O objetivo deste trabalho foi avaliar a flutuação de anticorpos séricos em cabras no período pré e imediatamente pós-parto e a eficiência do processo de aquisição de imunidade passiva em cabritos recém-nascidos utilizando colostro caprino e bovino, contribuindo para a avaliação de uma alternativa de manejo de colostro e o melhor conhecimento do mecanismo de formação e transferência de imunidade passiva. Foram utilizadas 18 cabras e suas respectivas crias, 33 animais, sendo 19 fêmeas e 14 machos. Os animais foram distribuídos ao acaso em dois grupos que receberam às 0, 12, 24 e 36 horas de vida colostro caprino (grupo A) e colostro bovino (grupo B). As coletas de sangue das cabras foram iniciadas 45 dias antes da data prevista para o parto, em intervalos de 5 dias, até o 5o dia após o parto. Amostras de sangue dos cabritos foram coletadas às 0, 12, 24 e 48 horas, e aos 5, 10, 15, 20, 25, 30, 35, 40, 50 e 60 dias de idade. As variáveis séricas analisadas foram proteína total (PT), através do método do biureto, e imunoglobulinas (Ig) através dos métodos de imunodifusão radial (IDR) e turvação por sulfato de zinco (ZST). O delineamento experimental utilizado foi o inteiramente casualizado e as variáveis séricas analisadas como medidas repetidas no tempo. A ausência de queda significativa na concentração sérica de PT e Ig no período pré-parto indica que a mobilidade destes componentes séricos para a glândula mamária ocorre em concentrações que permitem que o animal mantenha esses valores estáveis. O grupo A apresentou valores máximos de PT e Ig séricas mais tardiamente aos 20,05±1,36 e 20,11±1,72 dias de vida, e as médias foram de 5,91±0,22 g/dL e 28,17±2,05 unidades ZST, respectivamente. Neste grupo as variáveis PT e Ig após o acréscimo inicial, entre a primeira e segunda data, permanecem sem alteração ao longo do período experimental. No grupo B a data de concentração máxima de PT foi verificada nas 48,68±0,70 horas de vida, com média de 7,16±0,28 g/dL e a data de concentração máxima de Ig foi verificada às 48,75±0,73 horas de vida, com média de 37,56±2,38 unidades ZST. Em seguida, os valores decresceram gradativamente, em função do catabolismo das imunoglobulinas adquiridas. Os resultados indicam que o colostro caprino pode ser substituído pelo colostro bovino apresentando vantagens para cabritos recém-nascidos na aquisição inicial de Ig sérica. Determinou-se a correspondência dos valores de ZST em mg/mL de IgG através de análises comparativas de imunoglobulinas presentes em amostras séricas, obtidas do grupo B (colostro bovino) nas primeiras 48 horas de vida usando os métodos de IDR e ZST. Foi encontrado um coeficiente de correlação de 0,725, indicando a existência de associação entre unidades ZST e a concentração de imunoglobulina em mg/mL, permitindo, deste modo estimar o nível de imunoglobulina sérica através do método ZST. / The objective of this study was to evaluate the fluctuation of serum antibodies in goats pre and immediately post partum period and the passive immunity acquisition efficiency in goat kids fed caprine and bovine colostrum, contributing for an evaluation of colostrum management alternative and a better knowledge of passive immunity formation and transference mechanism. Eighteen goats and their respective offspring, 33 animals, 19 females and 14 males, were used. Animals were randomly distributed in two groups that received at 0, 12, 24 and 36 hours after birth, caprine colostrum (group A) and bovine colostrum (group B). Goat blood samples collection started 45 days before the birth predicted date, with intervals of 5 days until the 5th day post partum. Goat kids blood samples were collected at 0, 12, 24 and 48 hours, and at 5, 10, 15, 20, 25, 30, 40, 50 and 60 days of age. Serum variables analyzed were total protein (TP), by biurete method, and immunoglobulin (Ig) by radial immunodiffusion (IDR) and zinc sulphate turbidity (ZST) methods. A completely randomized desing was used with serum variables analyzed as repeated measures on time. The absence of significant fall on TP and Ig serum concentration in pre partum period, indicates that the mobility of these serum components to mammary gland occurs in concentrations that allow these animals to keep the stability of these values. Group A showed maximum values for serum TP and Ig later than group B, at 20.05±1.36 and 20.11±1.72 days after birth, and the means were 5.91±0.22 g/dL and 28.17±2.05 ZST units, respectively. In this group the variables TP e Ig after an initial increase, between first and second experimental date, remain without alterations through the experimental period. In group B, the highest concentration date for TP was observed on the 48.68±0.79 hours after birth, with means of 7.16±0.28 g/dL and the highest concentration date for Ig at 48±0.73 hours after birth, with mean of 37.56±2.38 ZST units. After this period the values gradually decreased, mainly function of acquired Ig catabolism. The results indicate that caprine colostrum can be used as a replacer for bovine colostrum showing advantages for the newborn in the initial acquisition of serum Ig status. ZST values correspondence in mg/mL of IgG was determined through comparative analyses of immunoglobulin present in serum samples from group B at first 48 hours after birth using the IDR and ZST methods. A correlation coefficient of 0.725 was found, indicating the existence of association between ZST units and Ig concentration in mg/mL, allowing to estimate the level of serum Ig through ZST method.

