BARBOSA, Francivandi Coêlho. Associação dos polimorfismos em genes de interleucinas (IL1B -511, IL1RN, IL6 -174G e IL8 -251) com lesões gástricas relacionadas ao Helicobacter pylori. 2012. 72 f. Dissertação (Mestrado em Microbiologia Médica) – Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2012. / Submitted by denise santos (denise.santos@ufc.br) on 2015-10-26T14:10:44Z No. of bitstreams: 1 2012_dis_fcbarbosa.pdf: 2039223 bytes, checksum: 21783f2c9101f5765736fd5e23312b27 (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2015-10-26T14:22:16Z (GMT) No. of bitstreams: 1 2012_dis_fcbarbosa.pdf: 2039223 bytes, checksum: 21783f2c9101f5765736fd5e23312b27 (MD5) / Made available in DSpace on 2015-10-26T14:22:16Z (GMT). No. of bitstreams: 1 2012_dis_fcbarbosa.pdf: 2039223 bytes, checksum: 21783f2c9101f5765736fd5e23312b27 (MD5) Previous issue date: 2012 / The gastric le sions ( G L ) develop from normal mucosa infected by Hel icobacter pylori ( H.pylori ) and can progress to gastric cancer ( GC ) through a multi - step process. The ability of H. pylori on cause G L is associated with some of their virulence factors that, when expressed , come in to c ontact with the gastric cells and pro moting activation of the innate immune response. This, in turn, activates the transcription of inflammatory cytokines such as interleukins (ILs) IL1B ( - 511), IL1RN (VTNR), IL6 ( - 174) and IL8 ( - 251). Such ILs have polymorphic genes that change expression an d , consequently , the intensity of the host inflammatory response. Therefore , this study aims to investigate the association between polymorphisms of ILs with the GC and G L in the progression to the G C . In this study DNA was obtained from 324 patients, 118 with CG and 206 with pre - malignant G L (LGPM), collected in four hospitals in Fortaleza - CE. The identification of polymorphisms was done by PCR - RFLP and PCR a nalysis and the detection of H. pylori by PCR. Genotypic analysis of the comparison between the G C a nd LGPM demonstrated an association of CG with IL8 TT genotypes (p = 0.0065), IL6 GG (p = 0.0012) and IL1RN LL (p = 0.0052 ) . The association of ILs 3x3 observed that the most inflammatory haplotypes were associated with GC , while in the analysis the 4x4 T - T - G - *2 (IL1BxIL8xIL6xIL1RN) haplotype was more related to GC (p = 2.89 × 10 5), which is a good marker for this type of cancer. In comparison with the CG and GCI it was observed the association between IL6 G allele with the G C (p = 0.0389). Comparison between GCA and GC showed that IL8 AT genotypes (p = 0.0016), IL1RN L * 2 (p = 0.0049) and IL6 GG (p = 0.0004) are associated with the GC , the analysis of the ILs association o n 4x4 showed that the T - T - G - * 2 haplotype (IL1BxIL8xIL6xIL1RN) was associated with G C (p = 5. 72 x10 - 5 ), confirming the importance of this haplotype as a good marker for the GC. In comparison with the MI CG observed a significant association with IL8 AT CG (p = 0.0426) and IL6 GG (p = 0.0475. So, it can be conclude that the genotypes of all inflammat ory IL s were associated with the CG and that, according to the h aplotype analysis , the mo st inflammatory haplotype (G - T - T - * 2 ) is a good marker for the G C . / As Lesões gástricas (LG) se desenvolvem a partir da mucosa normal infectada por Helicobacter pylori (H pylori) e podem progredir para o câncer gástrico (CG) através de um processo com múltiplas etapas A capacidade de H pylori provocar LG está associada com alguns fatores de sua virulência que quando expressa entra em contato com as células gástricas promovendo a ativação da resposta imune inata. Esta, por sua vez, ativa a transcrição de citocinas inflamatórias como as interleucinas (ILs) IL1B (-511) IL1RN (VTNR) IL6 (-174) e IL8 (-251) Tais ILs possuem genes polimórficos que alteram sua expressão e, consequentemente, a intensidade da resposta inflamatória do hospedeiro Assim este estudo objetiva verificar a associação entre os polimorfismos de ILs com as LG e o CG na progressão para o CG Neste estudo foi obtido DNA de 324 pacientes sendo 118 com CG e 206 com LG pré-malígnas (LGPM) coletadas em quatro hospitais de Fortaleza-CE A identificação dos polimorfismos foi feita por PCR-RFLP e PCR e a detecção de H pylori por PCR A análise genotípica da comparação entre as LGPM e CG demonstrou uma associação do CG com os genótipos IL8TT (p=0,0065) IL6GG (p=0,0012) e IL1RNLL (p=0,0052) Na associação das ILs 3x3 observou-se que os haplotipos mais inflamatórios estavam associados ao CG já na análise 4x4 o haplotipo T-T-G-*2 (IL1BxIL8xIL6xIL1RN) foi o mais associado ao CG (p=2,89x10-5) sendo este um bom marcador para este tipo de câncer Na comparação da GCI com o CG observou-se a associação do alelo IL6G com o CG (p=0,0389) A comparação entre GCA e CG demonstrou que os genótipos IL8AT (p=0,0016) IL1RNL*2 (p=0,0049) e IL6GG (p=0,0004) estão associados ao CG e a análise da associação das ILs 4x4 demonstrou que o haplotipo T-T-G-*2 (IL1BxIL8xIL6xIL1RN) foi associado ao CG (p=5,72x10-5) confirmando a importância deste haplotipo como bom marcador para o CG Na comparação da MI com CG observou-se associação significativa do CG com IL8AT (p=0,0426) e IL6GG (p=0,0475) Deste modo, pode-se concluir que os genótipos mais inflamatórios de todas as ILs estavam associados ao CG e que, de acordo com a análise haplotipica, o haplotipo mais inflamatório (T-T-G-*2) é um bom marcador para o CG

Helicobacter pylori (H. pylori) infection causes achlorydria, depressed gastric acid barrier, impaired immune response and is suspected in bacterial overgrowth and diarrhoea. These features of the infection are known to cause significant malabsorption of nutrients and impairment of linear growth in children. The prevalence of H. pylori infection in children is known to be much higher in developing countries, especially among the lower socio-economic groups. The true prevalence of infection in urban children in Mexico and its impact on their growth are largely unknown. This study examined the prevalence of H. pylori infection in school children from an urban area in Northwest Mexico and attempted to identify the risk factors that predispose individuals to infection in childhood; as well as to relate the presence of this infection to growth of children. The cross-sectional study was conducted in 1997/98 in the poorest socio-economic sectors of the city of Hermosillo, Sonora, among 178 children aged 9 and 10 years. H. pylori status was determined in children by the 13C-urea breath test. Anthropometric (weight and height) and haemoglobin measurements along with analysis of faecal samples and a 24-hour dietary recall were carried out in each child. Family sociodemographic/socio-economic status and living conditions data were elicited from parents by interview via structured questionnaires. The overall prevalence rate of H. pylori infection for the children in Hermosillo as determined by this study was 47.1%. The findings indicate that rural-born father, number of siblings, the type of main water supply (one tap in the yard) and the sharing of bed by the study child are important risk factors for acquiring the H. pylori infection. A borderline significant but small effect of H. pylori infection on height for-age was observed in this study. H. pylori infection was found to be positively highly associated with Hymenolepis nana. No differences in mean energy, protein and iron intakes between H. pylori positive and negative children were observed. However, significant differences in the mean energy, protein and iron intakes were observed between boys and girls. H. pylori infection and enteric parasites were not significantly correlated with the presence of anaemia.

