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Solid tumors are characterized by hypoxic areas, which are prone for macrophage infiltration. Once infiltrated, macrophages polarize to tumor associated macrophages (TAM) to support tumor progression. Therefore, the crosstalk between TAMs and tumor cells is of current interest for the development of novel therapeutic strategies. These may comprise induction of an iron- and lipid peroxidation-dependent form of cell death, known as ferroptosis. To study the macrophage - tumor cell crosstalk we polarized primary human macrophages towards a TAM-like phenotype, co-cultured them with HT1080 fibrosarcoma cells, and analyzed the tumor cell response to ferroptosis induction. In TAMs the expression of ceruloplasmin mRNA increased, which was driven by hypoxia inducible factor 2 and signal transducer and activator of transcription 1. Subsequently, ceruloplasmin mRNA was transferred from TAMs to HT1080 cells via extracellular vesicles. In tumor cells, mRNA was translated into protein to protect HT1080 cells from RSL3-induced ferroptosis. Mechanistically this was based on reduced iron abundance and lipid peroxidation. Interestingly, in naïve macrophages also hypoxia induced ceruloplasmin under hypoxia and a co-culture of HT1080 cells with hypoxic macrophages recapitulated the protective effect observed in TAM co-cultures. In conclusion, TAMs provoke tumor cells to release iron and thereby protect them from lipid peroxidation/ferroptosis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Marburg virus, a member of the Filoviridae, is the causative agent of Marburg virus disease (MVD), a hemorrhagic fever with a case fatality rate of up to 90 %. Acute kidney injury is common in MVD and is associated with increased mortality, but its pathogenesis in MVD remains poorly understood. Interestingly, autopsies show the presence of viral proteins in different parts of the nephron, particularly in proximal tubular cells (PTC). These findings suggest a potential role for the virus in the development of MVD-related kidney injury. To shed light on this effect, we infected primary human PTC with Lake Victoria Marburg virus and conducted transcriptomic analysis at multiple time points. Unexpectedly, infection did not induce marked cytopathic effects in primary tubular cells at 20 and 40 h post infection. However, gene expression analysis revealed robust renal viral replication and dysregulation of genes essential for different cellular functions. The gene sets mainly downregulated in PTC were associated with the targets of the transcription factors MYC and E2F, DNA repair, the G2M checkpoint, as well as oxidative phosphorylation. Importantly, the downregulated factors comprise PGC-1α, a well-known factor in acute and chronic kidney injury. By contrast, the most highly upregulated gene sets were those related to the inflammatory response and cholesterol homeostasis. In conclusion, Marburg virus infects and replicates in human primary PTC and induces downregulation of processes known to be relevant for acute kidney injury as well as a strong inflammatory response., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Introduction: Influenza A virus (IAV) infection can cause the often-lethal acute respiratory distress syndrome (ARDS) of the lung. Concomitantly, acute kidney injury (AKI) is frequently noticed during IAV infection, correlating with an increased mortality. The aim of this study was to elucidate the interaction of IAV with human kidney cells and, thereby, to assess the mechanisms underlying IAV-mediated AKI., Methods: To investigate IAV effects on nephron cells we performed infectivity assays with human IAV, as well as with human isolates of either low or highly pathogenic avian IAV. Also, transcriptome and proteome analysis of IAV-infected primary human distal tubular kidney cells (DTC) was performed. Furthermore, the DTC transcriptome was compared to existing transcriptomic data from IAV-infected lung and trachea cells., Results: We demonstrate productive replication of all tested IAV strains on primary and immortalized nephron cells. Comparison of our transcriptome and proteome analysis of H1N1-type IAV-infected human primary distal tubular cells (DTC) with existing data from H1N1-type IAV-infected lung and primary trachea cells revealed enrichment of specific factors responsible for regulated cell death in primary DTC, which could be targeted by specific inhibitors., Discussion: IAV not only infects, but also productively replicates on different human nephron cells. Importantly, multi-omics analysis revealed regulated cell death as potential contributing factor for the clinically observed kidney pathology in influenza., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Koch, Shehata, Müller-Ruttloff, Gouda, Wetzstein, Patyna, Scholz, Schmid, Dietrich, Münch, Ziebuhr, Geiger, Martinez-Sobrido, Baer, Mostafa and Pleschka.)

