Interacción del microbioma intestinal y el hueso / Interaction between the intestinal microbiome and bone

El "microbioma" no solo está constituido por los microbios, sino por todos los componen-tes que viven en el mismo hábitat conforman-do un nicho ecológico. Es decir, está conformado por los microorganismos (bacterias, hongos, protozoos, etc.), todo el espectro de moléculas producidas por ellos tales como sus componentes estructurales (ácidos nucleicos, proteínas, lípidos y glúcidos), meta-bolitos, toxinas, etc., y las moléculas producidas por el huésped. El microbioma intestinal (MI) ha emergido como un factor que tiene un gran efecto sobre la cantidad, calidad y fuerza del hueso. Las investigaciones revelan que la homeostasis ósea está ligada al micro-bioma saludable, mientras que la disbiosis (alteración en la biodiversidad microbiana) puede exacerbar la actividad osteoclástica y promover la osteoporosis. Los mecanismos potenciales involucrados en la interacción del microbioma intestinal y el hueso son la influencia del metabolismo del huésped, el mantenimiento de la integridad intestinal y regulación de la absorción de nutrientes, la regulación del eje intestino-sistema inmune y la modulación del sistema endocrino. Es decir que hay múltiples vías por las cuales el MI influye sobre el hueso, pero estos y otros mecanismos deben profundizarse más aún. También es necesario que se identifiquen y caractericen mejor los microorganismos que están asociados a las enfermedades óseas. El conocimiento de estos aspectos podría ser útil para el desarrollo de herramientas terapéuticas basadas en el MI que puedan mejorar la eficacia de los distintos tratamientos existentes. (AU)

The microbiome is not only constituted by microbes, but by all the components that live in the same habitat forming an ecological niche. It is conformed by the microorganisms ( bacteria, fungi, protozoa, etc), the entire spectrum of molecules produced by them (nucleic acids, proteins, lipid and carbohydrates, metabolites, toxins, etc) and the molecules produced by the host. The intestinal microbiome (IM) has emerged as a factor with great effects on the quantity, quality and strength of bone. The investigations reveal that bone homeostasis is linked to the healthy microbiome, while the dysbiosis (alteration in the microbial biodiversity) can exacerbate the osteoclastic activity and promote osteoporosis. The potential mechanisms involved in the interaction between IM and bone are the influence of the host metabolism, the maintenance of the intestinal integrity and regulation of the nutrient absorption, the regulation of the intestine/ immune system axis and the modulation of the endocrine system. That is, there are multiple ways through which IM influences on bone, but these and other mechanisms need to be further studied. It is also necessary to identify and characterize the microorganisms associated with the bone diseases. Knowledge of these aspects could be useful to develop therapeutical tools based on the IM that could improve the efficacy of the current treatments. (AU)

Diet, microbiota, and inflammatory bowel disease: lessons from Japanese foods

The incidence and prevalence of inflammatory bowel diseases (IBDs) including ulcerative colitis and Crohn disease are rapidly increasing in Western countries and in developed Asian countries. Although biologic agents targeting the immune system have been effective in patients with IBD, cessation of treatment leads to relapse in the majority of patients, suggesting that intrinsic immune dysregulation is an effect, not a cause, of IBD. Dramatic changes in the environment, resulting in the dysregulated composition of intestinal microbiota or dysbiosis, may be associated with the fundamental causes of IBD. Japan now has upgraded water supply and sewerage systems, as well as dietary habits and antibiotic overuse that are similar to such features found in developed Western countries. The purpose of this review article was to describe the association of diet, particularly Japanese food and microbiota, with IBD.

Expression of verocytotoxic Escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model

Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and possible adverse effects on the oral administration of obtained tobacco seeds were evaluated in a mouse model. Tobacco was transformed via Agrobacteium tumefaciens with chimeric constructs containing structural parts of the major subunit FedA of the F18 adhesive fimbriae and VT2e B-subunit genes under control of a seed specific GLOB promoter. We showed that the foreign Vt2e-B and F18 genes were stably accumulated in storage tissue by the immunostaining method. In addition, Balb-C mice receiving transgenic tobacco seeds via the oral route showed a significant increase in IgA-positive plasma cell presence in tunica propria when compared to the control group with no observed adverse effects. Our findings encourage future studies focusing on swine for evaluation of the protective effects of transformed tobacco seeds against E. coli infection.

