Identification and isolation of small CD44-negative mesenchymal stem/progenitor cells from human bone marrow using elutriation and polychromatic flow cytometry

The method of isolation of bone marrow (BM) mesenchymal stem/stromal cells (MSCs) is a limiting factor in their study and therapeutic use. MSCs are typically expanded from BM cells selected on the basis of their adherence to plastic, which results in a heterogeneous population of cells. Prospective identification of the antigenic profile of the MSC population(s) in BM that gives rise to cells with MSC activity in vitro would allow the preparation of very pure populations of MSCs for research or clinical use. To address this issue, we used polychromatic flow cytometry and counterflow centrifugal elutriation to identify a phenotypically distinct population of mesenchymal stem/progenitor cells (MSPCs) within human BM. The MSPC activity resided within a population of rare, small CD45⁻CD73⁺CD90⁺CD105⁺ cells that lack CD44, an antigen that is highly expressed on culture-expanded MSCs. In culture, these MSPCs adhere to plastic, rapidly proliferate, and acquire CD44 expression. They form colony forming units-fibroblast and are able to differentiate into osteoblasts, chondrocytes, and adipocytes under defined in vitro conditions. Their acquired expression of CD44 can be partially downregulated by treatment with recombinant human granulocyte-colony stimulating factor, a response not found in BM-MSCs derived from conventional plastic adherence methods. These observations indicate that MSPCs within human BM are rare, small CD45⁻CD73⁺CD90⁺CD105⁺ cells that lack expression of CD44. These MSPCs give rise to MSCs that have phenotypic and functional properties that are distinct from those of BM-MSCs purified by plastic adherence.

RP1 is a phosphorylation target of CK2 and is involved in cell adhesion

RP1 (synonym: MAPRE2, EB2) is a member of the microtubule binding EB1 protein family, which interacts with APC, a key regulatory molecule in the Wnt signalling pathway. While the other EB1 proteins are well characterized the cellular function and regulation of RP1 remain speculative to date. However, recently RP1 has been implicated in pancreatic cancerogenesis. CK2 is a pleiotropic kinase involved in adhesion, proliferation and anti-apoptosis. Overexpression of protein kinase CK2 is a hallmark of many cancers and supports the malignant phenotype of tumor cells. In this study we investigate the interaction of protein kinase CK2 with RP1 and demonstrate that CK2 phosphorylates RP1 at Ser(236) in vitro. Stable RP1 expression in cell lines leads to a significant cleavage and down-regulation of N-cadherin and impaired adhesion. Cells expressing a Phospho-mimicking point mutant RP1-ASP(236) show a marked decrease of adhesion to endothelial cells under shear stress. Inversely, we found that the cells under shear stress downregulate endogenous RP1, most likely to improve cellular adhesion. Accordingly, when RP1 expression is suppressed by shRNA, cells lacking RP1 display significantly increased cell adherence to surfaces. In summary, RP1 phosphorylation at Ser(236) by CK2 seems to play a significant role in cell adhesion and might initiate new insights in the CK2 and EB1 family protein association.

Molecular basis of Corynebacterium diphtheriae virulence and infection in the Caenorhabditis elegans model host

Corynebacterium diphtheriae is the causative agent of cutaneous and pharyngeal diphtheria in humans. While lethality is certainly caused by diphtheria toxin, corynebacterial colonization may primarily require proteinaceous fibers called pili, which mediate adherence to specific tissues. The type strain of C. diphtheriae possesses three distinct pilus structures, namely the SpaA, SpaD, and SpaH-type pili, which are encoded by three distinct pilus gene clusters. The pilus is assembled onto the bacterial peptidoglycan by a specific transpeptidase enzyme called sortase. Although the SpaA pili are shown to be specific for pharyngeal cells in vitro, little is known about functions of the three pili in bacterial pathogenesis. This is mainly due to lack of in vivo models of corynebacterial infection. As an alternative to mouse models as mice do not have functional receptors for diphtheria toxin, in this study I use Caenorhabditis elegans as a model host for C. diphtheriae. A simple C. elegans model would be beneficial in determining the specific role of each pilus-type and the literature suggests that C. elegans infection model can be used to study a variety of bacterial species giving insight into bacterial virulence and host-pathogen interactions. My study examines the hypothesis that pili and toxin are major virulent determinants of C. diphtheriae in the C. elegans model host.

