Genotipos de virulencia de Helicobacter pylori y su asociación con lesiones precursoras de malignidad gástrica y parámetros histológicos en pacientes colombianos / Helicobacter pylori virulence genotypes and their relationship with precursor lesions of gastric malignancy and histological parameters in infected patients in Colombia

Se determinó la presencia de los genotipos de virulencia de Helicobacter pylori y su asociación con las lesiones precursoras de malignidad gástrica y parámetros histológicos en pacientes con síntomas de dispepsia del suroccidente de Colombia. Se realizó reacción en cadena de polimerasa (PCR) para la caracterización genética de vacA, cagA, babA2 y sabA. Se empleó la prueba de chi cuadrado o Fischer para evaluar la asociación de cada genotipo sobre el desenlace clínico. En los pacientes con lesiones precursoras de malignidad gástrica se encontró que el 86,3% presentaron el genotipo vacA s1/m1, el 68,1% cagA+ y los genotipos babA2+ y sabA+ con el 68,8% y 55,8%, respectivamente. También, se demostró la asociación entre los genotipos de virulencia y el grado severo de infiltración de células polimorfonucleares. Además, se encontró una asociación entre la combinación de los genes vacA/cagA, vacA/sabA y babA2/sabA. Este estudio proporciona evidencia acerca de la asociación de los genotipos de virulencia del H. pylori y la inflamación gástrica en pacientes infectados.

The aim of this research was to determine the presence of Helicobacter pylori virulence genotypes and their association with precursor lesions of gastric malignancy and histological parameters in patients with dyspepsia symptoms in southwestern Colombia. Polymerase chain reaction (PCR) was used for the genetic characterization of vacA, cagA, babA2 and sabA. The chi-square or Fischer test were used to evaluate the association between each genotype and the clinical outcome. We found that 86.3% of the patients with precursor lesions of gastric malignancy presented the vacA s1/m1 genotype, 68.1% had the cagA+ genotype and 68.8% and 55.8% had the babA2+ and sabA+ genotypes, respectively. Our results show association between virulence genotypes and severe degree of polymorphonuclear cell infiltration. In addition, we found an association between the combination of vacA/cagA, vacA/sabA and babA2/sabA genes. This study provides evidence about the association of H. pylori virulence genotypes and gastric inflammation in infected patients.

Caracterização funcional de proteínas hipotéticas de Leptospira interrogans como adesinas e potenciais antígenos vacinais e para diagnóstico / Functional characterization of hypothetical proteins in Leptospira interrogans as adhesins and potential vaccine and diagnostic antigens

Leptospira spp. constitui um grupo de bactérias espiroquetas gram-negativas englobando espécies saprofíticas, intermediárias e patogênicas, sendo as últimas agentes causadores da leptospirose, doença zoonótica de alcance mundial e endêmica em regiões tropicais em desenvolvimento. O crescente número de espécies identificadas de leptospiras destaca ainda mais sua diversidade genética e mecanismos de virulência únicos, muitos deles com função ainda desconhecida. Esforços para o desenvolvimento de novas vacinas com proteção cruzada e efeito duradouro revelaram possíveis candidatos vacinais que necessitam ser adequadamente validados, sendo assim, há ainda uma urgente necessidade de uma vacina universal contra a leptospirose capaz de controlar e reduzir os surtos cada vez mais frequentes da doença. Adesinas são importantes fatores de virulência em diversos patógenos, constituindo antígenos promissores para o desenvolvimento de vacinas contra a leptospirose, assim como para o desenvolvimento de métodos diagnósticos mais rápidos e precisos. Previamente, foram identificadas três proteínas hipotéticas conservadas em L. interrogans pela técnica de phage display, denominadas arbitrariamente como LepA069, LepA962 e LepA388. A expressão do gene codificador da proteína LepA069 apresentou aumento de aproximadamente 70 % em animais infectados por leptospiras virulentas, representando a primeira evidência funcional desta proteína ainda desconhecida. Porções recombinantes da lipoproteína hipotética LepA962 (LepA962_Nt e LepA962_Phg) foram obtidos, sendo demonstrada a forte interação da proteína LepA962_Phg, contendo a sequência identificada por phage display, com laminina, fibronectina plasmática, colágeno I e fibrinogênio de maneira dose-dependente. Adicionalmente, LepA962_Phg apresentou ligação às células VERO e à sua matriz extracelular secretada, e o soro obtido a partir desta proteína recombinante foi capaz de se ligar à superfície de leptospiras virulentas, indicando que LepA962_Phg pode representar um importante domínio de interação entre as leptospiras e seu hospedeiro. Finalmente, a proteína LepA388 pertencente a uma extensa família de proteínas modificadoras de virulência com função desconhecida (DUF_61), presente apenas nas leptospiras patogênicas mais virulentas, apresentou aumento na expressão de seu gene codificador em animais infectados por leptospiras virulentas de acordo com dados na literatura. Além disso, porções recombinantes da região Nterminal desta proteína apresentaram ligação a laminina, colágenos I e IV, vitronectina e fibronectinas plasmática e celular, principalmente considerando a sequência identificada por phage display. Estes dados reforçam as predições de modelos tridimensionais da proteína LepA388 e de outros membros da família DUF_61, as quais identificam domínios semelhantes a toxinas (como abrina e CARDS) responsáveis pela ligação e internalização celulares nos hospedeiros. Dados recentes sugerem um possível papel citotóxico desempenhado pelas proteínas desta família em leptospiras, as quais podem também ser consideradas potenciais candidatas vacinais e para diagnóstico da leptospirose, devido à sua distribuição restrita em espécies e cepas patogênicas de importância para saúde humana.

