Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2009 Mar 8

In the post genomic era, proteomics has enormous potential in biology and medicine. Among the various bioanalytical tools developed, protein microarray is one of the recent advancements which offer high throughput profiling of cellular proteins to provide insights into the mechanisms of biological processes. Fundamentally, the protein microarray involves the immobilization of interacting elements, proteins, on a few square microns of a solid support and in principle, it is capable of detecting analytes with a higher sensitivity than conventional macroscopic immunoassays. Here in the present report we delineates the design, fabrication and functional analysis of protein microarray using semi-synthetic ssDNA tagged-proteins as capturing moiety as well as address on a solid support. Optimization of the platform has been carried out by investigating various parameters such as surface chemistry, signal amplification, and conditions for homogenous liguid phase protein-protein interaction. / Singapore-MIT Alliance (SMA)

Seroepidemiology aims to understand population-level exposure and immunity to infectious disease. Serological results are normally presented as binary outcomes describing the presence or absence of antibody, despite the fact that many assays measure continuous quantities. A population's antibody titers may include information on multiple serological states - naiveté, recent infection, non-recent infection, childhood infection - not just seropositivity or seronegativity. In the first part of this thesis, I investigate 20,152 general-population serum samples from southern Vietnam collected between 2009 and 2013 from which I report antibody titers to the influenza virus HA1 protein using a continuous titer measurement from a protein microarray assay. I describe titer distributions to subtypes 2009 H1N1 and H3N2, and using a model selection approach for mixture distributions, I determine that 2009 H1N1 is best described by four titer subgroups while H3N2 is best described by three titer subgroups. For H1N1, my interpretation is that the two highest-titer subgroups correspond to recent and historical infection, which is consistent with pandemic attack rates. For H3N2 however, right-censoring of titers makes interpretations difficult to validate. To move beyond this stationary interpretation of titers, I developed two methods for analyzing this serum collection as a time series. First, I attempted to analyze mixture categories in individual time windows. This approach did not lead to a consistent temporal picture of titer change; results differed by site and were sensitive to assumptions in the mixture fitting. Second, I attempted to fit a hybrid-dynamical model with free incidence parameters. This inference was robust to parameter assumptions, consistent across sites, and in agreement with the incidence reported in Vietnam's influenza surveillance network. In addition, my new approach showed evidence that there was a second silent wave of the 2009 influenza pandemic that was not recorded in national surveillance, which is this thesis' main novel result.

abstract: Immunosignaturing is a new immunodiagnostic technology that uses random-sequence peptide microarrays to profile the humoral immune response. Though the peptides have little sequence homology to any known protein, binding of serum antibodies may be detected, and the pattern correlated to disease states. The aim of my dissertation is to analyze the factors affecting the binding patterns using monoclonal antibodies and determine how much information may be extracted from the sequences. Specifically, I examined the effects of antibody concentration, competition, peptide density, and antibody valence. Peptide binding could be detected at the low concentrations relevant to immunosignaturing, and a monoclonal's signature could even be detected in the presences of 100 fold excess naive IgG. I also found that peptide density was important, but this effect was not due to bivalent binding. Next, I examined in more detail how a polyreactive antibody binds to the random sequence peptides compared to protein sequence derived peptides, and found that it bound to many peptides from both sets, but with low apparent affinity. An in depth look at how the peptide physicochemical properties and sequence complexity revealed that there were some correlations with properties, but they were generally small and varied greatly between antibodies. However, on a limited diversity but larger peptide library, I found that sequence complexity was important for antibody binding. The redundancy on that library did enable the identification of specific sub-sequences recognized by an antibody. The current immunosignaturing platform has little repetition of sub-sequences, so I evaluated several methods to infer antibody epitopes. I found two methods that had modest prediction accuracy, and I developed a software application called GuiTope to facilitate the epitope prediction analysis. None of the methods had sufficient accuracy to identify an unknown antigen from a database. In conclusion, the characteristics of the immunosignaturing platform observed through monoclonal antibody experiments demonstrate its promise as a new diagnostic technology. However, a major limitation is the difficulty in connecting the signature back to the original antigen, though larger peptide libraries could facilitate these predictions. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2011

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.