A bactéria Gram negativa Helicobacter pylori, além de estar associada ao câncer gástrico e duodenal, está relacionada a patologias extra gástricas. Entre essas estão as doenças cardiovasculares. Os mecanismos pelos quais o H. pylori pode causar, ou agravar essas doenças, ainda não são claros. A urease de H. pylori (HPU) é considerada um fator de virulência, visto que sua atividade catalítica cria um microambiente de pH mais elevado, possibilitando a sobrevivência do patógeno no estômago. A HPU é capaz de ativar plaquetas de coelho através da indução da secreção de seus grânulos densos com liberação de ADP, culminando na agregação plaquetária. Esse fenômeno ocorre via 12- lipoxigenase, rota de sinalização também utilizada pelo colágeno, um importante agonista intrínseco desse sistema. Demonstramos previamente que também as duas subunidades da HPU, HpUreB e HpUreA, interagem com membranas de plaquetas de coelho, sendo que a HpUreB contém o domínio da holoenzima responsável pela agregação plaquetária. Essa interação entre HPU e plaquetas pode ser mediada por GPVI, o principal receptor de colágeno dessas células No presente trabalho, estudamos a interação da HPU e suas subunidades com plaquetas humanas, através de citometria de fluxo. Demostramos que HPU ativa plaquetas humanas sem exposição significativa de P-selectina. O bloqueio com anticorpos específicos para o receptor de membrana GPIIbIIIa, mas não para GPVI, interfere na ativação plaquetária induzida por HPU. Em plaquetas ativadas por HPU ocorrem modificações do processamento pré-mRNA de proteínas pró-inflamatórias, aumentando os níveis de mRNAs que codificam IL-1 e CD14, indicando que plaquetas passam a apresentar um fenótipo pró-inflamatório após exposição à urease. No conjunto, nossos dados sugerem que a HPU, além de permitir a sobrevivência bacteriana na mucosa gástrica, pode ter um papel importante, e até agora negligenciado, nos estados inflamatórios associados com a infecção por H. pylori. / The Gram negative bacterium Helicobacter pylori, besides its association with gastric and duodenal cancer, correlates positively to several extragastric diseases suchas cardiovascular pathologies. However, the mechanisms by which H. pylori can cause or aggravate these diseases are still unclear. H. pylori urease (HPU) is considered a virulence factor, since its catalytic activity creates a microenvironment, of higher pH, that allows survival of the pathogen in the stomach. HPU is able to activate rabbit platelets by inducing the secretion of their dense granules with release of ADP, culminating in platelet aggregation. This phenomenon occurs with activation of the 12-lipoxygenase pathway, which is also used by collagen, an important intrinsic agonist of this system. We have previously demonstrated that both subunits of HPU, HpUreB and HpUreA, interact with rabbit platelet membranes, and that HpUreB contains the domain responsible for platelet aggregation This interaction of HPU and platelets could be mediated by GPVI, the main collagen receptor in these cells. In this work, by using flow cytometry assay, we have studied the interaction of HPU and of its subunits with human platelets. HPU was shown to activate human platelets without significant P-selectin exposure. Blockage with antibodies against the membrane receptor GPIIbIIIa, but not against GPVI, interfered on platelet activation induced by HPU. The processing of pre-mRNA of proinflammatory proteins was evaluated in HPU-activated platelets and increased levels of mRNAs encoding IL-1 and CD14 were found, indicating that platelets acquire a proinflammatory phenotype when exposed to the urease. Altogether, our data suggest that H. pylori urease, besides allowing bacterial survival within the gastric mucosa, may have an important, and so far overlooked role in the inflammation associated to the infection by H. pylori.