Microbes in the gut: a digestable account of host-symbiont interactions.

The human bowel is host to a diverse group of bacteria with over 500 different bacterial species contributing to this diversity. Until recently these bacteria were regarded as residents without any specific functions. The last two decades have seen a radical change in our understanding of the interactions between the gut flora and their eukaryotic hosts and there is a growing appreciation of the spectrum of functions performed by these symbionts. Intestinal bacteria are recognized for their role in nutrient absorption, mucosal barrier function, angiogenesis, morphogenesis and postnatal maturation of intestinal cell lineages, intestinal motility and more importantly maturation of gut associated lymphoid tissue (GALT). Although gut flora are implicated in certain pathological disorders, their remarkable contributions to health and homeostasis of the host need to be recognized and understood.

Association of IL-8-inducing strains of diffusely adherent Escherichia coli with sporadic diarrheal patients with less than 5 years of age

The role of diffusely adherent Escherichia coli (DAEC) in diarrheal disease has been controversial. However, DAEC strains were recently implicated in diarrheal disease in developing countries. To clarify whether DAEC are prevalent among sporadic cases of diarrheal illness in Osaka City, Japan, E. coli strains isolated between July 1997 and March 2000 during diarrheagenic E. coli (DEC) investigation were retrospectively examined. DAEC strains were recognized among 41 (4.4 percent) of 924 patients and formed the biggest subgroup of DEC. Previously, we reported that some DAEC strains caused epithelial cells to secrete as much IL-8 as enteroaggregative E. coli strains did. In this study, we attempted to evaluate epidemiologically whether the ability of DAEC to induce IL-8 was involved in the pathogenesis. Relationship among patient age, symptoms, Afa adhesins, season and IL-8 induction were examined. The subgroup of DAEC that possessed Afa genes and/or induced a high level of IL-8 was significantly prevalent among patients age 1 to 4 years; however total DAEC was not significantly high among the children compared to other age group. IL-8 inducing DAEC seems to play a role in causing sporadic diarrheal illnesses, particularly in pediatric fields. Investigations highlighting the relationship between IL-8 induction and enteropathogenicity are clearly necessary to confirm the role of DAEC in infectious enteritis.

Enhancement of protective immune responses by oral vaccination with Saccharomyces cerevisiae expressing recombinant Actinobacillus pleuropneumoniae ApxIA or ApxIIA in mice

We previously induced protective immune response by oral immunization with yeast expressing the ApxIIA antigen. The ApxI antigen is also an important factor in the protection against Actinobacillus pleuropneumoniae serotype 5 infection; therefore, the protective immunity in mice following oral immunization with Saccharomyces cerevisiae expressing either ApxIA (group C) or ApxIIA (group D) alone or both (group E) was compared with that in two control groups (group A and B). The immunogenicity of the rApxIA antigen derived from the yeast was confirmed by a high survival rate and an ApxIA-specific IgG antibody response (p < 0.01). The highest systemic (IgG) and local (IgA) humoral immune responses to ApxIA and ApxIIA were detected in group E after the third immunization (p < 0.05). The levels of IL-1beta and IL-6 after challenge with an A. pleuropneumoniae field isolate did not change significantly in the vaccinated groups. The level of TNF-alpha increased in a time-dependent manner in group E but was not significantly different after the challenge. After the challenge, the mice in group E had a significantly lower infectious burden and a higher level of protection than the mice in the other groups (p < 0.05). The survival rate in each group was closely correlated to the immune response and histopathological observations in the lung following the challenge. These results suggested that immunity to the ApxIA antigen is required for optimal protection.

