated that TNFR2, not TNFR1, was involved in mediating tissue damage in the fat pads of LCMV-immune mice infected with VV. We also found that TNF signaled through TNFR2 to up-regulate the expression of Fas on adipocytes. Thus, the engagement of Fas on the adipocytes with FasL expressed on activated VV-specific and cross-reactive LCMV-specific CD8 T cells in the fat pads could lead to panniculitis. Thus, our data may identify a potential mechanism in the pathogenesis of human panniculitis, and may suggest a possible treatment for this painful disease. Recent reports suggest that heterologous immunity may contribute to the tremendous variation in symptoms between individuals, from subclinical to death, upon viral infection. Even in genetically identical mice, variations in immunopathology from none to life-threatening levels of pathology are observed in LCMV-immune mice during VV infection. By adoptive transfer of splenocytes from a single LCMV-immune donor into two recipients, we showed that similar levels of pathology were generated in mice receiving the same splenocytes. However, the level of pathology varied among recipients receiving splenocytes from different LCMV-immune donors. The difference in levels of VV-induced pathology observed in individual LCMV-immune mice was a reflection of the private specificity of the T cell repertoire, which is a unique characteristic of each individual immune host. The goal of this doctoral thesis is to understand how heterologous immunity contributes to the pathogenesis of panniculitis. Our data demonstrate that TNF/LT and FasL directly contribute to development of panniculitis in LCMV-immune mice during VV infection, and suggest that anti-TNF treatment might be a useful treatment for diseases, such as erythema nodosum and lupus-induced acute fatty necrosis in humans.

This study was designed to observe the effect of a psychoeducational intervention on CD4/CD8 and viral loads in the HIV+ patients as well as to promote a space where patients could learn more about the HIV virus and direct their attention to aspects that need to be dealt with for their well-being. This study was also undertaken to evaluate the prognostic value of IL-16 serum levels, in comparison to CD4 counts, CD8 counts, viral load and 02 microglobulin levels (J32-MG). Two groups of HIV+ patients, one of women infected heterosexually (n=7) and another of male homo/bisexuals (n=11) participated in psychoeducational encounters. Blood samples were drawn at baseline (T1), approximately 3 months after baseline and immediately after group intervention (T2) and approximately 6 months after baseline (T3) to perform CD4/CD8 and viral load exams. Laboratory data from 55 HIV-infected subjects and 10 AIDS inpatients were analyzed to compare prognostic values. Encounters were reported as informative. General study results revealed that patients who attended less than 7 sessions had significantly lower CD4 numbers, between T1 and T3 ( p = 0.0039), patients who attended more than 7 sessions had significantly lower viral loads between T2 and e T3 ( p = 0.0234). Patients who were not offered any session had stable CD4 counts and viral loads over study period. The median values, in general, did not differ neither according to clinical classification nor to time (in months) of patients' diagnosis. An inverse correlation was detected between 1L-16 levels and viral load (r =-.269) and between 02-MG and CD4 (r =-610) while a positive correlation was observed between 132-MG and viral load (r = 0.433). Three conclusions may be stated. In relation to the psychoeducational intervention and immunological parameters, a larger number of participants is needed for significant statistical analysis. This study is favorable to the adoption of j32-MG, as a more useful predictor than 1L-16 levels for AIDS progression. Athough, 1L-16 is not confirmed as an useful marker in I IIV infection it still warrants future studies especially in regard to its feasibility as a therapeutic weapon. / Este estudo teve como objetivo avaliar o efeito da intervenção psicoeducativa sobre os parâmetros tradicionais, ou seja exames de CD4/CD8 e carga virai, de acompanhamento imunológico, em pacientes HW+, bem como proporcionar um espaço onde os pacientes poderiam aprender mais sobre o vírus 1-11V e pudessem dirigir a sua atenção a alguns aspectos que contribuam para o seu bem-estar. Ademais, o estudo visou