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Acute kidney injury (AKI) is one of the most important complications in hospitalized patients and its pathomechanisms are not completely elucidated. We hypothesize that signaling via toll-like receptor (TLR)-3, a receptor that is activated upon binding of double-stranded nucleotides, might play a crucial role in the pathogenesis of AKI following ischemia and reperfusion (IR). Male adult C57Bl6 wild-type (wt) mice and TLR-3 knock-out (-/-) mice were subjected to 30 minutes bilateral selective clamping of the renal artery followed by reperfusion for 30 min 2.5h and 23.5 hours or subjected to sham procedures. TLR-3 down-stream signaling was activated already within 3 h of ischemia and reperfusion in post-ischemic kidneys of wt mice lead to impaired blood perfusion followed by a strong pro-inflammatory response with significant neutrophil invasion. In contrast, this effect was absent in TLR-3-/- mice. Moreover, the quick TLR-3 activation resulted in kidney damage that was histomorphologically associated with significantly increased apoptosis and necrosis rates in renal tubules of wt mice. This finding was confirmed by increased kidney injury marker NGAL in wt mice and a better preserved renal perfusion after IR in TLR-3-/- mice than wt mice. Overall, the absence of TLR-3 is associated with lower cumulative kidney damage and maintained renal blood perfusion within the first 24 hours of reperfusion. Thus, we conclude that TLR-3 seems to participate in the pathogenesis of early acute kidney injury.
Background/Aims: Middle East respiratory syndrome coronavirus (MERS-CoV) and Marburg virus (MARV) are among the World Health Organization’s top 8 emerging pathogens. Both zoonoses share nonspecific early symptoms, a high lethality rate, and a reduced number of specific treatment options. Therefore, we evaluated extracorporeal virus and glycoprotein (GP) elimination by lectin affinity plasmapheresis (LAP).
Methods: For both MERS-CoV (pseudovirus) as well as MARV (GPs), 4 LAP devices (Mini Hemopurifiers, Aethlon Medical, San Diego, CA, USA) and 4 negative controls were tested. Samples were collected every 30 min and analyzed for reduction in virus infectivity by a flow cytometry-based infectivity assay (MERS-CoV) and in soluble GP content (MARV) by an immunoassay.
Results: The experiments show a time-dependent clearance of MERS-CoV of up to 80% within 3 h (pseudovirus). Up to 70% of MARV-soluble GPs were eliminated at the same time. Substantial saturation of the binding resins was detected within the first treatment hour.
Conclusion: MERS-CoV (pseudovirus) and MARV soluble GPs are eliminated by LAP in vitro. Considering the high lethality and missing established treatment options, LAP should be evaluated in vivo. Especially early initiation, continuous therapy, and timed cartridge exchanges could be of importance.
Adipose tissue as a stem cell source is ubiquitously available and has several advantages compared to other sources. It is easily accessible in large quantities with minimal invasive harvesting procedure, and isolation of adipose-derived mesenchymal stromal/stem cells (ASCs) yields a high amount of stem cells, which is essential for stem-cell-based therapies and tissue engineering. Several studies have provided evidence that ASCs in situ reside in a perivascular niche, whereas the exact localization of ASCs in native adipose tissue is still under debate. ASCs are isolated by their capacity to adhere to plastic. Nevertheless, recent isolation and culture techniques lack standardization. Cultured cells are characterized by their expression of characteristic markers and their capacity to differentiate into cells from meso-, ecto-, and entodermal lineages. ASCs possess a high plasticity and differentiate into various cell types, including adipocytes, osteoblasts, chondrocytes, myocytes, hepatocytes, neural cells, and endothelial and epithelial cells. Nevertheless, recent studies suggest that ASCs are a heterogeneous mixture of cells containing subpopulations of stem and more committed progenitor cells. This paper summarizes and discusses the current knowledge of the tissue localization of ASCs in situ, their characterization and heterogeneity in vitro, and the lack of standardization in isolation and culture methods.
