CD1a and Factor XIIIa Immunohistochemistry in Leprosy: A Possible Role of Dendritic Cells in the Pathogenesis of Mycobacterium leprae Infection

Leprosy is a curable chronic granulomatous infectious disease caused by the bacillus Mycobacterium leprae. This organism has a high affinity for skin and peripheral nerve cells. In the evolution of infections, the immune status of patients determines the disease expression. Dendritic cells are antigen-presenting cells that phagocytose particles and microorganisms. In skin, dendritic cells are represented by epidermal Langerhans cells and dermal dendrocytes, which can be identified by expression of CD1a and factor XIIIa (FXIIIa). In the present study, 29 skin samples from patients with tuberculoid (13 biopsies) and lepromatous (16 biopsies) leprosy were analyzed by immunohistochemistry using antibodies to CD1a and FXIIIa. Quantitative analysis of labeling pattern showed a clear predominance of dendritic cells in tuberculoid leprosy. Difference between the number of positive cells of immunohistochemistry for the CD1a and FXIIIa staining observed in this study indicates a role for dendritic cells in the cutaneous response to leprosy. Dendritic cells may be a determinant of the course and clinical expression of the disease.

Genital Manifestation of Graft-vs.-Host Disease: A Series of Case Reports

Introduction. After hematopoietic stem cell transplantation (HSCT), many patients present genital graft-vs.-host disease (GVHD) that can culminate with sexual problems, which are poorly dimensioned. Aim. We hope to draw attention to the need to perform genital biopsy to diagnose genital GVHD, and thus to call attention to the need to incorporate careful attention to sexual health in the treatment of these patients. Methods. Five allogeneic stem cell transplant recipients complaining of coital pain after HSCT were clinically diagnosed for genital GVHD. Genital biopsies were given for histological analysis, and microphotographs of the corresponding marked field in the slide were taken. Specimens were evaluated by the site pathologist and then sent to a reference pathologist, each blinded to the histological findings. A literature search was performed in PubMed/MEDLINE (1966-2009) for cross-sectional and cohort studies or trials related to genital GVHD. Expert opinions peer reviews and case reports were also considered. Main Outcome Measures. HSCT, genital GVHD, genital biopsy. Results. The biopsy showed evidence of dilated apoptotic cells in the basal layer and detachment of the epithelial lining of the mucosa, hyalinization and thickening of collagen fibers, capillary ectasia, and mononuclear inflammatory infiltrate of the submucosa. Three patients presented vulval lesion such as leucoplasia and ulcer on the large lip. Histological analyses showed evidence of epithelial hyperplasia and influx of inflammatory cells to the epithelial surface, intercellular edema and spongiosis, apoptotic bodies on the basal layer of the epithelium, spongiosis, and nuclear vacuolization. A common treatment based on corticotherapy resulted in complete remission of coetaneous or mucous genital lesions in all five patients. Conclusion. Genital biopsy is important to differentially diagnose GVHD and secondary symptoms due to hypoestrogenism. Prevention is the most important step in controlling the evolution GVHD in the vagina to prevent vaginal obstruction and sexual dysfunction. da Silva Lara LA, de Andrade JM, Mauad LMQ, Ferrarese SR, Marana HRC, Tiezzi DG, and de Sa Rosa e Silva ACJ. Genital manifestation of graft-vs.-host disease: A series of case reports. J Sex Med 2010;7:3216-3225.