avaliar o valor prognóstico de níveis séricos de IL-16 em comparação as contagens de CD4 e CDS, valores de carga virai e níveis de (32 microglobulina 032- MG). Dois grupos de pacientes 1-11V+, um de mulheres infectadas heterossexualmente (n=7) e outro de homo/bissexuais masculinos (n=11), participaram de encontros psicoeducativos no Hospital das Clínicas da Universidade Federal de Uberlândia (FIC-UFU), no período de maio a agosto de 2001. Amostras de sangue foram colhidas em três momentos: no tempo de linha de base (TI), aproximadamente três meses depois do tempo de linha de base e imediatamente após a intervenção grupal (T2) e, aproximadamente seis meses depois do tempo de linha de base (T3) para realizar os exames de CD4/CD8 e carga virai. Para a comparação entre as provas imunológicas, foram analisados os dados laboratoriais de 55 indivíduos HTV+ e de 10 pacientes internados com AIDS (HC¬UFU). Os encontros foram avaliados como sendo proveitosos. O estudo em geral revelou que os pacientes que participaram de menos de sete sessões tiveram números de CD4 significativamente mais baixos no T1 e T3 ( p = 0,0039), os pacientes que freqüentaram mais que sete sessões tiveram log de carga virai significativamente mais baixos entre T2 e T3 ( p = 0,0234) e os pacientes que não foram oferecidos nenhuma sessão tiveram números de CD4 e log de carga viral estáveis durante o período do estudo. Em geral, os valores medianos não diferiram de acordo com a classificação clínica nem com o tempo de diagnóstico (em meses). Urna correlação inversa foi encontrada entre níveis de IL-16 e carga viral (r = -0,269) e entre f32-MG e CD4 (r = - 0,610), enquanto uma correlação positiva foi observada entre f32-MG e carga viral (r 0,433). O estudo levou a três conclusões. Primeiramente,em relação à intervenção psicoeducativa e observação da evolução imunológica, há necessidade de um maior número de participantes para uma análise estatística significativa. Em segundo lugar, sugere-se que a 132-MG é um parâmetro mais útil do que a IL-16 para monitorar a progressão da AIDS. Finalmente a IL-16, apesar de não ser confirmado como um marcador útil na infecção pelo HIV, ainda constitui interessante objeto, para futuros estudos principalmente no que diz respeito à sua viablidade como arma terapêutica. / Mestre em Imunologia e Parasitologia Aplicadas

A epidemia causada pelo vírus da imunodeficiência humana (HIV) é a mais importante das ultimas décadas. A despeito dos avanços no conhecimento da patogenia do vírus e da resposta imune à infecção, até o momento não existe uma vacina eficaz contra a aquisição do HIV. Diversas linhas de evidência indicam que anticorpos neutralizantes ou ligadores, linfócitos T CD4+ e T CD8+ desempenham um papel importante na imunidade contra o HIV. Os anticorpos que são capazes de neutralizar o HIV são direcionados principalmente à glicoproteína do envelope do vírus (env), mas os candidatos vacinais baseados na proteína de envelope gp120 monomérica testados até hoje falharam em induzir proteção nos ensaios de eficácia. Avanços no entendimento da estrutura e função da glicoproteína env tem facilitado o desenvolvimento de uma nova geração de imunógenos baseada em trímeros mais estáveis e solúveis da glicoproteína gp140. Em uma formulação vacinal além da escolha do antígeno, os adjuvantes desempenham um papel fundamental. Os adjuvantes são conhecidos por aumentar a imunogenicidade das vacinas, e nos últimos anos vários compostos, incluindo agonistas de receptores do tipo Toll (TLR) e NOD (NLR) têm demonstrado eficácia em ensaios clínicos. Em estudos prévios, nosso grupo demonstrou que a imunização de camundongos com uma vacina de DNA codificando 18 epítopos para linfócitos T CD4+ do HIV-1 (HIVBr18), foi capaz de induzir resposta específica e ampla de linfócitos T CD4+ e T CD8+. Devido ao importante papel do efeito auxiliar de linfócitos T CD4+ na resposta humoral nas imunizações assistidas por diversos adjuvantes, o objetivo central do trabalho foi verificar se a imunização inicial com a vacina de DNA HIVBr18 seria capaz de aumentar a magnitude/qualidade de resposta imune humoral e celular induzida pelo trímero de gp140 na presença de diferentes adjuvantes. Para tal, camundongos BALB/c foram imunizados inicialmente com a vacina HIVBr18 ou com o vetor vazio e posteriormente com a proteína gp140 na presença dos adjuvantes: completo de Freund (ACF), poly IC, CpG ODN 