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Considerando os efeitos da radiação gama sobre proteínas e a capacidade do sistema imune de reconhecer macromoléculas modificadas, decidimos avaliar alguns aspectos imunológicos de camundongos B10.PL frente a ovalbumina e bothropstoxina–1 (BTHX-1), nas formas nativa e irradiada. Para avaliar prováveis modificações estruturais nas moléculas das proteínas após o processo de irradiação (radiação gama de 60Co), foi realizada a eletroforese em gel de poliacrilamida 15% para a ovalbumina e para a BTHX-1, nas formas nativa e irradiada. A ovalbumina também foi submetida à cromatografia de exclusão molecular, enquanto a BTHX-1 foi submetida à espectrometria de massa. Os resultados destes ensaios mostraram que a radiação gama foi capaz de promover alterações nas moléculas de ovalbumina e BTHX-1. A fim de avaliar a toxicidade da BTHX-1 irradiada em relação à nativa realizou-se um ensaio de citotoxicidade celular em células CHO. O resultado mostrou que a toxina na sua forma irradiada apresentou, aproximadamente, cinco vezes menos toxicidade do que a toxina na sua forma nativa. Com relação aos aspectos imunológicos, foram realizados ensaios de produção e identificação de anticorpos, nos quais, os animais foram imunizados com ovalbumina e BTHX-1, nas formas nativa ou irradiada. Observou-se que as proteínas nativas induziram, preferencialmente, uma resposta do tipo Th2, enquanto que as proteínas irradiadas induziram uma resposta do tipo Th1. Realizou-se um ensaio de proliferação celular para avaliar o comportamento de esplenócitos, retirados do baço de camundongos B10.PL, imunizados com ovalbumina e BTHX-1, nativas ou irradiadas, cultivados em presença de ambas as formas das proteínas. Em relação à ovalbumina, os resultados mostraram que tanto as células dos animais imunizados com a ovalbumina nativa como aquelas dos animais imunizados com a proteína na sua forma irradiada apresentaram crescimento semelhante. No caso da BTHX-1, os resultados mostraram que as células dos animais imunizados com a toxina irradiada foram capazes de reconhecer a forma nativa da toxina, pois apresentaram crescimento semelhante ao das células dos animais imunizados com a BTHX-1 nativa. / Considering the effects of gamma radiation on proteins and the capacity of the immune system to recognize modified macromolecules, we decided to evaluate some immunological aspects of B10.PL mice exposed to native or irradiated ovalbumin and bothropstoxin-1 (BthTx-1). In order to evaluate possible structural modifications of the molecules after being irradiated (60Co gamma rays), bothropstoxin-1 was analysed by electrophoresis, while ovalbumin was submitted to analytical size exclusion chromatography. The toxin was also analised by ESI-mass spectrometry. Our results indicate that radiation promoted modifications on both the molecules. Aiming to compare the toxicity of the native and irradiated forms of the toxin, an in vitro citotoxicity assay, using CHO cells, was performed. According to our results, the modified toxin was 5 folds less toxic than its native counterpart. Sera of animals immunized with the native and irradiated proteins were analyzed in order to evaluate levels of IgG, as well as to quantify specific isotypes. While the native proteins induced a predominant Th2 response, the irradiated molecules apparently promoted a switch towards a Th1 pattern. We also performed a cell proliferation assay with splenocites from mice immunized with either the native or the irradiated proteins, cultured in the presence of the antigens. Our results indicate that both the forms of the proteins induced a similar proliferative response. These data indicate a potential use of detoxified proteins as antigens for immunization.

