Master of Science in Biomedical Sciences / Department of Clinical Sciences / Elizabeth G. Davis / To protect horses from disease, equine practitioners typically prescribe a protocol of an initial primary vaccination followed by a booster vaccination 3-4 weeks later. Subsequent boosters are given every 6-12 months depending on the pathogen of concern. Each vaccination incurs an additional cost and increased chance for adverse reactions. Despite wide-spread protocol acceptance, duration of effectiveness of vaccines in protecting horses from disease is not well documented. It was hypothesized that horses vaccinated annually since birth have increased antibody production that remains consistent and sufficient for long-term protection from common diseases. This work resulted in the development of a novel, multiplex-magnetic bead-based indirect immunoassay to screen sera from vaccinated adult horses to measure antibody levels in response to vaccine administration. Antigens tested included West Nile Virus, Eastern Equine Encephalitis, Western Equine Encephalitis, Equine Influenza Virus, Equine Herpes Virus 1 and 4, Tetanus, and 7 different Rabies antigens (3 lab and 4 wild strains). The developed assay was a 7-plex capture antibody, which quantified equine IgG (Immunoglobulin G) that binds viral antigens derived from different rabies virus strains along with pure vaccine samples of the 7 different antigens. A 7-point standard curve was developed to quantify the viral-antigen reactive IgG concentration in vaccinated horse serum. Vaccinated horses increased serum antibody concentration for each antigen post-vaccination with the percent increase ranging between 34.0% for Equine Herpes Virus 4 and 257.3% for Equine Influenza Virus. Use of the novel assay will provide equine veterinarians with an economical method to measure immune activation toward common pathogens of concern. This methodology will provide foundation level information regarding antigen specific IgG concentrations that ultimately may be extrapolated to establish protective levels of immunity resulting in establishment of vaccine protocols.

Direct immunofluorescence (DIF) using frozen section material from a fresh/preserved perilesional biopsy is the gold standard for the immunopathologic diagnosis of bullous pemphigoid (BP). DIF in BP shows linear dermoepidermal junction (DEJ) staining for C3, with or without staining for IgG. In some situations, only a formalin-fixed lesional biopsy is obtained (with no fresh/preserved perilesional biopsy for DIF). In this setting, paraffin section C4d immunohistochemistry has proven to be diagnostically useful, demonstrating linear DEJ positivity for C4d. We present a novel use of C4d staining for the diagnosis of BP, specifically analyzing C4d perilesional frozen section DIF in a case where standard perilesional frozen section DIF for IgG/C3 was available, but was negative. An 80-year-old woman presented with a pruritic bullous lesion on her left upper extremity, clinically thought to represent BP. Lesional histologic findings were typical for BP, but perilesional frozen section DIF staining was negative for IgG and C3. A second set of biopsies processed at a different laboratory yielded the same result. A diagnosis of bullous scabies was considered. Subsequently, perilesional frozen section DIF for C4d was obtained, which showed strong linear DEJ positivity, confirming the diagnosis of BP. DIF for C4d is widely used in transplant pathology, since C4d is persistent in tissue, versus C3. Our case demonstrates that perilesional frozen section DIF staining for C4d may be positive and diagnostic in BP, even when conventional DIF staining for IgG and C3 is negative.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Newborn Holstein (n = 48) and Jersey (n = 30) calves were studied to compare the absorption of immunoglobulin G (IgG) from maternal colostrum (n = 39) or a colostrum replacement product derived from bovine serum (n = 39). Calves were also fed milk replacer with (n = 38) or without (n = 40) animal plasma to 29 d of age to determine the effect of plasma protein on IgG status, health, and growth. Colostrum or colostrum replacement was fed at 1.05 and 13.5 h of age and provided a total of 250, 180, 249, or 186 g IgG for Holsteins and Jerseys fed replacement or colostrum, respectively. Milk replacer (12.5% DM) was fed at 31% of metabolic birth weight (2 feedings/d). Jugular blood was sampled at 0 h, 24 h, and weekly to determine plasma IgG. At blood collection calves were weighed and measured to determine growth. Health scores, fecal scores, and grain intake were measured daily. Mean plasma IgG at 24 h did not differ between calves fed colostrum (13.78 ± 0.39 g/L) and replacement (13.96 ± 0.38 g/L). Average daily gain, wither height, hip height, body length, heart girth, health, and incidence of diarrhea were not different between treatment groups. Plasma IgG and performance were not affected by addition of animal plasma to milk replacer. The colostrum substitute successfully replaced colostrum as the source of IgG for newborn calves. Animal plasma was an acceptable source of protein, but did not enhance growth or immunity. / Master of Science

Streptococcus pneumoniae (pneumococcus) is a Gram positive opportunistic bacterium that is a primary cause of pneumonia in young children and immunocompromised individuals. This microorganism colonizes the nasopharynx of all age groups and health levels and is easily transmitted through respiratory droplets or aerosols. While capsule-based vaccines are available, these are becoming more obsolete as S. pneumoniae strains are undergoing serotype replacement, thus evading detection, and antibiotic resistant strains are increasing in prevalence. This study has taken a different approach by identifying immunogenic proteins that elicit antibodies which block attachment of bacteria to host epithelial cells. Proteins with the most potential were assessed by analyzing the reactivities of human sera with varying degrees of colonization blockage. Proteins which reacted with the primary protective antibody in mucosal sites, IgA, were prioritized. Identification of those proteins through mass spectrometry will be crucial in creating more effective, long-term protection against S. pneumoniae infections.

