Orientador: Hélio José Montassier / Banca: Aramis Augusto Pinto / Banca: Camillo Del Cistia Andrade / Resumo: A nucleoproteina do vírus da doença de Newcastle (VDN) é um dos componentes antigênicos ideais para fazer o imunodiagnóstico da DN, por ser mais conservada e possuir uma elevada imunogenicidade. A sequência completa do gene da nucleoproteína (NP) (1470 pb) da estirpe La Sota do VDN foi amplificada, nesse estudo, por RT-PCR e submetida a clonagem no vetor de expressão em Escherichia coli pETSUMO (Invitrogen). A proteína NP foi expressa sob a forma de uma proteína recombinante de fusão contendo o peptídeo SUMO e a sequência de poli-histidina, em seguida foi purificada em resina de níquel-agarose e caracterizada por SDSPAGE e Western-blotting, apresentando um peso molecular de cerca de 66kDa e reatividade com anticorpos policlonais de galinhas hiperimunizadas com esse mesmo vírus. Foi então desenvolvido um método indireto de ELISA com essa proteína (NPVDN- ELISA) para ser aplicado na detecção de anticorpos anti-virais específicos. O NP-VDN-ELISA revelou ser capaz de diferenciar amostras de soros positivos para o VDN das amostras de soros negativos e, na comparação dos resultados obtidos na análise de 125 soros de campo pelo NP-VDN-ELISA com os do teste de Inibição da Hemaglutinação (HI), foi encontrado um coeficiente de correlação significante entre estes métodos (r = 0,8345), bem como elevadas sensibilidade (89,3%), acurácia (90,4%) e especificidade (95,5%). Concluindo, a proteína NP recombinante expressa pelo sistema pET SUMO - E. coli compartilha os principais epítopos para interagir com anticorpos de galinhas produzidos contra a proteína NP do VDN, tendo, portanto, um bom potencial de ser aplicada de forma bem sucedida e com vantagens no teste de ELISA para realizar de forma mais rápida e prática, o imunodiagnóstico da DN de um maior número de amostras séricas de galinhas / Abstract: The nucleocapsid protein (NP) of Newcastle Disease Virus (NDV), is a preferred choice to develop a serologic assay on account of highly conserved sequences, and high immunogenicity. The whole open-reading-frame (orf) of NP gene from LaSota strain of NDV was amplified by RT-PCR and cloned in pETSUMO vector (Invitrogen) and Escherichia coli as cellular host. The NP protein was expressed as a fusion recombinant protein containing SUMO peptide and poly-histidine tags. This protein was easily purified in nickel-agarose resin, and characterized by SDS-PAGE and Western-blotting, showing a molecular weight of approximately 66 kDa and reactivity with polyclonal antibodies from NDV hiperimmunized chickens. The recombinant NP protein was used as antigen to develop an indirect ELISA (NP-NDVELISA) for the detection chicken anti-NDV antibodies. The capability of the recombinant NP protein to differentiate positive from normal chicken sera was evident in NP-NDV-ELISA, and by comparing this ELISA with haemagglutination-inhibition test (HI) a high and significant correlation with the haemagglutination-inhibition test (r = 0,8345), as well as high sensitivity (89,3%), specificity (95,5%) and accuracy (90,4%) were obtained. In conclusion the results indicated that the recombinant NP protein shared the main epitopes with the homologous viral protein and has a great potential to be advantageously used in the ELISA for the analysis of large number of samples in the DN immunodiagnosis / Mestre

Neurocysticercosis (NC) is a polymorphic disease and the immune response in human carrier is heterogenic. In this study, 35 primers were used for amplifications by Random Amplified Polymorphic DNA (RAPD) of Taenia solium metacestodes, from five different geographic areas in Brazil: 1) Distrito Federal (DF), Center West; 2) Barreiras (BA), Northeast and Southeast; 3) Hydro Basin of the Mosquito River (North of Minas Gerais, RM-MG), 4) São Paulo (SP) and 5) Uberaba (Minas Gerais, UB-MG). Metacestodes saline crude extracts of four populations (DF, BA, RM-MG e SP) were used for the detection of specific IgG antibodies by ELISA and Western Blotting (WB). A total of 157 serum samples of three groups, (G1): 49 NC patients; (G2): 68 patients with other helminthiasis: hydatidosis (10), taeniasis (20), strongyloidiasis (20), schistosomiasis (10) and hymenolepiasis (8); and (G3): 40 healthy individuals; were analyzed by ELISA. From these, the 98 serum samples were assayed by WB; G1 (49), G2 (39) and G3 (10). The genetic distances, in disagreement percentage, between the metacestode populations were calculated from of 15 RAPD markers and showed 49.5% (DF), 48% (BA), 38.5% (UBMG) and 28% (RM-MG and SP) of genetic distances. Six primers identified polymorphic fragments and the primers 26 (GGGTTTGGCA) and 29 (TCGCCAGCCA) allowed