Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 63-69).

Hemophilia A (HA) is a hemorrhagic disorder caused by a decrease/absence of coagulation Factor VIII (FVIII) in circulation. Management involves administration of FVIII to prevent bleeding episodes. The most serious complication of this replacement therapy is the development of inhibitory anti-FVIII antibodies which neutralize the infused FVIII. Low zone tolerance (LZT) is a state in which the immune system is unresponsive to an antigen induced by repeated prior exposure to low doses of said antigen. Previous animal studies exploring LZT demonstrated successful T-cell tolerance induction by this mechanism. This study investigated whether the administration of low-dose FVIII could induce immune tolerance to FVIII in a HA mouse model. HA mice received intravenous FVIII at doses ranging from 0.01 IU/kg - 5 IU/kg to determine the most promising doses (0.25 IU/kg, 2.5 IU/Kg) to further investigate. Naïve mice were treated with 0.25 IU/kg or 2.5 IU/kg weekly for 6 weeks, then immunized with 25 IU/kg FVIII weekly for 4 weeks. Following a two-week rest period (week 12), all mice received a 25 IU/Kg booster shot. Blood was collected on weeks 7, 11 and 13 and anti-FVIII antibody concentrations were measured by ELISA. Control mice received phosphate buffered saline (PBS) during weeks 1-6 of the experiment, followed by identical immunizations as stated above. Within the 2.5 IU/Kg FVIII treatment group, 11% of the mice developed tolerance to the treatment, indicated by undetectable anti-FVIII IgG titer by ELISA. The remaining 89% of these mice developed high titer antibodies, therefore they were not tolerized to FVIII. In the PBS treatment group, 62% of the mice developed high anti-FVIII antibodies. Conversely, 50% of the mice treated with 0.25 IU/kg were tolerized to FVIII and the remaining mice had significantly reduced antibody titers when compared to the controls. Moreover, upon booster dose injection, 100% of 0.25 IU/kg and 2.5 IU/Kg treated mice that were previously tolerized retained tolerance, suggesting that tolerance through low-dose injections is maintained upon FVIII re-exposure. The LZT experiment conducted here shines light on a new approach to preventing FVIII inhibitors. This study suggests that frequently administering low doses of FVIII effectively induces tolerance to FVIII in HA mice. Moreover, treatment through LZT induction may confer long lasting protection against inhibitor development as indicated by the retention of tolerance in mice subjected to a rest period and post-treatment booster shot. / Thesis / Master of Science (MSc)

Objectives: Tolerance is a hyporesponsive state caused by repeated exposure to a stimulus. In the intestine, dysregulation of tolerance to luminal stimuli may lead to chronic and deleterious inflammation, such as characterizes Inflammatory Bowel Disease. The role of T-cells in immune tolerance is well known, but that of the epithelium requires investigation. Epithelial tolerance is gene-specific and differentially regulated, but the role of and involvement of epigenetics in tolerance regulation is unknown. We hypothesized that prior stimulation may cause epithelial cells to become hyporesponsive (tolerized) and that modification of histone methylation may alter the response to pro-inflammatory stimulation. The aim of this work was to examine if known inhibitors of histone methylation modifying enzymes affected the expression of CXCL8 in response to IL-1β. Methods: CXCL8 production of intestinal epithelial cells was measured by ELISA after stimulation with the pro-inflammatory stimuli P3CK and IL-1β and small molecule epigenetic inhibitors. The CXCL8 production of cells stimulated with a pro-inflammatory stimulus was compared to pre-stimulated cells after a second stimulus. CXCL8 production of IL-1β-pre-stimulated cells was also compared to CXCL8 production when these cells were incubated with epigenetic inhibitors. The effects of these inhibitors on histone methylation levels were examined by Western blotting for the global effect and by ChIP-qPCR for specific effects at the CXCL8 locus. Results: Intestinal epithelial cells stimulated with pro-inflammatory stimuli produced a large CXCL8 response. Pre-stimulation significantly decreased CXCL8 production after a second stimulus. The time-course of CXCL8 expression was measured to ensure that CXCL8 expression due to pre-stimulation was over before the second IL-1β-stimulation. In the presence of specific epigenetic inhibitors, pre-stimulation by IL-1β did not reduce CXCL8 production after a second IL-1β- stimulation. The specific effect of these inhibitors on the epigenetic signature at the CXCL8 locus was confirmed by ChIP. Thus, histone methylation modification disrupted tolerization of intestinal epithelial cells to a pro-inflammatory stimulus. Conclusion: The inflammatory response of the intestinal epithelium can be tolerized by prior stimulation with pro-inflammatory cytokines. Tolerization is lost after incubation with inhibitors known to modify histone methylation status, indicating for the first time, the involvement of histone methylation in this phenomenon.