Maintenance of phosphatidylserine asymmetry in erythroid cells during differentiation, aging, and its significance for macrophage recognition

Phosphatidylserine (PS) is distributed almost entirely in the inner leaflet of the erythrocyte membrane bilayer, and appears to be maintained by a 32 kDa integral membrane protein (PS translocase). The expression of PS on the outer leaflet may serve as a recognition signal for macrophages, since insertion of PS into erythrocytes enhances their adherence to macrophages and clearance from the circulation. Therefore I have hypothesized that erythroid cells display PS on their outer leaflet early in differentiation and upon aging. Analysis of murine erythroleukemia cells (MELC, undifferentiated erythroid progenitor cells) showed high levels of PS on the outer leaflet that decreased during differentiation, correlating with the pattern of macrophage adherence. The activity of the PS translocase during differentiation appears to be unchanged although the equilibrium distribution of PS differs. This difference may be due to qualitative changes in the PS translocase. $\sp{125}$I-Bolton/Hunter-labeled-pyridyldithioethylamine ($\sp{125}$I-B/H-PDA), a radiolabeled probe for the PS translocase, labeled a 32 kDa protein in mature erythrocytes whereas in MELC a 45 kDa protein as well as a 32 kDa protein was identified. The abundance of the 45 kDa protein in relation to the 32 kDa protein declined during differentiation, possibly indicating this protein was a precursor of the 32 kDa protein. Analysis of the 45 kDa protein by N-glycosidase F and endoproteinase cleavage suggested this protein was not a glycosylated form of the 32 kDa protein but appeared to share some structural homology. Aged murine erythrocytes had elevated levels of PS on their outer leaflet, as well as decreased PS translocase activity. $\sp{125}$I-B/H-PDA labeled a 32 kDa protein in both normal and aged erythrocytes. However, the latter cells also contained a 28 kDa protein. Experimental evidence suggests that the appearance of the 28 kDa protein may be due to increased oxidation of aged erythrocytes. Examination of PS distribution showed that the levels of PS on the outer leaflet were elevated early in differentiation, decreased during the mature state, and returned to high levels as the erythrocyte aged. In conclusion,the levels of outer leaflet PS correlated with the differentiation status and macrophage recognition of erythroid cells. ^

Comparing different boosters of planning interventions on changes in fat consumption in overweight and obese individuals: A randomized controlled trial

Single planning interventions have been found to promote short-term dietary change. Repeated planning interventions may foster long-term effects on behavior change. It remains unknown whether there is a critical number of boosters to establish long-term maintenance of behavioral changes. This study aimed at investigating what social-cognitive variables mediate the effects of the interventions on dietary behavior change. Overall, 373 participants (n = 270 women, 72.4%; age M = 52.42, SD = 12.79) were randomly allocated to one of five groups: a control group, a single planning group, and three groups with 3, 6, or 9 weeks' repeated planning interventions. Follow-ups took place 4, 6, and 12 months after baseline. Change in fat consumption was not promoted by any of the interventions. In terms of social-cognitive variables, intentions, self-efficacy and coping planning displayed a time × group interaction, with the 9 weeks' planning group showing the most beneficial effects. Effect sizes, however, were very small. None of the tested planning interventions successfully promoted change in fat consumption across the 12 month period. This, however, could not be explained by problems with adherence to the intervention protocol. Potential explanations for this unexpected result are discussed.

