Human mesenchymal stem cells at the single-cell level: simultaneous seven-colour immunofluorescence

Extracellular, intracellular or surface proteins can be used as putative markers to characterize human mesenchymal stem cells (hMSC). However, these markers are also expressed by other cell types and primary cell pools reveal considerable heterogeneity. Therefore, the simultaneous detection of several markers on a single cell appears to be an attractive approach to identify hMSC. Here we demonstrate the specific distinction of human MSC from human osteoblasts via seven-colour fluorescence on the single cell level with simultaneous marker detection of CD44, CD105/endoglin, CD106/VCAM-1, collagen-IV, fibronectin, actin and DAPI nuclear staining. We performed spectral image acquisition using a Sagnac-type interferometer. Subsequent linear unmixing allowed for decomposition of each pixel in its spectral components. Our approach reveals a typical expression profile of the adherent singular cells, allowing the specific distinction between hMSC and osteoblasts on the single cell level.     

Expression of merozoite surface protein markers by Plasmodium falciparum-infected erythrocytes in peripheral blood and tissues of children with fatal malaria

Sequestration of Plasmodium falciparum-infected erythrocytes is a pathological feature of fatal cerebral malaria. P. falciparum is genetically diverse among, and often within, patients. Preferential sequestration of certain genotypes might be important in pathogenesis. We compared circulating parasites with parasites sequestered in the brain, spleen, liver, and lung in the same Malawian children with fatal malaria, classifying serotypes using antibodies to merozoite surface proteins 1 and 2 and immunofluorescence in order to differentiate parasites and to quantify the proportions of each serotype. We found (i) similar distributions of various serotypes in different tissues and (ii) concordance between parasite serotypes in peripheral blood and parasite serotypes in tissues. No serotypes predominated in the brain in cerebral malaria, and parasites belonging to a single serotype did not cluster within individual vessels or within single tissues. These findings do not support the hypothesis that cerebral malaria is caused by cerebral sequestration of certain virulent types.     

Molecular cytogenetic analysis of complex karyotypes with derivative chromosome der(1)t(1;5) found in two patients with myeloid leukemia

Dedifferentiated liposarcoma of the pleura is an extremely rare malignancy mimicking a variety of tumors, such as other sarcomas, mesothelioma, and malignant solitary fibrous tumor of the pleura. Liposarcoma of the pleura can be combined with mediastinal involvement, and in most cases it may be impossible to be certain where the primary tumor originated. In this report, we describe a very rare occurence of a dedifferentiated liposarcoma of the pleura in a 76-year-old woman associated with a distinct second dedifferentiated liposarcoma of the mediastinum. Histologically, the pleural tumor demonstrated spindle cells arranged in a fascicular pattern, whereas the mediastinal tumor was mostly adipocytic with small areas of spindle cells. Vimentin and protein S100 were focally expressed by the tumor cells. The differential diagnosis of the pleural mass included malignant solitary fibrous tumor. Cytogenetic analysis showed supernumerary ring chromosomes in the pleural tumor, as well as strong amplification of MDM2 and CDK4 genes in both tumors. Array comparative genomic hybridization showed amplifications of chromosome arms 6q, 12q, and 15q, shared by both tumors and strongly pointing to a common origin.     

Empowering senior citizens for healthy nutrition in Germany: A pilot study

Main goal of this study was to analyse how empowerment processes and bottom‐up activities aimed at healthier food choices and food environment could be initiated among a group of senior citizens (between 60 and 75 years old). The intervention was set up as a pilot study in a rural community (15,000 inhabitants) in the federal state of Bavaria, South Eastern Germany. A process evaluation documented how group formation and empowerment processes developed during the course of the intervention. Extensive field notes were taken in 27 meetings, interviews (n = 13) and focus groups (n = 4) were conducted with participants and key persons at different points of the intervention. Data were analysed using content analysis. The intervention succeeded in motivating senior citizens to participate in regular meetings over 11 months. During the intervention, the group members’ awareness of factors influencing their eating behaviour increased. Furthermore, they developed ideas to improve the community's food environment and accomplished duties needed to implement these ideas. However, initiating empowerment processes, especially in terms of fostering leadership and transferring responsibility, took longer than expected and could be realised only partially. The findings support a further use and evaluation of the empowerment approach for addressing nutritional aspects among senior citizens.     

The New Acetylcholinesterase Inhibitors PC‐37 and PC‐48 (7‐Methoxytacrine‐Donepezil‐Like Compounds): Characterization of Their Metabolites in Human Liver Microsomes,Pharmacokinetics and In Vivo Formation of the Major Metabolites in Rats

The objective of this study was to elucidate the pharmacokinetics and metabolite formation of newly developed non‐selective AChE/BChE 7‐MEOTA‐donepezil‐like inhibitors for potential therapeutic use in Alzheimer's disease (AD) patients. The chemical structures of metabolites were defined during incubation with human liver microsomes, and subsequently, the metabolization was verified in in vivo study. In vitro metabolic profiling revealed the formation of nine major metabolites in the case of PC‐37 and eight metabolites of PC‐48. Hydroxylation and the enzymatic hydrolysis of bonds close to the piperazine ring appeared to be the principal metabolic pathways in vitro. Of these metabolites, M1–M7 of PC‐37 and M1–M6 of PC‐48 were confirmed under in vivo conditions. Pilot pharmacokinetic experiments in rats were focused on the absorption, distribution and elimination of these compounds. Absorption after i.m. application was relatively fast; the bioavailability expressed as AUC_total was 28179 ± 4691 min.ng/mL for PC‐37 and 23374 ± 4045 min.ng/mL for PC‐48. Both compounds showed ability to target the central nervous system, with brain concentrations exceeding those in plasma. The maximal brain concentrations are approximately two times higher than the plasma concentrations. The relatively high brain concentrations persisted throughout the experiment until 24 hr after application. Elimination via the kidneys (urine) significantly exceeded elimination via the liver (bile). All these characteristics are crucial for new candidates intended for AD treatment. The principle metabolic pathways that were verified in the in vivo study do not show any evidence for formation of extremely toxic metabolites, but this needs to be confirmed by further studies.