Renal proximal tubular epithelial cells (PTCs) are central players during renal inflammation. In response to inflammatory signals, PTCs not only self-express altered mRNAs, microRNAs (miRNAs), proteins, and lipids, but also release altered extracellular vesicles (EVs). These EVs also carry inflammation-specific cargo molecules and are key players in cell-cell-communication. Understanding the precise molecular and cellular mechanisms that lead to inflammation in the kidney is the most important way to identify early targets for the prevention or treatment of acute kidney injury. Therefore, highly purified human PTCs were used as an in vitro model to study the cellular response to an inflammatory microenvironment. A cytokine-induced inflammatory system was established to analyze different miRNA expression in cells and their EVs. In detail, we characterized the altered miR expression of PTCs and their released EVs during induced inflammation and showed that 12 miRNAs were significantly regulated in PTCs (6 upregulated and 6 downregulated) and 9 miRNAs in EVs (8 upregulated and 1 downregulated). We also showed that only three of the miRNAs were found to overlap between cells and EVs. As shown by the KEGG pathway analysis, these three miRNAs (miR-146a-5p, miR-147b, and miR-155-5p) are functionally involved in the regulation of the Toll-like receptor signaling pathway and significantly correlated with the inflammatory mediators IL6 and ICAM1 released by stimulated PTCs. Especially with regard to a possible clinical use of miRs as new biomarkers, an accurate characterization of the miR expression altered during inflammatory processes is of enormous importance.

Inflammation is intimately involved in the pathogenesis of diabetic kidney disease. Inhibition of SGLT-2 by a specific class of drugs, gliflozins, has been shown to reduce inflammation and attenuate the progression of diabetic nephropathy, in addition to its main effect of inhibiting renal glucose reabsorption. We used highly purified human renal proximal tubular epithelial cells (PTCs) as an in vitro model to study the cellular response to a diabetic (high glucose) and inflammatory (cytokines) microenvironment and the effect of gliflozins. In this context, we investigated the influence of SGLT-2 inhibition by empa- and dapagliflozin (500 nM) on the expression of pro-inflammatory factors (IL-1β, IL-6, TNF-α, MCP-1, and ICAM-1). The results clearly indicate an anti-inflammatory effect of both gliflozins. Although induced expression of the four cytokines was only slightly attenuated, there was a clear effect on the expression of the adhesion molecule ICAM-1, a master regulator of cellular responses in inflammation and injury resolution. The induced expression of ICAM-1 mRNA was significantly reduced by approximately 13.5% by empagliflozin and also showed an inhibitory trend with dapagliflozin. However, induced ICAM-1 protein expression was significantly inhibited from 24.71 ± 1.0 ng/mL to 18.81 ± 3.9 (empagliflozin) and 19.62 ± 2.1 ng/mL (dapagliflozin). In conclusion, an additional anti-inflammatory effect of empa- and dapagliflozin in therapeutically observed concentrations was demonstrated in primary human PTCs in vitro.

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.

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.