The genetic structure of 3 ` untranslated region of the HLA-G gene: polymorphisms and haplotypes

The HLA-G gene is predominantly expressed at the maternal-fetal interface. It has been associated with maternal-fetal tolerance and in the inhibition of cytotoxic T lymphocyte and natural killer cytolytic functions. At least two variations in the 3` untranslated region (UTR) of HLA-G locus are associated with HLA-G expression levels, the 14-bp deletion/insertion polymorphism and the +3142 single-nucleotide polymorphism (SNP). However, this region has not been completely characterized yet. The variability of the 3`UTR of HLA-G gene and its haplotype structure were characterized in 155 individuals from Brazil, as well as HLA-G alleles associated with each of the 3`UTR haplotype. The following eight variation sites were detected: the 14-bp polymorphism and SNPs at the positions +3003T/C, +3010C/G, +3027A/C, +3035C/T, +3142G/C, +3187A/G and +3196C/G. Similarly, 11 different 3`UTR haplotypes were identified and several HLA-G alleles presented only one 3`UTR haplotype. In addition, a high linkage disequilibrium among the variation sites was detected, especially among the 14-bp insertion and the alleles +3142G and +3187A, all previously associated with low mRNA availability, demonstrating that their effects are not independent. The detailed analyses of 3`UTR of the HLA-G locus may shed some light into mechanisms underlying the regulation of HLA-G expression. Genes and Immunity (2010) 11, 134-141; doi: 10.1038/gene.2009.74; published online 1 October 2009

Anandamide prior to sensitization increases cell-mediated immunity in mice

The endocannabinoid system has become a topic of great interest in pharmacology due to its remarkable distribution in mammal organisms and capacity to play a modulatory role on several physiological systems, including modulation of immunity. Many studies have shown that administration of cannabinoids causes inhibitory effects on immune cells, including decreased proliferation and antigen-presenting cell (APC) costimulatory activity. In contrast, other groups have shown that some cannabinoids might present stimulatory actions on macrophage activity and T cell activation. Therefore, we aimed to investigate whether a treatment in vivo with a low dose of anandamide (0.1 mg/kg) immediately prior to sensitization would have an immunosuppressive or immunostimulatory effect on cell-mediated immunity (Th1 response) in mice. We report here that anandamide, prior to sensitization, was able to increase the Th1 response to ovalbumin in vivo and ex vivo. Anandamide increased delayed type hypersensitivity (DTH), splenocyte proliferation, and IFN-gamma production in a co-culture of adherent and non-adherent splenocytes. Moreover, anandamide prior to sensitization increased both the expression of DC co-stimulatory molecules (CD80/CD86) and IL-12/IL23 (p40) production ex vivo. We have also assessed direct effects of anandamide in the IFN-gamma/IL-4 balance of ConA-stimulated splenocytes in vitro. Anandamide at nanomolar concentrations increased the production of IFN-gamma, while such production decreased at micromolar range. Thus, anandamide induced both the increment of DC activation and IFN-gamma production, which are likely the mechanisms involved in the increase of Th1 response reported here. (C) 2010 Elsevier B.V. All rights reserved.

Regulation of Trypanosoma cruzi-Induced Myocarditis by Programmed Death Cell Receptor

Trypanosoma cruzi infection causes intense myocarditis, leading to cardiomyopathy and severe cardiac dysfunction. Protective adaptive immunity depends on balanced signaling through a T cell receptor and coreceptors expressed on the T cell surface. Such coreceptors can trigger stimulatory or inhibitory signals after binding to their ligands in antigen-presenting cells (APC). T. cruzi modulates the expression of coreceptors in lymphocytes after infection. Deregulated inflammation may be due to unbalanced expression of these molecules. Programmed death cell receptor 1 (PD-1) is a negative T cell coreceptor that has been associated with T cell anergy or exhaustion and persistent intracellular infections. We aimed to study the role of PD-1 during T. cruzi-induced acute myocarditis in mice. Cytometry assays showed that PD-1 and its ligands are strongly upregulated in lymphocytes and APC in response to T. cruzi infection in vivo and in vitro. Lymphocytes infiltrating the myocardium exhibited high levels of expression of these molecules. An increased cardiac inflammatory response was found in mice treated with blocking antibodies against PD-1, PD-L1, and to a lesser extent, PD-L2, compared to that found in mice treated with rat IgG. Similar results in PD-1(-/-) mice were obtained. Moreover, the PD-1 blockade/deficiency led to reduced parasitemia and tissue parasitism but increased mortality. These results suggest the participation of a PD-1 signaling pathway in the control of acute myocarditis induced by T. cruzi and provide additional insight into the regulatory mechanisms in the pathogenesis of Chagas` disease.