Indiana University-Purdue University Indianapolis (IUPUI) / SIMPL is a transcriptional co-activator that alters the activity of transcription factor, NF-κB. In response to pathogens, cytokines such as Interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF) signal through the IL-1 and TNF-α receptors, respectively, which are found on various cell types. Activation of these receptors can result in the nuclear localization of NF-κB where it enables the transcription of several different genes key in the innate immune response. Endogenous co-localization of the SIMPL protein with NF-κB in response to these same cytokine signals has yet to be demonstrated. Polyclonal antibody generated against a truncated version of the SIMPL protein was purified from the sera obtained from immunized rabbits using affinity chromatography. The antibody was found to have a high specificity for both the native and denatured form of the protein as demonstrated by the lack of nonspecific bands observed in immunoprecipitations and Western blotting. The antibody was utilized in immunofluorescence experiments on mouse endothelial cells that were either unstimulated or were stimulated (IL-1 or TNF-α). In the absence of cytokine, SIMPL was localized in both the cytoplasm and the nucleus as opposed to NF-κB which was almost exclusively localized in the cytoplasm. In the presence of IL-1, the concentration of SIMPL in the nucleus was increased, and in the presence of TNF-α, the concentration of SIMPL in the nucleus was even greater. Results of this study identified future routes for SIMPL antibody isolation as well as to demonstrate that endogenous SIMPL protein nuclear localization may not be solely dependent upon TNF-α signaling.

Polycyclic aromatic hydrocarbons are environmental chemicals that are produced during incomplete combustion of coal, oil, gas, and garbage. Toxic effects of these compounds are mediated via the ligand activated Aryl Hydrocarbon Receptor (AHR) signaling pathway. To enable the study of the AHR signaling mechanism, our lab has generated many human proteins using recombinant DNA technology. This thesis documents the design and synthesis of a number of proteins of the AHR deletion construct CΔ553. The bacterial expressed and purified fusion proteins could be utilized as antigen to generate antibodies and be used for cellular delivery. The purified protein was immunogenic in rabbits and produced significant amount of polyclonal antibodies. In western blot analysis, the antibodies were able to the detect baculovirus expressed AHR and different recombinant proteins of the AHR. The polyclonal antibodies were also used in the gel-shift assay to show the AHR dependent gel shift. Cellular delivery CΔ553 was achieved using the protein transduction domain from the HIV-1 virus transactivating protein (TAT). In order to deliver the CΔ553 into mammalian cells, an expression vector was constructed to generate the TAT-CΔ553 fusion protein. The TAT-CΔ553 fusion protein was successfully transduced into two mammalian cells-HeLa and HepG2. The in vivo function of TAT-CΔ553 was determined using the luciferase reporter plasmid assay. The transduced protein was functional; it competed with the AHR and heterodimerize with ARNT in both HeLa and HepG2 cells at a concentration of 3.8x103 nM and 18 nM respectively. Since there an apparent similarity between the basic region of TAT-PTD and CΔ553, we examined the transduction potential of CΔ553. Western blot analysis indicated that the extent of denatured protein transduction was comparable for CΔ553 and TAT-CΔ553 in HepG2 cells. Thus CΔ553 might have intrinsic transduction capability.

Helicobacter pylori ist ein humanpathogenes Bakterium, das den Magen kolonisiert und dadurch eine Immunantwort des Wirts induziert. Statt eine vollständige Eradikation von H. pylori durch die Immunreaktion zu erreichen, kommt es normalerweise zu einer lebenslangen Persistenz des Bakteriums und einer chronischen Infektion. Interessanterweise kommt es infolge einer Infektion zu einer Hochregulation der induzierbaren Stickstoffmonoxid-Synthase (iNOS), aber die zellulären Quellen und zugrundeliegenden Mechanismen von iNOS sind noch nicht vollständig verstanden. Der iNOS-abhängigen Produktion von Stickstoffmonoxid (NO) können sowohl antimikrobielle als auch pathologische Eigenschaften zugeschrieben werden. Daher wurden in der vorliegenden Arbeit iNOS-exprimierende Plasmazellen (PZ) aus der Magenmukosa H. pylori-infizierter Patienten isoliert und phänotypisch, vor allem mittels Durchflusszytometrie und molekularbiologisch hinsichtlich ihres Immunglobulin-Repertoires untersucht. Es wurde erstmals gezeigt, dass mukosale IgA-sezernierende PZ eine der wesentlichen iNOS+ Zelltypen während einer H. pylori-Infektion im Menschen darstellen und zusätzlich wurde ihre intrazelluläre NO-Produktion nachgewiesen. Da iNOS+ PZ in weiteren gastrointestinalen Infektionskrankheiten fehlten, scheint dies kein genereller Phänotyp von PZ der mukosalen Immunabwehr zu sein. Die Analyse der intrazellulären Zytokin-Expression der mukosalen B-Zellpopulationen in H. pylori-Patienten ergab eine Ko-Expression von IFN-γ und TNF-α in iNOS+ memory B-Zellen und eine TNF-α-Expression in iNOS+ PZ, aber nicht in den iNOS− Zellen. Die molekularbiologische Charakterisierung des Immunglobulin-Repertoires von iNOS+ und iNOS− PZ hinsichtlich der VHDJH-Regionen ergab keinen signifikanten Unterschied