Leptospira spp. constitutes a group of gram-negative spirochete bacteria comprising saprophytic, intermediate and pathogenic species, the last being causative agents of leptospirosis, a zoonotic disease of worldwide extent and endemic in developing tropical regions. The growing number of identified leptospiral species further highlights their genetic diversity and unique virulence mechanisms, many of them with unknown function. Efforts to develop new vaccines with cross-protection and long-lasting effect have revealed possible vaccine candidates that need to be properly validated. Therefore, there is still an urgent need for a universal vaccine against leptospirosis capable of controlling and reducing the increasing outbreaks of the disease. Adhesins are important virulence factors in several pathogens, constituting promising antigens for the development of vaccines against leptospirosis, as well as for the development of faster and more accurate diagnostic methods. Previously, three conserved hypothetical proteins in L. interrogans were identified by phage display technique, arbitrarily named as LepA069, LepA962 and LepA388. Expression of the LepA069 encoding gene showed an increase of approximately 70 % in animals infected by virulent leptospires, representing the first functional evidence of this still unknown protein. Recombinant portions of the hypothetical lipoprotein LepA962 (LepA962_Nt and LepA962_Phg) were obtained, demonstrating the strong interaction of the LepA962_Phg protein, containing the sequence identified by phage display, with laminin, plasma fibronectin, collagen I and fibrinogen in a dose-dependent manner. Furthermore, LepA962_Phg showed binding to VERO cells and its secreted extracellular matrix, and the serum obtained from this recombinant protein was able to bind to the surface of virulent leptospires, indicating that LepA962_Phg may represent an important domain of interaction between leptospires and its host. Finally, LepA388 protein belonging to an extensive family of virulence modifying proteins with unknown function (DUF_61), present only in the most virulent pathogenic leptospires, showed an increase in the expression of its encoding gene in animals infected by virulent leptospires according to data in literature. Moreover, recombinant portions of the N-terminal region of this protein showed binding to laminin, collagens I and IV, vitronectin and plasma and cell fibronectins, especially considering the sequence identified by phage display. These data support the predictions of three-dimensional models of the LepA388 protein and other members of the DUF_61 family, which identify toxin-like domains (such as abrin and CARDS) responsible for cellular binding and internalization in hosts. Recent data suggest a possible cytotoxic role played by proteins of this family in leptospires, which can also be considered potential vaccine candidates and antigens for diagnosis, due to their restricted distribution in pathogenic species and strains of importance to human health

Representações sociais de enfermeiras acerca da violência doméstica contra a mulher: estudo com abordagem estrutural / Representaciones sociales de enfermeras acerca de la violencia doméstica contra la mujer: estudio con enfoque estructural / Social representations of nurses concerning domestic violence against women: study with a structural approach

Resumo OBJETIVO Analisar a estrutura e os conteúdos das representações sociais de enfermeiras acerca da violência doméstica contra a mulher. MÉTODOS Estudo qualitativo realizado com 100 enfermeiras entre maio/setembro de 2014 em dois hospitais de Rio Grande/RS. Colheram-se os dados por meio de evocação-livre e entrevistas semiestruturadas. Foram tratados pelo software Evoc e análise contextual, respectivamente. RESULTADOS Observa-se uma representação negativa com elementos nucleares aludindo às formas de violência e ao seu julgamento, expresso em "agressão física" e "desrespeito". Na periferia, "medo" revela tanto o sentimento das profissonais quanto das vítimas frente ao agressor, e "submissão" é pontuada como causa da violência. Infere-se a possibilidade de um subgrupo com representação diferenciada, frente ao termo "agressão verbal" na zona de contraste. CONCLUSÕES A visão centralizada nos agravos físicos e na culpabilização da vítima pode limitar as ações de cuidado, portanto é fundamental problematizar este objeto com profissionais da saúde.

Resumen OBJETIVO Analizar la estructura y el contenidos de las representaciones sociales de los enfermeros sobre la violencia doméstica contra las mujeres. MÉTODOS Estúdio cualitativo cumplido em dos hospitales de Río Grande/RS. Los datos fueron recolectados entre mayo/septiembre de 2014 a través de entrevistas semi-estructuradas, cumplido con 100 enfermera, y evocaciones com 34. Fueron tratadas por un software Evoc y análisis contextual, respectivamente. RESULTADOS Se observa una representación negativa con elementos nucleares en alusión a las formas de violencia y su juicio, como "agresión física" y "falta de respeto". En la primera periferia, "miedo", revela tanto la sensación de profissonais como las víctimas contra el agresor, y "sumisión" se califica como una causa de la violencia. Se deduce de la posibilidad de una representación del subgrupo diferente, a través del la presencia del término "agression verbal", en la zona de contraste. CONCLUSIONES Una visión centralizada las lesiones físicas y culpabilidad a la víctima puede limitar las acciones de atención, por lo tanto es fundamental discutir este tema con profesionales de la salud.

Abstract OBJECTIVE To analyse the structure and contents of the social representations of nurses concerning domestic violence against women. METHODS This is a qualitative study conducted with 100 nurses between May and September 2014 in two hospitals of Rio Grande, RS, Brazil. Data were collected through evocations and semi-structured interviews. The data were processed in Evoc software and subjected to contextual analysis, respectively. RESULTS A negative representation was identified with core elements alluding to forms of violence and its judgment, expressed as "physical aggression" and "contempt". In the periphery, "fear" is how the professionals and the victims feel toward the aggressor and "submission" is mentioned as a cause of violence. The term "verbal aggression" in the contrast zone suggests the possibility of a subgroup with a different representation. CONCLUSIONS A centralised view regarding physical injuries and the culpabilisation of domestic abuse victims can limit care actions, revealing the need to discuss this subject with health workers.

