Aggregatibacter actinomycetemcomitans está associado à periodontite agressiva e produz CDT. Visando contribuir no entendimento do papel da CDT na regulação da resposta imune, foi determinada sua atividade sobre macrófagos e a resposta humoral contra a toxina. CDT inibiu a proliferação de células epiteliais OBA-9 e macrófagos Raw 264.7 e também a produção de NO por células Raw 264.7 e macrófagos peritoneais de camundongos C3H/HePas e C3H/HeJ, mas estimulou a produção de IL-12. Na imunidade humoral, 75% dos soros de indivíduos com PAgL e 81,8% dos PAgG foram reativos para A. actinomycetemcomitans. Não houve diferença na resposta contra CDTA e CDTB entre o soro de pacientes com diferentes condições periodontais. Todos os pacientes PAgG foram soropositivos para a CDTC, porém apenas 8,3% dos indivíduos com PAgL, nenhum dos PC e 25% dos saudáveis foram positivos. CDT tem atividade imunomodulatória e a resposta humoral difere entre indivíduos infectados pela bactéria. / Aggregatibacter actinomycetemcomitans is associated with aggressive periodontitis and produces CDT. Aiming to contribute in the understanding the CDT activity in the immune response regulation, its action on macrophages was determined and the response against the toxin analyzed. CDT inhibited the proliferation of OBA-9 epithelial cells and Raw 264.7 macrophages and also inhibited the NO production by Raw 264.7 cells and peritoneal macrophages of C3H/HePas and C3H/HeJ mice, however, stimulated the IL-12 production. In the humoral immunity, 75% of sera from LAgP subjects and 81.8% were reactive to A. actinomycetemcomitans. There was not difference in the response against CDTA and CDTB among sera of patients with different periodontal conditions. All GAgP subjects were sera-reactivity to CDTC, however only 8.3% LAgP subjects, none in CP and 25% of healthy subjects were positive. CDT has immunomodulatory activity and the humoral response differ among bacteria infected subjects.

Para elucidar os mecanismos envolvidos na inibição da hipersensibilidade do tipo I em proles murinas mediada pela imunização materna com Ovalbumina (OVA), as proles foram avaliadas quanto aos linfócitos Tγδ produtores de IL-17 (CD27-). A imunização materna com OVA reduziu a expressão de CD27, o que também se refletiu nos pulmões, e a produção de IL-17 por linfócitos Tγδ das proles. A redução da expressão de CD27 também foi evidenciada em linfócitos Tγδ intratímicos das proles após a transferência passiva de anticorpos IgG alérgeno específicos para fêmeas gestantes não imunizadas e in vitro em resposta a estes mesmos anticorpos, efeito que parece envolver a expressão de receptores para IgG expressos por outras células presentes no timo. As evidências obtidas indicam que a imunização materna influi na maturação tímica de linfócitos inibindo a população que colaborara com a inflamação alérgica. Aparentemente, os anticorpos IgG maternos são responsáveis por este fenômeno e a população estudada está envolvida na inibição da inflamação alérgica observada nas proles. / To elucidate the mechanisms involved in inhibition of type I hypersensitivity in murine offsprring mediated by maternal immunization with Ovalbumin (OVA), the offspring were evaluated as IL-17-producing γδT cells (CD27-). Maternal immunization with OVA reduced the expression of CD27, which was also reflected in the lungs, and the production of IL-17 by γδT cells of offspring. Reduction of CD27 expression was also evidenced in intrathymic γδT cells of offspring after passive transfer of allergen-specific IgG antibodies to non-immunized pregnant females and in vitro in response to these same antibodies, effect that seems to involve the expression of IgG receptors expressed by other cells present in the thymus. Evidence obtained indicates that maternal immunization influences the thymic maturation of lymphocytes by inhibiting the population that collaborate with allergic inflammation. Apparently, maternal IgG antibodies are responsible for this phenomenon and the population studied is involved in the inhibition of allergic inflammation observed in offspring.