hinsichtlich der Isotyp-Verteilungen, der Nutzung der VH- und JH-Segmente, CDRH3-Längen sowie somatischen Mutationen und alle Antikörper zeigten typische Charakteristika einer T-zellabhängigen Affinitätsreifung. / Helicobacter pylori is a human-pathogenic bacterium that colonizes the stomach and thereby initiates host immune response. Instead of a complete eradication of H. pylori by the induced immune response, a lifelong bacterial persistence leads to chronic infections. Interestingly, up-regulation of inducible nitric oxide synthase (iNOS) has been observed in gastric mucosal tissue during the course of H. pylori infection in humans, however the cellular sources and underlying mechanisms of iNOS induction are not fully understood. iNOS-dependent production of nitric oxide (NO) is one of the factors commonly linked to both, anti-microbial immunity and pathology. Therefore, in this thesis iNOS-expressing plasma cells (PCs) in the stomach mucosa of H. pylori-infected patients were isolated and phenotypically analyzed by flow cytometry, as well as screened using molecular techniques regarding their immunoglobuline (Ig) repertoires. For the first time, we identified mucosal IgA-producing PCs as a major iNOS+ cell population during H. pylori infection in humans, and additionally confirmed their intracellular nitric oxide production. Since iNOS+ PCs were not detectable in other gastrointestinal infectious diseases, this reaction does not seem to be a general feature of mucosal PCs under conditions of infection. Additionally, intracellular cytokine expression analyses of mucosal B-lineage cells isolated from H. pylori patients revealed a co-expression of IFN-γ and TNF-α in iNOS+ memory B cells and the expression of TNF-α in iNOS+ PCs, but not in iNOS− cells. Molecular analysis of the Ig repertoire of iNOS+ and iNOS− PCs of the VHDJH regions revealed no significant differences regarding the Ig isotype composition, VH and JH gene family usage, CDRH3 length, and frequency of somatic mutations and all antibodies were characterized by typical properties of T cell-dependent affinity maturation.

O objetivo deste trabalho foi estudar os efeitos de diferentes manejos de fornecimento de colostro sobre a aquisição sérica de imunoglobulina G (IgG) e do fator de crescimento semelhante à insulina-I (IGF-I), bem como as modificações morfológicas e morfométricas do epitélio intestinal em bezerros recém-nascidos. Foi utilizado o fornecimento prolongado de colostros de diferentes concentrações, incluindo colostro enriquecido com colostro liofilizado e acompanhada a flutuação dos níveis séricos de IgG e proteína total (PT) nas primeiras 72 horas de vida, com diferentes condições iniciais de aquisição de imunidade passiva. Também foi estudada a relação da massa de IGF-I ingerida com possíveis alterações na morfologia entérica e nos enterócitos. Oitenta e dois bezerros da raça Holandesa receberam, ao nascimento, colostro com concentrações variáveis de IgG. Com base na avaliação sérica das imunoglobulinas realizada na décima hora de vida, os animais foram distribuídos em dois grupos, IgG sérica abaixo ou acima de 12 mg mL-1. Cada grupo foi submetido à três tratamentos de acordo com a refeição fornecida às 12 horas de vida, colostro baixo (concentração de IgG inferior à 30 mg mL-1), colostro alto (superior à 100 mg mL-1) ou colostro enriquecido de colostro liofilizado (superior à 120 mg mL-1). Às 0, 10-12, 24, 36, 48 e 72 horas de vida foi coletado sangue dos bezerros para análise de IgG, PT e IGF-I, e amostras do duodeno; jejuno proximal, médio e distal; e íleo para análise histomorfológica. Para análise das variáveis séricas, considerou-se um delineamento experimental inteiramente casualizado com medidas repetidas no tempo. Para análise das variáveis morfométricas dos segmentos intestinais foi considerado um arranjo fatorial 3X3+1, correspondendo à massa total de IGF-I fornecida via colostro, às idades de abate (10, 24-36 e 72 horas de vida) e o grupo controle (nascimento). O fornecimento prolongado de colostro enriquecido em bezerros com alta aquisição inicial não apresentou efeito significativo sobre a flutuação sérica de IgG. A ingestão de sólidos totais acima de 150 g determinou menores valores de IgG sérica. Os animais com baixa aquisição inicial mostraram uma maior eficiência de absorção de anticorpos comparados aos com alta aquisição inicial. A massa de IGF-I consumida não influenciou os níveis séricos desta variável, porém influenciou o comportamento da densidade dos vilos ileais e a altura dos microvilos do duodeno, jejuno proximal e médio. Os valores do volume parcial da mucosa absortiva do nascimento às 10 horas de vida apresentaram diferenças significativas. O fornecimento prolongado de colostro de alta concentração de imunoglobulinas e IGF-I influenciou a morfologia da mucosa entérica. / The objective of this study was to evaluate the effects of different colostrum supply on the serum immunoglobulin G (IgG) acquisition and the insulin-like growth factor-I (IGF-I), as well as the morphologic and morphometric modifications of the intestinal epithelium in the newborn calves. The prolonged supply of colostrum with different concentrations was used, including colostrum enriched with lyophilized colostrum, and was accompanied the fluctuation of serum IgG and total protein (TP) of calves in the first 72 hours of life, according to initial passive immunity acquisition. It was