INTRODUÇÃO: Desde 1930, quando o primeiro caso de sensibilização alérgica a um veneno ofídico foi descrito na literatura, os venenos de todas as principais famílias de serpentes têm sido implicados como causadores de reações alérgicas. O desenvolvimento deste tipo de hipersensibilidade tem sido descrito após recorrentes exposições aos venenos através de picadas e, presumivelmente, de repetidas inalações ou contato desses venenos com a pele ou membranas mucosas. Esta condição tem sido observada entre os profissionais que manuseiam as serpentes e/ou os seus venenos. Entretanto, pouco se conhece a respeito da prevalência dessa doença entre esses trabalhadores, seus determinantes, e natureza molecular dos alérgenos envolvidos. Os objetivos deste estudo foram avaliar a prevalência da alergia ao veneno de Bothrops jararaca, apontando os fatores preditores para o seu desenvolvimento, entre trabalhadores expostos à serpente e/ou ao seu veneno, e demonstrar o envolvimento de um mecanismo IgE-mediado neste tipo de doença ocupacional. MÉTODOS: Sessenta e sete herpetologistas de um instituto de pesquisa, expostos à serpente Bothrops jararaca e/ou ao seu veneno, foram submetidos a um questionário e a testes imunológicos, para avaliar a presença de alergia ao veneno. A sensibilização ao veneno foi determinada pela quantificação de anticorpos da classe IgG e IgE, específicos ao veneno de Bothrops jararaca, através dos testes sorológicos ImmunoCAP e ELISA. Os alérgenos foram caracterizados através de Western blotting e de ensaios de inibição do ImmunoCAP. RESULTADOS: Doze dos 67 trabalhadores reportaram sintomas alérgicos variando desde urticária, rinoconjuntivite e asma, até anafilaxia. Sete indivíduos apresentaram anticorpos IgE específicos ao veneno de Bothrops jararaca (prevalência = 10,4%). Destes, seis apresentavam sintomas clássicos de reações alérgicas IgE-mediadas, quando expostos ao veneno botrópico. A presença de anticorpos IgG específicos também foi observada nos indivíduos com sensibilização alérgica ao veneno. O nível sérico elevado de IgE total (p=0,034), o tempo de exposição ocupacional (p=0,042), o manuseio de veneno em pó (p=0,014) e, possívelmente, a história pessoal de atopia (p=0,051), se constituíram em fatores preditores associados à sensibilização. Um componente do veneno de aproximadamente 32 kDa, que apresentou intensa ligação à IgE sérica de dois trabalhadores sensibilizados, pode ser um dos alérgenos principais desse veneno. Os ensaios de inibição do ImmunoCAP sugeriram a possibilidade de reações cruzadas ente os venenos de Bothrops jararaca e Crotalus durissus durissus. CONCLUSÕES: O veneno de Bothrops jararaca pode ser considerado uma importante fonte de alérgenos responsável pelo desenvolvimento de alergia ocupacional em trabalhadores que manuseiam as serpentes e/ou os seus venenos, através de mecanismo IgE-mediado. A prevalência da sensibilização alérgica a este veneno, neste grupo de trabalhadores, é da ordem de 10,4%, e o nível sérico elevado de IgE total, o tempo de exposição à serpente ou ao seu veneno, o manuseio do veneno em pó e, possívelmente, a história pessoal de atopia, são importantes fatores preditores. / BACKGROUND: Allergic sensitization to snake venom was first reported in 1930 and since that time, venoms from the major snake families have been implicated as cause of allergic reactions. The development of hypersensitivity to snake venom has been described after recurrent exposure through bites and, presumably, through repeated inhalations or repeated contact of the venom with skin or mucous membranes. This condition has been observed in amateur and professional snake handlers. However, only limited information is available on the prevalence of the disease among those workers, their determinants and the molecular nature of these allergens. The aim of this study was to evaluate the prevalence and the predictors of snake venom allergy among workers exposed to Bothrops jararaca venom and to demonstrate the involvement of IgE-mediated mechanisms in this occupational disease. METHODS: Sixty seven workers exposed to Bothrops jararaca snakes and/or their venoms in a research institute were assessed for snake venom-related allergy using questionnaires and immunological tests. Presence of snake venom sensitization was determined by quantification of specific IgE and IgG to Bothrops jararaca venom using the UniCAP® system and ELISA. Allergens were characterized by Western blotting and ImmunoCAP inhibition assays. RESULTS: Twelve of the 67 workers experienced symptoms ranging from urticaria, rhinoconjuctivitis and asthma to anaphylaxis. Seven individuals had specific IgE antibodies to Bothrops jararaca snake venom (prevalence = 10,4%). Six of them had typical symptoms of an IgE-mediated allergic reaction when exposed to Bothrops venom. Specific IgG antibodies could also be observed in sensitized individuals. High levels of total IgE (p=0,034), exposure level (p=0,042), handling of dried venom (p=0,014) and personal history of atopy (p=0,051) could be demonstrated as predictors for sensitization. A component of approximately 32 kDa which strongly bound to specific-IgE may be the major allergen present in this venom. Cross-reactivity between Bothrops jararaca snake venom and Crotalus durissus durissus snake venom was demonstrated using ImmunoCAP inhibition. CONCLUSIONS: Bothrops jararaca snake venom can be considered a potential source of allergens which may result in occupational allergy in snake handlers though IgE-mediated mechanisms. The prevalence rate of this condition is 10,4% among these workers and the handling of dried venom, high levels of total IgE, exposure level and probably personal history of atopy are important predictors.

