Expression of sTGFbetaRII-Fc-Jun from recombinant vaccinia virus TGFß has a biphasic role in tumorigenesis. In early phases it acts as tumor sup-pressor. However, in late phases when cells have escaped selectively from the antimito-genic response of TGFß, it may act as a promoter of tumor progression and invasion. One way of control tumor formation and progression is blocking of TGFß signalling pathways in late phases of tumorigenesis. We have constructed recombinant vaccinia virus P13 expressing soluble TGFbeta type II receptor fused with the Fc fragment of IgG1 and with Jun fragment (sTbetaRII-Fc-Jun). This sTbetaRII-Fc-Jun is supposed to increase the effect of antitumor vaccinia virus vaccine expressing SigE7LAMP, which is investigated for the treatment of the HPV-16 associated cervical cancer. Binding of sTbetaRII-Fc-Jun to protein G were tested by SDS-PAGE and by im-munoblotting. We found that Jun fragment and sTbetaRII fragment do not block Fc bind-ing site for protein G. sTbetaRII-Fc-Jun was characterised using SDS-PAGE and immunoblot analysis. We observed that the amount of sTbetaRII-Fc-Jun was higher in cell supernatans of in-fected cells in comparison to cell lysates. In cell lysates we observed higher amount of sTbetaRII than sTbetaRII-Fc-Jun. The expression of sTbetaRII-Fc-Jun was stronger under...

Submitted by Marcelo Andrade Silva (marcelo.andradesilva@ufpe.br) on 2015-03-09T14:18:41Z No. of bitstreams: 2 DISSERTAÇÃO CASSIA GISELLE DE OLIVEIRA NÓBREGA.pdf: 1673463 bytes, checksum: 460066c3a78447d401976dede5d04300 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-09T14:18:41Z (GMT). No. of bitstreams: 2 DISSERTAÇÃO CASSIA GISELLE DE OLIVEIRA NÓBREGA.pdf: 1673463 bytes, checksum: 460066c3a78447d401976dede5d04300 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2014 / A intensidade da resposta alérgica pode ser alterada em indivíduos sensibilizados por Ascaris lumbricoides, ou seja, com presença de anticorpos específicos no soro. Quanto à IgE anti-Asc, este anticorpo tem sido relatado como um fator de risco para asma, mas sobre a IgG1anti-Asc pouco é sabido. Este trabalho teve como objetivo verificar a associação entre IgE, IgG1 e IgG4 anti-Asc no soro e a presença de asma, bem como, entre asma e níveis de citocinas, valores absolutos de neutrófilos e eosinófilos, em pacientes com IgE ou IgG1 anti-Asc no soro. Para isto, crianças de 2 a 14 anos de idade, residentes na Região Metropolitana do Recife (n=104), asmáticas ou não asmáticos, sem infecção, tiveram as amostras de sangue coletadas. Foi realizado o leucograma e a cultura celular do sangue periférico. As células foram cultivadas, estimulados ou não com PHA, e os sobrenadantes submetidos à dosagem de citocinas por CBA. Os isótipos IgE, IgG1 e IgG4 anti-Asc, no soro, foram mensurados por ELISA. Foram formados 8 grupos de estudo: asma IgE anti-Asc positivo e negativo; controle (paciente não asmáticos) IgE anti-Asc positivo e negativo; asma IgG1 anti-Asc positivo e negativo; controle IgG1 anti-Asc positivo e negativo. Foi observado que não houve diferença na quantidade de indivíduos com IgE positivo e negativo entre os grupos asma e controle, bem como nos níveis deste anticorpo. O mesmo foi notado para a presença de IgG1 anti-Asc, porém com níveis mais elevados de anticorpo nos indivíduos controles. Não foram detectados níveis de IgG4 anti-Asc em pacientes asmáticos ou controles. Foram encontrados maiores níveis de IL-6, TNF-α e número de eosinófilos nos pacientes asmáticos em comparação aos controles. Este perfil se manteve nos pacientes asma IgE anti-Asc negativo, quando comparado aos controles negativo. Nos pacientes do grupo asma IgG1 anti-Asc positivo, foram observados maiores níveis de IL-6 e eosinófilos, em comparação aos do grupo controle positivo. No grupo asma IgG1 negativo houve maiores números de neutrófilos e eosinófilos, em comparação aos pacientes controle negativo. Os grupos asmáticos, independente da presença de IgE anti-Asc, apresentaram maior frequência de indivíduos com IL-10 e IFN-. Apenas os indivíduos do grupo asma IgG1 anti-Asc negativo apresentaram maior frequência de pacientes IL-10 e IFN-γ. Diante disso, o fato de ter asma favoreceu a produção de IL-6, TNF-α e eosinófilos, bem como IL-10 e IFN-γ, independente da presença da IgE anti-Asc. Contudo, em pacientes asmáticos a presença da IgG1 anti-Asc parece interferir melhorando a produção de IL-6, eosinófilos, mas não de IL-10 e IFN-γ.