a better differentiation of populations. The fragments of 1000, 500 and 326 pb (pairs of bases) in the UB-MG and of 600 and 244 pb in RM-MG were amplified by primer 26. The fragments generated by primer 29 were 500, 800 and 1191 pb, 300 and 1377 pb, 1000 pb and 244 and 434 pb in SP, UB-MG, DF and BA populations, respectively. In G1, the positivity by ELISA was: 90% (DF), 69% (BA), 71% (MG) and 67% (SP). The DF extract was more antigenic than others (p=0.02). In WB, the 64-68 kDa antigens were recognized in all extracts, exclusively, in serum samples from active NC patients (p=0.001). Variation in banding pattern was detected between the extracts (p<0.05). In G2, the serum samples of hydatidosis patients presented from 70 to 90% positivity by ELISA in antigenic extracts (p<0.05); however, the bands recognition pattern in WB was different from that presented in G1 samples. The 77 kDa band was significantly identified in hydatidosis samples (p=0.0001). In conclusion, the T. solium populations analyzed showed genetic variability and antigenic differences. / A neurocisticercose (NC) constitui doença polimórfica, apresentando heterogeneidade da resposta imune no hospedeiro humano. Nesse estudo, o teste Random Amplified Polymorphic DNA (RAPD) foi utilizado com 35 primers na detecção de polimorfismo em metacestódeos de Taenia solium provenientes de cinco áreas geográficas distintas do Brasil: 1) Distrito Federal (DF), região Centro-Oeste; 2) Barreiras (BA), região nordeste e da região sudeste: 3) Bacia Hidrográfica do Rio Mosquito (norte de Minas Gerais, RM-MG), 4) São Paulo (SP) e 5) Uberaba (Minas Gerais, UB-MG). Os extratos salinos totais de metacestódeos de quatro populações (DF, BA, RM-MG e SP) foram utilizados na detecção de anticorpos IgG específicos pelo ELISA e Western Blotting (WB). As 157 amostras de soro de três grupos (G) de indivíduos: G1: 49 pacientes com NC; G2: 68 pacientes com outras helmintíases, sendo hidatidose (10), teníase (20), estrongiloidíase (20), esquistossomose (10) e himenolepíase (8) e G3: 40 indivíduos saudáveis (controles); foram analisadas pelo ELISA. Foram ensaiadas 98 amostras de soro: G1 (49), G2 (39) e G3 (10) pelo WB. As distâncias genéticas, por porcentagem de desacordo, foram de 49,5% (DF), 48% (BA), 38,5% (UB-MG) e 28% (RM-MG e SP) nas populações de metacestódeos, calculadas a partir de 15 marcadores de RAPD. Seis primers geraram fragmentos polimórficos nos isolados analisados, sendo que os primers 26 (GGGTTTGGCA) e 29 (TCGCCAGCCA) permitiram melhor diferenciação entre as populações, o primer 26 gerou os fragmentos de 1000, 500 e 326 pb (pares de bases) na amostra de UB-MG, e de 600 e 244 pb em RM-MG. O 29 gerou fragmentos em quatro das populações analisadas, sendo 500, 800 e 1191 pb em SP, 300 e 1377 pb em UBMG, 1000 pb no DF e 244 e 434 pb na BA. No G1, as freqüências de positividade no ELISA, foram: 90% (DF), 69% (BA), 71% (MG) e 67% (SP), sendo o extrato do DF mais antigênico que os demais (p = 0,02). No WB, o peptídeo de 64-68 kDa foi reconhecido em todos os extratos, exclusivamente, em amostras de pacientes com NC ativa (p=0,001). Detectou-se variação no padrão de reconhecimento de bandas entre os extratos (p<0,05). No G2, as amostras de soro de pacientes com hidatidose apresentaram de 70 a 90% de positividade no ELISA frente aos extratos analisados (p<0,05); porém, o padrão de reconhecimento de bandas no WB diferiu do apresentado pelas amostras do G1, sendo que a banda de 77 kDa foi significativamente identificada pelas amostras de pacientes com hidatidose (p=0,0001). Concluiu-se que as populações de T. solium analisadas nesse estudo, apresentaram variabilidade genética e diferenças de antigenicidade. / Doutor em Imunologia e Parasitologia Aplicadas

Both Multiple Sclerosis (MS) and Guillan-Barré syndrome (GBS) are neurological inflammatory demyelinating autoimmune diseases, with a probable antibody contribution. Complement proteins in both MS and GBS does play a role in inflammation and demyelination at pathogenesis, according to earlier scientific evidence. The aim of this examination project work was to investigate systemic and intrathecal complement activation in MS and GBS, to gain further knowledge that might be useful for development of future therapeutics targeting immune responses during those diseases. An additional aim was to develop a new ELISA method for detection of complement iC3. By using sandwich ELISA, complement proteins C1q, C4, C3, fH and C3a were measured in plasma and cerebrospinal fluid (CSF) from persons within 4 different diagnostic groups; MS, other neurological diseases (OND), GBS and controls (C). An ELISA method to detect iC3 (hydrolysed C3) was also developed, including usage of SDS-PAGE. Results