Introduction: Periodontal disease is one of the most common inflammatory diseases in the world. A possible autoimmune aspect behind the local tissue destruction in periodontal disease, as a result of the invasion of oral pathogens over time has been reported in previous studies, but the correlation is yet unclear. Purpose: The aim of this literature review was to shed light on the topic if autoantibodies and autoimmune reactions are associated with gingivitis, periodontitis or peri-implantitis and the progression of these inflammatory diseases. Material and methods: A search in the Pubmed database was done resulting in 138 hits. To follow a systematic approach for selecting the studies to include, we used predefined inclusion and exclusion criteria Results: 26 articles studying a broad variety of different autoantibodies was included for this literature review. A vast majority of the included studies were of case-control design and, because of the broad variety and different variables and data, we decided that a meta-analysis could not be performed. Conclusion: Many studies where results could be compared due to similar comparisons, regarding the incidence of periodontal disease and the prevalence of certain autoantibodies, showed opposite results which makes it hard to reach a conclusion. The main part of the included studies were of small size and therefore more comparable studies are needed to clarify the possible association between periodontal disease and an autoimmune reaction mediated by autoantibodies.

Orientador: Valéria Marçal Félix Lima / Banca:Gisele Fabrino Machado / Banca:Sandra Helena Penha de Oliveira / Resumo: A leishmaniose é uma doença imunossupressora causada por protozoários do gênero Leishmania, tendo os cães como reservatório doméstico. A molécula Programmed Cell Death 1 (PD-1) é altamente expressa em células leucocitárias de cães com leishmaniose visceral e promove a exaustão dos linfócitos T e a supressão da secreção de citocinas. Como o PD-1 tem função supressiva em relação à imunidade celular, avaliamos o efeito de anticorpos bloqueadores de PD-1 na produção de NO, ROS, interleucina 17 (IL-17) e na carga parasitária em cultura de leucócitos esplênicos de cães naturalmente infectados. In vitro, o bloqueio de PD-1 promoveu aumento dos níveis de NO intracelular e reduziu os de IL-17 no sobrenadante da cultura, além de reduzir a carga parasitária, más não alterou os níveis de ROS. Concluímos que a PD-1 participa na regulação da resposta imune e que o anticorpo bloqueador é efetivo na restauração da atividade microbicida do hospedeiro. Isso pode ser investigado em estudos imuno-terapêuticos no futuro. / Abstract: Leishmaniasis is an immunosuppressive disease caused by protozoa of the genus Leishmania for which dogs are the domestic reservoir. The programmed cell death 1 molecule (PD-1) is highly expressed in leukocyte cells of dogs with visceral leishmaniasis, and it promotes T lymphocyte exhaustion and suppression of cytokine secretion. Because PD-1 has a suppressive function regarding cell immunity, we evaluated the effect of PD-1 blocking antibodies on NO, ROS and interleukin 17 (IL-17) production and on parasite load in spleen leukocyte cultures from naturally infected dogs. In vitro, PD-1 blocking promoted increased levels of intracellular NO and reduced the levels of IL-17 in the culture supernatant, in addition to reducing the parasite load, but do not change ROS levels. We conclude that PD-1 participates in regulation of the immune response and that the blocking antibody is effective in restoring host microbicidal activity. This can be investigated in an immuno-therapeutic study in the future. / Mestre