also studied the relation of IGF-I mass ingested with possible alterations in the intestinal morphology and enterocytes. Eighty two Holstein calves received at birth colostrum with different concentrations of IgG. Based on serum immunoglobulins evaluation at ten hour of life, the animals were distributed in two groups, serum IgG below or above 12 mg mL-1. Each group received three treatments in agreement with the meal supplied at 12 hours of life, low colostrum (IgG inferior to 30 mg mL-1), high colostrum (superior to 100 mg mL-1) or enriched with lyophilized colostrum (superior to 120 mg mL-1). At 0, 10-12, 24, 36, 48 and 72 hours of life blood was collected from calves for IgG, TP and IGF-I analysis, and samples from duodenum; proximal, middle and distal jejunum, and ileum for histomorphologic analysis. A completely randomized design was used with the serum variables analyzed as repeated measures on time. The morphometric variables of the intestinal segments were considered a factorial arrangement 3X3+1, corresponding to the total mass of IGF-I supplied in colostrum and the slaughter ages (10, 24-36 and 72 hours of life) plus the control group (birth). The prolonged supply of enriched colostrum in calves with high initial acquisition did not present significant effects on the serum IgG fluctuation. The ingestion of total solids above 150 g determined lower values of serum IgG. The animals with low initial acquisition showed better absorption efficiency of antibodies compared to high initial acquisition. The mass of IGF-I consumed did not influence the serum levels of this variable; however it influenced the behavior of the density of the ileum villi and the height of the microvillus from duodenum, proximal and middle jejunum. The values of the partial volume of the absorptive mucous membrane from birth to 10 hours of life presented significant differences. The prolonged supply of colostrum with high immunoglobulins and IGF-I concentrations influenced the morphology of the intestinal mucosa.

Há poucos dados na literatura sobre o transporte transplacentário de imunoglobulinas em gestações múltiplas. O objetivo deste estudo foi observar fatores que influenciam a concentração de imunoglobulina G (IgG) no cordão umbilical dos neonatos e a transferência transplacentária de IgG total e de IgG contra o Streptococcus grupo B (EGB), e lipopolissacarídeos (LPS) de Klebsiella spp. e Pseudomonas spp.. Métodos: estudo prospectivo realizado no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo no período de 2012 a 2013. Foram coletadas amostras de sangue materno e de cordão umbilical no momento do parto. Os critérios de inclusão foram gestações gemelares com ausência de sinais de infecção por HIV, citomegalovírus, Hepatites B e C, toxoplasmose e rubéola e ausência de doenças autoimunes, malformação fetal e síndromes genéticas. A análise multivariada foi realizada para avaliar a associação entre os níveis de IgG em cordão umbilical e as taxas de transferência de anticorpos com a concentração materna de IgG, a corionicidade da gestação, a presença de insuficiência placentária, a restrição de crescimento intrauterino, a idade gestacional de nascimento, o peso de nascimento, o tabagismo, a doença materna e a via de parto. Resultados: a concentração de IgG total em cordão umbilical apresentou correlação positiva com os níveis maternos séricos de IgG total e a idade gestacional do parto. Os níveis de IgG total em cordão umbilical foram significativamente menores em gestações monocoriônicas quando comparadas às dicoriônicas. A taxa de transferência de IgG total apresentou correlação positiva com a idade gestacional do parto, mas negativa com as concentrações maternas de IgG total. As concentrações de IgG contra EGB e LPS de Klebsiella spp. e Pseudomonas spp. apresentaram associação com os níveis maternos de IgG específicos contra esses antígenos e com o diabetes. Os níveis de IgG contra LPS de Klebsiella spp. também foram associados com o peso de nascimento e com hipertensão materna. As taxas de transferência de IgG contra EGB e LPS de Pseudomonas spp. apresentaram correlação com os níveis maternos de IgG específicos contra os antígenos referidos. A taxa de transferência de IgG contra EGB também esteve associada com a idade gestacional do parto, enquanto a taxa de transferência de IgG contra LPS de Pseudomonas spp. apresentou correlação com diabetes. Não houve correlação entre a taxa de transferência de IgG contra a LPS de Klebsiella spp. com nenhum fator analisado. Conclusão: em gestações gemelares, a concentração total de IgG em cordão umbilical foi influenciada pela concentração materna de IgG total, pela idade gestacional do parto e pela corionicidade placentária. As concentrações de IgG total foram significativamente menores em gestações monocoriônicas que em dicoriônicas. As concentrações séricas de IgG contra EGB e LPS de Klebsiella spp. e Pseudomonas spp. em cordão umbilical apresentaram associação com os níveis maternos de IgG específicos contra esses antígenos e com a presença de diabetes. Todos os outros parâmetros estudados apresentaram diferentes associações com as concentrações de IgG e com as taxas de transferências de IgG específicas contra cada antígeno investigado / There is a lack of data in the literature regarding the placental transport of immunoglobulins in twin pregnancies. The objective of this study was to examine factors that influence the