based on raw data and statistical analysis show significantly elevated levels of C3a (C3a/C3) in MS and decreased C3 in plasma. In CSF low levels of C4 and C3a/C3 in MS were detected, though correlation of C3a and C1q was positive. GBS reveal high levels of all complement proteins analysed in CSF except for C3, and a positive correlation of C3a and C1q as well as C3a and fH was found. These results indicate that MS patients have systemic complement activation; however the activation pathway is not determined. Complement activation in MS may also occur intrathecally, with correlation analysis indicating a possible activation via the classical pathway. MS patients suffering from a more acute relapsing-remitting (RR) MS have a more prominent systemic complement activation compared to MS patients responding to beta-interferon treatment. Systemic increased C3a/C3 ratio may be a possible biomarker to distinguish more acute RR MS in an earlier step of MS pathogenesis and should be further investigated. GBS patients have an intrathecal complement activation that seems to occur via the classical pathway.

Realizou-se revisão bibliográfica sobre a utilização do teste imunoenzimático, ELISA (enzyme-linked immunosorbent assay) na Vigilância Epidemiológica de infecções causadas por arbovírus da Família Flaviviridae, gênero Flavivirus e da Família Togaviridae, gênero Alphavirus. Foram consultados trabalhos publicados a partir de 1979, ano da introdução do teste em pesquisas de arboviroses. Observou-se que o teste tem sido empregado na pesquisa de anticorpos em humanos, de anticorpos e antígenos em reservatórios não humanos e na identificação de antígenos e da fonte alimentar de mosquitos vetores. Analisou-se o desempenho de ELISA comparando-o a técnicas tradicionalmente empregadas para identificação de anticorpos e antígenos de arbovírus. O teste apresentou 100,0 por cento de sensibilidade e especificidade média de 84,5 por cento na identificação de anticorpos anti-Alphayirus em humanos. A técnica também foi muito sensível para Flavivirus, com valor médio de 95,2 por cento e apresentou especificidade média de 77,6 por cento. Na identificação de anticorpos anti-arbovírus em resevatórios não humanos, ELISA mostrou sensibilidade de 100,0 por cento e especificidade de 97,4 por cento. Na pesquisa de antígenos vir ais em mosquitos vetores a técnica apresentou especificidade média de 93,6 por cento e sensibilidade média de 76,5 por cento. A técnica apresentou alto valor preditivo positivo, o que foi observado quando calculou-se a média dos valores apresentados em cada um dos trabalhos em que esse parâmetro foi pesquisado e obteve-se um resultado de 89,0 por cento. Nos trabalhos em que foi estudada a reprodutibilidade do teste observou-se coeficiente de variação de 3,0 a 14,0 por cento nos resultados. Observou-se grande diversidade quanto aos critérios de positividade adotados, impossibilitando a comparação dos resultados. Notou-se uma tendência a encurtar o tempo de realização do teste e torná-lo factível em condições de trabalho de campo. Verificou-se que o teste já está incorporado à rotina da Vigilância Epidemiológica de algumas arboviroses como encefalite Japonesa nos países asiáticos e encefalites do Leste, Oeste e de St.Louis, nos Estados Unidos da América. Os autores estudados foram unânimes em concluir que trata-se de teste rápido, apresenta simplicidade dos procedimentos técnicos e permite diagnóstico presuntivo de infecção aguda com apenas uma amostra de soro, características que o capacitam para uso na Vigilância Epidemiológica de arboviroses. / The author makes a review of the use of enzyme-linked immunosorbent assay, ELISA, in the surveillance of infections caused by arbovirus belonging to the Flaviviridae family (genus Flavivirus) and to the Togaviridae family (genus Alphavirus). Publications dating since 1979, when the use of ELISA in arbovirus research began, were consulted. It was noted that this assay was used for antibody identification on not human reservoirs, and for antigen and blood meal identification on mosquito vectors. ELISA\'s perfomance was compared to standard tests used for laboratorial diagnosis of arboviruses. The test presented 100.0 per cent sensitivity and an average specificity of 84.5 per cent in Alphavirus antibody identification in humans; and was also sensitive for Flavivirus with average values of 95.2 per cent and specificity average values of 77.6 per cent. ELISA furthermore showed 100.0 per cent sensitivity and 97.4 per cent specificity for antibody identification in not human reservoirs. For the antigen identification in mosquito vectors, the assay presented an average specificity of 93.6 per cent and an average sensitivity of 76.5 per