Made available in DSpace on 2016-08-29T15:38:47Z (GMT). No. of bitstreams: 1 tese_8485_DAMASCENO JPL- 2016 - Dissertação de mestrado - Estudo de solubilidade e das atividades antioxida.pdf: 2214333 bytes, checksum: cee189be1a37b594fdc4f8ef753ccb16 (MD5) Previous issue date: 2016-03-24 / Helicobacter pylori é uma bactéria Gram-negativa que infecta a mucosa do estômago levando ao surgimento de patologias, tais como gastrite, úlceras pépticas e câncer de estômago, sendo de difícil erradicação. O tratamento padrão nem sempre é bem sucedido em função de efeitos colaterais, altos custos e baixa adesão por parte do paciente, levando a necessidade da busca por novas moléculas mais efetivas contra este microrganismo. Neste contexto, a isocumarina paepalantina, 9,10-diidroxi-5,7-dimetoxi-1H-nafto(2,3c)-piran-1-ona, isolada dos capítulos de Paepalanthus bromelioides, Eriocaulaceae, tem demonstrado em estudos anteriores uma ampla variedade de atividades biológicas como antibacteriana, antioxidante, anti-inflamatória e citotóxica, dos quais se destacam a capacidade antimicrobiana e antioxidante, abrindo portas para a avaliação de seus efeitos sobre a infecção causada por H. pylori e modulação das espécies reativas de oxigênio (EROs) e espécies reativas de nitrogênio (ERNs) geradas neste processo. Como é uma molécula pouco estudada, ainda carece de dados sobre sua solubilidade a fim de se encontrar uma alternativa viável ao uso do DMSO considerando a toxicidade do mesmo em cultura celular e sua interferência em testes de atividade antioxidante. Dessa forma, o objetivo deste trabalho foi avaliar solventes como alternativas ao uso do DMSO, com validação de metodologia analítica espectrofotométrica própria, a fim de escolher um veículo adequado para ensaios de atividade antioxidante frente a radicais sintéticos (DPPH e ABTS) e espécies reativas de oxigênio (HOCl, OH●, O2-●, H2O2) e nitrogênio (NO●) e de atividade anti-H. pylori. Entre os solventes avaliados, propilenoglicol em pH 7,5 foi o solvente de escolha, sendo que os resultados sugerem uma menor interferência nos ensaios se comparado ao DMSO, tornando uma alternativa viável do ponto de vista tecnológico. Os resultados apontam uma forte atividade antioxidante da paepalantina, comparável ao Trolox®. Também foi observado um importante efeito sobre a cultura de H. pylori com CIM de 128 μg/ml e CBM de 256 μg/ml e sinergismo de sua sub-MIC com os antibióticos amoxicilina e metronidazol demonstrando possivelmente atuar sobre a permeabilidade da membrana bacteriana e por inibição das Penicillin-Bindind Proteins (PBPs), devido as alterações morfológicas observadas em H. pylori por microscopia eletrônica de varredura, tornando a paepalantina promissora para o desenvolvimento futuro de medicamento para o combate de H. pylori e seus males associados.

Ureases (3.5.1.5), enzimas níquel dependentes que catalisam a reação de hidrólise da uréia em amônia e dióxido de carbono, apresentam ampla distribuição em plantas, fungos e bactérias. A espiroqueta Helicobacter pylori causa úlceras pépticas e câncer gástrico por mecanismos ainda não totalmente conhecidos. H. pylori produz grande quantidade de urease, que neutraliza o meio ácido e permite sua sobrevivência no estômago. Nosso grupo demonstrou que as ureases de Canavalia ensiformis, soja e Bacillus pasteurii induzem agregação plaquetária independentemente de sua atividade ureolítica, por uma rota que requer ativação de canais de cálcio. ativação da rota do ácido araquidônico e secreção plaquetária. Estudos prévios mostraram ainda que a canatoxina, uma isoforma da urease de C.ensiformis, possui atividade pró-inflamatória, induzindo edema de pata em ratos. Neste trabalho, caracterizamos as propriedades ativadora de plaquetas e pró-inflamatória da urease recombinante de H. pylori (HPU). Em plaquetas, estudamos as vias recrutadas pela proteína na agregação plaquetária e comparamos com dados prévios para a canatoxina e a urease de Bacillus pasteurii. Em neutrófilos, demonstramos que a HPU, em doses nanomolares, induz quimiotaxia e produção de espécie reativas de oxigênio. A taxa de apoptose de neutrófilos ativados por HPU foi inibida, acompanhando alterações dos níveis de proteínas pró- e antiapoptóticas. Por último, mostramos que a resposta dos neutrófilos a HPU envolve aumento dos níveis de lipoxigenase(s), sem, contudo, haver alterações das ciclooxigenase( s). Concluímos que as propriedades não enzimáticas aqui descritas para a HPU podem potencialmente contribuir para o processo inflamatório promovido por H. pylori. / Ureases (EC 3.5.1.5), nickel-dependent enzymes that hydrolyze urea into ammonia and carbon dioxide, are widespread among plants, bacteria and fungi. The spirochete Helicobacter pylori is the etiological agent of gastric ulcers and gastric adenocarcinoma by mechanisms not yet fully understood. H. pylori produces high amounts of urease, which enables the bacterium to survive in the acidic medium of the stomach. We have previously reported that ureases from jackbean, soybean or Bacillus pasteurii induce blood platelet aggregation independently of their enzyme activity by a pathway requiring activation of calcium channels, lipoxigenase-derived eicosanoids and platelet secretion. We also showed that canatoxin, an isoform of C. ensiformis urease, presents pro-inflammatory property demonstrated by rat paw oedema. In this work we characterized the platelet aggregating and pro-inflammatory properties of the recombinant H. pylori urease (HPU). In platelets we studied the pathways recruited by the protein to induce platelet aggregation and compared the data to those previously reported for the plant urease canatoxin and for Bacillus pasteurii urease. Using neutrophils we demonstrated that nanomolar doses of HPU induce chemotaxis and production of oxygen reactive species in human neutrophils. The rate of apoptosis was decreased in HPU-treated neutrophils, accompanied by alterations in the levels of proand anti-apoptotic proteins. Moreover, we showed that the response of neutrophils to HPU requires increased levels of lipoxygenase(s) with no alterations of cyclooxygenase( s). We concluded that the non-enzymatic properties of HPU here described potentially contribute to the inflammatory process that underlies H. pylori infection.