concentration of immunoglobulin G (IgG) in cord serum and the placental transfer of total IgG and of IgGs against Klebsiella spp. LPS and Pseudomonas spp. LPS, and Group B Streptococcus (GBS). Methods: A prospective study was conducted at the Hospital das Clinicas in the São Paulo University Medical School between 2012 and 2013. Maternal and umbilical cord samples were collected at birth. The inclusion criteria were twin pregnancies with no evidence of infection with HIV, cytomegalovirus, hepatitis B or C, toxoplasmosis, or rubella. Twin pregnancies with evidence of autoimmune disease, fetal malformations or genetic syndromes were also excluded. Stepwise multivariate regression analysis was used to evaluate the association between cord serum concentrations of IgG and IgG transfer ratios as well as the associations between cord serum concentrations of IgG and maternal serum concentrations of IgG, pregnancy chorionicity, the presence of an abnormal umbilical artery pulsatility index, intrauterine growth restriction, gestational age at delivery (GAD), birth weight, placental weight, smoking during pregnancy, maternal disease, and mode of delivery. Results: Total IgG concentrations in cord sera were positively correlated with total IgG concentrations in maternal sera. Cord serum concentrations of IgG were also positively correlated with GAD. Cord serum concentrations of total IgG were significantly lower in monochorionic versus dichorionic pregnancies. The total IgG transfer ratio was positively correlated with GAD but was inversely correlated with total IgG concentration in maternal serum. Cord serum concentrations of IgGs against GBS, Klebsiella spp. LPS and Pseudomonas spp. LPS were significantly associated with maternal concentrations of specific IgGs and the presence of maternal diabetes. Cord serum concentrations of anti-Klebsiella spp. LPS IgG were also correlated with birth weight and the presence of maternal hypertension. The transfer ratios of IgGs against GBS and Pseudomonas spp. LPS were related to maternal concentrations of specific IgGs. The transfer ratios of IgGs against GBS and Pseudomonas spp. LPS were also associated with GAD and the presence of diabetes, respectively. None of the examined parameters were found to be correlated with the transfer ratio of IgG against Klebsiella spp. LPS. Conclusions: In twin pregnancies, in addition to the influences of maternal serum concentrations of total IgG and of GAD, chorionicity was also found to influence cord serum concentrations of total IgG. Compared with dichorionic twins, monochorionic twins were found to have lower concentrations of total IgG in cord sera. Umbilical cord serum concentrations of IgGs against GBS, Klebsiella spp. LPS and Pseudomonas spp. LPS were associated with maternal serum concentrations of specific IgGs and with maternal diabetes. All of the remaining parameters that were investigated had varying associations with concentrations of specific IgGs in cord serum and with placental transfer and were dependent on the antigen being studied

Avaliou-se o efeito de diferentes fontes de colostro, caprino e bovino in natura, e bovino liofilizado em 25 cabritas distribuídas aleatoriamente em cinco tratamentos: CCA - colostro caprino adequado (45 a 55 mg/mL de IgG); CCB - colostro caprino baixo (15 a 25 mg/mL de IgG); CBA - colostro bovino adequado (45 a 55 mg/mL de IgG); CBB - colostro bovino baixo (15 a 25 mg/mL de IgG) e CBL - colostro bovino liofilizado (45 a 55 mg/mL de IgG). Os animais receberam 5% do peso vivo de colostro às 0,12 e 24 horas após o nascimento e, em seguida, leite de vaca duas vezes ao dia e concentrado ad libitum até 60 dias de idade. Amostras de sangue foram coletadas nos dias 0, ½, 1, 2 , 5, 10,15, 20, 25, 30, 35, 40, 50 e 60 de idade. O sangue foi analisado quanto ao valor de hematócrito e a concentração de proteína total (PT). As frações protéicas separadas por eletroforese e a condição de diarréia foi avaliada através de cor, consistência, presença de sangue/muco. As variáveis sanguíneas foram analisadas como medidas repetidas no tempo. Com relação ao valor de hematócrito efeito de período foi observado, o valor no primeiro dia de idade, 28,76±1,76, foi maior que nos 10, 30, 35 e 60 dias de idade, com valores médios de 23,88±0,98, 24,18±0,70, 23,73±0,74 e 24,53±0,79%, respectivamente. Interação entre tratamento e período foi verificado para PT sérica, os tratamentos CCB, CBA, CBB e CBL apresentaram aumento gradativo nas concentrações séricas até os 60 dias de idade, enquanto CCA apresentou valores nos dias 10,15, 40, 50 e 60 dias de idade maiores que o valor ao nascimento. Os valores de albumina sérica foram afetados pelo tratamento e período, sendo o valor em CBL, 2,39±0,12 g/dL, significamente inferior ao dos tratamentos CCA, CCB, CBA e CBB, com 3,20±0,12, 2,94±0,12, 3,25±0,12, 3,08±0,12 g/dL, respectivamente. Um aumento gradativo nas concentrações desta variável foi observado até os 60 dias de idade. Ao 0 e ½ dia de idade os valores de globulina sérica foram menores que os observados na última data experimental, 60 dias de idade. A relação albumina/globulinas sérica foi afetada pelo efeito de tratamento e período, com os valores em CBL significativamente superiores do que em CCA, CCB, CBA e CBB. A relação foi maior ao nascimento diminuindo nas datas subseqüentes, ½ e 1 dia de idade. A variável gamaglobulina apresentou efeito de tratamento e período, com valores