Enzimas proteolíticas de origem vegetal têm sido amplamente empregadas em diversos estudos devido a sua capacidade de clivar anticorpos em fragmentos específicos, o que, para algumas aplicações, pode ser mais interessante do que a molécula de imunoglobulina como um todo. Além disso, elas podem ser utilizadas diretamente no paciente no papel de um \"fármaco\" (Há exemplos da aplicação de enzimas no estudo de várias patologias como: doenças auto-imunes, traumáticas ou neurológicas), no auxílio de técnicas clínicas para a detecção de anticorpos e como ferramentas para a produção de soros. Apesar de a utilização dessas enzimas ter grande importância e extensa aplicabilidade, o modo de ação das proteases ainda não é bem compreendido, entretanto, as mesmas são de fácil acesso e largamente encontradas na natureza. A ficina (encontrada nas figueiras) e bromelina (proveniente do abacaxi) são, por exemplo, enzimas que vem sendo exploradas em diversos trabalhos e demostram capacidade de digerir anticorpos. Neste projeto, propusemos verificar a atividade destas enzimas sobre anticorpos provenientes de equinos produção de fragmentos imunorreativos de imunoglobulina a partir de proteases de origem vegetal, diminuindo o risco potencial de transmissão de doenças de origem animal. Espera-se que os fragmentos gerados possam contribuir para diversos estudos de ordem clínica e para a produção de soros adequados contra as toxinas presentes nos venenos de animais. As amostras de soro foram obtidas a partir da imunização dos cavalos pertencentes ao Instituto Butantan, com \"pool\" de veneno de serpente do gênero Bothrops. Esses anticorpos foram purificados utilizando ácido caprílico, digeridos por bromelina ou ficina e diafiltrados. Os resultados obtidos indicam a formação do fragmento de imunoglobulina F(ab)\'2, como proposto no projeto, entretanto necessitando ainda de um delineamento experimental mais amplo a fim de otimizar o processo de digestão atingindo uma maior quantidade de fragmentos F(ab)\'2. / Proteolytic enzymes from vegetal origin have been largely employed in several studies due to its capacity to cleave antibodies into specific fragments, which for some applications, could be more relevant than the immunoglobulin molecule itself. Beyond that, they could be used directley into a patient playing the role of a \"drug\" (There are examples of enzyme application in studies of several pathologies such as autoimmune, traumatic and neurological diseases), in the aid of clinical techniques for antibodies detection and as tools for serum production. Despite the fact that those enzymes have been of great importance and large applicability, its ease of access and ubiquity in nature, the protease mode of action is still not fully understood. Ficin (found in Fig trees) and Bromelain (found in pineapples) are, for example, enzymes that are being explored in an array of articles and demonstrate the capacity to digest antibodies. In this project, we propose to verify the activity of such enzymes from vegetal origins over the antibodies obtained from equines in the production of immunoreactive fragments of immunoglobulin, lowering the potential risk of animal origin diseases transmission. It is hoped that the fragments generated can contribute to studies of clinical level and the production of proper serum against toxins present in animal poisons. Plasma samples were obtained from horse immunization with a venon \"pool\" from Bothrops snakes at Instituto Butantan. These antibodies were purified using caprilic acid, digested by bromelain or ficin and purified again using chromatographic techniques. The results indicate the formation of an immunoglobulin fragment - F(ab)\'2, however, more experiments are needed to optimize the digestive process in order to improve the reaction yield.

Neste trabalho, anticorpos anti-IgGh foram conjugados às nanopartículas de prata (NPAg) para detectar imunoglobulina G humana (IgGh). Um imunoensaio colorimétrico baseado na diminuição da agregação devido ao aumento da repulsão eletrostática após a interação ligante-alvo. A agregação é induzida pela variação da força iônica e uma mudança da coloração da suspensão coloidal de amarelo para vermelho pode ser observada. Na presença de IgGh, a agregação é inibida e a coloração da suspensão coloidal não se altera. As nanopartículas foram obtidas por meio de cinco procedimentos diferentes e caracterizadas por espectroscopia UV-Vis, espalhamento dinâmico de luz, difração de raios-X e microscopia eletrônica. Glicose e borohidreto de sódio foram utilizados como agentes redutores, enquanto CTAB e β-ciclodextrina foram utilizados como estabilizantes. Citrato de sódio foi utilizado como agente redutor e/ou estabilizante. Nanoesferas de carbono foram obtidas por tratamento hidrotérmico de uma solução aquosa de glicose e também foram utilizadas no preparo das nanopartículas. As nanopartículas foram funcionalizadas com ácido mercaptossuccínico e a conjugação ocorreu devido à interação entre grupos aminas e grupos carboxílicos ionizados, presentes no anticorpo e agente de acoplamento, respectivamente. A estabilidade dos conjugados e o efeito da adição de IgGh foram avaliados para todos os sistemas preparados. As nanopartículas de prata preparadas com borohidreto de sódio e citrato de sódio foram selecionadas para serem aplicadas no desenvolvimento do imunoensaio e as condições experimentais foram avaliadas. Em condições ótimas, observou-se uma correlação linear entre a diminuição da agregação do sistema (NPAg-anti-IgGh) e a concentração de IgGh (0 a 200 ng mL-1). O limite de detecção foi estimado em 25 ng mL-1. O método colorimétrico apresentou boa seletividade para a detecção de IgGh. Além disso, foi obtido um