Investigation of growth factors and cytokines that suppress adult stem cell asymmetric cell kinetics

Thesis (S.M.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005.Includes bibliographical references (leaves 40-43).Adult stem cells are potentially useful in many biomedical applications that can save lives and increase the quality of a patient's life, such as tissue engineering, cell replacement, and gene therapy. However, these applications are limited because of the difficulty in isolating and expanding pure populations of adult stem cells (ASCs). A major barrier to ASC expansion in vitro is their property of asymmetric cell kinetics. Our lab has developed a method, Suppression of Asymmetric Cell Kinetics (SACK), to expand ASCs in vitro by shifting their cell kinetics program from asymmetric to symmetric. We have found that guanine nucleotide precursors can be used to convert the kinetics of adult stem cells from asymmetric to symmetric, which promotes their exponential expansion. Previously, we have used the SACK method to derive hepatic and cholangiocyte stem cell strains from adult rat livers in vitro. These cell strains provide an assay to evaluate whether growth factors and cytokines previously implicated in proliferation of progenitor cells act by converting the kinetics of the stem cells in the population from asymmetric to symmetric, and thus identify new SACK agents. We are evaluating three agents, Wnt, IGF- 1, and Sonic hedgehog (Shh).(cont.) Wnt has been found to cause self-renewal and proliferation of hematopoietic stem cells (HSCs) in vitro. IGF- 1 also plays a role in hematopoietic progenitor self-renewal in vivo as well as in tissue maturation. Shh has been implicated in the proliferation of primitive neural cells as well as in cellular proliferation during invertebrate development. Thus far, we have found that Wnt peptide shifts the cell kinetics from asymmetric to symmetric and may reduce the generation time, whereas IGF-1 appears only to affect generation time. Studies involving Shh are currently underway. We are also currently investigating whether Wnt acts additively or synergistically with guanine nucleotide precursors to shift cell kinetic symmetry. Discovering new SACK agents will allow us to obtain purer populations of ASCs that can be used to study properties unique to stem cells. Furthermore, the observation that Wnt shifts the kinetics of adult rat hepatic stem cells from asymmetric to symmetric implicates the involvement of similar cell kinetics symmetry mechanisms in the proliferation effect of Wnt on murine and human HSCs.by Michal Ganz.S.M

Immune and inflammatory pathways in NASH

Immune and inflammatory pathways have a central role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Both the innate and adaptive immune systems contribute to the development of NAFLD. Pathogen-associated molecular patterns and danger-associated molecular patterns are known to activate a variety of pattern-recognition receptors that result in inflammation. The key features of the immune system and inflammatory pathways in the development of NAFLD are discussed in this review

High fat diet feeding results in gender specific steatohepatitis and inflammasome activation

AIM: To develop an animal model that encompasses the different facets of non-alcoholic steatohepatitis (NASH), which has been a challenge. METHODS: In this study, we used a high fat diet (HFD) feeding supplemented with fructose and sucrose in the water mimicking the high-fructose corn syrup that is abundant in the diet in the United States. We used C57Bl/6 wild-type mice for short and long-term feedings of 6 and 16 wk respectively, and evaluated the extent of liver damage, steatosis, and inflammasome activation. Our methods included histopathological analysis to assess liver damage and steatosis, which involved H and E and oil-red-o staining; biochemical studies to look at ALT and triglyceride levels; RNA analysis using quantitative polymerase chain reaction; and cytokine analysis, which included the enzyme-linked immunosorbent assay method to look at interleukin (IL)-1beta and tumor necrosis factor-alpha (TNFalpha) levels. Furthermore, at each length of feeding we also looked at insulin resistance and glucose tolerance using insulin tolerance tests (ITT) and glucose tolerance tests. RESULTS: There was no insulin resistance, steatosis, or inflammasome activation at 6 wk. In contrast, at 16 wk we found significant insulin resistance demonstrated by impaired glucose and ITT in male, but not female mice. In males, elevated alanine aminotransferase and triglyceride levels, indicated liver damage and steatosis, respectively. Increased liver TNFalpha and monocyte chemoattractant protein-1 mRNA and protein, correlated with steatohepatitis. The inflammasome components, adaptor molecule, Aim2, and NOD-like receptor 4, increased at the mRNA level, and functional inflammasome activation was indicated by increased caspase-1 activity and IL-1beta protein levels in male mice fed a long-term HFD. Male mice on HFD had increased alpha-smooth muscle actin and pro-collagen-1 mRNA indicating evolving fibrosis. In contrast, female mice displayed only elevated triglyceride levels, steatosis, and no fibrosis. CONCLUSION: Our data indicate gender differences in NASH. Male mice fed a long-term HFD display steatohepatitis and inflammasome activation, whereas female mice have steatosis without inflammation