A leishmaniose visceral é uma doença parasitária caracterizada por altos níveis de anticorpos IgG, importantes para o diagnóstico da infecção, mas sem discriminar doença ativa. Sem papel efetivo durante a resposta imune, estes anticorpos participam na formação de imunocomplexos circulantes, indicando a presença de antígenos circulantes. Apesar da alta especificidade diagnóstica, a detecção de antígenos em soro é pouco explorada na leishmaniose visceral. A detecção de antígenos e imunocomplexos dependem tanto da especificidade de anticorpos como das características dos antígenos, que podem ser desde grandes proteínas ou pequenos polissacarídeos. Neste estudo, avaliamos a detecção de antígenos circulantes livres ou em imunocomplexos, e as características e especificidade antigênica de anticorpos IgG a proteínas ou carboidratos de L. (L) infantum chagasi em leishmaniose visceral experimental. Amostras de hamster com infecção experimental por L. (L.) infantum chagasi foram avaliadas aos 15, 30, 45, 60 e 90 dias de infecção. Altas concentrações de anticorpos IgG específicos foram detectadas em amostras com leishmaniose visceral experimental e natural, sempre com alta avidez, mesmo em períodos iniciais da infecção. A purificação por imunoafinidade de IgG de baixa avidez também confirmou esta alta concentração. Proteínas e carboidratos foram isolados do extrato antigênico total de promastigotas por cromatografia de exclusão molecular ou precipitação com etanol e marcadas com biotina para aminas ou açucares. Para uso em ELISA, carboidratos foram conjugados a BSA, o que permitiu a detecção de altos níveis de anticorpos IgG específicos para carboidratos. A presença de carboidratos antigênicos também foi demonstrada através da detecção de incremento de IgG em amostras com infecção experimental, o que confirmou a presença de imunocomplexos circulantes específicos a carboidratos. Nossos dados sugerem, que a participação da resposta imune humoral na leishmaniose visceral deve ter o envolvimento de carboidratos do parasita. O desenvolvimento de novas ferramentas diagnósticas para a detecção de carboidratos antigênicos pode permitir a identificação de imunocomplexos e antígenos circulantes na leishmaniose visceral. / Visceral leishmaniasis is a parasitic disease characterized by high levels of IgG antibodies, important for the diagnosis of the infection, but without discriminating active disease. Without effective role during the immune response, these antibodies participate in the formation of circulating immune complexes, indicating the presence of circulating antigens. Despite the high diagnostic specificity, the detection of antigens in serum is poorly explored in visceral leishmaniasis. The detection of antigens and immunocomplexes depend on both the specificity of antibodies and the characteristics of the antigens, which may be from large proteins or small polysaccharides. In this study, we evaluated the detection of free circulating antigens or immunocomplexes, the characteristics and antigenic specificity of IgG antibodies to proteins or carbohydrates of L. (l) infantum in experimental visceral leishmaniasis. Hamster samples with experimental infection by L. (L.) infantum chagasi was evaluated at 15, 30, 45, 60 and 90 days of infection. High concentrations of specific IgG antibodies were detected in samples with experimental and natural visceral leishmaniasis, always with high avidity, even in initial periods of infection. Immunoaffinity purification of low avidity IgG also confirmed this high concentration. Proteins and carbohydrates were isolated from the total antigenic extract of promastigotes by molecular exclusion chromatography or ethanol precipitation and labeled with biotin for amines or sugars. For use in ELISA, carbohydrates were conjugated to BSA, which allowed the detection of high levels of carbohydrate-specific IgG antibodies. The presence of antigenic carbohydrates was also demonstrated by the detection of IgG increase in samples with experimental infection, which confirmed the presence of circulating immunocomplexes specific to carbohydrates. Our data suggest that the involvement of the humoral immune response in visceral leishmaniasis should have the involvement of carbohydrates of the parasite. The development of new diagnostic tools for the detection of antigenic carbohydrates may allow the identification of immunocomplexes and circulating antigens in visceral leishmaniasis.

