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Lipid mediators derived from omega (n)-3 and n-6 long-chain polyunsaturated fatty acids (LCPUFA) play key roles in bronchoconstriction, airway inflammation, and resolution processes in asthma. This study compared the effects of dietary supplementation with either a combination of LCPUFAs or eicosapentaenoic acid (EPA) alone to investigate whether the combination has superior beneficial effects on the outcome of asthmatic mice. Mice were sensitized with house dust mite (HDM) extract, and subsequently supplemented with either a combination of LCPUFAs or EPA alone in a recall asthma model. After the final HDM and LCPUFA administration, airway hyperresponsiveness (AHR), bronchoalveolar lavages, and lung histochemistry were examined. Lipid mediator profiles were determined by liquid chromatography coupled with tandem mass spectrometry (LC–MS–MS). The LCPUFA combination reduced AHR, eosinophilic inflammation, and inflammatory cytokines (IL-5, IFN-γ, and IL-6) in asthmatic mice, whereas EPA enhanced inflammation. The combination of LCPUFAs was more potent in downregulating EPA-derived LTB5 and LTC5 and in supporting DHA-derived RvD1 and RvD4 (2.22-fold and 2.58-fold higher levels) than EPA alone. Ex vivo experiments showed that LTB5 contributes to granulocytes’ migration and M1-polarization in monocytes. Consequently, the LCPUFA combination ameliorated airway inflammation by inhibiting adverse effects of EPA and promoting pro-resolving effects supporting the lipid mediator-dependent resolution program.

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.

Acute kidney injury is associated with mortality in COVID-19 patients. However, host cell changes underlying infection of renal cells with SARS-CoV-2 remain unknown and prevent understanding of the molecular mechanisms that may contribute to renal pathology. Here, we carried out quantitative translatome and whole-cell proteomics analyses of primary renal proximal and distal tubular epithelial cells derived from human donors infected with SARS-CoV-2 or MERS-CoV to disseminate virus and cell type-specific changes over time. Our findings revealed shared pathways modified upon infection with both viruses, as well as SARS-CoV-2-specific host cell modulation driving key changes in innate immune activation and cellular protein quality control. Notably, MERS-CoV infection-induced specific changes in mitochondrial biology that were not observed in response to SARS-CoV-2 infection. Furthermore, we identified extensive modulation in pathways associated with kidney failure that changed in a virus- and cell type-specific manner. In summary, we provide an overview of the effects of SARS-CoV-2 or MERS-CoV infection on primary renal epithelial cells revealing key pathways that may be essential for viral replication.

The biomedical consequences of allogeneic blood transfusions and the possible pathomechanisms of transfusion-related morbidity and mortality are still not entirely understood. In retrospective studies, allogeneic transfusion was associated with increased rates of cancer recurrence, metastasis and death in patients with colorectal cancer. However, correlation does not imply causation. The purpose of this study was to elucidate this empirical observation further in order to address insecurity among patients and clinicians. We focused on the in vitro effect of microparticles derived from red blood cell units (RMPs). We incubated different colon carcinoma cells with RMPs and analyzed their effects on growth, invasion, migration and tumor marker expression. Furthermore, effects on Wnt, Akt and ERK signaling were explored. Our results show RMPs do not seem to affect functional and phenotypic characteristics of different colon carcinoma cells and did not induce or inhibit Wnt, Akt or ERK signaling, albeit in cell culture models lacking tumor microenvironment. Allogeneic blood transfusions are associated with poor prognosis, but RMPs do not seem to convey tumor-enhancing effects. Most likely, the circumstances that necessitate the transfusion, such as preoperative anemia, tumor stage, perioperative blood loss and extension of surgery, take center stage.

Previous studies reported on the safety and applicability of mesenchymal stem/stromal cells (MSCs) to ameliorate pulmonary inflammation in acute respiratory distress syndrome (ARDS). Thus, multiple clinical trials assessing the potential of MSCs for COVID-19 treatment are underway. Yet, as SARS-inducing coronaviruses infect stem/progenitor cells, it is unclear whether MSCs could be infected by SARS-CoV-2 upon transplantation to COVID-19 patients. We found that MSCs from bone marrow, amniotic fluid, and adipose tissue carry angiotensin-converting enzyme 2 and transmembrane protease serine subtype 2 at low levels on the cell surface under steady-state and inflammatory conditions. We did not observe SARS-CoV-2 infection or replication in MSCs at steady state under inflammatory conditions, or in direct contact with SARS-CoV-2-infected Caco-2 cells. Further, indoleamine 2,3-dioxygenase 1 production in MSCs was not impaired in the presence of SARS-CoV-2. We show that MSCs are resistant to SARS-CoV-2 infection and retain their immunomodulation potential, supporting their potential applicability for COVID-19 treatment., Highlights • MSCs carry ACE2 and TMPRSS2 only at very low levels on the cell surface • Inflammatory conditions do not change ACE2 and TMPRSS2 expression on MSCs • MSCs are resistant to SARS-CoV-2 infection • MSCs retain their immunomodulation potential in the presence of SARS-CoV-2, Schäfer and colleagues found that human MSCs carry ACE 2 and TMPRSS2 at very low levels on the cell surface under steady-state and inflammatory conditions. Using their in vitro model for infection with SARS-CoV-2 from clinical isolates they show that MSC are resistant to SARS-CoV-2 infection and retain their immunomodulation potential supporting their potential applicability for COVID-19 treatment.

