Abstract -1 The immune response occurred in the mice infected with Angiostrongylus cantonensis was mainly humoral immune response. This study was designed to compare the systemic and localized humoral immune responses occurred after primary and secondary infections in C57BL/6J mice. Eight weeks after the primary infection with 20 third-stage larvae, each mouse received a second inoculation of the same dosage. Specific serum IgM, IgG and IgE were found in the second week after primary infection. However, the titers of IgG1 and IgG2b increased at the fourth week after primary infection. Antibodies of these mentioned increased continuously as the progress of infection. On the other hand, the IgM and IgG1 titers increased in brain tissue infusion since the forth week after primary infection, while the titer of IgG start to elevate at the sixth week. Nevertheless, the increase of IgG2B was only noticed at the sixth week and no significant change was observed for IgG2a and IgE. After the secondary infection, serum IgM titers increased while the titer of IgG1 in the brain tissue infusion decreased. Results of Western blot showed that IgG1and IgE in the brain tissue infusion lost the ability to recognize a 42 kDa molecule of the somatic and excreting-secreting antigens of fifth-stage larvae. These variations could be used in the diagnosis of the early stage of mice that re-infected with Angiostrongylus cantonensis. Abstract -2 The radiographic lateral views of the skulls of the mice infected with Angiostrongylus cantonensis were taken. Thus, the parietofrontal index ( PI ) was obtained by measuring and calculating the distances among specific positions on their skulls. Compared with the controls, a significant elevation over the top of the crania of the cases was observed sixty days post-infection. In addition, the phenomenon emerged apparently during the second to the fourth week post-infection. These findings are able to be applied as the external diagnostic references for the infection course of mice infected with Angiostrongylus cantonensis.

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.

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No. of bitstreams: 2 Tese - Benigno Alberto Moraes da Rocha - 2015.pdf: 2182475 bytes, checksum: 33c24230875842c1d6d2b9260ba26f6e (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-12-03 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Outro / Background: Dengue is the fastest growing arbovirosis in the world, infecting almost 390 million people. The dengue virus is the etiological agent of the disease, which has four serotypes (DENV1-4). All serotypes can cause dengue, varying from asymptomatic to severe forms and may lead to death. In Brazil, in 2010, there was the re-emergence of serotype DENV-4 after 28 years. In 2013, it was registered the largest epidemic in history with more than 1.4 million cases and circulation of the four serotypes. In the state of Goiás, Central Region of Brazil, after serotype DENV-4 entry in 2011, for the first time in the region, in 2013, there was a major epidemic which registered more than 10% of the cases in Brazil, more often DENV -4 followed by DENV-1. In this scenario, a study was conducted with the objective to characterize the clinical and molecular profile of patients with dengue; establish if there were associations between clinical and laboratory markers of severity with the infectious virus serotypes; and genetic characterization of the more frequent virus serotype during the 2012/2013 dengue epidemic in Goiânia, Goiás. Methods: A prospective clinical study with suspected dengue patients from eight reference health units (five public, two hospitals and three of secondary level; and three private hospitals) from January 2012 to July 2013 was conducted. The patients were evaluated in three different moments by clinicians who collected clinical and sociodemographic information. Blood sampling for laboratory testing (Hg, AST, and ALT) and confirmatory tests for dengue infection (IgM, IgG, NS1, and RT-PCR) were performed. The gene sequencing of dengue virus type 4 envelope was also carried out. Patients with confirmation of dengue infection were classified according to clinical signs according to the World Health Organization. Principal findings: Six hundred and fifty patients were recruited; 18 were excluded, and 632 were followed. Four hundred fifty-two patients (71.5%) had confirmed infection and 112 (24.8%) were hospitalized. More than 90% were over 15 years of age, and 235 were female (52.0%). In 328 patients, it was possible to classify the type of infection, and 248 (75.6%) of these had a secondary infection. 