O Lúpus Eritematoso Sistêmico (LES) é uma doença auto-imune caracterizada pela deposição de imunocomplexos nos tecidos. O clareamento de imunocomplexos está comprometido no LES, contribuindo para a patogênese da nefrite lúpica. Os receptores Fcg (FcgR) participam do clareamento dos imunocomplexos contendo IgG, pois se ligam à porção Fc desta molécula. O FcgRIIa é um receptor que tem dois alelos co-dominantemente expressos, o R131 e o H131, os quais diferem na sua eficiência em se ligar a subclasses de IgG. Células que expressam o homozigoto FcgRIIa-H/H131 são as únicas que se ligam eficientemente a imunocomplexos contendo IgG2, enquanto as que expressam FcgRIIa-R/R131 o fazem de forma menos eficaz. Este polimorfismo tem sido descrito como fator de risco para nefrite lúpica, embora ainda haja controvérsias. O propósito do nosso estudo foi o de analisar, em uma população de nefrite lúpica e em outra de glomerulopatias primárias, a associação entre o genótipo FcgRIIa-R/R131 e a gravidade da doença renal na sua instalação (definida pelo momento da biópsia renal) e ao final do seguimento, bem como possíveis relações com aspectos histológicos renais. A genotipagem do receptor FcgRIIa foi realizada em 76 pacientes com nefrite lúpica e 63 com glomerulopatias primárias através da extração do DNA genômico, seguido de reação de polimerização em cadeia (PCR) e nested PCR, utilizando-se primers específicos. Os pacientes foram avaliados por parâmetros clínicos e laboratoriais. Setenta e um pacientes com nefrite lúpica realizaram biópsia renal, enquanto 5 que já se encontravam em hemodiálise não a realizaram. Pacientes com glomerulonefrite membranoproliferativa, nefropatia da IgA e glomerulonefrite proliferativa mesangial foram agrupados como glomerulopatias proliferativas enquanto os com glomeruloesclerose segmentar e focal, glomerulopatia de lesões mínimas ou glomerulonefrite membranosa foram agrupados como glomerulopatias não proliferativas. O homozigoto FcgRIIa-R/R131 foi mais prevalente no grupo com nefrite lúpica (42,1% de R/R131 e 14,5% de H/H131) em relação ao grupo com glomerulopatias primárias (23,8% de R/R131 e 23,8% de H/H131), dado este estatisticamente significativo (p<0.05). Houve segregação do genótipo FcgRIIa- R/R131 nos pacientes com nefrite lúpica quando comparados aos com glomerulopatias não proliferativas, mas não quando comparados aos com glomerulopatias proliferativas (p<0.05). Não houve diferença na distribuição genotípica do receptor FcgRIIa em relação a classe histológica de nefrite lúpica, tampouco em relação aos que evoluíram ou não para insuficiência renal (Pcr = 1,4mg/dl ao final do seguimento). Um aumento na frequência do genótipo FcgRIIa- R/R131 foi encontrado nos pacientes com nefrite lúpica apresentando níveis mais elevados de FAN (FAN>1/100) e consumo de complemento C3 (p<0,05), mas não naqueles com presença de anticorpos anti-dsDNA ou anti-fosfolípide (p>0,05). Estes achados sugerem que uma distribuição anormal dos genótipos do receptor FcgRIIa com predomínio do homozigoto R/R131 é um fator importante que pode influenciar o desenvolvimento de nefrite lúpica e de glomerulopatias proliferativas. O genótipo FcgRIIa-R/R131 também está relacionado com maior atividade lúpica (FAN>1/100 e consumo de C3) em pacientes brasileiros. / Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by tissue deposition of immune complexes. Immune complex clearance is impaired in SLE, contributing to the pathogenesis of lupus nephritis. Fcg receptors (FcgR) participate in the clearance of the immune complexes containing immunoglobulin G, because they bind the Fc domain of this molecule. The FcgRIIa receptor has two co dominant alleles, R131 and H131. They differ in their efficiency to bind IgG subclasses. Cells expressing the homozygote FcgRIIa-H/H131 are the only ones, which bind efficiently immune complexes containing IgG2, whereas those expressing FcgRIIa-R/R131 do not. This polymorphism has been described as a risk factor for lupus nephritis. However, reports are still controversial. This study aims to establish the role of FcgRIIa polymorphism in the severity and prognosis of lupus nephritis compared to primary glomerulopathies, and whether it is related to histological findings or not. In 76 patients with lupus nephritis and 63 patients with primary glomerulopathies, genotyping of the FcgRIIa receptor was performed with standard PCR, followed by nested PCR using specific primers. The same patients were assessed according to clinical and laboratory patterns. Seventy-one patients with lupus nephritis underwent biopsy, while five did not since they were already under dialysis. Patients diagnosed as membranoproliferative glomerulonephritis, IgA glomerulonephritis and mesangial proliferative glomerulonephritis were grouped as proliferative glomerulopathies, while those with focal segmental glomerulosclerosis, membranous glomerulonephritis and minimal change disease were grouped as nonproliferative glomerulopathies. The homozygous FcgRIIa-R/R131 was more prevalent in lupus nephritis (42,1% being R/R131 and 14,5% H/H131) than in glomerulopathies (23,8% being R/R131 and 23,8% H/H131). These data were statistically significant (p<0.05). A segregation of the FcgRIIa-R/R131 genotype was found in patients with lupus nephritis compared to nonproliferative glomerulopathies, but not when compared to proliferative glomerulopathies (p<0.05). No relation was found between genotype distribution and histological class or renal insufficiency (end-study serum creatinine = 1.4 mg/dl). The genotype R/R131 was more prevalent in lupus nephritis patients presenting complement 3 (C3) consumption and higher antinuclear factor (ANF) titers, but not in those with antidouble- stranded DNA or antiphospholipid antibodies (p>0.05). We concluded that a skewed distribution of the FcgRIIa genotypes with R/R131 predominance may contribute to the development of lupus nephritis and proliferative glomerulopathy. In Brazilian patients, this polymorphism is also related to more intense lupus activity (ANF > 1/100 and C3 consumption).