Diseño de prácticas pedagógicas para la enseñanza en espacios educativos culturales y lingüísticamente diversos

Israel is a multi-cultural migration country and its education system face the challenges of equality and inclusion. This is comparative qualitative research based on a model that evaluates the development of intercultural competence. The purpose of the study is to examine the perceptions and attitudes of inter-cultural competence development in two groups of students, in which only one of them was involved in extra-curricular learning. The findings show differences between the two groups. Group A, had gained new knowledge in broader cultural contexts and had a deeper insight on creating a pluralistic professional identity, cultural-emotional commitment and strengthens the relationship between teaching and culture than the Group B. Moreover, the students in group A were more practical and dynamic and created a link between the content of the lesson and the children's origin culture. They allow discussion of controversial issues and encourage the children to share personal stories.Israel es un país de migración multicultural y su sistema educativo se enfrenta a los retos de la igualdad y de la inclusión. Esta es una investigación comparativa y cualitativa, basada en un modelo que evalúa el desarrollo de la competencia intercultural. El objeto del estudio es examinar las percepciones y actitudes sobre el desarrollo de la competencia intercultural en dos grupos de futuros maestros, uno de los cuales estaba involucrado en el aprendizaje extracurricular. Los resultados muestran diferencias entre los dos grupos. El Grupo A adquirió nuevos conocimientos sobre contextos culturales amplios y consiguió un conocimiento más profundo que el Grupo B sobre la creación de una identidad profesional pluralista, sobre el compromiso cultural-emocional sobre el vínculo entre enseñanza y cultura. Además, los estudiantes del Grupo A resultaron ser más prácticos y dinámicos, y establecieron un vínculo entre el contenido de la clase y la cultura originaria de los niños. Ello permite la discusión sobre temas controvertidos y alienta a los niños a contar historias personales

Increased oxidative capacity of circulating polymorphonuclear neutrophils (PMNs) in non-diabetic NASH patients

Background: Inflammation and oxidative stress are key factors in the pathogenesis of non-alcoholic steatohepatitis (NASH). Polymorphonuclear neutrophils are capable to produce significant amounts of reactive oxygen species (ROS) via the NADPH oxidase complex. Increased hepatic neutrophil infiltration has been described in steatohepatitis. We aimed to investigate the in vitro ROS generation by neutrophils of NASH patients and the hepatic NADPH oxidase activity in murine steatohepatitis. Material and methods: PMNs were isolated from peripheral blood of NASH patients (n=16) and healthy controls (n=16). In vitro ROS production was measured by luminol chemiluminescence after phorbol myristate acetate (PMA) or opsonized zymosan stimulation. Hepatic lipid peroxidation and NADPH oxidase activation were measured in mice fed with methionine-choline-deficient (MCD) or -supplemented (MCS) diets. Results: PMA activated oxidative burst both in patients and controls. However, ROS production was significantly increased in non-diabetic NASH patients (n=9) compared to controls 30 min after the PMA stimulation. PMNs from NASH patients with diabetes mellitus (n=7) did not have higher ROS production after PMA-stimulation compared to controls. The PMA-induced peak chemiluminescence was significantly higher in the non-diabetic NASH patients compared to controls and diabetic NASH patients. No significant difference was observed without any stimulation and in opsonized zymosan induced chemiluminescence. Consistent with the increased oxidative capacity of PMNs in NASH patients, we found increased hepatic lipid peroxidation, higher expression and activation of the NADPH oxidase complex in MCD-steatohepatitis. Conclusion: Our finding supports the role of neutrophil oxidative stress in NASH. Our novel data suggests that the increased oxidative capacity of the PMNs it is not only localized to the liver but can have systemic effects and serve as a potential biomarker of NASH