In regions endemic for malaria, natural exposure results in an acquired immunity which protects individuals from severe disease. However, no vaccine against the blood-stage of malaria, against which naturally-acquired immunity is targeted, currently exists that is capable of emulating, or out-performing, natural protection. To rationally direct the next generation of blood-stage malaria vaccine development, a greater understanding of the immunological mechanisms involved in clinical protection is required. To date, the assessment of naturally-acquired and vaccine-induced immunity to the blood-stage of malaria has suffered from a paucity of in vitro immunological assays that are both robust and reproducible, whilst allowing for assessment of anti-parasitic activity induced by antibodies, either alone, or in conjunction with immune cells. Thus this Thesis describes the development of the antibody-dependent respiratory burst (ADRB) assay, for assessment of blood-stage immunity against Plasmodium falciparum, as well as the Duffy antigen receptor for chemokines (DARC) – Duffy binding protein (DBP) binding inhibition assay, for assessing antibody mediated immunity to P. vivax. A reproducible and standardised assay of ADRB activity was developed here and applied to studies of immunity in both mice and humans. ADRB activity, which assesses antibodies' ability to activate oxidative burst in neutrophils via Fc receptor (FcR)-dependent pathways, was shown to associate with clinical protection in a cohort from Mali where FcR-independent immunological assays, such as the assay of growth inhibition activity, did not. This work thus elucidates the importance of FcR-dependent immunity to P. falciparum malaria and establishes the ADRB assay as a useful tool for future vaccine development. In addition, the DARC-DBP binding inhibition assay was established and utilised to assess inhibitory activity of antibodies induced in the first Phase I clinical trial of this antigen. Results identify the need for significant improvements in vaccine design, and show the utility of the assay as a tool for assessing future blood-stage vaccine development efforts against this neglected parasite.

Submitted by Gabriela Torres Rebech (gtr.torres@hotmail.com) on 2018-05-11T23:25:50Z No. of bitstreams: 1 Dissertação Gabriela.pdf: 515150 bytes, checksum: 8dcf7e5ca4b514e5c7c8ada92438306b (MD5) / Approved for entry into archive by Ederson Vasconcelos Pereira null (edersonpereira@fmva.unesp.br) on 2018-05-14T17:16:11Z (GMT) No. of bitstreams: 1 rebech_gt_me_araca_int.pdf: 515150 bytes, checksum: 8dcf7e5ca4b514e5c7c8ada92438306b (MD5) / Made available in DSpace on 2018-05-14T17:16:11Z (GMT). No. of bitstreams: 1 rebech_gt_me_araca_int.pdf: 515150 bytes, checksum: 8dcf7e5ca4b514e5c7c8ada92438306b (MD5) Previous issue date: 2018-04-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A leishmaniose é uma doença imunossupressora causada por protozoários do gênero Leishmania, tendo os cães como reservatório doméstico. A molécula Programmed Cell Death 1 (PD-1) é altamente expressa em células leucocitárias de cães com leishmaniose visceral e promove a exaustão dos linfócitos T e a supressão da secreção de citocinas. Como o PD-1 tem função supressiva em relação à imunidade celular, avaliamos o efeito de anticorpos bloqueadores de PD-1 na produção de NO, ROS, interleucina 17 (IL-17) e na carga parasitária em cultura de leucócitos esplênicos de cães naturalmente infectados. In vitro, o bloqueio de PD-1 promoveu aumento dos níveis de NO intracelular e reduziu os de IL-17 no sobrenadante da cultura, além de reduzir a carga parasitária, más não alterou os níveis de ROS. Concluímos que a PD-1 participa na regulação da resposta imune e que o anticorpo bloqueador é efetivo na restauração da atividade microbicida do hospedeiro. Isso pode ser investigado em estudos imuno-terapêuticos no futuro. / Leishmaniasis is an immunosuppressive disease caused by protozoa of the genus Leishmania for which dogs are the domestic reservoir. The programmed cell death 1 molecule (PD-1) is highly expressed in leukocyte cells of dogs with visceral leishmaniasis, and it promotes T lymphocyte exhaustion and suppression of cytokine secretion. Because PD-1 has a suppressive function regarding cell immunity, we evaluated the effect of PD-1 blocking antibodies on NO, ROS and interleukin 17 (IL-17) production and on parasite load in spleen leukocyte cultures from naturally infected dogs. In vitro, PD-1 blocking promoted increased levels of intracellular NO and reduced the levels of IL-17 in the culture supernatant, in addition to reducing the parasite load, but do not change ROS levels. We conclude that PD-1 participates in regulation of the immune response and that the blocking antibody is effective in restoring host microbicidal activity. This can be investigated in an immuno-therapeutic study in the future.