Caracterização bioquímica e funcional da proteína XadA3 de Xylella fastidiosa / Biochemistry and functional characterization of Xylella fastidiosa XadA3 adhesin

Gram-negativas e é utilizado por diversos patógenos para colonizar seus hospedeiros, sendo o primeiro passo do processo de desenvolvimento do biolfilme. Uma variedade de apêndices celulares e proteínas está envolvida na adesão bacteriana, tais como pili, fimbrias, adesinas fimbriais e afimbriais. O fitopatógeno Xylella fastidiosa, agente causal de importantes doenças como a doença de Pierce de videiras, a clorose variegada dos citros e a síndrome do rápido declínio de oliveiras, possui em sua superfície várias dessas estruturas que são potencialmente responsáveis pela colonização eficiente de insetos-vetores e plantas hospedeiras. Entre as adesinas afimbriais codificadas no genoma dessa bactéria, três XadA (XadA1, Hsf/XadA2 e XadA3) são classificadas como autotransportadores triméricos. Dados da literatura sugerem que XadA1 e XadA2 são importantes para a formação do biofilme, porém a função de XadA3 ainda não havia sido investigada. Nesse trabalho, tivemos como objetivo caracterizar bioquímica e funcionalmente a proteína XadA3 e obter informações adicionais sobre o papel desempenhado por XadA1 e XadA2 na adesão e virulência de X. fastidiosa. Utilizando imunodetecção com um anticorpo policlonal anti-XadA3 por nós obtido, demonstramos que essa proteína localiza-se na superfície bacteriana e medeia a adesão intercelular. A caracterização dos fenótipos de mutantes de deleção de cada um dos genes das adesinas XadA revelou que o mutante ΔxadA3 tem reduzida capacidade de agregação celular e formação de biofilme quando comparado tanto aos mutantes ΔxadA1 e ΔxadA2 como à cepa selvagem Temecula. A deleção dos genes xadA afeta marginalmente o perfil de expressão gênica global avaliado através de RNAseq das cepas mutantes comparativamente à cepa selvagem, porém destaca-se, nas cepas mutantes, o aumento nos níveis dos transcritos de lipases/esterases. Já foi descrito que essas enzimas parecem atuar na degradação do tecido vegetal associada aos sintomas da doença de Pierce de videiras. A deleção de xadA3 resulta em um fenótipo de hipervirulência em videiras, mas também de deficiência de transmissão pelo inseto-vetor. O conjunto dos resultados obtidos nesse trabalho evidenciam o importante papel desempenhado pelas adesinas XadAs, particularmente XadA3, na adesão intercelular, no desenvolvimento do biofilme e na virulência de X. fastidiosa

Adhesion is a widely conserved mechanism of virulence among Gram-negative bacteria that is used by several pathogens to colonize their hosts, being the first step in biolfilm development. A variety of appendages and proteins are involved in bacterial adhesion, such as pili, fimbriae, fimbrial and afimbrials adhesins. The phytopathogen Xylella fastidiosa, causal agent of important diseases such as Pierce's disease of grapevines, citrus variegated chlorosis and olive quick decline syndrome, harbours on its surface several of these structures that are potentially responsible for efficient colonization of insect vectors and plant hosts. Among the afimbrial adhesins encoded in the genome of this bacterium, three XadAs (XadA1, Hsf/XadA2 and XadA3) are classified as trimeric autotransporters. Data from the literature suggest that XadA1 and XadA2 are important for biofilm formation, but XadA3 function has not been yet investigated. In this work, we aimed to biochemically and functionally characterize the XadA3 protein and gather additional information about the role played by XadA1 and XadA2 in X. fastidiosa adhesion and virulence. Using immunodetection with a polyclonal anti-XadA3 antibody, we have demonstrated that this protein localizes to the bacterial surface and mediates intercellular adhesion. Phenotypic characterization of the deletion mutants of XadA adhesins encoded genes revealed that the ΔxadA3 mutant has reduced cell aggregation capacity and biofilm formation when compared to both ΔxadA1 and ΔxadA2 mutants as well as to Temecula wild type strain. Deletion of the xadA genes marginally affects the global gene expression profile assessed by RNA-seq of the mutant strains compared to the wild-type strain, eventhough an increase in lipase/esterase transcripts levels was observed in the mutant strains. It has been reported that these enzymes appear to participate in the degradation of plant tissue that is associated with symptoms of Pierce's disease of grapevines. The deletion of xadA3 results in a phenotype of hypervirulence in grapevines but also of deficiency in insect-vector transmission. The results obtained in this work evidenced the important role played by XadAs adhesins, particularly XadA3, in X. fastidiosa intercellular adhesion, biofilm development and virulence

Experimental research on Arginine-gingipain A gene vaccine from Porphyromonas gingivalis that prevents peri-implantitis in Beagle dogs / 华西口腔医学杂志

