Made available in DSpace on 2014-06-11T19:27:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-05-22Bitstream added on 2014-06-13T20:57:20Z : No. of bitstreams: 1 monteiro_ger_me_jabo.pdf: 4718837 bytes, checksum: 348326537ee49fc7b15073f8d7e90dfd (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / No presente estudo avaliou-se o desenvolvimento de resistência a ninfas do ixodídeo Amblyomma cajennense, induzida por infestações controladas, e a possível existência de reatividade cruzada com Amblyomma hebraeum. Para tal, caprinos sem contato prévio com carrapatos, com seis meses de idade, machos ou fêmeas, foram infestados três vezes sucessivamente com ninfas de A. cajennense. Ademais, outro grupo de caprinos foi infestado nas mesmas condições com ninfas de A. hebraeum e colhidos soros para pesquisa de possível reatividade cruzada com A. cajennense. Observou-se que caprinos desenvolveram resistência apenas parcial a ninfas de A. cajennense, como demonstrado pela alteração significativa dos seguintes parâmetros biológicos das ninfas, em relação à primoinfestação: redução de 41,7% e 37,1% no peso de ingurgitamento, respectivamente na 2ª e 3ª infestações; aumento de 20% no período de ecdise e redução de 25,7% na taxa de ecdise, respectivamente nas 3ª e 2ª infestações. Não houve alteração no período de ingurgitamento nem na porcentagem de recuperação de ninfas com as infestações sucessivas. A reação cutânea induzida pelas ninfas de A. cajennense durante as infestações em caprinos caracterizou-se por infiltrado de células inflamatórias, predominantemente neutrófilos e basófilos, estas últimas envolvidas na imunidade a carrapatos por meio de hipersensibilidade basofílica cutânea. Células apresentadoras de antígeno, nomeadamente macrófagos, linfócitos B e células dendríticas foram detectadas por imunoistoquímica em linfonodos drenantes de locais de fixação dos carrapatos, em maior número nos linfonodos dos animais infestados... / This study evaluated the acquision of resistance against Amblyomma cajennense nymphs in naïve goats, induced by repeated and controlled infestations, and a possible cross-reactivity with A. hebraeum. Ten naive goats, of both sexes, aged six months were used throughout the experiment. Animals were infested artificially thrice, 30 days intervals between infestations. In addition, goats were infested at same conditions with the tick A. hebraeum as well. Sera from these animals were collected looking for possible cross-reactivity between antigens from these ixodids. It was observed that goats developed partially resistance to A. cajennense nymphs from the 1st infestation on as shown by changes in some biological parameters, as follows: increase of 41.7% and 37.1% in engorgement weight, respectively at the 2nd and 3rd infestations, decrease in 25.7% in nymph s ecdise rate and increase of 20% in ecdise period, respectively at the 3rd and 2nd infestations. It was not observed alterations in engorgement period and percentage of yielded nymphs. Biopsies of tick bite lesions induced by A. cajennense nymphs during infestations in goats were characterized by inflammatory reaction with cellular influx by neutrophils and basophils predominantly, being the last one involved in the well-known cutaneous basophilia. Antigen presenting cells like macrophages, B-lymphocytes and dendritic cells were detected by immunohistochemical analysis of lymph nodes draining tick infested areas. It should be stressed that these cells were found in higher numbers at lymph nodes from infested goats than from the naïve ones. Western blotting analysis of nymphal A. cajennense extracts revealed shared polypeptides (160, 90 and 16kDa) when used sera from goats infested with A. cajennense either A. hebraeum, indicating cross reactivity between these two tick species.