resultado satisfatório ao aplicar o método para determinação do fator IX de coagulação. Foi desenvolvido também um método para determinação de ATP baseado na agregação de nanopartículas de ouro. Aptâmeros foram utilizados como elemento de reconhecimento. Em princípio, o método pode ser aplicável à determinação de outros analitos, por meio da substituição do aptâmero utilizado neste trabalho pelo oligonucleotídeo específico para o alvo de interesse. / In this work, antibodies to human immunoglobulin G (anti-IgGh) were used in combination with silver nanoparticle (NPAg) to detect IgGh. A colorimetric immunoassay based on the decrease of aggregation due to increased electrostatic repulsion upon ligand-target interaction. Aggregation was induced by varying the ionic strength and the solution of NPAg-anti-IgGh shows obvious visible color change from yellow to red. In the presence of IgGh aggregation the nanoparticle is inhibited and coloration of the colloidal solution does not change. The nanoparticles were obtained using five different procedures and they were characterized by UV-Vis spectroscopy, dynamic light scattering, X-ray diffraction and electron microscopy. Glucose and sodium borohydride were used as reducing agent, as CTAB and β-cyclodextrin reagents were used as stabilizers. Sodium citrate was used as reducing and stabilizing agent. Carbon nanospheres were prepared by hydrothermal treatment of glucose and used in the preparation of NPAg. The nanoparticles were functionalized with dimercaptosuccinic acid and their conjugation occurred due to the interaction of positively charged amine groups and anionic groups (-COO-) present on the antibody and coupling agent. The stability of conjugates and the variation of aggregation in the presence of IgGh were evaluated for all systems. The NPAg prepared by sodium borohydride were selected for use in the immunoassay and the optimum conditions of the assay were investigated. Under the optimal conditions, the ration between the absorbance at 396 nm and 564 nm was linearly proportional to the IgGh concentration on a range from 0 to 200 ng mL-1, with a detection limit of 25 ng mL-1. The colorimetric method showed good selectivity for IgGh detection. It was possible to adapt the method to the determination of other proteins, such as factor IX. In another approach, anti-aptamer ATP was used to develop a colorimetric method for the determination of ATP based on stabilization of gold nanoparticles provided by strands of DNA.The strategy was based on stabilization of nanoparticles due to interaction with single strands of DNA, and the change of the stability of the nanoparticles provided by the conformational change of the aptamer following recognition. This method could in principle be used to detect analytes by substituting the aptamer used in this study by the specific aptamer for the target of interest

by Shiu Kar Chi. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 61-67). / Abstract also in Chinese. / ACKNOWLEDGEMENTS / ABSTRACT / LIST OF TABLES / LIST OF FIGURES / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Overview --- p.2 / Chapter 1.2 --- Historical perspective --- p.2 / Chapter 1.3 --- Biochemistry of the IgG subclasses --- p.4 / Chapter 1.4 --- IgG subclasses and human diseases --- p.7 / Chapter 1.4.1 --- Glomerulonephritis --- p.7 / Chapter 1.4.2 --- Blistering skin lesions --- p.7 / Chapter 1.4.3 --- Insulin dependent diabetes mellitus (IDDM) --- p.8 / Chapter 1.5 --- Primary antibody deficiencies --- p.8 / Chapter 1.5.1 --- CVID --- p.8 / Chapter 1.5.2 --- X-linked antibody deficiency --- p.8 / Chapter 1.5.3 --- IgG subclass deficiency --- p.10 / Chapter 1.5.4 --- Specific antibody deficiencies --- p.10 / Chapter 1.5.5 --- Selective IgA deficiency --- p.10 / Chapter 1.6 --- IgG subclasses deficiency --- p.10 / Chapter 1.7 --- Clinical manifestation of IgG subclass deficiency --- p.11 / Chapter 1.8 --- Restriction of IgG subclass responses to exogenous antigens --- p.12 / Chapter 1.9 --- Expression of IgG subclasses --- p.14 / Chapter 1.10 --- Mechanisms of IgG subclass deficiency --- p.14 / Chapter 1.10.1 --- Gene deletion --- p.14 / Chapter 1.10.2 --- Immune dysregulation --- p.17 / Chapter 1.10.2.1 --- T-cell receptor defects --- p.18 / Chapter 1.10.2.2 --- Interferon gamma (IFN-y) --- p.18 / Chapter 1.10.2.3 --- Interleukin-4 (IL-4) --- p.19 / Chapter 1.10.2.4 --- Interleukin-6 (IL-6) --- p.19 / Chapter 1.11 --- Prevalence of IgG subclass deficiency --- p.19 / Chapter 1.12 --- Reference intervals for IgG subclass --- p.20 / Chapter 1.13 --- Methods for investigation of IgG subclass deficiency --- p.20 / Chapter 1.13.1 --- Radial-immunodiffusion --- p.21 / Chapter 1.13.2 --- Enzyme linked immunsorbent assay --- p.21 / Chapter 1.13.3 --- Nephelometry/turbidmetry --- p.21 / Chapter 1.14 --- Aim of study --- p.22 / Chapter CHAPTER 2 --- MATERIALS AND METHOD I The Binding Site IgG Subclass Assay --- p.23 / Chapter 2.1 --- Materials --- p.24 / Chapter 2.1.1 --- IgG subclass assay --- p.24 / Chapter 2.1.2 --- Evaluation of patients immune status --- p.24 / Chapter 2.1.3 --- Apparatus and equipment --- p.24 / Chapter 2.2 --- Evaluation of The Binding Site human IgG subclass assay on Beckman Array 360 protein system --- p.25 / Chapter 2.2.1 --- Principle of the Beckman Array Protein System --- p.25 / Chapter 2.2.2 --- Assay preparation and procedure --- p.25 / Chapter 2.2.3 --- Performance characteristic of the IgG subclasses assay --- p.28 / Chapter 2.2.3.1 --- Gain setting --- p.28 / Chapter 2.2.3.2 --- Within batch precision --- p.28 / Chapter 2.2.3.3 --- Interassay precision --- p.28 / Chapter 2.2.3.4 --- Linearity of the assay --- p.29 / Chapter 2.2.3.5 --- Interference of the IgG subclass assay --- p.29 / Chapter 2.2.3.6 --- Recovery experiment --- p.30 / Chapter CHAPTER 3 --- MATERIALS AND METHOD II IgG Subclass Deficiency in Hong Kong --- p.32 / Chapter 3.1 --- Patients and controls --- p.33 / Chapter 3.2 --- Blood samples --- p.33 / Chapter 3.3 --- Serum total haemolytic complement and alternative pathway haemolytic complement assay --- p.34 / Chapter 3.3.1 --- Total haemolytic complement --- p.34 / Chapter 3.3.2 --- Alternative pathway haemolytic complement --- p.34 / Chapter 3.4 --- Statistical analysis --- p.35 / Chapter CHAPTER 4 --- RESULTS I: Evaluation of The Binding Site IgG Subclass Array Kit --- p.36 / Chapter 4.1 --- Gain setting --- p.37 / Chapter 4.2 --- Within batch precision --- p.37 / Chapter 4.3 --- Inter-assay precision --- p.37 / Chapter 4.4 --- Linearity and lowest limit of detection --- p.37 / Chapter 4.5 --- Interference experiments --- p.37 / Chapter 4.6 --- Recovery experiment --- p.37 / Chapter CHAPTER 5 --- RESULTS II: IgG Subclass Deficiency in Hong Kong --- p.45 / Chapter 5.1 --- IgG subclass concentrations and humoral immune status evaluation results of patients and control subjects --- p.46 / Chapter 5.2 --- Statistical tests --- p.45 / Chapter CHAPTER 6 --- DISCUSSION I: The Binding Site IgG Subclass Array Kit --- p.52 / Chapter 6.1 --- IgG subclass assays --- p.53 / Chapter 6.2 --- Within batch and inter-assay precision --- p.53 / Chapter 6.3 --- Lowest limit of detection --- p.53 / Chapter 6.4 --- Interference --- p.54 / Chapter 6.5 --- Recovery of IgG --- p.54 / Chapter 6.6 --- Overall performance of the nephelometric assay --- p.55 / Chapter CHAPTER 7 --- DISCUSSION II: IgG Subclass Deficiency in Hong Kong --- p.56 / Chapter 7.1 --- IgG subclass deficiency in adults --- p.57 / Chapter 7.2 --- Paetiatric patients --- p.59 / Chapter 7.3 --- Recurrent infections and IgG subclass deficiency --- p.59 / Chapter 7.4 --- Summary --- p.60 / REFERENCES --- p.61

Enzimas proteolíticas de origem vegetal têm sido amplamente empregadas em diversos estudos devido a sua capacidade de clivar anticorpos em fragmentos específicos, o que, para algumas aplicações, pode ser mais interessante do que a molécula de imunoglobulina como um todo. Além disso, elas podem ser utilizadas diretamente no paciente no papel de um \"fármaco\" (Há exemplos da aplicação de enzimas no estudo de várias patologias como: doenças auto-imunes, traumáticas ou neurológicas), no auxílio de técnicas clínicas para a detecção de anticorpos e como ferramentas para a produção de soros. Apesar de a utilização dessas enzimas ter grande importância e extensa aplicabilidade, o modo de ação das proteases ainda não é bem compreendido, entretanto, as mesmas são de fácil acesso e largamente encontradas na natureza. A ficina (encontrada nas figueiras) e bromelina (proveniente do abacaxi) são, por exemplo, enzimas que vem sendo exploradas em diversos trabalhos e demostram capacidade de digerir anticorpos. Neste projeto, propusemos verificar a atividade destas enzimas sobre anticorpos provenientes de equinos produção de fragmentos imunorreativos de imunoglobulina a partir de proteases de origem vegetal, diminuindo o risco potencial de transmissão de doenças de origem animal. Espera-se que os fragmentos gerados possam contribuir para diversos estudos de ordem clínica e para a produção de soros adequados contra as toxinas presentes nos venenos de animais. As amostras de soro foram obtidas a partir da imunização dos cavalos pertencentes ao Instituto Butantan, com \"pool\" de veneno de serpente do gênero Bothrops. Esses anticorpos foram purificados utilizando ácido caprílico, digeridos por bromelina ou ficina e diafiltrados. Os resultados obtidos indicam a formação do fragmento de imunoglobulina F(ab)\'2, como proposto no projeto, entretanto necessitando ainda de um delineamento experimental mais amplo a fim de otimizar o processo de digestão atingindo uma maior quantidade de fragmentos F(ab)\'2. / Proteolytic enzymes from vegetal origin have been largely employed in several studies due to its capacity to cleave antibodies into specific fragments, which for some applications, could be more relevant than the immunoglobulin molecule itself. Beyond that, they could be used directley into a patient playing the role of a \"drug\" (There are examples of enzyme application in studies of several pathologies such as autoimmune, traumatic and neurological diseases), in the aid of clinical techniques for antibodies detection and as tools for serum production. Despite the fact that those enzymes have been of great importance and large applicability, its ease of access and ubiquity in nature, the protease mode of action is still not fully understood. Ficin (found in Fig trees) and Bromelain (found in pineapples) are, for example, enzymes that are being explored in an array of articles and demonstrate the capacity to digest antibodies. In this project, we propose to verify the activity of such enzymes from vegetal origins over the antibodies obtained from equines in the production of immunoreactive fragments of immunoglobulin, lowering the potential risk of animal origin diseases transmission. It is hoped that the fragments generated can contribute to studies of clinical level and the production of proper serum against toxins present in animal poisons. Plasma samples were obtained from horse immunization with a venon \"pool\" from Bothrops snakes at Instituto Butantan. These antibodies were purified using caprilic acid, digested by bromelain or ficin and purified again using chromatographic techniques. The results indicate the formation of an immunoglobulin fragment - F(ab)\'2, however, more experiments are needed to optimize the digestive process in order to improve the reaction yield.

