Dysgenesis of enteroendocrine cells in Aristaless-Related Homeobox polyalanine expansion mutations.

OBJECTIVES: Severe congenital diarrhea occurs in approximately half of patients with Aristaless-Related Homeobox (ARX) null mutations. The cause of this diarrhea is unknown. In a mouse model of intestinal Arx deficiency, the prevalence of a subset of enteroendocrine cells is altered, leading to diarrhea. Because polyalanine expansions within the ARX protein are the most common mutations found in ARX-related disorders, we sought to characterize the enteroendocrine population in human tissue of an ARX mutation and in a mouse model of the corresponding polyalanine expansion (Arx). METHODS: Immunohistochemistry and quantitative real-time polymerase chain reaction were the primary modalities used to characterize the enteroendocrine populations. Daily weights were determined for the growth curves, and Oil-Red-O staining on stool and tissue identified neutral fats. RESULTS: An expansion of 7 alanines in the first polyalanine tract of both human ARX and mouse Arx altered enteroendocrine differentiation. In human tissue, cholecystokinin, glucagon-like peptide 1, and somatostatin populations were reduced, whereas the chromogranin A population was unchanged. In the mouse model, cholecystokinin and glucagon-like peptide 1 populations were also lost, although the somatostatin-expressing population was increased. The ARX protein was present in human tissue, whereas the Arx protein was degraded in the mouse intestine. CONCLUSIONS: ARX/Arx is required for the specification of a subset of enteroendocrine cells in both humans and mice. Owing to protein degradation, the Arx mouse recapitulates findings of the intestinal Arx null model, but is not able to further the study of the differential effects of the ARX protein on its transcriptional targets in the intestine.

Imunolocalização de enteroglucagon em células endócrinas presentes no estômago do muçuã Kinosternon scorpioides (Reptilia, Chelonia, Kinosternidae) / Immunolocalization of enteroglucagon in endocrine cells in the stomach of scorpion mud turtle Kinosternon scorpioides (Reptilia, Chelonia, Kinosternidae)

The scorpion mud turtle (Kinosternon scorpioides) is a small chelonian, typical of the floodplains of the Amazon. This species is an important source of food and income and it has being studied because of its vulnerability to indiscriminate hunting, deforestation and burning; for this reason, it has been researched in order to supply data for the preservation of the species. The immunohistochemistry activity of endocrine cells present in the stomach of scorpion mud turtle is not totally known. Therefore, the aim of this work was to identify the presence of enteroglucagon hormone and to classify the endocrine cells in the stomach of the scorpion mud turtle. The fragments were submitted to Hematoxylin-Eosin technique and streptavidin peroxidase for staining and detection of antigen, respectively. Immunoreactivity to enteroglucagon in cells was found on the three portions of the stomach (gastric, fundica and pyloric); however, the immunoreactivity was more evident in the first two regions than in the last one. Endocrine cells of stomach were classified as argyrophil and argentaffin and were found among cells that comprise the gastric glands and were classified as open type and closed type. There is no difference between the biochemistry and immunohistochemistry of enteroglucagon K. scorpioides and other animal species.

O muçuã ou jurará (Kinosternon scorpioides) é um quelônio de pequeno porte de água doce, nativo das varzéas da região amazônica. Essa espécie é importante fonte de alimentação e renda e vem sendo estudada por apresentar vulnerabilidade em consequência da caça indiscriminada, queimadas e desmatamento e, por esta razão, tem sido fonte de pesquisa para o fornecimento de dados para a preservação da espécie. Ainda não é totalmente compreendida a atividade imunohistoquímica das células endócrinas presentes no estômago do muçuã. Sendo assim, este trabalho tem por objetivo identificar a presença do hormônio enteroglucagon e classificar as células endócrinas do estômago de muçuã. Os fragmentos de estômago foram submetidos à técnica de Hematoxilina-Eosina e da Estreptovidina peroxidase para coloração e detecção do antígeno, respectivamente. Foram encontradas células imunorreativas ao enteroglucagon em uma das três porções gástricas (cárdica, fúndica e pilórica); contudo, nas duas primeiras regiões, a imunoreatividade foi mais evidente do que na última. As células endócrinas do estômago de muçuã foram classificadas em argirófilas e argentafins e encontradas entre as células que compõem as glândulas gástricas, sendo classificadas como do tipo aberta e do tipo fechada. Não houve diferença bioquímica e imunohistoquímica entre o enteroglucagon do K. scorpioides com as de outras espécies animais.