Lung failure is responsible for significant morbidity and is a frequent cause of death in ataxia-telangiectasia (A-T). Disturbance in the redox balance of alveolar epithelial cells must be considered as a causal factor for respiratory disease in A-T. To investigate bronchoalveolar sensitivity to reactive oxygen species (ROS) and ROS-induced DNA damage, we used bleomycin (BLM) to induce experimental inflammation and fibrotic changes in the Atm-deficient mouse model.BLM or saline was administered by oropharyngeal instillation into the lung of Atm-deficient mice and wild-type mice. Mice underwent pulmonary function testing at days 0, 9, and 28, and bronchoalveolar lavage (BAL) was analysed for cell distribution and cytokines. Lung tissue was analysed by histochemistry.BLM administration resulted in a tremendous increase in lung inflammation and fibrotic changes in the lung tissue of Atm-deficient mice and was accompanied by irreversible deterioration of lung function. ATM (ataxia telangiectasia mutated) deficiency resulted in reduced cell viability, a delay in the resolution of γH2AX expression and a significant increase in intracellular ROS in pulmonary epithelial cells after BLM treatment. This was confirmed in the human epithelial cell line A549 treated with the ATM-kinase inhibitor KU55933.Our results demonstrate high bronchoalveolar sensitivity to ROS and ROS-induced DNA damage in the Atm-deficient mouse model and support the hypothesis that ATM plays a pivotal role in the control of oxidative stress-driven lung inflammation and fibrosis. Keywords: Pulmonary inflammation, Lung fibrosis, Mice, Oxidative stress

Acute kidney injury remains an important cause of renal dysfunction. In this context, Toll-like receptors have been demonstrated to play a critical role in the induction of innate and inflammatory responses. Among these, Toll-like receptor 2 (TLR2) is constitutively expressed in tubular epithelial cells (TECs) of the kidney and is also known to mediate ischaemia reperfusion (IR) injury. Adult male C57BL/6JRj mice were randomized into seven groups (n = 8): a non-operative control group (CTRL) and six interventional groups in which mice were subjected to a 30 min. bilateral renal ischaemia. Immediately before reperfusion, mice were treated either with saline or with TLR2 antibody (clone T2.5) and harvested after ischaemia and reperfusion for 3, 24 and 48 hr. Analysed kidney homogenates of TLR2 antibody-treated mice displayed significantly decreased levels of TLR2 protein after 3 hr of IR compared to saline-treated mice. Accordingly, the degree of AKT phosphorylation was significantly decreased after 3 hr of IR compared to saline-treated animals. TUNEL staining revealed significantly higher apoptosis rates in TLR2 antibody-treated animals compared to saline-treated mice after 3 and 24 hr of IR. Further, a positive correlation between TLR2 protein expression and phosphorylation of AKT as well as a negative correlation with the number of TUNEL-positive cells could be observed. Inhibition of TLR2 and its signalling pathway by a single application of TLR2 antibody results in reduced phosphorylation of AKT and consecutively increased apoptosis.

Lipocalin-2 (Lcn-2) is rapidly upregulated in macrophages after renal tubular injury and acts as renoprotective and pro-regenerative agent. Lcn-2 possesses the ability to bind and transport iron with high affinity. Therefore, the present study focuses on the decisive role of the Lcn-2 iron-load for its pro-regenerative function. Primary mouse tubular epithelial cells were isolated from kidney tissue of wildtype mice and incubated with 5&mu, M Cisplatin for 24h to induce injury. Bone marrow-derived macrophages of wildtype and Lcn-2-/- mice were isolated and polarized with IL-10 towards an anti-inflammatory, iron-release phenotype. Their supernatants as well as recombinant iron-loaded holo-Lcn-2 was used for stimulation of Cisplatin-injured tubular epithelial cells. Incubation of tubular epithelial cells with wildtype supernatants resulted in less damage and induced cellular proliferation, whereas in absence of Lcn-2 no protective effect was observed. Epithelial integrity as well as cellular proliferation showed a clear protection upon rescue experiments applying holo-Lcn-2. Notably, we detected a positive correlation between total iron amounts in tubular epithelial cells and cellular proliferation, which, in turn, reinforced the assumed link between availability of Lcn-2-bound iron and recovery. We hypothesize that macrophage-released Lcn-2-bound iron is provided to tubular epithelial cells during toxic cell damage, whereby injury is limited and recovery is favored.