188 (41.6%) were classified as FD, 238 (52.6%) as DwC and 26 (5.8%) with DG. Compared to those aged xvi over 15 years, spontaneous bleeding, severe abdominal pain and plasma extravasation were more frequent in children under 15 years of age (p <0.05). There was one death of a 16-year-old. During two years of follow-up, viral RNA was identified in 243 (40.5%) of 600 patients evaluated in these tests, where the four serotypes were identified: 91 (37.4%) with DENV-1, 4 (1.6%) with DENV-2, 13 (5.3%) with DENV-3 and 135 (55.5%) with DENV-4. A higher frequency of some symptoms of severity was observed (thrombocytopenia, spontaneous bleeding and severe abdominal pain) in DENV-1 serotype compared to DENV-4. Eighty percent of patients with DENV-4 had a secondary infection. In the phylogenetic study of DENV-4 envelope gene, genotype II was identified. Conclusion: The present study described clinically and molecularly a large dengue epidemic that occurred in a state of Brazil's Central Region, which revealed the concurrent circulation of the four serotypes, the frequency of clinical signs of disease severity in children aged under 15 years and in people with DENV-1 infection. It also identified the current genotype of the most common serotype in the epidemic. These findings should serve as a warning sign for the health authorities because we are experiencing a significant dispersion of serotypes situation worldwide. / Contexto: Dengue é a arbovirose que mais cresce no mundo, infectando quase 390 milhões de pessoas. Ela é causada pelos vírus dengue que possui quatro sorotipos (DENV1-4). Todos os sorotipos podem causar a doença, que varia de formas assintomáticas a graves e podem levar a óbito. No Brasil em 2010 houve a reemergência do sorotipo DENV-4 após 28 anos. No ano de 2013, foi registrada a maior epidemia da história com mais de 1,4 milhão de casos e circulação do quatro sorotipos. Na Região Central do Brasil, Estado de Goiás, após a entrada sorotipo DENV-4, em 2011, pela primeira vez na região, em 2013, aconteceu uma grande epidemia onde foi registrado mais de 10% dos casos do Brasil, com maior frequência do DENV-4 seguido pelo DENV-1. Neste cenário foi realizado um estudo com os objetivos de caracterizar o perfil clínico e molecular dos pacientes com dengue, estabelecer se houve associação entre marcadores de gravidade clínica e laboratorial com os sorotipos virais infectantes e caracterizar geneticamente o vírus da dengue mais frequente que circulou na epidemia de 2012/2013 em Goiânia – GO. Metodologia: Foi realizado um estudo clínico prospectivo com pacientes suspeitos de dengue atendidos em oito unidades de saúde (cinco públicas, sendo dois hospitais e três de nível secundário; três hospitais privados) de referência de janeiro de 2012 a julho de 2013. Os pacientes foram abordados em três momentos diferentes e avaliados por clínicos que coletaram informações clínicas e sócio-demográficas. Também houve coleta de sangue para realização de exames complementares (Hg, AST e ALT) e exames confirmatórios da infecção por dengue (IgM, IgG, NS1 e RT-PCR). Foi realizado ainda o sequenciamento do gene do envelope do vírus dengue tipo 4. Os pacientes com confirmação da infecção por dengue foram classificados quanto à forma clínica da doença de acordo com a Organização Mundial de Saúde, Principais achados: Foram recrutados 650 pacientes, dos quais 18 foram excluídos e 632 foram seguidos. Quatrocentos e cinquenta e dois pacientes (71,5%) tiveram a infecção confirmada. Destes, 112 (24,8%) foram hospitalizados. Mais de 90% tinham idade superior a 15 anos e 235 (52,0%) eram do sexo feminino. Para 328 pacientes foi possível classificar o tipo de infecção e destes, 248 (75,6%) tiveram infecção xiv secundária. Classificou-se como FD 188 indivíduos (41,6%), 238 (52,6%) com DwC e 26 (5,8%) com DG. Sangramento espontâneo, dor abdominal intensa e extravasamento de plasma foram mais freqüentes em menores de 15 anos de idade, quando comparado aos maiores de 15 anos (p<0,05). Houve um óbito de um adolescente de 16 anos de idade. O RNA viral foi identificado em 243 (40,5%) de 600 pacientes investigados por estes exames, onde foi identificados os quatros sorotipos DENV-1 em 91 pacientes (37,4%) DENV-2 em 4 (1,6%), DENV-3 em 13 (5,3%) e DENV-4 em 135 (55,5%) nos dois anos de acompanhamento. Foi observada uma frequência maior de alguns sintomas de gravidade (plaquetopenia, sangramentos espontâneos e dor abdominal intensa) no sorotipo DENV-1 comparado ao DENV-4. Oitenta por cento dos pacientes com DENV- 4 tiveram infecção secundária. No estudo de filogenia do gene do envelope do DENV-4 foi identificado o genótipo II. Conclusão: Foi descrito clínica e molecularmente uma grande epidemia que ocorreu em um Estado da Região Central do Brasil que revelou a circulação concomitante dos quatros sorotipos virais, frequência de sinais de gravidade em menores de 15 anos de idade e em pessoas com infecção pelo DENV-1. Identificou-se ainda o genótipo circulante do sorotipo mais freqüente na epidemia. Estas constatações devem servir de sinal de alerta para as autoridades de saúde, pois nos encontramos em situação de grande dispersão dos sorotipos em todo o mundo.

Suite à une infection, des lymphocytes T CD8+ naïfs (LTn CD8+) spécifiques d’un antigène du pathogène sont activés. Après, cette activation, ces lymphocytes se différencient en lymphocytes T effecteurs CD8+ (LTe CD8+) chargés d’éliminer le pathogène. Une fois que l’infection est résolue, la grande majorité des effecteurs meurent par apoptose et les survivants se différencient en lymphocytes T mémoires CD8+ (LTm CD8+). Ces derniers protègeront l’organisme à long terme contre une réinfection par le même pathogène. Il est ainsi primordial d’avoir une meilleure compréhension des mécanismes impliqués dans le développement et la maintenance des LTm CD8+. Plusieurs études ont déjà montré que certaines cytokines de la famille γC (IL-2, IL-7, IL-15 et IL-21) influençaient individuellement le développement des LTe et LTm CD8+. Cependant, nous pensons que ces cytokines ont un impact bien plus grand sur l’homéostasie des LTe et LTm CD8+ que la littérature actuelle le laisse entendre. En effet, nous émettons l’hypothèse que ces cytokines de la famille γC agissent en synergie entre elles afin de promouvoir le développement des LTe et LTm CD8+. Pour tester notre hypothèse, nous avons dans un premier temps étudié l’impact combiné des signaux IL-2 et IL-15 sur la génération des LTe et LTm CD8+ dans un modèle d’infection LCMV Armstrong. Ensuite, dans le même modèle expérimental, nous avons étudié l’effet d’une déficience en signaux IL-2, IL-15 et IL-21 sur le développement des LTe et LTm CD8+. Nos résultats montrent que ces trois cytokines collaborent afin de soutenir l’expansion et la différenciation des LTe CD8+. Plus précisément, l’IL-2, l’IL-15 et l’IL-21 sont essentielles pour l’homéostasie d’une population particulière de LTe CD8+ : les Short-Lived Effector Cells (SLECs). Nous avons également mis en évidence que ces trois cytokines sont toutes les trois requises afin de générer un nombre maximum de LTm CD8+. De plus, la différenciation et le maintien de la population effecteur mémoire (TEM) sont particulièrement réduites en l’absence des signaux combinés de l’IL-2, l’IL-15 et l’IL-21. Nos résultats mettent pour la première fois en lumière les rôles redondants et synergiques de trois cytokines dépendantes de la chaine γc dans le développement et la maintenance des LTe et LTm CD8+. / Over the course of an infection, antigenic signals trigger a specific CD8+ T cells (LTn CD8+) response. Upon antigen recognition, LTn CD8+ are activated and undergo a massive proliferation wave. This leads them to differentiate into effector cells (LTe CD8+) in charge of pathogen elimination. While most effector T cells die after the infection is resolved, a small part of this population persists and differentiates into memory T cells (LTm CD8+). These cells provide long term protection to the organism against