em CBL significativamente superiores do que em CCB e CBB, não diferindo dos encontrados em CCA e CBA. Os valores ao 0 e ½ dia de idade foram menores que aos 60 dias de idade. Observando a condição de diarréia nos tratamentos verificou-se que os animais CCB e CBB apresentaram maior frequência de diarréia que os animais dos demais tratamentos, indicando uma relação inversa entre anticorpos no colostro e condição de diarréia. Os resultados indicam que o colostro bovino liofilizado pode ser utilizado para caprinos recém-nascidos como fonte alternativa para a aquisição de proteção inicial. / It was to evaluated the effect of different sources of colostrum, goat and bovine in natura, and lyophilized bovine in 25 newborn female goat kids were randomly allocated to five treatments: GCA - goat colostrum adequate (45 to 55 mg/mL of IgG), GCL - goat colostrum low (15 to 25 mg/mL of IgG), BCA - bovine colostrum adequate (45 to 55 mg/mL of IgG), LBC - bovine colostrum low (15 to 25 mg / mL of IgG) and of treatment CBL, lyophilized bovine colostrum (45 to 55 mg / mL of IgG). The animals received 5% of body weight of colostrum at 0, 12, 24 hr after birth and then, bovine milk twice a day and concentrate ad libitum until 60 days of life. Blood samples were collected in 0, ½, 1, 2 , 5, 10,15, 20, 25, 30, 35, 40, 50 e 60 days of age. Blood was analyzed for hematocrit value and total protein (TP). Protein fractions were separated by electrophoresis and the condition of diarrhea was evaluated by color, consistency, blood/mucus presence. Blood variables analyzed as repeated measures on time.Related to hematocrit values effect of period was observed, the value at one day of age, 28.76±1.76, was higher than at 10, 30, 35 and 60 days of age, with average at 23.88±0.98, 24.18±0.70, 23.73±0.74 and 24.53 ± 0.79%, respectively. Interaction between treatment and period was observed for serum TP, treatments GCL, BCA, BCL, and LBC showed a gradual increase in serum concentrations up to 60 days of age, while GCA showed values on days 10, 15, 40, 50 and 60 days of age, greater than the value at birth. The values of serum albumin were affected by treatment and period, being the value in LBC, 2.39±0.12 g/dL, significantly lower than the GCA treatment, GCL, BCA and BCL, with 3.20±0.12 , 2.94±0.12, 3.25±0.12 and 3.08± 0.12 g/dL, respectively. A gradual increase in the concentrations of this variable was observed until 60 days of age. To 0 and ½ day of age values of serum globulin were significantly lower than the observed at last experimental date, 60 days of age. Serum albumin/globulin relation was affected by the treatment and period with the values in CBL significantly higher than observed in GCA, GCL, BCA and BCL. The relation was higher at birth decreasing in the following dates, ½ and 1 day of age. The variable gamma globulin was affected by treatment and period, with values in LBC significantly higher than in GCL and BCL, not differing from the found in GCA and BCA. The values at 0 and ½ day of age were lower than 60 days of age. Observing the diarrhea condition, in the treatments, it was verified that the GCL and BCL animal had showed higher diarrhea incidence than the animals from the other treatments, indicating an inverse relationship between antibodies in the colostrum and condition of diarrhea. The results indicate that lyophilized bovine colostrum can be used to newborns goat kids as an alternative source for the acquisition of initial protection.

Aggregatibacter actinomycetemcomitans está associado à periodontite agressiva e produz CDT. Visando contribuir no entendimento do papel da CDT na regulação da resposta imune, foi determinada sua atividade sobre macrófagos e a resposta humoral contra a toxina. CDT inibiu a proliferação de células epiteliais OBA-9 e macrófagos Raw 264.7 e também a produção de NO por células Raw 264.7 e macrófagos peritoneais de camundongos C3H/HePas e C3H/HeJ, mas estimulou a produção de IL-12. Na imunidade humoral, 75% dos soros de indivíduos com PAgL e 81,8% dos PAgG foram reativos para A. actinomycetemcomitans. Não houve diferença na resposta contra CDTA e CDTB entre o soro de pacientes com diferentes condições periodontais. Todos os pacientes PAgG foram soropositivos para a CDTC, porém apenas 8,3% dos indivíduos com PAgL, nenhum dos PC e 25% dos saudáveis foram positivos. CDT tem atividade imunomodulatória e a resposta humoral difere entre indivíduos infectados pela bactéria. / Aggregatibacter actinomycetemcomitans is associated with aggressive periodontitis and produces CDT. Aiming to contribute in the understanding the CDT activity in the immune response regulation, its action on macrophages was determined and the response against the toxin analyzed. CDT inhibited the proliferation of OBA-9 epithelial cells and Raw 264.7 macrophages and also inhibited the NO production by Raw 264.7 cells and peritoneal macrophages of C3H/HePas and C3H/HeJ mice, however, stimulated the IL-12 production. In the humoral immunity, 75% of sera from LAgP subjects and 81.8% were reactive to A. actinomycetemcomitans. There was not difference in the response against CDTA and CDTB among sera of patients with different periodontal conditions. All GAgP subjects were sera-reactivity to CDTC, however only 8.3% LAgP subjects, none in CP and 25% of healthy subjects were positive. CDT has immunomodulatory activity and the humoral response differ among bacteria infected subjects.