Novel therapeutic strategies are urgently needed for advanced metastatic prostate cancer (PCa). Phytochemicals used in Traditional Chinese Medicine seem to exhibit tumor suppressive properties. Therefore, the therapeutic potential of artesunate (ART) on the progressive growth of therapy-sensitive (parental) and docetaxel (DX)-resistant PCa cells was investigated. Parental and DX-resistant PCa cell lines DU145, PC3, and LNCaP were incubated with artesunate (ART) [1-100 µM]. ART-untreated and 'non-cancerous' cells served as controls. Cell growth, proliferation, cell cycle progression, cell death and the expression of involved proteins were evaluated. ART, dose- and time-dependently, significantly restricted cell growth and proliferation of parental and DX-resistant PCa cells, but not of 'normal, non-cancerous' cells. ART-induced growth and proliferation inhibition was accompanied by G0/G1 phase arrest and down-regulation of cell cycle activating proteins in all DX-resistant PCa cells and parental LNCaP. In the parental and DX-resistant PC3 and LNCaP cell lines, ART also promoted apoptotic cell death. Ferroptosis was exclusively induced by ART in parental and DX-resistant DU145 cells by increasing reactive oxygen species (ROS). The anti-cancer activity displayed by ART took effect in all three PCa cell lines, but through different mechanisms of action. Thus, in advanced PCa, ART may hold promise as a complementary treatment together with conventional therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vakhrusheva, Erb, Bräunig, Markowitsch, Schupp, Baer, Slade, Thomas, Tsaur, Puhr, Culig, Cinatl, Michaelis, Efferth, Haferkamp and Juengel.)

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., (© 2022 Kohli et al.)

Hematopoietic stem cell transplantation (HSCT) has been proposed as a promising therapeutic opportunity to improve immunity and prevent hematologic malignancies in Ataxia-telangiectasia (A-T). However, experience in the transplantation strategy for A-T patients is still scarce. The aim of this study was to investigate whether different approaches of HSCT are feasible in regard to graft versus host response and sufficient concerning functional immune reconstitution. Atm -deficient mice were treated with a clinically relevant non-myeloablative host-conditioning regimen and transplanted with CD90.2-depleted, green fluorescent protein (GFP)-expressing, and ataxia telangiectasia mutated (ATM)-competent bone marrow donor cells in a syngeneic, haploidentical or allogeneic setting. Like syngeneic HSCT, haploidentical HSCT, but not allogeneic HSCT extended the lifespan of Atm -deficient mice through the reduction of thymic tumors and normalized T-cell numbers. Donor-derived splenocytes isolated from transplanted Atm -deficient mice filled the gap of cell loss in the naïve T-cell population and raised CD4 cell functionality up to wild-type level. Interestingly, HSCT using heterozygous donor cells let to a significantly improved survival of Atm -deficient mice and increased CD4 cell numbers as well as CD4 cell functionality equivalent to HSCT using with wild-type donor cells. Our data provided evidence that haploidentical HSCT could be a feasible strategy for A-T, possibly even if the donor is heterozygous for ATM. However, this basic research cannot substitute any research in humans., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Duecker, Gronau, Baer, Zielen and Schubert.)

The kidneys play a vital role in the basic physiological functions of the body [...].

Cell-free therapy using extracellular vesicles (EVs) from adipose-derived mesenchymal stromal/stem cells (ASCs) seems to be a safe and effective therapeutic option to support tissue and organ regeneration. The application of EVs requires particles with a maximum regenerative capability and hypoxic culture conditions as an in vitro preconditioning regimen has been shown to alter the molecular composition of released EVs. Nevertheless, the EV cargo after hypoxic preconditioning has not yet been comprehensively examined. The aim of the present study was the characterization of EVs from hypoxic preconditioned ASCs. We investigated the EV proteome and their effects on renal tubular epithelial cells in vitro. While no effect of hypoxia was observed on the number of released EVs and their protein content, the cargo of the proteins was altered. Proteomic analysis showed 41 increased or decreased proteins, 11 in a statistically significant manner. Furthermore, the uptake of EVs in epithelial cells and a positive effect on oxidative stress in vitro were observed. In conclusion, culture of ASCs under hypoxic conditions was demonstrated to be a promising in vitro preconditioning regimen, which alters the protein cargo and increases the anti-oxidative potential of EVs. These properties may provide new potential therapeutic options for regenerative medicine.

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., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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 5μM 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., Competing Interests: The authors declare no conflict of interest.

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) [...]., Competing Interests: The authors declare no conflict of interest.

