Würzburg, Univ., Diss., 2001. / Erscheinungsjahr an der Haupttitelstelle: 2000

Thesis (Ph. D.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Molecular Biology and Microbiology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Techn. Hochsch., Diss., 2000--Aachen.

Universiẗat, Diss., 2003--Bonn.

<p>B lymphocytes are multifunctional and play important roles in both innate and adaptive immunity. The diverse roles of B cells can be attributed to the various and distinct types of B cells as determined by their origin, developmental stage, antigen specificity, and function.</p><p>Evidence suggests that human innate-like B cells (i.e., marginal zone and/or B1-like B cells) develop during fetal life. However, the characteristics of human fetal B-lineage cells are less understood. Recent studies of fetal and human umbilical cord B cells indicated that CD27, a well-established marker of human memory B cells, may also be expressed on human B1-like B cells. Indeed, CD27+ B cells are present in patients with hyper-IgM 1 (HIGM1) syndrome who are unable to generate GCs or memory B cells. In order to define the origin of naïve CD27+IgD+ human B cells, I studied B-cell development in both fetal and adult tissues.</p><p>In human fetal liver, most CD19+ cells co-express CD10, a marker of human developing B cells. Some CD19+CD10+ B cells express CD27, and these fetal CD27+ cells are present in the pro-B, pre-B, and immature/transitional B-cell compartments. Lower frequencies of phenotypically identical cells are also identified in adult bone marrow. CD27+ pro-B, pre-B, and immature/transitional B cells express recombination activating gene-1, terminal deoxynucleotidyl transferase, and Vpre-B mRNA comparable to their CD27− counterparts. CD27+ and CD27− developing B cells show similar immunoglobulin heavy chain gene usage with low levels of mutations, suggesting that CD27+ developing B cells are distinct from mutated memory B cells. Despite these similarities, CD27+ developing B cells differ from CD27− developing B cells by their increased expression of LIN28B, a transcription factor associated with the fetal lymphoid lineages of mice. Furthermore, CD27+ pro-B cells efficiently generate IgM+IgD+ immature/transitional B cells in vitro. Our observations suggest that CD27 expression during B-cell development identifies a physiologic state or lineage for human B-cell development distinct from the memory B-cell compartment.</p><p>Regarding B-cell repertoire, due to the random recombination of immunoglobulin V, D, and J gene segments during B-cell development, B cells are highly diversified in their antigen specificity. Through their specific B-cell antigen receptors (BCRs), B cells recognize foreign (and self-) antigens, and present these antigens to cognate T cells to elicit/establish humoral responses, such as germinal centers, immunological memory, and long-lasting circulating antibodies. Some bacteria and viruses escape the host’s immune system by mimicking host antigens, as B cells that recognize shared epitopes on self- and foreign antigens may provide protection against such pathogens; however, these B cells are normally eliminated by tolerance mechanisms during development. The extent of tolerization manifest among human B cells that recognize both self- and foreign antigens is unknown. Here, I and my colleagues use an efficient single B-cell culture method and multiplexed antigen-binding assays to determine the specificity of about 2,300 clonal IgG antibodies produced by the progeny of single transitional and mature B cells. We show that in healthy individuals, half of the self-reactive B cells crossreact with foreign antigen, and that the frequencies of crossreactive B cells decrease by half between the transitional and mature B-cell stages, indicating that a substantial fraction of foreign specificities is lost by the second tolerance mechanisms. In SLE patients, who show defective peripheral tolerance, frequencies of crossreactive B cells are unchanged between the B-cell stages. The crossreactive, mature B cells in SLE patients show distinct reactivity to foreign antigens. We propose that activating forbidden B cells may be a good strategy for protection against host-mimicking pathogens if we can control tolerance. </p><p>Activated B cells can present antigen to T cells, as well as differentiate into memory B cells and plasma cells. Indeed, activated B cells express high levels of MHCII and are considered to be professional antigen presenting cells (APC), along with dendritic cells and macrophages. APC can be used to discover the epitopes targeted in T-cell responses; T cells are co-cultured with autologous APC in the presence of antigens and T-cell responses are evaluated. With numerous epitope candidates, mapping T-cell epitopes requires large numbers of APC; the availability of APC in blood is a limiting component and leukapheresis is often required. Since B cells can be expanded in vitro more easily than other APC, they represent a solution for the challenge of isolating adequate numbers of APC from blood in order to determine T-cell antigen specificity. I modified our single B-cell culture to support efficient activation and proliferation of both naïve and memory human B cells for the purpose of generating large numbers of autologous APC. Briefly, naïve or memory B cells recovered from blood are cultured with recombinant human IL-2, IL-4, IL-21, and BAFF on CD154+ feeder cells; this culture supports extensive B-cell proliferation, with approximately 103 fold increases following 8 days in culture, and 106 fold increases when cultures are split and cultured for 8 more days. The capacity for continued proliferation is stable for at least another week. In culture, a significant fraction of naïve B cells undergo isotype switching and terminally differentiate into plasmacytes. Culture-derived (CD) B cells are readily cryopreserved, and when recovered, retain their ability to proliferate and differentiate. Significantly, proliferating CD B cells express high levels of MHCII, CD80, and CD86. I have examined the APC function of CD B cells and found that they present both allo- and microbial antigens to autologous T cells with comparable efficiency to PBMC. Moreover, I am able to activate and expand antigen-specific memory B cells; these cultured cells are highly effective in presenting antigen to T cells. This culture method provides a platform for studying the BCR and TCR repertoires within a single individual.</p> / Dissertation