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-03-16T19:41:01Z No. of bitstreams: 1 anamarciamenezesdemattos.pdf: 1900352 bytes, checksum: 4c5592f85b3e69efcd500c09d135cdcd (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-03-18T11:49:02Z (GMT) No. of bitstreams: 1 anamarciamenezesdemattos.pdf: 1900352 bytes, checksum: 4c5592f85b3e69efcd500c09d135cdcd (MD5) / Made available in DSpace on 2017-03-18T11:49:02Z (GMT). No. of bitstreams: 1 anamarciamenezesdemattos.pdf: 1900352 bytes, checksum: 4c5592f85b3e69efcd500c09d135cdcd (MD5) Previous issue date: 2016-11-10 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A tuberculose (TB) permanece como um dos maiores problemas de saúde pública no mundo. A identificação de novos antígenos de Mycobacterium tuberculosis e de biomarcadores de infecção, capazes de auxiliar no diagnóstico da doença ativa e da infecção latente são metas importantes para o controle da TB. No presente trabalho foram estudados pacientes TB antes e após receberem o tratamento, e indivíduos contatos TB com diagnóstico de infecção latente (PPD+). Foram investigados os níveis séricos de IgG e IgG1 e a produção de citocinas e quimiocinas contra os seguintes antígenos: 1) antígenos da fase de infecção ativa (ESAT-6/CFP-10, Rv0717 e Rv3353); 2) infecção latente ou DosR (Rv1733, Rv2628, Rv2029 e Rv1737); e, 3) antígenos da fase de reativação da infecção ou Rpf (Rv0867 e Rv2389). A produção de IFN-ꝩ, TNF-α,IL-17, IL-10 e IL-4 foi avaliada em culturas de células mononucleares de sangue periférico (PBMC) estimuladas com antígenos do M. tuberculosis, através de ELISA e/ou CBA-citometria. Níveis séricos de CCL-2, CXCL-8, CXCL-9 e CXCL10 foram mensurados em pacientes TB e controles. Nossos resultados mostram níveis séricos elevados de anticorpos IgG1 contra ESAT-6/CFP-10, Rv0717, Rv3353, Rv1733, Rv2029, Rv2628 e Rv0867 em pacientes com TB ativa em comparação ao grupo controle (p<0.001). Após completada a quimioterapia, a resposta de IgG1 frente aos antígenos ESAT-6/CFP-10, Rv3353, Rv2628, Rv2029, e Rv0867 foi reduzida a níveis de controle (p<0,01). A análise da curva ROC confirmou o bom desempenho dos antígenos Rv0717, Rv1733, Rv3353, Rv2628, Rv2029 e Rv0867 no diagnóstico da TB. Interessantemente, Rv0717 e Rv1733 induziram um pico de resposta de IgG1 após 1-3 meses de quimioterapia (p<0,01). Neste grupo de pacientes (1-3M), uma alta produção de IFN-ꝩ e TNF-α foi observada após estimulação de PBMC com ESAT6/CFP-10, Rv1733 e Rv2029. Os grupos não diferiram na produção de IL-17, IL-10 e IL-4, indicando uma predominante resposta com perfil Th1 nos pacientes estudados. Níveis elevados de CXCL-8, CXCL-9 e CXCL-10 foram detectados no soro de pacientes com TB ativa. Após 6 meses de tratamento os níveis séricos de CXCL-9 e CXCL-10 foram reduzidos a níveis de controle. Níveis séricos elevados de anticorpos IgG e IgG1 específicos para os antígenos ESAT-6/CFP-10 e Rv1733 foram observados em pacientes com TB ativa e em contatos TB, em comparação com o grupo controle (p< 0,05). De forma semelhante, pacientes TB e contatos TB também não diferiram quanto a produção de IFN-ꝩ em resposta ao antígeno ESAT-6/CFP-10, indicando haver dificuldade em diferenciar pacientes com TB ativa de indivíduos com TB latente. Interessantemente, em resposta ao antígeno DosR Rv1733, níveis elevados de IFN-ꝩ foram observados nas culturas de PBMC dos contatos TB, em comparação aos grupos TB ativa e controle sadio (p<0,05). Em conjunto, esses resultados sugerem a existência de biomarcadores da tuberculose, representados por: 1) detecção de níveis elevados de anticorpos IgG1 contra ESAT-6/CFP-10, Rv0717, Rv3353, Rv1733, Rv2628, Rv2029 e Rv0867; 2) alta produção de IFN-ꝩ e TNF-α em resposta a diferentes antígenos do M. tuberculosis; e, 3) níveis séricos aumentados de CXCL-9 e CXCL-10. Esse perfil de resposta imune pode auxiliar no diagnóstico da TB e no monitoramento do tratamento. Além disso, a detecção de níveis elevados de IFN-ꝩ em resposta ao antígeno Rv1733 pode contribuir no diagnóstico da doença em contatos TB que apresentam tuberculose assintomática. / Tuberculosis (TB) remains a major public health problem in the world. The identification of new Mycobacterium tuberculosis antigens and infection biomarkers capable of assisting in the diagnosis of active disease and latent infection are important targets for TB control. In the present study, TB patients before and after receiving treatment and contacts TB diagnosed with latent infection (PPD +), were studied. We investigated the levels of serum IgG and IgG1 and the production of cytokines and chemokines against the following antigens: 1) antigens from the active phase of infection (ESAT-6/CFP-10, Rv0717 and Rv3353); 2) latent infection antigens or DosR (Rv1733, Rv2628, Rv2029 and