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: Ataxia-telangiectasia (A-T) is a multisystem disorder with progressive cerebellar ataxia, immunodeficiency, chromosomal instability, and increased cancer susceptibility. Cellular immunodeficiency is based on naïve CD4 + and CD8 + T-cell lymphopenia. Hematopoietic stem cell transplantation (HSCT) offers a potential to cure immunodeficiency and cancer due to restoration of the lymphopoietic system. The aim of this investigation was to analyze the effect of HSCT on naïve CD4 + as well as CD8 + T-cell numbers in A-T. Methods: We analyzed total numbers of peripheral naïve (CD45RA + CD62L + ) and memory (CD45RO + CD62L - ) CD4 + and CD8 + T-cells of 32 A-T patients. Naïve (CD62L high CD44 low ) and memory (CD62L low CD44 high ) T-cells were also measured in Atm-deficient mice before and after HSCT with GFP-expressing bone marrow derived hematopoietic stem cells. In addition, we analyzed T-cells in the peripheral blood of two A-T patients after HLA-identic allogeneic HSCT. Results: Like in humans, naïve CD4 + as well as naïve CD8 + lymphocytes were decreased in Atm -deficient mice. HSCT significantly inhibited thymic lymphomas and increased survival time in these animals. Donor cell chimerism increased up to more than 50% 6 months after HSCT accompanied by a significant increase of naïve CD4 and CD8 T-cell subpopulations, but not of memory T-cells. This finding was also identified in the blood of the A-T patients after HSCT. Conclusion: HSCT seems to be a feasible strategy to overcome immunodeficiency and might be a conceivable strategy to avoid T-cell driven cancer in A-T at higher risk for malignancy. Naïve CD4 and CD8 T-cells counts are suitable markers for monitoring immune reconstitution post-HSCT. However, risks and benefits of HSCT in A-T have to be properly weighted., (Copyright © 2019 Duecker, Baer, Buecker, Huenecke, Pfeffermann, Modlich, Bakhtiar, Bader, Zielen and Schubert.)

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., Competing Interests: The authors declare no conflict of interest.

Determining the cell fate and the distribution of mesenchymal stromal/stem cells (MSCs) after transplantation are essential parts of characterizing the mechanisms of action and biosafety profile of stem cell therapy. Many recent studies have shown that MSCs migrate into injured tissues, but are only detectable at extremely low frequencies. We investigated the cell fate of MSCs after transplantation in an acute kidney injury (AKI) mouse model using in vivo bioluminescence imaging (BLI) and subsequent verification of cell migration using quantitative real-time polymerase chain reaction (qRT-PCR). The AKI was induced by a single injection of cisplatin (8 or 12 mg/kg). One day later, adipose-derived mesenchymal stromal/stem cells isolated from luciferase transgenic mice (Luc⁺-mASCs, 5 × 10⁵) were intravenously transplanted. Migration kinetics of the cells was monitored using BLI on day 1, 3, and 6, and finally via quantitative real-time PCR at the endpoint on day 6. Using BLI, infused Luc⁺-mASCs could only be detected in the lungs, but not in the kidneys. In contrast, PCR endpoint analysis revealed that Luc-specific mRNA could be detected in injured renal tissue; compared to the control group, the induction was 2.2-fold higher for the 8 mg/kg cisplatin group ( p < 0.05), respectively 6.1-fold for the 12 mg/kg cisplatin group ( p < 0.001). In conclusion, our study demonstrated that Luc-based real-time PCR rather than BLI is likely to be a better tool for cell tracking after transplantation in models such as cisplatin-induced AKI.

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.

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., (The Author(s). Published by S. Karger AG, Basel.)

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 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.