El rol de los prebióticos, probióticos y simbióticos en gastroenterología / Role of prebiotics, probiotics and sinbiotics in gastroenterology

El tracto gastrointestinal es indudablemente el área más expuesta a microorganismos y antígenos dietarios. El epitelio intestinal es un importante componente de la barrera de la mucosa intestinal, el cual debe discriminar adecuadamente entre bacterias patogénicas y no-patogénicas. La flora bacteriana intestinal tiene un efecto condicionador sobre la homeostasis del intestino, entregando señales que regulan el epitelio, el sistema inmune de la mucosa y la actividad neuromuscular del intestino. Estudios han demostrado que la flora bacteriana comensal y sus componentes, son factores importantes en la patogénesis de varias enfermedades gastrointestinales, tales como la enfermedad inflamatoria intestinal, síndrome intestino irritable, cáncer de colon, enfermedad hepática crónica. Aunque estudios experimentales han demostrado que prebióticos, probióticos y simbióticos ejercen efectos antibacterianos, modulación inmune y antiinflamatorios, lo cual puede ser beneficioso en algunas enfermedades gastrointestinales, su real papel en el ser humano aún debe ser evaluado. Porque no todos poseen el mismo efecto terapéutico, colonización con específicos probióticos y simbióticos (incluyendo la ingeniería bacteriana para secretar citokinas anti-inflamatorias y restaurar la flora comensal y la tolerancia intestinal) podría ser la próxima estrategia para el tratamiento de las enfermedades gastrointestinales y otras enfermedades inmunológicas.

Interaction of bifidobacteria with the gut and their influence in the immune function

Bifidobacteria are predominant in the lumen of the large intestine and confer various health benefits on the host. They are also used in the preparation of new fermented milks (bioyogurts) or added to conventional yogurt to generate probiotic effects. The colonization of the gut by bacteria tends to be host specific due partly to the way in which bacteria adhere to the intestinal wall. Using a homologous strain of Bifidobacterium animalis in an experimental mouse model, we analyzed by immunofluorescence labelled-bacteria and transmission electronic microscopy the importance of the bacterial interaction with epithelial an immune cells associated to the gut, and the effect of feeding of B. animalis in the immune response. It was able to adhere and interact with both small and large intestine. In spite of this interaction with the gut, no modifications in the immune state (secretory or systemic response) were observed. A heterologous strain of Bifidobacterium adolescentis from human faeces, was neither incapable of binding to the intestine, nor influence the immune system activation, when it was administered during 2, 5 or 7 consecutive days; we believe that using a homologous strain, oral tolerance is developed even when the microorganism interacts with the immune cells associated with the intestine. However, we cannot ignore the beneficial effect of these microorganisms, especially in the prevention of intestinal infections. We think that this property exerted by bifidobacteria is more related to other mechanisms such as competitive inhibition, acid production or others, than enhancement of the immune state.

Identification of gut antigens from female boophilus microplus ticks

Un extracto de intestino de hembras semi-repletas de garrapata Boophilus microplus, fue separado mediante electroforesis SDS-PAGE, identificándose al menos 18 bandas en un rango de peso molecular de 17 a 258 kDa; al practicar el ensayo inmunoenzimático de Dig-glicanos, las proteínas por arriba de 58 kDa, mostraron estar asociadas con carbohidratos. El análisis de Western blot permitió identificar cinco antígenos de intestino con un peso molecular en un rango de 89 a 208 kDa, utilizando un suero de conejo anti-intestino de garrapata. Estos antígenos fueron localizados en la superficie del intestino de la garrapata mediante inmunofluorescencia indirecta en cortes histológicos del mismo intestino. Los tres antígenos (99, 141, 189 KDa) identificados, son diferentes de los previamente reportados en la literatura y podrían ser utilizados en pruebas para inducir inmunidad en contra de las garrapatas en ganado bovino

Determinación de la velocidad de tránsito intestinal en el ratón BALB/c / Determinaton of the velocity of intestinal transit in the BALB/c mouse