Glycosylation is among the most common post-translational protein modifications. Glycans are complex carbohydrates attached to the surface of many proteins, but are rarely extensively studied in a high-throughput manner. However, there is an increasing evidence of their involvement in various physiological processes and diseases. Glycosylation of Immunglobulin G was shown to be important in adaptive immunity, where it can act as a "safety switch" for different types of the immune response. Although the main enzymes of the glycosylation pathway are known, little is understood about how this template-independent process is regulated to result in a faithful synthesis of a specific glycoform. This question was previously addressed using genome-wide association studies (GWAS) and 9 loci were identified as being significantly associated with IgG N-glycosylation. Only 4 of these loci were the known glycosylation enzymes. An additional five loci were discovered by applying a newly developed multivariate GWAS method on the same dataset. Here, by performing a GWAS on 77 IgG N-glycan traits measured by ultra-performance liquid chromatography in more than 8000 samples from four European cohorts the number of genome-wide significant (p? ≤ 2.4 x 10−9) loci increased to 27, 15 of which are novel, with 6 additional loci being suggestively associated (p? ≤ 2.4 x 10−8). To assess which of the genes from the associated loci are more likely to be regulating IgG glycosylation, different gene prioritising strategies were employed. For 7 loci evidence of a non-synonymous amino acid change was found, two of which were predicted to be deleterious. Evidence of regulation through changes in gene expression levels in B-cells, the cell lineage responsible for production of IgG, was found for 4 genes, with an additional 11 genes exhibiting the same evidence with expression in peripheral blood or other immune cells. For the remaining loci the most likely candidate gene was proposed based on co-expression with genes from the enriched gene-sets or based on a physical proximity to the variant with the strongest association. To narrow down the most important loci for a functional follow-up, the omics nature of this data was used to compare glycome-wide SNP effects and suggest how newly discovered loci form a functional network that regulates the established members of the glycosylation pathway. The potential role of IgG glycosylation in various complex traits and diseases was explored by assessing the pleiotropy of the associated SNPs. The inflation of SNPs related to autoimmune, digestive and neurological diseases was observed in glycosylation SNPs. To assess whether IgG N-glycosylation is likely to share the same causal variant as the identified pleiotropic traits and diseases, regional association patterns were compared using summary data based Mendelian Randomisation analyses. This work demonstrates that an increased sample size empowered the identification of novel loci, enabling further insights into the molecular mechanisms underlying protein glycosylation and its relationship with complex human diseases. It also shows that such analyses of omic traits can assist in creating a functional network of the identified loci, prioritising the most important genes and allowing a more focused approach to future experimental functional follow-up.

Considerando os efeitos da radiação gama sobre proteínas e a capacidade do sistema imune de reconhecer macromoléculas modificadas, decidimos avaliar alguns aspectos imunológicos de camundongos B10.PL frente a ovalbumina e bothropstoxina–1 (BTHX-1), nas formas nativa e irradiada. Para avaliar prováveis modificações estruturais nas moléculas das proteínas após o processo de irradiação (radiação gama de 60Co), foi realizada a eletroforese em gel de poliacrilamida 15% para a ovalbumina e para a BTHX-1, nas formas nativa e irradiada. A ovalbumina também foi submetida à cromatografia de exclusão molecular, enquanto a BTHX-1 foi submetida à espectrometria de massa. Os resultados destes ensaios mostraram que a radiação gama foi capaz de promover alterações nas moléculas de ovalbumina e BTHX-1. A fim de avaliar a toxicidade da BTHX-1 irradiada em relação à nativa realizou-se um ensaio de citotoxicidade celular em células CHO. O resultado mostrou que a toxina na sua forma irradiada apresentou, aproximadamente, cinco vezes menos toxicidade do que a toxina na sua forma nativa. Com relação aos aspectos imunológicos, foram realizados ensaios de produção e identificação de anticorpos, nos quais, os animais foram imunizados com ovalbumina e BTHX-1, nas formas nativa ou irradiada. Observou-se que as proteínas nativas induziram, preferencialmente, uma resposta do tipo Th2, enquanto que as proteínas irradiadas induziram uma resposta do tipo Th1. Realizou-se um ensaio de proliferação celular para avaliar o comportamento de esplenócitos, retirados do baço de camundongos B10.PL, imunizados com ovalbumina e BTHX-1, nativas ou irradiadas, cultivados em presença de ambas as formas das proteínas. Em relação à ovalbumina, os resultados mostraram que tanto as células dos animais imunizados com a ovalbumina nativa como aquelas dos animais imunizados com a proteína na sua forma irradiada apresentaram crescimento semelhante. No caso da BTHX-1, os resultados mostraram que as células dos animais imunizados com a toxina irradiada foram capazes