Quality of care after acute coronary syndromes in a prospective cohort with reasons for non-prescription of recommended medications

BACKGROUND Adherence to guidelines is associated with improved outcomes of patients with acute coronary syndrome (ACS). Clinical registries developed to assess quality of care at discharge often do not collect the reasons for non-prescription for proven efficacious preventive medication in Continental Europe. In a prospective cohort of patients hospitalized for an ACS, we aimed at measuring the rate of recommended treatment at discharge, using pre-specified quality indicators recommended in cardiologic guidelines and including systematic collection of reasons for non-prescription for preventive medications. METHODS In a prospective cohort with 1260 patients hospitalized for ACS, we measured the rate of recommended treatment at discharge in 4 academic centers in Switzerland. Performance measures for medication at discharge were pre-specified according to guidelines, systematically collected for all patients and included in a centralized database. RESULTS Six hundred and eighty eight patients(54.6%) were discharged with a main diagnosis of STEMI, 491(39%) of NSTEMI and 81(6.4%) of unstable angina. Mean age was 64 years and 21.3% were women. 94.6% were prescribed angiotensin converting enzyme inhibitors/angiotensin II receptor blockers at discharge when only considering raw prescription rates, but increased to 99.5% when including reasons non-prescription. For statins, rates increased from 98% to 98.6% when including reasons for non-prescription and for beta-blockers, from 82% to 93%. For aspirin, rates further increased from 99.4% to 100% and from to 99.8% to 100% for P2Y12 inhibitors. CONCLUSIONS We found a very high adherence to ACS guidelines for drug prescriptions at discharge when including reasons for non-prescription to drug therapy. For beta-blockers, prescription rates were suboptimal, even after taking into account reason for non-prescription. In an era of improving quality of care to achieve 100% prescription rates at discharge unless contra-indicated, pre-specification of reasons for non-prescription for cardiovascular preventive medication permits to identify remaining gaps in quality of care at discharge.

Standardized definitions and clinical endpoints in trials investigating endovascular repair of aortic dissections.

OBJECTIVES Endovascular therapy is a rapidly expanding option for the treatment of patients with aortic dissection (AD) and various studies have been published. These trials, however, are often difficult to interpret and compare because they do not utilize uniform clinical endpoint definitions. METHODS The DEFINE Group is a collaborative effort of an ad hoc multidisciplinary team from various specialties involved in AD therapy in Europe and the United States. DEFINE's goal was to arrive at a broad based consensus for baseline and endpoint definitions in trials for endovascular therapy of various vascular pathologies. In this project, which started in December 2006, the individual team members reviewed the existing pertinent literature. Following this, a series of telephone conferences and face-to-face meetings were held to agree upon definitions. Input was also obtained from regulatory (United States Food and Drug Administration) and industry (device manufacturers with an interest in peripheral endovascular revascularization) stakeholders, respectively. RESULTS These efforts resulted in the present document containing proposed baseline and endpoint definitions for clinical and morphological outcomes. Although the consensus has inevitably included certain arbitrary consensus choices and compromises, adherence to these proposed standard definitions would provide consistency across future trials, thereby facilitating evaluation of clinical effectiveness and safety of various endovascular revascularization techniques. CONCLUSIONS This current document is based on a broad based consensus involving relevant stakeholders from the medical community, industry and regulatory bodies. It is proposed that the consensus document may have value for study design of future clinical trials in endovascular AD therapy as well as for regulatory purposes.

Quantitative Myocardial Contrast Echocardiography during Pharmacological Stress for Diagnosis of Coronary Artery Disease: A Systematic Review and Meta-analysis of Diagnostic Accuracy Studies

