Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 185-219).

Severe lymphopenia is a clinical feature of Severe Acute Respiratory Syndrome (SARS) patients. However, lymphocytes do not express receptor for SARS-CoV, neither the widely accepted viral receptor angiotensin converting enzyme 2 (ACE2) nor the putative receptors Dendritic Cell- and Liver/lymph-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN and L-SIGN). Our group previously showed in vitro that, SARS-CoV Spike pseudotyped particles (SARSCoVpp) could infect human B cells only when inoculated in presence of anti-SARSCoV Spike immune serum. Such observations raised concerns about the possible occurrence of antibody-dependent enhancement (ADE) of infection, a phenomenon during which a virus bounded by antibodies could gain entry into cells through mechanisms involving complement receptors or Fc receptors. Recently, we have demonstrated the participation of the human Fc gamma receptor II (hFcγRII) molecules in granting SARS-CoV an opportunity to infect human immune cells. The aim of this study was to decipher the molecular mechanism leading to antibodymediated, FcγRII-dependent infection of immune cells by SARS-CoV. By using transduction experiment, I highlighted that different members of the hFcγRII family (namely hFcγRIIA, hFcγRIIB1 and hFcγRIIB2) could confer susceptibility to ADE of SARS-CoVpp infection. I further demonstrated that purified anti-viral immunoglobulin G, but not other soluble factor(s) from heat-inactivated immune serum, was the determinant for occurrence of ADE infection. Additionally, with the development of a cell-cell fusion assay, I illustrated that in contrast to the ACE2- dependent pathway, ADE infection did not occur at the plasma membrane, but rather require internalization of virus/antibodies immune complexes by the target cells. In line with this hypothesis, my results using a panel of FcγRII-expressing mutants demonstrated that binding of immune complexes to cell surface FcγRII was a prerequisite but was not sufficient to trigger ADE infection. In these experiments, only FcγRII signaling-competent constructions conferred susceptibility to ADE of SARS-CoVpp infection. Altogether my results point toward a role of the anti-SARS-CoV Spike IgG in vitro in granting SARS-CoV an opportunity to infect cells bearing signaling-competent FcγRII receptors. If further confirmed, such observations could have implications for understanding SARS-CoV tropism and SARS pathogenesis, as well as warrant for careful design of SARS vaccines and immunotherapy based on anti-viral antibodies. / published_or_final_version / Microbiology / Master / Master of Philosophy

No description available.

Dissertation (Ph.D.)--University of Toledo, 2010. / "Submitted to the Graduate Faculty In partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Science." Title from title page of PDF document. "A Dissertation entitled"--at head of title. Non-Latin script record Bibliography: p. 68-101.

No description available.