Objetivo: O objetivo desse estudo foi avaliar alterações na resposta clínica e imunológica ao camarão após a imunoterapia com Dermatophagoides pteronyssinus. Métodos: Selecionou-se 35 indivíduos alérgicos a Dermatophagoides pteronyssinus (Der p), os quais foram submetidos a testes cutâneos de leitura imediata para ácaros, baratas, camarão, tropomiosina recombinante, além de cão, gato e fungos. A detecção de IgE espcífica in vitro foi feita para o ácaro, camarão, barata americana e para suas tropomiosinas. Em todos, avaliou-se reatividade clínica ao camarão através de provocação oral. Dez pacientes foram alocados para o grupo controle, e 25 foram submetidos à imunoterapia alérgeno específica para o ácaro. Os testes cutâneos e a dosagem de IgE sérica específica foram repetidas após a indução da imunoterapia, e após 1 ano do início. A reatividade clínica ao camarão foi reavaliada no final do estudo pela provocação oral. Resultados: No grupo dos pacientes que foram submetidos à imunoterapia, observamos diminuição na reatividade nos testes cutâneos e dosagem de IgE específica para Der p, camarão e tropomiosina recombinante. Dos 10 pacientes com testes cutâneos positivos para camarão, 4 foram negativos na dosagem após um ano de imunoterapia (p= 0,04). Quanto à dosagem sérica de IgE para camarão, dos 9 positivos no início, 6 ficaram negativos (p= 0,014). Nenhum paciente submetido a imunoterapia desenvolveu nova sensibilização para camarão. Não houve alteração na reatividade clínica ao camarão após imunoterapia. Conclusão: A imunoterapia para Dermatophagoides pteronyssinus foi acompanhada de diminuição da reatividade imunológica para camarão e clinicamente não houve alteração da sensibilidade a camarão / Objective: The objective of this study was to determine changes in clinical and immunological response to shrimp after immunotherapy with Dermatophagoides pteronyssinus. Methods: We studied 35 allergic subjects to Dermatophagoides pteronyssinus (Der p), submitted to skin tests to mites, cockroach, shrimp, recombinant tropomyosin, and dog, cat and fungi. The detection of serum specific IgE was performed to mite, shrimp, and tropomyosin from American cockroach. In all patients, the clinical reactivity to shrimp was assessed through oral challenge. Ten patients were allocated to the control group, and 25 were submitted to immunotherapy for mite. Skin tests and determination of serum specific IgE were repeated after the induction of immunotherapy (3-4 months) and 1 year after of beginning of the treatment. The clinical reactivity to shrimp was assessed again at the end of the study by oral challenge. Results: In the group of patients who were undergoing immunotherapy, we observed decreased reactivity in the skin tests and specific IgE levels to Der p, shrimp and recombinant tropomyosin. Among the 10 patients with positive skin tests to shrimp, 4 were negative when assessed after one year of immunotherapy (p = 0.04). About serum specific IgE to shrimp, from the 9 positive reactors in the beginning of treatment, 6 became negative (p= 0.014). There was no change in clinical reactivity to shrimp after immunotherapy. Conclusion: The immunotherapy for Dermatophagoides pteronyssinus was accompanied by decreased immune reactivity to shrimp and clinically there was no change in sensitivity to shrimp

Objetivo: O objetivo desse estudo foi avaliar alterações na resposta clínica e imunológica ao camarão após a imunoterapia com Dermatophagoides pteronyssinus. Métodos: Selecionou-se 35 indivíduos alérgicos a Dermatophagoides pteronyssinus (Der p), os quais foram submetidos a testes cutâneos de leitura imediata para ácaros, baratas, camarão, tropomiosina recombinante, além de cão, gato e fungos. A detecção de IgE espcífica in vitro foi feita para o ácaro, camarão, barata americana e para suas tropomiosinas. Em todos, avaliou-se reatividade clínica ao camarão através de provocação oral. Dez pacientes foram alocados para o grupo controle, e 25 foram submetidos à imunoterapia alérgeno específica para o ácaro. Os testes cutâneos e a dosagem de IgE sérica específica foram repetidas após a indução da imunoterapia, e após 1 ano do início. A reatividade clínica ao camarão foi reavaliada no final do estudo pela provocação oral. Resultados: No grupo dos pacientes que foram submetidos à imunoterapia, observamos diminuição na reatividade nos testes