Among the complications of current treatments for hemophilia A, the development of anti-FVIII antibodies including “FVIII inhibitors” remains the major clinical problem in treating hemophiliacs. Factor VIII inhibitors work through neutralizing the coagulation cofactor activity of the infused FVIII and preventing the restoration of normal hemostasis. This thesis explains the influence of genetic background on the generation of FVIII inhibitors, introduces a new pre-clinical approach that reduces the immunological response towards FVIII and predicts the in vivo behavior of recombinant and plasma-derived FVIII products in hemophilic patients. First, we studied the influence of the genetic background on the formation of FVIII antibodies by treating hemophilia A Balb/c and C57BL/6 mice with repetitive FVIII infusions. We observed that the C57BL/6 mice developed higher FVIII antibody titers than the Balb/c mice. Our results suggest that differences in the cytokine immune responses due to FVIII in Balb/c and C57BL/6 mice are responsible for the different FVIII antibody titers in each of these strains. Second, we investigated the use of FVIII-pulsed immature dendritic cells in inducing immune tolerance against FVIII prior to the FVIII treatment. We showed that in vivo, FVIII does not induce the activation and proliferation of hemophilic T cells. Furthermore, infusing FVIII-pulsed immature dendritic cells into hemophilic mice resulted in a long-term reduction in immune reactivity towards FVIII. Also, we have proposed methods on how to improve the tolerogenic abilities of dendritic cells. Our results indicate that the immature dendritic cells induced the formation of T regulatory cells and that these T regulatory cells were responsible for the observed reduction in immune reactivity. Finally, we were able to identify the mechanisms behind the immune system activation in mice treated with either recombinant or plasma-derived FVIII products. We showed that plasma-derived FVIII results in reduced FVIII antibody titer formation in hemophilic mice. Our results demonstrate that the differences in antibody formation in hemophilic mice treated with either recombinant or plasma- derived FVIII products are due to the distinct cytokine micro-environment induced by each product. This thesis contributes to the current knowledge on FVIII immunology and the in vivo behavior of FVIII in hemophilic mice. The results generated from this thesis can be used to modify the available FVIII treatments in order to minimize the immunological complications of FVIII and improve the quality of life of hemophilic patients. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2008-08-14 18:22:51.56

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Cornell University, August, 2008. / Vita. Includes bibliographical references (leaves 135-160).

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 69-79).

Thesis (Ph.D. in Immunology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 91-100). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-15T13:52:56Z No. of bitstreams: 1 Luciana Souza de Aragão França Redução....pdf: 995559 bytes, checksum: ba08fbe35c691051eb1d16b7657f0655 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-15T13:53:13Z (GMT) No. of bitstreams: 1 Luciana Souza de Aragão França Redução....pdf: 995559 bytes, checksum: ba08fbe35c691051eb1d16b7657f0655 (MD5) / Made available in DSpace on 2016-02-15T13:53:13Z (GMT). No. of bitstreams: 1 Luciana Souza de Aragão França Redução....pdf: 995559 bytes, checksum: ba08fbe35c691051eb1d16b7657f0655 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / As alergias afetam cerca de 20 a 30% da população mundial e sua prevalência, bem como a gravidade dos sintomas, tem aumentado nas últimas décadas. As terapias existentes para as desordens do trato respiratório ocorrem por períodos prolongados, apresentam efeitos colaterais, muitas vezes não são efetivas para pacientes graves e dependem do afastamento do alérgeno. Uma alternativa para esses pacientes seria a indução de tolerância imunológica, através da terapia celular com células dendríticas pulsadas com o alérgeno. O presente trabalho objetivou avaliar o efeito de células dendríticas mielóides sensibilizadas in vitro com extrato de B. tropicalis em modelo murino de alergia respiratória. Em modelos experimentais de alergia respiratória, células T auxiliares (Th2) alérgeno-específica produzem citocinas que regulam a síntese de anticorpos IgE alérgeno-específicos e controlam a inflamação eosinofílica e a remodelação do tecido das vias aéreas, e esses parâmetros foram analisados neste trabalho. Células dendríticas tolerogênicas de camundongos A/J foram obtidas na presença de GM-CSF e dexametasona e caracterizadas por apresentarem baixa expressão de MHC-II e moléculas coestimulatórias, redução acentuada de PD-L2 e um perfil antiinflamatório de produção de citocina.A inoculação prévia de células dendríticas sensibilizadascom Blomia reduziu especificamente e significativamente o número total de leucócitos e o número de eosinófilos no fluido de lavagem broncoalveolar e reduziu a produção de IgE, em comparação à inoculação de células sensibilizadas com ovalbumina, e estes efeitos foram mais intensos com uma segunda inoculação de células tolerogênicas. Além disso, redução do infiltrado inflamatório pulmonar foi observada em animais que tinham sido tratados com células dendríticas, independente do antígeno utilizado na sensibilização das mesmas. Estes resultados indicam que células dendríticas geradas como descrito neste trabalho podem inibir uma resposta alérgica Th2. A especificidade do tratamento, entretanto, deve ser melhor investigada. / Allergies affect about 20-30% of world population and its prevalence and severity of symptoms has increased in recent decades. Existing therapies to respiratory tract disorders are extense, with side effects, not effective for severe patients and depending on the allergen removal. An alternative for these patients is the induction of immune tolerance by cell therapy with dendritic cells pulsed with the allergen. This study aimed to evaluate the effect of myeloid dendritic cells sensitized in vitro with B. tropicalis extract in a murine model of respiratory allergy. In experimental models of respiratory allergy, T helper cells (Th2) produce allergen-specific cytokines which regulate the synthesis of allergen-specific IgE antibodies and control eosinophilic inflammation and tissue remodeling of the airways, these parameters were evaluated in this study. Tolerogenic dendritic cells from A / J mice were obtained in the presence of GM-CSF and dexamethasone and were characterized by low MHC-II expression and costimulatory molecules, marked reduction of PD-L2 and antiinflammatory profile of cytokine production. Prior inoculation of sensitized dendritic cells with Blomiasignificantly and specifically reduced the total number of leukocytes and eosinophils in the bronchoalveolar lavage fluid and reduces IgE production compared to inoculation with cells sensitized with ovalbumin, and these effects were improved with a second inoculation of tolerogenic cells. Furthermore, reducing the pulmonary inflammatory infiltrate was seen in animals that had been treated with dendritic cells regardless of the antigen used for sensitization. These results indicate that dendritic cells generated as described here can inhibit an allergic Th2 response. The specificity of the treatment, however, should be examined.