Sepse é a primeira causa de morte em Unidades de Terapia Intensiva. A gravidade dessa doença é considerada conseqüência de um desequilíbrio da resposta inflamatória e, apesar dos avanços em diagnóstico e tratamento, os índices de mortalidade se mantêm inalterados. O papel dos receptores Fc de immunoglobulinas nesta situação é pouco esclarecido. Tais receptores deflagram respostas imunes opostas, que dependem do receptor envolvido e podem ser tanto ativatórias, quanto inibitórias. As respostas ativatórias são atribuídas a um motivo chamado ITAM, enquanto as inibitórias são relacionadas ao motivo ITIM. Camundongos apresentam dois receptores de IgG ativatórios (FcγRI e FcγRIII), que portam motivos ITAM, associados a uma sub-unidade conhecida como cadeia gamma, assim como um receptor de IgG que apresenta um motivo ITIM na sua porção intra-citoplasmática (FcγRII). Este trabalho teve como objetivo o estudo do papel destes receptores em bacteremias e sepse. Para isso, utilizamos um modelo de peritonite induzida por ligadura e punção cecal. Este projeto descreve pela primeira vez, um papel importante do FcRγII na indução de apoptose em linfócitos B, durante infecção bacteriana severa. Nossos resultados colocaram em evidência, ainda, o fato de que animais deficientes em cadeia gamma apresentam mortalidade diminuída, quando submetidos a esse modelo de peritonite, e que essa diminuição é associada a menores valores de TNFα no soro e nos fluidos peritoneais, menor recrutamento peritoneal de células inflamatórias, assim como a um surpreendente aumento na fagocitose de E. coli. Hemocultura e cultura do lavado peritoneal desses animais revelaram uma flora multimicrobiana, enquanto camundongos selvagens apresentaram uma forte predominância de E. coli e um número total bastante superior de bactérias. Esse papel inibitório da cadeia gamma pode estar relacionado a mecanismos de auto-tolerância. Lisado total de células peritoneais de camundongos deficientes em cadeia gamma apresentam fosforilação aumentada de diversas proteínas, quando comparados a lisados obtidos, a partir de camundongos selvagens. Estudos semelhantes realizados com camundongos transgênicos para o receptor de IgA (FcαRI), entretanto, não demonstraram um papel crucial desse receptor nesta doença. Este trabalho abre, portanto, novas perspectivas para o tratamento de doenças infecciosas, através de intervenção sobre a cadeia gamma e coloca em rediscussão os conceitos atuais de ITAM e ITIM. / Sepsis is the first cause of death in Critical Care Units and despite the development in its diagnosis and treatment, mortality remains unaffected. The role of immunoglobulin Fc receptors in sepsis is not clearly understood. These receptors initiate opposing responses, depending on their aggregation by the ligand and can induce activating or inhibitory responses. The activating responses are attributed to a motif known as ITAM, and the inhibitory responses to another one called ITIM. Mice express two activating IgG receptors (FcγRI et FcγRIII) which have ITAM motifs in the intracytoplasmic domain of an associated subunit, called the FcRγ chain, as well as an inhibitory IgG receptor which possesses an ITIM motif in its intracytoplasmic domain. The objective of this work is to study the importance of these receptors in bacteremia and in sepsis. To this aim, we have used a peritonitis model, induced by cecal ligation and puncture (CLP). This project describes for the first time, an important role of FcγRII in B lymphocytes apoptosis. Moreover, our results show that FcRγ chain knockout mice have a decreased mortality in this model, which is associated to diminished TNFα serum and peritoneal fluids levels, to a reduced recruitment of peritoneal inflammatory cells and to a surprising increase in E. coli phagocytosis. Blood and peritoneal fluid cultures have shown a polymicrobial flora 24 hours post-CLP for FcRγ-chain deficient mice, whereas wild-type mice present a strong predominance of E. coli in the same cultures and an increased bacteria total count. Lysates from FcRγ-chain deficient peritoneal cells revealed augmented phosphorylation of many proteins, as compared to wild-type cells. This FcRγ chain inhibitory role could be related to self-tolerance mechanisms. This work opens new perspectives for the treatment of bacterial diseases, proposing FcRγ chain targeting and the reexamination of the actual concepts of ITAM and ITIM.

<p>Induction of collagen-induced arthritis (CIA), an animal model for human rheumatoid arthritis, is dependent on anti-collagen type II (CII) antibodies. The effector mechanism by which autoantibodies contribute to inflammatory reactions in autoimmune diseases is not well understood. In this thesis I have studied the effector pathways used by IgG anti-CII antibodies to initiate arthritis, namely the IgG Fc receptors (FcγRs) and the complement system. We have found that FcγRIII is crucial for development of CIA, as CII-immunized mice lacking this receptor do not develop arthritis and IgG1 and IgG2b anti-CII antibodies require FcγRIII to trigger arthritis when transferred to naïve mice. The antibody-mediated arthritis was further enhanced in mice deficient in the inhibitory FcγRIIB, indicating that FcγRIIB regulates the activation of FcγRIII. Furthermore, we demonstrate that FcγRIII exist as three distinct haplotypes in mice, FcγRIII:H, FcγRIII:V and FcγRIII:T. Mice expressing the FcγRIII:H haplotype are more susceptible to CIA than mice expressing the FcγRIII:V haplotype, indicating that certain FcγRIII haplotype predisposes for CIA. We also show that the most likely FcγRIII-expressing effector cell in CIA is the macrophage, since FcγRIII-expressing macrophages exclusively can induce arthritis in FcγRIII-deficient mice challenged for CIA.</p><p>The complement system was also investigated in development of CIA. We found that this effector pathway is also necessary for onset of arthritis, as CIA was inhibited by treatment with anti-complement factor 5 (C5) antibodies. C5-deficient mice could neither develop CIA unless provided with C5-containing sera. </p><p>Taken together, the work presented in this thesis indicates that FcγRs and the complement system are crucial for the induction of experimental arthritis. These findings are important for understanding the mechanisms behind rheumatoid arthritis and blocking of these effector pathways may in the future be used as treatment of rheumatoid arthritis. </p>

