Embora existam vários mecanismos propostos, o papel das moléculas de histocompatibilidade na susceptibilidade ao DM1 ainda não está totalmente esclarecido. Existem várias evidências de que o número de células apresentadoras de antígenos e a densidade de expressão das moléculas de histocompatibilidade nessas células pode influenciar o resultado da resposta imune. Assim, neste estudo, foram avaliadas as porcentagens das células CD3+, CD4+, CD8+, CD19+ e CD14+, coexpressando as moléculas de histocompatibilidade de classe I e II, a densidade de expressão das moléculas de histocompatibilidade de classe I e II nessas populações linfomonocitárias e a correlação entre densidade de expressão dessas moléculas com o perfil imunogenético do DM1. Para esse fim, foram avaliados 20 pacientes com DM1, recentemente diagnosticados, metabolicamente compensados, sendo 12 do sexo masculino. Como controles, foram avaliados 20 indivíduos saudáveis, pareados com os pacientes em termos de idade, sexo e raça, procedentes da mesma região geográfica dos pacientes. A densidade de expressão das moléculas HLA nas diversas subpopulações linfomonocitárias foi avaliada por citometria de fluxo. Os marcadores imunogenéticos foram tipados utilizando-se iniciadores de oligonucleotídeos seqüência específicos. Os resultados foram analisados usando o teste não paramétrico de Mann Whitney U. Foi observado aumento da densidade de expressão das moléculas de histocompatibilidade de classe I em linfócitos T CD3+, CD4+ e CD8+ de pacientes com DM1 quando comparados aos controles. Em relação às moléculas HLA de classe II, o número e a porcentagem dos linfócitos T CD4+, coexpressando as moléculas HLA-DQ de pacientes estavam diminuídos em relação aos controles. Os resultados referentes à correlação do perfil genotípico dos pacientes revelam que pacientes portadores dos alelos HLA-DQB1*02 apresentaram diminuição no número e porcentagem das células CD3+, CD4+, CD8+, CD19+ e CD14+ coexpressando as moléculas HLA-DQ, e ainda, o aumento da densidade de expressão da molécula HLA-DQ nas células CD19+, em relação aos pacientes sem esses alelos. Pacientes com o alelo HLA-DQB1*0302 apresentaram aumento do número de células CD14+ e CD19+ coexpressando as moléculas HLA-DQ, e ainda, o aumento da densidade de expressão dessas molécula nas células CD14+ em relação aos pacientes negativos para esse alelo. Além da instabilidade de ligação dos peptídeos com as moléculas de susceptibilidade ao DM1, este estudo reafirma a importância da densidade de expressão das moléculas de classe II na susceptibilidade ao DM1. / Althougth the role of MHC molecules in the susceptibility to DM1 has not been elucidated, the density of MHC molecules on cell surface may influence the outcome of the immune response. In this study, the number of CD3+, CD4+, CD8+, CD19+ and CD14+ cells coexpressing MHC class I and II, and the correlation between the density of MHC molecules and the immunogenetic profile of DM1 patients were studied. A total of 20 recently diagnosed patients (12 males) and 20 control individuals matched to the patients in terms of age, sex and race were studied. MHC molecules on cell sufaces were evaluated using flow cytometry. MHC aleles were typed using sequence specific probes. Statistical analysis was performed by the non-parametric Mann Whitney-U test. Increased expression of MHC class I molecules was observed in patients T CD3+, CD4+ and CD8+ cells in relation to controls. The number and porcentage of double-positive CD4+/HLA-DQ+ cells were significantly decreased in patients. Compared to DM1 patients who were not typed as HLA-DQB1*02, DM1 patients typed as HLA-DQB1*02 exhibited decreased numbers of CD3+, CD4+, CD8+, CD19+ and CD14+ cells expressing HLA-DQ molecules, whereas the density of HLA-molecules was increased in CD19+ cells. Compared to non-HLA-DQB1*0302 patients, those typed as HLA-DQB1*0302 presented increased number of CD14+ and CD19+ cells expressing HLA-DQ molecules. Besides the instability of peptide ligation with susceptibility molecules, this study stresses the relevance of the density of MHC class II molecules on the susceptibility to DM1.