Chemokines in human periodontal disease tissues

An immunoperoxidase technique was used to examine IP-10 (interferon-gamma inducible protein 10), RANTES (regulated on activation normal T cell expressed and secreted), MCP-1 (monocyte chemoattractant protein-1), and MIP-1alpha (macrophage inflammatory protein-1alpha) in gingival biopsies from 21 healthy/gingivitis and 26 periodontitis subjects. The samples were placed into 3 groups according to the size of infiltrate. MIP-1alpha+ cells were more abundant than the other chemokines with few MCP-1+ cells. The mean percent MIP-1alpha+ cells was higher than the percent MCP-1+ cells (P = 0.02) in group 2 (intermediate size infiltrates) lesions from periodontitis subjects, other differences not being significant due to the large variations between tissue samples. Analysis of positive cells in relation to CD4/CD8 ratios showed that with an increased proportion of CD8+ cells, the mean percent MIP-1alpha+ cells was significantly higher in comparison with the mean percent RANTES+ and MCP-1+ cells (P < 0.015). Endothelial cells were MCP-1+ although positive capillaries were found on the periphery of infiltrates only. Keratinocyte expression of chemokines was weak and while the numbers of healthy/gingivitis and periodontitis tissue sections positive for IP-10, RANTES and MCP-1 reduced with increasing inflammation, those positive for MIP-1alpha remained constant for all groups. In conclusion, fewer leucocytes expressed MCP-1 in gingival tissue sections, however, the percent MIP-1alpha+ cells was increased particularly in tissues with increased proportions of CD8 cells and B cells with increasing inflammation and also in tissues with higher numbers of macrophages with little inflammation. Further studies are required to determine the significance of MIP-1alpha in periodontal disease.

Cytokines in periodontal disease: where to from here?

Numerous studies have attempted to elucidate the cytokine networks involved in chronic periodontitis, often with conflicting results. A variety of techniques were used to study cells in situ, cells extracted from gingival tissues, peripheral blood mononuclear cells, purified cell populations, and T cell lines and clones. Bacterial components, including sonicates, killed cells, outer membrane components, and purified antigens, have all been used to stimulate cells in vitro, making comparisons of cytokine profiles difficult. As it is likely that different cells are present at different disease stages, the inability to determine disease activity clinically is a major limitation of all these studies. In the context of tissue destruction, cytokines such as IL-1, IL-6 and IL-18 are likely to be important, as are their regulating cytokines IL-10 and IL-11. In terms of the nature of the inflammatory infiltrate, two apparently conflicting hypotheses have emerged: one based on direct observations of human lesions, the other based on animal experimentation and the inability to demonstrate IL-4 mRNA in gingival extracts. In the first of these, Th1 responses are responsible for the stable lesion, while in the second Th2 responses are considered protective. Using Porphyromonas gingivalis specific T cell lines we have shown a tendency for IFN-gamma production rather than LL-I or IL-10 when antigen is presented with peripheral blood mononuclear cells which may contain dendritic cells. It is likely that the nature of the antigen-presenting cell is fundamental in determining the nature of the cytokine profile, which may in turn open up possibilities for new therapeutic modalities.

The immunology of Epstein-Barr virus infection

Epstein-Barr virus is a classic example of a persistent human virus that has caught the imagination of immunologists, virologists and oncologists because of the juxtaposition of a number of important properties. First, the ability of the virus to immortalize B lymphocytes in vitro has provided an antigen presenting cell in which all the latent antigens: of the virus are displayed and are available for systematic study. Second, the virus presents an ideal system for studying the immune parameters that maintain latency and the consequences of disturbing this cell-virus relationship. Third, this wealth of immunological background has provided a platform for elucidating the role of the immune system in protection from viral-associated malignancies of B cell and epithelial cell origin. Finally attention is now being directed towards the development of vaccine formulations which might have broad application in the control of human malignancies.