cutâneos e dosagem de IgE específica para Der p, camarão e tropomiosina recombinante. Dos 10 pacientes com testes cutâneos positivos para camarão, 4 foram negativos na dosagem após um ano de imunoterapia (p= 0,04). Quanto à dosagem sérica de IgE para camarão, dos 9 positivos no início, 6 ficaram negativos (p= 0,014). Nenhum paciente submetido a imunoterapia desenvolveu nova sensibilização para camarão. Não houve alteração na reatividade clínica ao camarão após imunoterapia. Conclusão: A imunoterapia para Dermatophagoides pteronyssinus foi acompanhada de diminuição da reatividade imunológica para camarão e clinicamente não houve alteração da sensibilidade a camarão / Objective: The objective of this study was to determine changes in clinical and immunological response to shrimp after immunotherapy with Dermatophagoides pteronyssinus. Methods: We studied 35 allergic subjects to Dermatophagoides pteronyssinus (Der p), submitted to skin tests to mites, cockroach, shrimp, recombinant tropomyosin, and dog, cat and fungi. The detection of serum specific IgE was performed to mite, shrimp, and tropomyosin from American cockroach. In all patients, the clinical reactivity to shrimp was assessed through oral challenge. Ten patients were allocated to the control group, and 25 were submitted to immunotherapy for mite. Skin tests and determination of serum specific IgE were repeated after the induction of immunotherapy (3-4 months) and 1 year after of beginning of the treatment. The clinical reactivity to shrimp was assessed again at the end of the study by oral challenge. Results: In the group of patients who were undergoing immunotherapy, we observed decreased reactivity in the skin tests and specific IgE levels to Der p, shrimp and recombinant tropomyosin. Among the 10 patients with positive skin tests to shrimp, 4 were negative when assessed after one year of immunotherapy (p = 0.04). About serum specific IgE to shrimp, from the 9 positive reactors in the beginning of treatment, 6 became negative (p= 0.014). There was no change in clinical reactivity to shrimp after immunotherapy. Conclusion: The immunotherapy for Dermatophagoides pteronyssinus was accompanied by decreased immune reactivity to shrimp and clinically there was no change in sensitivity to shrimp

SANTRAUKA Serologinių sifilio ir Laimo boreliozės reakcijų kryžminis reaktyvumas Darbo autorė: Tatjana Subočiūtė Darbo vadovas: med. m. dr. Andrius Vagoras Darbo tikslas: nustatyti VULSK diagnostikos centre rutiniškai naudojamų serologinių sifilio ir Laimo boreliozės testų galimai klaidingai teigiamų rezultatų dažnumą dėl kryžminio viena iš šių infekcijų užsikrėtusiųjų pacientų kraujo serumų reaktyvumo. Darbo metodai: Tyrimai buvo atlikti Vilniaus universiteto ligoninės Santariškių klinikos (VULSK) Laboratorinės diagnostikos centro Mikrobiologijos laboratorijos Infekcijų žymenų tyrimų padalinyje. Serumai buvo surinkti ir ištirti nuo 2005 m. vasario iki 2006 m. kovo mėn. Tyrimo medžiagą sudarė 40 serumų. 23 serumai dėl sifilio tyrimų buvo gauti iš VULSK Dermatovenerologijos centro. 8 serumai dėl Laimo ligos tyrimų buvo gauti iš VULSK Dermatovenerologijos centro. UAB „Endemik” atrinkti 9 serumai ištirti dėl Laimo ligos. Visi į tyrimą įtraukti serumai buvo ištirti šiomis reakcijomis: 1) RPR su titru, 2) TPHA, 3) Treponema pallidum ELISA, 4) Treponema pallidum imunofluorescencija, 5) Borrelia burgdorferi imunofluorescencija. Tyrimo rezultatai: Ištyrus penkiomis reakcijomis serumus (RPR, TPHA, Treponema pallidum IFR, Treponema pallidum IFA bei Borrelia burgdorferi IFR), buvo pastebėtas kryžminis serologinis reaktyvumas tarp Borrelia burgdorferi IFR ir Treponema pallidum IFR reakcijų. Mūsų darbo duomenimis, tiriant 17 serumų (teigiamus dėl Laimo ligos) kryžminis reaktyvumas... [toliau žr. visą tekstą] / SUMMARY Cross - reactivity in serological test for Lyme disease and syphilis Author: Tatjana Subočiūtė Supervisor: Andrius Vagoras, MD Work purpose: to determine the incidence of possibly false positive results of serological syphilis and Lyme borreliosis tests, routinely used in VULSK Diagnostics centre, occurring because of cross reactivity of patients, having one of these disease, blood. Work methods: the research was carried out in Vilnius University Hospital Santariškių Klinikos (VULSK) Infection Markers Research Department of Microbiological Laboratory in Laboratory Diagnostics Centre. Serum samples were collected and tested from February, 2005, till March, 2006. The investigation material was composed of 40 serum samples. 