The migration of T lymphocytes is a vital component of the immune system, with roles in immunosurveillance and inflammation. The role of Phosphoinositide 3-kinase within T lymphocyte migration is unclear, with some evidence that it may be a disposable signal. Here, using Staphylococcal Enterotoxin B activated peripheral blood mononuclear cells and the T cell line CEM cells, the role of Phosphoinositide 3-kinase and its downstream kinases was investigated. CCL22 mediated CEM cell migration and CXCL12 mediated peripheral blood mononuclear cell migration were shown to be independent of Phosphoinositide 3-kinase using several different broad-spectrum Phosphoinositide 3-kinase inhibitors. However, these cells were Akt-dependent, as demonstrated by incubation with the Akt inhibitor Akti-1/2. Differences in the effect of the inhibitors on Akt activity were discovered, indicating that either Akt can be activated in the absence of Phosphoinositide 3-kinase, or differences exist regarding the relative abundance of each protein within the cell. Th17 cells are a subtype of the T helper cell family and have been shown to be involved in inflammation and immune diseases. Mouse splenocytes were polarised to a Th17 phenotype and analysed for the surface expression of chemokine receptors. CCR2, CCR6 and CCR9 were shown to be expressed on Th17 cells and upregulated under Th17 polarising conditions. However, only CCR2 and CCR6 induced migration of Th17 cells. This migration was sensitive to Phosphoinositide 3-kinase and Akt inhibitors. This data reveals a model for the migration of Th17 cells to areas of inflammation, and sheds light on the role of Phosphoinositide 3-kinase during this process.

Benzene and toluene are known groundwater contaminants . Male CD-I mice were continuously exposed to 0, 31, 166, and 790 mg/ L benzene and 0, 17, 80, and 405 mg/L toluene, respectively, in drinking water for four weeks. Benzene caused a reduction of leukocytes, lymphocytes and erythrocytes, and resulted in a macrocytic anemia. Lymphocyte response to both B- and T-cell mitogens, mixed lymphocyte response to alloantigens, and the ability of cytotoxic lymphocytes to lyse tumor cells were enhanced at the lowest dose of benzene and depressed in the higher dosage animals. Benzene at doses of 166 and 790 mg/L decreased the number of sheep red blood cell (SRBC) -specific plaque-forming cells, the level of serum anti-SRBC antibody, and the activity of interleukin-2 (IL -2). Benzene treatment increased endogenous concentrations of the brain biogenic amines norepinephrine (NE), dopamine (DA) and serotonin (5-HT), and concomitantly, elevated the levels of their respective major metabolites vanillymandelic acid (VMA), 3,4-dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA), in several brain regions . In most cases, the changes were dose related; in several instances, maximum effects occurred at the 166 mg/L benzene dose. Toluene did not adversely affect the hematological parameters. Depression of immune function was evident at the highest dose (405 mg/L), except for mitogeneses. Increased neurochemical concentrations caused by toluene displayed a dose-dependent biphasic manner which began at a dose of 17 mg/L, peaked at 80 mg/L, and decreased at 405 mg/L. Toluene treatment had more selective effects on NE, 5-HT ,VMA and 5-HIAA, than DA, DOPAC and HVA. Both compounds, by increasing concentrations of the hypothalamic NE and its major metabolite VMA, stimulated the hypothalamic-pituitary-adrenocortical axis activity, resulting in an elevated plasma adrenocorticotropic hormone and serum corticosterone which had an additive adverse effect on IL-2 synthesis. Toluene, 325 mg/ L, completely inhibited benzene-induced cytopenia and immunosuppression when it was coadministered with benzene (166 mg/L). The low dose of toluene (80 mg/L ) did not antagonize benzene immunotoxicity. Mice given the combined exposures exhibited raised levels of regional neurochemicals when compared to the untreated controls. Increased levels of monoamine metabolites in several brain regions were greater in the combined treatments of benzene and toluene than when either chemical was used alone. The results of the interaction studies support the known metabolic interaction mechanisms of benzene and toluene.