A bactéria Gram negativa Helicobacter pylori, além de estar associada ao câncer gástrico e duodenal, está relacionada a patologias extra gástricas. Entre essas estão as doenças cardiovasculares. Os mecanismos pelos quais o H. pylori pode causar, ou agravar essas doenças, ainda não são claros. A urease de H. pylori (HPU) é considerada um fator de virulência, visto que sua atividade catalítica cria um microambiente de pH mais elevado, possibilitando a sobrevivência do patógeno no estômago. A HPU é capaz de ativar plaquetas de coelho através da indução da secreção de seus grânulos densos com liberação de ADP, culminando na agregação plaquetária. Esse fenômeno ocorre via 12- lipoxigenase, rota de sinalização também utilizada pelo colágeno, um importante agonista intrínseco desse sistema. Demonstramos previamente que também as duas subunidades da HPU, HpUreB e HpUreA, interagem com membranas de plaquetas de coelho, sendo que a HpUreB contém o domínio da holoenzima responsável pela agregação plaquetária. Essa interação entre HPU e plaquetas pode ser mediada por GPVI, o principal receptor de colágeno dessas células No presente trabalho, estudamos a interação da HPU e suas subunidades com plaquetas humanas, através de citometria de fluxo. Demostramos que HPU ativa plaquetas humanas sem exposição significativa de P-selectina. O bloqueio com anticorpos específicos para o receptor de membrana GPIIbIIIa, mas não para GPVI, interfere na ativação plaquetária induzida por HPU. Em plaquetas ativadas por HPU ocorrem modificações do processamento pré-mRNA de proteínas pró-inflamatórias, aumentando os níveis de mRNAs que codificam IL-1 e CD14, indicando que plaquetas passam a apresentar um fenótipo pró-inflamatório após exposição à urease. No conjunto, nossos dados sugerem que a HPU, além de permitir a sobrevivência bacteriana na mucosa gástrica, pode ter um papel importante, e até agora negligenciado, nos estados inflamatórios associados com a infecção por H. pylori. / The Gram negative bacterium Helicobacter pylori, besides its association with gastric and duodenal cancer, correlates positively to several extragastric diseases suchas cardiovascular pathologies. However, the mechanisms by which H. pylori can cause or aggravate these diseases are still unclear. H. pylori urease (HPU) is considered a virulence factor, since its catalytic activity creates a microenvironment, of higher pH, that allows survival of the pathogen in the stomach. HPU is able to activate rabbit platelets by inducing the secretion of their dense granules with release of ADP, culminating in platelet aggregation. This phenomenon occurs with activation of the 12-lipoxygenase pathway, which is also used by collagen, an important intrinsic agonist of this system. We have previously demonstrated that both subunits of HPU, HpUreB and HpUreA, interact with rabbit platelet membranes, and that HpUreB contains the domain responsible for platelet aggregation This interaction of HPU and platelets could be mediated by GPVI, the main collagen receptor in these cells. In this work, by using flow cytometry assay, we have studied the interaction of HPU and of its subunits with human platelets. HPU was shown to activate human platelets without significant P-selectin exposure. Blockage with antibodies against the membrane receptor GPIIbIIIa, but not against GPVI, interfered on platelet activation induced by HPU. The processing of pre-mRNA of proinflammatory proteins was evaluated in HPU-activated platelets and increased levels of mRNAs encoding IL-1 and CD14 were found, indicating that platelets acquire a proinflammatory phenotype when exposed to the urease. Altogether, our data suggest that H. pylori urease, besides allowing bacterial survival within the gastric mucosa, may have an important, and so far overlooked role in the inflammation associated to the infection by H. pylori.

Ureases (3.5.1.5), enzimas níquel dependentes que catalisam a reação de hidrólise da uréia em amônia e dióxido de carbono, apresentam ampla distribuição em plantas, fungos e bactérias. A espiroqueta Helicobacter pylori causa úlceras pépticas e câncer gástrico por mecanismos ainda não totalmente conhecidos. H. pylori produz grande quantidade de urease, que neutraliza o meio ácido e permite sua sobrevivência no estômago. Nosso grupo demonstrou que as ureases de Canavalia ensiformis, soja e Bacillus pasteurii induzem agregação plaquetária independentemente de sua atividade ureolítica, por uma rota que requer ativação de canais de cálcio. ativação da rota do ácido araquidônico e secreção plaquetária. Estudos prévios mostraram ainda que a canatoxina, uma isoforma da urease de C.ensiformis, possui atividade pró-inflamatória, induzindo edema de pata em ratos. Neste trabalho, caracterizamos as propriedades ativadora de plaquetas e pró-inflamatória da urease recombinante de H. pylori (HPU). Em plaquetas, estudamos as vias recrutadas pela proteína na agregação plaquetária e comparamos com dados prévios para a canatoxina e a urease de Bacillus pasteurii. Em neutrófilos, demonstramos que a HPU, em doses nanomolares, induz quimiotaxia e produção de espécie reativas de oxigênio. A taxa de apoptose de neutrófilos ativados por HPU foi inibida, acompanhando alterações dos níveis de proteínas pró- e antiapoptóticas. Por último, mostramos que a resposta dos neutrófilos a HPU envolve aumento dos níveis de lipoxigenase(s), sem, contudo, haver alterações das ciclooxigenase( s). Concluímos que as propriedades não enzimáticas aqui descritas para a HPU podem potencialmente contribuir para o processo inflamatório promovido por H. pylori. / Ureases (EC 3.5.1.5), nickel-dependent enzymes that hydrolyze urea into ammonia and carbon dioxide, are widespread among plants, bacteria and fungi. The spirochete Helicobacter pylori is the etiological agent of gastric ulcers and gastric adenocarcinoma by mechanisms not yet fully understood. H. pylori produces high amounts of urease, which enables the bacterium to survive in the acidic medium of the stomach. We have previously reported that ureases from jackbean, soybean or Bacillus pasteurii induce blood platelet aggregation independently of their enzyme activity by a pathway requiring activation of calcium channels, lipoxigenase-derived eicosanoids and platelet secretion. We also showed that canatoxin, an isoform of C. ensiformis urease, presents pro-inflammatory property demonstrated by rat paw oedema. In this work we characterized the platelet aggregating and pro-inflammatory properties of the recombinant H. pylori urease (HPU). In platelets we studied the pathways recruited by the protein to induce platelet aggregation and compared the data to those previously reported for the plant urease canatoxin and for Bacillus pasteurii urease. Using neutrophils we demonstrated that nanomolar doses of HPU induce chemotaxis and production of oxygen reactive species in human neutrophils. The rate of apoptosis was decreased in HPU-treated neutrophils, accompanied by alterations in the levels of proand anti-apoptotic proteins. Moreover, we showed that the response of neutrophils to HPU requires increased levels of lipoxygenase(s) with no alterations of cyclooxygenase( s). We concluded that the non-enzymatic properties of HPU here described potentially contribute to the inflammatory process that underlies H. pylori infection.

