Charles University Faculty of Pharmacy in Hradec Králové Study program: Pharmacy Author: Marianna Šamajová Supervisor: RNDr. Klára Konečná, Ph.D. Consultant: plk. gšt. doc. RNDr. Zuzana Kročová, Ph.D. Title of diploma thesis: Entry of bacteria Mycobacterium bovis BCG into B lymphocytes Background: The objective of this work was to evaluate the entry of bacterium Mycobacterium bovis BCG into B lymphocytes and the role of selected receptors in this process. Methods: Peritoneal cell suspensions with unblocked and/or blocked receptors on BALB/c mouse B lymphocytes we infected by bacterium M. bovis BCG-GFP unopsonized and/or opsonized by fresh murine serum ("complement") or immune serum ("antibodies"). Using flow cytometry we evaluated the entry of bacterium M. bovis BCG-GFP into B lymphocytes and their subpopulations B1a, B1b and B2. Results: M. bovis BCG-GFP actively enters into B lymphocytes. Depending on the subpopulation, it most infects B1a, less B1b and at least B2 lymphocytes. Only the subpopulation B2 responds significantly to the opsonization by complement. Opsonization by antibodies had no significant effect on the infection. Entry into CD19+ cells is mediated through the BCR receptor, especially in subpopulations B1a and B1b. Under the opsonized conditions, the CR1/2 complement receptor is...

B cells get help in the antibody response by presenting processed antigen to helper T cells. We asked whether the antigen presenting B cell must induce T helper functions before receiving help, or whether B cell activation is a direct consequence of T cell antigen recognition on the B cell surface. Although antigen-dependent increases in B cell c-myc expression occur as early as two hours after conjugation, the B cell response depends on induction of a contact-dependent helper function in the T cell, which is inhibitable by cyclosporin A. Induction but not delivery of contact help is blocked by anti-class II MHC antibody, indicating that the delivery of T cell help is not Ag dependent or MHC restricted. Also, contact with activated helper T cells induces a different pattern of immediate early gene expression from signals transduced through the B cell antigen receptor. Egr-1 is rapidly upregulated in response to mitogenic signals induced by receptor crosslinking on murine B lymphocytes, and its expression closely correlates with B cell proliferation in several models of B cell activation and tolerance. We compared egr-1 expression during B cell stimulation with Fab'2 and IgG anti-Ig, since it is known that Fab'2 anti-Ig is mitogenic while IgG is not, due to a dominant inhibitory effect of crosslinking the B cell FcγRII to membrane Ig. While mitogenic doses of Fab'2 anti-Ig induce large and rapid increases in egr-1 expression, intact anti-Ig results in only small increases in egr-1 mRNA, comparable to that seen with submitogenic concentrations of Fab'2 anti-Ig. However, when IL-4 is added as a comitogen to induce B cell proliferation with submitogenic concentrations of Fab'2 anti-Ig or IgG anti-Ig, no concomitant increases in egr-1 are observed. The regulation of egr-1 therefore, is similar to that of c-myc in this system, since neither correlates with IL-4 induced DNA synthesis.

No description available.

DNA encodes the genetic instructions for the development and function of organisms and hence maintaining genomic integrity is essential for the propagation of life. However, DNA molecules are under constant threat of metabolic and environmental insults resulting in DNA damages including DNA double strand breaks (DSB), which are considered as a serious threat to cell survival. The majority of these DSB are repaired by Non-homologous end joining (NHEJ). Unrepaired DSB can lead to genomic instability resulting in cell cycle arrest, apoptosis, and mutations. Thus, delineating this DNA repair process is important in understanding the molecular mechanisms of aging and malignant progression. B lymphocytes undergo physiological DNA breaks and NHEJ-mediated DNA repair during their bone marrow differentiation and peripheral class switch recombination (CSR), thus lending them as a good model system in which to delineate the DNA repair mechanisms. To determine the effect of aging on NHEJ, B lymphocytes from old mice were analyzed. The results showed compromised DNA repair in cells from old mice compared to cells from adult mice. These results suggest that NHEJ is compromised during aging and might play critical roles in the aging process and age-associated conditions. To delineate the role of a CT in regulating the immune system, transgenic mice expressing NUP98-HOXD13 (NHD13) were analyzed for B lymphocyte differentiation, peripheral development, CSR, and antibody production. The results showed impaired B cell development and antibody production, which worsened with antigenic stimulation, suggesting the role of NHD13 in immune regulation. These studies explored the possibility of altered NHEJ-mediated DNA repair as a contributing reason for aging process and age-associated conditions. Also, the results from NHD13 study suggested that a primary CT can result in impaired NHEJ and regulate immune cell development and function. Furthermore, the results pointed to the possibility that a primary CT may lead to secondary mutations through altered NHEJ. Thus, these studies shed insight into the molecular mechanisms of altered NHEJ and may help in developing preventive or therapeutic strategies against accumulation of DNA damage, aging process and secondary mutations. / Ph. D.