1826, Monofosforil lipídeo A (MPL), Muramildipeptídeo (MDP), Imiquimod (R837), e Resiquimod (R848). Observamos que a imunização inicial com HIVBr18 foi capaz de fornecer um auxílio cognato para a proliferação específica de linfócitos T CD4+ e T CD8+ e também para a produção da citocina IFNy. A análise da xx resposta humoral mostrou que a imunização inicial com a vacina HIVBr18, foi capaz de influenciar a produção das subclasses de imunoglobulinas, independente do adjuvante testado. No presente trabalho, também analisamos a influência dos adjuvantes na imunogenicidade da gp140. Os animais que receberam os adjuvantes MPL, poly IC e CpG ODN 1826 apresentaram títulos de anticorpos estatisticamente superiores quando comparados aos animais que receberam os adjuvantes Alum, MDP, R837 e R848. Observamos que os animais imunizados com a gp140 na presença dos diferentes adjuvantes desenvolveram células B do centro germinativo e células TCD4+ auxiliar foliculares. Estes resultados nos permitem concluir que a imunização inicial com HIVBr18 é capaz de alterar a qualidade da resposta humoral e celular gp140- específica. Assim, essa formulação poderia ser utilizada para auxiliar e/ou direcionar a resposta imune induzida por outros imunógenos como por exemplo o trímero de gp140. Podemos concluir também que diferentes formulações de adjuvantes que se encontram em ensaios clínicos como poly IC, CpG ODN e MPL podem ser capazes de induzir um resposta imune humoral e celular tão ou mais potente que aquela induzida pelo ACF / The epidemic caused by the human immunodeficiency virus (HIV) is the most important in the last decades. Despite advances in the knowledge about virus pathogenesis and immune response to infection, until now there is not an effective vaccine against HIV acquisition. Several evidences indicate that neutralizing or binding antibodies, CD4+ and CD8+ T lymphocytes play an important role in immunity against HIV. The antibodies that are able to neutralize HIV are primarily directed against the virus envelope glycoprotein (env), but the vaccine candidates based on monomeric gp120 envelope protein tested so far failed to induce protection in efficacy trials. Advances in understanding the structure and function of the env glycoprotein have facilitated the development of a new generation of immunogens based on trimers, a more stable and soluble form of gp140 glycoprotein. In a vaccine formulation, in addition to the antigen, adjuvants play a pivotal role. Adjuvants are known to increase the immunogenicity of vaccines and, in the last years, several compounds, including agonists of Toll-like receptors (TLR) and NOD (NLR), have presented efficacy in clinical trials. In previous work, our group demonstrated that immunization of mice with a DNA vaccine (HIVBr18) encoding 18 CD4+ T cells epitopes from HIV-1 was able to induce a broad CD4+ T and CD8+ T cells specific response.. Given the important role of CD4+ T cells in the humoral response after adjuvant-assisted immunization, the aim of the study was to verify whether an initial immunization with the DNA vaccine HIVBr18 could increase the magnitude/quality of humoral and cellular immune response induced by gp140 trimer in the presence of different adjuvants. Therefore, BALB/c mice were initially immunized with the vaccine HIVBr18 or empty vector and then with gp140 in the presence of the following adjuvants: Freund\'s complete (CFA), poly IC, CpG ODN 1826, monophosphoryl lipid A (MPL), Muramyl dipeptide (MDP), Imiquimod (R837), and Resiquimod (R848). We observed that initial immunization with HIVBr18 was able to provide cognate help for specific CD4+ and CD8+ T cells proliferation and also for IFN-y production. Analysis of humoral response showed that initial immunization with the HIVBr18 vaccine was able to alter the production of immunoglobulin subclasses independent of the adjuvant tested. This work also analyzed the influence of adjuvants on the immunogenicity of gp140. Mice that received the adjuvant MPL, poly IC and CpG ODN 1826 presented higher antibody titers when compared to animals that received Alum, MDP, R837 and R848. We observed that mice immunized with gp140 in the presence of all adjuvants tested developed germinal center B cells and follicular helper T cells (TFH). We conclude that initial immunization with HIVBr18 is able to alter the quality of specific humoral and cellular immune responses.. Therefore, this formulation could be used in combination with other immunogens, such as gp140, to help/redirect the immune response. We also conclude that the adjuvants that are in clinical trials such as poly IC, MPL and CpG ODN 1826 may be able to induce stronger humoral and cellular response than CFA