OBJECTIVE@#This study aims to use Arginine-gingipain A gene vaccine (pVAX1-rgpA) to immunize adult Beagle dogs and to evaluate its effect during peri-implantitis progression and development.@*METHODS@#Plasmid pVAX1-rgpA was constructed. The second and third bilateral mandible premolars of 15 adult Beagle dogs were extracted, and the implants were placed immediately. After 3 months, the animals were randomly divided into groups A, B, and C. Afterward, the animals were immunized thrice with plasmid pVAX1-rgpA, with heat-killed Porphyromonas gingivalis, or pVAX1, respectively. IgG in the serum and secretory IgA (sIgA) in saliva were quantitatively analyzed by enzyme-linked immunosorbent assay before and after 2 weeks of immunization. Peri-implantitis was induced with cotton ligatures fixed around the neck of implants. Probing depth (PD) and bleeding on probing were recorded. All animals were sacrificed after ligaturation for 6 weeks. Decalcified sections with thickness of 50 μm were prepared and dyed with methylene blue to observe the bone phenotype around implants.@*RESULTS@#Levels of serum IgG and sIgA in saliva were higher in groups A and B after immunization than before the process (P0.05). At 4 and 6 weeks after ligaturation, PD of the ligatured side in group C was higher than that in groups A and B (P0.05). Bone loss in group A was significantly lower than that of the other groups (P<0.05). Abundant inflammatory cells and bacteria were present in the bone loss area around the implants in the three groups, as identified through hard tissue section observation. However, group C presented the most number of inflammatory cells and bacteria in the bone loss area around the implants.@*CONCLUSIONS@#IgG and sIgA can be generated by immunity with rgpA DNA vaccine, which can significantly slow down bone loss during experimental peri-implantitis in dogs.

Placental and colostral transfer of antibodies reactive with enteropathogenic Escherichia coli intimins α, ß, or γ / Transferência placentária e colostral de anticorpos reativos a Escherichia coli enteropatogênica com expressão das intiminas α, ß, or γ

Abstract Objective: Intimins are protein adhesins of enteropathogenic Escherichia coli and enterohemorrhagic E. coli capable of inducing attachment and effacement lesions in enterocytes. Anti-intimin antibodies are important for the protection from enteropathogenic E. coli and enterohemorrhagic E. coli infections because these antibodies inhibit bacterial adhesion and impair the initial step of the pathogenesis. We studied the transfer of maternal anti-intimin antibodies from healthy Brazilian mothers to their newborns through the placenta and colostrum. Methods: Serum immunoglobulin G and secretory immunoglobulin A antibodies against conserved and variable regions of intimins α, β, and γ were analyzed using an enzyme linked-immunosorbent assay in the blood and colostrum from 45 healthy women as well as cord blood serum samples from their newborns. Results: The concentrations of antibodies reactive with α intimin were significantly lower than those of anti-γ and anti-conserved intimin antibodies in the colostrum samples. IgG serum antibodies reactive with all the subtypes of intimins were transferred to the newborns, but the concentrations of anti-conserved intimin serum antibodies were significantly higher in mothers and newborns than concentrations of antibodies against variable regions. The patterns of IgG transfer from mothers to newborns were similar for all anti-intimin antibodies. These values are similar to the percentage transference of total IgG. Conclusions: Anti-intimin antibodies are transferred from mothers to newborns through the placenta, and reinforce the protection provided by breastfeeding against diarrheagenic E. coli infections.

Resumo Objetivo: As intiminas são adesinas proteicas de Escherichia coli enteropatogênicas (EPEC) e enterro-hemorrágicas (EHEC) capazes de induzir as lesões attaching and effacing nos enterócitos. Anticorpos anti-intiminas são importantes para a proteção contra infecções por EPEC e EHEC porque esses anticorpos inibem a adesão bacteriana e impedem o passo inicial do mecanismo patogênico dessas bactérias. Nós estudamos a transferência de anticorpos maternos anti-intiminas de mães brasileiras saudáveis para os seus recém-nascidos através da placenta e do colostro. Métodos: Anticorpos séricos da classe IgG e secretórios da classe IgA (SIgA) reativos com as porções conservada (cons) e variáveis das intiminas α (vα), β (vβ) e γ (vγ) foram analisados pelo teste de ELISA no sangue e no colostro de 45 parturientes saudáveis e no sangue de cordão umbilical dos seus respectivos recém-nascidos. Resultados: As concentrações de anticorpos reativos com intimina vα foram significativamente mais baixas que as dos anticorpos anti-vγ e anti-cons nas amostras de colostro. Anticorpos IgG séricos reativos com todas as intiminas foram transferidos para os recém-nascidos, mas as concentrações de anti-cons foram significativamente mais altas tanto nas mães como nos recém-nascidos do que os anticorpos reativos com as regiões variáveis das intiminas. O padrão de transferência de IgG das mães para os recém-nascidos foi muito semelhante para todos os anticorpos anti-intiminas. Os valores de porcentagem de transferência foram semelhantes à transferência de IgG total. Conclusões: Anticorpos anti-intimina são transferidos das mães para os recém-nascidos pela placenta e corroboram a proteção contra infecções por Escherichia coli diarreiogênicas (DEC) conferida pelo aleitamento materno.

Parenteral adjuvant potential of recombinant B subunit of Escherichia coli heat-labile enterotoxin

