ABSTRACT
Aims: Sepsis is a leading cause of mortality in intensive care units. Development of new strategies such as the therapy with mesenchymal stem cells (MSC) appears beneficial for the treatment of sepsis. In this study we evaluated anti-septic effects of rat MSCs and recombinant erythropoietin (EPO) in a rat experimental model of endotoxemia. Study Design: Controlled in vitro and in vivo studies. Place and Duration of Study: Federal Research Clinical Center of FMBA of Russia, Moscow, Russia, between June 2014 and May 2015. Methodology: Endotoxemia was induced by intraperitoneal administration of bacterial lipopolysacharide (LPS). The animals were then treated either with allogeneic MSCs alone, with recombinant EPO alone, or with a combination of EPO and MCS. After 3 days , the animals were euthanized, and a pathology study of their liver, spleen, thymus, lung, and kidney was performed. The serum levels of IL-1β, IL-6, and TNF-α were quantified. A histochemical analysis of splenic and thymic expression of CD3, CD57, p53, and Bcl-2 was performed. Results: Essential positive effects of the combined MSC-EPO treatment were observed: 1) The animals treated with MSCs and EPO had the lowest serum concentrations of IL-1β in respect to that of the rats treated with LPS alone (58 ± 22 pg/mL vs. 155±90 pg/mL, P = .01); 2) The treatment of endotoxemic rats with a combination of MSCs and EPO also caused production of the anti-apoptotic factor Bcl-2, while its expression was markedly down-regulated in the other groups of animals; 3) In the MSC + EPO group, the degree of interstitial pulmonary edema was the lowest as compared to the other groups, and a minimal renal injury was detected . Conclusion: These findings suggest that EPO generally improves anti-inflammatory and anti-apoptotic effects of MSCs injected in the acute phase of experimental endotoxemia.
ABSTRACT
Downregulation of gap junctions by monoclonal antibodies against the second extracellular loop of connexin-43 (E2Cx43) was studied in a passaged culture of astrocytes. The results of confocal laser scanning microscopy demonstrated that, after two hours of coincubation of cells loaded with Calcein AM and Dil according to Goldberg et al. and unlabelled cells, the cytoplasmic dye Calcein AM was actively transferred to unlabelled cells through newly formed gap junctions. This transfer could be almost completely blocked by addition of 60 μg/ml of anti-E2Cx43 antibodies. Flow cytometric analysis showed that, in experiments carried out according to Goldberg et al., with approximately 2% of labeled cells added to unlabelled ones, about 2.5% of the total cell population took up Calcein AM through gap junctions, thus forming a cell pool characterized by low-intensity green fluorescence. In the presence of antibodies, the proportion of these cells was no more than 0.6%, which indicates an at least fourfold suppression of the gap junction function by E2Cx43 antibodies. The data obtained were reproduced in several independent series. Thus, we obtained monoclonal antibodies capable of modulating the gap junction function in cultures of Cx43-positive cells.