La determinación de la velocidad del tránsito intestinal en un modelo animal es de primordial importancia, sobre todo cuando se pretende utilizar la vía digestiva del mismo como sitio para la presentación de antígenos. En el presente trabajo se determinó la velocidad del tránsito intestinal en el ratón BALB/c, uno de los animales de laboratorio frecuentemente utilizado en la investigación de la respuesta inmune. Se administró un colorante vegetal por medio de una sonda orogástrica, a 45 ratones, mayores de 8 semanas de edad. Posteriormente se sacrificaron los animales a diferentes tiempos en un rango de 10 min a 24 h. El tracto intestinal se dividió de manera arbitraria, el intestino delgado en tres tercios y el intestino grueso, en dos partes. Las determinaciones mostraron que 25 min después de administrado el colorante, éste se localizó a nivel del primer tercio y en menor proporción, a nivel del segundo tercio del intestino delgado. A los 90 min, el colorante alcanzó la primera mitad del intestino grueso, y a las 2 h comenzó a ser eliminado en las heces. Sin embargo, 3 h después de la administración aún se encontró colorante en el estómago, con lo anterior se demostró que el colorante se emilinó de manera intermitente a partir del estómago hasta las 3 h. A las 10 h se encontró colorante en intestino grueso, pero no en intestino delgado ni en estómago, a las 24 h se había eliminado por completo. Se concluye que en el intestino, el tránsito del colorante es constante, aunque la liberación a partir del estómago es intermitente, y que la totalidad del proceso de tránsito intestinal se lleva a cabo antes de 24 h

Flora autóctona intestinal: Revisión Bibliográfica / Autochthonous intestinal flora: bibliographic review

Se revisan diversos aspectos sobre la flora autóctona intestinal en el niño sano y en el enfermo. Su asociación con la antibioticoterapia y la dieta, los efectos metabólicos sobre la flora intestinal, su manipulación y su relación con el sistema inmunológico

Correlacíon de anticuerpos antigliadina y antiendomisiales clase IgA (AGA-IgA - EMA-IgA) con la histologia indestinal en la enfermedad celíaca (E C) / Correlation of antigliadin, antiendomysial antibodies IgA class (AGA-IgA - EMA-IgA) with intestinal histology en celiac disease

Se evaluó la correlación de los AGA-IgA y EmA-IgA con la histología intestinal de pacientes celíacos en distintas etapas de la enfermedad a los fines de establecer su valor diagnóstico, de seguimiento y de control terapéutico. Se estudiaron 56 niños (27 mujeres y 29 varones)de m. a 12 años. 39 se hallaban en distintas etapas diagnósticas de la E.C. Los controles fueron 17 niños con biopsia normal que consultaron por síntomas compatibles con malabsorción. Se obtuvieron 60 muestras y se determinó AGA-IgA (ELISA) y EmA-IgA (inmunofluorescencia indirecta en cortes por congelación de 1/3 inferior de esófago do mono Rhesus). Simultáneamente se efectuó biopsia peroral de intestino delgado. De las 34 muestras con histología grado III-IV, sólo 2 fueron AGA-IgA negativo y 1 EmA-IgA negativo. De las 26 muestras con histología normal, se halló sólo 2 AGA-IgA positivo y 4 EmA-IgA positivo. De estas cifras resultan: EmA-IgA: sensibilidad 97% especificidad 84,6% valor predictivo positivo 89,2%, valor predictivo negativo 95%. AGA-IgA: sensibilidad 94,1%, especificidad 92,3, valor predictivo positivo 94,1% y valor predictivo negativo 92,3%. Los AGA-IgA y EmA-IgA tienen estrecha correlación con la histología intestinal y son complementarios para diagnóstico y seguimiento de la E.C. Orientan sobre la oportunidad de biopsia intestinal, permiten controlar el cumplimiento dietético y posibilitan la detección de familiares asintomáticos

