Die Rolle des wnt-5a in der Determinierung humaner mesenchymaler Stammzellen zu Preadipozyten

In der Pathogenese der Adipositas und des Diabetes mellitus Typ 2 spielt neben der Vergrößerung bestehender Adipozyten auch die Generierung neuer Fettzellen aus undifferenzierten mesenchymalen Vorläuferzellen eine wichtige Rolle. Dieser Prozess, auch Adipogenese genannt, besteht aus zwei Teilschritten, der Determinierung mesenchymaler Stammzellen zu Preadipozyten und der Differenzierung von Preadipozyten zu reifen Adipozyten. Ziel der vorliegenden Arbeit war es zu untersuchen, ob das Signalmolekül wnt-5a eine Rolle in der molekularen Regulation der Determinierung spielt. Dazu wurden humane mesenchymale Stammzellen aus Nabelschnurblutproben und humane Preadipozyten aus Fettgewebebiopsien isoliert. Diese beiden primären Zellpopulationen wiesen eine vergleichbare Morphologie sowie eine übereinstimmende Expression mesenchymaler Zelloberfächenmarker (CD29, CD44, CD73) auf. Allerdings zeigten sich deutliche Unterschiede im Entwicklungspotenzial. Während mesenchymale Stammzellen sowohl zu Adipozyten als auch zu Osteozyten differenziert werden konnten, waren Preadipozyten nur in der Lage sich zu Adipozyten zu entwickeln. Mittels Expressionsanalysen wurde nachgewiesen, dass multipotente Stammzellen signifikant mehr wnt-5a exprimieren als Preadipozyten, was nahe legt, dass dieses Molekül eine wichtige Rolle in der Determinierung spielt. In der Tat führte die Behandlung humaner mesenchymaler Stammzellen mit neutralisierenden anti-wnt-5a Antikörpern zu einer verminderten Osteogenese und einer gesteigerten Adipogenese. Neben diesen loss of function Experimenten zeigten gain of function Untersuchungen mit rekombinatem wnt-5a gegenteilige Effekte hinsichtlich Osteo- und Adipogenese, was die funktionelle Bedeutung dieses Signalmoleküls unterstreicht. Auf molekularer Ebene wurde nachgewiesen, dass wnt-5a den nicht-kanonischen JNK Signalweg in humanen mesenchymalen Stammzellen aktiviert. Die JNK vermittelte wnt-5a Signaltransduktion führte zu einer Aktivierung des Osteopontinpromotors und zu einer gesteigerten Osteopontin Expression. Diese Experimente legen den Schluss nahe, dass wnt-5a zur Aufrechterhaltung des Osteogenesepotentials beiträgt und dass die Inhibierung dieses Signalweges ein wichtiger Mechanismus in der Determinierung multipotenter mesenchymaler Stammzellen zu Preadipozyten beim Menschen ist. Immunhistochemische Untersuchungen an Fettgewebebiopsien zeigten nachfolgend, dass wnt-5a nicht nur von mesenchymalen Vorläuferzellen sondern auch von Makrophagen exprimiert wird, die bei Patienten mit Adipositas vermehrt im Fettgewebe vorzufinden sind. Auf zellulärer Ebene wurde nachgewiesen, dass Makrophagen über die Sekretion von wnt-5a die Adipogenese von mesenchymalen Vorläuferzellen hemmen. Zusammenfassend konnte in der vorliegenden Arbeit gezeigt werden, dass die proosteogenen Eigenschaften von wnt-5a für die Aufrechterhaltung der Multipotenz mesenchymaler Stammzellen von Bedeutung sind, während die antiadipogenen Effekte von wnt-5a in der Interaktion von Makrophagen und dem Fettgewebe beim Menschen eine funktionelle Relevanz haben

The Role of Paracrine and Autocrine Signaling in the Early Phase of Adipogenic Differentiation of Adipose-derived Stem Cells.

INTRODUCTION: High cell density is known to enhance adipogenic differentiation of mesenchymal stem cells, suggesting secretion of signaling factors or cell-contact-mediated signaling. By employing microfluidic biochip technology, we have been able to separate these two processes and study the secretion pathways. METHODS AND RESULTS: Adipogenic differentiation of human adipose-derived stem cells (ASCs) cultured in a microfluidic system was investigated under perfusion conditions with an adipogenic medium or an adipogenic medium supplemented with supernatant from differentiating ASCs (conditioned medium). Conditioned medium increased adipogenic differentiation compared to adipogenic medium with respect to accumulation of lipid-filled vacuoles and gene expression of key adipogenic markers (C/EBPα, C/EBPβ, C/EBPδ, PPARγ, LPL and adiponectin). The positive effects of conditioned medium were observed early in the differentiation process. CONCLUSIONS: Using different cell densities and microfluidic perfusion cell cultures to suppress the effects of cell-released factors, we have demonstrated the significant role played by auto- or paracrine signaling in adipocyte differentiation. The cell-released factor(s) were shown to act in the recruitment phase of the differentiation process