23 serum samples for syphilis test came from VULSK Centre of Dermatovenereology. 8 serum samples for Lyme borreliosis test were received from VULSK Centre of Dermatovenereology. 9 serum samples for Lyme borreliosis test were picked by UAB “Endemik”. All the samples, included into the research, were tested by following reactions: 1) RPR with titer, 2) TPHA, 3) Treponema pallidum ELISA, 4) Treponema pallidum immunofluorescence, 5) Borrelia burgdorferi immunofluorescence. Research results: After testing the serums by five reactions (PRP, TPHA, Treponema pallidum FTA – ABS, Treponema pallidum ELISA and Borrelia burgdorferi immunofluorescence), a serological cross reactivity between Borrelia burgdorferi immunofluorescence and Treponema pallidum FTA - ABS... [to full text]

Antibodies play an important role in the natural immune response to invading pathogens. The strong and specific binding to their antigens also make them indispensable tools for research, diagnostics and therapy. This thesis describes the development of methods for characterization of an- tibody specificity and the use of these methods to investigate the polyclonal antibody response after immunization. Paper I describes the development of an epitope-specific serum fractionation technique based on epitope map- ping using overlapping peptides followed by chromatographic separation of polyclonal serum. This technique together with another epitope mapping technique based on bacterial display of protein fragments were then used to generate antibody sandwich pairs (Paper I), investigate epitope variations of repeated immunizations (Paper II) and to determine the ratio of antibodies targeting linear and conformational epitopes of polyclonal antibodies (Paper III). Paper IV describes the optimization of in situ-synthesized high-density peptide arrays for epitope mapping and how different peptide lengths influ- ence epitope detection and resolution. In Paper V we show the development of planar peptide arrays covering the entire human proteome and how these arrays can be used for epitope mapping and off-target binding analysis. In Paper VI we show how polyclonal antibodies targeting linear epitopes can be used for peptide enrichment in a rapid, absolute protein quantification protocol based on mass spectrometry. Altogether these investigations demonstrate the usefulness of peptide arrays for fast and straightforward characterization of antibody specificity. The work also contributes to a deeper understanding of the polyclonal anti- body response obtained after immunization with recombinant protein frag- ments. / <p>QC 20141111</p>

Background: Insulin is an important hormone for glucose homeostasis. It is released from β-cells in the endocrine pancreas as a response to increased concentrations of plasma glucose. The major effect of insulin is the facilitation of cellular uptake and storage of glucose as glycogen. Insulinomas are tumours that produce excessive amounts of insulin resulting in hypoglycaemia. The condition has been observed in dogs and cats and is often malignant. One part of establishing the diagnosis is confirmation of elevated concentrations of insulin in a hypoglycaemic sample. Aim: The aim of this study was to evaluate if an equine-optimized insulin ELISA (Mercodia AB, Uppsala, Sweden) is useful for analysis of insulin in canine and feline serum samples when insulinoma is suspected. Material and methods: All samples were analysed with Equine insulin ELISA. Precision, linearity and effects of haemolysis were studied. The stability of insulin was evaluated after storage in 4°C, room temperature and after repeated freezing and thawing. A reference interval was constructed for both canine and feline samples. Results: Total precision expressed as CV was 4.4 – 18.9 %. The method was linear up to at least 100 mU/L for dogs and 15 mU/L for cats. Reference interval for cats was &lt;11.6 mU/L, due to few healthy animals no reference interval for dogs could be established. Stability was acceptable for up to four days. No effects of haemolysis were detected. Conclusion: Mercodia Equine insulin ELISA is suitable for analysis of insulin in serum from dogs and cats when suspecting insulinoma.