INTRODUÇÃO: As infecções causadas por bactérias ou vírus são frequentes em pacientes com imunodeficiência comum variável (ICV) devido à deficiência de anticorpos e associação com alteração da função das células T. OBJETIVOS: Avaliar o efeito da ativação de receptores Toll-like (TLR) utilizando ligantes de TLRs em células T monofuncionais ou polifuncionais em pacientes com ICV. MÉTODOS: Foram selecionados 16 pacientes com ICV do Ambulatório de Manifestações Dermatológicas das Imunodeficiências Primárias ADEE3003 HC-FMUSP e 16 controles saudáveis. Os métodos utilizados de citometria de fluxo foram: a) análise em sangue periférico de linfócitos B, linfócitos T quanto ao perfil de ativação/maturação, linfócitos T foliculares (Tfh) e células T reguladoras (Treg); b) dosagem de citocinas e quimiocinas em amostras de soro e em sobrenadante de culturas de células mononucleares do sangue periférico (CMNs) estimuladas com agonistas de TLRs; c) avaliação das células TCD4+ mono e polifuncionais secretoras de IL-17a, IL-22, TNF, IFN- e IL-10, e expressão de marcador de ativação crônica de CD38 estimuladas por agonistas de TLR2, TLR3 e TLR7/8 e estímulos policlonais como enterotoxina B de Staphylococcus aureus (SEB) e acetato miristato de forbol e ionomicina (PMA/IONO); d) células Th22 e Tc22 estimuladas com TLR e SEB. RESULTADOS: Na ICV, os linfócitos B do sangue periférico mostram diminuída frequência, sendo em maior frequência de linfócitos B naïve (CD19+IgD+CD27-), e ausência de células B de memória. Além disto, um aumento na expressão do marcador de exaustão PD-1 foi observado nas células TCD4+ de memória efetora (CD45RA-CCR7-) e na expressão de CD38, em células TCD8+ terminalmente diferenciadas (CD45RA + CCR7-). Em contraste, houve diminuição na frequência de células T reguladoras naïve nos pacientes com ICV. Nos indivíduos com ICV foi observado aumento na frequência de células TCD4+ TNF+ sob estímulo TLR2 e TLR7/8 comparado ao grupo controle, enquanto que sob estímulo com PMA/IONO houve menor frequência de células TCD4+ e TCD8+ secretoras de IFN-y IL-17a, IL-22 ou TNF. Já em células TCD8+ houve importante redução na ativação via TLR3 na resposta de IL-22, IFN-y e IL-17a. Contudo, os estímulos com TLR7/8 e SEB foram capazes de aumentar a frequência de células Th22 e Tc22 nos pacientes com ICV. Em geral, as células TCD4+, que secretam simultaneamente 4 a 5 citocinas induzidas por TLR foram preservadas em ICV. Embora as células TCD4+ polifuncionais secretoras de 3 citocinas, foram capazes de responder a estímulos via TLR2 e TLR7/8, as células TCD8+ não responderam para qualquer estímulo via TLRs. Além disso, as células T que expressam CD38 mostraram menor polifuncionalidade aos estímulos via TLRs e PMA/IONO. O perfil inflamatório nos pacientes com ICV foi observado pela elevação sérica de IL-6, CCL-2, CCL-5, CXCL8, CXCL-9, CXCL-10. Alteração na resposta aos agonistas de TLRs em ICV pode ser observada com a ativação dos agonistas de TLRs em CMNs, que mostrou maior produção de TNF e diminuição de CCL2 e CXCL8 após ativação via TLR4. Em contraste, o agonista de TLR7/8, teve ação oposta induzindo CXCL10 e reduzindo os níveis de CXCL9. Chama atenção no ICV, à reduzida secreção de IFN-alfa induzida por TLR7/8, que não foi observada com a ativação via TLR9. CONCLUSÕES: Até o momento, os achados em ICV mostram alterações nas células T, seja quanto à baixa frequência de células T reguladoras naïve e a reduzida resposta efetora, em especial das células TCD8+. Contudo, enfatiza o potencial de adjuvante dos agonistas de TLR7/8 na ativação das células T / INTRODUCTION: Infections caused by bacteria or viruses are common in patients with Common Variable Immunodeficiency (CVID), due to antibody deficiency and association with altered function of T cells. OBJECTIVES: To evaluate the effect of Toll-like receptors (TLR) activation using TLR agonists on the monofunctional or polyfunctional T cells in patients with CVID. METHODS: We selected 16 patients with ICV from the Dermatologic Manifestations of Primary Immunodeficiencies Clinic ADEE3003 HC-FMUSP and 16 healthy controls. The methods used for flow cytometry were: a) analysis of peripheral blood B lymphocytes, T lymphocytes were assessed by the activation/maturation profile, follicular T cells (Tfh) and regulatory T cells (Treg); b)evaluation of cytokines and chemokines serum levels and in supernatants of mononuclear cell cultures from peripheral blood (PBMC) stimulated with TLR agonists; c) evaluation of mono and polyfunctional CD4+ T cells secreting IL-17a, IL-22, TNF, IFN-y and IL-10, and expression chronic activation marker of CD38 stimulated by agonists of TLR2, TLR3 and TLR7/8 and polyclonal stimuli such as