the initial infectious agent. It is thus crucial to have a better understanding of all mechanisms governing the development and the maintenance of LTm CD8+. Several studies have already shown that some members of the "C-dependent cytokines family (IL-2, IL-7, IL-15 and IL-21) individually regulate LTe and LTm CD8+ development. However, we believe that these cytokines have a far greater impact on the homeostasis of LTe and LTm CD8+ than the current literature suggests. Indeed, we hypothesized that "C-dependant cytokines act in synergy to promote the development of LTe and LTm CD8+. To assess their effect, we first studied the combined impact of IL-2 and IL-15 signals on the generation of LTe and LTm CD8+ during an LCMV Armstrong infection in mice. In this same experimental model, we also studied the effect of a deficiency in IL-2, IL-15 and IL-21 signals on the development of LTe and LTm CD8+. Our results show that these three cytokines cooperate to support the expansion and differentiation of LTe CD8+. More accurately, IL-2, IL-15 and IL-21 are essential for the homeostasis of a particular LTe CD8+ subset : Short- Lived Effector Cells (SLECs). We also demonstrated that all three cytokines are required to generate a maximal number of LTm CD8+. In addition, differentiation and maintenance of the memory effector population (TEM) are substantially reduced in the combined absence of IL-2, IL-15 and IL-21 signals. These results are the first to our knowledge to highlight redundant and synergistic functions of three "C-dependent cytokines as promoters of the development and maintenance of LTe and LTm CD8+.

The aim of this study was to isolate and characterize antifungal compounds from the most active medicinal plant species that could be used to address secondary infection problems in immunocompromised patients. An ethnobotanical study was conducted and 45 medicinal plant species used traditionally to treat candidiasis and related infections in HIV/AIDS patients were identified and documented. The most popular plant species used included Acacia caffra, Clerodendrum glabrum, Croton gratissimus, Elaeodendron transvaalense, Faurea saligna, Hippocratea longipetiolata, Osyris lanceolata, Richardia brasiliensis, Schkuhria pinnata, Schotia brachypetala, Spilanthes acmella, Strychnos potatorum, Vangueria infausta subsp. infausta and Withania somnifera. The plant parts used in the therapeutic preparations were roots (26.7%), bark (22.2%), and a combination of roots and bark (17.7%). Decoctions (44.4%), infusions (20%) and macerations (17.7%) were used. Most of the herbal remedies were administered orally. Chemical profiles of the plant species were established by using thin layer chromatography. Leaf extracts of these plant species were tested for antimicrobial activity against two common pathogenic fungal species in humans (Candida albicans and Cryptococcus neoformans) and four nosocomial bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa) using a two-fold serial microdilution method and bioautography. All plant species investigated had some degree of antimicrobial activity against the test microorganisms. The hexane and the acetone extracts of Clerodendrum glabrum, Hippocratea longipetiolata, Schkuhria pinnata and Withania somnifera were the most active with MIC values ranging from 0.06 to 0.08 mg/ml. The most susceptible pathogen to the test samples was C. neoformans while C. albicans was resistant to most of the plant extracts. The water extracts of Withania somnifera and Hippocratea longipetiolata (14%) had MIC < 1 mg/ml against C. albicans. C. neoformans was susceptible to nine water plant extracts (64%) with MIC < 1 mg/ml and the promising activity was observed in Hippocratea longipetiolata and Faurea saligna extracts with MIC values of 0.16 and 0.31 mg/ml respectively. The hexane extract of C. glabrum was the most active against C. albicans with an MIC value of 0.06 mg/ml and total activity of 550 ml/g. In the bioautography, most plant extracts tested had few active compounds, others had no active components at all and this may be attributed to the disruption of synergism by the thin layer chromatography. C. glabrum had eight active antifungal compounds on bioautograms