Rv1737); and 3) antigens from the reactivation phase of infection or Rpf (Rv0867 and Rv2389). The production of IFN-ꝩ, TNF-α, IL-17, IL-10 and IL-4 was evaluated in peripheral blood mononuclear cell (PBMC) cultures stimulated with M. tuberculosis antigens, by using ELISA and/or CBA-cytometry. Serum levels of CCL-2, CXCL-8, CXCL-9 and CXCL-10 were measured in TB patients and controls. Our results show elevated serum levels of IgG1 antibodies against ESAT6/CFP-10, Rv0717, Rv3353, Rv1733, Rv2628, Rv2029 and Rv0867 in patients with active TB, in comparison with the control group (p<0.001). After completion of the chemotherapy, the IgG1 response to ESAT-6/CFP-10, Rv3353, Rv2628, Rv2029 and Rv0867 was reduced to control levels (p <0.01). The ROC curve analysis confirmed the good performance of Rv0717, Rv3353, Rv1733, Rv2628, Rv2029 and Rv0867, and antigens in the diagnosis of TB. Interestingly, Rv0717 and Rv1733 induced an IgG1 peak response after 1-3 months of chemotherapy (p<0.01). In this patient group (1-3m), a high IFN-ꝩ and TNF-α response was observed after stimulation of PBMC with ESAT-6/CFP-10, Rv1733 and Rv2029. The groups did not differ in IL-17, IL-10 and IL4, indicating a predominant Th1 response in the patients studied. High levels of CXCL8, CXCL-9 and CXCL-10 were detected in the serum of patients with active TB. After 6 months of treatment, levels of both CXCL-9 and CXCL-10 were reduced to control levels. Elevated serum levels of IgG and IgG1 specific for both ESAT-6/CFP-10 and Rv1733 were observed in patients with active TB and in TB contacts, in comparison with the control group (p<0.05). Similarly, TB patients and TB contacts also did not differ in the production of IFN-ꝩ in response to ESAT-6/CFP-10, indicating that the discrimination between patients with active TB and individuals with latent TB is difficult. Interestingly, in response to the DosR Rv1733 antigen, high levels of IFN-ꝩ were observed in PBMC cultures of TB contacts in comparison to both active TB and healthy control groups (p<0.05). Together, these results suggest the existence of tuberculosis biomarkers represented by: 1) elevated serum levels of IgG1 antibodies against ESAT6/CFP-10, Rv0717, Rv3353, Rv1733, Rv2628, Rv2029 and Rv0867; 2) high IFN-ꝩ and TNF-α production in response to different M. tuberculosis antigens; and 3) increased serum levels of CXCL-9 and CXCL-10. This profile of immune response may assist in the diagnosis of TB and monitoring drug therapy. Furthermore, detection of high levels of IFN-ꝩ in response to the Rv1733 antigen may contribute to the diagnosis of TB disease in contacts presenting asymptomatic tuberculosis.

As espécies Klebsiella pneumoniae, Escherichia coli e Pseudomonas aeruginosa são responsáveis por infecções neonatais hospitalares. Lipopolissacarídeo (LPS) é o principal indutor de respostas inflamatórias. Os objetivos foram avaliar a transferência placentária de IgG reativa ao LPS de K. pneumoniae, E. coli O111, O26 e O6 e P. aeruginosa empregando ELISA para dosar IgG em soro materno e de cordão de 29 neonatos pré-termos e 32 a termo; analisar IgM total e específica no soro materno; e investigar a influência das patologias apresentadas pelas mães na transferência placentária. Concentrações de IgG total foram reduzidas em pré-termos como esperado, porem índices de transferência placentária de IgG total e IgG anti-LPS foram sistematicamente reduzidos quando comparados aos neonatos a termo. Níveis de IgM total e anti-LPS foram equivalentes em mães de ambos os grupos. As patologias das mães influenciaram os níveis de IgM no grupo de mães de pré-termos. Estes resultados indicam uma imunidade adquirida deficiente pelo grupo pré-termo aumentando os riscos de infecção. / Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa species are responsible for neonatal nosocomial infections. Bacterial lipopolysaccharide (LPS) is the major inducer of the inflammatory responses. The aims were to evaluate the placental transfer of IgG reactive to LPS present in K. pneumoniae, in E. coli O111, O26 and O6 and in P. aeruginosa employing ELISA to detect IgG in maternal and cord sera from 29 preterm and 32 term neonates; to analyze total and specific IgM on the mothers sera; and to investigate the influence of the pathologies presented by some mothers in the placental transfer. Total IgG concentrations were reduced in preterm neonates as expected, but placental transfer indexes of total and anti-LPS IgG were systematically reduced when compared with term neonates. Total and anti-LPS IgM levels were equivalent on mothers of both groups. The mothers pathologies influenced only the IgM levels in the preterm mothers group. These results indicate a deficient acquired immunity by the preterm group increasing the risk of infection.

