The glutathione S-transferase (GST) supergene family encodes isoenzymes that appear to be critical in protection against oxidative stress. Certain GST loci are polymorphic, demonstrating alleles that are null (GSTMI/GSTT1), encode low activity variants (GSTPI), or are associated with variable inducibility (GSTM3). Interleukin-1 (IL- 1) alpha and beta are cytokines involved in recruitment of inflammatory cells, the process of inflammation, and blood-brain barrier breakdown and nerve regeneration. Polymorphisms of both GST and of a complementary interleukin-1 receptor antagonist have been associated with severity and susceptibility to other inflammatory conditions. This thesis examines the influence of the GST and IL-1 genes on both the susceptibility to Multiple Sclerosis (MS), and the course of disease progression. The population examined consisted of four hundred patients with clinically definite MS. Disease severity was measured using the Kurtzke Expanded Disability Status Scale (EDSS), a robust established ranking scale. PCR-based genotyping was performed using DNA extracted from lymphocytes. Significant associations between genotypes and clinical outcome were corrected for known demographic factors influencing prognosis, these being; gender, onset age, and disease duration using the statistical method of logistic regression. Significant associations, withstanding multiple testing corrections, with certain IL-I genotypes and disease severity were found. There was also a significant trend with the GST isoenzymeM 3 that is expressedin nervous tissue. No robust findings suggest that these genes influence susceptibility to MS, but the results suggest that long-term prognosis is genetically influenced by the modulation of inflammatory cytokines and also by the ability to remove the toxic products of oxidative stress.

Incidence of dengue fever (DF) has been estimated a 30 fold increase in the past 50 years. Clinical manifestations of DF range from a simple febrile illness with physical soreness to life-threatening dengue hemorrhagic fever (DHF). The need for a better classification of the severity in DEN infections has been proposed to clarify the immunopathogenesis for the prevention and management of serious DEN infections. We attempted to investigate whether different mechanisms involved in the varied manifestations of bleeding tendency and vascular leakage in DF. In a hospital-based study, we first compared clinical features as well as laboratory data including virus load, T helper (Th1/Th2) cytokines, and vascular leakage-related mediators between patients with DHF and DF. Moreover, we defined another class of patients associated with bleeding tendency but not fulfilled with DHF criteria, called DF w/B, for a further comparison. The virus load in blood was not significantly different among DF, DHF and DF w/B. DF patients had a higher Th1 cytokine, IFNr, expression (70.0 ¡Ó 10.7 vs. 33.1 ¡Ó 8.0 vs. 33.0 ¡Ó 7.1 pg/ml; DF vs. DF w/B, p = 0.009; DF vs. DHF, p = 0.002), and both DHF and DF w/B patients had a significantly higher IL-10 levels (14.3 ¡Ó 4.1 vs. 26.2 ¡Ó 3.3 vs. 26.0 ¡Ó 3.5 pg/ml; DF vs. DF w/B, p = 0.023; DF vs. DHF, p = 0.016) than DF patients. Both DHF and DF w/B patients also had a higher rate of secondary dengue infection (DF w/B vs. DHF vs. DF: 50.0%, 74.4% and 14.3%¡A p < 0.001). By contrast, DHF but not DF w/B patients had significantly higher vascular leakage-related mediators: sVCAM-1, PGE2 and TNF£\ levels than DF patients. Patients with DF w/B had a higher platelet counts (DF w/B vs. DHF: 66.0 ¡Ó 8.3 vs. 20.7 ¡Ó 2.1 x109/L, p < 0.001) but lower ALT levels than those with DHF (DF w/B vs. DHF: 56.3 ¡Ó 7.7 and 144.7 ¡Ó 20.5 IU/L). This study provides new insight to different immune mechanisms involved in patients with DF, DF w/B, and DHF. DF involves augmented Th1 reaction, and DF w/B involves altered Th2 reaction, but DHF involves both altered Th2 reaction and augmented vascular insult. Clarification of the immune mechanisms among DF, DFw/B and DHF will facilitate certain specific treatment and prevention of DF patients from varied bleeding tendency and vascular leakage manifestations.