AIMS: We conducted a meta-analysis to evaluate the accuracy of quantitative stress myocardial contrast echocardiography (MCE) in coronary artery disease (CAD). METHODS AND RESULTS: Database search was performed through January 2008. We included studies evaluating accuracy of quantitative stress MCE for detection of CAD compared with coronary angiography or single-photon emission computed tomography (SPECT) and measuring reserve parameters of A, beta, and Abeta. Data from studies were verified and supplemented by the authors of each study. Using random effects meta-analysis, we estimated weighted mean difference (WMD), likelihood ratios (LRs), diagnostic odds ratios (DORs), and summary area under curve (AUC), all with 95% confidence interval (CI). Of 1443 studies, 13 including 627 patients (age range, 38-75 years) and comparing MCE with angiography (n = 10), SPECT (n = 1), or both (n = 2) were eligible. WMD (95% CI) were significantly less in CAD group than no-CAD group: 0.12 (0.06-0.18) (P < 0.001), 1.38 (1.28-1.52) (P < 0.001), and 1.47 (1.18-1.76) (P < 0.001) for A, beta, and Abeta reserves, respectively. Pooled LRs for positive test were 1.33 (1.13-1.57), 3.76 (2.43-5.80), and 3.64 (2.87-4.78) and LRs for negative test were 0.68 (0.55-0.83), 0.30 (0.24-0.38), and 0.27 (0.22-0.34) for A, beta, and Abeta reserves, respectively. Pooled DORs were 2.09 (1.42-3.07), 15.11 (7.90-28.91), and 14.73 (9.61-22.57) and AUCs were 0.637 (0.594-0.677), 0.851 (0.828-0.872), and 0.859 (0.842-0.750) for A, beta, and Abeta reserves, respectively. CONCLUSION: Evidence supports the use of quantitative MCE as a non-invasive test for detection of CAD. Standardizing MCE quantification analysis and adherence to reporting standards for diagnostic tests could enhance the quality of evidence in this field.

BMZI-HEA. Adaption des Berner Motiv- und Zielinventars im Freizeit- und Gesundheitssport für Menschen im höheren Erwachsenenalter

Die Förderung regelmäßiger Bewegungs- und Sportaktivitäten bei älteren Menschen gewinnt zunehmend an Bedeutung. Für eine effiziente Bewegungs- und Sportförderung werden zielgruppenspezifische Maßnahmen gefordert. Sportbezogene Motive und Ziele von Älteren werden aktuell selten systematisch in die Konzeption von Interventionen miteinbezogen, wenngleich sie für das Wohlbefinden und die Aufrechterhaltung des Sportverhaltens eine zentrale Rolle einnehmen. Das bereits bestehende BMZI ermöglicht die Individualdiagnose von Motiven und Zielen im Freizeit- und Gesundheitssport bei Personen im mittleren Erwachsenenalter. Der vorliegende Beitrag zielt auf eine Adaption des Fragebogens für Menschen im höheren Erwachsenenalter. Das BMZI-HEA deckt mit insgesamt 27 Items folgende Motive und Ziele ab: Figur/Aussehen, Kontakt, Wettkampf/Leistung, Alltagskompetenz/Gesundheit, Positive Bewegungserfahrungen, Kognitive Funktionsfähigkeit, Stimmungsregulation. Der Fragebogen wurde an drei Stichproben explorativ und konfirmatorisch überprüft. Der globale Modell-Fit des BMZI-HEA ist als zufriedenstellend zu beurteilen. Die erwartungskonformen Zusammenhänge mit der Selbstkonkordanz verweisen auf eine gute Konstruktvalidität des Instruments. Das BMZI-HEA empfiehlt sich als ökonomisches Instrument für die Individualdiagnose der psychischen Handlungsvoraussetzungen für das Sporttreiben von Menschen im höheren Erwachsenenalter.

Antiretrovirale Therapie

Antiretroviral therapy to treat HIV, as we know it today, is nothing less than a huge success story in modern medical history. What used to be an almost certain death-sentence was transformed into a very manageable chronic disease by means of highly efficient und mostly well tolerated drugs. Today, HIV-infected patients treated according to international recommendations have a very good chance to outgo the negative effects of HIV-1 and are therefore able to reach an almost normal life expectancy. Furthermore, patients successfully treated with antiretroviral drugs are no longer infectious, which is an essential aspect of global strategies to overcome the pandemic. Nevertheless, due to the complexity of HIV, physicians treating patients with antiretroviral therapy require profound knowledge of aspects such as viral resistance mechanisms and immune reconstitution, as well as drug-toxicity und drug-drug-interactions. Many other aspects such as long-term side-effects of antiretroviral drugs are still unknown. Strict adherence to treatment is of utmost importance.

Integrated care programmes for adults with chronic conditions: a meta-review.