A doença de Chagas, causada pelo parasita protozoário Trypanosoma cruzi, afeta milhões de pessoas, a maioria delas vivendo na América latina. Apesar dos avanços da medicina e da biotecnologia, ainda existem poucas opções de tratamento para indivíduos com a doença. Assim, é importante compreendermos os detalhes moleculares da infecção parasitária, para que novas alternativas terapêuticas e de diagnóstico possam ser desenvolvidas para esses pacientes. Neste trabalho estudamos esta doença em duas frentes, uma do ponto de vista do parasita, e a outra, da resposta do hospedeiro. Utilizando bioinformática, identifcamos um peptídeo conservado (denominado TS9) presente nas proteínas de superfície gp85/transsialidases do parasita. Este peptídeo é capaz de promover adesão celular e, na sua forma sintética, inibe a entrada do T. cruzi na célula hospedeira. Análise da estrutura proteica revelou que o peptídeo TS9 encontra-se num domínio do tipo laminina-G, lado-a-lado com o peptídeo FLY, outro peptídeo conservado desta grande família, previamente descrito pelo nosso grupo. Juntos, eles formam um sítio de adesão a citoqueratinas e proteínas de flamento intermediário. Na segunda parte, investigamos os antígenos e epítopos reconhecidos pelas imunoglobulinas de pacientes portadores da doença nas suas diferentes formas clínicas: assintomática e cardiomiopatias, leve ou grave. Criamos uma biblioteca de phage display contendo, virtualmente, todos os fragmentos proteicos existentes no T. cruzi, que foi varrida contra imunoglobulinas para a construção de um mapa da resposta humoral dos pacientes com a doença de Chagas. Nossos resultados mostram que a resposta dos pacientes é complexa, e mais de dois mil epítopos foram mapeados. Muitos deles, como os antígenos B13, SAPA e FRA já foram previamente descritos, validando nosso método. Porém, um grande número de novos epítopos, inclusive contra proteína descritas como hipotéticas ou sem função conhecida, também foram encontrados. Seus papéis na infecção e resposta imune da doença merecem, portanto, atenção. Em resumo, as abordagens e técnicas utilizadas nesta tese são inovadoras, e permitiram a identifcação de peptídeos e moléculas que poderão ser úteis para o desenvolvimento de novos métodos diagnósticos e terapêuticos para a doença de Chagas. / Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, afects millions of people, most of them living in Latin America. Despite advances in medicine and biotechnology, there are still few treatment options for individuals with the disease. Thus, it is important to understand the molecular details of the parasitic infection, so that new therapeutic and diagnostic alternatives can be developed for these patients. In this work, we study this disease in two fronts, one from the point of view of the parasite, and the other, of the response of the host. Using bioinformatics, we identifed a conserved peptide (called TS9) present in the surface proteins gp85 / trans-sialidases of the parasite. This peptide is capable of promoting cell adhesion and, in its synthetic form, inhibits the entry of T. cruzi into the host cell. Analysis of the protein structure revealed that the TS9 peptide is in a laminin-G-like domain, side-by-side with the peptide FLY, another conserved peptide of this large family, previously described by our group. Together, they form an adhesion site to cytokeratins and intermediate flament proteins. In the second part, we investigated the antigens and epitopes recognized by the immunoglobulins of patients with the disease in their diferent clinical forms: asymptomatic and cardiomyopathies, mild or severe. We created a phage display library containing virtually all existing protein fragments in T. cruzi. This library was screened against immunoglobulins for the construction of a humoral response map of patients with Chagas disease. Our results show that the response of the patients is complex, and more than 2,000 epitopes have been mapped. Many of them, such as the B13, SAPA and FRA antigens have been previously described, validating our method. However, a large number of new epitopes, including many against proteins described as hypothetical or with no known function, were also found. Their roles in infection and immune response of the disease deserve, therefore, attention. In summary, the approaches and techniques used in this thesis are innovative and have allowed the identifcation of new peptides and molecules that may be useful for the development of new diagnostic and therapeutic methods for Chagas disease.