Prediction of arterial pressure increase after fluid challenge

<p>Abstract</p> <p>Background</p> <p>Mean arterial pressure above 65 mmHg is recommended for critically ill hypotensive patients whereas they do not benefit from supranormal cardiac output values. In this study we investigated if the increase of mean arterial pressure after volume expansion could be predicted by cardiovascular and renal variables. This is a relevant topic because unnecessary positive fluid balance increases mortality, organ dysfunction and Intensive Care Unit length of stay.</p> <p>Methods</p> <p>Thirty-six hypotensive patients (mean arterial pressure < 65 mmH) received a fluid challenge with hydroxyethyl starch. Patients were excluded if they had active bleeding and/or required changes in vasoactive agents infusion rate in the previous 30 minutes. Responders were defined by the increase of mean arterial pressure value to over 65 mmHg or by more than 20% with respect to the value recorded before fluid challenge. Measurements were performed before and at one hour after the end of fluid challenge.</p> <p>Results</p> <p>Twenty-two patients (61%) increased arterial pressure after volume expansion. Baseline heart rate, arterial pressure, central venous pressure, central venous saturation, central venous to arterial PCO₂difference, lactate, urinary output, fractional excretion of sodium and urinary sodium/potassium ratio were similar between responder and non-responder. Only 7 out of 36 patients had valuable dynamic indices and then we excluded them from analysis. When the variables were tested as predictors of responders, they showed values of areas under the ROC curve ranging between 0.502 and 0.604. Logistic regression did not reveal any association between variables and responder definition.</p> <p>Conclusions</p> <p>Fluid challenge did not improve arterial pressure in about one third of hypotensive critically ill patients. Cardiovascular and renal variables did not enable us to predict the individual response to volume administration.</p> <p>Trial registration</p> <p>ClinicalTrials.gov: <a href="http://www.clinicaltrials.gov/ct2/show/NCT00721604">NCT00721604</a>.</p

Oral abstracts of the 21st International AIDS Conference 18-22 July 2016, Durban, South Africa

The rate at which HIV-1 infected individuals progress to AIDS is highly variable and impacted by T cell immunity. CD8 T cell inhibitory molecules are up-regulated in HIV-1 infection and associate with immune dysfunction. We evaluated participants (n=122) recruited to the SPARTAC randomised clinical trial to determine whether CD8 T cell exhaustion markers PD-1, Lag-3 and Tim-3 were associated with immune activation and disease progression.Expression of PD-1, Tim-3, Lag-3 and CD38 on CD8 T cells from the closest pre-therapy time-point to seroconversion was measured by flow cytometry, and correlated with surrogate markers of HIV-1 disease (HIV-1 plasma viral load (pVL) and CD4 T cell count) and the trial endpoint (time to CD4 count <350 cells/μl or initiation of antiretroviral therapy). To explore the functional significance of these markers, co-expression of Eomes, T-bet and CD39 was assessed.Expression of PD-1 on CD8 and CD38 CD8 T cells correlated with pVL and CD4 count at baseline, and predicted time to the trial endpoint. Lag-3 expression was associated with pVL but not CD4 count. For all exhaustion markers, expression of CD38 on CD8 T cells increased the strength of associations. In Cox models, progression to the trial endpoint was most marked for PD-1/CD38 co-expressing cells, with evidence for a stronger effect within 12 weeks from confirmed diagnosis of PHI. The effect of PD-1 and Lag-3 expression on CD8 T cells retained statistical significance in Cox proportional hazards models including antiretroviral therapy and CD4 count, but not pVL as co-variants.Expression of ‘exhaustion’ or ‘immune checkpoint’ markers in early HIV-1 infection is associated with clinical progression and is impacted by immune activation and the duration of infection. New markers to identify exhausted T cells and novel interventions to reverse exhaustion may inform the development of novel immunotherapeutic approaches

Stromal-vascular fraction content and adipose stem cell behavior are altered in morbid obese and post bariatric surgery ex-obese women

