Labeling of mesenchymal stromal cells with iron oxide-poly(l-lactide) nanoparticles for magnetic resonance imaging: uptake, persistence, effects on cellular function and magnetic resonance imaging properties

Background aims. Mesenchymal stromal cells (MSC) are the focus of research in regenerative medicine aiming at the regulatory approval of these cells for specific indications. To cope with the regulatory requirements for somatic cell therapy, novel approaches that do not interfere with the natural behavior of the cells are necessary. In this context in vivo magnetic resonance imaging (MRI) of labeled MSC could be an appropriate tool. Cell labeling for MRI with a variety of different iron oxide preparations is frequently published. However, most publications lack a comprehensive assessment of the noninterference of the contrast agent with the functionality of the labeled MSC, which is a prerequisite for the validity of cell-tracking via MRI. Methods.We studied the effects of iron oxide-poly(L-lactide) nanoparticles in MSC with flow cytom-etry, transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), Prussian blue staining, CyQuant® proliferation testing, colony-forming unit-fibroblast (CFU-F) assays, flow chamber adhesion testing, immuno-logic tests and differentiation tests. Furthermore iron-labeled MSC were studied by MRI in agarose phantoms and Wistar rats. Results. It could be demonstrated that MSC show rapid uptake of nanoparticles and long-lasting intracellular persistence in the endosomal compartment. Labeling of the MSC with these particles has no influence on viability, differentiation, clonogenicity, proliferation, adhesion, phenotype and immunosuppressive properties. They show excellent MRI properties in agarose phantoms and after subcutaneous implantation in rats over several weeks. Conclusions. These particles qualify for studying MSC homing and trafficking via MRI

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Migration of progenitor cells between blood, bone marrow and the tumor microenvironment

It has been shown that stem and progenitor cells are therapeutically effective after i.v application. Yet, many aspects regarding intracellular signaling pathways which are involved in the homing and local action of these cells still have to be elucidated. In this work, it was aimed to investigate the role of the small GTPase Rap1 in adhesion activation in Hematopoietic Stem and Progenitor Cells (HSC/HPC) and in Mesenchymal Stem Cells (MSC). The potential role of Rap1 was assessed in, mice which were homozygote negative for the expression of the Rap1a gene. Peripheral blood lymphocyte counts as well as numbers of HPCs in the blood were decreased in Rap1a-/- mice compared to wild-type controls. Additionally the adhesion capability of HPCs from Rap1a-/- to the endothelial ligand, Vascular Cell Adhesion Molecule – 1 under shear stress was decreased. The hematopoietic repopulation potential of Rap1a-/- HPC was however not decreased in a competitive bone marrow transplantation model, indicating that deficiency of Rap1a in HSC/HPC does not negatively affect their ability to interact with the bone marrow microenvironment. In contrast, the isolation of MSC was not possible from Rap1a-/- bone marrow, indicating an altered situation in the bone marrow niche through changed stromal cell behaviour. Instead, Rap1a+/- MSC could be isolated and showed an adhesion deficit under shear stress. In contrast, no differences were noted in their differentiation potential. In a mouse homing model, the overall ability of the Rap1a+/- MSC to home to different tissues was found preserved. Finally, in a murine subcutaneous carcinoma model, cells with an HPC phenotype were observed to be present in the tumor microenvironment, and it was shown that they home directly to tumors. Since HPC isolated from bone marrow were able to differentiate into cells with a pro-angiogenic phenotype in vitro, HPC may be of relevance for neovascularization, as tumor-infiltrating progenitor cells. The results of the study should contribute to the understanding of the regulation of progenitor cell homing behaviour in situations simulating cell therapy approaches in preclinical situations.Es wurde gezeigt, dass Stamm- und Vorläufer zellen nach i.v. Anwendung therapeutisch wirksam sind. Jedoch müssen noch viele Aspekte bezüglich intrazellulärer Signalwege, die am Homing und der lokalen Wirkung dieser Zellen beteiligt sind, aufgeklärt werden. Ziel dieser Arbeit war es, die Rolle der kleinen GTPase Rap1a in bezug auf das Homing- und Adhäsionsverhalten von hämatopoietischen Stamm- und Vorläuferzellen (HSC / HPC) wie auch von mesenchymalen Stromazellen (MSC) zu untersuchen. Die Untersuchungen zum Einfluss von Rap1a auf das Homing und die Adhäsion wurde in Mäusen durchgeführt, die homozygot negativ für die Expression von Rap1a waren. In Rap1a-/- Mäusen war die Anzahl an peripheren Blutlymphozyten im Vergleich zu Wildtypmäusen ebenso erniedrigt wie die Bindung von HPCs an den endothelialen Liganden VCAM-1 (Vascular Cell Adhesion Molecule 1) unter Scherdruckbedingungen. Das hämatopietische Repopulierungspotential von Rap1a-/- HPCs war jedoch bei Verwendung eines kompetitven Knochenmark-Transplantationsmodells nicht verändert, was darauf hindeutet, dass die Defizienz von Rap1a in HSCs bzw. HPCs deren Interaktionen mit der Knochenmark-Mikroumgebung nicht negativ beeinflusst. Dagegen war es nicht möglich, MSC aus Knochenmark von Rap1a-/- Mäusen zu isolieren. Dies deutet auf eine veränderte Situation in der Knochenmarkniche durch eine Veränderung der stromalen Zellen hin. Stattdessen konnten aus Rap1a+/- Mäusen MSCs isoliert werden. Diese zeigten eine verminderte Adhäsion unter Scherdrücken. Jedoch wurden keine Unterschiede in ihrem Differenzierungspotential gefunden. In einem murinen Homingmodell war die Fähigkeit der Rap1a+/- MSCs in verschiedene Organe zu homen unverändert. Abschließend konnten in einem murinen subcutanen Tumormodell Zellen mit einem hämatopoietischen Vorläuferphenotyp im Tumor gefunden werden, welche in den Tumor einwandern. Da hämatopoietische Vorläuferzellen aus Knochenmark die Fähigkeit besitzen in Zellen mit einem proangiogenen Phenotyp in vitro zu differenzieren, können die in den Tumor eingewanderten HPCs bei der Neovaskularisierung eine entscheidende Rolle spielen. Die Ergebnisse dieser Arbeit können zum Verständnis der Regulation des Homingverhaltens von Vorläuferzellen und somit zur Verbesserung von Zelltherapieansätzen beitragen

Bisexuality among a cohort of university students: prevalence and psychological distress

Sociocultural prejudices and pressures may impair the mental health of bisexual people. We aim to evaluate psychological status according to sexual orientation in a sample of Italian university students, with specific attention to bisexuality and its frequency. Among a recruited sample of 551 university students, we found the following percentages for sexual orientation: heterosexuals 96.9% (n = 534), homosexuals 1.1% (n = 6), bisexuals 2% (n = 11). The cross-sectional analysis for psychological symptoms, with the Symptoms Check List-90 Revised (SCL-90-R), revealed that bisexual subjects have statistically significant higher scores on some symptomatic scales compared to heterosexuals. In particular, obsession-compulsion, paranoid ideation, hostility were significantly higher in bisexuals. Therefore, if heterosexual or homosexual orientation are not specified by particular psychological symptoms, bisexuality is characterized by a strong link with some facets of psychological distress, which are likely caused by a peculiar double stigma. In conclusion, through a specific psychometric tool, we found an association between bisexuality and various forms of psychological suffering. This evidence should further encourage clinicians to accurately assess the psychological health in young bisexual people