CD40 and CD86 upregulation with divergent CMRF44 expression on blood dendritic cells in inflammatory bowel diseases

OBJECTIVE: Dendritic cells (DC) are the only antigen-presenting cells that can activate naive T lymphocytes and initiate a primary immune response. They are also thought to have a role in immune tolerance. DC traffic from the blood to peripheral tissue where they become activated. They then present antigen and the costimulating signals necessary to initiate an immune response. In this study, we investigated the number, subsets, and activation pattern of circulating and intestinal DC from patients with clinically mild ulcerative colitis (UC) or Crohn's disease. METHODS: Patients were recruited, if they were not taking immunosuppressive therapy, and were assessed for clinical severity of their disease using for UC, the Clinical Activity Index, and for Crohn's disease, the Crohn's Disease Activity Index. Blood CD11c(+) and CD11c(-) DC subsets, expression of costimulatory antigens, CD86 and CD40, and the early differentiation/activation antigen, CMRF44, were enumerated by multicolor flow cytometry of lineage negative (lin(-) = CD3(-), CD19(-), CD14(-), CD16(-)) HLA-DR+ DC. These data were compared with age-matched healthy and the disease control groups of chronic noninflammatory GI diseases (cGI), acute noninflammatory GI diseases (aGI), and chronic non-GI inflammation (non-GI). In addition, cryostat sections of colonoscopic biopsies from healthy control patients and inflamed versus noninflamed gut mucosa of inflammatory bowel disease (IBD) patients were examined for CD86(+) and CD40(+)lin(-) cells. RESULTS: Twenty-one Crohn's disease and 25 UC patients, with mean Crohn's Disease Activity Index of 98 and Clinical Activity Index of 3.1, and 56 healthy controls, five cGI, five aGI, and six non-GI were studied. CD11c(+) and CD11c(-) DC subsets did not differ significantly between Crohn's, UC, and healthy control groups. Expression of CD86 and CD40 on freshly isolated blood DC from Crohn's patients appeared higher (16.6%, 31%) and was significantly higher in UC (26.6%, 46.3%) versus healthy controls (5.5%, 25%) (p = 0.004, p = 0.012) and non-GI controls (10.2%, 22.8%) (p = 0.012, p = 0.008), but not versus cGI or aGI controls. CD86(+) and CD40(+) DC were also present in inflamed colonic and ileal mucosa from UC and Crohn's patients but not in noninflamed IBD mucosa or normal mucosa. Expression of the CMRF44 antigen was low on freshly isolated DC, but it was upregulated after 24-h culture on DC from all groups, although significantly less so on DC from UC versus Crohn's or healthy controls (p = 0.024). The CMRF44(+) antigen was mainly associated with CD11c(+) DC, and in UC was inversely related to the Clinical Activity Index (r = -0.69, p = 0.0002). CONCLUSIONS: There is upregulation of costimulatory molecules on blood DC even in very mild IBD but surprisingly, there is divergent expression of the differentiation/activation CMRF44 antigen. Upregulation of costimulatory molecules and divergent expression of CMRF44 in blood DC was also apparent in cGI and aGI but not in non-GI or healthy controls, whereas intestinal CD86(+) and CD40(+) DC were found only in inflamed mucosa from IBD patients. Persistent or distorted activation of blood DC or divergent regulation of costimulatory and activation antigens may have important implications for gut mucosal immunity and inflammation. (Am J Gastroenterol 2001;96:2946-2956. (C) 2001 by Am. Coll. of Gastroenterology).