Orientador: Jose Pedrazzoli Junior / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-04T15:29:11Z (GMT). No. of bitstreams: 1 Becker_TaglianeLiza_M.pdf: 5325289 bytes, checksum: d08598655314f45f1785148d9eb3b6b0 (MD5) Previous issue date: 2005 / Resumo: As principais causas de ulceração da mucosa gástrica são o uso prolongado de antiinflamatórios não-esteroidais (AINEs) e a infecção pelo Helicobacter pylori. Pacientes com ulcerações gástrica são tratados, principalmente, com antagonistas de receptor de histamina-2 e inibidores de bomba protônica, que neutralizam elou reduzem a secreção ácida. Estudos têm mostrado que estas drogas possuem propriedades adicionais de inibirem funções leucocitárias. Estas propriedades são de relevante importância, visto que a lesão na mucosa gástrica induzida tanto por AINEs quanto pelo H. pylori, envolve diretamente a participação de neutrófilos. Baseado nestes relatos, os objetivos deste trabalho foram: 1) padronizar o modelo de inflamação induzida por H. pylori em bolsa de ar na pele dorsal de ratos; 2) avaliar a possível atividade antiinflamatória de inibidores de secreção ácida modelo experimental. A inoculação de H. pylori na bolsa de ar em ratos causou um significativo infiltrado neutrofílico, sendo este, dependente da concentração e, independente da viabilidade e do genótipo das linhagens inoculadas. Nota-se que após o tratamento com os inibidores de secreção ácida, durante 7 ou 28 dias, a produção de exsudato inflamatório e migração de leucócitos na bolsa de ar incubada com H. pylori ou carragenina não foi alterada. Em contraste, a dexametasona, antiinflamatório esteroidal, inibe significantemente a resposta inflamatória. Ao contrário dos estudos anteriores, esses resultados indicam que os inibidores de bomba protônica e os antagonistas de receptor H2 não possuem atividade antiinflamatória no modelo de inflamação in vivo induzido pelo H. pylori em bolsa de ar em ratos, quando utilizados por curto ou longo período / Abstract: Helicobacter pylori infection and the administration of nonsteroidal antiinflammatory drugs are the main causes of gastric ulcerations. In the clinical setting, patients with gastric ulcerations are treated with histamine-2 (H2) receptor antagonists and proton pump inhibitors (PPI) to neutralize or reduce the acid secretion. Recently, studies have shown that these drugs have additional properties related to their capacity to inhibit some neutrophil functions. Since the damage to gastric mucosa, associated with H. pylori or nonsteroidal anti-inflammatory drugs, is also induced by neutrophils these properties could be beneficial. In the present study, we characterized the inflammatory response induced by H. pylori in the rat air pouch model. We also evaluated the anti-inflammatory activity of acid secretion inhibitors such as omeprazole, lanzoprazole, pantoprazole and cimetidine in this experimental model. The injection of H. pylori into rat air pouch caused pronounced neutrophil infiltration. This response was dependent upon the number of bacteria injected and independent of bacterial viability and genotype. The duration of treatment with the acid secretion inhibitors did not affect the ability of neutrophils to migrate in response to H. py/ori or carrageenan. On the other hand, dexamethasone, a classic anti-inflammatory drug, reduced the exudate formation and leukocyte migration. Our results indicate that proton pump inhibitors or histamine-2 receptor antagonists have no anti-inflammatory activity in vivo / Mestrado / Mestre em Farmacologia

Helicobacter pylori is one of the most genetically diverse bacterial species that successfully colonizes at least 50% of the world population. It has been associated with humans for thousands of years and most probably evolved from ancestral gastric Helicobacter species in early mammals. One of the important characteristics of this pathogen is the degree of allelic diversity and genetic variability which helps it to adapt and colonize. Phase variation is one of the mechanisms used by H. pylori to generate variation. The presence of homopolymeric nucleotide or dinucleotide repeats in an ORF make it prone to frequent length changes as a consequence of slipped strand mispairing mediated mutagenesis. Interestingly, R-M genes comprise a significant percentage of H. pylori strain-specific genes and are more prevalent in H. pylori than in other bacterial species whose genomes have been fully sequenced. R-M systems in H. pylori have been identified on the basis of sequence similarity to known restriction endonucleases and methyltransferases, genetic organization, and specific enzyme isolation and characterization. Analysis of genome sequences of H. pylori strains 26695, J99, HPAGI and 26 others has revealed the presence of more than 20 R-M systems in each stain, which are far more than detected in any other bacterial genome sequence till date. hp1369 and hp1370 are two ORFs in stain 26695 coding for hypothetical proteins. hp 1369 has a stretch of poly-G repeats, thus making hp1369-hp1370, a candidate of phase variation. hpag1_1313 is homolog of hp1369-hp1370 which got up-regulated, in a person suffering from acute gastritis, thus making these genes an interesting subject of investigation. This study was therefore initiated with the following objectives: 1. Cloning, over-expression and purification of Type III MTase (ORF- hp1369- hp1370) and its cognate restriction enzyme (hp1371). 2. Biochemical characterization of MTase (HP1369-HP1370): Determination of oligomeric status, kinetic properties, binding affinities for AdoMet and DNA. Sequence analysis shows the presence of a poly-G track (10 Gs) at 3’-end of hp1369 which is a signature sequence for phase variation. Addition of a single nucleotide can place both hp1369 and hp1370 in-frame, which could code for a single polypeptide. hp1369 and hp1370 in H. pylori strain 26695 alone do not code for any functional protein but with the fusion of hp1369 and hp1370 can code for a protein with all the nine motifs of a DNA MTase. Interestingly, on the basis of arrangement of Motifs, it is probably the first example of ε type of methyltransferase. By site-directed mutagenesis a single G nucleotide was inserted in the poly-G track and both the ORFs (hp1369 and hp1370 ) became in-frame, coding for fully functional HP1369-HP1370 MTase. Kinetic parameters for functional HP1369-HP1370 MTase were determined, and has shown that there was substrate inhibition in methylation reaction at higher concentrations of AdoMets. When preincubation studies were done, enzyme-DNA complex was found to be more competent than enzyme-AdoMet complex. HP1369-HP1370 MTase exists as dimer in solution, having affinity for duplex DNA and does not bind to single-stranded DNA. Binding affinity for ligand (AdoMet) was determined by Isothermal Titration Calorimetry method. H. pylori has evolving restriction-modification systems. It is capable of taking new R-M systems from the environment in the form of DNA released from other bacteria or other Helicobacter strains. H. pylori genome is dynamic with high mutation rates. Random mutations in R-M genes can result in a non-functional R-M systems or R-M systems with new properties. The dynamics of R-M system plays a vital role in shaping up the genome.