OBJECTIVE To review systematic reviews and meta-analyses of integrated care programmes in chronically ill patients, with a focus on methodological quality, elements of integration assessed and effects reported. DESIGN Meta-review of systematic reviews and meta-analyses identified in Medline (1946-March 2012), Embase (1980-March 2012), CINHAL (1981-March 2012) and the Cochrane Library of Systematic Reviews (issue 1, 2012). MAIN OUTCOME MEASURES Methodological quality assessed by the 11-item Assessment of Multiple Systematic Reviews (AMSTAR) checklist; elements of integration assessed using a published list of 10 key principles of integration; effects on patient-centred outcomes, process quality, use of healthcare and costs. RESULTS Twenty-seven systematic reviews were identified; conditions included chronic heart failure (CHF; 12 reviews), diabetes mellitus (DM; seven reviews), chronic obstructive pulmonary disease (COPD; seven reviews) and asthma (five reviews). The median number of AMSTAR checklist items met was five: few reviewers searched for unpublished literature or described the primary studies and interventions in detail. Most reviews covered comprehensive services across the care continuum or standardization of care through inter-professional teams, but organizational culture, governance structure or financial management were rarely assessed. A majority of reviews found beneficial effects of integration, including reduced hospital admissions and re-admissions (in CHF and DM), improved adherence to treatment guidelines (DM, COPD and asthma) or quality of life (DM). Few reviews showed reductions in costs. CONCLUSIONS Systematic reviews of integrated care programmes were of mixed quality, assessed only some components of integration of care, and showed consistent benefits for some outcomes but not others.

Ultrathin strut biodegradable polymer sirolimus-eluting stent versus durable polymer everolimus-eluting stent for percutaneous coronary revascularisation (BIOSCIENCE): a randomised, single-blind, non-inferiority trial

BACKGROUND Refinements in stent design affecting strut thickness, surface polymer, and drug release have improved clinical outcomes of drug-eluting stents. We aimed to compare the safety and efficacy of a novel, ultrathin strut cobalt-chromium stent releasing sirolimus from a biodegradable polymer with a thin strut durable polymer everolimus-eluting stent. METHODS We did a randomised, single-blind, non-inferiority trial with minimum exclusion criteria at nine hospitals in Switzerland. We randomly assigned (1:1) patients aged 18 years or older with chronic stable coronary artery disease or acute coronary syndromes undergoing percutaneous coronary intervention to treatment with biodegradable polymer sirolimus-eluting stents or durable polymer everolimus-eluting stents. Randomisation was via a central web-based system and stratified by centre and presence of ST segment elevation myocardial infarction. Patients and outcome assessors were masked to treatment allocation, but treating physicians were not. The primary endpoint, target lesion failure, was a composite of cardiac death, target vessel myocardial infarction, and clinically-indicated target lesion revascularisation at 12 months. A margin of 3·5% was defined for non-inferiority of the biodegradable polymer sirolimus-eluting stent compared with the durable polymer everolimus-eluting stent. Analysis was by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT01443104. FINDINGS Between Feb 24, 2012, and May 22, 2013, we randomly assigned 2119 patients with 3139 lesions to treatment with sirolimus-eluting stents (1063 patients, 1594 lesions) or everolimus-eluting stents (1056 patients, 1545 lesions). 407 (19%) patients presented with ST-segment elevation myocardial infarction. Target lesion failure with biodegradable polymer sirolimus-eluting stents (69 cases; 6·5%) was non-inferior to durable polymer everolimus-eluting stents (70 cases; 6·6%) at 12 months (absolute risk difference -0·14%, upper limit of one-sided 95% CI 1·97%, p for non-inferiority <0·0004). No significant differences were noted in rates of definite stent thrombosis (9 [0·9%] vs 4 [0·4%], rate ratio [RR] 2·26, 95% CI 0·70-7·33, p=0·16). In pre-specified stratified analyses of the primary endpoint, biodegradable polymer sirolimus-eluting stents were associated with improved outcome compared with durable polymer everolimus-eluting stents in the subgroup of patients with ST-segment elevation myocardial infarction (7 [3·3%] vs 17 [8·7%], RR 0·38, 95% CI 0·16-0·91, p=0·024, p for interaction=0·014). INTERPRETATION In a patient population with minimum exclusion criteria and high adherence to dual antiplatelet therapy, biodegradable polymer sirolimus-eluting stents were non-inferior to durable polymer everolimus-eluting stents for the combined safety and efficacy outcome target lesion failure at 12 months. The noted benefit in the subgroup of patients with ST-segment elevation myocardial infarction needs further study. FUNDING Clinical Trials Unit, University of Bern, and Biotronik, Bülach, Switzerland.