de reconhecer a forma nativa da toxina, pois apresentaram crescimento semelhante ao das células dos animais imunizados com a BTHX-1 nativa. / Considering the effects of gamma radiation on proteins and the capacity of the immune system to recognize modified macromolecules, we decided to evaluate some immunological aspects of B10.PL mice exposed to native or irradiated ovalbumin and bothropstoxin-1 (BthTx-1). In order to evaluate possible structural modifications of the molecules after being irradiated (60Co gamma rays), bothropstoxin-1 was analysed by electrophoresis, while ovalbumin was submitted to analytical size exclusion chromatography. The toxin was also analised by ESI-mass spectrometry. Our results indicate that radiation promoted modifications on both the molecules. Aiming to compare the toxicity of the native and irradiated forms of the toxin, an in vitro citotoxicity assay, using CHO cells, was performed. According to our results, the modified toxin was 5 folds less toxic than its native counterpart. Sera of animals immunized with the native and irradiated proteins were analyzed in order to evaluate levels of IgG, as well as to quantify specific isotypes. While the native proteins induced a predominant Th2 response, the irradiated molecules apparently promoted a switch towards a Th1 pattern. We also performed a cell proliferation assay with splenocites from mice immunized with either the native or the irradiated proteins, cultured in the presence of the antigens. Our results indicate that both the forms of the proteins induced a similar proliferative response. These data indicate a potential use of detoxified proteins as antigens for immunization.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Orientador: Sônia Maria Alves Bueno / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-26T10:22:21Z (GMT). No. of bitstreams: 1 Silva_LuanaCristinaAndradeda_D.pdf: 3401459 bytes, checksum: 94b29258ef88b106fed9841a38d11161 (MD5) Previous issue date: 2014 / Resumo: As imunoglobulinas do tipo G humana (IgG) são amplamente empregadas em tratamentos terapêuticos contra câncer, doenças auto-imunes e imunodeficiência adquirida e na área analítica, em testes diagnósticos. Os fragmentos Fab podem ser obtidos por clivagem enzimática da molécula de IgG nativa ou por tecnologia de DNA recombinante. O fragmento Fab é composto por regiões constantes e variáveis, sendo que as regiões variáveis de cadeias leve e pesada compõem o fragmento Fv. Tanto os fragmentos Fab quanto os fragmentos Fv foram citados como segunda e terceira geração de anticorpos, respectivamente, e têm sido crescente o interesse em empregá-los em tratamentos terapêuticos, visto que ambos possuem menor massa molar em relação a IgG intacta e preservam a capacidade de se ligar ao antígeno. Como a utilização destas proteínas nas áreas terapêutica e analítica requer um alto grau de pureza, estratégias de purificação vêm sendo desenvolvidas. No intuito de contribuir para o desenvolvimento de processos de purificação de fragmento Fab, estudou-se o emprego de ligantes alternativos aos bioespecíficos proteínas A, G e L, comumente empregados para este fim. Neste trabalho, a purificação dos fragmentos Fab foi realizada por cromatografia de afinidade com íons metálicos imobilizados (IMAC) e fenil boronato (FB). Quanto a IMAC, avaliou-se o efeito dos agentes quelantes IDA (ácido iminodiacético) e TREN (Tris 2(aminoetil) amina), os íons metálicos Cu2+, Ni2+ e Co2+ e os tampões Tris-HCl, fosfato de sódio e HEPES na ausência e na presença de NaCl para a purificação de Fab a partir de solução de clivagem enzimática de IgG humana policlonal. Os resultados foram analisados conjuntamente por Western blot e imunodifusão radial. No que se refere à pureza dos fragmentos Fab obtidos na etapa de flowthrough, a ordem crescente obtida foi TREN-Ni2+ (100%) > IDA-Ni2 (88,6%) > IDA-Co2+ (71,1%) > IDA-Cu2+ (63,9%). Em relação ao rendimento, obteve-se a seguinte ordem: TREN-Ni2+ (86,2%) = IDA-Co2+ (86,2%) > IDA-Ni2+ (74,3%) = IDA-Cu2+ (73,7%). No tocante à capacidade de adsorção de proteína total por mL de gel, obteve-se a ordem 14,3 mg mL-1 ( TREN-Ni2+) > 11,2 mg mL-1 (IDA-Ni2+) > 5,65 mg mL-1 (IDA-Cu2+) > 4,3 mg mL-1 (IDA-Co2+). O ligante FB adsorveu os fragmentos Fab e Fc e não promoveu a separação destes. Este trabalho possibilitou concluir que os agentes quelantes IDA e TREN com os íons metálicos empregados, possibilitam separar os fragmentos Fab de fragmentos Fc e obtê-los com grau de pureza satisfatório / Abstract: Polyclonal human G immunoglobulins are widely applied in therapeutic treatments for cancer, autoimmune diseases and immunodeficiency and in the analytical area of diagnostic testing. The human IgG Fab fragments can be obtained via enzymatic cleavage of the native molecule or recombinant DNA technology. The Fab is composed of constant regions (CL and CH) and variable (VL and VH). The variable regions of the light and heavy chains comprise the Fv fragment. Fab fragments and Fv fragments were cited as second and third generation of antibodies, respectively, and has been an increasing interest in employing them in therapeutic treatments, as both have molecular weight less than the intact IgG and preserve the affinity for the antigen. The applied these proteins in therapy and analytical investigation require a high degree of purity, so, different purification strategies