Doctor en Farmacología / Helicobacter pylori (H. pylori) infecta la mucosa gástrica de la mitad de la población mundial, y es el único microorganismo conocido capaz de sobrevivir exitosamente en el estómago humano. Muchos estudios han establecido a H. pylori como agente etiológico del cáncer gástrico, linfoma del tejido linfoide asociado a la mucosa (MALT), y la úlcera péptica. En efecto, en 1994, la Agencia Internacional para la Investigación sobre el Cáncer (IARC) de la Organización Mundial de la Salud (OMS), definió a H. pylori como un agente carcinógeno definitivo (tipo I) en el cáncer gástrico. Es importante destacar que en Chile la prevalencia de la infección por H. pylori normalizada para la población mundial de entre 20 y 60 años es aproximadamente de un 80%. Este alto nivel de infección por H. pylori explicaría por qué Chile tiene la mayor tasa de mortalidad por cáncer gástrico en América, y se ubica entre los cinco primeros países del mundo con respecto a la mortalidad por cáncer gástrico. Sin embargo, la gran mayoría de las personas infectadas con H. pylori permanece asintomática. El tratamiento de la infección por H. pylori requiere el uso de una combinación de antibióticos con un inhibidor de la bomba de protones. La literatura muestra un aumento en la aparición de cepas resistentes a los antibióticos, por lo que hoy en día es importante buscar sustancias no-antibióticas con actividad anti-H. pylori. Entre éstas, un número importante de diferentes productos del reino vegetal han sido probados, incluyendo antimicrobianos pertenecientes a diferentes grupos de fitoquímicos, tales como los aceites esenciales y los polifenoles. Los polifenoles son componentes ubicuos de plantas medicinales y nutricionales. Estos compuestos, no sólo actuarían como antioxidantes, sino que también podrían ejercer actividades antimicrobianas en diferentes porciones del tracto digestivo. Numerosos antecedentes sugieren que el consumo regular de alimentos o bebidas ricas en polifenoles, podría ayudar a prevenir y atenuar el daño a la mucosa gástrica inducido por H. pylori. En relación con esto último, las manzanas poseen muchas propiedades beneficiosas para la salud humana asociadas con su alto contenido en compuestos fenólicos. Entre las diversas variedades, las manzanas Granny Smith se exportan tanto como producto fresco como para la producción de jugo concentrado. Aunque una gran parte de estas exportaciones considera el uso de la fruta entera, en la última década, los productos de manzana deshidratada han tenido un aumento significativo. Puesto que la deshidratación requiere la eliminación de la cáscara, se llega a acumular importantes cantidades de ésta, las que constituyen un residuo agroindustrial que genera problemas a las empresas con las autoridades de la CONAMA. Algunas variedades de manzana podrían tener entre un 40 a 50% de los polifenoles totales del fruto en la cáscara. De hecho, la concentración de polifenoles en la cáscara puede triplicar al observado en la pulpa. Los principales grupos de polifenoles de la manzana incluyen los glucósidos de flavonoides, ésteres fenólicos de ácidos carboxílicos, dihidrochalconas, catequinas y procianidinas. Los polifenoles de la cáscara de manzana podrían actuar contra H. pylori no sólo como agentes antimicrobianos, sino también neutralizando algunos de sus factores de virulencia. También estos compuestos podrían reducir el daño inducido por las especies reactivas del oxígeno y nitrógeno (ERON) generadas durante el proceso inflamatorio asociado a la infección. En esta tesis se realizó un estudio farmacológico de los efectos in vitro e in vivo de un extracto enriquecido en polifenoles de cáscara de manzana sobre H. pylori. La preparación del extracto de cáscara de manzana (APPE) se llevó a cabo utilizando adsorción de los polifenoles en la resina Sepabeads SP-850. De esta forma, los polifenoles de los extractos acuosos de cáscara de manzana fueron adsorbidos en forma exitosa. Los polifenoles fueron recuperados con etanol y concentrados en rotavapor, llegando a un rendimiento de 3.50 ± 0.7 g por 1000 g de cáscara de manzana fresca. El contenido de polifenoles totales fue de ~ 60%, con un perfil HPLC-DAD casi idéntico al obtenido en la cáscara fresca de manzana Granny Smith. El análisis específico de los compuestos polifenólicos de APPE obtenidos por RP-HPLC, permitieron identificar la presencia de: (1) ácido clorogénico, (2) procianidina B1, (3) procianidina B2, (4) (-)-epicatequina, (5) procianidina C1, ( 6) rutina, (7) hiperósido, (8) isoquercitrina (9), quercetina-3-O-pentósido, (10), quercetina-3-O-pentósido, (11) quercitrina, (12) floretina-O-xiloglucósido, y (13) floridzina. Para confirmar la identidad de los 13 principales componentes se desarrolló un método de separación por HPLC-ESI/MS/MS, el cual permitió obtener la huella digital de APPE caracterizada por el perfil de polifenoles. Usando RP-HPLC, se logró una muy buena separación de los glucósidos quercetina y algunas procianidinas desde los monómeros hasta los trímeros. Mediante RP-HPLC no fue posible separar los oligómeros superiores a los trímeros, probablemente debido al gran número de isómeros que interfieren con la separación. Por lo tanto, para el análisis de oligómeros de prociandinas superiores de los trímeros se consideró a la NP-HPLC como la técnica más adecuada. Así, con el uso de la NP-HPLC, las procianidinas desde los monómeros a los undecámeros fueron separadas según su grado de polimerización, siendo éste el método de elección para la separación de oligómeros. Con el objetivo de fraccionar los polifenoles de APPE de acuerdo a su tamaño molecular, se utilizó una combinación de cromatografía en columnas de Sephadex LH-20 y Toyopearl HW-40. Como resultado de esta estrategia, se obtuvieron las fracciones de polifenoles de bajo peso molecular (LMW) y alto peso molecular (HMW). La determinación del grado de polimerización (mDP) de APPE, HMW y LMW se llevó a cabo mediante depolimerización por floroglucinólisis y tiólisis con cisteamina y tolueno--tiol. La depolimerización generada por dichos nucleófilos permitió obtener perfiles donde claramente se