The proposed thesis contributes to the knowledge about tick-borne encephalitis and its pathogenesis. The thesis describes pathogenesis and immunopathogenesis of tick-borne encephalitis, impact of host's genotype in clinical course determination, immune response of patients with acute tick-borne encephalitis, the mechanism of tick-borne encephalitis virus migration into central nervous system and virus interaction with cells of neurovascular unit as well as potential medical interventions.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Open reading frame 2 (ORF2) of porcine circovirus type 2 (PCV2) codes for the 233 amino acid capsid protein (CP). Baculovirus-based vaccines that express only ORF2 are protective against clinical disease following experimental challenge or natural infection. The goal of this study was to identify regions in CP preferentially recognized by sera from experimentally infected and vaccinated pigs, and compare these responses to pigs diagnosed with porcine circovirus-associated disease (PCVAD). The approach was to react porcine sera with different CP polypeptide fragments that each contained one or more immunoreactive regions. Expression of polypeptides was performed using E.coli. Initial results showed that sera from vaccinated pigs preferentially recognized only the largest CP(43-233) polypeptide fragment and showed low levels of binding to other CP polypeptide fragments. The results of sera from pigs diagnosed with PMWS showed only minimal reactivity with CP polypeptide fragments, including the largest CP(43-233). PCV2 infected or PDNS diagnosed pigs reacted to all CP polypeptides: however, the strongest reactivity was primarily directed towards CP polypeptides containing residues in the 160-180 region. For this purpose, finer mapping studies were performed. These experiments involved reacting sera from experimentally infected PCV2 pigs and PDNS pigs with overlapping oligopeptides that covered amino acids 141-200. Overall, the results showed a subset of experimentally infected pigs and pigs with PDNS preferentially recognized the CP oligopeptide, 169-STIDYFQPNNKR-180. Alanine scanning identified Y-173, F-174, Q-175 and K-179 as important for antibody recognition. The results from this study support the notion of PCV2 modulation of immunity, including antibody responses that may represent a precursor for disease. The results from this study support the notion of PCV2 modulation of immunity. Furthermore, the methods incorporated in this study provide a means for characterizing the immune response upon vaccination, natural infection and disease.

A malária asociada à gavidez pode gerar complicações para a mãe e para o feto. Receptores do tipo Toll (TLR) TLR2, TLR4 e TLR9, podem reconhecer componentes do Plasmódio. Estes receptores sinalizam via proteína MyD88. Contudo existem poucos dados sobre os TLR na malária placentária. Assim, o objetivo desse trabalho foi estudar o papel dos TLR2, 4, 9 e da MyD88 na malária placentária. Dentre fêmeas C57BL/6, TLR2-/-, TLR9-/- e MyD88-/-, a linhagem MyD88-/- apresentou maiores níveis de parasitemia, sobrevivência e cuidado parental, e ainda placentas de fêmeas MyD88-/- infectadas, ao contrario das TLR2-/- e TLR9-/-, não tiveram diminuição do espaço vascular em relação aos controles. Animais C57BL/6 infectados apresentaram aumento do mRNA de IL1-<font face=\"Symbol\">b e IL-6 na placenta, o que não ocorreu nos MyD88-/-. Gestantes C57BL/6 e MyD88-/- infectadas tiveram mais esplenócitos, com expansão preferencial de linfócitos B (CD19+). Também foi evidenciado nos animais C57BL/6 infectados um aumento da expressão do marcador de ativação CD69 nos linfócitos TCD8+. Em conjunto, estes resultados sugerem que a sinalização via MyD88 é importante para o desenvolvimento da malária placentaria e esta pode estar relacionada com a resposta inflamatória exacerbada induzida pelo parasita. / Pregnancy associated malaria can lead to complications both for the mother and the fetus. Toll like receptors (TLR) TLR2, TLR4 and TLR9 can recognize components of the Plasmodium sp. These receptors signal through the MyD88 protein. However there are few data on TLR in placental malaria. Thus, The objective of this work was to study the role of TLR2, 4, 9 and MyD88 in placental malaria. Among female mice C57BL/6, TLR2-/-, TLR9-/- and MyD88-/-, the lineage MyD88-/- showed higher levels of parasitemia, survival and parental care, and still placentas of MyD88-/- infected female, differently of TLR2-/- and TLR9-/-, had no decrease in the vascular space compared to controls. Animals C57BL/6 infected showed increased mRNA for IL1-<font face=\"Symbol\">b and IL-6 in the placenta, which did not occur in MyD88-/-. Pregnant infected C57BL/6 and MyD88-/- had more splenocytes, with preferential expansion of B lymphocytes (CD19+). in infected C57BL/6 was also demonstrated an increased expression of the activation marker CD69 on CD8+ T lymphocytes. Together, these results suggest that signaling through MyD88 is important for the development of placental malaria and this may be related with an increased inflammatory response induced by the parasite.