Imunolocalização de enteroglucagon em células endócrinas presentes no estômago do muçuã Kinosternon scorpioides (Reptilia, Chelonia, Kinosternidae) / Immunolocalization of enteroglucagon in endocrine cells in the stomach of scorpion mud turtle Kinosternon scorpioides (Reptilia, Chelonia, Kinosternidae)

The scorpion mud turtle (Kinosternon scorpioides) is a small chelonian, typical of the floodplains of the Amazon. This species is an important source of food and income and it has being studied because of its vulnerability to indiscriminate hunting, deforestation and burning; for this reason, it has been researched in order to supply data for the preservation of the species. The immunohistochemistry activity of endocrine cells present in the stomach of scorpion mud turtle is not totally known. Therefore, the aim of this work was to identify the presence of enteroglucagon hormone and to classify the endocrine cells in the stomach of the scorpion mud turtle. The fragments were submitted to Hematoxylin-Eosin technique and streptavidin peroxidase for staining and detection of antigen, respectively. Immunoreactivity to enteroglucagon in cells was found on the three portions of the stomach (gastric, fundica and pyloric); however, the immunoreactivity was more evident in the first two regions than in the last one. Endocrine cells of stomach were classified as argyrophil and argentaffin and were found among cells that comprise the gastric glands and were classified as open type and closed type. There is no difference between the biochemistry and immunohistochemistry of enteroglucagon K. scorpioides and other animal species.(AU)

O muçuã ou jurará (Kinosternon scorpioides) é um quelônio de pequeno porte de água doce, nativo das varzéas da região amazônica. Essa espécie é importante fonte de alimentação e renda e vem sendo estudada por apresentar vulnerabilidade em consequência da caça indiscriminada, queimadas e desmatamento e, por esta razão, tem sido fonte de pesquisa para o fornecimento de dados para a preservação da espécie. Ainda não é totalmente compreendida a atividade imunohistoquímica das células endócrinas presentes no estômago do muçuã. Sendo assim, este trabalho tem por objetivo identificar a presença do hormônio enteroglucagon e classificar as células endócrinas do estômago de muçuã. Os fragmentos de estômago foram submetidos à técnica de Hematoxilina-Eosina e da Estreptovidina peroxidase para coloração e detecção do antígeno, respectivamente. Foram encontradas células imunorreativas ao enteroglucagon em uma das três porções gástricas (cárdica, fúndica e pilórica); contudo, nas duas primeiras regiões, a imunoreatividade foi mais evidente do que na última. As células endócrinas do estômago de muçuã foram classificadas em argirófilas e argentafins e encontradas entre as células que compõem as glândulas gástricas, sendo classificadas como do tipo aberta e do tipo fechada. Não houve diferença bioquímica e imunohistoquímica entre o enteroglucagon do K. scorpioides com as de outras espécies animais.(AU)

Descrubrimientos sobre reprogramación nuclear merecen el premio Nobel en 2012 / Discoveries on nuclear reprogramming deserves the Nobel prize in 2012

Este artículo fue escrito para honrar a J.B Gurdon y S. Yamanaka, laureados con el Premio Nobel en Fisiología p Medicina 2012 "por el descubrimiento de que las células maduras pueden ser reprogramadas para volverse pluripotentes". Se presentan en forma concisa sus aportes científicos y reseñas biográficas. J.B. Gardon, en Inglaterra, demostró hace 50 años en anfibios que al trasplantar el núcleo de una célula intestinal a un huevo u ovocito enuncleado se obtiene una célula totipotente que se convierte en un embrión y se desarrolla hasta convertirse en una rana adulta, lo cual implica la conservación de genoma en el proceso de diferenciación y la resersibilidad de dicho proceso. Estos descubrimientos llevaron a que otros autores realizaran la clonación de mamiferos utilizando el núcleo de células somáticas y la obtención de células madre pluripotentes a partir de los embrines que se producen in vitro por el desarrollo de las células totipotentes. Se mencionan varias aplicaciones y las contribuciones de Gurdon para comprender el proceso de reprogramación. S. Yamanaka, en Japón, hace seis años, reprogramó al estado embrionario fibroblastos cutáneos de ratones y humanos adultos insertando mediante vectores retrovirales una combinación de los genes de cuatro factores de transcripción: Oct3/4, Sox2, Klf4 y c-Myc. Las células reprogramadas fueron denominadas células madre pluripotentes inducidas. Utilizando la técnica desarrollada por Yamanaka y otras surgidas a raiz de sus descrubrimientos, miles de personas obtienen ahora células madre pluripotentes inducidas a partir de muchas especies y tejidos, incluyendo seres humanos sanos y enfermos. Las células madre pluripotentes o sus derivadas tienen un amplio potencial de aplicación, entre ellas, estudios de embriología y fisiopatología, modelos de enfermedades, descubrimiento de drogas y terapias celulares

This paper was written to honor J.B Gurdon y S. Yamanaka, 2012 Nobel Prize laureates in Physiology or Medicine for "the discovery that mature cells can be reprogrammed to become pluripotent". Their main scientific contributions and biography are presented in a concise manner. JB Gurdon, in England, showed fifty years ago in amphibians that the transplantation of the nucleus of an intestinal cell to an enucleated egg or oocyte produces a totipotent cell that develops into an embryo and adult frog. This implies that cellular differentiation is reversible and the genome is conserved in that process. The discoveries led to the cloning of mammals by other authors using the nucleus of somatic cells and to obtain pluripotent stem cells in vitro from the embryos produced by development of the totipotent cells. Some applications are considered. Gurdon's contribution to the understanding of the reprogramming process is mentioned. S. Yamanaka six years ago in Japan reprogrammed skin fibroblastis from adult mice and humans to the embryonic state by introducing via retroviral vectors a combination of the genes of 4 transcription factors, Oct3/4. Sox2, Klf4 and c-Myc. The reprogammed cells were named induced pluripontent stem cells. Throusands of people are now producing induced pluripotent stem cells from many tissues and species, including healthy and ill humans, using Yamanaka's methods and other techniques stimulated by his work. Pluripotent stem cells or their derivatives have great potential for a wide range of applications including research in embryology and pathophysiology, disease modeling, drug discovery and cell transplantation therapies

Insulin is secreted upon glucose stimulation by both gastrointestinal enteroendocrine K-cells and L-cells engineered with the preproinsulin gene.