As the principal antigen-presenting cells to T cells, dendritic cells (DCs) have a key role in the balance of immunity and autoimmunity. They are essential in two major, converse roles - eliciting T cell immune responses to pathogenic material, and maintaining peripheral tolerance to self-tissue by inhibiting self-reactive T cells. These functions involve the processing of pathogenic or self antigens and subsequent presentation of antigenic peptides on MHC to antigen-specific T cells. DC recognition of conserved pathogenic markers induces a mature phenotype that governs immunogenic presentation to T cells and, consequently, the adaptive immune response. In contrast, DC recognition of self tissue suppresses maturation, instead inducing a tolerogenic phenotype that induces self antigen-specific T cell to die, become anergised, or converted to T regulatory cells. Apoptotic cells are the major source of self-antigen for the maintenance of peripheral tolerance, and their defective clearance by DCs is implicated in autoimmunity. Apoptotic cells are thought to actively suppress maturation of DCs and inhibit the possible immune responses promoted by proinflammatory mediators released from necrotic cells. However, the immune function of apoptotic cells and their relative influence over necrotic cells are highly contested, partially due to the complex nature of immunogenicity arising from the sourcing and generation of apoptotic cells. In this investigation, various methods of inducing apoptosis and necrosis are evaluated. Definitive methods of inducing well-characterised cell death are then employed to compare the effects of apoptotic and necrotic cells on dendritic cells and in vitro and in vivo immune responses. Reported here are in vitro findings that support previous reports of the anti-inflammatory response of DCs to apoptotic cells, and the inflammatory response of DCs to necrotic cells. The previously-reported inhibitory effect of apoptotic cells on LPS-induced secretion of Th1 cytokines is supported here, but the inhibitory effect of apoptotic cells on LPS-induced upregulation of co-stimulatory molecules is contested. Novel findings describe the upregulation of DC expression of co-inhibitory molecules induced by both apoptotic cells and necrotic cells. Apoptotic cells, but not necrotic cells, had a suppressive effect on CpG-induced upregulation of co-stimulatory molecules and pro-inflammatory cytokines. Apoptotic cells suppressed the capacity of untreated and CpG-treated, but not LPS-treated, DCs to elicit IFNγ production by T cells. Apoptotic cells, but not necrotic cells, induced regulatory T cells and partially restored their CpG-suppressed induction. Finally, apoptotic cell-modulation of DCs inhibited the induction of autoimmunity in a novel modification of an in vivo model of diabetes. Interestingly, novel evidence for the possibility of necrotic cell-induced tolerance by means of direct T cell killing is addressed.