INTRODUCTION: Subcutaneous adipose tissue is an interesting source of autologous stem cells with a fundamental role in the pathophysiology of obesity, metabolic syndromes and insulin resistance. We hypothesize that obesity could alter the stromal-vascular fraction (SVF) and adipose stem cell (ASCs) functions, which could compromise its regenerative behavior. Furthermore, we aimed to evaluate whether ASCs derived from post bariatric surgery ex-obese women maintain their functions in a similar fashion as do those from individuals who have never been obese. METHODS: The SVF of subcutaneous adipose tissue from control (n = 6, body mass index – BMI - 27.5 ± 0.5 kg/m(2)), obese (n = 12, BMI 46.2 ± 5.1 kg/m(2)) and post bariatric surgery ex-obese (n = 7, initial BMI 47.8 ± 1.3 kg/m(2); final BMI 28.1 ± 1.1 kg/m(2)) women were isolated and evaluated by flow cytometry. ASCs were tested for lipid accumulation by perilipin, adipose differentiation-related protein (ADRP) and Oil Red O staining after adipogenic stimulus. The cytokines secreted by the ASCs and after lipid accumulation induction were also evaluated. RESULTS: The subcutaneous adipose tissue of obese and post bariatric surgery ex-obese women was enriched in pericytes (p = 0.0345). The number of supra-adventitial cells was not altered in the obese patients, but it was highly enriched in the post bariatric surgery ex-obese women (p = 0.0099). The ASCs of the post bariatric surgery ex-obese patients secreted more MCP-1 (monocyte chemoattractant protein-1; p = 0.0078). After lipid accumulation induction, the ASCs of the patients in all groups secreted less IL-6 than the ASCs with no adipogenic stimulus (p < 0.0001). Obese ASCs with lipid accumulation secreted the highest amount of IL-6 (p < 0.001) whereas the ASCs from the controls secreted the highest amount of adiponectin (p < 0.0001). The ASCs from the post bariatric surgery ex-obese patients showed the highest levels of lipid accumulation whereas those from the obese women had the lowest levels (p < 0.0001). CONCLUSIONS: SVF content and ASC behavior are altered in the subcutaneous adipose tissue of morbid obese women; these changes are not completely restored after bariatric surgery-induced weight loss. The cellular alterations described in this study could affect the regenerative effects of adipose stem cells. Further investigations are required to avoid jeopardizing the development of autologous stem cell-based therapies

SRM・CRMの観点から考察する商社営業のコンピテンシー

Stem cells have captured the imagination of the general public by their potential as new therapeutic tools in the fight against degenerative diseases. This potential is based on their capability for self-renewal and at the same time for producing progenitor cells that will eventually provide the building blocks for tissue and organ regeneration. These processes are carefully orchestrated in the organism by means of a series of molecular cues. An emerging molecule which is responsible for some of these physiological responses is adrenomedullin, a 52-amino acid regulatory peptide which increases proliferation and regulates cell fate of stem cells of different origins. Adrenomedullin binds to specific membrane receptors in stem cells and induces several intracellular pathways such as those involving cAMP, Akt, or MAPK. Regulation of adrenomedullin levels may help in directing the growth and differentiation of stem cells for applications (e.g., cell therapy) both in vitro and in vivo. © 2012 Elsevier Inc.Peer Reviewe

Vampires in the village Žrnovo on the island of Korčula: following an archival document from the 18th century

Središnja tema rada usmjerena je na raščlambu spisa pohranjenog u Državnom arhivu u Mlecima (fond: Capi del Consiglio de’ Dieci: Lettere di Rettori e di altre cariche) koji se odnosi na događaj iz 1748. godine u korčulanskom selu Žrnovo, kada su mještani – vjerujući da su se pojavili vampiri – oskvrnuli nekoliko mjesnih grobova. U radu se podrobno iznose osnovni podaci iz spisa te rečeni događaj analizira u širem društvenom kontekstu i prate se lokalna vjerovanja.The main interest of this essay is the analysis of the document from the State Archive in Venice (file: Capi del Consiglio de’ Dieci: Lettere di Rettori e di altre cariche) which is connected with the episode from 1748 when the inhabitants of the village Žrnove on the island of Korčula in Croatia opened tombs on the local cemetery in the fear of the vampires treating. This essay try to show some social circumstances connected with this event as well as a local vernacular tradition concerning superstitions

Comparison: Flu prescription sales data from a retail pharmacy in the US with Google Flu trends and US ILINet (CDC) data as flu activity indicator.

The potential threat of bioterrorism along with the emergence of new or existing drug resistant strains of influenza virus, added to expanded global travel, have increased vulnerability to epidemics or pandemics and their aftermath. The same factors have also precipitated urgency for having better, faster, sensitive, and reliable syndromic surveillance systems. Prescription sales data can provide surrogate information about the development of infectious diseases and therefore serve as a useful tool in syndromic surveillance. This study compared prescription sales data from a large drug retailing pharmacy chain in the United States with Google Flu trends surveillance system data as a flu activity indicator. It was found that the two were highly correlated. The correlation coefficient (Pearson 'r') for five years' aggregate data (2007-2011) was 0.92 (95% CI, 0.90-0.94). The correlation coefficients for each of the five years between 2007 and 2011 were 0.85, 0.92, 0.91, 0.88, and 0.87 respectively. Additionally, prescription sales data from the same large drug retailing pharmacy chain in the United States were also compared with US Outpatient Influenza-like Illness Surveillance Network (ILINet) data for 2007 by Centers for Disease Control and Prevention (CDC). The correlation coefficient (Pearson 'r') was 0.97 (95% CI, 0.95-0.98)