Damage to kidney cells can occur due to a variety of ischemic and toxic insults and leads to inflammation and cell death, which can result in acute kidney injury (AKI) [...]

Gliflozins are inhibitors of the renal proximal tubular sodium-glucose co-transporter-2 (SGLT-2), that inhibit reabsorption of urinary glucose and they are able to reduce hyperglycemia in patients with type 2 diabetes. A renoprotective function of gliflozins has been proven in diabetic nephropathy, but harmful side effects on the kidney have also been described. In the current project, primary highly purified human renal proximal tubular epithelial cells (PTCs) have been shown to express functional SGLT-2, and were used as an in vitro model to study possible cellular damage induced by two therapeutically used gliflozins: empagliflozin and dapagliflozin. Cell viability, proliferation, and cytotoxicity assays revealed that neither empagliflozin nor dapagliflozin induce effects in PTCs cultured in a hyperglycemic environment, or in co-medication with ramipril or hydro-chloro-thiazide. Oxidative stress was significantly lowered by dapagliflozin but not by empagliflozin. No effect of either inhibitor could be detected on mRNA and protein expression of the pro-inflammatory cytokine interleukin-6 and the renal injury markers KIM-1 and NGAL. In conclusion, empa- and dapagliflozin in therapeutic concentrations were shown to induce no direct cell injury in cultured primary renal PTCs in hyperglycemic conditions.

Background We aimed at clarifying the role of lipocalin-2 (LCN-2) in clear-cell renal cell carcinoma (ccRCC). Since LCN-2 was recently identified as a novel iron transporter, we explored its iron load as a decisive factor in conferring its biological function. Methods LCN-2 expression was analysed at the mRNA and protein level by using immunohistochemistry, RNAscope® and qRT-PCR in patients diagnosed with clear-cell renal cell carcinoma compared with adjacent healthy tissue. We measured LCN-2-bound iron by atomic absorption spectrometry from patient-derived samples and applied functional assays by using ccRCC cell lines, primary cells, and 3D tumour spheroids to verify the role of the LCN-2 iron load in tumour progression. Results LCN-2 was associated with poor patient survival and LCN-2 mRNA clustered in high- and low-expressing ccRCC patients. LCN-2 protein was found overexpressed in tumour compared with adjacent healthy tissue, whereby LCN-2 was iron loaded. In vitro, the iron load determines the biological function of LCN-2. Iron-loaded LCN-2 showed pro-tumour functions, whereas iron-free LCN-2 produced adverse effects. Conclusions We provide new insights into the pro-tumour function of LCN-2. LCN-2 donates iron to cells to promote migration and matrix adhesion. Since the iron load of LCN-2 determines its pro-tumour characteristics, targeting either its iron load or its receptor interaction might represent new therapeutic options.

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 disease., publishedVersion

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.

Multiple preclinical and clinical studies demonstrate the potential of mesenchymal stem/stromal cells (MSCs) to support organ and tissue regeneration. Besides transplantation of cellular MSC grafts, conditioned media from MSC cultures or extracellular vesicles (EVs) derived from MSCs show similar regenerative effects. In vitro pretreatment of MSCs affects their paracrine properties and can improve their reparative and regenerative capacities. In this chapter effects of MSC pretreatment on MSC-EVs and different strategies to increase their release and to improve their therapeutic efficacy will be discussed.

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 &alpha, (TNF&alpha, ) 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 TNF&alpha, 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.

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.

Early and adequate restoration of endothelial and tubular renal function is a substantial step during regeneration after ischemia reperfusion (IR) injury, occurring, e.g., in kidney transplantation, renal surgery, and sepsis. While tubular epithelial cell injury has long been of central importance, recent perception includes the renal vascular endothelium. In this regard, the fibrin cleavage product fibrinopeptide Bβ15-42 mitigate IR injury by stabilizing interendothelial junctions through its affinity to VE-cadherin. Therefore, this study focused on the effect of Bβ15-42 on post-acute physiological renal regeneration. For this, adult male C57BL/6 mice were exposed to a 30 min bilateral renal ischemia and reperfusion for 24 h or 48 h. Animals were randomized in a non-operative control group, two operative groups each treated with i.v. administration of either saline or Bβ15-42 (2.4 mg/kg) immediately prior to reperfusion. Endothelial activation and inflammatory response was attenuated in renal tissue homogenates by single application of Bβ15-42. Meanwhile, Bβ15-42 did not affect acute kidney injury markers. Regarding the angiogenetic players VEGF-A, Angiopoietin-1, Angiopoietin-2, however, we observed significant higher expressions at mRNA and trend to higher protein level in Bβ15-42 treated mice, compared to saline treated mice after 48 h of IR, thus pointing toward an increased angiogenetic activity. Similar dynamics were observed for the intermediate filament vimentin, the cytoprotective protein klotho, stathmin and the proliferation cellular nuclear antigen, which were significantly up-regulated at the same points in time. These results suggest a beneficial effect of anatomical contiguously located endothelial cells on tubular regeneration through stabilization of endothelial integrity. Therefore, it seems that Bβ15-42 represents a novel pharmacological approach in the targeted therapy of acute renal failure in everyday clinical practice.

The development of new strategies to preserve renal function after acute kidney injury (AKI) is necessary due to limited clinical intervention options. The organ-protective effects of mesenchymal stromal/stem cells (MSCs) and their conditioned medium (CM) have been investigated demonstrating that both separately promoted tubular recovery and ameliorated the outcome of AKI. Nevertheless, strategies to optimise the regenerative potential of both are highly needed. Here we investigated the effects of CM from adipose-derived MSCs (ASCs) preincubated in a hypoxic environment (Hyp). Protective factors were investigated by PCR analysis and a protein array in vitro. The expression of 64 of the 308 proteins assayed was found to be more than two-fold increased after Hyp. CM of Hyp-pretreated ASCs (pCM) was used to enhance regeneration in a mouse model of cisplatin-induced AKI (cisAKI). Renal function was assessed by measurements of markers for AKI and serum cytokine levels. The pCM significantly ameliorated serum creatinine and neutrophil gelatinase-associated lipocalin values, and also the levels of inflammatory cytokines IL-1β and IL-6 in the serum of mice with AKI. Our work clearly showed that a Hyp preconditioning significantly increases the release of protective factors in ASCs and enhances the therapeutic effects of CM in cisAKI in mice.

Mesenchymal stem/stromal cells (MSCs) are becoming increasingly important for the development of cell therapeutics in regenerative medicine. Featuring immunomodulatory potential as well as secreting a variety of trophic factors, MSCs showed remarkable therapeutic effects in numerous preclinical disease models. However, sustainable translation of MSC therapies to the clinic is hampered by heterogeneity of MSCs and non-standardized in vitro culture technologies. Moreover, potent MSC therapeutics require MSCs with maximum regenerative capacity. There is growing evidence that in vitro preconditioning strategies of MSCs can optimize their therapeutic potential. In the following we will discuss achievements and challenges of the development of MSC therapies in regenerative medicine highlighting specific in vitro preconditioning strategies prior to cell transplantation to increase their therapeutic efficacy.

Ataxia telangiectasia (A-T) is a highly pleiotropic disorder. Patients suffer from progressive neurodegeneration, severe bronchial complications, immunodeficiency, hypersensitivity to radiotherapy and elevated risk of malignancies. Leukemia and lymphoma, along with lung failure, are the main causes of morbidity and mortality in A-T patients. At present, no effective therapy for A-T exists. One promising therapeutic approach is bone marrow transplantation (BMT) that is already used as a curative therapy for other genomic instability syndromes. We used an established clinically relevant non-myeloablative host-conditioning regimen and transplanted green fluorescent protein (GFP)-expressing ataxia telangiectasia mutated (ATM)-competent bone marrow-derived cells (BMDCs) into Atm-deficient mice. GFP expression allowed tracking of the potential migration of the cells into the tissues of recipient animals. Donor BMDCs migrated into the bone marrow, blood, thymus, spleen and lung tissue of Atm-deficient mice showing an ATM-competent phenotype. BMT inhibited thymic lymphomas, normalized T-lymphocyte populations, improved weight gain and rearing activity of Atm-deficient mice. In contrast, no GFP(+) cells were found in the cerebellum or cerebrum, and we detected decreased size index in MRI imaging of the cerebellum in 8-month-old transplanted Atm-deficient mice in comparison to wild-type mice. The repopulation with ATM-competent BMDCs is associated with a prolonged lifespan and significantly improved the phenotype of Atm-deficient mice.

Isolation of epithelial cells for cell culture is based on destruction of epithelial integrity. The consequences are manifold: cell polarity and specific cell functions are lost; cells acquire non-epithelial characteristics and start to proliferate. This situation may also occur in situ when parts of the epithelium are lost, either by apoptosis or necrosis by organ or tissue injury. During recovery from this injury, surviving epithelial cells proliferate and may restore epithelial integrity and finally re-differentiate into functional epithelial cells. In vitro, this re-differentiation is mostly not complete due to sub-optimal culture conditions. Therefore cultured epithelial cells resemble wounded or injured epithelia rather than healthy and well differentiated epithelia. The value of an in vitro cell model is the extent to which it helps to understand the function of the cells in situ. A variety of parameters influence the state of differentiation of cultured cells in vitro. Although each of these parameters had been studied, the picture how they co-ordinately influence the state of differentiation of epithelial cells in vitro is incomplete. Therefore we discuss the influence of the isolation method and cell culture on epithelial cells, and outline strategies to achieve highly differentiated epithelial cells for the use as an in vitro model.

Although many studies using stem cells as therapeutic agents after renal failure have been published in recent years, our knowledge of the factors involved and the cellular mechanisms underlying their beneficial effect on organ regeneration is incomplete. A growing insight into these interactions would help to utilize the biological potential of stem cells for therapeutic approaches. It is here hypothesized that soluble factors released by tubular epithelial cells (TECs) induce epithelial differentiation in adipose-derived adult mesenchymal stem cells (ASCs). ASCs were therefore cultured in conditioned medium (CM) derived from TECs and the changes in expression genes towards an epithelial pattern were determined by microarray and qPCR analyses. The changes in gene expression were evaluated using Affymetrix HG-U133 Plus 2.0 arrays. Microarray-based screening revealed 117 genes differentially expressed in a significant manner after short-time incubation (3 days) of ASCs with CM, and four of these were solute carriers (SLCs). Changes in mRNA expression of these SLCs were verified by qPCR at several time points, additionally with four stem cell factors and five epithelial markers. qPCR analyses showed that expression of three of the SLCs rose significantly, whereas three of the four stem cell markers analysed decreased during 7 days of CM incubation. Moreover, a robust expression of three characteristic epithelial markers (cytokeratin 18, ZO-1 and ZO-2) was observed after 17 days. These changes in the expression patterns strongly indicate differentiation towards the epithelial lineage. The capability of ASCs to differentiate into epithelial cells may be important in organ repair mechanisms. Copyright © 2011 John Wiley & Sons, Ltd.

Adipose-derived stem cells (ASCs) possess a multilineage differentiation potential, can be used from an autologous origin, and are, therefore, attractive candidates for clinical applications to repair or regenerate damaged tissues and organs. Adipose tissue as a stem cell source is ubiquitously available and has several advantages compared with other sources. It is easily accessible in large quantities with a minimal invasive harvesting procedure, and the isolation of ASCs yields a high amount of stem cells, which is essential for stem cell-based therapies and tissue engineering. Differentiation of ASCs into cell types of mesodermal origin has been shown in a variety of studies. The plasticity of ASCs toward cells of the mesodermal lineage has been shown by their differentiation into chondrocytes, osteoblasts, adipocytes, and myocytes. Their potential to differentiate into lineages with nonmesodermal origin is even more exciting: ASCs are also able to differentiate into cells of ecto- and endodermal origin. Various in vitro and in vivo studies documented the induced differentiation into neural cells, hepatocytes, pancreatic islet cells, endothelial cells, and epithelial cells. Epithelial cells can embryologically arise from each of the 3 germ layers. This article summarizes and discusses the current knowledge of the potential of ASCs to differentiate into the epithelial lineage. The differentiation of ASCs into different types of epithelial cells, including hepatocytes, pancreatic cells, and endothelial cells, is highlighted together with a view on current clinical trials and future options.

Background aims The potential of cultured adipose-derived stem cells (ASC) in regenerative medicine and new cell therapeutic concepts has been shown recently by many investigations. However, while the method of isolation of ASC from liposuction aspirates depending on plastic adhesion is well established, a standard expansion medium optimally maintaining the undifferentiated state has not been described. Methods We cultured ASC in five commonly used culture media (two laboratory-made media and three commercially available media) and compared them with a standard medium. We analyzed the effects on cell morphology, proliferation, hepatocyte growth factor (HGF) expression, stem cell marker profile and differentiation potential. Proliferation was measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a fluorescent assay. Release of HGF was assessed by an immunoassay. Expression of characteristic stem cell-related transcription factors and markers was evaluated by quantitative polymerase chain reaction (qPCR) (Nanog, Sox-2, Rex-1, nestin and Oct-4) and flow cytometry (CD44, CD73, CD90, CD105 and CD166), and differentiation was shown by adipogenic medium. Results The morphology and expansion of ASC were significantly affected by the media used, whereas none of the media influenced the ASC potential to differentiate into adipocytes. Furthermore, two of the media induced an increase in expression of transcription factors, an increased secretion of HGF and a decrease in CD105 expression. Conclusions Culture of ASC in one of these two media before using the cells in cell therapeutic approaches may have a benefit on their regenerative potential.

High hopes are put into the use of mesenchymal stem cells (MSCs) in various approaches for tissue engineering and regenerative medicine. MSCs are derived from different tissues with only small differences in their phenotype or their differentiation potential, but higher differences in the cell yield. Since fat is easily accessible and contains a high amount of MSCs to be isolated, adipose-derived stem cells (ASCs) are very promising for clinical approaches. ASCs are not a completely homogeneous cell population. Our study was initiated to explore an easy and convenient method to reduce heterogeneity. We tested different isolation methods: (1) the standard isolation method for ASCs based on plastic attachment, (2) the standard method with an initial washing step after 60 min of adherence and (3) immunomagnetic isolation by 4 typical markers (CD49a, CD90, CD105 and CD271). Cells isolated by these methods were evaluated using quantitative PCR and flow cytometry as well as by their differentiation potential. Washing led to a significantly lower expression of desmin, smA and six2, and a higher expression of the stem cell markers nestin, oct-4 and sall-1, compared to standard isolated cells, while the immunomagnetically isolated cells showed no significant changes. All cells independent of the isolation method could be induced to differentiate into adipocytes and osteoblasts. Our study demonstrates that a simple washing step reduces heterogeneity of cultured ASCs according to PCR analysis, whereas the immunomagnetic isolation only showed minor advantages compared to the standard method, but the disadvantage of significantly lower cell yields in the primary isolates.

Background: Cluster specific immunotherapy (SIT) is a modern form of allergen immunotherapy allowing safe administration of high allergen doses in a short time interval compared to classic SIT. In the current study, we investigated the safety profile and immunological effect of cluster SIT in children with allergic asthma due to house dust mite allergy. Methods: A total of 34 children (6–18 years) with allergic asthma were assigned to cluster (n = 22) or classic SIT (n = 12). To achieve a maintenance dose of allergen extract, cluster patients received 14 injections of house dust mite allergen within 6 weeks, whereas the classic SIT group received 14 injections within 14 weeks. Safety was monitored by recording adverse events. Immunogenicity was measured by specific IgGMite and IgG4Mite, by antibody-blocking properties on basophil activation, and by the T cell subset transcription factors Foxp3, T-bet, and GATA-3. Results: There were no significant differences in local and systemic side effects between the two groups. In the cluster group, serum levels of specific IgGMite (p < 0.001) and specific IgG4Mite (p < 0.001) significantly increased after 8 weeks, while it took 12 weeks in the classic SIT group. These data were confirmed by blocking CD63 expression as well as release of cysteinyl leukotrienes after in vitro basophil stimulation. No differences in transcription factor expression were found in the two groups. Conclusion: Cluster SIT is safe in children. Additionally, our data demonstrated an even more rapid induction of specific immune tolerance. Cluster SIT is an attractive alternative to conventional up-dosing schedules with fewer consultations for the patients.

Objective: Stem cell-based therapy seems to be a promising option to support regeneration after organ failure and tissue injury. The transplantation of MSCs requires cells with a maximum regenerative capacity. Therefore, the research on new strategies to improve the release of regenerative factors of MSCs is urgently needed. Methods: Human adipose-derived stromal/stem cells (ASC) were isolated from lipoaspirates, characterized, and cultured. Cells were either cultured under standard conditions or preconditioned by incubation in a hypoxic environment (0.5% O2) or in normoxia in the presence of recombinant human tumor necrosis factor-α (TNFα) or recombinant human epidermal growth factor (EGF) for 48 hours. First, seven selected regeneration promoting factors were evaluated by qPCR analysis. Afterwards the secretome of ASCs was estimated using a commercially available protein array for 507 proteins. Results: PCR analysis showed a differential induction of ASCs` gene expression by the three pretreatments. Whereas ASCs in hypoxia showed a significant mRNA induction of VEGF, FGF-7, and IGF-II, the other pretreatments induced no significant change in VEGF expression. The gene expression of HB-EGF and M-CSF was significantly induced in hypoxia and by incubation with TNFα, but not EGF. Angiopoietin-like 1 mRNA was not significantly induced by all three preconditioning regimens. Evaluation by the protein array revealed that from the 507 proteins investigated 21.9% were found to be more than five-fold increased after incubation in hypoxia (111 of 507 proteins). Preincubation with EGF resulted in an upregulation of 32.3% (164/507), whereas TNFα upregulated 28.8% of all proteins evaluated (146/507). Conclusion: The findings indicate that all three preconditioning regimens induced a wide variety of proteins. However, short-term pretreatment with EGF induced the highest quantity of proteins, and, therefore, appears to be the best preconditioning regime for cell therapeutic approaches.

Objective The aim of the present study was to examine the immune-modulating effect of two different fat blends enriched with a low dose of anti- or proinflammatory polyunsaturated fatty acids on the fatty acid status and subsequently on the immune response of healthy volunteers. Methods Thirty healthy volunteers were randomly assigned to group A (anti-inflammatory blend rich in polyunsaturated fatty acids: α-linolenic acid, 240 mg/d; eicosapentaenoic acid, 120 mg/d; stearidonic acid, 49 mg/d; and γ-linolenic acid, 73 mg/d) or group B (arachidonic acid, 40 mg/d; containing an inflammatory fat blend) for a 2-wk dietary supplementation period. Concentrations of interleukin-8, interleukin-10, tumor necrosis factor-α, prostaglandins E 1 and E 2 , and leukotriene B 4 were investigated before, after 2 wk of supplementation, and 2 wk after stopping supplementation using a whole blood ex vivo lipopolysaccharide-stimulation assay. Results Plasma concentrations of α-linolenic acid and eicosapentaenoic acid were significantly increased in group A. In addition, dietary fat blends influenced eicosapentaenoic acid concentration in erythrocyte membranes. Supplementation of the fat blends resulted in contrasting effects on the expression of lipid mediators and cytokines after ex vivo lipopolysaccharide stimulation. Release of prostaglandin E 1 and leukotriene B 4 were significantly decreased in group A, whereas prostaglandin E 2 and interleukin-10 concentrations were significantly increased in group B. No effect on interleukin-8 or tumor necrosis factor-α release was found after supplementation with either fat blend. Conclusions These results show an immune-modulating effect of a low-dose dietary polyunsaturated fatty acid supplementation. However, further studies regarding fat-blend composition and period of supplementation in patients with inflammatory conditions are required.

Ataxia telangiectasia is a genetic instability syndrome characterized by neurodegeneration, immunodeficiency, severe bronchial complications, hypersensitivity to radiotherapy and an elevated risk of malignancies. Repopulation with ATM-competent bone marrow-derived cells (BMDCs) significantly prolonged the lifespan and improved the phenotype of Atm-deficient mice. The aim of the present study was to promote BMDC engraftment after bone marrow transplantation using low-dose irradiation (IR) as a co-conditioning strategy. Atm-deficient mice were transplanted with green fluorescent protein-expressing, ATM-positive BMDCs using a clinically relevant non-myeloablative host-conditioning regimen together with TBI (0.2-2.0 Gy). IR significantly improved the engraftment of BMDCs into the bone marrow, blood, spleen and lung in a dose-dependent manner, but not into the cerebellum. However, with increasing doses, IR lethality increased even after low-dose IR. Analysis of the bronchoalveolar lavage fluid and lung histochemistry revealed a significant enhancement in the number of inflammatory cells and oxidative damage. A delay in the resolution of γ-H2AX-expression points to an insufficient double-strand break repair capacity following IR with 0.5 Gy in Atm-deficient splenocytes. Our results demonstrate that even low-dose IR results in ATM activation. In the absence of ATM, low-dose IR leads to increased inflammation, oxidative stress and lethality in the Atm-deficient mouse model.

Background: The culture of human renal tubular cells of well-defined nephron origin is an important basis in the research of various physiological and pathophysiological mechanisms in the kidney. In vitro differentiation of cultured cells is affected by cell isolation and culture conditions. Our study describes in vitro differentiation of cultured human renal proximal and distal (thick ascending limb and early distal) tubular epithelial cells. Methods: Proximal tubular cells (PTC) and early distal tubular cells (DTC) were isolated immunomagnetically and cultured. In vitro differentiation was assessed by Western blot analysis and reverse transcriptase polymerase chain reaction using characteristic markers. Morphologic characterization was shown by fluorescence and scanning electron microscopy. Results: E-cadherin was highly expressed in DTC, whereas expression was low in PTC. In contrast to DTC, in PTC, intercellular adhesion molecule 1 and aquaporin 1 were constitutively expressed at high levels. Both cell types expressed cytokeratin 18 and Na-K-ATPase but not α-smooth muscle actin. Morphological analysis showed that PTC develop long microvilli, whereas DTC formed short microvilli in culture, and both express the tight junction protein zona occludens protein 1. Conclusions: We demonstrate differential expression of E-cadherin, intercellular adhesion molecule 1 and aquaporin 1 in cultured PTC and DTC as described for the renal tubule in vivo. We could show the in vitro differentiation of the cells, and thus, their usefulness as an in vitro model of the human proximal and early distal tubule.

Background. Caspofungin (CAS) has recently been approved for treatment of invasive aspergillosis. In clinical trials, CAS-induced nephrotoxicity was markedly less pronounced compared to amphotericin B (AmB). Nevertheless, in a recent trial, nephrotoxicity in CAS-treated patients was considerably more pronounced than in preceding studies. Therefore, the aim of this study was to assess toxic effects of CAS on human renal proximal and distal tubular epithelial cells (PTC and DTC) in vitro, and to compare them to those of AmB. Methods. Cells were isolated from human kidney tissue, and exposed to clinically relevant concentrations of CAS and AmB for 24 h. Total DNA content and cell viability were determined by DAPI staining and a modified MTT assay. For testing of cytotoxicity, LDH activity was measured in cell culture supernatants. To assess apoptotic effects, AnnexinV-binding assay and DAPI staining for detection of fragmented DNA were performed. Results. DTC were more vulnerable towards the antifungal agents than PTC. In contrast to AmB, cell-damaging effects of CAS were less severe. DAPI staining revealed slight and dose-dependent antiproliferative effects of CAS at concentrations reflecting relevant plasma levels. At these concentrations, cell viability, determined by MTT assay, was not decreased in PTC and DTC. LDH release was marginally increased in a dose-dependent manner; apoptosis was not detected. Nevertheless, at CAS concentrations reflecting potential tissue concentrations, cell damaging effects were considerably more pronounced. Conclusion. Our results suggest that CAS is less nephrotoxic than AmB in vitro. The antiproliferative and cytotoxic effects of CAS predominantly affect DTC, which seem to be more susceptible to CASinduced damage.

Initial in vitro investigations demonstrated type I interferons (IFNs: IFN-alpha, IFN-beta) to inhibit replication of SARS coronavirus (SARS-CoV), but found the nucleoside analogue ribavirin ineffective in Vero cells. In this report, ribavirin was shown to inhibit SARS-CoV replication in five different cell types of animal or human origin at therapeutically achievable concentrations. Since clinical anti-SARS-CoV activity of type I interferons or ribavirin is limited, we investigated the combination of IFN-beta and ribavirin. Determination of the virus yield indicated highly synergistic anti-SARS-CoV action of the combination suggesting the consideration of ribavirin plus IFN-beta for the treatment of SARS.

BACKGROUND Interleukin-6 (IL-6) is a multifunctional cytokine which regulates immune responses and host defence mechanisms. IL-6 has been found to be increased in certain inflammatory conditions of the kidney, in which tubular epithelial cells play a pivotal role. Human renal tubular cells express IL-6. Until now no data about the effect of the immunosuppressant drug mycophenolic acid (MPA) on IL-6 expression were available. METHODS Proximal and distal tubular epithelial cells (PTC/DTC) have been isolated immunomagnetically. Confluent monolayers were stimulated with interleukin-1beta (IL-1beta; 25 U/ml), IL-1beta+ MPA (0.25-50 micro M) or MPA alone for 48 h. Release of IL-6 protein into the supernatant was evaluated with an enzyme immunoassay, IL-6 mRNA expression was evaluated using the Quantikine mRNA kit. RESULTS After IL-1beta stimulation, a highly significant 2.6- (PTC) and 3.8-fold (DTC) upregulation of IL-6 expression was detectable. IL-6 mRNA was upregulated by IL-1beta [1.57- (PTC) and 2.03-fold (DTC)]. MPA inhibited this cytokine-induced IL-6 expression in a dose-dependent manner. Incubation with the lowest MPA concentration had no effect on the stimulated upregulation, whereas all higher doses significantly decreased IL-6 expression. Dexamethasone significantly inhibited the cytokine-induced IL-6 protein release in PTC, but not in DTC. CONCLUSIONS In this study we demonstrated for the first time an inhibitory effect of MPA on the stimulated IL-6 expression of renal tubular epithelial cells. In contrast to older data, which showed a synergistic upregulation of the expression of a CC-chemokine by a combination of cytokines and MPA, in the present study we could demonstrate an immunosuppressive effect of MPA on the expression of an important cytokine.

BACKGROUND Expression of proinflammatory molecules by tubular epithelial cells plays an important role in renal allograft rejection and inflammatory kidney diseases. Different studies from patients with acute rejection point to the involvement of distal tubular segments. At present no in vitro system for the human distal tubule is established. METHODS Human distal tubular cells were isolated immunomagnetically. Cultured cells were stimulated with cytokines (interferon-gamma, tumor necrosis factor-alpha, interleukin-1beta, or a cytokine mix). Secretion of RANTES (regulated upon activation, normal T-cell expressed and secreted) was evaluated with an enzyme-linked immunoassay. Expression of HLA-DR and intercellular adhesion molecule (ICAM)-1 was assessed by flow cytometric analysis and immunofluorescence studies. RESULTS Our data clearly indicate that distal tubular cells express RANTES, HLA-DR, and ICAM-1 in response to a mixture of specific cytokines. Dexamethasone inhibited the induced expression of RANTES and HLA-DR significantly, but not that of ICAM-1. CONCLUSIONS We demonstrate an appropriate in vitro system for the human distal tubule. The present study proves the involvement of the distal tubular segment during inflammatory kidney diseases.