Broadsheet - Dendritic cells and their emerging clinical applications

Dendritic cells (DC) are now recognised as a unique leukocyte type, consisting of two or more subsets. The origins and functional inter-relationships of these cells are the subject of intense basic scientific investigation. They play important roles in initiating and directing immune responses, defending the host from pathogens and maintaining self tolerance. Fundamental studies are defining new molecules and mechanisms associated with DC function. The first methods for counting these rare blood cell populations are already providing interesting new clinical data. Indeed, abnormal DC function may contribute to deficiencies in the immune response against malignancies. Phase I trial data suggests that DC-based cancer vaccination protocols may contribute an important new biological approach to cancer therapy. Manipulation of DC to facilitate allogeneic transplantation and even to manage autoimmune disease are likely developments.

P-gingivalis-specific T-cell lines produce Th1 and Th2 cytokines

Cytokines produced by T-cells in periodontal lesions may determine the nature of the adaptive immune response. Since different antigen-7 presenting cells (APC) may direct the Th1/Th2 response, P. gingivalis-specific T-cell lines were established by different APC subpopulations, and their cytokine profiles were determined. Peripheral blood mononuclear cells induced similar percentages of IL-4+ and IFN-gamma+ T-cells and lower percentages of IL-10+ T-cells, Epstein-Barr virus-trans formed B-cells (LCL) induced higher percentages of IL-4+ cells than IFN-gamma+ cells, with lower percentages of IL-10+ cells. Peripheral blood mononuclear cells induced a higher percent of IFN-gamma+ CD8 cells than LCL (p = 0.004). Purified B-cells, monocytes, and dendritic cells induced similar percentages of IL-4+ and IFN-gamma+ cells, although again, the percentage of IL-10+ cells was lower. The results of the present study have demonstrated that, as measured by FACS analysis of intracytoplasmic cytokines, P. gingivalis-specific T-cells produce both Th1 and Th2 cytokines, regardless of the APC population.

The number of long-lasting functional memory CD8(+) T cells generated depends on the nature of the initial nonspecific stimulation

The mechanism of generation of memory cytotoxic T cells (CTL) following immunization remains controversial. Using tumor protection and IFN-gamma ELISPOT assays in mice to detect functional CTL, we show that the initial effector CTL burst size after immunization is not directly related to the amount of functional memory CTL formed, suggesting that memory CTL are unlikely to arise stochastically from effector CTL. Induction of MHC class II-restricted T helper cells at the time of immunization by inclusion of a T helper peptide or protein in the immunogen, is necessary to generate memory CTL, although no T helper cell induction is required to generate effector CTL to a strong MHC class I-binding peptide. Host protective T cell memory correlates with the number of CTL epitope responsive IFN-gamma-secreting memory T cells as measured in an ELISPOT assay at the time of tumor challenge. We conclude that a different antigen presenting environment is required to induce long-lasting functional memory CTL, and non-cognate stimulation of the immune system is essential to allow generation of a long-lasting host protective memory CTL response.

Chitosan-alginate microparticulate delivery system for an alternative route of administration of BCG vaccine

Immunisation against M. tuberculosis with current available BCG vaccine lacks efficacy in preventing adult pulmonary tuberculosis. Targeting nasal mucosa is an attractive option for a more effective immunization. The delivery of BCG via the intranasal route involves overcoming barriers such as crossing the physical barrier imposed by the mucus layer and ciliar remotion, cellular uptake and intracellular trafficking by antigen presenting cells. Due to its biodegradable, immunogenic and mucoadhesive properties, chitosan particulate delivery systems can act both as vaccine carrier and adjuvant, improving the elicited immune response. In this study, different combinations of Chitosan/Alginate/TPP microparticles with BCG were produced as vaccine systems. The developed microparticle system successfully modulates BCG surface physicochemical properties and promotes effective intracellular uptake by human macrophage cell lines Preliminary immune responses were evaluated after s.c. and intranasal immunisation of BALB/c mice. BCG vaccination successfully stimulated the segregation of IgG2a and IgG1, where intranasal immunisation with chitosan/alginate particulate system efficiently elicited a more equilibrated cellular/humoral immune response.