Inmunología y aparato digestivo / Immunology and digestive tract

El intestino es un órgano inmunológico mayor, que está relacionado con la producción de IgA secretora, pero también con la inmunidad celular. La IgA secretora juega un rol muy importante en la protección del organismo contra antígenos bacterianos y alimentarios que ingresan por vía digestiva. Cálculos relacionados con la determinación de la vida media de las diferentes inmunoglobulinas y con su pérdida por vía digestiva, indican que el intestino sintetiza diariamente una mayor cantidad de inmunoglubulinas que todo el resto del organismo. El sistema inmunológico intestinal representa un mecanismo defensivo y protector de vital importancia, cuya deficiencia predispone a diversas enfermedades y en especial a numerosas infecciones crónicas bacterianas y/o parasitarias. A la inversa, la respuesta inmune intestinal excesiva o inapropiada es capaz de desencadenar procesos linfomatosos locales e incluso afecciones inmunológicas de carácter sistémico

The current status of cholera vaccine development and experience with cholera vaccine trials in volunteers.

In recent years notable advances have been made in the development of improved vaccines to prevent cholera. These new vaccines are administered orally to maximally stimulate intestinal secretory immunity. Killed vibrios, given in conjunction with purified B subunit or administered alone, in three spaced doses, caused no adverse reactions and have conferred significant protection in volunteer challenge studies and in field trials. Two attenuated mutants of V. cholerae, prepared by recombinant DNA techniques, CVD 103 and CVD 103-HgR are well-tolerated and elicit prominent immune responses and protective immunity after ingestion of a single oral dose. Other modern approaches being pursued include the development of auxotrophic strains and of modifying attenuated S. typhi strain Ty21a to express V. cholerae Inaba and Ogawa LPS antigens.

Systemic and intestinal immunities after different typhoid vaccinations.

The comparative studies of systemic and intestinal immunities to S. typhi were performed in 29 healthy volunteers during 2 years after receiving oral vaccination with attenuated S. typhi Ty21a in gelatin capsule, parenteral vaccination with acetone inactivated or heat inactivated-phenol preserved S. typhi Ty2. The methods used were immunobead ELISA for total secretory IgA and indirect ELISA for specific secretory IgA in the intestinal lavage fluid. The specific systemic IgG, IgM and anti-O, anti-H agglutinins were measured by indirect ELISA and Widal test respectively. The leukocyte migration inhibition test was used for the measurement of systemic cell mediated immunity. The results indicate that the oral S. typhi Ty21a stimulated intestinal immunity better than both parenteral vaccines but evoked less systemic antibody response. The stimulation of systemic cell-mediated immunity by the live attenuated and acetone inactivated vaccine was comparable while stimulation by heat inactivated-phenol preserved vaccine was less pronounced. The same studies were performed in 26 healthy volunteers during 6 months following different doses of oral vaccination with S. typhi Ty21a in enteric-coated capsule. The results suggest that the stimulation of intestinal and systemic immunities by this vaccine is dosage dependent. Three doses of vaccine provide better stimulation than two doses and one dose, respectively.

Antibodies against Vibrio cholerae lipopolysaccharide, cell-bound haemagglutinin and toxin in the intestinal fluid during convalescence.

Specific antibodies to V. cholerae lipopolysaccharide (LPS), cell-bound haemagglutinin (CHA) and toxin (CT) in the intestinal lavages of healthy Thais and Thai cholera patients during the convalescence period were determined by enzyme-linked immunosorbent assay. Only IgM and IgA specific antibodies were detectable in the specimens. All of the persons who were just recovered from cholera had IgA anti-CT and IgA anti-LPS and 82.4% had IgA anti-CHA. The IgA anti-CT, anti-LPS and anti-CHA were detected also in the gut fluids of 70.6%, 94.1% and 88.2%, respectively, of the healthy controls. The mean levels of the IgA antibodies of all specificities between the two groups of individuals were not different. However, the IgM anti-CT and IgM anti-LPS of the cholera patients increased during the convalescence period. The levels, therefore, were significantly higher than those of the controls. The ratios of IgA anti-CT: IgM anti-CT and IgA anti-LPS: IgM anti-LPS among the patients were 2.93:1 and 2.02:1, respectively while those of the controls were 10:1 and 34:1, respectively. IgA antibodies predominated in the lavages of both groups of the individuals.