Mesenchymal stromal/stem cells and their derivates are the most promising cell source for cell therapies in regenerative medicine. The application of extracellular vesicles (EVs) as cell-free therapeuticals requires particles with a maximum regenerative capability to enhance tissue and organ regeneration. The cargo of mRNA and microRNA (miR) in EVs after hypoxic preconditioning has not been extensively investigated. Therefore, the aim of our study was the characterization of mRNA and the miR loading of EVs. We further investigated the effects of the isolated EVs on renal tubular epithelial cells in vitro. We found 3131 transcripts to be significantly regulated upon hypoxia. Only 15 of these were downregulated, but 3116 were up-regulated. In addition, we found 190 small RNAs, 169 of these were miRs and 21 were piwi-interacting RNAs (piR). However, only 18 of the small RNAs were significantly altered, seven were miRs and 11 were piRs. Interestingly, all seven miRs were down-regulated after hypoxic pretreatment, whereas all 11 piRs were up-regulated. Gene ontology term enrichment and miR-target enrichment analysis of the mRNAs and miR were also performed in order to study the biological background. Finally, the therapeutic effect of EVs on human renal tubular epithelial cells was shown by the increased expression of three anti-inflammatory molecules after incubation with EVs from hypoxic pretreatment. In summary, our study demonstrates the altered mRNA and miR load in EVs after hypoxic preconditioning, and their anti-inflammatory effect on epithelial cells.
The thrombopoietin receptor agonist eltrombopag was successfully used against human cytomegalovirus (HCMV)-associated thrombocytopenia refractory to immunomodulatory and antiviral drugs. These effects were ascribed to the effects of eltrombopag on megakaryocytes. Here, we tested whether eltrombopag may also exert direct antiviral effects. Therapeutic eltrombopag concentrations inhibited HCMV replication in human fibroblasts and adult mesenchymal stem cells infected with six different virus strains and drug-resistant clinical isolates. Eltrombopag also synergistically increased the anti-HCMV activity of the mainstay drug ganciclovir. Time-of-addition experiments suggested that eltrombopag interfered with HCMV replication after virus entry. Eltrombopag was effective in thrombopoietin receptor-negative cells, and the addition of Fe3+ prevented the anti-HCMV effects, indicating that it inhibits HCMV replication via iron chelation. This may be of particular interest for the treatment of cytopenias after hematopoietic stem cell transplantation, as HCMV reactivation is a major reason for transplantation failure. Since therapeutic eltrombopag concentrations are effective against drug-resistant viruses, and synergistically increase the effects of ganciclovir, eltrombopag is also a drug-repurposing candidate for the treatment of therapy-refractory HCMV diseas.
Mesenchymal stromal/stem cells (MSCs) are immature multipotent cells, which represent a rare population in the perivascular niche within nearly all tissues. The most abundant source to isolate MSCs is adipose tissue. Currently, perirenal adipose tissue is rarely described as the source of MSCs. MSCs were isolated from perirenal adipose tissue (prASCs) from patients undergoing tumor nephrectomies, cultured and characterized by flow cytometry and their differentiation potential into adipocytes, chondrocytes, osteoblasts and epithelial cells. Furthermore, prASCs were stimulated with lipopolysaccharide (LPS), lipoteichoic acid (LTA) or a mixture of cytokines (cytomix). In addition, prASC susceptibility to human cytomegalovirus (HCMV) was investigated. The expression of inflammatory readouts was estimated by qPCR and immunoassay. HCMV infection was analyzed by qPCR and immunostaining. Characterization of cultured prASCs shows the cells meet the criteria of MSCs and prASCs can undergo trilineage differentiation. Cultured prASCs can be induced to differentiate into epithelial cells, shown by cytokeratin 18 expression. Stimulation of prASCs with LPS or cytomix suggests the cells are capable of initiating an inflammation-like response upon stimulation with LPS or cytokines, whereas, LTA did not induce a significant effect on the readouts (ICAM-1, IL-6, TNFα, MCP-1 mRNA and IL-6 protein). HCMV broadly infects prASCs, showing a viral load dependent cytopathological effect (CPE). Our current study summarizes the isolation and culture of prASCs, clearly characterizes the cells, and demonstrates their immunomodulatory potential and high permissiveness for HCMV
The kidneys play a vital role in the basic physiological functions of the body. Kidney dysfunction impairs these physiological functions and can lead to a wide range of diseases. Damage to the kidney cells can be caused by a variety of ischemic, toxic or immunological complaints that lead to inflammation and cell death, which can lead to organ damage and, ultimately, complete failure. Although the mechanisms underlying acute kidney injury (AKI) and chronic kidney disease (CKD) are quite distinct, clinical evidence suggests that the two conditions are inextricably interconnected [1]. AKI and CKD, regardless of the underlying cause, have inflammation and activation of the immune system as the common underlying mechanisms. Inflammation, a process aimed, in principle, at detecting and fighting harmful pathogens, is, therefore, a major pathogenic mechanism for both AKI and CKD [1]. While the kidney has the remarkable ability to regenerate after an acute injury and can recover completely, depending on the type of kidney lesion, the options for clinical interventions are currently limited to fluid management and extracorporeal kidney support. However, persistent chronic inflammation can trigger renal fibrosis and chronic kidney disease. The investigation of the molecular mechanisms involved in each individual injury is currently insufficiently understood.
Pulmonary failure is the main cause of morbidity and mortality in the human chromosomal instability syndrome Ataxia-telangiectasia (A-T). Major phenotypes include recurrent respiratory tract infections and bronchiectasis, aspiration, respiratory muscle abnormalities, interstitial lung disease, and pulmonary fibrosis. At present, no effective pulmonary therapy for A-T exists. Cell therapy using adipose-derived mesenchymal stromal/stem cells (ASCs) might be a promising approach for tissue regeneration. The aim of the present project was to investigate whether ASCs migrate into the injured lung parenchyma of Atm-deficient mice as an indication of incipient tissue damage during A-T. Therefore, ASCs isolated from luciferase transgenic mice (mASCs) were intravenously transplanted into Atm-deficient and wild-type mice. Retention kinetics of the cells were monitored using in vivo bioluminescence imaging (BLI) and completed by subsequent verification using quantitative real-time polymerase chain reaction (qRT-PCR). The in vivo imaging and the qPCR results demonstrated migration accompanied by a significantly longer retention time of transplanted mASCs in the lung parenchyma of Atm-deficient mice compared to wild type mice. In conclusion, our study suggests incipient damage in the lung parenchyma of Atm-deficient mice. In addition, our data further demonstrate that a combination of luciferase-based PCR together with BLI is a pivotal tool for tracking mASCs after transplantation in models of inflammatory lung diseases such as A-T.
Stem cell-based therapies require cells with a maximum regenerative capacity in order to support regeneration after tissue injury and organ failure. Optimization of this regenerative potential of mesenchymal stromal/stem cells (MSC) or their conditioned medium by in vitro preconditioning regimens are considered to be a promising strategy to improve the release of regenerative factors. In the present study, MSC were isolated from inguinal adipose tissue (mASC) from C57BL/6 mice, cultured, and characterized. Then, mASC were either preconditioned by incubation in a hypoxic environment (0.5% O2), or in normoxia in the presence of murine epidermal growth factor (EGF) or tumor necrosis factor α (TNFα) for 48 h. Protein expression was measured by a commercially available array. Selected factors were verified by PCR analysis. The expression of 83 out of 308 proteins (26.9%) assayed was found to be increased after preconditioning with TNFα, whereas the expression of 61 (19.8%) and 70 (22.7%) proteins was increased after incubation with EGF or in hypoxia, respectively. Furthermore, we showed the proliferation-promoting effects of the preconditioned culture supernatants on injured epithelial cells in vitro. Our findings indicate that each preconditioning regimen tested induced an individual expression profile with a wide variety of factors, including several growth factors and cytokines, and therefore may enhance the regenerative potential of mASC for cell-based therapies.