Progression of non-alcoholic steatosis to steatohepatitis and fibrosis parallels cumulative accumulation of danger signals that promote inflammation and liver tumors in a high fat-cholesterol-sugar diet model in mice

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is becoming a pandemic. While multiple \u27hits\u27 have been reported to contribute to NAFLD progression to non-alcoholic steatohepatitis (NASH), fibrosis and liver cancer, understanding the natural history of the specific molecular signals leading to hepatocyte damage, inflammation and fibrosis, is hampered by the lack of suitable animal models that reproduce disease progression in humans. The purpose of this study was first, to develop a mouse model that closely mimics progressive NAFLD covering the spectrum of immune, metabolic and histopathologic abnormalities present in human disease; and second, to characterize the temporal relationship between sterile/exogenous danger signals, inflammation, inflammasome activation and NAFLD progression. METHODS: Male C57Bl/6 mice were fed a high fat diet with high cholesterol and a high sugar supplement (HF-HC-HSD) for 8, 27, and 49 weeks and the extent of steatosis, liver inflammation, fibrosis and tumor development were evaluated at each time point. RESULTS: The HF-HC-HSD resulted in liver steatosis at 8 weeks, progressing to steatohepatitis and early fibrosis at 27 weeks, and steatohepatitis, fibrosis, and tumor development at 49 weeks compared to chow diet. Steatohepatitis was characterized by increased levels of MCP-1, TNFalpha, IL-1beta and increased liver NASH histological score. We found increased serum levels of sterile danger signals, uric acid and HMGB1, as early as 8 weeks, while endotoxin and ATP levels increased only after 49 weeks. Increased levels of these sterile and microbial danger signals paralleled upregulation and activation of the multiprotein complex inflammasome. At 27, 49 weeks of HF-HC-HSD, activation of M1 macrophages and loss of M2 macrophages as well as liver fibrosis were present. Finally, similar to human NASH, liver tumors occurred in 41% of mice in the absence of cirrhosis and livers expressed increased p53 and detectable AFP. CONCLUSIONS: HF-HC-HSD over 49 weeks induces the full spectrum of liver pathophysiologic changes that characterizes the progression of NAFLD in humans. NAFLD progression to NASH, fibrosis and liver tumor follows progressive accumulation of sterile and microbial danger signals, inflammasome activation, altered M1/M2 cell ratios that likely contribute to NASH progression and hepatic tumor formation

Caring international research collaborative: A five-country partnership to measure perception of nursing staffs' compassion fatigue, burnout, and caring for self

Partnering in research across disciplines and across countries can be challenging due to differing contexts of practice and culture. This study sought to demonstrate how central constructs that have application across disciplines and countries can be studied while concurrently considering context. Groups of nurses from Botswana, Ireland, Israel, New Zealand, and Spain partnered to identify how to measure the constructs of caring for self, burnout, and compassion fatigue, replicating a study by Johnson (2012), who found that caring for self had a moderately strong negative relationship with both compassion fatigue and burnout. While these constructs were of interest to all five groups, the conversation of contextual influences varied. All five groups used the same instruments to measure the central constructs. Levels of burnout and compassion fatigue varied by country but were moderated by caring for self. Partnering across countries made it possible to understand that caring for self moderates the negative impact of burnout and compassion fatigue in all five countries. This study gives insight into methods for partnering across disciplines and contexts