A produção de kits para diagnóstico in vitro deve ser feita seguindo-se a legislação vigente de Boas Práticas de Fabricação e Controle (BPF). O objetivo deste trabalho foi elaborar um procedimento para desenvolvimento, produção e validação de um produto para diagnóstico in vitro, de acordo com a legislação vigente. Adotamos como modelo um kit imunoenzimático para Doença de Chagas. Dentro dos requisitos de BPF, a validação é uma etapa importante, pois tem por objetivos, dentre outros: auxiliar no estabelecimento de procedimentos de produção e controle de qualidade, avaliar desvios e dimensionar possíveis erros, avaliar o desempenho quanto à utilidade médica dos resultados obtidos e estabelecer condições ideais de uso. No estabelecimento dos requisitos para validação devem-se considerar as características do método utilizado, a utilidade clínica e diagnóstica dos resultados e as condições de uso do kit. Os parâmetros para validação devem ser definidos considerando a finalidade do uso do produto. Os resultados obtidos em três lotes pilotos demonstraram que o kit pode ser utilizado tanto com soro como com plasma, as amostras podem ser congeladas e descongeladas antes do uso por até 5 ciclos, o índice de concordância com kit comercial é de 0,9 (ótimo) e o kit mantém-se estável por pelo menos 7 dias à 37ºC, o que neste trabalho foi equivalente a pelo menos um ano na sua condição ideal de armazenamento de 2 a 8ºC. Além disso, o kit apresentou 100% de sensibilidade, 99% de especificidade, com coeficiente de variação 15,2% tanto na repetitividade como na reprodutibilidade de amostras positivas. Quanto à análise de interferentes, amostras hemolisadas e a presença de fator reumatóide podem interferir nos resultados e anticorpos anti-Leishmania na amostra podem dar reação cruzada. Conclui-se que o procedimento elaborado e o kit desenvolvido e validado atenderam aos requisitos pré-estabelecidos, de acordo com as regras de BPF vigentes. / The production of an in vitro diagnostic kit should be done following current Good Manufacturing Practices (GMP). The objective of this work was to establish a procedure for the development, production and validation of an in vitro diagnostic product in accordance with current regulations governing Medical Devices. An enzyme-linked immunoassay kit for Chagas\' disease was used as a model. Validation is a very important step contained within GMP requirements. Validation provides documented evidence that processes and product batches are consistent, it aids in the establishment of production and quality control procedures, evaluate deviations and identify possible mistakes, evaluate the performance and medical usefulness of the product based on the obtained results, and establish ideal conditions of use and storage. In order to establish validation requirements for product development, it is necessary to consider the characteristics of the assay method, the clinical and diagnostic usefulness of the results and the conditions of use of the kit. The parameters for validation should be defined considering the purpose of the use of the product. In the case of this Chagas assay, results obtained in three pilot lots demonstrated that the kit could be used with both serum and plasma, samples could be frozen and thawed before use for up to 5 cycles. The agreement index when compared with a commercially licensed kit is 0,9 (optimum correlation). The kit remained stable for at least 7 days at 37ºC, which is equivalent to at least one year stability in its ideal storage condition of 2 to 8ºC. The kit presented 100% sensitivity and 99% specificity, with variation coefficient of 15,2% for both repeatability and reproducibility of the positive samples. Interference analysis indicated that: hemolyzed samples and the presence of reumathoid factor could interfere with test results. Antibodies anti-Leishmania in the test sample can cross react with T. cruzi proteins. In conclusion: the established procedure for development and validation of chagas kit, and the actual developed and validated kit are in accordance with pre-established current GMP requirements.