identificó como principal unidad de extensión y de término a la epicatequina. Por medio del estudio de los productos de despolimerización y los perfiles de NP-HPLC, fue posible confirmar que en su mayoría LMW posee unidades monoméricas (mDP = 1). APPE posee un mDP = 2-4, mientras que para HMW mDP = 8-10. Se evaluó el efecto del extracto APPE y las fracciones LMW y HMW sobre la viabilidad de H. pylori, la actividad de la ureasa y el estallido respiratorio de neutrófilos. Los resultados sugieren, en base a los valores de sus IC50, que la actividad inhibitoria de la ureasa mostrada por APPE estuvo asociada principalmente a la presencia de polifenoles de alto peso molecular (HMW) y, en menor grado, con de la presencia de los componentes de bajo peso molecular (LMW). Tal inhibición fue concentración-dependiente, tiempo-independiente, reversible y competitiva. Por el contrario, el efecto de APPE tanto sobre la viabilidad de H. pylori, como sobre la producción de las especies reactivas del oxígeno (ERO) por parte de los neutrófilos, se asoció principalmente con los componentes de LMW. APPE pudo inhibir el estallido respiratorio de neutrófilos inducido por H. pylori, PMA y fMLP en forma concentración-dependiente. Este efecto se observó tanto en el interior como en el exterior de los neutrófilos. Este resultado indicó que APPE podría atenuar el daño a la mucosa gástrica asociado a las ERO producidas por los neutrófilos, particularmente cuando H. pylori despliega sus mecanismos de evasión. Se investigaron los efectos inhibitorios de APPE contra la infección por H. pylori, y la vacuolación inducida por VacA. APPE disminuyó significativamente la vacuolación en células HeLa con un valor de IC50 de 450 g GAE / mL. APPE también mostró un efecto anti-adherente in vitro contra H. pylori. Se observó una inhibición significativa de la adhesión de H. pylori con una reducción del 20-60% para concentraciones de entre 0.250 y 5 mg GAE / mL. En un modelo a corto plazo de infección (ratones C57BL6 / J), dos niveles de dosis de APPE (150 y 300 mg / kg / día) mostraron un efecto inhibitorio sobre el asentamiento de H. pylori. Después de 7 días de cultivo, no fue posible recuperar bacterias con la morfología típica de H. pylori, tanto en los grupos tratados con APPE como en los controles no infectados. Paralelamente, el análisis por PCR de tiempo real de los estómagos de animales tratados con APPE, no mostró diferencias significativas con el control no infectado. Estos resultados concordaron con la visible reducción de la carga de H. pylori observada en las evaluaciones histológicas. Pese al corto plazo de interacción huésped-patógeno y el bajo índice de gastritis observado en los animales infectados, los polifenoles de APPE mostraron un efecto anti-inflamatorio sobre la gastritis asociada a la presencia de H. pylori, reduciendo los niveles de malondialdehído. En esta tesis se ha demostrado que APPE podría ejercer sus efectos anti-H. pylori mediante múltiples mecanismos. Así, los polifenoles de alto peso molecular inhibieron la actividad de la ureasa, el proceso de adhesión de las bacterias a la mucosa gástrica y la actividad de la toxina VacA. Por otro lado, las propiedades anti-H. pylori y atrapadoras de ERO fueron ligadas a la presencia de los polifenoles de bajo peso molecular. Como era de esperar, el efecto antioxidante de APPE y sus propiedades anti-VacA, probablemente cumplan un papel en la disminución del daño inflamatorio in vivo causados por H. pylori a la mucosa gástrica de los ratones infectados. En resumen, en este estudio se observó que APPE puede prevenir eficazmente las primeras etapas del proceso de colonización gástrica por H. pylori y suprimir algunas de sus consecuencias patológicas más relevantes como la inflamación / Helicobacter pylori (H. pylori) infects the gastric mucosa of half of the world´s population, and is the only microorganism known to successfully inhabit the human stomach. Many studies have established H. pylori as an etiologic agent of gastric cancer, mucosa-associated lymphoid tissue lymphoma (MALT), and peptic ulcer. In fact, in 1994, the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) defined H. pylori as a definite carcinogen in gastric cancer (type I). Importantly, in Chile the prevalence of H. pylori infection standardised for world population for ages 20 to 60 is approximately 80%. This high level of H. pylori infection may explain why Chile has the highest rate of gastric cancer mortality in the Americas, and is among the top five countries in the world with regard to gastric cancer mortality. However, the vast majority of people infected with H. pylori has no symptoms and will never develop problems. The treatment of H. pylori infection requires the use of two or more antibiotics combined with a proton pump inhibitor. The occurrence of strains resistant to antibiotics has been to increasing, and it is nowadays important to search for new alternatives to antibiotics with anti-H. pylori activity. Among these, a number of different products from vegetable origin have been tested, including antimicrobials belonging to phytochemical such as essential oils and polyphenols. Polyphenols are ubiquitous constituents of medicinal and nutritional plants. Such compounds, not only would act as antioxidants, but also they could exert antimicrobial activities in different portions of the digestive tract. Growing evidence suggests that regular intake of foods or polyphenol-rich beverages might help to prevent and attenuate H. pylori-induced damage to the gastric mucosa. Regarding the latter, apples possess many beneficial properties for the human health related to their high content in phenolic compounds. Among several varieties, Granny Smith apples are exported both as fresh product and as juice concentrates. Although a great part of such exports considers the whole fruit, during the past decade, exports of value added dehydrated apple products have had a significant increase. Since dehydration requires peel removal, important amounts of apple peel accumulate to be largely classified as an agro-industrial waste. Some apple varieties concentrate in the