Common Breast Cancer Susceptibility Alleles and the Risk of Breast Cancer for BRCA1 and BRCA2 Mutation Carriers: Implications for Risk Prediction

The known breast cancer (BC) susceptibility polymorphisms in FGFR2, TNRC9/TOX3, MAP3K1,LSP1 and 2q35 confer increased risks of BC for BRCA1 or BRCA2 mutation carriers. We evaluated the associations of three additional SNPs, rs4973768 in SLC4A7/NEK10, rs6504950 in STXBP4/COX11 and rs10941679 at 5p12 and reanalyzed the previous associations using additional carriers in a sample of 12,525 BRCA1 and 7,409 BRCA2 carriers. Additionally, we investigated potential interactions between SNPs and assessed the implications for risk prediction. The minor alleles of rs4973768 and rs10941679 were associated with increased BC risk for BRCA2 carriers (per-allele Hazard Ratio (HR)=1.10, 95%CI:1.03-1.18, p=0.006 and HR=1.09, 95%CI:1.01-1.19, p=0.03, respectively). Neither SNP was associated with BC risk for BRCA1 carriers and rs6504950 was not associated with BC for either BRCA1 or BRCA2 carriers. Of the nine polymorphisms investigated, seven were associated with BC for BRCA2 carriers (FGFR2, TOX3, MAP3K1, LSP1, 2q35, SLC4A7, 5p12, p-values:7×10−11-0.03), but only TOX3 and 2q35 were associated with the risk for BRCA1 carriers (p=0.0049, 0.03 respectively). All risk associated polymorphisms appear to interact multiplicatively on BC risk for mutation carriers. Based on the joint genotype distribution of the seven risk associated SNPs in BRCA2 mutation carriers, the 5% of BRCA2 carriers at highest risk (i.e. between 95th and 100th percentiles) were predicted to have a probability between 80% and 96% of developing BC by age 80, compared with 42-50% for the 5% of carriers at lowest risk. Our findings indicated that these risk differences may be sufficient to influence the clinical management of mutation carriers

Designing pedagogical practices for teaching in educational spaces culturally and linguistically diverse

Israel is a multi-cultural migration country and its education system face the challenges of equality and inclusion. This is comparative qualitative research based on a model that evaluates the development of intercultural competence. The purpose of the study is to examine the perceptions and attitudes of inter-cultural competence development in two groups of students, in which only one of them was involved in extra-curricular learning. The findings show differences between the two groups. Group A, had gained new knowledge in broader cultural contexts and had a deeper insight on creating a pluralistic professional identity, cultural-emotional commitment and strengthens the relationship between teaching and culture than the Group B. Moreover, the students in group A were more practical and dynamic and created a link between the content of the lesson and the children's origin culture. They allow discussion of controversial issues and encourage the children to share personal stories.Israel es un país de migración multicultural y su sistema educativo se enfrenta a los retos de la igualdad y de la inclusión. Esta es una investigación comparativa y cualitativa, basada en un modelo que evalúa el desarrollo de la competencia intercultural. El objeto del estudio es examinar las percepciones y actitudes sobre el desarrollo de la competencia intercultural en dos grupos de futuros maestros, uno de los cuales estaba involucrado en el aprendizaje extracurricular. Los resultados muestran diferencias entre los dos grupos. El Grupo A adquirió nuevos conocimientos sobre contextos culturales amplios y consiguió un conocimiento más profundo que el Grupo B sobre la creación de una identidad profesional pluralista, sobre el compromiso cultural-emocional sobre el vínculo entre enseñanza y cultura. Además, los estudiantes del Grupo A resultaron ser más prácticos y dinámicos, y establecieron un vínculo entre el contenido de la clase y la cultura originaria de los niños. Ello permite la discusión sobre temas controvertidos y alienta a los niños a contar historias personales