Immune reconstitution after autologous hematopoietic stem cell transplantation

Abstract The investigation of the web of relationships between the different elements of the immune system has proven instrumental to better understand this complex biological system. This is particularly true in the case of the interactions between B and T lymphocytes, both during cellular development and at the stage of cellular effectors functions. The understanding of the B–T cells interdependency and the possibility to manipulate this relationship may be directly applicable to situations where immunity is deficient, as is the case of cancer or immune suppression after radio and chemotherapy. The work presented here started with the development of a novel and accurate tool to directly assess the diversity of the cellular repertoire (Chapter III). Contractions of T cell receptor diversity have been related with a deficient immune status. This method uses gene chips platforms where nucleic acids coding for lymphocyte receptors are hybridized and is based on the fact that the frequency of hybridization of nucleic acids to the oligonucleotides on a gene chip varies in direct proportion to diversity. Subsequently, and using this new method and other techniques of cell quantification I examined, in an animal model, the role that polyclonal B cells and immunoglobulin exert upon T cell development in the thymus, specifically on the acquisition of a broader repertoire diversity by the T cell receptors (Chapter IV and V). The hypothesis tested was if the presence of more diverse peptides in the thymus, namely polyclonal immunoglobulin, would induce the generation of more diverse T cells precursors. The results obtained demonstrated that the diversity of the T cell compartment is increased by the presence of polyclonal immunoglobulin. Polyclonal immunoglobulin, and particularly the Fab fragments of the molecule, represent the most diverse self-molecules in the body and its peptides are presented by antigen presenting cells to precursor T cells in the thymus during its development. This probably contributes significantly to the generation of receptor diversity. Furthermore, we also demonstrated that a more diverse repertoire of T lymphocytes is associated with a more effective and robust T cell immune function in vivo, as mice with a more diverse T cell receptors reject minor histocompatiblility discordant skin grafts faster than mice with a shrunken T cell receptor repertoire (Chapter V). We believe that a broader T cell receptor diversity allows a more efficient recognition and rejection of a higher range of external and internal aggressions. In this work it is demonstrated that a reduction of TCR diversity by thymectomy in wild type mice significantly increased survival of H-Y incompatible skin grafts, indicating decrease on T cell function. In addiction reconstitution of T-cell diversity in mice with a decreased T cell repertoire diversity with immunoglobulin Fab fragments, lead to a increase on TCR diversity and to a significantly decreased survival of the skin grafts (Chapter V). These results strongly suggest that increases on T cell repertoire diversity contribute to improvement of T cell function. Our results may have important implications on therapy and immune reconstitution in the context of AIDS, cancer, autoimmunity and post myeloablative treatments. Based on the previous results, we tested the clinical hypothesis that patients with haematological malignancies subjected to stem cell transplantation who recovered a robust immune system would have a better survival compared to patients who did not recover such a robust immune system. This study was undertaken by the examination of the progression and overall survival of 42 patients with mantle cell non-Hodgkin lymphoma receiving autologous hematopoietic stem cell transplantation (Chapter VI). The results obtained show that patients who recovered higher numbers of lymphocytes soon after autologous transplantation had a statistically significantly longer progression free and overall survivals. These results demonstrate the positive impact that a more robust immune system reconstitution after stem cell transplantation may have upon the survival of patients with haematological malignancies. In a similar clinical research framework, this dissertation also includes the study of the impact of recovering normal serum levels of polyclonal immunoglobulin on the survival of patients with another B cell haematological malignancy, multiple myeloma, after autologous stem cell transplantation (Chapter VII). The relapse