BACKGROUND The B subunit of Escherichia coli heat-labile enterotoxin (LTB) is a potent mucosal immune adjuvant. However, there is little information about LTB's potential as a parenteral adjuvant. OBJECTIVES We aimed at evaluating and better understanding rLTB's potential as a parenteral adjuvant using the fused R1 repeat of Mycoplasma hyopneumoniae P97 adhesin as an antigen to characterise the humoral immune response induced by this construct and comparing it to that generated when aluminium hydroxide is used as adjuvant instead. METHODS BALB/c mice were immunised intraperitoneally with either rLTBR1 or recombinant R1 adsorbed onto aluminium hydroxide. The levels of systemic anti-rR1 antibodies (total Ig, IgG1, IgG2a, and IgA) were assessed by enzyme-linked immunosorbent assay (ELISA). The ratio of IgG1 and IgG2a was used to characterise a Th1, Th2, or mixed Th1/Th2 immune response. FINDINGS Western blot confirmed rR1, either alone or fused to LTB, remained antigenic; anti-cholera toxin ELISA confirmed that LTB retained its activity when expressed in a heterologous system. Mice immunised with the rLTBR1 fusion protein produced approximately twice as much anti-rR1 immunoglobulins as mice vaccinated with rR1 adsorbed onto aluminium hydroxide. Animals vaccinated with either rLTBR1 or rR1 adsorbed onto aluminium hydroxide presented a mixed Th1/Th2 immune response. We speculate this might be a result of rR1 immune modulation rather than adjuvant modulation. Mice immunised with rLTBR1 produced approximately 1.5-fold more serum IgA than animals immunised with rR1 and aluminium hydroxide. MAIN CONCLUSIONS The results suggest that rLTB is a more powerful parenteral adjuvant than aluminium hydroxide when administered intraperitoneally as it induced higher antibody titres. Therefore, we recommend that rLTB be considered an alternative adjuvant, even if different administration routes are employed.

Correlation between virulence markers of Helicobacter pylori in the oral cavity and gastric biopsies / Correlação entre marcadores de virulência de Helicobacter pylori na cavidade oral e em biópsias gástricas

ABSTRACT BACKGROUND: The clinical outcome of Helicobacter pylori infection has been associated with virulence factors. The presence of these factors is useful as molecular markers in the identification of the high risk for developing severe gastric pathologies. OBJECTIVE: To correlate the presence of virulence markers cagA and bab2A of H. pylori in oral and gastric biopsy samples. METHODS: An observational, prospective, descriptive, and cross-sectional study was carried out between September 2011 and September 2012. Patients suffering dyspepsia with indication for upper gastrointestinal video endoscopy who attended the Gastroenterology Service of the Hospital Dr. Julio C. Perrando were included. Epidemiological investigation was completed. To detect the bacteria and their virulence genes, samples of saliva, dental plaque and gastric biopsy were taken and processed by PCR. RESULTS: Sixty-one patients were selected for this study (30 women and 31 men). H. pylori was detected in 31 gastric biopsies and 31 oral samples. Significant difference between oral and gastric samples was found in cagA genotype. Agreement between oral and gastric genotypes was found in 38.7% of samples from the same patient. CONCLUSION: This study is the first in provide information about the genotypes of the Argentinean Northeast H. pylori strains. Despite the high prevalence of H. pylori infection, the most of patients had less virulent genotypes in oral cavity and gastric tissue. The cagA / babA2 combination was not frequent in the samples studied. There was not a statistical correlation between the virulence genes and gastroduodenal or oral diseases. Although in some patients the same genotype was found both in oral and gastric samples, it cannot be ensure that they corresponding to the same strain because a DNA sequencing was not performed.

RESUMO CONTEXTO: O resultado clínico da infecção por Helicobacter pylori tem sido associado com fatores de virulência. A presença desses fatores como marcadores moleculares é útil na identificação do risco elevado para o desenvolvimento de graves patologias gástricas. OBJETIVOS: Correlacionar a presença de marcadores de virulência cagA e bab2A do H. pylori em amostras de biópsias gástricas e orais. MÉTODOS: Um estudo observacional, prospectivo, descritivo e transversal foi realizado entre setembro de 2011 e setembro de 2012. Foram incluídos pacientes com sintomas de dispepsia com indicação de endoscopia gastrointestinal que compareceram ao Serviço de Gastroenterologia do Hospital Dr. Julio C. Perrando . Investigação epidemiológica foi concluída. Para detectar a bactéria e seus genes de virulência, amostras de saliva, placa dentária e biópsia gástrica foram tomadas e processadas pelo PCR. RESULTADOS: Sessenta e um pacientes foram selecionados para este estudo (30 mulheres e 31 homens). H. pylori foi detectado em 31 biópsias gástricas e 31 amostras orais. Foi encontrada diferença significativa entre as amostras orais e gástricas no genótipo cagA . A ocorrência simultânea entre genótipos orais e gástricos do mesmo paciente foi encontrada em 38,7% das amostras. CONCLUSÃO: Este é o primeiro estudo a fornecer informações sobre os genótipos das cepas do H. pylori no Nordeste Argentino. Apesar da alta prevalência da infecção pelo H. pylori , a maioria dos pacientes tinha genótipos menos virulentos na cavidade oral e tecido gástrico. A combinação cagA / babA2 não foi frequente nas amostras estudadas. Não houve correlação estatística entre os genes de virulência e doenças gastroduodenais ou orais. Embora em alguns pacientes o mesmo genótipo tenha sido encontrado tanto nas amostras orais quanto gástricas, não se pode garantir que correspondam à mesma variação, pois um sequenciamento de DNA não foi realizado.

PAR-2 expression in the gingival crevicular fluid reflects chronic periodontitis severity

Abstract Recent studies investigating protease-activated receptor type 2 (PAR-2) suggest an association between the receptor and periodontal inflammation. It is known that gingipain, a bacterial protease secreted by the important periodontopathogen Porphyromonas gingivalis can activate PAR-2. Previous studies by our group found that PAR-2 is overexpressed in the gingival crevicular fluid (GCF) of patients with moderate chronic periodontitis (MP). The present study aimed at evaluating whether PAR-2 expression is associated with chronic periodontitis severity. GCF samples and clinical parameters, including plaque and bleeding on probing indices, probing pocket depth and clinical attachment level, were collected from the control group (n = 19) at baseline, and from MP patients (n = 19) and severe chronic periodontitis (SP) (n = 19) patients before and 6 weeks after periodontal non-surgical treatment. PAR-2 and gingipain messenger RNA (mRNA) in the GCF of 4 periodontal sites per patient were evaluated by Reverse Transcription Polymerase Chain Reaction (RT-qPCR). PAR-2 and gingipain expressions were greater in periodontitis patients than in control group patients. In addition, the SP group presented increased PAR-2 and gingipain mRNA levels, compared with the MP group. Furthermore, periodontal treatment significantly reduced (p <0.05) PAR-2 expression in patients with periodontitis. In conclusion, PAR-2 is associated with chronic periodontitis severity and with gingipain levels in the periodontal pocket, thus suggesting that PAR-2 expression in the GCF reflects the severity of destruction during periodontal infection.