Acute viral infections induce CD8 memory T cells that play an important role in the protection of the host upon re-infection with the same pathogen. These virus epitope-specific memory CD8 T cells develop complex TCR repertoires that are specific for that epitope. As individuals age virus-specific immunity appears to wane. Older people have difficulty controlling infection with common viruses such as influenza A (IAV), a RNA virus which causes recurrent infections due to a high rate of genetic mutation, and Epstein Barr virus (EBV), a DNA virus which persists in B cells for life in the 95% of people that become acutely infected. Many factors may contribute to this waning immunity including changes in virus-specific TCR repertoires. We hypothesize that epitope-specific memory CD8 TCR repertoires to these two common viruses change with increasing age and that CD8 T cell cross-reactivity may be one of the mechanisms mediating these changes. To address this hypothesis in our first study, we compared epitope-specific CD8 memory TRBV repertoires directly ex vivo for these two common human viruses. In cross-sectional and longitudinal studies of EBV seropositive, HLA-A2+, young (18-22 years), middle age (25-59 years), and older (>60 years) donors, we demonstrated that CD8 memory TCR repertoires to three immunodominant epitopes, known to have cross-reactive responses, IAV-M158-66, EBV-BM280-288, and EBV-BR109-117 co-evolve as individuals age. Cross-sectional studies showed that IAV-M1-and both EBV-specific repertoires narrowed their TRBV usage by middle-age. In fact, narrowing of EBV-BM and EBV-BR-specific TRBV usage correlated with increasing age. Although narrowing of IAV-M1-specific TRBV did not directly correlate with increasing age there was clear evidence that the TRBV usage was changing with age. The dominant TRBV19 usage appeared to become bimodal in the older age group and interestingly TRBV30 usage did directly correlate with age. For the EBV epitope-specific responses there was preferential usage of particular TRBV and changes in the hierarchy of TRBV usage in the different age groups. Longitudinal studies tracking 3 donors over 10-15 years (middle age to older) showed that there were changes in the TCR repertoire of IAV-M1, EBV-BM and -BR-specific responses over time. In two of the donors who experienced acute IAV infection there was evidence these repertoire changes may be influenced by TCR cross-reactivity, which is enhanced during acute IAV infection. The results of this first ex vivo study are consistent with our hypothesis. They suggest that virus-specific TCR repertoires change over time as an individual ages leading to narrowing of the repertoire and may co-evolve in the presence of CD8 T cell cross-reactivity. To further test our hypothesis in a second study we compared CD8 memory TRAV and TRBV repertoires to the three immunodominant epitopes IAV-M1, EBV-BM, and EBV-BR in the two extreme age groups, young donors (YSP) (18-22 years) and older donors (OSP) (>60 years) using the same donors as in the first study. Since these three epitopes are known to generate cross-reactive CD8 T cell responses and humans during their lifetime are frequently infected with both viruses at the same time these studies were also designed to more closely examine if TCR cross-reactivity could contribute to changes in TCR repertoire with increasing age. We examined the differences in both TRAV and TRBV in these two age groups by monoclonal antibody (mAb) staining and by deep sequencing and single cell sequencing in tetramer positive sorted cells from short-term cultures. Our initial studies showed that there were strong correlations in TRBV usage between short-term cultured and ex vivo antigen-specific responses; functional differences as well as differences in TRBV usage and diversity as measured by mAb staining particularly for the EBV epitope-specific responses between YSP and OSP donors. The TCR deep sequencing data also showed significant differences in TRBV usage between YSP and OSP. However, there were many more differences in TRAV and TRAJ usage than TRBV between the age groups suggesting that TRAV may play a greater role in evolution of the TCR repertoire. With increasing age, there was a preferential selection or retention of TCR for all three epitopes that have features in their CDR3a and b that increase their ease of generation, such as greater usage of convergent recombinant amino acids, and increase cross-reactive potential, such as multiple glycines. YSP and OSP differed in the patterns of TRAV/AJ and TRBV/BJ pairings and usage of dominant CDR3 motifs in all three epitope responses. Both YSP and OSP had cross-reactive responses between these 3 epitopes which were unique and differed from the cognate responses. Analyses of single cell