Diarrhoe ist eines der häufigsten Probleme beim equinen Neonaten. Nahezu alle Fohlen entwickeln Durchfall innerhalb der ersten Lebenswochen. Unterschiedliche virale, bakterielle und parasitäre Ursachen werden diskutiert. In diesen Zeitraum fällt ebenfalls die erste Rosse der Stute, sodass der Durchfall um den 5. - 15. Lebenstag (LT) bei den Fohlen als „Fohlenrossedurchfall“ bezeichnet wird. Es wird vermutet, dass die Entwicklung der intestinalen Mikroflora und die Reifung der Darmschleimhaut im Wesentlichen für das Durchfallgeschehen verantwortlich sind. Bisher ist jedoch wenig bekannt über die Entwicklung der intestinalen Mikroflora bei Fohlen. Einige Probiotika sind als Darmflorastabilisatoren bei Tieren zugelassen. Studien belegten positive Effekte von Toyocerin® (B. cereus var. toyoi) auf die Darmgesundheit bei anderen Tierarten wie z.B. Kälbern, Ferkeln, Broilern, Puten und Mastkaninchen. Die vorliegende Arbeit sollte klären, ob die Supplementierung von B. cereus var. toyoi zu einer Stabilisierung der sich entwickelnden intestinalen Mikroflora und damit zu einer Verringerung der Durchfälle bei Fohlen führt. Die Untersuchung wurde an 25 Mutterstuten eines Vollblutgestüts und ihren Fohlen durchgeführt. Alle Fohlen wurden von Februar bis Mai 2011 geboren. Von Geburt an wurden die Fohlen randomisiert in drei Behandlungsgruppen eingeteilt: Placebo-Gruppe (10 ml isotone Kochsalzlösung, n=8), 50 mg Toyocerin-Gruppe (5 x 108 KbE B. cereus var. toyoi gelöst in 10 ml isotoner Kochsalzlösung, n=7) und 200 mg Toyocerin-Gruppe (2 x 109 KbE B. cereus var. toyoi gelöst in 10 ml isotoner Kochsalzlösung, n=10). Die Placebo- und Behandlungsgruppen wurden einmal täglich vom 1. – 58. LT supplementiert. Herz- und Atemfrequenz, Körperinnentemperatur und die Körpermasseentwicklung wurden nach einem standardisierten Protokoll erhoben. Kotproben konnten mit Hilfe von Kotsammelbeuteln oder durch rektale Entnahme innerhalb von 24 Stunden nach der Geburt sowie an LT 9, 16, 23, 30, 44, 58 und am ersten Durchfalltag gewonnen werden. Blutproben wurden aus der Vena jugularis externa am 1., 9., 16., 30., 58. LT sowie am ersten Durchfalltag entnommen. Die bakteriologische Untersuchung erfolgte mit Hilfe des Kulturverfahrens. Die Bestimmung der Gesamt-IgG-Werte wurde mit einem kompetitiven ELISA, die Bestimmung der spezifischen Antikörper IgG-anti-LPS von E. coli J5 und IgG-anti-PLC-von-C. perfringens-1a mit einem indirekten ELISA durchgeführt. 88 % der Fohlen entwickelten Durchfall (Placebo 7/8, 50 mg Toyocerin 5/7, 200 mg Toyocerin 10/10) mit einer hohen Inzidenz zwischen dem 8. und 16. LT. Das Allgemeinbefinden und die Bewegungs- und Sauglust blieben dabei unbeeinflusst. Zum Zeitpunkt des ersten Östrus der Stute zeigten 59 % der Fohlen Diarrhoe. Unter den 41 %, die keinen Durchfall zum Zeitpunkt der Fohlenrosse hatten, waren Fohlen, die nie Durchfall vom 1. – 58. LT zeigten, aber auch welche die Diarrhoe entwickelten, als die Mutterstute sich nicht in Rosse befand. Ein Zusammenhang zwischen der Fohlenrosse der Stute und Durchfall bei ihrem Fohlen konnte nicht hergestellt werden. Es zeigte sich eine Tendenz, dass hohe Spiegel der Gesamt-IgG (>20 mg/ml) und IgG-anti-LPS von E. coli J5 (>120 RE/ml) nach der Kolostrumaufnahme im Zusammenhang mit einer geringeren Anzahl von Durchfalltagen innerhalb der ersten zwei Lebensmonate standen. C. perfringens und Enterobakterien waren gleichermaßen nachweisbar bei Fohlen mit Durchfall als auch bei unauffälligen Fohlen. Aus der Supplementierung von B. cereus var. toyoi ergab sich kein Effekt auf die Kotflora der Fohlen, außer auf die Gesamtkeimzahlen (GKZ) der aeroben Bakterien. Bei den Aerobiern im Fohlenkot konnte ein signifikanter Behandlungseffekt (p=0,012) festgestellt werden. Im ersten Milchkot der Fohlen waren GKZ von 4,5 x 104 KbE/g (200 mg Toyocerin-Gruppe) bis 5,0 x 105 KbE/g (50 mg Toyocerin-Gruppe) bei den aeroben Bakterien und GKZ von 2,4 x 105 KbE/g (200 mg Toyocerin-Gruppe) bis 4,7 x 106 KbE/g (50 mg Toyocerin-Gruppe) median bei den Anaerobiern nachweisbar. Danach stieg der Gehalt der aeroben und anaeroben Bakterien weiter bis zum 3. LT und stagnierte bis zum 16. LT. Während dieser Stagnationsphase trat bei 92 % der Fohlen (23/25) eine Veränderung der Kotkonsistenz bis hin zu Durchfällen auf. Vom 16. bis zum 58. LT sanken die Gehalte moderat bei den Aerobiern median am 58. LT auf 2,7 x 105 KbE/g (Placebo-Gruppe) bis 2,2 x 106 KbE/g (50 mg Toyocerin-Gruppe) und bei den Anaerobiern median am 58. LT auf 3,8 x 105 KbE/g (Placebo-Gruppe) bis 2,9 x 106 KbE/g (200 mg Toyocerin-Gruppe). Bis zum 58. LT näherte sich der Medianwert der aeroben und anaeroben Bakterien im Kot der Placebo-Gruppe dem Wert der Mutterstuten (gemessen am ersten Tag nach der Geburt) an. Innerhalb der ersten Lebenstage war eine hohe aerobe sowie anaerobe Keimzahl im Kot der Fohlen nachzuweisen, die sich oberhalb der Keimzahlen befand, die im Kot der Mutterstuten zum Zeitpunkt der Geburt gemessen wurde. Im Rahmen der Entwicklung