No description available.

In the history of cancer treatment, the immunotherapy is considered to be the most promising treatment approach. The idea behind this breakthrough is to stimulate the patient’s own immune system to recognize the cancer cells and destroy them. In this therapy, the antibodies are known to be powerful medications to activate the immune system in different ways. They circulate throughout the body until they discover a substance that body recognize as alien i.e. antigen and bind to them. Similarly, cancer cells often have molecules on their surface known as tumor-associated antigens. The researchers can design many clones of the antibody that only target a certain antigen type such as one found on tumors or cancer cells. Then, these are used as an effective drug for treating cancer. Thus, the antigen specific antibody interactions play a vital role in cancer immunotherapy. In this study, we propose a dynamic model to represent the population of antigens and antibodies in cancer patients; in particular we focus on the antigen-specific-antibody interactions to elicit an immune response that leads to the death of cancer cells. We formulate a terminal control problem where the schedule and doses of these antibodies are considered as control variables. The objective functional has been formulated as a measure of antigen population at the end of the treatment period. Pontryagin minimum principle (PMP) has been used to obtain the optimal control policies. For illustration, a series of numerical results is presented showing the effectiveness of immune therapy for cancer treatment corresponding to the different scenarios, choices of parameters and treatment periods. The results indicate that the control doses are followed by the emergence of antigen population. This approach would be potentially applicable to determine and prescribe the optimal doses and schedules for cancer patients.

PURPOSE: In the mouse, the macrophage adhesion G protein-coupled receptor (ad-GPCR) molecule, F4/80, is required for the development of regulatory T cells in two models of tolerance, the eye and gut. Since F4/80 is not expressed in humans, the purpose of this research is to determine the human analog of F4/80. F4/80 belongs to a novel family of Epidermal growth factor-seven transmembrane (EGF-TM7) molecules, which include the EGF module-containing mucin-like hormone receptor (EMR) molecules. In the human, EMR1 has sequential homology with F4/80 and EMR2 has shown immune suppressing function in tumor cells. Thus, we investigate the possible suppressor role of the EMR family in human ocular tolerance. METHODS: Human peripheral blood mononuclear cells (huPBMC) were treated with porcine TGF&beta;2 and LPS or an antigenic stimulant for at least six hours to generate tolerogenic antigen presenting cells (APC). Cells were characterized by flow cytometric analysis for expression of CD14, CD40, PDL1, ILT3, and EMR2. Later, T regulatory cells were generated by incubating tolerogenic APCs with autologous huPBMC for five to seven days. Post culture, the T cells were stained and characterized for expression of CD4, CD25, and FoxP3. RESULTS: Post treatment of huPBMC with TGFβ2 and antigen, the resulting tolerogenic APCs expressed PDL1, ILT3, and EMR2. CD40 remained unchanged and CD14 was constitutively expressed. Post five to seven day culture, tolerogenic APCs treated with TGFβ2 increased the CD4+ CD25+ FoxP3+ lymphocyte populations. CONCLUSIONS: The upregulation of EMR2 on human tolerogenic APCs suggests that EMR2 may have a role in inducing tolerance in humans. Much like its mouse counterpart, F4/80, EMR2 is an adhesion molecule that may facilitate the induction of naïve T lymphocytes to regulatory T lymphocytes. Once the F4/80 analog is established for humans, novel therapies may be developed to interfere or encourage signaling in the treatment of tumors or immune inflammatory diseases, respectively.

