Warren Antonio Vieira

Activated intestinal macrophages in patients with cirrhosis release NO and IL- 6 that may disrupt intestinal barrier function

BACKGROUND & AIMS Bacterial infections commonly occur in decompensated cirrhosis resulting from bacterial translocation from the intestine. We studied the role of intestinal macrophages and the epithelial barrier in cirrhosis. METHODS Forty-four patients with NASH/ASH cirrhosis (decompensated n=29, compensated n=15) and nineteen controls undergoing endoscopy were recruited. Serum was obtained and LPS and LBP levels determined. Intestinal macrophages were characterized by flow cytometry, immunohistochemistry, and nitric oxide (NO) production measured in supernatant of cultured duodenal samples. Quantitative RT-PCR was performed on duodenal biopsies assessing 84 inflammatory genes. Protein levels of cytokines/chemokines were assessed in serum and supernatant. The duodenal wall was assessed by electron microscopy, tight junction protein expression determined by RT-PCR, immunohistochemistry, and Western blot and, functional analysis performed by transepithelial resistance measurement and permeability studies. RESULTS Increased plasma LPS, LBP levels and higher numbers of duodenal CD33(+)/CD14(+)/Trem-1(+) macrophages, synthesizing iNOS and secreting NO were present in decompensated cirrhosis. Upregulation of IL-8, CCL2, CCL13 at the transcriptional level, and increased IL-8, and IL-6 were detected in supernatant and serum in cirrhosis. IL-6 and IL-8 co-localised with iNOS(+) and CD68(+), but not with CD11c(+) cells. Electron microscopy demonstrated an intact epithelial barrier. Increased Claudin-2 was detected by Western blot and immunohistochemistry, while decreased transepithelial resistance and increased duodenal permeability were detected in decompensated cirrhosis. CONCLUSIONS Our study shows the presence of activated CD14(+)Trem-1(+)iNOS(+) intestinal macrophages, releasing IL-6, NO, and increased intestinal permeability in patients with cirrhosis, suggesting that these cells may produce factors capable of enhancing permeability to bacterial products.

Sulphamoylated 2-Methoxyestradiol Analogues Induce Apoptosis in Adenocarcinoma Cell Lines

2-Methoxyestradiol (2ME2) is a naturally occurring estradiol metabolite which possesses antiproliferative, antiangiogenic and antitumor properties. However, due to its limited biological accessibility, synthetic analogues have been synthesized and tested in attempt to develop drugs with improved oral bioavailability and efficacy. The aim of this study was to evaluate the antiproliferative effects of three novel in silico-designed sulphamoylated 2ME2 analogues on the HeLa cervical adenocarcinoma cell line and estrogen receptor-negative breast adenocarcinoma MDA-MB-231 cells. A dose-dependent study (0.1–25 μM) was conducted with an exposure time of 24 hours. Results obtained from crystal violet staining indicated that 0.5 μM of all 3 compounds reduced the number of cells to 50%. Lactate dehydrogenase assay was used to assess cytotoxicity, while the mitotracker mitochondrial assay and caspase-6 and -8 activity assays were used to investigate the possible occurrence of apoptosis. Tubulin polymerization assays were conducted to evaluate the influence of these sulphamoylated 2ME2 analogues on tubulin dynamics. Double immunofluorescence microscopy using labeled antibodies specific to tyrosinate and detyrosinated tubulin was conducted to assess the effect of the 2ME2 analogues on tubulin dynamics. An insignificant increase in the level of lactate dehydrogenase release was observed in the compounds-treated cells. These sulphamoylated compounds caused a reduction in mitochondrial membrane potential, cytochrome c release and caspase 3 activation indicating apoptosis induction by means of the intrinsic pathway in HeLa and MDA-MB-231 cells. Microtubule depolymerization was observed after exposure to these three sulphamoylated analogues.

The impact of asthma on the gastrointestinal tract (GIT).

The gastrointestinal tract (GIT) of vertebrates is composed of several distinct compartments and glands as well as an extensive mucosal surface. Its primary function is that of chemical and physical digestion of food and the absorption of nutrients; however, due to its continual antigen exposure, the GIT also has an important defensive immunological function. The GIT’s immunological participation is facilitated by the mucosa-associated lymphoid tissues, thought to share the mucosal immunological system with the respiratory mucosa-associated lymphoid tissues. As a result of this shared mucosal immunity, it has been hypothesized that bronchial asthma may be able to affect the body’s GIT in the same pathophysiological manner as the airways and lungs. Here we discuss the link between bronchial asthma and pathophysiological features in the GIT – including leukocyte influx, goblet cell alterations, fibrosis, and epithelial and villous atrophy.