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Adipose tissue is a rich, ubiquitous and easily accessible source for multipotent stromal/stem cells and has, therefore, several advantages compared to other sources of mesenchymal stromal/stem cells. Several studies have tried to identify the origin of the stromal/stem cell population within adipose tissue in situ. This is a complicated attempt because no marker has currently been described which unambiguously identifies native adipose-derived stromal/stem cells (ASCs). Isolated and cultured ASCs are a non-uniform preparation consisting of several subsets of stem and precursor cells. Cultured ASCs are characterized by their expression of a panel of markers (and the absence of others), whereas their in vitro phenotype is dynamic. Some markers were expressed de novo during culture, the expression of some markers is lost. For a long time, CD34 expression was solely used to characterize haematopoietic stem and progenitor cells, but now it has become evident that it is also a potential marker to identify an ASC subpopulation in situ and after a short culture time. Nevertheless, long-term cultured ASCs do not express CD34, perhaps due to the artificial environment. This review gives an update of the recently published data on the origin and phenotype of ASCs both in vivo and in vitro. In addition, the composition of ASCs (or their subpopulations) seems to vary between different laboratories and preparations. This heterogeneity of ASC preparations may result from different reasons. One of the main problems in comparing results from different laboratories is the lack of a standardized isolation and culture protocol for ASCs. Since many aspects of ASCs, such as the differential potential or the current use in clinical trials, are fully described in other recent reviews, this review further updates the more basic research issues concerning ASCs' subpopulations, heterogeneity and culture standardization.

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.)

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.

The characterization of adipose-derived stromal/stem cells (ASCs) remains difficult due to the lack of a definitive and unique cellular marker. Therefore, a combination of markers is necessary to identify the cells. No comprehensive analysis of the immunophenotype of expanded plastic adherent ASCs has been published. Therefore, the aim of this study was to characterize the general phenotype of cultured ASCs and to further analyze cellular subsets. ASCs were isolated from lipoaspirates from patients undergoing cosmetic liposuction and cultured in standard cell culture. A comprehensive phenotype characterization was done with the BD Lyoplate™ Human Cell Surface Marker Screening Panel containing 242 antibodies and isotype controls. Cultured ASCs not only showed the characteristic expression profile of mesenchymal stem cells (MSCs), but also revealed donor-specific variability in the expression of 49 other markers. We further detected markers with a scattering in the fluorescence intensity, indicating subpopulations with different expression profiles. Therefore, a multi-color flow cytometric analysis was done after staining the cells with direct-labeled antibodies against CD73, CD90, CD105, and either CD34, CD140b, CD200, CD201, or CD36 to verify the selected subpopulations of ASCs. We detected no CD34-CD36 double-positive population, but CD34(+)-CD36(-) and CD34(-)CD36(+) subpopulations, both of which are positive for the 3 main MSC markers, CD73, CD90, and CD105. All other detected subpopulations also co-expressed the 3 main MSC markers, and therefore fulfill the minimal phenotypic criteria for the definition of cultured MSCs. Our study demonstrates the first comprehensive phenotypic characterization of ASCs and clearly highlights donor-specific variability in ASC preparations.

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., (© The Author(s) Journal compilation © 2012 International Federation for Cell Biology.)

Background Aims: Adipose-derived stromal/stem cells (ASC) 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. Beside their well-known differentiation into cells of mesodermal origin, ASC are able to differentiate into cells of ecto- and endodermal origin., Methods: Previous studies have shown that all trans retinoic acid (ATRA) induces the expression of cytokeratin 18 (CK18), indicating the beginning of differentiation into the epithelial lineage. Nevertheless, ATRA does not induce the expression of other epithelial markers. Therefore, we tested the additional influence of two growth factors on the onset of epithelial differentiation of ASC. The cells were cultured with ATRA, Activin A (ActA) and bone morphogenetic protein-7 (BMP-7), either alone or in combination. Differentiation into the epithelial lineage was assessed by the expression of the characteristic epithelial markers CK18 and zonula occludens protein 1 (ZO-1) using Western blot, immunofluorescence staining and polymerase chain reaction (PCR) analysis., Results: The mixture of all three factors induced epithelial differentiation of ASC without enhancing cell proliferation. Upon induction, the ASC showed phenotypic changes consistent with an epithelial phenotype. The addition of the growth factors ActA and BMP-7 enhanced the inductive effect of ATRA, as shown by the de novo expression of ZO-1 in addition to CK18 expression., Conclusions: Our study highlights the onset of the epithelial differentiation of ASC induced by culture with a combination of ATRA, ActA and BMP-7.

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.

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.