Immunoprotective effect of combined pneumococcal endopeptidase O and pneumococcal surface adhesin A vaccines against Streptococcus pneumoniae infection / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the prokaryotic expression of proteins pneumococcal endopeptidase O (PepO) and pneumococcal surface adhesin A (PsaA) in Streptococcus pneumoniae and their immunoprotective effect as vaccine candidate proteins.</p><p><b>METHODS</b>Specific primers of target gene fragments were designed, and then PCR amplification was performed to establish recombinant plasmids pET28a(+)-pepO and pET28a(+)-psaA, which were transformed into host cells, Escherichia coli BL21 and DE3, respectively, to induce expression. Highly purified target proteins PepO and PsaA were obtained after purification. Mucosal immunization was performed for BALB/c mice and specific antiserum was prepared. ELISA was used to measure the antibody titer, and Western blot was used to analyze the specificity of the antiserum of target proteins. The mice were randomly divided into negative control group, PepO group, PsaA group, and PepO+PsaA combined immunization group, with 18 mice in each group. The models of different serotypes of Streptococcus pneumoniae infection were established to evaluate the immunoprotective effect of target proteins used alone or in combination.</p><p><b>RESULTS</b>The target proteins PepO and PsaA were successfully obtained and Western blot demonstrated that the antiserum of these proteins had good specificity. There was no significant difference in the titers of IgA in saliva and IgG in serum between the PepO group and the combined immunization group (P>0.05); however, these two groups had significantly higher antibody titers than the PsaA group (P<0.05). The PepO, PsaA, and combined immunization groups had significantly higher protection rates for mice infected with Streptococcus pneumoniae D39 and CMCC31436 in the nasal cavity than the negative control group (P<0.05). The PepO and combined immunization groups had a significantly higher protection rate for mice infected with Streptococcus pneumoniae D39 than the PsaA group (P<0.05). The results of colonization experiment showed that compared with the control group, the PepO, PsaA, and combined immunization groups showed a significant reduction in the colonization of Streptococcus pneumoniae (CMCC31693 and CMCC31207) in the nasopharynx and lung (P<0.05). The combined immunization group showed a better effect on reducing the colonization of CMCC31207 in the lung than the PepO and PsaA alone groups.</p><p><b>CONCLUSIONS</b>Combined PepO/PsaA vaccines may produce a better protective effect by mucosal immunization compared with the vaccine used alone in mice. The combined vaccines can effectively reduce the colonization of Streptococcus pneumoniae in the nasopharynx and lung. Therefore, such protein vaccines may have a great potential for research and development.</p>

Fusobacterium nucleatum. ¿un patógeno periodontal promotor de carcinogénesis colorrectal? / Fusobacterium nucleatum. is this periodontal pathogen a promoter of colorectal carcinogenesis?

El Fusobacterium nucleatum es una bacteria anaerobia Gram negativa,es un residente común en el biofi lm oral y se ha encontrado una estrechaasociación entre las fusobacterias y las periodontitis. El Fusobacteriumnucleatum se ha asociado con el cáncer colorrectal, pero la causalidad y el mecanismo subyacente aún no se han establecido. La microbiota intestinal humana tiene un papel reconocido en el cáncer colorrectal. Se ha encontrado que el Fn se adhiere, invade, e induce respuestas inflamatorias oncogénicas que estimulan el crecimiento de las células de cáncer colorrectal a través de un factor de la adhesina FadA.

The anaerobic, Gram-negative bacterial species Fusobacterium nucleatumis common in oral biofi lm and the association between it andperiodontitis is well-established. Fusobacterium nucleatum has beenassociated with colorectal cancer, though causality and the underlyingmechanism have yet to be determined. The role of the human gutmicrobiota in colorectal cancer has been acknowledged. Fusobacteriumnucleatum has been found to adhere to, invade, and induce oncogenicand infl ammatory responses that stimulate the growth of colorectalcancer cells through its unique FadA adhesin.

The expression of heterologous MAM-7 in Lactobacillus rhamnosus reduces its intrinsic capacity to inhibit colonization of pathogen Vibrio parahaemolyticus in vitro