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No. of bitstreams: 2 Dissertação - Isabela Cinquini Junqueira - 2018.pdf: 1854672 bytes, checksum: 9118e1e74d08a31917a913e8d1a77416 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-09-22 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Variations in IgG subclass concentrations are associated with numerous infectious diseases. Observations on dengue hemorrhagic fever pathogenesis suggest a high degree of immune stimulation with extensive activation of complement system preceding the onset of shock, signaling a possible role of complement activation products in endothelium damage and plasma leakage. Antibodies IgG1 and IgG3 are known to activate the complement system efficiently while IgG2 and IgG4 are weak activators. Moreover, these are the subclasses that exhibit cytophilic properties. Studies have demonstrated that antibodies in association with FcRs receptors, present on the surface of numerous cells of the organism, are involved with both, protection and dengue imunopathogenesis. In the present study, IgG subclass antibodies antigen-specific and also non DENV specific were determined in dengue seropositive patients through respectively semi-quantitative and quantitative enzyme immunoassays “in house” procedures. Serum samples of individuals participating in this study were collected in the epidemic period of october 2005 to march 2006 on dengue reference health care units in the city of Goiânia-GO. Study groups were divided as follows: 30 – dengue fever (DF); 30- dengue with complications (DCC) and 30- dengue hemorrhagic fever (DHF). Overall, it was observed an increase in total IgG1 and IgG3 in dengue more severe clinical forms, except in the age group of 25-45 years in which the highest concentration of IgG1 was observed in subjects with DF and not DCC nor DHF. As for antigen-specific subclasses, it was found an IgG1 raise and IgG3 decrease in the most severe clinical forms of the disease. It was also observed a significant difference in IgG1 and IgG3 concentration between the most severe clinical forms, DCC and DHF. DCC clinical form was characterized by a larger concentration of IgG3 DENV-specific while the clinical form of DHF was characterized by an increased amount of total and specific IgG1. The results of this study indicates that it may be a correlation between IgG subclasses concentrations, mainly IgG1 and IgG3, and dengue prognosis. / Variações nas concentrações das subclasses de IgG são associadas com numerosas doenças infeciosas. Observações na patogêneses da febre hemorrágica do dengue demonstram alto grau de estimulação imune com extensa ativação do sistema complemento precedendo o início do choque, sinalizando um possível papel dos produtos da ativação do complemento nos danos ao endotélio e extravasamento de plasma. Os anticorpos IgG1 e IgG3 são conhecidos como eficientes ativadores do complemento enquanto que a IgG2 e IgG4 são ativadores fracos. Além disso, são as subclasses que apresentam propriedades citofílicas. Estudos tem mostrado que anticorpos em associação com os receptores FcRs, presentes na superfície de inúmeras células do organismo, estão envolvidos tanto com proteção a dengue como com imunopatogênese. No presente estudo, anticorpos das subclasses de IgG, antígeno específicos e também não DENV específicos, foram determinados em pacientes soropositivos para dengue através de ensaio imunoenzimático semi-quantitativo e quantitativo, respectivamente, com procedimentos “in house”. Amostras de soro dos indivíduos participantes do estudo foram coletadas no período epidêmico de outubro de 2005 a março de 2006 em unidades de saúde de referência em dengue na cidade de Goiânia-GO. Os indivíduos do estudo foram divididos como se segue: 30 – febre do dengue (FD); 30 – dengue com complicações (DCC) e 30 – febre hemorrágica do dengue (FHD). Observou-se, no geral, um aumento de IgG1 e IgG3 totais nas formas clínicas de maior gravidade de dengue, exceto na faixa etária de 25-45 anos em que a maior concentração de IgG1 foi observada nos pacientes com DC e não DCC ou FHD. Quanto às subclasses antígeno-específicas, foi encontrado um aumento de IgG1 e uma diminuição de IgG3 nas formas clínicas mais graves da doença. Foi possível também observar uma diferença significativa na concentração de IgG1 e IgG3 entre as formas clínicas de maior gravidade, DCC e FHD. A forma clínica DCC foi caracterizada por maior quantidade de IgG3 DENV específica enquanto que a forma clínica FHD foi caracterizada por maior quantidade de IgG1 DENV específica e total. Os resultados desse estudo indicam a existência de uma possível correlação entre as concentrações das subclasses de IgG, principalmente IgG1 e IgG3, e o prognóstico da dengue.