Master of Science / Department of Diagnostic Medicine and Pathobiology / Melinda J. Wilkerson / Failure of passive transfer (FPT) is one of the main reasons for increased mortality rate in newborn calves and diagnosis is dependent on determination of serum IgG concentrations (diagnosis is based on < 1 g/dL of total IgG). Several qualitative assays are available, but the reference method, single radial immunodiffusion assay (SRID), albeit quantitative measures only one subclass at a time. We set out to develop a competitive multiplex microsphere flow cytometry assay to measure bovine IgG1 and IgG2 concentrations in 30 serum samples acquired from newborn Holstein calves prior to and 24 hours after ingestion of colostrum and to compare the values with SRID. A triplex bead assay was created by mixing three distinct sets of Quantum plex carboxylated fluorescent microspheres that were coated with purified bovine IgG1, IgG2 or albumin using a two step chemical reaction. The triplex protein coated beads were reacted with a cocktail of sheep anti-bovine IgG1 and IgG2. Evaluation of analytical specificity demonstrated cross reactivity between anti-bovine IgG2 and IgG1 coated beads that precluded determination of IgG2 > 0.5 g/dL. Cross reactivity between anti-IgG1 and IgG2 coated beads was minimal and did not affect IgG1 concentrations between 0.15 to 1.2 g/dL. A competitive linear decrease in the fluorescence intensity was observed in the triplex assay when 2-fold dilutions spanning a concentration range of 12 mg/dL – 100 mg/dL of either purified bovine IgG1 or IgG2 were included as a competitive inhibitor of the reaction. Precolostral serum samples from 29 calves were determined to be < 0.4 g/dL by SRID. Standard calibrants for the flow assay were prepared from two fold serial dilutions of purified bovine IgG (stock concentration 10 g/dL) using a precolostral calf serum pool as the diluent. The standard calibrants (IgG1 was 1.0- 0.16 g/dL and IgG2 was 3.4 – 0.22 g/dL) were used as the inhibitors in a triplex assay to develop a standard curve for unknown samples. Dilutions of bovine reference serum containing known amounts of IgG1 (1.2 – 0.15 g/dL) and IgG2 (1.6 – 0.2 g/dL) was used as positive control. The intra Intra-assay and inter-assay precision of the mutiplex assay was good (coefficient of variation < 10%). Since the IgG2 concentrations of post colostral samples were below detection limit, only IgG1 values were compared to the SRID. The agreement between triplex microsphere assay and SRID for IgG1 was poor with a mean bias of 0.743 g/dL towards triplex microsphere assay (95% confidence interval of 0.382 to 1.105 g/dL). Method comparison studies between total IgG determined by SRID and the gamma-globulin fraction determined by serum electrophoresis indicated that the SRID calculated higher values than the protein method (mean bias of -1.4 g/dL, 95% confidence interval was -1.8 to -1.05 g/dL). We hypothesized that the positive bias for the microsphere assay was explained in part by the use of dilution factors, use of standards that had a low analytical range, and erroneously high standards used in the SRID method.