sequencing data suggested that unique combinations of TRAV and TRBV are occurring, where one chain has features consistent with interaction with antigen one and the other chain had features consistent with interaction with antigen two. Interestingly, both the deep sequencing and single cell data show an increased tendency for the classic IAV-M1 specific clone BV19-IRSS-BJ2.7/AV27-CAGGGSQGNLIF-AJ42 to appear among the cross-reactive clones, suggesting that the dominance of this highly public clone may relate to its cross-reactivity with EBV. These results suggest that although OSP and YSP retain some of the classic TCR features for each epitope the TCR repertoire is gradually changing with age retaining TCR that are cross-reactive between these two common human viruses that we are exposed to frequently, one with recurrent infections and the other a persistent virus which frequently reactivates. These results are highly supportive our hypothesis and their importance in relation to viral immune-pathogenesis and potential novel immunotherapies will be discussed. These studies further emphasize the complexity and potential importance of human virus-specific T cell responses and TCR repertoires as people age and the need for a better understanding of TCR cross-reactivity between different viruses. For instance, at the present time these studies are highly relevant to better understanding the immune-pathogenesis observed during the COVID19 pandemic.

Hepatitis C virus (HCV) is a major global pathogen that infects an estimated 170 million people worldwide, and for which currently no vaccine is available. HCV is a highly diverse viral pathogen and exists as 6 major genotypes sharing only 75% sequence homology; developing a vaccine that is cross-reactive between genotypes is a major challenge. Defining immune responses that target different HCV genotypes will facilitate pan-genotypic T cell vaccine development. HCV genotype 3 (gt3) is now the most common infecting genotype in the United Kingdom and large parts of Asia; however, data regarding the T cell antigenic targets of this genotype is very limited. In this thesis, HCV gt3 specific T cell targets were defined in acute, chronic and spontaneously resolved infection: in chronic gt3 infection, T cell responses were low in magnitude and narrowly focused in specificity, similar to those previously reported for gt1; in contrast, resolved infection was associated with a higher magnitude and broader specificity of CD4+ and CD8+ T cell responses across the genome. Overall, T cell specificity in gt3 infection was markedly different to that previously described for gt1, confirming that sequence differences between genotypes result in distinct immunological profiles. Previous work from our laboratory demonstrated that, though T cell responses induced by a potent T cell vaccine containing HCV gt1b non-structural regions do target epitopes dominant in natural infection, induced T cells show limited cross-reactivity against other genotypes. In this thesis, it was assessed whether T cells primed in natural gt3 infection are able to recognize viral sequence variants at dominant epitopes, which would make these potential targets in cross-reactive vaccine design. For seven gt3-specific T cell epitopes identified here as dominant, major sequence variability was observed within and between genotypes, and limited T cell cross-reactivity observed against identified viral variants. This suggests that regions frequently targeted in natural infection may not serve as attractive targets for cross-reactive vaccine design. These results informed the subsequent design of a cross-reactive vaccine based on fragments of HCV that are conserved between genotypes. A generic algorithm was developed to define viral regions conserved between major HCV genotypes (for 1a/1b, 1/3a, 1-6), and these were joined to form immunogens between 819 and 1543 AA long. Possible artificial, non-HCV epitopes formed by junctions were identified using online epitope prediction servers, and abrogated through the insertion of 2-6 amino acid linkers. To address the concern that conserved regions may not be immunogenic, epitopes described in natural HCV infection were mapped on HCV sequences, showing that conserved segments are well populated with epitopes; additionally, strong binding peptides were predicted for conserved segments using online epitope prediction programs, suggesting potential in vivo immunogenicity. In conclusion, HCV T cell specificity is distinct between genotypes, with limited T cell cross-reactivity between viral variants. Leading from this result, vaccine immunogens were designed entirely based on conserved viral regions. This work paves the way for future studies of novel HCV immunogens based on conserved viral segments between genotypes.