and most of these components were observed in the EMW solvent system. Based on this and its wide distribution in rural areas, C. glabrum was chosen for further study. The antioxidant activity and possible immune boosting potential of the species were determined using 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH), 2, 2’ azinobis-(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and ferric reducing antioxidant power (FRAP) assays. In the DPPH qualitative assay, the aqueous plant extracts had several prominent antioxidant components than the organic plant extracts. The aqueous plant extracts which had the most prominent antioxidant activity were F. saligna with 8 compounds, followed by E. transvaalense, H. longipetiolata O. lanceolata, R. brasiliensis and S.brachypetala, with five compounds each and their Rf values ranged from 0;06 to 0.94. This appears to validate the ethnomedicinal use of the plant species to some extent because decoction is the most common method used in the preparation of the remedy by the traditional healers. With regard to the organic plant extracts, only one plant extract, F. saligna had two prominent antioxidant components at Rf values 0.81 and 0.88. A third of the plant species had a high level of free radical scavenging activities in the DPPH, ABTS and FRAP assays. However, all plant extracts had lower antioxidant activity than the positive control (Trolox) used. The selected plant species were also evaluated for their in vitro toxicity against the Vero monkey kidney cell line using 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. The acetone plant extracts of O. lanceolata, S. acmella, S. pinnata and S. brachypetala had high cytotoxic activity against Vero cells with IC50 values of 13.7±0, 19.9±0.001, 21.6±0.001 and 28.34±0.001 μg/ml respectively. However, their IC50 values were higher than that of the positive control, doxorubicin (IC50 = 9.9±0 μg/ml). The rest of the acetone plant extracts (64%) had moderate cytotoxic activity (30 < IC50<100 μg/ml). The aqueous plant extracts were relatively non-toxic to the Vero cells with IC50 values ranging from 137 to > 500 µg/ml. This supports the use of aqueous extracts in the traditional medicine. However, their low selectivity index values ranging from 0.26 to 1.68 suggest that the plant extracts are probably suitable for external use only. Fractionation of the hexane extract of the leaves of C. glabrum by chromatographic techniques yielded six fractions of which fractions C and D had significant antifungal activity (average MIC value = 0.1 mg/ml) against C. albicans and C. neoformans. From these fractions, one new triterpenoid, 3-(1-oxobutyl)-11α-hydroxytaraxast-20(30)-ene-24,28-dioic acid (clerodendrumic acid) (1) was isolated along with known heptadecanoic acid (2). C. albicans was relatively insensitive to clerodendrumic acid (1) (MIC value = 125 µg/mL) and was resistant to heptadecanoic acid (2) (MIC value = 188 µg/ml). Compounds 1 and 2 were non-toxic against monkey kidney Vero cells in vitro with IC50 values of 202.6 and 108.4 µg/ml respectively. Due to its low antifungal activity, the novel compound clerodendrumic acid (1) is not a viable candidate for drug development which could be used to combat candidiasis and related fungal infections. However, due to its relative safety, it may possibly be used as a lead compound to produce new chemically modified active derivatives or could be used together with known antibiotics to mitigate their undesirable side effects. To the best of our knowledge, the isolation of a novel, clerodendrumic acid (1) and a known heptadecanoic acid (2) compounds from leaf extracts of C. glabrum is reported herein for the first time. The results obtained from this study generally substantiate the rationale behind the use of the selected plant species in the traditional medicine to treat candidiasis and related infections to some extent. This study showed the potential of studying traditional medicine in the search for effective plant extracts or new lead compounds that could be developed into drugs for combating microbial infections among the rural poor people. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Paraclinical Sciences / Unrestricted