Transgenic mice carrying the human insulin gene driven by the K-cell glucose-dependent insulinotropic peptide (GIP) promoter secrete insulin and display normal glucose tolerance tests after their pancreatic p-cells have been destroyed. Establishing the existence of other types of cells that can process and secrete transgenic insulin would help the development of new gene therapy strategies to treat patients with diabetes mellitus. It is noted that in addition to GIP secreting K-cells, the glucagon-like peptide 1 (GLP-1) generating L-cells share/ many similarities to pancreatic p-cells, including the peptidases required for proinsulin processing, hormone storage and a glucose-stimulated hormone secretion mechanism. In the present study, we demonstrate that not only K-cells, but also L-cells engineered with the human preproinsulin gene are able to synthesize, store and, upon glucose stimulation, release mature insulin. When the mouse enteroendocrine STC-1 cell line was transfected with the human preproinsulin gene, driven either by the K-cell specific GIP promoter or by the constitutive cytomegalovirus (CMV) promoter, human insulin co-localizes in vesicles that contain GIP (GIP or CMV promoter) or GLP-1 (CMV promoter). Exposure to glucose of engineered STC-1 cells led to a marked insulin secretion, which was 7-fold greater when the insulin gene was driven by the CMV promoter (expressed both in K-cells and L-cells) than when it was driven by the GIP promoter (expressed only in K-cells). Thus, besides pancreatic p-cells, both gastrointestinal enteroendocrine K-cells and L-cells can be selected as the target cell in a gene therapy strategy to treat patients with type 1 diabetes mellitus.

Insulin is secreted upon glucose stimulation by both gastrointestinal enteroendocrine K-cells and L-cells engineered with the preproinsulin gene

Transgenic mice carrying the human insulin gene driven by the K-cell glucose-dependent insulinotropic peptide (GIP) promoter secrete insulin and display normal glucose tolerance tests after their pancreatic p-cells have been destroyed. Establishing the existence of other types of cells that can process and secrete transgenic insulin would help the development of new gene therapy strategies to treat patients with diabetes mellitus. It is noted that in addition to GIP secreting K-cells, the glucagon-like peptide 1 (GLP-1) generating L-cells share/ many similarities to pancreatic p-cells, including the peptidases required for proinsulin processing, hormone storage and a glucose-stimulated hormone secretion mechanism. In the present study, we demonstrate that not only K-cells, but also L-cells engineered with the human preproinsulin gene are able to synthesize, store and, upon glucose stimulation, release mature insulin. When the mouse enteroendocrine STC-1 cell line was transfected with the human preproinsulin gene, driven either by the K-cell specific GIP promoter or by the constitutive cytomegalovirus (CMV) promoter, human insulin co-localizes in vesicles that contain GIP (GIP or CMV promoter) or GLP-1 (CMV promoter). Exposure to glucose of engineered STC-1 cells led to a marked insulin secretion, which was 7-fold greater when the insulin gene was driven by the CMV promoter (expressed both in K-cells and L-cells) than when it was driven by the GIP promoter (expressed only in K-cells). Thus, besides pancreatic p-cells, both gastrointestinal enteroendocrine K-cells and L-cells can be selected as the target cell in a gene therapy strategy to treat patients with type 1 diabetes mellitus.

Esquistossomose murina esperimental: estudo imunohistoquímicos das células ganglionares mioentéricas / Experimental murine Schistosomiasis: an immunohistochemical study of the ganglionic myoenteric cells

Para estudar a influência do processo granulomatoso esquistossomóticosobre as células ganglionares mioentéricas, foram utilizados 30 camundongos albinos Swiss infectadoscom 50 cercárias da cepa SLM do S. mansoni.O grupo controle foi constituído por dez animais näo infectados. Após sessenta dias de infecçäo, cortes histológicos do intestino delgadocorados por hematoxilina-eosina e P.A.S. demonstraram granulomas periovulares em todas as camadas da parede intestinal. Através do método imunohistoquímico indireto, usando-se a enolase neurônio-específica como marcador, observou-se desorganizaçäo do plexo mioentêrico em áreas contendo granulomas. Além disso, ocorreu rarefaçäo das estaçöes ganglionares, com aparente destruiçäo de células neuronais. A possível contribuiçäo dessas alteraçöes para a sintomatologia da esquistossomose humana é avaliada