have been developed. In order to contribute to the development of Fab fragment purification processes, we studied the use of alternative biospecific binding to proteins A, G and L, commonly applied for this purpose. In this work, the purification of the Fab fragments was performed by affinity chromatography with chelated metals (IMAC) and phenyl and boronate (PB) ligand immobilized on agarose gel. In this investigation, the metal chelates Cu 2+, Ni 2+ and Co 2+ were studied when tested in IDA (iminodiacetic acid) and TREN (Tris 2 (aminoethyl) amine) and buffer system Tris-HCl, sodium phosphate and HEPES with or without NaCl for purification of Fab fragments from enzymatic cleavage solution of polyclonal human IgG. The results of Western blot and radial immunodiffusion were analyzed together. For the purity of the Fab fragments obtained in flowthrough, the increasing order was obtained TREN-Ni2+ (100%) > IDA-Ni2+ (88.6%) > IDA-Co2+ (71.1%) > IDA-Cu2+ (63.9%). In relation to yield, we obtained the following order: TREN-Ni2+ (86.2%) = IDA-Co2+ (86.2%) > IDA-Ni2+ (74.3%) = IDA-Cu2+ (73.7% ). With regard to the total capacity protein adsorption per ml of gel, was obtained the order 14.3 mg mL-1 (TREN-Ni2+) > 11.2 mg mL-1 (IDA-Ni2+) > 5.65 mg ml -1 (IDA-Cu2+) > 4.3 mg ml-1 (IDA-Co2+). The binder FB adsorbed Fab and Fc did not cause separation thereof. This work led us to conclude that the chelating agents IDA and TREN with the metal ions used, enable separate the Fab fragments of Fc fragments and get them with satisfactory degree of purity / Doutorado / Desenvolvimento de Processos Biotecnologicos / Doutora em Engenharia Quimica

No description available.

Leishmaniasis is protozoan diseases, which is transport into the host during the feeding of sand fly. During the feeding of infected sand flies not only the leishmania but also the sand fly saliva are inoculated into the hosts. Sand fly saliva can strongly affect the response of the immune system. If the host hadn't met sand fly saliva yet, the course of infection is usually worse. In cutaneous leishmaniasis, the lesions developed early, being more destructive and perstiting longer, if not healed. The hosts living in endemic areas of leishmaniasis and the vector hosts are often exposed to feeding uninfected sand flies. To hosts are repeatedly inoculated the sand fly saliva antigen and induced specific cellular and antibody responses. Cellular and antibody responses are different for different hosts, attempts were made most frequently in murine and canine models. In humans, as host sis it difficult to monitor development leishamnia infectipon after previous exposure, that's why in humans mainly it is monitors the levels of antibodies, according to which we can determine the extent of sand fly bited and the risk of transmission of leishmaniasis. The specifity of immune responses against sand fly saliva is important for the testing new type of controlling and healing programs against sand fly and...

Low affinity Fcγ receptors (FcγR) mediate key immune effector mechanisms through the engagement of the Fc portion of immunoglobulin G (IgG). These receptors are involved in multiple biological processes, including clearance of antigen/antibody immune complexes, enhancement of antigen presentation, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, regulation of antibody production, and activation of inflammatory cells. FcγR phenotypic variability modulates these processes through altering receptor IgG subclass binding affinity (FcγRIIa-H131R and FcγRIIIa-F158V), subcellular localization (FcγRIIb-I232T), post-translational modification (FcγRIIIb-HNA1a/b/c), expression of an otherwise pseudogene (FcγRIIc), and receptor surface density (gene copy number variability and promoter haplotypes). Accumulating data suggest that FcγR-mediated effector functions play a significant role in HIV-1 protective immunity, which is substantiated by the association of FcγR phenotypic variants with HIV-1 disease outcome. This study set out to characterize FcγR functional variability in the South African population, and to investigate the potential role thereof in HIV-1 transmission and disease progression in South African Black individuals. Since the only known determinant of FcγRIIIa surface density – FCGR3A gene copy number – is rare, this study investigated novel genetic determinants of FcγRIIIa expression by flow cytometry and nucleotide sequencing. FcγRIIIa expression on peripheral blood mononuclear cells was characterized for 32 South African Caucasian individuals and 22 South African Black individuals (Chapter 3). Significant differences in the proportion of FcγRIIIa-positive monocytes and FcγRIIIa expression levels on natural killer (NK) cells were observed between the population groups. A novel four-variant FCGR3A intragenic haplotype that associated with increased surface expression of FcγRIIIa on NK cells was detectable in Caucasian individuals, but not Black individuals and may account for the observed population differences. Further exploration of genetic diversity at the low affinity FCGR gene locus was extended to include all currently known functional variants of FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb using a commercial multiplex ligation-dependent probe amplification assay (Chapter 4). Thirty-two South African Caucasian individuals and 131 South African Black