Effects of systematic patient education in skin care and protection in a hand eczema clinic.

BACKGROUND Hand eczema has a high impact on patients' quality of life. The treatment focuses on improving skin barrier function. OBJECTIVES To evaluate the effects and acceptance of a novel educational program for patients with hand eczema. METHODS Retrospectively, the records of 36 patients who attended the prevention program and follow-up visits were analyzed. Physician global assessment (PGA) scores, acceptance and behavioral changes were assessed. RESULTS In 67% of patients, an improvement of the hand eczema could be attributed to the effects of our educational program. The mean PGA score significantly decreased from 3 before education to 2.2 during follow-up. Behavioral changes in both skin care and protection were reported in 81 and 86%, respectively. CONCLUSIONs: Our educational program had a positive effect on clinical outcome as well as adherence to skin care and protection measures. Its integration in a hand eczema clinic was feasible and well accepted by the patients.

Systemic therapy for atopic dermatitis.

Systemic therapy for atopic dermatitis (AD) is indicated in patients with severe disease refractory to adequate topical treatment. Currently available drugs aim to decrease inflammation by suppressing and/or modulating immune responses and thus may indirectly improve skin barrier function, resulting in a decrease in clinical signs and symptoms in particular pruritus. Before considering systemic treatment, patient adherence to topical treatment including skin care has to be ensured. The selection of the drug depends on the disease severity, localization, complications, concomitant diseases, and age of the patient, but also on their availability and costs as well as the doctor's experience. Bearing in mind the potential risk of resistance, systemic therapy with antibiotics should be exclusively considered in clinically manifest infections such as in children. Here, we review recently published clinical trials and case reports on systemic therapy of pediatric and adult patients with AD to draw conclusions for clinical practice. Although AD is a common disease, controlled clinical studies investigating the efficacy of systemic drugs are scarce, except for cyclosporine, which has been approved for the therapy of severe AD.

Global phylogenomic analysis of nonencapsulated Streptococcus pneumoniae reveals a deep-branching classic lineage that is distinct from multiple sporadic lineages.

The surrounding capsule of Streptococcus pneumoniae has been identified as a major virulence factor and is targeted by pneumococcal conjugate vaccines (PCV). However, nonencapsulated Streptococcus pneumoniae (Non-Ec-Sp) have also been isolated globally, mainly in carriage studies. It is unknown if Non-Ec-Sp evolve sporadically, if they have high antibiotic non-susceptiblity rates and a unique, specific gene content. Here, whole genome sequencing of 131 Non-Ec-Sp isolates sourced from 17 different locations around the world was performed. Results revealed a deep-branching classic lineage that is distinct from multiple sporadic lineages. The sporadic lineages clustered with a previously sequenced, global collection of encapsulated S. pneumoniae (Ec-Sp) isolates while the classic lineage is comprised mainly of the frequently identified multi-locus sequences types ST344 (n=39) and ST448 (n=40). All ST344 and nine ST448 isolates had high non-susceptiblity rates to β-lactams and other antimicrobials. Analysis of the accessory genome reveals that the classic Non-Ec-Sp contained an increased number of mobile elements, than Ec-Sp and sporadic Non-Ec-Sp. Performing adherence assays to human epithelial cells for selected classic and sporadic Non-Ec-Sp revealed that the presence of a integrative conjugative element (ICE) results in increased adherence to human epithelial cells (P=0.005). In contrast, sporadic Non-Ec-Sp lacking the ICE had greater growth in vitro possibly resulting in improved fitness. In conclusion, Non-Ec-Sp isolates from the classic lineage have evolved separately. They have spread globally, are well adapted to nasopharyngeal carriage and are able to coexist with Ec-Sp. Due to continued use of pneumococcal conjugate vaccines, Non-Ec-Sp may become more prevalent.