The versatile differentiation potential of adipose-derived stem cells (ASC) into cells of mesodermal, entodermal, and ectodermal origin places these cells at the forefront of cell-based therapies and cell transplantation. Epithelial differentiation of ASC may either be initiated by direct cell-cell or cell-matrix contacts, by chemical factors like retinoic acid, or via secreted cellular factors like cytokines, interleukins, or growth factors included in conditioned media.This protocol describes methods to induce the in vitro differentiation of ASC from human adipose tissue into the epithelial lineage, and describes the methods used to verify this induced differentiation. We present two differentiation protocols based on either retinoic acid or conditioned medium of cultured epithelial cells.

Background: For biotechnological use of cells in tissue engineered applications, such as biohybrid renal devices, optimal culture conditions are required. Oxygen delivery is one of the most important cell determined system criterion for ex vivo applications. It is involved in the maintenance of highly oxygen-dependent renal tubular epithelial cells, affecting metabolic state, differentiation, and desired transport functions. The purpose of this study was to examine respiratory patterns such as basal oxygen consumption, solute transport-related oxygen demand, and oxygen concentration-dependent oxygen uptake of renal tubular epithelial cells in vitro., Methods: Respiratory patterns of highly purified human primary renal proximal (hPTC) and early distal tubular cells (hTALDC) were analyzed by perfusion respirometry. Spontaneous oxygen consumptions and maximum respirations after carbonyl cyanide m-chlorophenyl hydrazone (CCCP) uncoupling were measured. Respiration fractions contributing to basolateral Na(+) /K(+) -ATPase transport activities were assessed via ouabain inhibition and Na(+) -free medium. Furthermore, we determined oxygen uptake in dependency of oxygen concentration and morphology in various culture conditions (shaken, static)., Results: Respiration of solely hPTC strongly depended on oxygen concentration in a Michaelis-Menten pattern at noncritical oxygen concentrations. Respiration of both cell types was significantly increased by CCCP, whereas average Na(+) /K(+) -ATPase-based oxygen uptake fractions differ significantly between the two cell types. Nevertheless, no significant differences were found in spontaneous respiration between hPTC and hTALDC., Conclusions: Our results clearly indicate that cell-specific oxygen consumption parameters have to be considered in the design of biotechnological devices intended to support kidney function by cell-supported renal replacement therapy., (Copyright © 2011 American Institute of Chemical Engineers (AIChE).)

Lung diseases like cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are associated with chronic airway inflammation. The aim of our study was to compare a complex biomarker profile in order to characterize specific inflammatory patterns in sputum of patients with CF and COPD. Induced sputum samples of 19 CF-, 26 COPD patients and 21 healthy controls were analyzed for concentrations of IL-1beta, IL-2, IL-6, IL-8, IL-13, IP-10, MCP-1, IFN-gamma and TNF-alpha using the new cytometric bead array (CBA) technology. Significant differences in airway biomarker profiles of CF and COPD were detected. Patients with CF showed a significant increase in IL-1beta, IL-6, IL-8, IL-13, TNF-alpha, IFN-gamma and MCP-1. COPD patients showed an increase in IL-6, IL-8, IL-13 and MCP-1 compared to healthy controls. CF and COPD compared to each other exhibited differences in IL-1beta, IL-2, IL-8, TNF-alpha, IFN-gamma and MCP-1 levels. Significant correlations between the parameters of lung function and sputum biomarker levels were found. Analyzing induced sputum allows characterization of specific airway biomarker profiles in CF and COPD and can be related to the clinical status of the patient. CBA of induced sputum seems to be a pivotal tool to characterize pulmonary inflammation., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

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., (Copyright 2010 S. Karger AG, Basel.)