und Etablierung der bakteriellen intestinalen Mikroflora wurde das Fohlenrossedurchfallgeschehen bei den Fohlen beobachtet. B. cereus var. toyoi hatte dabei keinen Einfluss auf die Anzahl der Fohlen mit Durchfall und den Gesundheitsstatus der Fohlen. / Diarrhoea is probably one of the most common problems in equine neonates. Almost all foals develop transient diarrhoea within the first weeks of life. Different viral, bacterial and parasitic causes are discussed. Between the 5th and the 15th day of the foal’s life, when their dam’s first post partum (p.p.) oestrus is expected, diarrhoea in foals is observed quite often. That is why it’s called “foal heat diarrhoea”. In literature establishment of intestinal microflora and maturation of the intestinal mucosa is responsible for the occurrence of diarrhoea in this period of life. But little is known about the development of the intestinal microflora in foals. Many probiotics are authorised as gut flora stabilisers in animal nutrition. Some studies proved positive effects of Bacillus (B.) cereus var. toyoi (Toyocerin®) on intestinal health in other species e.g. calves, piglets, broiler chicken, poultry and growing rabbits. The present study deals with the question if a supplementation of B. cereus var. toyoi lead to a stabilisation of the developing intestinal microflora and therefore to a reduction of diarrhoea in foals. A total of 25 mares and foals of a thoroughbred stud were included into the study. Foals were born between February and May 2011. From birth, the foals were randomly assigned to three treatment groups: placebo group (10 ml isotonic saline solution, n=8), 50 mg Toyocerin group (5 x 108 cfu B. cereus var. toyoi solved in 10 ml isotonic saline solution, n=7) and 200 mg Toyocerin group (2 x 109 cfu B. cereus var. toyoi solved in 10 ml isotonic saline solution, n=10). Placebo- and treatment groups were orally supplemented once a day starting on the 1st through to the 58th day of life. Determination of heart and respiratory rate, body temperature, body weight was realised according to a standardised protocol. Within the first day of life, on day 9, 16, 23, 30, 44, 58 and on the first day of diarrhoea faecal samples has been taken from the rectum or by the use of a collection bag. Blood samples were taken via jugular venipuncture on day 1, 9, 16, 30, 58 and on the first day of diarrhoea. Culture-depend methods were used to analyse the bacterial microflora. Serum IgG was analysed by a competitive ELISA, IgG-anti-LPS from E. coli J5 and IgG-anti-PLC-from-C. perfringens-1a by an indirect ELISA. 88 % of the foals developed diarrhoea (placebo 7/8, 50 mg Toyocerin 5/7, 200 mg Toyocerin 10/10) with a high incidence between the 8th and the 16th day of the foal’s life. Meanwhile, foals remained bright and alert and continued to nurse. At the time point of the first p.p. oestrus in the mares, 59 % of their foals showed signs of diarrhoea. Within the remaining 41 % there are foals that had no diarrhoea but there are also foals which had diarrhoea when the mare had not been in heat. Neonatal diarrhoea in foals is not linked to p.p. oestrus in their mares. There was a tendency, that high serum-IgG (> 20 mg/ml) and IgG-anti-LPS from E. coli J5 (> 120 RE/ml) after colostrum uptake were associated with lower diarrhoea severity in the first 58 days of the foal’s life. C. perfringens and enterobacteria can be found equally in foals with diarrhoea and in foals which are not afflicted. B. cereus var. toyoi supplementation had no effect on faecal bacteria in foals, except on aerobic bacteria (p=0,012). In the first milk faeces aerobic bacteria were detected in median from 4,5 x 104 cfu/g (200 mg Toyocerin-group) to 5,0 x 105 cfu/g (50 mg Toyocerin-group) and anaerobic bacteria were detected in median from 2,4 x 105 cfu/g (200 mg Toyocerin-group) to 4,7 x 106 cfu/g (50 mg Toyocerin-group). Afterwards the counts increased towards the 3rd day of life and stayed on a high level till the 16th day of life. During this stagnation in 92 % of the foals a change in faecal consistency and diarrhoea was observed. Afterwards, from the 16th though to the 58th day of life, the bacteria counts in the faeces moderately decreased in median for the aerobic bacteria on the 58th day of life down to 3,8 x 105 cfu/g (placebo-group) till 2,9 x 106 cfu/g (200 mg Toyocerin-group). On the 58th day of life the counts of aerobic and anaerobic bacteria in the faeces of the placebo-group approached the counts in the faeces of the mare (measured at the time point of birth). In the first days of foals’ life detection of aerobic and anaerobic bacteria in the faeces were high, and above the level of the bacteria counts in the faeces of the mare at the time point of birth. Foal heat diarrhoea is observed as a part of the development and establishment of bacterial intestinal microflora. B. cereus var. toyoi had no effect on the percentage of foals with diarrhoea and health status in the foals at that point.