free survival of the 110 patients with multiple myeloma analysed was associated with their ability to recover normal serum levels of the polyclonal compartment of immunoglobulin. These results suggest again the important effect of polyclonal immunoglobulin for the (re)generation of the immune competence. We also studied the impact of a robust immunity for the response to treatment with the antibody anti CD20, rituximab, in patients with non- Hodgkin’s lymphoma (NHL) (Chapter VIII). Patients with higher absolute counts of CD4+ T lymphocytes respond better (in terms of longer progression free survival) to rituximab compared to patients with lower number of CD4+ T lymphocytes. These observations highlight again the fact that a competent immune system is required for the clinical benefit of rituximab therapy in NHL patients. In conclusion, the work presented in this dissertation demonstrates, for the first time, that diverse B cells and polyclonal immunoglobulin promote T cell diversification in the thymus and improve T lymphocyte function. Also, it shows that in the setting of immune reconstitution, as after autologous stem cell transplantation for mantle cell lymphoma and in the setting of immune therapy for NHL, the absolute lymphocyte counts are an independent factor predicting progression free and overall survival. These results can have an important application in the clinical practice since the majority of the current treatments for cancer are immunosuppressive and implicate a subsequent immune recovery. Also, the effects of a number of antineoplastic treatments, including biological agents, depend on the immune system activity. In this way, studies similar to the ones presented here, where methods to improve the immune reconstitution are examined, may prove to be instrumental for a better understanding of the immune system and to guide more efficient treatment options and the design of future clinical trials. Resumo O estudo da rede de inter-relações entre os diversos elementos do sistema immune tem-se mostrado um instrumento essencial para uma melhor compreensão deste complexo sistema biológico. Tal é particularmente verdade no caso das interacções entre os linfócitos B e T, quer durante o desenvolvimento celular, quer ao nível das funções celulares efectoras. A compreensão da interdependência entre linfócitos B e T e a possibilidade de manipular esta relação pode ser directamente aplicável a situações em que a imunidade está deficiente, como é o caso das doenças neoplásicas ou da imunossupressão após radio ou quimioterapia. O trabalho apresentado nesta dissertação iniciou-se com o desenvolvimento de um novo método laboratorial para medir directamente a diversidade do reportório celular (Capítulo III). Reduções da diversidade do reportório dos receptores de células T têm sido relacionadas com um estado de imunodeficiência. O método desenvolvido utiliza “gene chips”, aos quais hibridizam os ácidos nucleicos codificantes das cadeias proteicas dos receptores linfocitários. A diversidade é calculada com base na frequência de hibridização do ácido nucleico da amostra aos oligonucleótidos presentes no “gene chip”. De seguida, e utilizando este novo método e outras técnicas de quantificação celular examinei, num modelo animal, o papel que as células policlonais B e a imunoglobulina exercem sobre o desenvolvimento linfocitário T no timo, especificamente na aquisição de um reportório diverso de receptores T (Capítulos IV e V). Testei, então, a hipótese de que a presença no timo de péptidos mais diversos, como a imunoglobulna policlonal, induzisse a génese de precursores T mais diversos. Demonstrámos que a diversidade do compartimento T é aumentado pela presença de imunoglobulina policlonal. A imunoglobulina policlonal, e particularmente os fragmentos Fab desta molécula, representam as moléculas autólogas mais diversas presentes nos organismos vertebrados. Estes péptidos são apresentados por células apresentadoras de antigénio às células precursoras T no timo, durante o desenvolvimento celular T. Tal, provavelmente, contribui para a génese da diversidade dos receptores. Também demonstrámos que a presença de um reportório mais diverso de linfócitos T se associa a um incremento da função imunológica T in vivo. Uma diversidade de receptores T mais extensa parece permitir um reconhecimento e rejeição mais eficientes de um maior número de agressores internos e externos. Demonstrámos que ratinhos com receptores de células T (RCT) com maior diversidade rejeitam transplantes cutâneos discordantes para antigénios minor de histocompatibilidade mais