A disponibilidade de hemoderivados é um parâmetro importante para medir a qualidade da saúde em um país. Dentre os produtos hemoderivados, imunoglobulinas tem alto valor agregado. O Instituto Butantan tem por objetivo o estabelecimento de uma planta industrial para fracionamento de plasma, com um processo produtivo baseado principalmente em cromatografias. Eventos tromboembólicos a partir de infusões de imunoglobulinas por via intravenosa (IgIV) foram relacionados com presença de Fator XI de coagulação (FXI) como contaminantes nas preparações de IgIVs. Com objetivo de detectar o FXI nas frações, o processo cromatográfico foi testado em escala piloto, bancada, e em cromatografia direta e o FXI foi dosado nas frações iniciais e no produto final IgIV. Foram estabelecidos os métodos de dosagem de atividade de FXI por tempo de coagulação e ensaio cromogênico. Concluímos que o FXI acompanha a IgG nas etapas iniciais dos processos cromatográficos e verificamos que ocorre a presença de FXI nos produtos finais. Este trabalho contribui para o desenvolvimento do conjunto de testes de controle de qualidade de biofármacos derivados de plasma humano. / The availability of hemoderivatives is an important parameter to measure a countrys health quality. Among hemoderivatives products, immunoglobulin have high value.. Instituto Butantan, aims the establishment of an industrial plant for plasma fractionation with a process drawn, based mainly in chromatographies. Thromboembolic events from infusions of intravenous immunoglobulins (IVIg) have been related with the presence of coagulation Factor XI (FXI) as contaminant in the IVIG preparations. With an objective of tracking FXI in the chromatographic fractions, the process was tested in pilot and bench scales and in direct chromatography and the FXI was measured in the initial fractions and in the final product IVIg. The methods of measurement of FXI activity thru coagulation time and chromogenic assay were established. We have concluded that FXI accompanies IgG in the early stages of the chromatographic processes and verified that the presence occurs in the final products, This work contributes to the development of the set of quality control tests of biopharmaceuticals derivatives from human plasma.

Antibodies are the defining characteristic of the humoral immune response. Their functions are diverse, including direct neutralization of pathogens and recruitment of other immune molecules or cells. While most successful vaccines induce protective neutralizing antibody responses, effective vaccine-elicited neutralizing antibodies against some pathogens, including HIV, HCV, malaria, and TB, remain elusive. Thus, researchers have begun to focus on how vaccines can elicit strong non-neutralizing antibody functions, including recruitment of innate immune factors for antibody-dependent cellular cytotoxicity, complement deposition, and anti\-body-dependent phagocytosis. The antibody's constant region (Fc) mediates most effector functions through isotype and subclass selection or alteration of the structure of the Fc-attached N-glycan, which controls function with exquisite specificity. Glycan modifications are naturally induced during inflammatory conditions such as autoimmune disease and natural infection however, the specific signals that regulate Fc-glycosylation remain unknown. This dissertation sought to understand how antibody glycosylation is regulated and how it can be programmed through vaccination. To do this, we first developed a technique to analyze antibody glycan structures both of bulk Fc and antigen-specific antibodies. Using this technique, we observed significant modulation of antibody glycans during viral infection as well as in vaccine-elicited antibodies. To identify specific signals important for altering the antibody glycan, we transcriptionally profiled stimulated B cells and identified a set of innate and adaptive stimuli that regulate the genes responsible for antibody glycosylation. The results described in this dissertation begin to define the specific mechanism(s) by which infection and vaccination modulate antibody glycosylation to elicit functional antibodies that can ultimately provide effective and sustained protection from infection.