HLA-DR1 transgenic (DR1 Tg) mice provide a model for evaluating the breadth and specificity of CD4 T cell responses that may develop in humans following influenza infection or vaccination. Recent studies identified a tremendously broad HLA-DR1-restricted CD4 T cell responses in DR1 Tg mice infected intranasally with influenza A/New Caledonia/20/99 (NC). In this study, our goals were to characterize B cell responses after NC infection in DR1 Tg mice and establish the correlation between B cell responses and CD4 T cell responses in this system. Influenza-specific B cell responses following virus administration were analyzed in DR1 Tg mice and in the genetically matched H-2b strain C57BL/10J (B10). Following intranasal (i.n.) NC infection, B cell responses in B10 mice featured strong IgG2b and IgG2c production and were typical of previously described B cell responses to a variety of mouse-adapted influenza strains. In contrast, B cell responses in DR1 Tg mice followed delayed kinetics and were strongly skewed to IgG1 production, suggesting the Th2 polarization of CD4 T cell responses. The different antibody isotype profile in DR1 Tg mice compared to B10 mice was evident in antibody secreting cells (ASCs) frequencies and in circulating Abs levels. Surprisingly, although DR1 Tg mice had lower influenza-specific Abs levels, they exhibited higher neutralizing Abs titers early in the response. B cell responses following intranasal infection of influenza A/Puerto Rico/8/1934 (PR8) or intramuscular vaccination of inactivated NC in DR1 Tg mice were different from the observed IgG1 bias after i.n. NC infection. After i.n. PR8 infection, B cell responses were similar in DR1 Tg mice and B10 mice, characterized by predominant IgM/IgG3 production. Additionally, following intramuscular administration of inactivated NC, B cell responses were skewed towards IgG2c production in both DR1 Tg mice and B10 mice, suggesting the Th1 polarization of CD4 T cell responses. A mechanistic understanding of IgG1/Th2 biased B cell responses and better neutralizing Abs production in DR1 Tg mice following i.n. NC infection may have implications for the optimal control of influenza infection.

HLA-DR1 transgenic (DR1 Tg) mice provide a model for evaluating the breadth and specificity of CD4 T cell responses that may develop in humans following influenza infection or vaccination. Recent studies identified a tremendously broad HLA-DR1-restricted CD4 T cell responses in DR1 Tg mice infected intranasally with influenza A/New Caledonia/20/99 (NC). In this study, our goals were to characterize B cell responses after NC infection in DR1 Tg mice and establish the correlation between B cell responses and CD4 T cell responses in this system. Influenza-specific B cell responses following virus administration were analyzed in DR1 Tg mice and in the genetically matched H-2b strain C57BL/10J (B10). Following intranasal (i.n.) NC infection, B cell responses in B10 mice featured strong IgG2b and IgG2c production and were typical of previously described B cell responses to a variety of mouse-adapted influenza strains. In contrast, B cell responses in DR1 Tg mice followed delayed kinetics and were strongly skewed to IgG1 production, suggesting the Th2 polarization of CD4 T cell responses. The different antibody isotype profile in DR1 Tg mice compared to B10 mice was evident in antibody secreting cells (ASCs) frequencies and in circulating Abs levels. Surprisingly, although DR1 Tg mice had lower influenza-specific Abs levels, they exhibited higher neutralizing Abs titers early in the response. B cell responses following intranasal infection of influenza A/Puerto Rico/8/1934 (PR8) or intramuscular vaccination of inactivated NC in DR1 Tg mice were different from the observed IgG1 bias after i.n. NC infection. After i.n. PR8 infection, B cell responses were similar in DR1 Tg mice and B10 mice, characterized by predominant IgM/IgG3 production. Additionally, following intramuscular administration of inactivated NC, B cell responses were skewed towards IgG2c production in both DR1 Tg mice and B10 mice, suggesting the Th1 polarization of CD4 T cell responses. A mechanistic understanding of IgG1/Th2 biased B cell responses and better neutralizing Abs production in DR1 Tg mice following i.n. NC infection may have implications for the optimal control of influenza infection.