No interesse de reduzir os efeitos adversos relacionados às vacinas veterinárias e induzir tipos específicos de resposta imune, levou-se o desenvolvimento de numerosos novos adjuvantes. Recentemente, houve o descobrimento de um peptídeo intracelular funcional obtido através do estímulo celular com INF- γ, denominado EL28, tendo como principais características a hidrossolubilidade e a biodegradebilidade e, porém, no momento carece de ensaios de imunoestimulação associados com vacinas com potencial de mercado \"in vivo\". Neste sentido, o presente estudo teve como objetivo verificar a atividade imunoestimulante do EL28, associado ao vírus rábico inativado (VRI). Realizou-se a produção do antígeno VRI, e prepararam-se formulações vacinais: VRI associado ao EL28, VRI associado com Al(OH)3 e VRI sem presença de adjuvante, para imunização de camundongos Balb/c e avaliação da resposta imune humoral. A produção de anticorpos IgG totais, IgG1 e IgG2a foram determinados pelo ensaio de ELISA indireto. E os anticorpos neutralizantes para o vírus rábico foram quantificados através do método RIFFT. Os resultados do ELISA indireto foram expressos como Dose Imunoenzimática 50% (DI50) e mostraram que não houve aumento na produção de anticorpos na resposta imune humoral (IgG totais) entre a vacina associada ao EL28 (DI50= 3,0 X 10-3µL) e a vacina sem adjuvante (DI50=2,0 X 10-3µL). As IgG2a e IgG1 estão relacionados ao perfil da resposta Th1 e Th2 respectivamente, e mostraram diferença significativa em relação ao título da IgG1 entre a vacina associada ao EL28 (DI50= 5,9x10-4µL) e a vacina sem adjuvante (DI50=5,2x10-4µL), enquanto para os títulos de IgG2a não houve diferença significativa entre essas vacinas. O perfil da resposta Th foi obtido através da razão entre IgG2a e IgG1, o qual demonstrou que em todas as imunizações, com e sem adjuvantes, tiveram resposta predominante do tipo Th2. O método de RIFFT mostrou um aumento na potência de anticorpos neutralizantes na vacina associada ao EL28 em relação à imunização dos animais sem nenhum adjuvante. Portanto, esses resultados demonstram aumento da especificidade na resposta imunológica da vacina associada ao EL28. Sendo assim, pelos resultados apresentados, a vacina de raiva associada ao EL28 possivelmente teria uma maior eficiência e segurança. / In the interest of reducing the adverse effects related to veterinary vaccines and inducing specific types of immune response, numerous new adjuvants have been developed. Recently, a functional intracellular peptide obtained through cellular stimulus with the INF-γ, named EL28, it have as main characteristics the water solubility and the biodegradability, but at the moment it lacks immunostimulation assays associated with potencial market vaccine \"in vivo\". In this sense, the present study aimed to verify the immunostimulatory activity of EL28, associated with inactivated rabies virus (IRV). It carried out the production of IRV antigen, and prepared vaccine formulations: IRV with EL28, IRV with Al(OH)3 and IRV without the presence of adjuvant, for immunization of Balb/c mice and evaluation of the humoral immune response. The production of total IgG antibodies, IgG1 and IgG2a were determined by the indirect ELISA assay. And neutralizing antibodies to rabies virus were quantified using the RIFFT method. Indirect ELISA results were expressed as 50% Immunoenzymatic Dose (ID50) and showed no increase in antibody humoral immune response (IgG total) between the EL28 associated vaccine (ID50 = 3.0 X 10-3µL) and the vaccine without adjuvant (ID50 = 2.0 X 10-3µL). The IgG2a and IgG1 are related to the profile of Th1 and Th2 respectively, and showed a significant difference in relation to the IgG1 titer between the EL28 associated vaccine (ID50 = 5,9x10-4µL) and the non-adjuvant vaccine (ID50 = 5,2x10-4µL), while for IgG2a titers there was no significant difference between these vaccines. The Th response profile was obtained by the ratio of IgG2a and IgG1, which showed that in all immunizations with and without adjuvant, were predominantly Th2 response. The RIFFT method showed an increase of the potency of neutralizing antibodies in the vaccine with EL28 in relation to the immunization of the animals without any adjuvant. Therefore, these results demonstrate increased specificity in the immune response associated with EL28 vaccine. Therefore, from the results presented, the rabies vaccine associated with EL28 would possibly have a greater efficiency and safety.