<p>Antibodies have the ability to influence their own production in a process called antibody feedback regulation. Depending on the type of antigen and the subclass of the antibody, the outcome of feedback regulation can be complete suppression or several hundred-fold enhancement of the antibody response.</p><p>IgE and IgG3 enhance responses to soluble protein antigens. Previous results suggest that IgG3-mediated enhancement of antibody responses is dependent on complement and not Fc receptors for IgG. However, the Fc receptor-deficient animals used did not completely lack the IgG3-binding FcγRI. We re-examined the role of this receptor in a new mouse strain completely lacking FcγRI and found that IgG3-mediated enhancement was unperturbed, thus confirming a role for complement. </p><p>To investigate the early responses resulting in IgE-mediated enhancement of antibody responses we used biotinylated antigen and found that mature follicular B cells and to a lesser extent transitional type 2 B cells capture IgE/antigen complexes. Adoptive transfer of CD4+ T cells expressing a transgenic TCR specific for ovalbumin demonstrated that these T cells localize near the B-cell follicle after 6-12 hours and that IgE, in contrast to IgG3, significantly increased specific T cell proliferation. After 3 days the T cells had gone through several rounds of divisions and showed an activated phenotype. Additional cell transfer studies identified CD23+ B cells as the responsible effector cells. These results indicate that the mechanism underlying IgE-mediated enhancement is rapid transport of IgE/antigen complexes by follicular B cells into B-cell follicles, followed by antigen presentation by CD23+ B cells to naïve CD4+ T cells. IgG3, inducing poor T cell responses, is more likely to depend on lowering the threshold for B-cell activation by co-ligating the B-cell receptor with the complement receptor 2/CD19 complex on the surface of the B cell.</p>

Induction of collagen-induced arthritis (CIA), an animal model for human rheumatoid arthritis, is dependent on anti-collagen type II (CII) antibodies. The effector mechanism by which autoantibodies contribute to inflammatory reactions in autoimmune diseases is not well understood. In this thesis I have studied the effector pathways used by IgG anti-CII antibodies to initiate arthritis, namely the IgG Fc receptors (FcγRs) and the complement system. We have found that FcγRIII is crucial for development of CIA, as CII-immunized mice lacking this receptor do not develop arthritis and IgG1 and IgG2b anti-CII antibodies require FcγRIII to trigger arthritis when transferred to naïve mice. The antibody-mediated arthritis was further enhanced in mice deficient in the inhibitory FcγRIIB, indicating that FcγRIIB regulates the activation of FcγRIII. Furthermore, we demonstrate that FcγRIII exist as three distinct haplotypes in mice, FcγRIII:H, FcγRIII:V and FcγRIII:T. Mice expressing the FcγRIII:H haplotype are more susceptible to CIA than mice expressing the FcγRIII:V haplotype, indicating that certain FcγRIII haplotype predisposes for CIA. We also show that the most likely FcγRIII-expressing effector cell in CIA is the macrophage, since FcγRIII-expressing macrophages exclusively can induce arthritis in FcγRIII-deficient mice challenged for CIA. The complement system was also investigated in development of CIA. We found that this effector pathway is also necessary for onset of arthritis, as CIA was inhibited by treatment with anti-complement factor 5 (C5) antibodies. C5-deficient mice could neither develop CIA unless provided with C5-containing sera. Taken together, the work presented in this thesis indicates that FcγRs and the complement system are crucial for the induction of experimental arthritis. These findings are important for understanding the mechanisms behind rheumatoid arthritis and blocking of these effector pathways may in the future be used as treatment of rheumatoid arthritis.

Antibodies have the ability to influence their own production in a process called antibody feedback regulation. Depending on the type of antigen and the subclass of the antibody, the outcome of feedback regulation can be complete suppression or several hundred-fold enhancement of the antibody response. IgE and IgG3 enhance responses to soluble protein antigens. Previous results suggest that IgG3-mediated enhancement of antibody responses is dependent on complement and not Fc receptors for IgG. However, the Fc receptor-deficient animals used did not completely lack the IgG3-binding FcγRI. We re-examined the role of this receptor in a new mouse strain completely lacking FcγRI and found that IgG3-mediated enhancement was unperturbed, thus confirming a role for complement. To investigate the early responses resulting in IgE-mediated enhancement of antibody responses we used biotinylated antigen and found that mature follicular B cells and to a lesser extent transitional type 2 B cells capture IgE/antigen complexes. Adoptive transfer of CD4+ T cells expressing a transgenic TCR specific for ovalbumin demonstrated that these T cells localize near the B-cell follicle after 6-12 hours and that IgE, in contrast to IgG3, significantly increased specific T cell proliferation. After 3 days the T cells had gone through several rounds of divisions and showed an activated phenotype. Additional cell transfer studies identified CD23+ B cells as the responsible effector cells. These results indicate that the mechanism underlying IgE-mediated enhancement is rapid transport of IgE/antigen complexes by follicular B cells into B-cell follicles, followed by antigen presentation by CD23+ B cells to naïve CD4+ T cells. IgG3, inducing poor T cell responses, is more likely to depend on lowering the threshold for B-cell activation by co-ligating the B-cell receptor with the complement receptor 2/CD19 complex on the surface of the B cell.