peel from 40 to 50% of the total fruit polyphenols. In fact, the amount of polyphenols in the apple peel could be up to three times higher than that found in the pulp. The principal classes of whole apple polyphenols include flavonoid glycosides, phenol carboxylic acid esters, dihydrochalcones, catechins, and procyanidins. Apple peel polyphenols might act against H. pylori exerting not only an antimicrobial action but also neutralizing some of its virulence factors. These compounds could also reduce the damage induced by the Reactive Oxygen and Nitrogen Species (RONS) generated during the inflammatory process associated to the infection. In this thesis a pharmacological study was undertaken considering the in vitro and in vivo effects of an apple peel polyphenols-enriched extract upon H. pylori. The preparation of apple peel extract (APPE) was carried out using the adsorption resin Sepabeads SP-850. Thus, the polyphenols from the aqueous extracts of apple peel were successfully adsorbed. Recovery of the polyphenols was carried out with ethanol and concentrated through evaporative rotation with a yield of 3.50± 0.7g from 1000 g of fresh apple peel. Total polyphenolic content was ~60 % with a HPLC-DAD profile remarkably similar to that obtained in the fresh peel of Granny Smith apples. Specific analysis of APPE polyphenolic compounds by RP-HPLC allowed to identify the presence of (1) chlorogenic acid, (2) procyanidin B1, (3) procyanidin B2, (4) (−)-epicatechin, (5) procyanidin C1, (6) rutin, (7) hyperoside, (8) isoquercitrin, (9) quercetin-3-O-pentoside, (10) quercetin-3-O-pentoside, (11) quercitrin, (12) phloretin-O-xyloglucoside, and (13) phloridzin. To confirm the identity of the 13 main components of APPE a fingerprint profile method was developed using HPLC-ESI/MS/MS method. Using RP-HPLC, the extremely good separation of quercetin glycosides and individual procyanidins from monomers to trimers was achieved. RP-HPLC method was not able to separate oligomers higher than trimers probably due to the higher number of isomers interfering with the separation. Hence, NP-HPLC was better suited for analysis of procyandin oligomers higher than trimers. Using NP-HPLC, procyanidin monomers through undecamers were separated by the degree of polymerization and the separation of higher oligomers was preferable. With the aim to fractionate APPE polyphenols according to their molecular size, a combination of Sephadex LH-20 and Toyopearl HW-40s column chromatography was used. As result of this strategy, low molecular weight (LMW) and high molecular weight (HMW) polyphenolic fractions were obtained. Determination of mDP of APPE, HMW and LMW was carried out by depolymerization via phloroglucinolysis and thiolysis with cysteamine and toluene--thiol. Depolymerization with nucleophile generated profiles where it is clearly seen that the main extension and terminal unit was epicatechin. By means of the study of depolymerization products and NP-HPLC profiles, it was possible to confirm that LMW possesses mostly monomeric units (mDP = 1). APPE possesses an mDP = 2-4, while for HMW mP = 8-10. The effect of APPE, LMW y HMW against H. pylori viability, urease activity and neutrophil respiratory burst was evaluated. On the basis of the IC50 ranking order, the results suggested that the urease-inhibitory activity displayed by APPE is associated, primarily, with the presence of HMW polyphenols and, to a lower degree, with the presence of monomeric components (LMW). Such urease inhibition was concentration-dependent, time-independent, reversible and competitive. On the contrary, inhibitory effect for both viability and reactive oxygen species (ROS) production by stimulated neutrophils was associated mainly with LMW components. Thus, APPE inhibited the respiratory burst of neutrophils induced by H. pylori, PMA and fMLP in concentration-dependent form. This effect was observed on both the interior and exterior of the neutrophil. This result suggests that APPE have an attenuating effect on the damage to gastric mucosa caused by neutrophil generated ROS and, particularly, when H. pylori display its evasion mechanism. The inhibitory effects of APPE against H. pylori infection, and VacA-induced vacuolation were investigated. APPE significantly prevented vacuolation in HeLa cell with an IC50 value of 450 g GAE/mL. APPE also displayed an in vitro anti-adhesive effect against H. pylori. A significant inhibition was observed with a 20-60% reduction of H. pylori attachment at concentrations between 0.250 and 5 mg GAE/mL. In a short-term infection model (C57BL6/J mice), two levels of APPE doses (150 and 300 mg/kg/day) showed an inhibitory effect on H. pylori attachment. After 7 days of culture, it was not possible to recover bacteria with the typical H. pylori morphology neither in the groups treated with APPE nor in the controls. Real-time PCR analysis of the stomachs from animals treated with APPE did not show significant differences with non-infected control mice. These results were in agreement with the visible reduction in H. pylori load observed in the histology evaluations. Although the short-term pathogen-host interaction period and the low gastritis score observed in the infected animals, APPE showed an anti-inflammatory effect on the H. pylori-associated gastritis lowering malondialdehyde levels. In this thesis it has been demonstrated that APPE could exert their anti-H. pylori effects by multiple mechanism. So, high molecular weight polyphenols inhibit the urease activity, the process of adherence of the bacteria to the gastric mucosa and the activity of VacA toxin. On another side, low molecular weight polyphenols were involved in the anti-H. pylori and ROS scavenging properties of APPE. As expected, the antioxidant and anti-VacA properties of APPE probably played a role in diminishing the in vivo inflammatory damage caused by H. pylori to the mice gastric mucosa. In summary, in this study it was observed that APPE can effectively prevent the initial steps in the H. pylori colonization process and suppress some of their most relevant pathological consequences such as mucosal inflammation