Allergic asthma is a chronic inflammatory airway disease that is dominated by Th2 immune responses, with accumulation of eosinophils, IgE and IgG1 production, and airway hyperresponsiveness. We reported previously that treatment of OVA-asthmatic mice with allergen-presenting IL-10-differentiated dendritic cells (DC) (DC10) leads to progressive and long-lasting full-spectrum asthma tolerance. However, little has been done in investigating a role for antigen-specific B cells in DC10-induced tolerance. In this study, we characterized the surface markers of DC10 and found that these cells expressed lower levels of CD40, CD80, MHC II, PD-L1 and PD-L2 relative to immunostimulatory LPS-differentiated DCs (DCLPS). Co-culturing DC10 or DC10-induced regulatory T cells (iTreg) with CD4+ Th2 effector T cells from asthmatic mice led to a marked suppression of DCLPS-induced T effector cell proliferation. Moreover, DC10 treatment of asthma phenotype mice down-regulated airway eosinophilic inflammation as determined 48 h after a recall allergen challenge, and reduced pulmonary parenchymal tissue OVA-specific IgG1-secreting (OVA-IgG1) plasma cell numbers. The number of lung OVA-specific IgG1 plasma cells decreased by 46.7% over a 2 week period in the absence of repeated allergen challenge, while the numbers of bone marrow OVA-specific IgG1 plasma cells stayed relatively stable over a 6 week period, as determined 48 h after a single allergen challenge of asthmatic mice. DC10 treatment had a significant impact on the serum of IgG1/IgE response. To address the question of how DC10 influence OVA-IgG1 plasma cells responses, we co-cultured enzymatically-dispersed lung total cells from asthmatic mice with or without DC10, and found that the DC10 significantly suppressed OVA-IgG1 plasma cell antibody production. To determine whether DC10 required input from T cells to accomplish this, we co-cultured CD4 T cell-depleted, B cell-enriched populations from the lungs of asthmatic mice with or without DC10, and found that DC10 strongly (65.4+/-3.5%) suppressed OVA-IgG1 plasma cells in CD4 T cell-depleted lung cell cultures. To assess whether DC10-induced Treg also suppress IgG1-secretion, we co-cultured lung CD4+ T cells from untreated or DC10-tolerized asthmatic mice with total lung cells from asthmatic donors, and found that the DC10-induced Tregs effectively (52.2+/-8.7%) suppressed OVA-IgG1 plasma cell responses. In summary, DC10 treatment strongly down-regulate OVA-specific IgG1 plasma cell responses of asthmatic mice, both in vivo and in vitro by at least two mechanisms: directly via DC10 as well as indirectly through DC10-induced Tregs.

No description available.

Conventional techniques within the beef cattle industry involve weaning the calf from the dam when the calf is about 205 days of age. Weaning induces a stress-response that is implicated in reducing the health and productivity of newly weaned calves. Our goal was to evaluate the impact of weaning on the stress immune responses of beef calves. To that end, we 1) evaluated novel methods to quantify physiological markers of stress, 2) compared immune function and growth of calves grazing legume versus grass forages, and 3) compared the effects of abrupt versus two-stage weaning on calves. In study 1, calf, yearling, and adult beef cattle were used to assess the accuracy and precision of handheld glucometers in quantifying bovine blood glucose concentration. Precision Xtra® and ReliOn® glucometers were used chute side to quantify blood glucose concentrations in cattle and were compared to an accepted plasma glucose analysis on the same samples for validation. The Precision Xtra® glucometer was more accurate and precise than the ReliOn® glucometer. In study 2, weaned heifers were used to compare the immunomodulatory effects of grazing alfalfa versus fescue over a 30 day grazing period. No differences were detected in the interferon gamma (IFNγ) production and weight gain between the heifers on alfalfa and fescue. In study 3, effects of two-stage (fenceline) and abrupt weaning were compared. Calf weights, immune cell function, antibody production, blood glucose concentrations, fecal cortisol concentrations, and gene expression (FAS, IL-4,IL-10, and IFNγ) were measured pre- and post-weaning. On the day after weaning, the abruptly weaned calves had higher blood glucose concentrations than fenceline weaned calves. Fecal cortisol concentration and gene expression of FAS and IL-4 increased in both groups after weaning, but no differences were detected between the weaning treatments. Gene expression of IL-10 and IFNγ did not change over time. No date, treatment or treatment*date effect was detected for total weight gain or IFNγ production within the non-stimulated and the mitogen-stimulated whole blood samples. / Master of Science

L'embolisation artérielle a récemment émergé sur le plan interventionnel comme un traitement sûr et efficace pour arrêter une hémorragie ou induire la dévascularisation d'un tissu cible. La visualisation directe des agents d'occlusions vasculaires n'est pas toujours possible. L'estimation de leur position dans la branche vasculaire en se basant sur la distribution de produit opaque aux rayons X est partiellement incorrecte. Un marquage des agents d'occlusion vasculaire est souhaitable pour leur suivi par IRM pendant l'intervention ou a posteriori, ce, notamment en raison de la diffusion de l'IRM comme modalité d'imagerie anatomique et interventionnelle. Dans ce travail, plusieurs protocoles ont été établis pour repérer des agents d'occlusion vasculaire marqués avec le SPIO, in situ, in vitro et dans des « conditions » in vivo, sous IRM 1,5T et 3T. Les agents d'occlusion étudiés sont des microsphères de trisacryl (Embosphère®), et des microparticules ou microcapsules utilisées éventuellement comme vecteurs de principes actifs : parmi celles-ci, des microsphères gonflables (Hépasphère™) non dégradables ont été marquées, et des microbilles d'alginate et des systèmes d'émulsions dégradables réalisés par nos soins. L'établissement de protocoles de détectabilité sous IRM a impliqué le choix de séquences et l'optimisation des paramètres de ces séquences, le choix de l'antenne, ainsi que la mise au point d'une quantité suffisante de marqueur dans les agents d'occlusion afin qu'ils soient visibles à l'IRM. Une méthodologie de mesure de faible variation de l'intensité du signal des images IRM a été développée. Un modèle expérimental in vitro a été conçu avec la présence des microparticules marquées dans un environnement simulant grossièrement une vascularisation tumorale et son drainage veineux. La fixation des immunoglobulines (IgG1), équivalentes aux anti-VEGF, sur des microsphères (Hépasphères™) a été étudiée. Enfin, les propriétés mécaniques et électriques des systèmes de microémulsions ont été explorées par ultrasons et par impédancemétrie.