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Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Introdução: A tuberculose (TB), doença crônica causada por Mycobacterium tuberculosis (Mtb), é uma das doenças infecciosas que mais acomete a população brasileira, com 2.832 óbitos em 2010. Na infecção causada pelo Mtb, a interação das células T com os macrófagos (MØs) infectados é fundamental na imunidade protetora contra o bacilo. A Mce1A é uma proteína da parede celular do Mtb que confere grande capacidade de aderência, invasão e sobrevivência em MØs. Contudo, a caracterização da proteína Mce1A pode fornecer um biomarcador para diagnóstico e monitoramento do tratamento. Nosso objetivo é avaliar a produção de IgG total anti-Mce1A em pacientes com TB e seus comunicantes domiciliares (CDTB). Material e métodos: Indivíduos diagnosticados com TB pulmonar e CDTB foram submetidos a coleta de sangue por punção venosa. O diagnóstico da TB foi baseado em quadro clínico e/ou radiografia sugestiva e/ou baciloscopia do escarro positiva. Nos CDTB, a infecção foi determinada a partir da reação do teste tuberculínico (TT) e avaliação radiográfica. Soro dos três grupos foi coletado e armazenado a -20ºC, até a determinação dos níveis de IgG total anti-Mce1A por meio de um ensaio imunoenzimático (ELISA). Resultados: Entre janeiro de 2012 e outubro de 2013 foram identificados 50 pacientes com TB pulmonar e 50 CDTB, dentre os quais 23 foram TT positivo e 27 foram TT negativo. A média de idade da população estudada foi de 37,8 anos (DP ± 20,4). O gênero masculino prevaleceu entre os pacientes com TB (68%), porém nos CDTB, o gênero feminino prevaleu (62%). A maioria dos indivíduos incluídos foram vacinados com BCG (71,4%). Cerca de 20% dos CDTB, TT negativo (18,5%) e TT positivo (20,8%), relataram que tiveram contato prévio com um doente com TB. Pacientes com TB tinham níveis de IgG anti-Mce1A (1,380±0,2950) estatisticamente maiores que indivíduos TT negativo (IgG anti-Mce1A: 1,049±0,2666). A diferença entre os grupos TT positivo (IgG anti-Mce1A 2.018±0.2807) e TT negativo (IgG anti-Mce1A: 1,049±0,2666) também foi estatisticamente diferente. Todos os grupos estudados tiverem diferença estatística na produção de anticorpos (p<0,0001). Não houve diferença estatística entre os pacientes com TB e indivíduos TT positivo (p>0,05). Conclusão: A produção de IgG total anti-Mce1A ocorreu em pacientes com TB e seus comunicantes domiciliares. Pacientes com TB e indivíduos TT positivo apresentam maior produção de anticorpos IgG total anti-Mce1A em comparação aos indivíduos TT negativo, sugerindo o potencial papel desta imunoglobulina como biomarcador de doença e de infecção tuberculosa. / Introduction: Tuberculosis (TB), a chronic disease caused by Mycobacterium tuberculosis (Mtb), is an infectious disease that affects the Brazilian population, with 2,832 deaths in 2010. In Mtb infection, the interaction of T cells with infected macrophages (MØs) is critical in protective immunity against the bacillus. The Mce1A is a cell wall protein of Mtb which gives great adhesion characteristics, invasion and survival in MØs. However, the characterization of protein Mce1A can provide a biomarker for diagnosis and monitoring of treatment. Our goal is to evaluate the production of total IgG anti-Mce1A TB patients and their household contacts (HHC). Material and Methods: Individuals diagnosed with pulmonary TB and HHC were subjected to blood collection by venipuncture. The diagnosis of TB was based on clinical and/or suggestive radiography and/or positive sputum smear. In HHC, infection was determined from the reaction of the tuberculin skin test (TST) and radiographic evaluation. Three groups of serum was collected and stored at -20 ° C until determination of the levels of anti-Mce1A total IgG by an enzyme-linked immunosorbent assay (ELISA). Results: Between January 2012 and October 2013, 50 patients were identified with pulmonary TB and 50 HHC, of which 23 were positive TST and 27 were negative TST. The average age of the study population was 37.8 years (SD ± 20.4). The males predominated among patients with TB (68%), but the HHC, the prevailed females (62%). Most individuals vaccinated with BCG were included (71.4%). About 20% of HHC, negative TST (18.5%) and positive TST (20.8%) reported that they had previous contact with a TB patient. TB patients had levels of IgG anti-Mce1A (1.380 ± 0.2950) statistically higher than individuals negative TST (anti-Mce1A IgG: 1.049 ± 0.2666). The difference between the positive TST groups (anti-Mce1A IgG 2.018 ± 0.2807) and negative TST (anti-Mce1A IgG: 0.2666 ± 1.049) was also statistically different. All groups had statistically difference in antibody production (p <0.0001). There was no statistical difference between patients with TB and positive TST individuals (p> 0.05). Conclusion: The production of anti-Mce1A total IgG occurred in TB patients and their household contacts. TB patients and TST positive individuals have a higher production of anti- Mce1A IgG antibodies overall compared to individuals negative TST, suggesting the potential role of immunoglobulin biomarker for TB disease and infection.