Thesis (Ph.D. in Immunology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 103-118). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Student Number : 9708297R - MSc (Med) dissertation - School of Medicine - Faculty of Health Sciences / B chronic lymphocytic leukaemia (B-CLL) is a monoclonal haematopoietic disorder with expansion of small lymphocytes of B-cells. B-CLL cells accumulate in blood, bone marrow, lymph nodes and spleen, resulting in enlargement of these organs and decreased bone marrow function. B-CLL is the most common leukaemia, with an annual incidence of 1.8 to 3.0 per 100 000 population in the United States. It is characterised by the accumulation of long-lived monoclonal CD5+ B lymphocytes. In vivo normal B-lymphocytes derive growth factors through interactions with T-cells and monocytes. In culture however, survival and growth of activated B-cells depends on the availability of external factors such as interleukins. B-CLL cells populations are unable to survive in culture long enough to respond to the addition of growth factors. Such factors are important for the proliferation and survival of many cell types and in the absence of cytokines, these cells die as a result of apoptosis. Chronic lymphocytic leukaemia cells are influenced in vitro by a number of exogenously added cytokines that include IFN- α, IFN-γ, IL-2, IL-4, IL-10, IL-13, IL-15, TGF- β and TNF- α. The aim of this study was to investigate the effect of cytokines e.g., IFN, IL-2, IL-6, IL7 and IL-10 on the proliferation and survival of B-CLL cells and furthermore to compare the induction and reduction of spontaneous and induced apoptosis in vitro. Patients with B-CLL were recruited from three centres. Thirty blood samples were collected, separated using Ficoll Hypaque Gradient and purified by rosetting with AET treated SRBC. The proliferation and survival of B-CLL cells were studied in vitro in response to GM-CSF, IFN, IL-2, IL-6, IL7 and IL-10,. The survival and apoptosis of B-CLL cells in cultures with or without interleukins and other growth factors were studied under microscopic examinations and DNA agarose gel electrophoresis. It was observed in B-CLL cells cultures that IFN and IL-2 enhanced proliferation significantly. IL6, IL-7 and GM-CSF also enhanced proliferation of B-CLL cells but not to the greater extent than IL2 and IFN. IL-10 inhibited proliferation of B-CLL cells when compared to controls. In a long-term (5-day) culture, survival of B-CLL cells was greatly enhanced by IFN and followed by IL-2. Therefore it appeared that IFN and IL-2 are the two most potent growth factors tested in this study to promote B-CLL cells proliferation and survival. The combination of these mitogens did not further enhanced proliferation. IL-6 and GM-CSF enhanced proliferation and survival of B-CLL cells. IL-7 promoted proliferation but had no effect on survival of B-CLL cells in-vitro. IL-10 enhanced apoptosis and did not promote survival of B-CLL cells in-vitro. IFN and IL2 are survival and promoting growth factors for B-CLL cells in culture. In contrast, IL-10 has demonstrated to induce apoptotic cell death of B-CLL cells. In conclusion B-CLL cells proliferated equally well with IFN and IL-2. IL-6, IL-7 and GM-CSF had a much lower proliferation and survival effect with noticeable antiapototic activity when compared to IFN and IL-2. IL-7 was found not to promote survival of B-CLL cells and IL-10 enhanced cell death by apoptosis.

A via Unfolded Protein Response (UPR) é uma via de sinalização ativada pelo estresse do Retículo endoplasmático (ER). Anteriormente descrevemos um Paciente com Imunodeficiência Comum Variável (CVID) que apresenta um atraso na ativação da via UPR associado com o acumulo de imunoglobulinas dentro do ER e uma taxa de proliferação diminuída. Nossos resultados demonstram que a ativação crônica da UPR interrompe o ciclo celular de EBV-B através da quebra da natureza cíclica da ciclina A1. Essa parada é depende da linhagem EBV-B estudada e da droga utilizada. Além disso, a ativação crônica da UPR aumenta a apoptose através da ativação do braço da PERK da via UPR. Células ex-vivo e EBV-B do Paciente P apresentaram uma taxa metabólica muito baixa e numero aumentado de células em apoptose. A deficiência da resposta do paciente P frente a ativação da via UPR parece ser somente no reconhecimento de proteínas não dobradas. Nossos resultados sugerem que a proliferação deficiente observada em diversos paciente com CVID pode ser resultado de uma ativação deficiente da via UPR. / The unfolded protein response (UPR) is a signaling pathway activated by endoplasmic reticulum (ER) stress. Previously we described a patient (Patient P) with Common Variable Immunodeficiency (CVID) whose delayed activation of the UPR associates with accumulation of immunoglobulins and slower rate of proliferation. Our results showed that chronic UPR stress interrupted cell cycling of EBV-B cells through dysruption of the cyclic nature of cyclin A1. This interrption is depend of the cell type and drug. Furthermore, chronic ER stress triggered early apoptosis through activation of the PERK branch of the UPR. EBV-B and ex vivo cells from patient presented low metabolic rate and a high apoptosis rate even in the absence of ER stressors.. We noted that the deficiency of UPR pathway activation by Patient P apears to be on the recognition of unfolded proteins. Our results support the hypothesis that deficient proliferation observed in some CVID patients might be the result of deficient UPR activation.