BACKGROUND: Vibrio parahaemolyticus (V. parahaemolyticus) is a Gram-negative, halophilic bacterium recognized as one of the most important foodborne pathogen. When ingested, V. parahaemolyticus causes a self-limiting illness (Vibriosis), characterized mainly by watery diarrhoea. Treatment is usually oral rehydration and/or antibiotics in complicated cases. Since 1996, the pathogenic and pandemic V. parahaemolyticus O3:K6 serotype has spread worldwide, increasing the reported number of vibriosis cases. Thus, the design of new strategies for pathogen control and illness prevention is necessary. Lactobacillus sp. grouped Gram positive innocuous bacteria, part of normal intestinal microbiota and usually used as oral vaccines for several diarrheic diseases. Recombinants strains of Lactobacillus (RL) expressing pathogen antigens can be used as part of an anti-adhesion strategy where RL block the pathogen union sites in host cells. Thus, we aimed to express MAM-7 V. parahaemolyticus adhesion protein in Lactobacillus sp. to generate an RL that prevents pathogen colonization RESULTS: We cloned the MAM-7 gene from V. parahaemolyticus RIMD 2210633 in Lactobacillus expression vectors. Recombinant strains (Lactobacillus rhamnosus pSEC-MAM7 and L. rhamnosus pCWA-MAM7) adhered to CaCo-2 cells and competed with the pathogen. However, the L. rhamnosus wild type strain showed the best capacity to inhibit pathogen colonization in vitro. In addition, LDH-assay showed that recombinant strains were cytotoxic compared with the wild type isogenic strain CONCLUSIONS: MAM-7 expression in lactobacilli reduces the intrinsic inhibitory capacity of L. rhamnosus against V. parahaemolyticus.

Association of number of tandem repeats in two important adhesins in Mycoplasma hyopneumoniae / Associação entre os números de repetições em tandem em duas importantes adesinas de Mycoplasma hyopneumoniae

Diversidade genética de Mycoplasma hyopneumoniae tem sido relatada em análise múltipla de repetições em tandem em número variável (MLVA). O objetivo deste estudo foi descrever a distribuição espacial e a heterogeneidade genética de tipos de M. hyopneumoniae no Brasil, bem como investigar a correlação entre regiões de repetição 1 (RR1) e 3 (RR3) de duas adesinas importantes (P97 e P146). Foram identificados 39 tipos de MLVA baseados no número de repetições em tandem em P97 RR1 e RR3 P146. A correlação negativa significativa (Spearman's rho = -0,26; P = 0,022) entre P97 RR1 e RR3 P146 foi observada, o que sugere um possível mecanismo compensatório que permitiria a bactéria manter a sua capacidade de adesão. Os resultados contribuem para compreender a epidemiologia das M. hyopneumoniae no quarto maior país produtor de suínos do mundo.(AU)

Does the bracket composition material influence initial biofilm formation.

Context: Orthodontic treatment has been reported to contribute to the development and accumulation of dental biofilm, which is commonly found on bracket and adjacent surfaces. Aims: The aim of this work is to test the hypothesis if there are differences in dental biofilm formation on the surface of orthodontic brackets according to the type of composition material. Subjects and Methods: Three bracket types (metallic, composite, and ceramic) had been evaluated. Subjects wore acrylic palatal orthodontic appliances, containing 6 brackets each, for two 3‑day cycles. On the end of first cycle, the amount of dental biofilm formed on the samples was extracted using 1.0‑M NaOH and analyzed by spectrophotometry for quantification. An additional cycle was carried out to verify the dental biofilm formation using scanning electronic microscope analysis. Statistical Analysis Used: Three‑way ANOVA was used to analyze the difference among the materials (metallic, ceramic and composite) concerning the dental biofilm absorbance spectrum. Multiple comparisons were made using the Tukey’s test (α =0.05). Results: Composite brackets showed greater values concerning biofilm formation, when compared with the metallic and ceramic ones, both of which presented similar scores. The hypothesis is accepted. There are differences on the biofilm formation according to the type of material. Conclusions: The in situ model tested was found to be effective in evaluating the accumulation and development of biofilm on orthodontic brackets. In the quantitative analysis, composite brackets showed greater biofilm adhesion values while metallic and ceramic presented similar biofilm absorbance spectrum.

sialic acid binding adhesin [SabA] of Helicobacter pylori isolated from Egypt is associated with gastric cancer

Background: Helicobacter pylori [H. pylori] expressed outer membrane proteins [OMP[s]] that assist in bacterial adherence to the gastric epithelium promoting successful colonization. One of these OMPs is the blood group antigen binding adhesin A [BabA] which bind to the fucosylated Lewis[b] blood group antigen [Le[b]] on the surface of gastric epithelial cells. Another OMP[s] is the sialic acid binding adhesin [SabA] that mediates H. pylori binding the specific sialyl dimeric Lewis[x] glycosphingolipid [Le[x]] on the gastric epithelium. A lot of discrepancies about the correlation between the presence of both babA and sabA genes and the apparent clinical outcome of H. pylori infection were reported

Objectives: The present study was to disclose the relationship between the presence of these genes and the clinical outcomes in Egyptian H. pylori patients

Methodology: Forty three H. pylori strains were isolated from patients with different clinical findings. Polymerase chain reaction [PCR] for detecting the presence of babA and sabA genes was performed using different sets of primers for detecting different regions of the gene. Further bioinformatics analysis for the sabA product was done using KEGG and Pfam websites

Results: evincing striking correlation between sabA presence and the gastric cancer disease. However, we could not find any correlation between presence of babA and the associated diseases

Conclusions: SabA is one of the H. pylori OMPs adhesins involving in increasing the risk of H. pylori associated gastric cancer in H. pylori Egyptian patients

Comparative possession of Shiga toxin, intimin, enterohaemolysin and major extended spectrum beta lactamase [ESBL] genes in Escherichia coli isolated from backyard and farmed poultry

The present work was conducted to compare the occurrence of Escherichia coli possessing virulence and ESBL genes in backyard and farmed poultry. Three hundred and sixty samples from the poultry kept in backyard system and 120 samples from the farmed birds were collected from West Bengal, India. Among the E. coli isolates of backyard poultry [O2, O10, O25, O55, O60, O106, UT], none of them possessed any of the Shiga toxin genes and eight E. coli isolates [8/272; 2.9%] harboured eaeA gene alone. Whereas among the E. coli isolated from the farmed poultry [O17, O20, O22, O102, O114, O119, rough, UT], four isolates [4/78, 5.1%] harboured stx[1]/stx[2] gene and 11 isolates [11/78, 14.1%] possessed eaeA gene. None of the E. coli isolates from the backyard poultry harboured any studied ESBL gene. Whereas 29.4% of E. coli isolates from the farmed poultry were found to possess the ESBL genes