cent. The immunoassay presented high predictive values (average of 89.0 per cent) and it was reproducible when this characteristic was studied with a coefficient variation index ranging from 3.0 to 14.0 per cent There was a great variety in the positivity criteria, making the comparison of results difficult. An attempt to process the test in the shortest time and in field conditions is noted in many publications. The assay is used routinely on surveillance of Japanese encephalitis in Asian countries and in eastern and western equine encephalitis and St.Louis encephalitis in the USA. ELISA was considered a rapid assay with sirnple procedures when it is cornpared to tradicional tests and provides presuntive diagnosis of an acute infection with a single serurn sarnple. These characteristics suggest that the test is a useful tool for arbovirus surveillance.

O carrapato Rhipicephalus (Boophilus) microplus é um ectoparasito hematófago que infesta os rebanhos bovinos de regiões tropicais e subtropicais, e é um dos principais causadores de prejuízos econômicos à pecuária. O principal método de controle baseia-se no uso de acaricidas. No entanto, devido à crescente preocupação com os problemas criados pela poluição química do meio ambiente, ao alto custo e toxicidade das drogas e ao aparecimento de carrapatos resistentes aos acaricidas, métodos alternativos para o controle do R. microplus devem ser encontrados. Um dos métodos alternativos estudados é o uso de vacinas. As glutationa S-transferases (GST) são enzimas que estão presentes em organismos animais e vegetais e entre suas funções podem-se destacar transporte intracelular, participação em processos digestivos, síntese de prostaglandinas, e detoxificação de substâncias tóxicas e proteção contra estresse oxidativo. Neste estudo foi analisada a resposta imunológica comparativa de camundongos para a GST de R. microplus (GST-Bm) e para a GST de Haemaphysalis longicornis (GST-Hl). Ambas as proteínas foram expressas em Escherichia coli linhagem AD494, e purificadas por cromatografia de afinidade por glutationa utilizando a coluna GSTrap FF. Para verificar a imunogenicidade das proteínas, foram utilizados 25 camundongos Balb/c divididos em 12 grupos. As condições testadas foram as inoculações das proteínas GST-Bm e GST-Hl com os adjuvantes Montanide, saponina ou sem adjuvantes. Camundongos controle foram inoculados com extrato de E. coli ou somente com os adjuvantes. Foi coletado sangue de todos os animais com intervalos de sete dias durante 70 dias. Os soros foram analisados por ELISA para acompanhar a cinética da produção de anticorpos de todos os animais imunizados. Os camundongos inoculados com GST-Hl emulsificada com Montanide mostraram aumento dos níveis de anticorpos a partir do dia 21. O nível máximo de anticorpos foi detectado no dia 42, e diminuiu após o dia 56. Todos os outros animais não apresentaram aumento nos níveis de anticorpos. / Rhipicephalus (Boophilus) microplus tick is a hematophagous ectoparasite that infests cattle in tropical and subtropical regions and is one of the principal causes of economic losses in the cattle farm. The principal method of control is the use of acaricides. However, due to increased worry about environmental chemical pollution, high costs and drugs toxicity and the selection of ticks resistant to acaricides, alternative methods for R. microplus control should be developed. One of these methods is vaccination. Glutathione S-transferases (GSTs) enzymes are present in animal and vegetal organisms and the functions are intracellular transport, participation in digestive process, synthesis of prostaglandins and detoxification of toxic substances and protection against oxidative stress. In this study the immunological response of mice inoculated with R. microplus GST (GST-Bm) and with Haemaphysalis longicornis GST (GST-Hl) was analysed. Both proteins were expressed in Escherichia coli strain AD494 and were purified by affinity chromatography using GSTrap FF columm. To verify the protein immunogenicity, 25 Balb/c mice divided into 12 groups were used. The tested conditions were inoculation of GST-Bm, GST-Hl proteins with Montanide and saponin adjuvants or without adjuvant. Control mice were inoculated with E. coli extract or the adjuvant alone. Blood from all animals were collected with intervals of seven days during seventy days, the sera were analyzed by ELISA to verify the kinetic of antibodies production of all immunized animals. Mice inoculated with GST-Hl emulsified with Montanide showed an increase in the antibodies levels from day 21. The maximum level of antibodies was detected on day 42 and decreased after day 56. The other animals did not show an increase in antibodies levels.

Orientadores: Getulio da Rocha Nogueira Filho, Maria Cristina Foss de Freitas / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-14T01:50:30Z (GMT). No. of bitstreams: 1 Tunes_RobertaSantos_D.pdf: 3904894 bytes, checksum: 5bfa07e25946e02ccbf23c44e8160e08 (MD5) Previous issue date: 2009 / Resumo: Este estudo objetiva verificar a influência sistêmica da periodontite crônica severa frente ao controle metabólico bem como do tratamento periodontal na resposta imunológica de indivíduos diabéticos tipo 2 através da análise da expressão gênica e protéica de citocinas por células mononucleares aderentes de sangue periférico. Foram selecionados 20 pacientes divididos em: grupo teste - 10 diabéticos tipo 2 com periodontite crônica severa e controle metabólico inadequado; grupo controle - 10 diabéticos tipo 2 sem periodontite e controle metabólico inadequado. Primeiramente, os pacientes foram hospitalizados por 8 dias para a obtenção de um controle metabólico adequado, permanecendo inalterada a condição periodontal nesta fase. Posteriormente, o grupo teste foi submetido à terapia mecânica em estágio único associada à antibioticoterapia sistêmica com amoxicilina e metronidazol. Amostras sanguíneas foram obtidas no primeiro e último dias de hospitalização, e 4 a 6 semanas após o tratamento periodontal para a análise imunológica. Para avaliação dos parâmetros laboratoriais relacionados ao controle metabólico, como HbA1c, foram obtidas amostras sanguíneas antes e após a hospitalização, e 3 a 6 semanas após o tratamento periodontal. Culturas de um total de 2,5x106 células/mL mononucleares aderentes, foram realizadas por 24h na presença e ausência de LPS (5µg/mL). As citocinas TNF- a, IL-1 ß, IL-8 e IL-6 foram detectadas nos sobrenadantes das culturas através do ELISA, enquanto que a sua expressão gênica foi verificada por PCR em tempo real. Enquanto o controle metabólico diminuiu a secreção de TNF- a (p=0,02), IL-1ß(p=0,03), IL-8 (p=0,04) e IL-6 (p=0,02) nos sobrenadantes das células estimuladas do grupo controle, os pacientes do grupo teste apresentaram níveis maiores destas citocinas após o controle metabólico quando comparados ao grupo controle (p<0,01 ou p=0,01). O controle metabólico bem como a infecção periodontal severa, apesar de não terem influenciado a expressão gênica das citocinas próinflamatórias pelas células estimuladas de ambos os grupos, aumentou a expressão gênica em 2 vezes da IL-8 (p=0,04) pelas células basais do grupo teste, evidenciando-se uma hipoexpressão, respectivamente, de 4, 3 e 11 vezes, da IL-1 ß, IL-8 e IL-6 (p<0,01 ou p=0,01) por estas células no grupo teste em relação ao grupo controle, antes do controle metabólico. Enquanto o tratamento periodontal promoveu uma diminuição da secreção de TNF-?(p=0,04) pelas células estimuladas do grupo teste, o mesmo não influenciou a expressão gênica das citocinas nestas condições. O controle metabólico associado ao tratamento periodontal promoveu um aumento da expressão gênica em 6 e 5 vezes, respectivamente da IL-1 a e IL-6 (p<0,01), pelas células basais do grupo teste. Enquanto, o controle metabólico promoveu reduções na HbA1c de 1% no grupo controle (p=0,04) e 1,1% no grupo teste (p=0,03), o tratamento periodontal promoveu uma redução adicional de 1,6% na HbA1c do grupo teste. Assim, demonstrou-se o papel modulador da periodontite crônica severa na resposta imunológica dos pacientes diabéticos tipo 2, mantendo a capacidade reativa próinflamatória das células mononucleares frente ao controle metabólico, e que o tratamento desta infecção local pode exercer efeitos sistêmicos, diminuindo o potencial próinflamatório das células mononucleares circulantes, contribuindo para a melhor sensibilidade insulínica e facilitando o controle metabólico destes indivíduos / Abstract: The purpose of this study was to evaluate the systemic influence of chronic severe periodontitis in relation to metabolic control and of periodontal therapy in the immune response of diabetic type 2 patients through the evaluation of cytokine protein and gene expression in human adherent peripheral blood mononuclear cells. Twenty patients were studied, distributed in 2 groups: test group - 10 type 2 diabetic patients with severe chronic periodontitis and inadequate metabolic control; control group - 10 type 2 diabetic patients without periodontitis and inadequate metabolic control. First, all participants were hospitalized for 8 to 10 days to obtain adequate metabolic control, without any periodontal condition alterations. Afterwards, the test group received one-stage non-surgical eriodontal therapy associated with Amoxicilin and Metronidazole. Blood samples were obtained on he first and last day of hospitalization, and 4 to 6 weeks after periodontal therapy for immunological analyses. For metabolic parameters analyses, like HbA1c, blood samples were obtained before and after hospitalization, and 3 to 6 weeks after periodontal therapy. Mononuclear cells were isolated by gradient density using Ficoll-Hypaque?. A total of 2.5 X 106 adherent cells/mL were cultivated during 24hs in the presence or absence of LPS. The cytokines TNF-a, IL-1ß, IL-8, IL-6 were quantified in cell culture supernatants using ELISA, while their gene expression was verified by Real Time PCR. It was demonstrated that metabolic control promoted a reduction of TNF- a (p=0,02), IL-1? (p=0,03), IL-8 (p=0,04) and IL-6 (p=0,02) levels in control group supernatants stimulated cells, while the test group showed increased levels of that cytokines compared to the control group after metabolic control (p<0,01 or p=0,01). The metabolic control as the presence of chronic severe periodontal infection, although did not affect the gene expression of that cytokines by stimulated cells from both groups, increased IL-8 gene expression 2 times (p=0,04) by test group unstimulated cells while it was verified a lower gene expression respectively of IL-1 ß, IL-8 and IL-6 (p<0,01 or p=0,01) by 4, 3, and 11 times related to the control group, before metabolic control. Moreover, periodontal therapy promoted a reduction of TNF- a (p=0,04) levels in test group supernatants stimulated cells, although did not affect the gene expression of the cytokines studied by these cells. There was observed combined effect of metabolic control and periodontal therapy on IL-1 ß and IL-6 (p<0,01) gene expression, that were increased by 6 and 5 times, respectively, by test group unstimulated cells. While metabolic control promoted reductions of 1% and 1,1% in HbA1c levels of control and test group respectively, periodontal therapy promoted an additional reduction of 1,6% in HbA1c levels of test group. Therefore, it was suggested that chronic severe periodontal infection can modulate the immunological response of type 2 diabetic patients, through the maintenance of the inflammatory reactive capacity of mononuclear cells even if in the presence of metabolic control, and that the local infection treatment can promote systemic effects, through decreasing the pro-inflammatory potential of mononuclear circulating cells, helping to restore insulin sensitivity, resulting in an improved glycemic control in those individuals / Doutorado / Periodontia / Doutor em Clínica Odontológica

Made available in DSpace on 2014-08-20T13:32:54Z (GMT). No. of bitstreams: 1 tese_carina_moraes.pdf: 479524 bytes, checksum: 9bdbed7287bcee59185e68ac89b72108 (MD5) Previous issue date: 2008-10-13 / Strangles is a contagious disease of the upper respiratory tract of horses caused by Streptococcus equi subsp. equi. Asymptomatic carriers responsible for maintaining the infection in the herd can only be detected by serological or microbiological methods and vaccines used for the control of the disease induce levels of protection generally not exceeding 50%. Considering that S. equi SeM protein is considered the most promising antigen to protect against the disease, this research aimed to produce and evaluate as antigen for vaccines and for ELISA, a recombinant S. equi SeM protein (rSeM). rSeM was produced by cloning and expression in Escherichia coli and purified by affinity chromatography. To test its immunogenicity isogenic female Balb-c mice 4-6 weeks-old were randomly divided and inoculated with 1 / 20th of the estimated dose of the vaccine for horses by the SC route, on days 0 and 21 of the experiment. One group was vaccinated with 250mL (12 mg mL-1) of rSeM without adjuvant, another with 300mL of vaccine containing 12 mg mL-1 of rSeM plus 20% of aluminiun hydroxide, two other groups were vaccinated with two commercial bacterins against Strangles, other two groups were vaccinated with the same commercial vaccines containing 12 mg mL-1 of rSeM and the remaining group was inoculated with a bacterin produced with a field strain. The control group was inoculated the same dose of sterile saline. Blood samples were collected from the retro-orbital venous plexus on days 0, 21, 42. The antibodies were titrated by ELISA using rSeM as antigen. rSeM was immunogenic for mice with a protection index of 100%. For the standardization of an ELISA, groups of 20 negative, vaccinated and positive animals were used. Using as Cut-off the mean plus two SD of the Optical Densities of the negatives, the test showed 100% sensitivity and specificity. / A Adenite Eqüina é uma enfermidade contagiosa do trato respiratório superior dos eqüídeos causada por Streptococcus equi subesp. equi. Animais portadores assintomáticos responsáveis pela permanência da infecção nos rebanhos só podem ser detectados por métodos microbiológicos ou sorológicos e as vacinas utilizadas no controle da doença induzem níveis de proteção geralmente não superiores a 50 %. Considerando que a proteína SeM de S. equi é o antígeno mais promissor na proteção contra a doença, este trabalho objetivou produzir a proteína SeM recombinante de S. equi, visando sua utilização como antígeno em vacinas e em ELISA. Proteína SeM recombinante (rSeM) foi produzida mediante a clonagem e expressão em Escherichia coli e purificada por cromatografia de afinidade. Para testar sua capacidade imunogênica, vacinas elaboradas com rSeM foram aplicadas a camundongos. Fêmeas Balb/c isogênicas com 4-6 semanas foram divididas aleatoriamente e inoculadas por via SC com 1/20 da dose vacinal estimada para cavalos, nos dias 0 e 21 do experimento. Um grupo foi vacinado com 250 mL (12 mg mL-1) de proteína recombinante sem adjuvante, outro com 300 mL de vacina contendo 12 mg mL-1 rSeM adicionada de 20% de hidróxido de alumínio, outros dois grupos com duas bacterinas comerciais contra Adenite Eqüina; dois grupos com as vacinas comerciais, acrescidas de 12 mg mL-1 de rSeM e o grupo restante com uma bacterina contendo cepas de campo. O grupo controle foi inoculado com o mesmo volume de solução salina estéril. Coletou-se sangue por punção do plexo venoso retro-ocular nos dias 0, 21 e 42. Os anticorpos foram titulados por ELISA utilizando a proteína rSeM como antígeno. A rSeM foi imunogênica em camundongos com índices de proteção de 100%. Para a padronização de um ELISA, utilizaram-se grupos de 20 soros equinos de animais negativos, vacinados e positivos. Utilizando um ponto de corte de média das densidades ópticas dos soros negativos acrescidos de dois desvios padrão, o teste teve 100% de sensibilidade e especificidade.

Philosophiae Doctor - PhD / More than 20 million infants in the world (15.5 % of all births) are born with low birth weight. Ninety-five % of them are in developing countries. Oral colonization of Gramnegative anaerobes has been implicated as a risk factor for preterm delivery of low birth weight (PLBW) infants. The objective of this study was to investigate the association between periodontal pathogens and pre-term delivery of low birth weight (PLBW) infants. The study sample included 200 randomly selected women admitted to the department of obstetrics-gynecology of the teaching hospital of Butare in Rwanda. Mothers were asked to complete a questionnaire in order to identify factors which might pose a health risk to them and their infants. Gingival crevicular fluid (GCF) was collected from each quadrant of the mother’s month (using paper points) within 24 hours of delivery. Ten ml of foetal cord serum samples were collected at delivery and 10 ml of maternal serum samples were collected within 48 of delivery. GCF was examined by PCR for the presence of 5 periodontopathogens and ELISA was used for the evaluation of cytokines (IL-6 and IL-10) and immunoglobulins (IgM, IgG) in foetal cord and maternal blood against the periodontopathogens. P. intermedia showed significant associations either on its own or in combinations with most indicators of periodontal disease used in this study, while Aa and members of the red complex were significantly associated with gum bleeding and reduced frequency of tooth brushing. A strong association between PLBW and maternal and foetal cord serum sample levels of IL-10 was observed. Also, a good association was observed between PLBW and FCB sample levels of IL-6. Significant associations were observed between PLBW and maternal IgG against the different peridontopathogens. The findings of this study may suggest that the levels of maternal IgG and foetal IgM against the different periodontopathogens are associated with dissemination of maternal periodontopathogens to the foetus thereby illiciting an inflammatory response which contributes to PLBW. / South Africa

Philosophiae Doctor - PhD / More than 20 million infants in the world (15.5 % of all births) are born with low birth weight. Ninety-five % of them are in developing countries. Oral colonization of Gramnegative anaerobes has been implicated as a risk factor for preterm delivery of low birth weight (PLBW) infants. The objective of this study was to investigate the association between periodontal pathogens and pre-term delivery of low birth weight (PLBW) infants. The study sample included 200 randomly selected women admitted to the department of obstetrics-gynecology of the teaching hospital of Butare in Rwanda. Mothers were asked to complete a questionnaire in order to identify factors which might pose a health risk to them and their infants. Gingival crevicular fluid (GCF) was collected from each quadrant of the mother’s month (using paper points) within 24 hours of delivery. Ten ml of foetal cord serum samples were collected at delivery and 10 ml of maternal serum samples were collected within 48 of delivery. GCF was examined by PCR for the presence of 5 periodontopathogens and ELISA was used for the evaluation of cytokines (IL-6 and IL-10) and immunoglobulins (IgM, IgG) in foetal cord and maternal blood against the periodontopathogens. P. intermedia showed significant associations either on its own or in combinations with most indicators of periodontal disease used in this study, while Aa and members of the red complex were significantly associated with gum bleeding and reduced frequency of tooth brushing. A strong association between PLBW and maternal and foetal cord serum sample levels of IL-10 was observed. Also, a good association was observed between PLBW and FCB sample levels of IL-6. Significant associations were observed between PLBW and maternal IgG against the different peridontopathogens. The findings of this study may suggest that the levels of maternal IgG and foetal IgM against the different periodontopathogens are associated with dissemination of maternal periodontopathogens to the foetus thereby illiciting an inflammatory response which contributes to PLBW.

New designer drugs are constantly emerging onto the illicit drug market and it is often difficult to validate and maintain comprehensive analytical methods for accurate detection of these compounds. Generally, toxicology laboratories utilize a screening method, such as immunoassay, for the presumptive identification of drugs of abuse. When a positive result occurs, confirmatory methods, such as gas chromatography (GC) or liquid chromatography (LC) coupled with mass spectrometry (MS), are required for more sensitive and specific analyses. In recent years, the need to study the activities of these compounds in screening assays as well as to develop confirmatory techniques to detect them in biological specimens has been recognized. Severe intoxications and fatalities have been encountered with emerging designer drugs, presenting analytical challenges for detection and identification of such novel compounds. The first major task of this research was to evaluate the performance of commercially available immunoassays to determine if designer drugs were cross-reactive. The second major task was to develop and validate a confirmatory method, using LC-MS, to identify and quantify these designer drugs in biological specimens. Cross-reactivity towards the cathinone derivatives was found to be minimal. Several other phenethylamines demonstrated cross-reactivity at low concentrations, but results were consistent with those published by the assay manufacturer or as reported in the literature. Current immunoassay-based screening methods may not be ideal for presumptively identifying most designer drugs, including the “bath salts.” For this reason, an LC-MS based confirmatory method was developed for 32 compounds, including eight cathinone derivatives, with limits of quantification in the range of 1-10 ng/mL. The method was fully validated for selectivity, matrix effects, stability, recovery, precision, and accuracy. In order to compare the screening and confirmatory techniques, several human specimens were analyzed to demonstrate the importance of using a specific analytical method, such as LC-MS, to detect designer drugs in serum as immunoassays lack cross-reactivity with the novel compounds. Overall, minimal cross-reactivity was observed, highlighting the conclusion that these presumptive screens cannot detect many of the designer drugs and that a confirmatory technique, such as the LC-MS, is required for the comprehensive forensic toxicological analysis of designer drugs.