Histological assessment of SJL/J mice treated with the antioxidants coenzyme Q10 and resveratrol.

The muscular dystrophies (MDs) are genetic disorders of muscle degeneration due to mutations in genes that encode a wide variety of proteins. Dysferlinopathy are characterized by the absence of dysferlin in skeletal muscle and an autosomal recessive mode of inheritance. Both histological and ultrastructural pathology have been well established in dysferlinopathy patients and dysferlin-deficient animal models. To our knowledge the effect of antioxidant supplementation on this level has not been described previously. This article therefore focuses on the histopathology to reveal the effect of antioxidant supplementation. The study aimed to determine, at cellular level, the histopathological changes in the SJL/J mouse model following a 90 day trial with antioxidant supplementation. Markedly reduced inflammatory insult in the more affected quadriceps muscles of animals treated with high doses of CoQ10 and a combination of resveratrol/CoQ10 were observed. The outcome provides evidence that high doses of antioxidant supplementation resulted in decreased dystrophic markers and enhanced tissue integrity at cellular level.

Comparative scanning electron microscopy of platelets and fibrin networks of human and differents animals

El proposito del presente estudio fue comparar la ultraestructura de plaquetas y las redes de fibrina de los seres humanos y de ocho diferentes especies de animales, con el fin de determinar las diferencias morfologicas de estas estructuras y si las diferencias pueden ser observadas por microscopia electronica de barrido. Las plaquetas y las redes de fibrina desempenan un papel importante tanto en el proceso de coagulacion como, fisiologicamente en procesos alergicos y mecanismos inmunologicos. Elgrosor de las redes de fibrina humana fue comparado con las del raton (Mus musculus), equino (Equus caballus), mono vervet (Chlorocebus aethiops, anteriormente Cercopithecus aethiops, antilope Africano (Oryx gazella), ovino (Ovis aries), pinguino (Spheniscus demersus), conejo (Oryctolagus cuniculus) y tortuga marina (Caretta caretta). Las fibras fueron medidas y agrupadas en fibras delgadas (menor), fibras intermedias y fibras gruesas (grandes). Los resultados obtenidos indicaron que para cada una de las tres clases de fibrina, los rangos de su tamano en el mono, antilope africano y en equino no fueron significativamente diferentes entre si, mientras que en humano, pinguino, antilope africano y ovino no fueron significativamente diferentes entre estos. De estos resultados se pudo concluir que mamiferos y aves poseen una distribucion tri-modal de fibras de fibrina, distinta a la de las especies de reptiles estudiadas, donde la tortuga de mar posee una distribucion bimodal de fibras de fibrina. Se puede sugerir que la utilizacion de los modelos mamiferos y aviar, en terminos de patrones de distribucion de fibras de fibrina, pueden ser una alternativa adecuada para los estudios ultraestructurales.

Investigating the effect of the homeopathic immunomodulator, MODUL8® on blood count, bronchial lavage and fibrin ultrastructure on experimental asthmatic BALB/c mice

Modul8® es una mezcla compuesta de productos naturales que es conocida por ser un inmunomodulador. En el presente estudio, el efecto de este inmunomodulador se prueba de forma experimental en el modelo de raton asmaticos BALB/c, para investigar sus propiedades sobre el conteo de globulos blancos en la sangre y lavado bronquial de los animales, ya que los globulos blancos desempenan un papel fundamental en el proceso de respuesta inflamatoria implicado en el asma. Como es sabido, tambien las plaquetas desempenan un papel importante en el sistema inmunologico, asi, la ultraestructura de las plaquetas y las redes de fibrina tambien fueron investigadas por microscopia electronica de barrido. Los animales fueron sensibilizados, nebulizados y tratados durante un periodo de 43 dias hasta el termino. Los resultados de los frotis de sangre, asi como los de lavado bronquial revelaron un numero significativamente mayor de eosinofilos en el grupo de asmaticos en comparacion con el control y grupos tratados. Cambios en la ultraestructura de las plaquetas y redes de fibrina tambien pueden ser observados, donde el grupo tratado con la Modul8® aparece similar a el grupo control, donde los fibras de mayor grosor y menor grosor pueden ser claramente distinguidas y ademas, puede ser observada una apretada masa de plaquetas aglutinadas. Considerando las redes de la fibrina en animales asmaticos parecen endebles con una apretada masa de fibras de menor grosor que cubren las fibras de mayor grosor. Los agregados de plaquetas en asmaticos aparecen granulares sin el aspecto apretado del agregado plaquetario que rodea al grupo control. Por tanto, se concluye que Modul8® positivamente influye en el conteo de globulos blancos mediante la alteracion del perfil de asmaticos a un aspecto similar al del control. Ademas, parece como si Modul8® tuviera un efecto estabilizador en las plaquetas y las redes de fibrina. De estos resultados se puede sugerir que Modul8® puede ser utilizado con exito en el tratamiento de procesos inflamatorios como el asma.