Parmi les contaminants environnementaux reconnus pour leur toxicité et leur propagation à travers le monde, les métaux lourds sont certainement d'une première préoccupation. Des études démontrent que les métaux lourds peuvent altérer différents systèmes dont le système immunitaire. Ils peuvent induire des effets immunomodulateurs qui pourront avoir comme conséquences, l'augmentation de la susceptibilité à des agents infectieux, l'apparition de maladies néoplasiques et de maladies auto-immunes. Afin de se protéger de cette agression, les cellules immunitaires induisent des thiols (cystéines, glutathion, métalloprotéines) qui sont des molécules capables de neutraliser les métaux en s'y liant et ainsi protéger l'organisme des effets néfastes. La pré-activation de ces cellules augmenterait leur contenu intracellulaire en thiols et les rendrait donc plus résistantes à l'effet néfaste des métaux lourds. Dans cette étude, nous avons voulu démontrer, l'effet de la pré-activation des lymphocytes avec la concanavaline A sur la toxicité de certains métaux lourds tels que le mercure (organique et inorganique), le cadmium, le zinc et le sélénium. Nous avons mesuré la prolifération Iymphoblastique, la viabilité, l'apoptose ainsi que le niveau de thiols intracellulaires dans ces cellules pré-activées ou non pré-activées. Pour la prolifération lymphoblastique, les cellules pré-activées ont été significativement plus résistantes que les cellules au repos pour les deux formes de mercure, le cadmium et le zinc. Le niveau de thiols a été significativement augmenté chez les lymphocytes pré-activés de même que la viabilité, alors que l'apoptose a été augmentée dans les lymphocytes pré-activés par rapport à leur contrôle respectif en présence de mercure inorganique, de zinc et de sélénium. Les résultats suggèrent que la pré-activation des lymphocytes entraîne une augmentation du niveau de thiols intracellulaires leur procurant une meilleure protection contre l'effet néfaste des métaux lourds.

Les infections par Plasmodium induisent des changements importants sur la réponse effectrice et la régulation des cellules B. Ces effets se caractérisent par une suppression de la production d'anticorps contre des antigènes hétérologues et par une activation polyclonale non spécifique des cellules B. Nous avons centré notre attention sur le facteur inhibiteur de la migration des macrophages (MlF), une cytokine secrétée de façon constitutive chez plusieurs types cellulaires et intensifiée durant l'infection par Plasmodium. Le MlF inhibe l'apoptose des cellules B «in vitro» et son rôle dans les altérations des cellules B durant la malaria n'a pas encore été évalué. En utilisant des souris BALB/c infectées avec P. chabaudi adami DK avant et suite à un régime d'immunisation avec de la γ-globuline humaine, nous avons observé que l'infection après l'immunisation réduit significativement les titres d'lgG totaux et IgGI anti-γ-globuline humaine. Le possible rôle du MlF a été vérifié en neutralisant des souris BALB/c (immunisées avec de la γ-globuline humaine) avec anti-MIF. Les résultats démontrent une augmentation significative d'lFN-γ; une diminution de cellules B anexine +; mais aucun effet significatif sur la production d'anticorps. Étant donné que le MlF est présent pendants les infections nous avons neutralisé le MlF avant et après l'infection par P. chabaudi DK. L'inhibition dans les titres d'anticorps spécifiques à la γ-globuline induit par l'infection était comparable chez des souris traitées avec l'anti-MlF, ce qui suggère que le MlF n'y participe pas. Malgré les effets suggérés «in vitro» sur des cellules B, nos études ont démontré une diminution de cellules B, PI+ chez des souris infectées et neutralisées. En conclusion, nous pouvons dire que le MlF ne semble pas participer dans l'activation polyclonale non spécifique des cellules B durant la malaria et nous suggérons d'évaluer le temps de vie des anticorps en évaluant l'expression du récepteur néonatale Fc (FcRn) qu'y est impliqué à la régulation, le rôle des cellules T régulatrices ainsi que la modulation de l'expression du complexe CD74-CD44 durant les infections par Plasmodium. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Malaria, Cellules B, Réponse d'anticorps, MlF, Activation polyclonale.