Few cancers arising in fluid organ systems can be cured with localized therapeutic modalities, such as radiation or surgical organ removal. Chemotherapy and hematopoietic stem cell transplants have long been employed as the standard of care for patients diagnosed with leukemias and lymphomas. Though research continues to propose new, more potent chemotherapeutic agents, a new paradigm of treating cancerous malignancies with tumor-specific monoclonal antibodies, adoptively transferred tumor-fighting cells, and other exogenously administered immunomodulatory agents, has emerged over the past decade. These immunotherapies have dramatically improved the outcomes of patients diagnosed with cancers of B lymphocytes, referred to as B-cell malignancies. Though curative FDA-approved therapies for patients diagnosed with B-cell malignancies have yet to be established, recent research in the field of adoptive T-cell therapy has produced promising results. Tumor infiltrating lymphocyte therapy (TIL therapy), T-cell Receptor Therapy (TCR therapy) and Chimeric Antigen Receptor T-cell Therapy (CAR T-cell therapy) are the three most extensively studied adoptive T-cell immunotherapies in the context of B-cell malignancies. TIL and TCR therapies, in which patients are provided with either the patient’s own tumor-specific T-cells or T-cells expressing engineered, tumor-specific TCRs, respectively, enhance the patient’s immune system to mount a more potent, anti-tumor response. However, these adoptive T-cell therapies do not change the mechanisms of the immune response. Cancerous cells can evade immune attack and dampen immune responses to survive and thrive in the body. By down-regulating their expression of human major histocompatibility complex I (MHC I), for example, cancer cells escape T-cell recognition, which is dependent on MHC expression. A chimeric antigen receptor (CAR), is composed of an antibody-derived (B-cell derived) extracellular, antigen-recognition domain, and T-cell derived intracellular domains. CAR T-cells, therefore, exploit the cytotoxic nature of CD8+ T-cells, and the MHC independent recognition of B-cell receptors, to identify and destroy all cells expressing a specific target. Consequently, many of the cancer cell’s mechanisms of immune evasion are less effective in the presence of CAR T-cells. Progressive generations of CAR T-cell designs couple these receptors with costimulatory molecules to amplify the activation, efficacy, and potency of these cells in-vivo. Over the past five years, phase I and IIa clinical trials have produced remarkable results in the treatment of advanced stage, high-risk B-cell malignancies, namely Acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), and Non-Hodgkin’s Lymphoma (NHL). However, the significant oncogenic risks and fatal adverse events associated with this therapy necessitate further research to improve safety and reliable clinical efficacy of CAR T-cell therapy. In spite of these risks, the adoptive transfer of CD19-targeting, CAR expressing, cytotoxic T-cells (anti-CD19 CAR-T-cells) has produced sustained, complete remissions in patients diagnosed with progressive, advanced-stage, B-cell malignancies, for whom alternative treatments were not available. The unprecedented results of early clinical trials, as well as ongoing preclinical studies aimed at improving the design and production of CAR T-cells suggest a promising future for CAR T-cell therapy as a cure for B-cell malignancies.

Made available in DSpace on 2014-09-24T12:58:28Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 154.pdf: 7447537 bytes, checksum: f05caacd8152f680cccd33520b65caea (MD5) Previous issue date: 2004 / O teste intradérmico para o diagnóstico da Leishrnaniose foi preconizado por Montenegro em 1926. Tratava-se de um extrato alcalino de formas promastigotas de Leishmania que se acreditava livre de partículas e de parasitos inteiros. Atualmmte, os antígenos mais utilizados constituem-se ainda de formas promastigotas, algumas íntegras e outras sonicadas, a partir de "pool " cepas de Leishmania ou cepas únicas, diluídas em soluções preservadoras como salinas fenoladas 0,4 por cento ou mertiolatadas 1:10.000. A produção do antígeno, não tem portanto uma padronização definida no Brasil e no Mundo e vários preparações antigênicos em diferentes apresentações são descritas comprometendo a qualidade e a comparabilidade das respostas. Justificou-se assim, a necessidade de padronização da metodologia para a produção do antígeno de Montenegro, em diferentes fases. Este estudo propôs definições de padrões na produção do antígeno para a intradermorreação de Montenegro em relação a melhor inóculo padronizado de promastigotas, métodos de quantificação de concentração de proteínas, prazo de validade e controles de qualidade, universalmente aceitos. / Montenegro’s antigen was advocated publicly by Montenegro in 1926 for leishmaniasis diagnosis through intradermal test. It was an alkaline extract of Leishmania promastigotes forms, which was thought to be particles of and parasites free. However, nowadays the most used antigens are of promastigotes forms, some of which are integral and others are sonicated as from pool of Leishmania strains or single ones diluted in preserving solutions like phenol or merthiolate saline. The production of this antigen has not been standardized in Brazil or worldwide; thus several antigen preparations, in different doses, are described jeopardizing the quality and comparison of answers. Therefore, it was made necessary a standardized methodology to produce Montenegro’s antigen in different phases. This study propose antigen definitions for Montenegro’s intradermal reactions related to the best promastigotes inoculum, protein concentration checking methods, validity and quality controls equivalent to the vaccines ones.