Imunodeficiência Comum Variável (ICV) é uma imunodeficiência primária de igual distribuição entre os sexos e que afeta crianças e adultos, caracterizada por hipogamaglobulinemia com susceptibilidade aumentada a infecções e ampla variedade de complicações não infecciosas, como autoimunidade, malignidade, hiperplasia linfoide, doenças gastrointestinais, dentre outras. Os objetivos deste estudo foram: avaliar as manifestações clínicas, infecciosas e não infecciosas, mais frequentes em portadores de ICV (antes e após início da terapia com reposição de imunoglobulina humana) acompanhados em ambulatórios de imunologia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (HCFMRP-USP), além dos níveis séricos de imunoglobulinas (IgG, IgA e IgM) ao diagnóstico, bem como as alterações quantitativas de células CD19+, CD3+CD4+, CD3+CD8+ e CD3-CD16+CD56+ desses pacientes. Neste estudo descritivo foram obtidas informações de pacientes com ICV acompanhados no HCFMRPUSP, através de registros de prontuários médicos. Foram avaliados 32 pacientes: 19 do sexo masculino e 13 do sexo feminino. A mediana da idade de início dos sintomas foi de 8,5 anos, com um pico de incidência precoce. O tempo médio de atraso para o diagnóstico foi de 7,7 anos. Todos os pacientes apresentaram infecções recorrentes, que levaram ao diagnóstico da ICV. As infecções mais frequentes foram as respiratórias, sendo que antes do diagnóstico as pneumonias foram as mais observadas (gerando, inclusive, grande número de internações) e durante o primeiro ano de uso regular da terapia de reposição com imunoglobulina humana as rinossinusites foram as que mais ocorreram. Houve redução na incidência de infecções após início do tratamento. Todos os pacientes apresentaram níveis séricos de IgG, IgA e IgM reduzidos ao diagnóstico, sendo que as medianas dos níveis séricos foram de 158 mg/dL, 10,15 mg/dL e 17 mg/dL, respectivamente. De 30 pacientes que haviam realizado imunofenotipagem, cerca de 73% apresentaram número absoluto reduzido de células CD19+ e 40% apresentaram número absoluto reduzido de linfócitos T CD4+. A relação CD4/CD8 foi invertida em aproximadamente 53% dos pacientes. Em 18 pacientes as células natural killer foram quantificadas e cerca de 56% deles apresentaram número absoluto reduzido. A maioria (97%) dos pacientes manifestou, no mínimo, uma comorbidade não infecciosa no tempo médio de seguimento de 8,2 anos, sendo que hiperplasia linfoide e doença pulmonar crônica foram as mais frequentes, cada uma ocorrendo em cerca de metade dos pacientes. O atraso para o diagnóstico da ICV foi importante, sugerindo que a presença de infecções recorrentes, especialmente do trato respiratório, deveria levar à investigação de deficiências de anticorpos, com dosagem de imunoglobulinas. Complicações não infecciosas foram extremamente comuns nesta casuística, ressaltando o amplo espectro clínico da doença. / Common variable immunodeficiency (CVID) is a primary immunodeficiency that is equally distributed between men and women and affects children and adults, characterized by hypogammaglobulinemia with increased susceptibility to infections and a wide variety of noninfectious complications such as autoimmunity, malignancy, lymphoid hyperplasia, gastrointestinal diseases, among others. The purposes of this study were to evaluate infectious and non-infectious clinical manifestations (before and after immunoglobulin replacement therapy) of CVID patients attended at immunology outpatient clinics of the Clinical Hospital of Ribeirão Preto Medical School - University of São Paulo (HCFMRP-USP), in addition to immunoglobulins (IgG, IgA and IgM) serum levels at diagnosis, as well as quantitative differences in CD19+, CD3+CD4+, CD3+CD8+ and CD3-CD16+CD56+ cells. In this descriptive study, data of CVID patients followed up at HCFMRP-USP were collected through medical records. Thirty-two patients were found: 19 males and 13 females. The median age of onset of symptoms was 8.5 years, with an early peak of incidence. The mean delay for diagnosis was 7.7 years. All patients had recurrent infections, which led to the diagnosis of CVID. The most frequent infections were respiratory tract infections. Pneumonias were more observed before the diagnosis (generating a large number of hospitalizations) and rhinosinusitis were more frequent during the first year under regular use of immunoglobulin replacement therapy. There was a reduction in the incidence of infections after initiation of treatment. All patients had low IgG, IgA and IgM serum levels (lower than the 3th percentile for age) at diagnosis and the median of serum levels were 158 mg/dL, 10.15 mg/dL and 17 mg/dL, respectively. Among 30 patients that had been immunophenotyped, approximately 73% had a reduced absolute number of CD19+ cells and 40% had a reduced absolute number of T CD4+ lymphocytes. The CD4/CD8 ratio was inverted in approximately 53% of the patients. Natural killer cells were quantified in 18 patients and about 56% of them had reduced absolute number. The majority (97%) of patients manifested at least one noninfectious comorbidity at a mean follow-up time of 8.2 years, with lymphoid hyperplasia and chronic lung disease being the most common, each occurring in about half of the patients. The delay for the diagnosis of CVID was important, suggesting that the presence of recurrent infections, especially of the respiratory tract, should lead to the investigation of antibody deficiencies with dosage of immunoglobulins. Noninfectious complications were extremely common in this series, highlighting the broad clinical spectrum of the disease.