Neste trabalho foi avaliado o potencial antigênico das vesículas de membrana externa, OMV, de N. meningitidis, das OMV e componetes protéicos selecionados de N. lactamica. Para tal foram realizados cultivos de ambas as espécies em biorreatores, ensaios de imunização em camundongos, análises de espectrometria de massas, e análises de tamanho das partículas, polidispersabilidade e potencial zeta. As análises da cinética de cultivos levaram a dados inéditos possibilitando uma nova discussão sobre o metabolismo e sobre a produtividade de OMV destas bactérias. N. lactamica obteve valores 5 vezes maiores de concentração máxima de OMV de 152 mg/L e 2,5 vezes maiores de produtividade de OMV de 0,32 g/L.h comparado aos obtidos para N. meningitidis nas mesmas condições de cultivo. OMV obtidas nos cultivos de ambas e componentes proteicos de N. lactamica foram utilizadas para imunizar camundongos com 3 doses subcutâneas. Ensaios de imunoblote e ELISA demonstraram que soros gerados contra as proteínas isoladas de N. lactamica foram reativos com proteínas de N. meningitidis, assim como o soro anti-OMV de N. lactamica que reagiu com 6 proteínas de N. meningitidis, as proteínas de membrana App, Omp85, PilQ, PorA, PorB, e ComL ou Opa/Opc. Análises de espectrometria de massas identificaram 229 proteínas na OMV de N. lactamica, sendo 77 proteínas de membrana e 243 proteínas de N. meningitidis, sendo 54 proteínas de membrana. Os resultados obtidos neste trabalho sugerem a possibilidade do uso das OMV de Neisseria lactamica como abordagem alternativa para o desenvolvimento de vacinas contra a doença meningocócica. / In these studies we evaluated the antigenic potential of outer membrane vesicles (OMV) from N. meningitidis, OMV from N. lactamica and proteic components from N. lactamica. Outer membrane vesicles were obtained from cultures of both species in bioreactors. Immunization tests were conducted in mice. Western-blotting and ELISA techniques were used to evaluate the cross-reactivity of murine sera against outer-membrane proteins from Neisseria meningitidis and Neisseria lactamica. Analysis of mass spectrometry and determination of particle size, polydispersity and zeta potential were also performed. Analysis of bacterial growth kinetics led to new data enabling a discussion about metabolism and OMV productivity. When both species were cultured in the same medium OMV concentration of N. lactamica (152 mg/L) is 5 times higher than that in N. meningitidis and OMV productivity of N. lactamica (0.32 g/L.h) is 2.5 times higher. Mice were vaccinated subcutaneously with 3 doses of OMV from both species and proteins from N. lactamica. These vaccines induced antibodies against N. meningitidis proteins. By mass spectrometry it was possible to identify these membrane proteins as App, Omp85, PilQ, PorA, PorB, and ComL or Opa/Opc. Mass spectrometry analyses also identified 229 proteins in N. lactamica OMV, with 77 predicted as membrane proteins and 243 in N. meningitidis OMV with 54 predicted as membrane proteins. Ours results suggested that OMV from Neisseria lactamica provides protection against N. meningitidis and could be used as an alternative approach for the development of a vaccine against meningococcal disease.