A malária asociada à gavidez pode gerar complicações para a mãe e para o feto. Receptores do tipo Toll (TLR) TLR2, TLR4 e TLR9, podem reconhecer componentes do Plasmódio. Estes receptores sinalizam via proteína MyD88. Contudo existem poucos dados sobre os TLR na malária placentária. Assim, o objetivo desse trabalho foi estudar o papel dos TLR2, 4, 9 e da MyD88 na malária placentária. Dentre fêmeas C57BL/6, TLR2-/-, TLR9-/- e MyD88-/-, a linhagem MyD88-/- apresentou maiores níveis de parasitemia, sobrevivência e cuidado parental, e ainda placentas de fêmeas MyD88-/- infectadas, ao contrario das TLR2-/- e TLR9-/-, não tiveram diminuição do espaço vascular em relação aos controles. Animais C57BL/6 infectados apresentaram aumento do mRNA de IL1-<font face=\"Symbol\">b e IL-6 na placenta, o que não ocorreu nos MyD88-/-. Gestantes C57BL/6 e MyD88-/- infectadas tiveram mais esplenócitos, com expansão preferencial de linfócitos B (CD19+). Também foi evidenciado nos animais C57BL/6 infectados um aumento da expressão do marcador de ativação CD69 nos linfócitos TCD8+. Em conjunto, estes resultados sugerem que a sinalização via MyD88 é importante para o desenvolvimento da malária placentaria e esta pode estar relacionada com a resposta inflamatória exacerbada induzida pelo parasita. / Pregnancy associated malaria can lead to complications both for the mother and the fetus. Toll like receptors (TLR) TLR2, TLR4 and TLR9 can recognize components of the Plasmodium sp. These receptors signal through the MyD88 protein. However there are few data on TLR in placental malaria. Thus, The objective of this work was to study the role of TLR2, 4, 9 and MyD88 in placental malaria. Among female mice C57BL/6, TLR2-/-, TLR9-/- and MyD88-/-, the lineage MyD88-/- showed higher levels of parasitemia, survival and parental care, and still placentas of MyD88-/- infected female, differently of TLR2-/- and TLR9-/-, had no decrease in the vascular space compared to controls. Animals C57BL/6 infected showed increased mRNA for IL1-<font face=\"Symbol\">b and IL-6 in the placenta, which did not occur in MyD88-/-. Pregnant infected C57BL/6 and MyD88-/- had more splenocytes, with preferential expansion of B lymphocytes (CD19+). in infected C57BL/6 was also demonstrated an increased expression of the activation marker CD69 on CD8+ T lymphocytes. Together, these results suggest that signaling through MyD88 is important for the development of placental malaria and this may be related with an increased inflammatory response induced by the parasite.

Chronic myelogenous leukemia (CML) is a clonal hematopoetic stem cell disorder characterized by proliferation of cells expressing BCR-ABL fusion protein. In the BCR-ABL+ leukemia murine model, 12B1, we explored the therapeutic applicability of chaperone-rich cell lysate (CRCL) in the context of syngeneic hematopoietic cell transplantation (HCT) to treat pre-existing leukemia. Our results demonstrate that tumor growth is significantly delayed in mice receiving syngeneic HCT from 12B1 tumor CRCL immunized donors compared to animals receiving HCT from non-immunized donors. CRCL immunization post-immune HCT further hindered tumor growth when compared to immune HCT without post-transplant vaccination. The magnitude of the immune response was consistent with the anti-tumor effects observed in vivo. We also demonstrated that cured mice had developed long-term tumor specific immunity against 12B1 tumor cells. In addition, we documented that both T cells and NK cells contributed to the anti-tumor effect of CRCL vaccination as depletion of either subset hampered tumor growth delay. Thus, our results suggest that CRCL represents a promising vaccine capable of generating specific immune responses. This anti-tumor immunity can be effectively transferred to a host via HCT and further enhanced post-HCT with additional tumor CRCL immunizations.CD4+CD25+ regulatory T cells (Treg) play a central role in the prevention of autoimmunity and in the control of immune responses by down-regulating the function of effector CD4+ or CD8+ T cells. The role of Treg in Mycobacterium tuberculosis infection and persistence is inadequately documented. Therefore, the current study was designed to determine whether CD4+CD25+ FoxP3+ regulatory T cells may modulate immunity against human tuberculosis (TB). Ourresults indicate that the number of CD4+CD25+FoxP3+ Treg increases in the blood or at the site of infection in active TB patients. The frequency of CD4+CD25+FoxP3+ Treg in pleural fluid inversely correlates with local MTB-specific immunity(p<0.002). These CD4+CD25+FoxP3+ T lymphocytes isolated from the blood and pleural fluid are capable of suppressing MTB-specific IFN-γ and IL-10 production in TB patients. Therefore, CD4+CD25+FoxP3+ Treg expanded in TB patients suppress Mycobacterium tuberculosis immunity and may therefore contribute to the pathogenesis of human TB.