INTRODUÇÃO: O câncer constitui-se na principal causa de mortalidade entre indivíduos de 45 a 84 anos, configurando-se em um dos principais problemas de saúde pública dos países em desenvolvimento. As reações de hipersensibilidade aos quimioterápicos têm aumentado, impedindo muitas vezes a utilização de terapias de primeira linha no tratamento de neoplasias primárias ou recidivantes. O procedimento de dessensibilização é uma abordagem alternativa, por meio do qual o paciente passa a tolerar a medicação que antes desencadeava reações potencialmente letais. Quimioterápicos do grupo das platinas são exemplos de drogas passíveis de readministração por meio do processo de dessensibilização, no entanto faltam biomarcadores preditivos de reações durante o procedimento. OBJETIVOS: O objetivo principal do estudo foi avaliar o papel do teste de ativação de basófilos (BAT) como biomarcador para reações de hipersensibilidade ocorridas durante a dessensibilização em pacientes alérgicas às platinas. Como objetivo secundário, avaliou-se a prevalência e o impacto da mutação dos genes BRCA 1 e 2 em pacientes com hipersensibilidade imediata à carboplatina submetidas à dessensibilização. MÉTODOS: Padronizou-se o BAT, com análise da expressão de CD63 e CD203c na superfície de basófilos de pacientes com hipersensibilidade imediata às platinas submetidas à dessensibilização. Foram realizados BATs em 15 pacientes portadoras de neoplasias malignas submetidas a 27 dessensibilizações devido à anafilaxia a quimioterápico do grupo das platinas e em 12 indivíduos de dois grupos controle (Grupo 1: seis pacientes tolerantes às platinas e Grupo 2: seis voluntários sadios que nunca foram expostos às platinas). Os resultados dos BATs foram comparados entre os três grupos. Correlacionou-se o BAT com a ocorrência ou não de reação durante a dessensibilização e com os níveis de triptase sérica. Para análise da prevalência e impacto da mutação dos genes BRCA 1 e 2 nas dessensibilizações, realizou-se análise retrospectiva de prontuários de 138 portadoras de neoplasias malignas ginecológicas submetidas à dessensibilização à carboplatina. RESULTADOS: O BAT foi positivo em 11 das 15 pacientes alérgicas (n= 11; 73,3%), com aumento de expressão de CD203c e CD63 em 11 (73,3%) e 6 (40%) pacientes, respectivamente. Todos os participantes dos grupos controles apresentaram testes negativos. Maior expressão de CD63 foi observada em pacientes com reações iniciais mais graves. O BAT foi positivo em 92,3% das reações ocorridas durante as dessensibilizações (n=12/13), sendo positivo em todas as reações que apresentaram aumento concomitante de triptase sérica (n=5). Com relação à mutação dos genes BRCA 1 e 2, sua prevalência foi de 34% nas pacientes com hipersensibilidade às platinas (n=47/138), sendo que 51% das portadoras reagiram durante a dessensibilização. CONCLUSÕES: O BAT positivo, com aumento da expressão de CD63 e/ou CD203c na superfície do basófilo, identificou pacientes alérgicos às platinas com especificidade de 100% e sensibilidade de 73,3%. O BAT e a mutação dos genes BRCA 1 e 2 identificaram pacientes mais propensos a reagir durante o procedimento de dessensibilização. A utilização de biomarcadores preditores de reações durante a dessensibilização aos quimioterápicos do grupo das platinas pode aumentar a segurança do procedimento e auxiliar na manutenção do esquema quimioterápico de primeira linha do paciente / INTRODUCTION: Cancer is the leading cause of death in the age group of 45 to 84 years, and one of the main public health issues in developing nations. Hypersensitivity reactions to chemotherapeutic agents have been increasing, sometimes hindering the use of first-line therapies in the treatment of primary or relapsed tumors. Rapid drug desensitization (RDD) is an alternative approach, through which a patient becomes tolerant to the medication that once triggered a potentially lethal hypersensitivity reaction. Platinum-based compounds are examples of drugs that can be readministered through the desensitization procedure, but currently there are no known biomarkers that could help predict reactions during RDD. OBJECTIVES: The main goal of our study was to assess the basophil activation