Orientador: Gervásio Henrique Bechara / Banca: Romário Cerqueira Leite / Banca: Rosângela Zacarias Machado / Resumo: No presente estudo avaliou-se o desenvolvimento de resistência a ninfas do ixodídeo Amblyomma cajennense, induzida por infestações controladas, e a possível existência de reatividade cruzada com Amblyomma hebraeum. Para tal, caprinos sem contato prévio com carrapatos, com seis meses de idade, machos ou fêmeas, foram infestados três vezes sucessivamente com ninfas de A. cajennense. Ademais, outro grupo de caprinos foi infestado nas mesmas condições com ninfas de A. hebraeum e colhidos soros para pesquisa de possível reatividade cruzada com A. cajennense. Observou-se que caprinos desenvolveram resistência apenas parcial a ninfas de A. cajennense, como demonstrado pela alteração significativa dos seguintes parâmetros biológicos das ninfas, em relação à primoinfestação: redução de 41,7% e 37,1% no peso de ingurgitamento, respectivamente na 2ª e 3ª infestações; aumento de 20% no período de ecdise e redução de 25,7% na taxa de ecdise, respectivamente nas 3ª e 2ª infestações. Não houve alteração no período de ingurgitamento nem na porcentagem de recuperação de ninfas com as infestações sucessivas. A reação cutânea induzida pelas ninfas de A. cajennense durante as infestações em caprinos caracterizou-se por infiltrado de células inflamatórias, predominantemente neutrófilos e basófilos, estas últimas envolvidas na imunidade a carrapatos por meio de hipersensibilidade basofílica cutânea. Células apresentadoras de antígeno, nomeadamente macrófagos, linfócitos B e células dendríticas foram detectadas por imunoistoquímica em linfonodos drenantes de locais de fixação dos carrapatos, em maior número nos linfonodos dos animais infestados... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study evaluated the acquision of resistance against Amblyomma cajennense nymphs in naïve goats, induced by repeated and controlled infestations, and a possible cross-reactivity with A. hebraeum. Ten naive goats, of both sexes, aged six months were used throughout the experiment. Animals were infested artificially thrice, 30 days intervals between infestations. In addition, goats were infested at same conditions with the tick A. hebraeum as well. Sera from these animals were collected looking for possible cross-reactivity between antigens from these ixodids. It was observed that goats developed partially resistance to A. cajennense nymphs from the 1st infestation on as shown by changes in some biological parameters, as follows: increase of 41.7% and 37.1% in engorgement weight, respectively at the 2nd and 3rd infestations, decrease in 25.7% in nymph’s ecdise rate and increase of 20% in ecdise period, respectively at the 3rd and 2nd infestations. It was not observed alterations in engorgement period and percentage of yielded nymphs. Biopsies of tick bite lesions induced by A. cajennense nymphs during infestations in goats were characterized by inflammatory reaction with cellular influx by neutrophils and basophils predominantly, being the last one involved in the well-known cutaneous basophilia. Antigen presenting cells like macrophages, B-lymphocytes and dendritic cells were detected by immunohistochemical analysis of lymph nodes draining tick infested areas. It should be stressed that these cells were found in higher numbers at lymph nodes from infested goats than from the naïve ones. Western blotting analysis of nymphal A. cajennense extracts revealed shared polypeptides (160, 90 and 16kDa) when used sera from goats infested with A. cajennense either A. hebraeum, indicating cross reactivity between these two tick species. / Mestre