Leprosy is a chronic infection caused by Mycobacterium leprae which affects skin and peripheral nerves. Various aspects related to genetic and phenotypic variability of the pathogenesis of immune response need to be further studied. In leprosy several polymorphisms of genes involved in the immune response of the host to pathogen were associated with leprosy per se and its clinical course. This present case-control study aimed to examine the possible association between polymorphisms rs2275913 of IL-17 and rs1024611 of MCP-1 with leprosy. The sample was composed of 199 patients and 85 healthy individuals unrelated households of the cases. The polymorphisms were analyzed using the PCR technique Taqman. For statistical analysis we used the software SPSS version 22.0 with employment of the Student T test for independent samples, Chi-square and Fisher's exact test where appropriate and analysis thereof. There were differences in the distribution of genotypes of MCP-1 and IL-17 between leprosy and contactant controls (p = 0.015 and p = 0.031 respectively) with greater frequency of genotype GG in the leprosy patients. The comparison of the distribution of genotypes between the groups stratified by multibacillary and paucibacillary clinical forms, did not show any difference in the distribution of genotypes of either MCP-1 or IL-17 in the patients with the operacional forms (p = 0.46 and p=0.14 respectively). The analysis of sick and healthy individuals according to the variables age, sex and genotypes of MCP-1 and IL-17 allowed to discriminate five groups. Groups 1 and 5 were composed of patients who had only the GG genotype of IL-17 while the GG genotype of MCP-1 was more frequent in Group 1 which was composed of mostly patients (p = 0.0001). Possibly the presence of genotype GG of MCP-1 and IL-17 GG genotype contributed to the outcome of the disease in the studied subjects. / A hanseníase é uma infecção crônica causada pelo Mycobacterium leprae o qual afeta pele e nervos periféricos. Vários aspectos genéticos relacionados à patogênese e à variabilidade fenotípica da resposta imune do hospedeiro precisam ser mais estudados em hanseníase uma vez que polimorfismos de genes envolvidos na resposta imune ao patógeno podem estar associados à manifestação ou não da doença e sua evolução clínica. Trata-se de um estudo de caso-controle que teve como objetivo analisar a possível associação entre os polimorfismos rs2275913 de IL-17 e rs1024611 de MCP-1 com a hanseníase. A amostra foi composta de 199 pacientes e 85 contactantes saudáveis não consanguíneos coabitantes dos casos. Os polimorfismos foram analisados utilizando a técnica de reação em cadeia de polimerase em tempo real Taqman. Para análise estatística foi utilizado o software SPSS versão 22.0 com emprego do teste T de Student para amostras independentes, teste do qui-quadrado e teste exato de Fisher quando apropriado e análise de agrupamentos. Houve diferença na distribuição de genótipos GG de MCP-1 (p=0,015) e GG de IL-17 (p=0,031) entre doentes e contactantes. Não houve diferença quando da comparação da distribuição dos genótipos de MCP-1 e IL-17 entre os doentes estratificados pelas formas clínicas paucibacilar e multibacilar (p=0,46 3 p=0,14 respectivamente). A análise dos indivíduos doentes e contactantes segundo as variáveis idades, sexo e genótipos de MCP-1 e IL-17 permitiu discriminar cinco grupos. Quando da análise de agrupamento segundo frequência genotípica e características clínicas, os grupos 1 e 5 foram constituídos por doentes que apresentavam somente o genótipo GG de IL-17 e maior frequência dos genótipos AG e GG de MCP-1 (p=0,0001). Possivelmente a presença do genótipo GG de MCP-1 e genótipo GG de IL-17 contribuíram para a manifestação da doença nos indivíduos estudados.