One of the purposes of allergen-specific immunotherapy (SIT) is to modulate the humoral immune response against allergens with significant increases in allergen-specific IgG1 and IgG4 levels. These antibodies are associated with blocking activity by preventing IgE binding to allergen and leading to reduced inflammatory responses. This study aimed to investigate in vitro blocking activity of allergen-specific IgG antibodies on IgE reactivity to D. pteronyssinus (Dpt) in sera from atopic patients. Dpt-specific IgG antibodies were obtained from atopic sera and irrelevant IgG from non-atopic sera. IgG antibodies were purified by ammonium sulfate precipitation followed by Protein-G affinity chromatography and evaluated with regards to purity by SDS-PAGE and immunoreactivity by slot-blot and immunoblot assays. The blocking activity was evaluated by inhibition ELISA. The electrophoretical profile after salting-out precipitation showed an enrichment of high molecular weight proteins in the precipitated fraction and strongly stained bands in the ligand fraction after chromatography, compatible with molecular weight of human IgG. It was detected strong immunoreactivity to IgG, negligible to IgA, and no reactivity to IgE and IgM. Dpt-specific IgG fraction was capable to significantly reduce levels of IgE anti-Dpt, resulting in 35-51% inhibition of IgE reactivity to Dpt in atopic patient sera. Allergen-specific IgG antibodies purified using available and standardized methodology are able to inhibit IgE reactivity to Dpt allergen extract. In addition to the clinical symptoms improvement (subjective parameter), this approach reinforces that the intermittent measurement of serum allergen-specific IgG antibodies will be an important objective laboratorial parameter that will help specialists to follow their patients under SIT. / Uma das propostas da imunoterapia alérgeno específica é a de modular a resposta imune humoral contra alérgenos, com aumento significativo nos níveis de IgG1 e IgG4 específicos. Esses anticorpos estão associados com uma atividade bloqueadora, impedindo a ligação de anticorpos IgE ao alérgeno e levando a uma redução nas respostas inflamatórias. Esse estudo objetivou investigar a atividade bloqueadora, in vitro, de anticorpos IgG específicos sobre a reatividade de IgE a D. pteronyssinus (Dpt) em soros de pacientes atópicos. Anticorpos IgG específicos foram obtidos de soros de pacientes atópicos, e IgG irrelevante a partir de soros de não atópicos, e depois purificados por precipitação com sulfato de amônio, seguido de cromatografia de afinidade em Proteina G-agarose. A pureza desses anticorpos foi avaliada por SDS-PAGE, a imunoreatividade por ensaios de slot-blot e immunoblot, e a atividade bloqueadora por ELISA inibição. O perfil eletroforético, após precipitação com sulfato de amônio, mostrou um enriquecimento de proteínas de alto peso molecular na fração precipitada,e bandas fortemente coradas na fração ligante após a cromatografia, compatíveis com o peso molecular de IgG humana. Foi detectada uma forte imunoreatividade para IgG, leve para IgA, e nenhuma reatividade para IgE e IgM. A Fração IgG específica foi capaz de reduzir significantemente os níveis de IgE anti-Dpt, resultando em 35-51% de inibição da reatividade de IgE a Dpt em pools de soros de pacientes atópicos. Anticorpos IgG específicos purificados, através de uma metodologia disponível e padronizada, são capazes de inibir a reatividade de IgE ao extrato alergênico Dpt. Além da melhoria da sintomatologia clínica, considerada um parâmetro subjetivo, essa abordagem reforça que a avaliação intermitente de anticorpos IgG alérgeno-específicos pode ser uma ferramenta importante, auxiliando especialistas a acompanharem seus pacientes em processo de imunoterapia específica. / Mestre em Ciências da Saúde