Made available in DSpace on 2014-06-11T19:29:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-02Bitstream added on 2014-06-13T18:39:10Z : No. of bitstreams: 1 oliveira_jcv_me_botfmvz.pdf: 876846 bytes, checksum: 0964099d5611eb445b184c0c142d8ec3 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A cisticercose é uma das principais causas de perdas econômicas na pecuária de corte brasileira devido às condenações da carne para o controle do complexo teníase-cisticercose. O tratamento quimioterápico pode ser utilizado para o controle da infecção nos animais, mas sua eficácia deve ser monitorada por testes de diagnóstico que detectem parasitas vivos ou seus produtos. Na presente pesquisa, anticorpos monoclonais contra antígenos totais (TAEB) e de fluido vesicular (TAEF) de metacestóides de Taenia saginata foram produzidos através de fusão celular, utilizando-se células de baço de camundongos BALB/c imunizados. Cinco fusões TAEB e quatro TAEF foram realizadas e os sobrenadantes de cultura foram triados por teste ELISA indireto. Dez e nove híbridos pertencentes aos protocolos TAEB e TAEF, respectivamente, foram selecionados e clonados. As linhagens celulares clonadas foram mantidas em meio de cultura para a obtenção dos anticorpos monoclonais, resultando em 18 clones IgG1 e 32 IgM reativos para TAEB e 9 IgG1 e 9 IgM para TAEF. Dentre esses clones, 5 do protocolo TAEB e 5 TAEF foram selecionados para produção de líquido ascítico, através da injeção dos clones em camundongos BALB/c. A avaliação da identificação antigênica dos anticorpos produzidos aos antígenos homólogos foi realizada por meio de western blotting, resultando em reatividade com frações protéicas de baixo peso molecular (< 18kDa), 43, 55, 66 e 100kDa. A imunofluorescência indireta demonstrou que os anticorpos monoclonais avaliados reconhecem antígenos presentes tanto do escólex quanto da membrana vesicular de metacestóides de bovinos naturalmente infectados. Os anticorpos monoclonais produzidos poderão ser utilizados para detecção de antígenos circulantes na avaliação da eficácia do tratamento de bovinos infectados, evitando assim perdas econômicas e danos à saúde pública. / Cysticercosis is a major cause of economic losses in the Brazilian bovine chain production due to meat condemnation for the taeniasis-cysticercosis complex control. Chemotherapy can be used to control infection in cattle but treatment efficacy should be monitored by diagnostic tests that can detect live parasites or their products. Monoclonal antibodies against Taenia saginata metacestode crude (TAEB) and cyst fluid (TAEF) antigens were produced by cell fusion procedures using spleen cells from immunized BALB / c mice. Five TAEB and four TAEF fusions were performed and the culture supernatants from these cell cultures were screened by indirect ELISA assay. Ten TAEB and nine TAEF hybrids were selected and cloned. Cloned cell lines were grown in culture medium to collect the monoclonal antibodies, resulting in 18 IgG1 and 32 IgM clones reactive to TAEB and 9 IgG1 and 9 IgM clones reactive to TAEF. Five TAEB and five TAEB clones were selected for ascitic fluid production by injection in BALB/c mice and further harvesting. Western blotting was performed to test the antigenic identification by the antibodies produced resulting in reactivity to protein fractions of low molecular weight (<18kDa) and to 43, 55, 66 and 100kDa. The indirect immunofluorescence test showed that monoclonal antibodies recognize antigens from both the scolex and the bladder wall of metadestodes from naturally infected bovine. The monoclonal antibodies obtained can be useful for detection of circulant antigens in treatment efficacy evaluation preventing economic losses and public health injury.