Staphylococcus aureus enterotoxin B (SEB) and phorbol myristate acetate and ionomycin (PMA / IONO); d) analysis of Tc22 and Th22 cells stimulated with TLR and SEB. RESULTS: In the CVID, the peripheral blood B cells show decreased frequency, being higher frequency of naïve B cells (IgD+ CD19+ CD27-) and lack of memory B cells. Moreover, an increased expression of PD-1, an exhaustion marker, was detected in the CD4+ T cell effector memory (CD45RA- CCR7-) and expression of CD38 on CD8+ T terminally differentiated cells (CD45RA+ CCR7-). In contrast, a decreased frequency of naïve regulatory T cells was detected in the patients with CVID. In CVID patients it was observed increased frequency of T CD4+ TNF+ cells upon TLR2 and TLR7/8 agonists compared to the control group, while under stimulation with PMA /IONO there was a lower frequency of CD4+ and CD8+T cells secreting IFN-y, IL-17a, IL-22 or TNF. The CD8+T cells showed a significant reduction of in the IL-22 response, IFN-? and IL-17a induced by TLR3 activation. However, stimulation with TLR7/8 and SEB were able to increase the frequency of Th22 and TC22 cells in the patients with CVID. In CVID patients it was observed increased frequency of T CD4+ TNF+ cells upon TLR2 and TLR7/8 agonists compared to the control group, while under stimulation with PMA /IONO there was a lower frequency of CD4+ and CD8+ T cells secreting IFN-y, IL-17a, IL-22 or TNF. The CD8+ T cells showed a significant reduction of in the IL-22 response, IFN-y and IL-17a induced by TLR3 activation. However, stimulation with TLR7/8 and SEB were able to increase the frequency of Th22 and TC22 cells in the patients with CVID. In general, CD4+ T cells that secrete simultaneously 4 to 5 cytokines induced by TLR were preserved in CVID. Although polyfunctional CD4+ T cells secreting 3 cytokines were able to respond to TLR2 and TLR7/8 agonists, the CD8+ T cells did not respond to any stimuli. In addition, T cells expressing CD38, showed lower polyfunctionality to the stimuli via TLRs and PMA/IONO. Furthermore, the inflammatory status in the patients with CVID was observed by the increased serum levels of IL-6, CCL-2, CCL-5, CXCL8, CXCL-9, CXCL-10. In contrast, the agonist of TLR7/8 had opposite action inducing CXCL10 and reducing the CXCL9 levels. Noteworthy in CVID, that the reduced secretion of IFN-alfa induced by TLR7/8 was not observed with TLR9 activation. CONCLUSIONS: To date, the CVID findings shows alterations in the T cells, as the low frequency of naïve regulatory T cells and reduced effector response, mainly of CD8+ T cells. However, it emphasizes the adjuvant potential of the TLR7/8 agonist in the T cells activation

submitted by Liu Dan. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 66-80). / Abstract also in Chinese. / CONTENTS --- p.i / LIST OF TABLES --- p.iii / LIST OF FIGURES --- p.iv / LIST OF ABBREVIATIONS --- p.v / ACKNOWLEDGEMENTS --- p.vi / ABSTRACT --- p.vii / Chapter Chapter 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- History --- p.1 / Chapter 1.2 --- Immunoglobulins --- p.3 / Chapter 1.2.1 --- Structure --- p.3 / Chapter 1.2.2 --- Properties of immunoglobulins --- p.7 / Chapter 1.3 --- Monoclonal proteins and monoclonal gammopathies --- p.12 / Chapter 1.3.1 --- Monoclonal proteins --- p.12 / Chapter 1.3.2 --- Monoclonal gammopathies --- p.14 / Chapter 1.4 --- Laboratory investigation of monoclonal immunoglobulin --- p.17 / Chapter 1.4.1 --- The current procedure of investigation in laboratory --- p.17 / Chapter 1.4.2 --- Problems in identifying monoclonal immunolgobuin --- p.19 / Chapter 1.5 --- Comparison of different techniques --- p.20 / Chapter 1.5.1 --- Immunoelectrophoresis --- p.20 / Chapter 1.5.2 --- Immunofixation electrophoresis --- p.22 / Chapter 1.5.3 --- Isoelectric focusing and immunoisoelectric focusing --- p.24 / Chapter 1.6 --- Aim of the present study --- p.27 / Chapter 1.7 --- Design of experiment --- p.27 / Chapter Chapter 2 --- MATERIALS AND METHODS --- p.30 / Chapter 2.1 --- Study subjects --- p.30 / Chapter 2.2 --- Apparatus --- p.30 / Chapter 2.2 --- Apparatus --- p.30 / Chapter 2.3 --- Reagents and materials --- p.32 / Chapter 2.4 --- Preparation of gels --- p.35 / Chapter 2.5 --- Isoelectric focusing procedure --- p.36 / Chapter 2.6 --- Acid fixation and staining --- p.37 / Chapter 2.7 --- Technical factors affecting results --- p.38 / Chapter Chapter 3 --- RESULTS --- p.40 / Chapter 3.1 --- Interpretation of results in isoelectric focusing --- p.40 / Chapter 3.2 --- Affecting factors --- p.47 / Chapter 3.3 --- Comparison of the results between IEF and IFE --- p.53 / Chapter Chapter 4 --- DISCUSSION --- p.59 / Chapter Chapter 5 --- CONCLUSION --- p.65 / References --- p.66