Ultrastructure of platelets and fibrin networks of asthmatic mice exposed to selenium and Withania somnifera

Platelets and fibrin play an important role in allergic processes, including allergic asthma. The asthmatic BALB/c mouse model was used to induce asthma, and asthmatic mice were treated with the anti-inflammatory plant Withania somnifera, separately and in combination with the antioxidant selenium. Selenium is an important supplement in asthma, because asthmatics may have a selenium deficiency. Hydrocortisone was used as positive control. Results indicate control mice possess major thick fibers, minor thin fibers, and tight round activated platelets with typical pseudopodia formation. Minor fibers of asthmatic mice have a netlike appearance covering the major fibers whereas the platelets form loosely connected, granular aggregates. Hydrocortisone made the fibrin more fragile and platelet morphology changes from a tight activated platelet to a more granular activated platelet, not closely fused to each other. The plant extracts, separately and in combination with selenium did not affect the fragility of the fibrin and reversed the formation of the dense minor netlike layer over the major fibers, and the platelets formed a dense aggregate. Asthmatic mice treated with selenium showed a dense minor fibrin layer; however, the platelets formed a dense aggregate. We conclude that the anti-inflammatory products affect the stability of fibrin networks but not platelet stability (seen with hydrocortisone). Selenium does not affect the stability of the fibrin networks, but affects platelet stability. These results suggests that asthmatic patients should indeed use an antioxidant supplement, e.g. selenium, because it stabilizes activated platelets, together with anti-inflammatory products.

A new and improved algorithm for the quantification of chromatin condensation from microscopic data shows decreased chromatin condensation in regenerating axolotl limb cells

The nuclear landscape plays an important role in the regulation of tissue and positional specific genes in embryonic and developing cells. Changes in this landscape can be dynamic, and are associated with the differentiation of cells during embryogenesis, and the de-differentiation of cells during induced pluripotent stem cell (iPSC) formation and in many cancers. However, tools to quantitatively characterize these changes are limited, especially in the in vivo context, where numerous tissue types are present and cells are arranged in multiple layers. Previous tools have been optimized for the monolayer nature of cultured cells. Therefore, we present a new algorithm to quantify the condensation of chromatin in two in vivo systems. We first developed this algorithm to quantify changes in chromatin compaction and validated it in differentiating spermatids in zebrafish testes. Our algorithm successfully detected the typical increase in chromatin compaction as these cells differentiate. We then employed the algorithm to quantify the changes that occur in amphibian limb cells as they participate in a regenerative response. We observed that the chromatin in the limb cells de-compacts as they contribute to the regenerating organ. We present this new tool as an open sourced software that can be readily accessed and optimized to quantify chromatin compaction in complex multi-layered samples.

The Morphological Effects of Asthma, As Well As Conventional and Alternative Asthma Therapies on Parietal and Chief Cells in the Stomach of BALB/c Mice

La literatura cientifica, aunque limitada en esta area, apoya la hipotesis de que el asma, por medio del trafico selectivo de leucocitos entre los diferentes sitios y la mucosa glandular del cuerpo, puede tener los mismos efectos fisiopatologicos en el estomago y las vias respiratorias. Este estudio tuvo como objetivo determinar si el asma, en ausencia y presencia de diversos tratamientos para el asma (hidrocortisona y Modul8 TM), genero alguna alteracion morfologica en las celuals parietales y principales del estomago. El modelo murino BALB/c del raton asmatico fue el modelo de eleccion en este estudio. El protocolo de induccion de asma, asi como el tratamiento del asma demostro ser eficaz con la ayuda de lavado bronquial y cuantificacion leucocitaria del fluido. Biopsias de las regiones fundica y pilorica fueron extraidas y evaluadas por medio de microscopia electronica de transmision, de barrido y de luz. Las biopsias extraidas de la region fundica y pilorica revelaron que el asma solamente induce hipertrofia de las celulas parietales (aumento del tamano de las celulas parietales P <0,00001 en ambas regiones del estomago) e hiperfuncionamiento de las celulas principales. El uso de hidrocortisona y Modul8 TM, como una terapia para corregir las alteraciones gastricas fue disimil, solo en el caso de las celulas parietales fundicas ambos tratamientos fueron capaces de compensar el efecto hipertrofico causado por el asma, mientras que en la celula parietal pilorica la hipertrofia inducida por el asma solamente se vio compensada por Modul8TM.