Leprosy is a chronic infection caused by Mycobacterium leprae which affects skin and peripheral nerves. Various aspects related to genetic and phenotypic variability of the pathogenesis of immune response need to be further studied. In leprosy several polymorphisms of genes involved in the immune response of the host to pathogen were associated with leprosy per se and its clinical course. This present case-control study aimed to examine the possible association between polymorphisms rs2275913 of IL-17 and rs1024611 of MCP-1 with leprosy. The sample was composed of 199 patients and 85 healthy individuals unrelated households of the cases. The polymorphisms were analyzed using the PCR technique Taqman. For statistical analysis we used the software SPSS version 22.0 with employment of the Student T test for independent samples, Chi-square and Fisher's exact test where appropriate and analysis thereof. There were differences in the distribution of genotypes of MCP-1 and IL-17 between leprosy and contactant controls (p = 0.015 and p = 0.031 respectively) with greater frequency of genotype GG in the leprosy patients. The comparison of the distribution of genotypes between the groups stratified by multibacillary and paucibacillary clinical forms, did not show any difference in the distribution of genotypes of either MCP-1 or IL-17 in the patients with the operacional forms (p = 0.46 and p=0.14 respectively). The analysis of sick and healthy individuals according to the variables age, sex and genotypes of MCP-1 and IL-17 allowed to discriminate five groups. Groups 1 and 5 were composed of patients who had only the GG genotype of IL-17 while the GG genotype of MCP-1 was more frequent in Group 1 which was composed of mostly patients (p = 0.0001). Possibly the presence of genotype GG of MCP-1 and IL-17 GG genotype contributed to the outcome of the disease in the studied subjects. / A hanseníase é uma infecção crônica causada pelo Mycobacterium leprae o qual afeta pele e nervos periféricos. Vários aspectos genéticos relacionados à patogênese e à variabilidade fenotípica da resposta imune do hospedeiro precisam ser mais estudados em hanseníase uma vez que polimorfismos de genes envolvidos na resposta imune ao patógeno podem estar associados à manifestação ou não da doença e sua evolução clínica. Trata-se de um estudo de caso-controle que teve como objetivo analisar a possível associação entre os polimorfismos rs2275913 de IL-17 e rs1024611 de MCP-1 com a hanseníase. A amostra foi composta de 199 pacientes e 85 contactantes saudáveis não consanguíneos coabitantes dos casos. Os polimorfismos foram analisados utilizando a técnica de reação em cadeia de polimerase em tempo real Taqman. Para análise estatística foi utilizado o software SPSS versão 22.0 com emprego do teste T de Student para amostras independentes, teste do qui-quadrado e teste exato de Fisher quando apropriado e análise de agrupamentos. Houve diferença na distribuição de genótipos GG de MCP-1 (p=0,015) e GG de IL-17 (p=0,031) entre doentes e contactantes. Não houve diferença quando da comparação da distribuição dos genótipos de MCP-1 e IL-17 entre os doentes estratificados pelas formas clínicas paucibacilar e multibacilar (p=0,46 3 p=0,14 respectivamente). A análise dos indivíduos doentes e contactantes segundo as variáveis idades, sexo e genótipos de MCP-1 e IL-17 permitiu discriminar cinco grupos. Quando da análise de agrupamento segundo frequência genotípica e características clínicas, os grupos 1 e 5 foram constituídos por doentes que apresentavam somente o genótipo GG de IL-17 e maior frequência dos genótipos AG e GG de MCP-1 (p=0,0001). Possivelmente a presença do genótipo GG de MCP-1 e genótipo GG de IL-17 contribuíram para a manifestação da doença nos indivíduos estudados.

This diploma thesis deals with methods of determining and treatment of coeliac disease. In the theoretical part of the work sums up informations about coeliac disease - history, prevalence, etiopathogenesis, immunopathogenesis, possibilities of diagnosis, discusses about gluten-free diet, mentions legislation of the Czech Republic and the European Union and compares expensiveness of gluten-free diet and normal diet. The experimental part of this thesis compares PCR, ELISA and indirect immunofluorescence methods for assesment of determination of genetic predisposition and antibodies in coeliac disease (greatest emphasis was placed on the comparison of antibodies against gliadin and deamidated gliadin antibodies in IgA and IgG).