rapidamente do que ratinhos com um menor reportório T (Capítulo V). Por outro lado, uma redução da diversidade do RCT, causada por timectomia de ratinhos de estirpes selvagens, mostrou aumentar significativamente a sobrevivência de transplantes cutâneos incompatíveis para o antigénio H-Y (antigénio minor de histocompatibilidade), indicando uma diminuição da função linfocitária T. Além disso, a reconstituição da diversidade dos linfócitos T em ratinhos com uma diversidade de reportório T diminuída, induzida pela administração de fragmentos Fab de imunoglobulina, conduz a um aumento da diversidade dos RCT e a uma diminuição significativa da sobrevivência dos enxertos cutâneos (Capítulo V). Estes resultados sugerem que o aumento do reportório de células T contribui para uma melhoria das funções celulares T e poderão ter implicações importantes na terapêutica e reconstitutição imunológica em contexto de SIDA, neoplasias, autoimunidade e após tratamentos mieloablativos. Baseado nos resultados anteriores, decidimos testar a hipótese clínica de que doentes com neoplasias hematológicas sujeitos a transplantação de precursores hematopoiéticos e com recuperação imunológica precoce após transplante teriam uma sobrevivência mais longa do que doentes que não recuperassem tão bem a sua imunidade. Analisámos a sobrevivência global e sobrevivência sem doença de 42 doentes com linfoma não Hodgkin de células do manto sujeitos a transplante autólogo de precursores hematopoiéticos (Capítulo VI). Os resultados obtidos mostraram que os doentes que recuperaram contagens mais elevadas de linfócitos imediatamente após o transplante autólogo, apresentaram uma sobrevivência global e sem progressão mais longa do que doentes que não recuperaram contagens linfocitárias tão precocemente. Estes resultados demonstram o efeito positivo de uma reconstitutição imunológica robusta após transplante de presursores hematopoiéticos, sobre a sobrevivência de doentes com neoplasias hematológicas. Do mesmo modo, estudámos o efeito que a recuperação de níveis séricos normais de imunoglobulina policlonal tem na sobrevivência de doentes com outras neoplasias hematológicas de linfócitos B, como o mieloma múltiplo,após transplante autólogo de precursos hematopoiéticos (Capítulo VII). A sobrevivência livre de doença dos 110 doentes com mieloma múltiplo analizados está associada com a sua capacidade de recuperar níveis séricos normais do compartmento policlonal de imunoglobulina. Estes resultados pioneiros indicam a importância da imunoglobulina policlonal para a génese de competência imunológica. Também estudámos o impacto de um sistema imunitário eficiente sobre a resposta ao tratamento com o anticorpo anti CD20, ituximab, em doentes com linfoma não Hodgkin (LNH) (Capítulo VIII). Os resultados mostram que doentes com valores mais elevados de linfócitos T CD4+ respondem melhor (em termos de maior sobrevida livre de doença) ao rituximab, do que doentes com valores mais baixos. Estas observações ilustram a necessidade de um sistema imunitário competente para o benefício clínico da terapêutica com rituximab em doentes com LNH. Em conclusão, o trabalho apresentado nesta dissertação demonstra que as células B e a imunoglobulina policlonal promovem a diversidade das células T no timo e melhoram a função linfocitária T periférica. Concomitantemente, também demonstrámos que, no contexto de reconstituição imune, por exemplo, após transplante autólogo de precursores hematopoiéticos em doentes com linfomas de células do manto, o número absoluto de linfócitos é uma factor independente da sobrevivência. Os resultados demonstram, também, a importância dos valores de linfocitos T na resposta ao tratamento com rituximab no caso de doentes com LNH. O mesmo princípio se prova pelo facto de que doentes com mieloma múltiplo sujeitos a transplante autólogo de precursores hematopoiéticos que recuperam valores normais séricos de imunoglobulinas policlonais, terem melhores taxas de resposta em comparação com doentes que não recuperam valores normais de imunoglobulinas policlonais. Estes resultados podem ter importantes aplicações na prática clínica dado que a maioria dos tratamentos de doenças neoplásicas implica imunossupressão e, subsequente, recuperação imunológica. Estes estudos podem ser um instrumento fundamental para uma melhor compreensão do sistema imune e guiar uma escolha mais eficiente de opções terapêuticas bem como contribuir para a concepção de futuros estudos clínicos.

Regulation of autoimmune neuroinflammation by stress-responsive genes

Dissertation presented to obtain the Ph.D degree in Biology