When exposed to light, metal nanoparticles exhibit a phenomenon known as LSPR, Localized Surface Plasmon Resonance. The wavelengths at which LSPR occurs is very dependent on the refractive index of the surrounding medium. Binding of biomolecules to the surface of gold nanoparticles result in a change in the refractive index that can be detected spectrophotometrically by monitoring the LSPR peak shift. When functionalized with the corresponding ligand(s), gold nanoparticles can be utilized in biosensors to detect the presence and concentration of a predetermined analyte. However, the system must exhibit high specificity and give rise to a detectable shift for analytes in the desired concentration range to be of commercial interest. The aim of the diploma project was to investigate and optimize the biofunctionalization and performance of a fiber optics based LSPR biosensor.  Three ligand systems were investigated for detection of antibodies (IgG), insulin and avidin. Binding of the analyte to the ligand caused a shift of a few nanometers when using spherical gold nanoparticles. The shifts were significantly larger when using gold nanorods. When using the IgG and insulin ligands, only minor unspecific binding was observed. The setup thus shows great potential for use in a wide range of sensing applications.

<p>Njurarna är ett organsystem med viktiga funktioner som exempelvis utsöndring av flertalet vattenlösliga substanser. För att sjukdomssymtom ska uppträda krävs mer än tre fjärdedelars bortfall av njurfunktionen, eftersom njurarna har en enorm reservkapacitet. Genom att analysera bland annat proteinerna albumin, immunoglobulin G, kappa och lambda i urin utreds om njurfunktionen fungerar som den ska. Analys av dessa proteiner kan ske med analysinstrumenten IMMAGE 800 från Beckman Coulter och BN ProSpec från DADE BEHRING. Båda dessa analysinstrument använder sig av metoden nefelometri, som är en metod där ljusspridning i en vätska eller gas kan mätas.</p><p>Syftet med föreliggande studie var att analysera urinprover på både IMMAGE 800 och BN ProSpec och sedan jämföra resultaten. Under denna studie kalibrerades standardkurvor, genomfördes kvalitetskontroller och 37 prov analyserades. Samma prov analyserades flera gånger, både under samma dag och vid ett antal kommande dagar för att erhålla precisionen. Korrelationskoefficienten blev 0,999 för U-albumin; 0,998 för U-IgG; 0,947 för U-kappa och 0,883 för U-lambda. ProSpec kan således användas vid analys av U-albumin, U-IgG, U-kappa och U-lambda då den uppfyller EQUALIS kvalitetsmål.</p>

The pathogen Streptococcus pyogenes is a significant cause of human morbidity and mortality. Most of the work in this thesis is focused on streptococcal virulence factor IdeS, but the thesis also features work on SpeB, another streptococcal virulence factor. Both IdeS and SpeB are secreted cysteine proteases and both have previously been shown to degrade human IgG. IgG is the only known substrate for IdeS while SpeB is a more promiscuous protease with a larger number of identified substrates. A significant part of the data presented in this thesis is the result of designing and optimizing methods to detect and accurately measure the proteolytic degradation of IgG. Methods aimed at measuring the binding interactions between enzyme and substrate have also been frequently utilized. I show that IdeS is a monomeric protease, as opposed to previously published data that suggested it to be dimeric. IdeS cleaves the two heavy chains of IgG in a two-step reaction and I demonstrate that the first cleavage is magnitudes faster than the second one. This means that IdeS is a more efficient enzyme than previously thought. The difference in rate cannot completely be explained by a loss of affinity between IdeS and IgG after the cleavage of the first heavy chain. The velocity of IdeS is further increased by the presence of human Cystatin C, via an unknown mechanism. Cystatin C is normally a protease inhibitor and it having an opposite effect is puzzling.The synthesis and evaluation of novel inhibitors are also described. Peptide analogues mimicking the sequence surrounding the scissile bond on IgG - with an amino acid replaced with a more rigid motif - act as specific, but low-affinity, inhibitors of IdeS. The peptide analogues’ inhibitory capacity for SpeB and papain was also assayed.When it comes to SpeB, I show that it does not have IgG as a substrate under physiological conditions, in contrast to what was previously thought. This thesis does not only present findings on the IgG degrading capacity of IdeS and SpeB but also include data on fundamental enzymatic properties for these proteases.