test (BAT) as a biomarker of breakthrough reactions occurred during RDD in patients allergic to platinum-based agents. As a secondary goal, we evaluated the prevalence and impact of the BRCA 1/2 mutation in carboplatin-allergic patients undergoing RDD. METHODS: We standardized the BAT by evaluating CD63 and CD203c expressions on the basophils of patients with immediate hypersensitivity reactions to platinum-based agents undergoing RDD. We analyzed BATs of 15 patients with malignant neoplasms who had undergone 27 RDD procedures due to anaphylaxis to platinum-based agents, and of 12 control subjects (Group 1: six patients tolerant to platinum-based agents, and Group 2: six healthy volunteers who had never been exposed to platinum-based agents). BAT results were compared among the three groups. We correlated BAT results with the occurrence of breakthrough reactions during RDD and with serum tryptase levels. To conduct the analysis of the BRCA 1/2 mutation prevalence and its impact on RDD, a retrospective review of 138 medical records of patients with gynecological malignancies who underwent RDD to carboplatin was performed. RESULTS: BAT was positive in 11/15 allergic patients (73.3%), with increased expression of CD203c and CD63 in 11 (73.3%) and 6 (40%) patients, respectively. All control subjects presented negative BATs. A higher CD63 expression was observed in patients with severe initial reactions. BAT was positive in 92.3% of the breakthrough reactions occurred during RDD (n=12/13), and in all reactions with concomitant increased tryptase levels (n=5). Regarding the BRCA1/2 mutation, its prevalence was 34% in patients allergic to platinum-based agents (n=47/138), and 51% of the mutation carriers had breakthrough reactions during RDD. CONCLUSIONS: A positive BAT, with an increased expression of CD63 and/or CD203c, identified patients allergic to platinum-based agents with a specificity of 100% and a sensitivity of 73.3%. The BAT and the BRCA 1/2 mutation helped identify patients at risk of breakthrough reactions during RDD. The use of predictive biomarkers of breakthrough reactions during RDD to platinum-based agents might enhance RDD safety and help maintain a patient`s first-line treatment

The failure of the immune system to provide efficient protection against tumour cells has been considered as a major issue in immunology. It is now well established that inadequate function of the host immune system is one of the main mechanisms by which tumours escape from immune control contributing to the limited success of cancer immunotherapy. Several cell populations have been described which display immunosuppressive properties and may impede tumor-specific immunity. Among them, GR1+CD11b+ immature myeloid suppressor cells and CD4+CD25+ regulatory T cells seem to play an important role. These cells accumulate in the spleens of tumour bearing mice and patients with cancer and contribute to immunosuppression by inhibiting the function of CD8+ T cells and/or by promoting tumour angiogenesis.<p><p>The aim of our work was to define the mechanisms by which a single dose of cyclophosphamide (CTX), a chemical agent commonly used in chemotherapy treatment, induces the rejection of established P815 mastocytoma. <p><p>Our data show that CTX treatment leads to the selective loss of GR1medCD11b+ splenic myeloid cell producing TGF-â, a cytokine which is known to suppress antitumoral response. Furthermore, injection of CTX causes a decrease in the number of naturally occurring regulatory T cells (CD4+CD25+Foxp3+) in the spleen and the tumor. Finally, CTX treatment induces the differentiation of GR1highCD11b+ splenic myeloid cells into mature GR1highCD11b+CD11c+ (possibly dendritic cells?) which express high levels of CD11c, MHC class II and CD86 molecules. Of note, these cells are mainly detected in tumour necrosis areas. <p><p>Collectively, these results suggest that CTX prevents suppressive mechanisms and induces a population of CD11c+ myeloid cells which may present tumor antigens and activate T lymphocytes, an hypothesis in line with the requirement for CD4+ cells in CTX-induced long term resistance. <p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished