Magister Scientiae - MSc / The aim of this study was to investigate the effects of Sutherlandia frutescens on the inflammatory response and T cell differentiation in vitro using cytokines as biomarkers. Whole blood cells containing various concentrations of Sutherlandia frutescens were stimulated in vitro with either Lipopolysaccharide (LPS) or Phytohaemagglutinin (PHA). Results show that Sutherlandia frutescens is not toxic at any of the concentrations tested. The addition of Sutherlandia frutescens at high concentrations to the stimulated whole blood cell cultures reflects a significant down regulation of Interleukin(IL) 6 and IL-10 compared to the control (P<0.05) hence suppressed the inflammatory and humoral immune response. Results obtained for Inteferon-gamma (IFN ) shows that Sutherlandia frutescens is donor specific as it reflects both up and down regulation in the release of IFN at the concentrations tested. The in vitro data generated by this study supports the use of Sutherlandia frutescens in the management of inflammatory conditions and allergies such as asthma. However the effects of Sutherlandia frutescens on cell mediated immunity was found to be donor specific. Further investigation of Sutherlandia frutescens on cellular immunity is advised. / South Africa

Agamaglobulinemia ligada ao X (XLA) é uma imunodeficiência primária caracterizada por ausência ou número reduzido de células B maduras em sangue periférico, de todos os isotipos de imunoglobulina e um aumento da susceptibilidade a infecções bacterianas e enterovirais graves. XLA é causada por mutações no gene Bruton\'s tirosino quinase, que codifica um membro da proteína da família das tirosino quinases citoplasmáticas que tem papel vital na modulação de muitos processos celulares. Neste estudo foram analisados trinta e três pacientes quanto à presença de mutações de BTK, por SSCP/HA e seqüenciamento. A análise da expressão foi realizada pela técnica por PCR em tempo real. Foram encontradas mutações do tipo stop codons, substituições de aminoácido, defeitos de splicing, pequenas deleções/inserções e frameshift nestes pacientes afetando os domínios PH, SH3, SH2 e o domínio da quinase da proteína. A análise da expressão mostrou níveis baixos nos pacientes com mutação do tipo stop codon, e nas outras mutações, os níveis de expressão foram de aproximadamente 15% e se correlacionaram com os tipos de mutação. / X-linked Agammaglobulinemia (XLA) is a primary immunodeficiency characterized by the absence or decreased numbers of mature B cells in peripheral blood, and by a lack of all immunoglobulin isotypes, leading to an increased susceptibility to severe bacterial and enteroviral infections. XLA is caused by mutations in the gene encoding for Bruton\'s tyrosine kinase (BTK), a protein member of the Tec family of cytoplasmic tyrosine kinases and plays a vital modulation role in many cellular processes. In this study thirty-three patients were analyzed for the presence of BTK mutations by SSCP/HA and sequencing. The expression analysis was carried out by the technique of Real-Time PCR. It was found mutations of the stop codons type, amino acid substitutions, splice defects, small deletions/insertions and frameshift in these patients affecting the PH, SH3, SH2 and tyrosine kinase domains of protein. The expression levels were very low in the patients with stop codon mutations, and in the other mutations, the expression levels were about 15% and were correlated with the mutation types.

Cryptococcus neoformans, an opportunistic fungal pathogen ubiquitously present in the environment, causes cryptococcal meningitis (CM) mainly in immunocompromised patients, such as AIDS patients. We aimed to identify disease-associated cryptococcal protein antigens targeted by the human humoral immune response. Therefore, we used sera from Colombian CM patients, with or without HIV infection, and from healthy individuals living in the same region. Serological analysis revealed increased titers of anti-cryptococcal IgG in HIV-negative CM patients, but not HIV-positive CM patients, compared to healthy controls. In contrast, titers of anti-cryptococcal IgM were not affected by CM. Furthermore, we detected pre-existing IgG and IgM antibodies even in sera from healthy individuals. The observed induction of anti-cryptococcal IgG but not IgM during CM was supported by analysis of sera from C. neoformans-infected mice. Stronger increase in IgG was found in wild type mice with high lung fungal burden compared to IL-4Ra-deficient mice showing low lung fungal burden. To identify the proteins targeted by human anti-cryptococcal IgG antibodies, we applied a quantitative 2D immunoproteome approach identifying cryptococcal protein spots preferentially recognized by sera from CM patients or healthy individuals followed by mass spectrometry analysis. Twenty-three cryptococcal proteins were recombinantly expressed and confirmed to be immunoreactive with human sera. Fourteen of them were newly described as immunoreactive proteins. Twelve proteins were classified as disease-associated antigens, based on significantly stronger immunoreactivity with sera from CM patients compared to healthy individuals. The proteins identified in our screen significantly expand the pool of cryptococcal proteins with potential for (i) development of novel anticryptococcal agents based on implications in cryptococcal virulence or survival, or (ii) development of an anti-cryptococcal vaccine, as several candidates lack homology to human proteins and are localized extracellularly. Furthermore, this study defines preexisting anti-cryptococcal immunoreactivity in healthy individuals at a molecular level, identifying target antigens recognized by sera from healthy control persons.

A faringite estreptocócica desencadeada pelo Streptococcus pyogenes pode resultar em uma série de doenças humanas e complicações como a febre reumática (FR) em indivíduos predispostos não tratados. A FR é uma doença autoimune que afeta mais de 20 milhões de crianças em países em desenvolvimento. A proteína M presente na membrana do S. pyogenes representa o maior fator de virulência da bactéria, e é objetivo de estudos para o desenvolvimento de uma vacina contra essa patologia. Atualmente mais de 200 tipos de proteínas M foram descritos na literatura e a sua porção Cterminal é conservada entre os diferentes tipos. Desenvolvemos um protótipo de vacina que compreende 55 resíduos de aminoácido da porção C-terminal, denominado StreptInCor. Neste trabalho analisamos a resposta humoral e celular específica contra o peptídeo sintético StreptInCor, usando camundongos transgênicos portadores de HLA de classe II humanos DR2, DR4, DQ6 e DQ8. O protocolo de imunização consistiu em administrar 50 g do StreptInCor adsorvido em 300 g de hidróxido de alumínio nos dias 0 e 14. Os grupos controles foram injetados com salina nas mesmas condições. O soro obtido no 28º dia foi testado por ensaio imunoenzimático (ELISA) para verificarmos a presença de anticorpos contra o StreptInCor e os esplenócitos destes animais, obtidos nessa data, foram utilizados para ensaios de proliferação celular na presença do StreptInCor. Testes de segurança foram efetuados e não observamos reação cruzada contra a miosina cardíaca e após 12 meses de acompanhamento, amostras de tecidos desses animais foram submetidas à análise histológica. Em conclusão não verificamos indícios de reações autoimunes nos animais imunizados com o StreptInCor e os resultados obtidos mostram a capacidade do StreptInCor em desencadear uma resposta imune, duradoura e segura em camundongos portadores de moléculas HLA de classe II / Streptococcal pharyngitis triggered by Streptococcus pyogenes throat infection can result in rheumatic fever (RF) and rheumatic heart disease (RHD) in untreated susceptible individuals. RF is an autoimmune disease that affects more than 20 million children in developing countries. M protein is the major factor of virulence of the bacteria, and it has been studied to develop a vaccine. Currently more than 200 M protein types have been described and its Cterminal domain is conserved in many different serotypes. We developed a vaccine epitope (StreptInCor) composed by 55 amino acid residues of the Cterminal portion of the M protein. In the present work we analyze the ability of the StreptInCor of induce immune response in HLA class II transgenic mice. The transgenic mice harboring the HLA Class II DR2, DR4, DQ6 and DQ8 were immunized subcutaneously with 50 g StreptInCor adsorbed onto 300 g of aluminum hydroxide gel on days 0 and 14. Control groups were immunized with vehicle (Saline) in same conditions. The sera were obtained on day 28 and tested by ELISA to verify the presence of antibodies. The specific cellular immune response was evaluated by proliferation assay using splenocytes. No cross reaction with cardiac myosin were observed. Tissue samples from immunized mice followed by 12 months were analyzed in order to verify if StreptInCor induces some histological damage. No autoimmune or deleterious reactions were observed. In conclusion our results indicate that StreptInCor Induces a good and prolonged and safe immune response in HLA class II transgenic mice

T. gondii é um protozoário amplamente disseminado pelo mundo que pode causar doença em animais e humanos. A evolução e a gravidade da doença dependem de características genéticas do parasita e do hospedeiro. A prevalência varia geograficamente, em Erechim, RS, 88% da população é soropositiva e 18% destes apresentam toxoplasmose ocular (TO). A resposta imune humoral contra T. gondii é persistente em todas as fases da infecção. O objetivo deste retrospectivo estudo foi correlacionar a imunidade humoral e a resposta contra peptídeos cepa-específicos com a gravidade da TO em pacientes de Erechim. 327 amostras de soro foram testadas (ELISA) para a pesquisa dos isótipos específicos e contra peptídeos cepa-específicos de regiões polimórficas (GRA6 e GRA7) do parasita. Nossos resultados sugerem que IgG2 e IgG3 estão associados à infecção adquirida recente, porém não há associação entre os isótipos e a evolução clínica da TO. Entretanto, embora seis diferentes sorotipos infectem estes pacientes, a gravidade da TO está associada a um novo padrão sorotípico (Atípico D). / T. gondii is a widespread protozoan parasite that is associated with a large spectrum of diseases in both humans and animals. The progression and severity of disease is quite variable and presumably due to some combination of host and parasite genetics. Prevalence varies with geography. In Erechim, Brazil, it is 88% prevalent and is related with a high incidence (18%) of ocular toxoplasmosis (OT). Humoral immune response against the parasite is effective. The aim of this retrospective study was to correlate the humoral immunity and response against the strain-specific peptides with the severity of the TO in Erechim`s patients. 327 sera were evaluated by ELISA to isotypes, IgG avidity and serotyped using strain-specific polymorphic peptides (GRA6 and GRA7). Our results suggest that IgG2 and IgG3 were associated with recent acquired infection. However, there is no association between isotypes and clinical evolution of OT, and also 6 different serotype-strains were detected in this population, but only one of these (Atypical D) was strongly associated with severe OT.

Introdução: A pandemia de gripe A H1N1 em junho de 2009 resultou em elevadas taxas de hospitalização entre pacientes imunodeprimidos, incluindo pacientes com artrite idiopática juvenil (AIJ). Embora a vacinação seja uma medida eficaz contra complicações da gripe pandêmica, não há estudos na literatura sobre seus efeitos na AIJ. Objetivos: Avaliar a resposta resposta da vacina contra influenza A H1N1/2009 sem adjuvante na AIJ, como uma extensão do estudo anterior de imunogenicidade e segurança em uma grande população de pacientes com doenças reumáticas juvenis. Além disso, avaliar a possível influência de dados demográficos, subtipos de AIJ, atividade da doença e do tratamento sobre a imunogenicidade e o potencial efeito deletério da vacina sobre a doença, particularmente sobre o número de articulações ativas e os marcadores inflamatórios. Métodos: 95 pacientes com AIJ e 91 controles saudáveis foram avaliados antes e 21 dias após a vacinação contra influenza A H1N1/2009 e a sorologia anti-H1N1 foi realizada por ensaio de inibição de hemaglutinação. A avaliação global de atividade da artrite por uma escala visual analógica (EVA) pelo paciente e pelo médico, o Childhood Health Assessment Questionnaire (CHAQ), o número de articulações ativas, as provas de fase aguda (VHS e PCR) e o tratamento foram avaliados antes e após a vacinação. Os eventos adversos foram também reportados. Resultados: Pacientes com AIJ e controles foram comparáveis em relação à média de idade atual (14,9 ± 3,2 vs. 14,6 ± 3,7 anos, p=0,182). A taxa de soroconversão após a vacinação foi significantemente menor nos pacientes com AIJ em relação aos controles (83,2% vs. 95,6%, p=0,008), particularmente no subtipo poliarticular (80% vs. 95,6%, p=0,0098). Os subtipos de AIJ, o número de articulações ativas, as provas de fase aguda, a EVA do paciente e do médico, o CHAQ e a frequencia de uso de DMARDs/imunossupressores foram semelhantes entre os pacientes que soroconverteram versus os que não soroconverteram (p>0,05). Em relação à segurança da vacina, não foi observada piora no número de articulações ativas e nas provas de fase aguda durante o período de estudo. Conclusão: A vacinação contra influenza A H1N1/2009 na AIJ induziu uma resposta humoral reduzida com adequado efeito protetor, independente de parâmetros da doença e tratamento, e com um perfil adequado de segurança da doença. / Introduction: The influenza H1N1 pandemic in June 2009 resulted in high hospitalization rates among immunocompromised patients, including patients with juvenile idiopathic arthritis (JIA). Although vaccination is an effective tool against pandemic flu complications, there are no studies in the literature on its effects in JIA. Objectives: To assess the immune response against the influenza A H1N1/2009 vaccine without adjuvant in JIA as an extension of previous observation of its immunogenicity and safety in a large population of patients with juvenile rheumatic diseases. Moreover to assess the possible influence of demographic data, subtypes of JIA, disease activity and treatment on the immunogenicity and the potential deleterious effect of vaccine on disease itself, particularly on the number of active joints and inflammatory markers. Methods: 95 JIA patients and 91 healthy controls were evaluated before and 21 days after vaccination against influenza A and serology for anti-H1N1 was performed by hemagglutination inhibition assay. The overall assessment of arthritis activity by a visual analogue scale (VAS) by patient and physician, the Childhood Health Assessment Questionnaire (CHAQ), the number of active joints, the acute phase reactants (ESR and CRP) and treatment were evaluated before and after vaccination. Adverse events were also reported. Results: JIA patients and controls were comparable regarding mean current age (14.9 ± 3.2 vs. 14.6 ± 3.7 years, p=0.182). After vaccination seroconversion rate was significantly lower in JIA patients compared to controls (83.2% vs. 95.6%, p=0.008), particularly in polyarticular subtype (80% vs. 95.6%, p=0.0098). JIA subtypes, number of active joints, acute phase reactants, patient and the physician VAS, CHAQ and frequency of use of DMARDs/Immunosuppressants were similar between patients with and without seroconversion (p>0.05). Regarding vaccine safety, no deterioration was observed in the number of active joints and the acute phase reactants during the study period. Conclusion: Influenza A H1N1/2009 vaccination in JIA induces a lower but effective antibody response, probably independent of disease parameters and treatment with an adequate disease safety profile.

Introdução: A pandemia de gripe A H1N1 em junho de 2009 resultou em elevadas taxas de hospitalização entre pacientes imunodeprimidos, incluindo pacientes com artrite idiopática juvenil (AIJ). Embora a vacinação seja uma medida eficaz contra complicações da gripe pandêmica, não há estudos na literatura sobre seus efeitos na AIJ. Objetivos: Avaliar a resposta resposta da vacina contra influenza A H1N1/2009 sem adjuvante na AIJ, como uma extensão do estudo anterior de imunogenicidade e segurança em uma grande população de pacientes com doenças reumáticas juvenis. Além disso, avaliar a possível influência de dados demográficos, subtipos de AIJ, atividade da doença e do tratamento sobre a imunogenicidade e o potencial efeito deletério da vacina sobre a doença, particularmente sobre o número de articulações ativas e os marcadores inflamatórios. Métodos: 95 pacientes com AIJ e 91 controles saudáveis foram avaliados antes e 21 dias após a vacinação contra influenza A H1N1/2009 e a sorologia anti-H1N1 foi realizada por ensaio de inibição de hemaglutinação. A avaliação global de atividade da artrite por uma escala visual analógica (EVA) pelo paciente e pelo médico, o Childhood Health Assessment Questionnaire (CHAQ), o número de articulações ativas, as provas de fase aguda (VHS e PCR) e o tratamento foram avaliados antes e após a vacinação. Os eventos adversos foram também reportados. Resultados: Pacientes com AIJ e controles foram comparáveis em relação à média de idade atual (14,9 ± 3,2 vs. 14,6 ± 3,7 anos, p=0,182). A taxa de soroconversão após a vacinação foi significantemente menor nos pacientes com AIJ em relação aos controles (83,2% vs. 95,6%, p=0,008), particularmente no subtipo poliarticular (80% vs. 95,6%, p=0,0098). Os subtipos de AIJ, o número de articulações ativas, as provas de fase aguda, a EVA do paciente e do médico, o CHAQ e a frequencia de uso de DMARDs/imunossupressores foram semelhantes entre os pacientes que soroconverteram versus os que não soroconverteram (p>0,05). Em relação à segurança da vacina, não foi observada piora no número de articulações ativas e nas provas de fase aguda durante o período de estudo. Conclusão: A vacinação contra influenza A H1N1/2009 na AIJ induziu uma resposta humoral reduzida com adequado efeito protetor, independente de parâmetros da doença e tratamento, e com um perfil adequado de segurança da doença. / Introduction: The influenza H1N1 pandemic in June 2009 resulted in high hospitalization rates among immunocompromised patients, including patients with juvenile idiopathic arthritis (JIA). Although vaccination is an effective tool against pandemic flu complications, there are no studies in the literature on its effects in JIA. Objectives: To assess the immune response against the influenza A H1N1/2009 vaccine without adjuvant in JIA as an extension of previous observation of its immunogenicity and safety in a large population of patients with juvenile rheumatic diseases. Moreover to assess the possible influence of demographic data, subtypes of JIA, disease activity and treatment on the immunogenicity and the potential deleterious effect of vaccine on disease itself, particularly on the number of active joints and inflammatory markers. Methods: 95 JIA patients and 91 healthy controls were evaluated before and 21 days after vaccination against influenza A and serology for anti-H1N1 was performed by hemagglutination inhibition assay. The overall assessment of arthritis activity by a visual analogue scale (VAS) by patient and physician, the Childhood Health Assessment Questionnaire (CHAQ), the number of active joints, the acute phase reactants (ESR and CRP) and treatment were evaluated before and after vaccination. Adverse events were also reported. Results: JIA patients and controls were comparable regarding mean current age (14.9 ± 3.2 vs. 14.6 ± 3.7 years, p=0.182). After vaccination seroconversion rate was significantly lower in JIA patients compared to controls (83.2% vs. 95.6%, p=0.008), particularly in polyarticular subtype (80% vs. 95.6%, p=0.0098). JIA subtypes, number of active joints, acute phase reactants, patient and the physician VAS, CHAQ and frequency of use of DMARDs/Immunosuppressants were similar between patients with and without seroconversion (p>0.05). Regarding vaccine safety, no deterioration was observed in the number of active joints and the acute phase reactants during the study period. Conclusion: Influenza A H1N1/2009 vaccination in JIA induces a lower but effective antibody response, probably independent of disease parameters and treatment with an adequate disease safety profile.