Prokaryotic expression of Staphylococcus aureus Clumping factor B and evaluation of the antiserum-mediated opsonic activity / 生物工程学报

Staphylococcus aureus is a major cause of hospital-acquired infection. Because the bacteria are very easy to become resistant to antibiotics, vaccination is a main method against S. aureus infection. Clumping factor B (ClfB) is an adhesion molecule essential for S. aureus to colonize in the host mucosa and is regarded as an important target antigen. In this study, we successfully used Escherichia coli to express a segment encoding the N1-N3 regions of ClfB protein (Truncated-ClfB) cloned from S. aureus. The protein was purified by affinity and ion exchange chromatographies and gel filtration. Rabbits were immunized three times with purified Truncated-ClfB. After that, blood was collected to prepare serum which were then used for measurement of antibody level. Phagocytosis of S. aureus opsonized by the serum was determined by a flow cytometry. Results show that the serum IgG titer reached 1:640 000. Phagocytosed S. aureus by polymorphonuclear leukocytes were significantly more when the bacteria were opsonized by the serum from Truncated-ClfB immunized rabbits than those from no immunized group (P < 0.01). Therefore, the results indicated that Truncated-ClfB could be a promising vaccine candidate against S. aureus infection.

Expressions of protease-activated receptors in human gingival fibroblasts and its functions in periodontitis / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the expression types of protease-activated receptors(PAR) in human gingival fibroblasts(HGF) and the functions of PAR in periodontitis.</p><p><b>METHODS</b>Primary HGF were cultured.Reverse transcription PCR(RT-PCR) was used to detect the expression of PAR in HGF. Recombinant gingipain R (rRgp) was applied to HGF. The change of PAR expression on the cell surface was analyzed by real-time quantitative RT-PCR, and enzyme-linked immunosorbent assay (ELISA) was used to detect the change of the interleukin (IL)-6 production from HGF. The results of RT-PCR and ELISA were statistically analyzed using the two independent samples t-test of SPSS10.0 software.</p><p><b>RESULTS</b>HGF expressed PAR-1 and PAR-3. The expression of PAR-1 and PAR-3 changed after two rRgp treatment with HGF cells. The relative expression of PAR-1 was decreased from 1.04 ± 0.31 to 0.67 ± 0.11 and 0.31 ± 0.11. The relative expression of PAR-3 was decreased from 1.01 ± 0.44 to 0.79 ± 0.13 and 0.44 ± 0.12(P < 0.05). The level of IL-6 was increased after rRgp treatment for 8 h. The control group was (18.77 ± 4.09) µg/L, the rRgp treatment groups were (179.36 ± 15.81) and (320.56 ± 26.19) µg/L respectively.</p><p><b>CONCLUSIONS</b>HGF expressed PAR-1 and PAR-3 and were involved in periodontal inflammation.</p>

Expression of integrin α5 and β1 in osteoblast in the process of gingipains-induced apoptosis / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the regulatory mechanisms of integrin α5 and β1 in osteoblast in the process of gingipains-induced apoptosis.</p><p><b>METHODS</b>Gingipains were isolated and purified from supernatants of Porphyromonas gingivalis W83 which was cultured under standard anaerobic conditions. MC3T3-E1 was challenged with or without 8.3480 U/L gingipains for 48 h and apoptosis was examined by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (TUNEL-DAPI) staining. The expression of integrin α5 and β1 was analyzed by Western blotting after MC3T3-E1 was treated under different conditions.</p><p><b>RESULTS</b>Arginine-specific proteinases(Rgp) activity was (41.74 ± 2.11) U/L and lysine-specific proteinase(Kgp) was (1.02 ± 0.25) U/L.Gingipains induced MC3T3-E1 cells apoptosis after 48 h. Compared with control group, expression of integrin α5 and β1 was down-regulated by gingipains in a time-dependent manner within short periods ( ≤ 72 h), integrin α5 and β1 relative expression was (0.485 ± 0.039),(0.504 ± 0.002) at 48 h,(0.398 ± 0.058),(0.179 ± 0.001) at 72 h respectively (P < 0.05). After 72 h, integrin α5 expression in MC3T3-E1 cells was stable compared with control group while integrin β1 was still lower(control group:1.000 ± 0.000, 96 h:0.604 ± 0.003, 120 h: 0.357 ± 0.002) (P < 0.05). Proteinase inhibitor tosyl- L- lysine-chloromethyl-ketone(TLCK) effectively blocked the activity of gingipain and inhibited down-regulation of integrin α5 and β1 induced by gingipains from (0.398 ± 0.058,0.179 ± 0.001 ) to (0.781 ± 0.012, 0.857 ± 0.060) (P < 0.05). TLCK alone did not have any effect on integrin α5 and β1(P > 0.05). Gingipains also decreased integrin α5 and β1 in a dose-dependent manner.When cells were treated with 20.8700 U/L gingipains, integrin α5 and β1 relative expression reached to the lowest(0.105 ± 0.004,0.020 ± 0.000) (P < 0.05).</p><p><b>CONCLUSIONS</b>Gingipains inhibited the expression of integrin α5 and β1 in a time- and dose- dependent manner in osteoblasts in the process of apoptosis, which may not be mediated by direct proteolytic effect.</p>