Intravenous immunoglobulin (IVIG) is a common treatment for the neurological autoimmune diseases multiple sclerosis, multifocal motor neuropathy, myasthenia gravis, and chronic inflammatory demyelinating polyneuropathy. However, there is scant literature regarding the psychological effects of this treatment on quality of life (QOL). Using illness intrusiveness theory and personal control theory, this correlational, cross-sectional study examined the relationship between the IVIG treatment experience and QOL in neurological autoimmune patients. Surveys were employed to collect data from 79 patients at a neurological infusion center in Phoenix, AZ. Quantitative analyses included correlation, multiple regression, and mediation analyses to determine whether (a) IVIG treatment experience predicted QOL measured by 10 Neuro-QOL scales, (b) illness intrusiveness mediated the relationship between IVIG treatment experience and QOL, and (c) personal control mediated the relationship between illness intrusiveness and QOL. IVIG treatment experience predicted QOL in 1 Neuro-QOL subscale; illness intrusiveness mediated 9 of the Neuro-QOL subscales using bias-corrected bootstrapping for statistical significance; and personal control did not mediate the relationship between illness intrusiveness and QOL. These results may affect social change by increasing the understanding of physicians, nurses, and patients regarding the psychosocial impact of IVIG treatment. Results from the study may provide insight for interventions to assist patients in adjusting to this form of treatment.

L’immunoglobuline A (IgA) est l’immunoglobuline la plus abondamment synthétisée chez les mammifères. Ses propriétés ambivalentes l’impliquent non seulement dans des fonctions de protection contre les agents pathogènes mais aussi dans des phénomènes de tolérance immunitaire vis-à-vis des germes commensaux du microbiote. Toutefois, les IgA peuvent développer des propriétés pathogènes. Dans la première partie de mon travail de thèse, nous avons étudié les effets pathogènes de l’IgA. Les dépôts d’IgA sur le mésangium sont la caractéristique de l’IgAN. La physiopathologie de cette maladie est mal connue. L’hypothèse d’un défaut de glycosylation de l’IgA est souvent retenue ; ce défaut peut être la cause de sa polymérisation et de son antigénicité, il peut aussi favoriser le clivage du récepteur CD89. Nous avons analysé l’effet du défaut d’affinité de la région variable des IgA, de la substitution de la chaîne légère ainsi que de l’association des IgA à leur récepteur, le CD89 sur l’induction des lésions et le dysfonctionnement rénal chez quatre modèles murins différents générés au laboratoire et suivis pendant 12 mois. Nous avons également étudié les propriétés physico-chimiques des IgA de 28 patients ayant une dysglobulinémie et de 28 IgA produites par des hybridomes ; la relation entre ces propriétés et la capacité des IgA à se déposer a été observée. Dans une seconde partie, nous avons étudié l’aspect immunomodulateur et les propriétés antiinflammatoires conférées par l’IgA humaine surexprimée chez un modèle murin de lupus systémique (souris MRL/lpr). Dans la dernière partie du travail, nous avons contribué à la caractérisation d’un modèle de souris transgénique exprimant l’IgA de classe 2 et à l’étude de l’effet de signalisation médiée par cette IgA2 sur le développement des populations lymphocytaires. L’ensemble de ces travaux a montré l’effet pathogène des IgA naturelles ayant une faible affinité sur le développement de la néphropathie à IgA ; ainsi les analyses des IgA des patients et des hybridomes montrent que c’est la stabilité moléculaire de préférence au profil de glycosylation qui joue un rôle crucial dans leur capacité de dépôt. L’expression des IgA humaines chez les souris lupiques a considérablement prolongé leur durée de vie et a ralenti la survenue de l’auto-immunité et de l’atteinte rénale ce qui témoigne du rôle anti-inflammatoire des IgA. L’étude du modèle murin exprimant l’IgA2 humaine a montré que la signalisation via l’IgA2 joue un rôle inhibiteur sur le développement précoce de certaines sous-populations de cellules B. L’ensemble de ces résultats montrent la multitude d’effets de l’IgA lui permettant d’intervenir d’une part dans la pathogenèse d’une maladie complexe (l’IgAN) et d’autre part dans la protection de l’auto-immunité, témoignant de la complexité des interactions mises en jeu et du caractère régulateur de cette immunoglobuline. / Immunoglobulin A (IgA) is the most synthetized immunoglobulin in mammals. IgA has ambivalent properties: it is implicated in the mechanisms of defense against pathogens but also in the immune tolerance of commensal microbiota. However, IgA can develop pathogenic properties. In the first part of my thesis, we studied the pathogenic effects of IgA. IgA deposits are the main characteristic of IgA nephropathy (IgAN). IgAN physiopathology is not yet clearly understood. The hypothesis of a glycosylation defect is strongly adapted. This defect can be due to IgA polymerization or antigenicity. It can also induce shedding of CD89 (IgA Fc receptor) or other factors. We studied the effect of variable region altered affinity, the light chain substitution and the association of IgA with CD89 on the development of kidney lesions and impairment of kidney function in four mouse models followed up during 12 months. In addition, we studied the physico-chemical properties of 28 IgA purified from patients with dysglobulinaemia and 28 chimeric IgA produced by hybridomas. The effect of these properties on the propensity of IgA for mesangial deposition was explored. In the second part, we studied the immunomodulatory and anti-inflammatory properties conferred by the overexpression of human IgA in a mouse model with systemic lupus (MRL/lpr model). In the last part, we contributed to the characterization of a transgenic mouse model producing IgA class 2 and to the study of the effect of IgA2-mediated signaling on B lymphocyte development. Altogether, obtained results show the pathogenic effect of low affinity-IgA on the development of IgA nephropathy. In addition, different analyses showed that molecular stability but not glycosylation profile is the determining factor for IgA deposition. On the other hand, IgA expression in lupus-prone mice extended their survival, delayed the onset of auto-immunity and ameliorated kidney functions in these animals which supports IgA anti-inflammatory properties. The study of IgA2-mediated signaling in the transgenic model showed the inhibitory effect of IgA2 on the early development of several B cell sub-populations. All of these results show the multiple effects of IgA which contribute on one hand to the pathogenesis of a complex disease (IgAN) and on the other hand to protection from autoimmunity, demonstrating the complexity of interactions and the regulatory character of this immunoglobulin.