The ƒÑ-toxin of Clostridium perfringens is a toxin involved in numerous diseases of humans and agriculturally important animals. One of these diseases is necrotic enteritis (NE), a sporadic enteric disease which affects avian species world-wide. This study involved the inactivation of alpha-toxin (ƒÑ-toxin) for use as a potential vaccine candidate to combat NE in chickens, and other diseases caused by C. perfringens type A. During the course of this research a number of ƒÑ-toxin recombinant proteins were developed through molecular inactivation of the ƒÑ-toxin gene, plc. Proteins plc316 and plc204 were developed by the deletion of the first three and seven ƒÑ-helices of the N-terminal domain respectively. These deletions resulted in proteins which were unstable in solution, constantly aggregated into insoluble masses and elicited lower overall antibody responses when administered to mice. A third protein, plcInv3 was developed from the deletion of part of the catalytic domain of the ƒÑ-toxin. PlcInv3 was highly soluble and upon immunisation of mice elicited a significant antibody response which was also capable of protecting mice against a live challenge of C. perfringens. The fourth and final protein developed was plc104. The smallest of the recombinant ƒÑ-toxin proteins, it consisted entirely of the C-terminal domain of ƒÑ-toxin. Its small size did not affect its ability to induce a strong antibody response when administered to mice, the antibodies of which were also protective during a challenge with C. perfringens. STM1, an attenuated strain of S. Typhimurium was used in the development of a vectored vaccine for the expression and oral delivery of plcInv3 and plc104 within the mouse host. The proteins were expressed within STM1 from expression plasmids containing the in vivo inducible promoters PhtrA and PpagC. A measurable humoral immune response against ƒÑ-toxin was absent following three oral vaccinations with the vectored vaccines, although, cytokine profiling of splenocytes from vaccinated mice revealed an increase in the number of interleukin-4 (IL-4)secreting cells and the lack of interferon-gamma (IFN-ƒ×) secreting cells. This indicated the stimulation of a T-helper type 2 (TH2) immune response which also lead to partial protection against a live C. perfringens challenge. This study demonstrates the feasibility of using STM1 as a carrier for the in vivo expression of the C. perfringens ƒÑ-toxin recombinant proteins plcInv3 and plc104. It is the first study to express C. perfringens antigens within an attenuated strain of S. Typhimurium, STM1.The partial protection of mice immunised with these vaccines indicates there is potential for this vectored vaccine system to be used in the protection of diseases caused by the ƒÑ-toxin of C. perfringens.

<p>This study shows that herbs can be effectively screened for potiential bio-activity using in vitro methods. Further studies will be needed to better explore Artemisia afra&rsquo / s effect on immunoregulation, particularly long term effects of the herb on the immune system and its effect on other disease states.</p>

<p>This study shows that herbs can be effectively screened for potiential bio-activity using in vitro methods. Further studies will be needed to better explore Artemisia afra&rsquo / s effect on immunoregulation, particularly long term effects of the herb on the immune system and its effect on other disease states.</p>