Dans les organes lymphoïdes secondaires, les lymphocytes B acquièrent des antigènes immobilisés à la surface de cellules voisines. L’engagement du BCR (récepteur des cellules B) avec de tels antigènes induit la formation d’une synapse immunologique et la polarisation des lymphocytes B. Cette polarisation inclut le repositionnement du centrosome à la synapse immunologique ainsi que le recrutement et la sécrétion locale des lysosomes qui sont nécessaires à l’extraction, l’apprêtement et la présentation des antigènes sur les molécules du complexe majeur d’histocomptabilité de classe II (CMH-II) aux lymphocytes T CD4+ pré-activés. Des travaux précurseurs menés dans le laboratoire ont permis de mettre en évidence les premiers acteurs moléculaires impliqués dans ce processus. Cependant, le mécanisme précis gouvernant la polarisation du centrosome demeure encore aujourd’hui inconnu. Le travail réalisé pendant cette thèse avait pour objectif d’identifier de nouveaux régulateurs contrôlant la polarisation du centrosome dans les lymphocytes B après engagement du BCR avec des antigènes immobilisés. De plus, au regard du rôle grandissant joué par le microenvironnement tissulaire dans l’activation des lymphocytes B ainsi que dans la modulation de leurs fonctions, nous avons étudié l’effet de la protéine extracellulaire Galectine-8 sur la régulation de la capacité des lymphocytes B à se polariser et à extraire et présenter des antigènes immobilisés. Le travail présenté dans ce manuscrit montre que la présence du complexe Arp2/3 au centrosome des lymphocytes B non activés permet la nucléation locale de filaments d’actine qui permettent, grâce à leur interaction avec le complexe LINC, de lier le centrosome au noyau. L’activation des lymphocytes B induit la déplétion partielle du complexe Arp2/3 du centrosome qui est recruté à la synapse immunologique par la protéine HS1. Ceci induit une diminution de la nucléation d’actine au centrosome entraînant la séparation entre le centrosome et le noyau et permettant la polarisation du centrosome vers la synapse. De plus, nous montrons que la présence de la protéine Galectine-8 dans le milieu extracellulaire favorise le recrutement et la sécrétion des lysosomes à la synapse immunologique, conférant aux lymphocytes B une meilleure capacité à extraire et présenter des antigènes immobilisés. Nos résultats mettent en évidence des mécanismes inattendus régulant la polarisation des lymphocytes B en réponse à une stimulation antigénique et soulèvent des questions intéressantes concernant la régulation coordonnée de ces mécanismes qui confèrent aux lymphocytes B la capacité d’extraire, d’apprêter et de présenter des antigènes immobilisés efficacement. / In secondary lymphoid organs, B cells acquire antigens that are tethered at the surface of neighboring cells. Engagement of the B cell receptor (BCR) with such immobilized antigens leads to the formation of an immune synapse and the subsequent polarization of B cells. This includes the repositioning of the centrosome towards the immune synapse as well as the recruitment and local secretion of lysosomes required for efficient antigen extraction, processing and presentation onto class II major histocompatibility complex (MHC-II) molecules to primed CD4+ T cells. Pioneer work performed in the lab has highlighted the first molecular players involved in this process. However, the precise mechanism governing centrosome polarization remains to be fully elucidated. The work performed during this thesis aimed at identifying new regulators supporting centrosome polarization in B lymphocytes upon BCR engagement with immobilized antigens. In addition, in view of the emerging role played by the tissue microenvironment in shaping B cell activation and functions we investigated whether extracellular Galectin-8 modulates the ability of B cells to polarize, extract and present immobilized antigens. We show here that, in resting lymphocytes, centrosome-associated Arp2/3 (actin related protein-2/3) locally nucleates F-actin, which is needed for centrosome tethering to the nucleus via the LINC (linker of nucleoskeleton and cytoskeleton) complex. Upon lymphocyte activation, Arp2/3 is partially depleted from the centrosome as a result of its HS1-dependent recruitment to the immune synapse. This leads to a reduction in F-actin nucleation at the centrosome and thereby allows its detachment from the nucleus and polarization to the synapse. In addition, we show that extracellular Galectin-8 favors lysosome recruitment and secretion at the immune synapse, hence providing B cells with an enhanced capacity to extract and present immobilized antigens. Our findings highlight unexpected mechanisms that tune B cell polarity in response to antigenic stimulation and raise exciting questions concerning the coordinated regulation of these mechanisms to provide B cells with the capacity to efficiently extract, process and present surface-tethered antigens.