Altered signaling pathways, which are mediated by post-translational modifications and changes in protein expression levels, are key regulators of cancer initiation, progression, and therapeutic escape. Many aspects of cancer progression, including early carcinogenesis and immediate response to drug treatment, are beyond the scope of genetic profiling and non-invasive monitoring techniques. Global protein profiling of cancer cell line models, tumor tissues, and biofluids (e.g. serum or urine) using mass spectrometry-based proteomics produces novel biological insights, which support improved patient outcomes. Recent technological advances resulting in next-generation mass spectrometry instrumentation and improved bioinformatics workflows have led to unprecedented measurement reproducibility as well as increased depth and coverage of the human proteome. It is now possible to interrogate the cancer proteome with quantitative proteomics to identify prognostic cancer biomarkers, stratify patients for treatment, identify new therapeutic targets, and elucidate drug resistance mechanisms. There are, however, numerous challenges associated with protein measurements. Biological samples have a high level of complexity and wide dynamic range, which is even more pronounced in samples used for non-invasive disease monitoring, such as serum. Cancer biomarkers are generally found in low abundance compared to other serum proteins, particularly at early stages of disease where cancer detection would make the biggest impact on improving patient survival. Additionally, the large-scale datasets generally require bioinformatics expertise to produce useful biological insights. These difficulties converge to create obstacles for down-stream clinical translation. This dissertation research demonstrates how proteomics is applied to develop new resources and generate novel workflows to improve protein quantification in complex biosamples, which could improve translation of cancer research to benefit patient care. The studies described in this dissertation move from assessment of quantitative mass spectrometry platforms, to analytical assay development and validation, and ending with personalized biomarker development applied to patient samples. As an example, four different quantitative mass spectrometry acquisition platforms are explored and comparisons of their ability to quantify low abundance peptides in a complex background are explored. Lung cancers frequently have aberrant signaling resulting in increased kinase activity and targetable signaling hubs; kinase inhibitors have been successfully developed and implemented clinically. Therefore, changes in amounts of kinase peptides in the complex background of peptides from all ATP-utilizing enzymes in a lung cancer cell line model after kinase inhibitor treatment was selected as a model system. Traditional mass spectrometry platforms, data dependent acquisition and multiple reaction monitoring, are compared to the two newer methods, data independent acquisition and parallel reaction monitoring. Relative quantification is performed across the four methods and analytical performance as well as downstream applications, including drug target identification and elucidation of signaling changes. Liquid chromatography – multiple reaction monitoring (LC-MRM) was selected for development of multiplexed quantitative assays based on superior sensitivity and fast analysis times, allowing for larger peptide panels. Method comparison results also provide guidelines for quantitative proteomics platform selection for translational cancer researchers. Next, a multiplexed quantitative LC-MRM assay targeting a panel of 30 RAS signaling proteins was developed and described. Over 30% of all human cancers have a RAS mutation and these cancers are generally aggressive and limited treatment options, leading to poor patient prognosis. Many targeted inhibitors have successfully shut down RAS signaling, leading to tumor regression, however, acquired drug resistance is common. The multiplexed LC-MRM assays characterized and validated are a publically available resource for cancer researchers to interrogate the RAS signal transduction network. Feasibility has been demonstrated in cell line models in order to identify signaling changes that confer BRAF inhibitor resistance and biomarkers of sensitivity to treatment. This analytical LC-MRM panel could support meaningful development of new therapeutic options and identification of companion biomarkers, with the end goal of improving patient outcomes. Multiplexed LC-MRM assays developed for personalized disease biomarkers using an integrated multi-omics approach are described for Multiple Myeloma, an incurable malignancy with poor patient outcomes. This disease is characterized by clonal expansion of the plasma cells in the bone marrow, which secrete a monoclonal immunoglobulin, or M-protein. Clinical treatment decisions are based on multiple semi-quantitative assays that require manual evaluation. In the clinic, minimal residual disease quantification methods, including multi-parameter flow cytometry and immunohistochemistry, are applied to bone marrow aspirates, which is a highly invasive technique that does not provide a systemic evaluation of the disease. To address these issues, we hypothesized that unique variable region peptides could be identified and LC-MRM assays developed specific to each patient’s M-protein to improve specificity and sensitivity in non-invasive disease monitoring. A proteogenomics approach was used to design personalized assays for each patient to monitor their disease progression, which demonstrate improved specificity and up to a 500-fold increase in sensitivity compared to current clinical methods. Assays can be developed from marrow aspirates collected when the patient was at residual disease stage, which is useful if no sample with high disease burden is available. The patient-specific tests are also multiplexed with constant region peptide assays that monitor all immunoglobulin heavy and light chain classes, which could reduce analysis to a single test. In conclusion, highly sensitive patient-specific assays have been developed that could change the paradigm for patient evaluation and clinical decision-making, increasing the ability of clinicians to continue first line therapy in the hopes of achieving a cure, or to intervene at an earlier time point in disease recurrence. This study also provides a blueprint for future development of personalized diagnostics, which could be applied to biomarkers of other cancer types. Overall, these studies demonstrate how quantitative proteomics can be used to support translational cancer research, from the impact of different mass spectrometry platforms on elucidating signaling changes and drug targets to the characterization of multiplexed LC-MRM assays applied to cell line models for translational research purposes and in patient serum samples optimized for clinical translation. We believe that mass spectrometry-based proteomics is poised to play a pivotal role in personalized diagnostics to support implementation of precision medicine, an effort that will improve the quality and efficiency of patient care.