Non-Hodgkin’s lymphoma is the fifth most common cancer in the UK, accounting for 4% of all new cases. The control of lymphomagenesis still remains a challenge. Early growth response gene (Egr) 2 and 3 are zinc finger transcription factors. Egr2 plays an important role in the development of both central nervous system and lymphocytes. However the mechanism of action in lymphocytes is still unknown. In order to fully understand the function of Egr2, in lymphocytes, we developed Egr2 and 3 double knockout mice (Egr2-/-Egr3-/-) by crossbreeding lymphocyte specific Egr2 knockout mice (CD2-Egr2-/-) with Egr3 knockout mice (Egr3-/-), as previous reports suggested that Egr3 compensates for the role of Egr2. In the absence of Egr2 and 3, the homeostasis of T cells is dysregulated with hyper-homeostatic proliferation of effector like phenotype cells. More importantly the development of spontaneous B and T cell lymphoma was found in more than 70% of Egr2-/-Egr3-/- mice. The lymphoma cells from Egr2-/-Egr3-/- mice were highly proliferative and metastatically spread into other non-lymphoid organs, such as lung, liver and kidney. In additional to this lymphoma development the Egr2-/-Egr3-/- mice showed signs of chronic inflammatory disorder. This inflammatory disorder was characterised by glomerulonephritis and an increase in serum cytokines, which may provide the microenvironment for the lymphoma development. To explore the molecular mechanism of tumour development in Egr2-/-Egr3-/- mice, the transcriptional profile of Egr2 was studied by microarray and ChIP-on-chip. We found firstly that Egr2 directly binds to the promoter regions of Ikaros and FOXO3. The deletion of Egr2 and 3 in lymphocytes led to the downregulation of Ikaros, Aiolos and FOXO3 expression. The impaired expression was found to be associated with proliferative disorder and the development of T and B cell lymphoma. Secondly Egr2 strongly inhibits STAT3 transcriptional activity by regulating SOCS3, which is a known inhibitor of STAT3. The breakdown of this regulation could be an important mechanism in lymphomagenesis. A model is proposed which defines Egr2 and Egr3 as the backbone of important tumour suppressor genes that control cell fate decision and regulates homeostasis in the lymphoid system. Thus, our results suggest that Egr2 and 3 are important regulators of lymphocyte function by their involvement in multiple cell signalling pathways, which could potentially be key genes for future cancer therapy.

Piperine, the major alkaloid derived from black pepper corns, has played an important role in traditional medicine worldwide. Current research has demonstrated piperine to have several anti-inflammatory properties, however, little is known concerning the effect of piperine on B cells. Spleen-derived murine B cells were cultured in the presence or absence of piperine during T-dependent or T-independent activation. Piperine reversibly inhibited B cell proliferation in a dose-dependent manner. This was due to a G0/1-phase cell cycle arrest, and was associated with a reduction in phospho-ERK, phospho-AKT, and Cyclin D1, D2, and D3. Piperine also inhibited antibody and cytokine production. Furthermore, piperine treatment diminished B cell-mediated antigen presentation determined by measuring OT-II transgenic T cell proliferation in response to OVA, which was attributed to the decreased MHC-II ad co-stimulatory molecule expression observed. This in vitro study shows that piperine has potent immuno-suppressive effects on B cell activation and effector function.

T-lymphocytes (T-cells) trafficking in the lymph nodes (LNs) is key for T-cells activation and their effector functions in adaptive immune responses. T-cells enter the LNs through high endothelial venules (HEVs) and interact with dendritic cells (DCs) for cognate antigens in the T-cell zone (TCZ). After scanning the TCZ for antigens, T-cells leave the LNs through efferent lymphatic vessel. CCR7 and its ligands, CCL19 and CCL21 are involved in the recruitment and compartmentalization of T-cells in LNs. However, their specific role(s) in mediating T-cells migration in LNs sub-regions remain unclear. In addition, the mechanism behind the passage of T-cells from the TCZ to the abluminal side of medullary sinuses (for their exit through medullary sinuses) is not well understood. Here, I hypothesize that different CCL19 and CCL21 fields in LNs sub-regions, orchestrate T-cells sub-regional migration in LNs.. In this study, I examined the CCL19 and CCL21 distribution profiles in mouse LNs sub-regions by immunofluoroscence staining and confocal microscopy. Using microfluidic devices that can flexibly configure well-defined single and co-existing chemical concentration gradients, I quantitatively analyzed the migration of activated human blood T-cells in LNs relevant CCL19 and CCL21 fields. The results suggested a novel CCL19 and CCL21 based combinatorial guiding mechanism for T-cells migration in different LNs sub-regions. In particular, this mechanism operates in the TCZ periphery region to guide T-cells migration away from the TCZ. Furthermore, the CCL19 and CCL21 fields mimicking the region beyond the TCZ toward the medulla result in disturbed chemotaxis, which prevents T-cells from being attracted back to the TCZ. Taken together, this microfluidics-based in vitro study shows the coordinated T-cells migration in different single and combined CCL19 and CCL21 fields, leading to interesting new insights into the guiding mechanisms for T-cells trafficking in LNs sub-regions.

Thesis (doctoral)--Universität Hamburg, 1979.

Thesis (Ph.D.) -- Virginia Commonwealth University, 2006. / Prepared for: Dept. of Microbiology and Immunology. Bibliography: p. 152-184. Also available online.