As células T CD4+ do baço são importantes na proteção frente à malária através de mecanismos mediados pelas citocinas do perfil Th1. Neste trabalho observamos o aumento das células NK1.1+TCR<font face=\"Symbol\">a<font face=\"Symbol\">b por baço nos camundongos C57BL/6, que apresentam o fenótipo de células ativadas, produzem IFN-<font face=\"Symbol\">g, expressam de níveis altos de Fas e PD-L1 que correlaciona com a baixa capacidade proliferativa na fase aguda da infecção pelo Plasmodium chabaudi AS. As células T CD4+ convencionais são a principal subpopulação de células T ativadas na resposta imune frente à infecção, que se desenvolve em duas fases consecutivas concomitantemente com as parasitemias aguda e crônica. Na fase aguda da infecção, a resposta das células T CD4+ convencionais é intensa e de curta duração, com produção grandes quantidades citocinas com cinética semelhante às células T CD4+ não-convencionais. Dessa maneira, as células T CD4+ convencionais possuem um papel central no início da resposta ao P. chabaudi, respondendo em paralelo com as células T não-convencionais como uma ponte entre a imunidade inata e adquirida. / Spleen CD4+ T cells have an important role for protection against malaria through mechanisms mediated by Th1 cytokines. We observed that the increase in NK1.1+TCR<font face=\"Symbol\">a<font face=\"Symbol\">b cell numbers per spleen in C57BL/6 mice, show an activated cell phenotype, with high expression of Fas and PD-L1 correlating with their low proliferative capacity and produce IFN-<font face=\"Symbol\">g during the acute P. chabaudi infection. We show that conventional CD4+ T cells are the main activated T cells subpopulation in the immune response to infection, which develops in two consecutive phases concomitantly with acute and chronic parasitemias. In the acute phase, the conventional CD4+ T cell response is intense and short-lasting, rapidly providing proinflammatory. Taken together, these results indicated the central role of conventional CD4+ T cells during P. chabaudi malaria, acting in parallel with non-conventional CD4+ T cells as a link between innate and acquired immunity.

Neste trabalho nós estudamos qual seria o impacto da indução de resposta imune a epítopos CD8 subdominantes na imunidade gerada pela vacinação genética. Durante a infecção experimental apenas um epítopo imunodominante presente no antígeno ASP-2 é reconhecido. Já os linfócitos T CD8+ induzidos nos animais vacinados com o gene da ASP-2 são capazes de reconhecer além deste mais dois outros epítopos (subdominantes). A identificação desses epítopos permitiu que estudássemos o papel da resposta imune a epítopos subdominantes na imunidade protetora. Após imunização genética com o gene da ASP-2 mutado, sem resposta para o epítopo dominante, confirmamos que a resposta imune aos epítopos subdomiantes pode contribuir na proteção contra a infecção experimental. Apesar do papel critico dos linfócitos T CD8+ na resposta imune protetora induzida pela vacinação genética do tipo imunização e reforço heterólogo, não se sabe ao certo se após o desafio experimental estes linfócitos T CD8+ necessitam proliferar ou recircular para mediar a imunidade protetora. Nossos resultados desafiam o paradigma de ação das vacinas tradicionais de que a imunidade é dependente da proliferação e não da recircular dos linfócitos T de memória e para mediar a imunidade protetora. / In the present study, we evaluated the impact of the immune response to sub-dominant CD8 epitopes on immunity generated by genetic vaccination. During experimental infection only a single dominant epitope is recognized on the antigen ASP-2. In contrast, the CD8+ T lymphocytes induced in animals genetically vaccinated with ASP-2 recognized, in addition to the dominant epitope, two other epitopes (sub-dominants). The identification of these epitopes allowed us to test the role of immune response to sub-dominant epitopes in protective immunity. After genetic vaccination with ASP-2 mutated gene, without the response to dominant epitope, we concluded that the immune response to the sub-dominant epitopes can be important to protective immunity. In spite of the critical role of CD8+ T lymphocytes in protective immune response induced by genetic vaccination using the heterologous prime-boost regimen, it is unclear whether after the experimental challenge these CD8+ T lymphocytes need to proliferate or recirculate to mediate protective immunity. Our results challenge the paradigm of action of traditional vaccines that immunity is dependent on the proliferation of memory T lymphocytes and that these cells do not need to recirculate.