Purpose of Review: To review the current knowledge of the contribution of mesenchymal stem cells (MSCs) during renal repair and the factors and mechanisms underlying these effects., Recent Findings: Although many studies using MSCs as therapeutic agents have been published in recent years, our understanding is incomplete how migration, growth, and differentiation of adult MSCs are governed by interactions with resident renal cells, growth factors, and cytokines. Different laboratories have shown that extrarenal stem cells contribute to renal regeneration: MSCs migrate into injured renal tissue, are integrated into the tubular system, and differentiate into epithelial cells. Nevertheless, a couple of studies could not verify these results in all, but despite this they have shown a beneficial effect of administered MSCs on renal regeneration. It is well established that renal tubular epithelial cells secrete growth factors and cytokines. These factors participate in renal repair as paracrine regulators or chemoattractants to induce migration of extrarenal stem cells, whereas only limited data are available about the interactions between all key players during renal repair mechanisms., Summary: Although different studies demonstrate the effectiveness of MSC therapy, there are still open questions defining the mechanism of action during homing and differentiation and the long-term safety on the way to a therapeutic option.

Despite expanded research in stem cell biology, little is known about the mechanisms underlying migration, growth, and differentiation of adipose-derived adult mesenchymal stem cells (ASC). The simultaneous measurement of intracellular pathways opens new avenues to gain further insights in these processes. We used the Cytometric Bead Array (CBA) Flex Set technology to simultaneously analyze protein phosphorylation after stimulation of ASC and compared the results with data generated by corresponding Western blots. Signal transduction of ASC was stimulated by epidermal growth factor (EGF) and analyzed by determining phosphorylation of mitogen-activated protein kinases (MAPKs) ERK, p38, and JNK by Western blotting and CBA. After incubation with EGF, all MAPKs were significantly but differentially phosphorylated depending on time and dose. Furthermore, the ERK-response was abolished by EGF-R antagonist AG 1478 and kinase inhibitor PD98059, whereas p38 and JNK were only inhibited by AG1478. The stimulation and inhibition profiles between the two assays were highly comparable and the data were significantly correlated. In the present study we demonstrated that the CBA technology offers a reliable and convenient method for multiplexing of phospho-proteins in the evaluation of signal transduction pathways of adipose-derived mesenchymal stem cells.

Adult stem cells act as a pluripotent source of regenerative cells during tissue injury. Despite expanded research in stem cell biology, understanding how growth and migration of adipose-derived adult mesenchymal stem cells (ASC) are governed by interactions with growth factors is very limited. One important property of ASC is the presence of the epidermal growth factor (EGF) receptor and the cellular response to soluble EGF. Expression of the EGF receptor was proven by PCR and Western blotting. Signal transduction was analyzed by Western blotting and PhosFlow assay. EGF caused robust phosphorylation of SHC and ERK1/2, which could be inhibited by EGF receptor antagonist AG1478 and MEK inhibitor PD98059. ASC proliferation was determined by MTT assay. Stem cell migration was analyzed in a modified Boyden chamber. Incubation with EGF led to cell proliferation and induced cell migration, but did not change the undifferentiated state of the cells. In the kidney, injured renal tubular cells express high amounts of EGF. Therefore, our results may highlight a mechanism underlying renal regeneration. Thus, future in vivo studies that focus on the effects of EGF on recruitment of ASC to sites of injury are necessary.

Objectives: Mesenchymal-epithelial interactions play a pivotal role in tubular morphogenesis and in maintaining the integrity of the kidney. During renal repair, similar mechanisms may regulate cellular reorganization and differentiation. We have hypothesized that soluble factors from proximal tubular epithelial cells (PTC) induce differentiation of adipose-derived adult mesenchymal stem cells (ASC). This hypothesis has been tested using cultured ASC and PTC., Material and Methods: Conditioned medium was prepared from injured PTC and transferred to ASC cultures. ASC proliferation was analysed by a fluorometric and photometric assay. Signal transduction was analysed by phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2). Grade of ASC differentiation was assessed by morphological analysis and cell expression of characteristic markers., Results: Conditioned medium significantly induced proliferation and phosphorylation of ERK1/ERK2 of ASC. After 12 days of incubation, cell morphology changed to an epithelial-like monolayer. Expression of cytokeratin 18 was induced by conditioned medium, while alpha-smooth muscle actin, CD49a and CD90 expression decreased. These alterations strongly indicate onset of the differentiation process to the epithelial lineage. In summary, soluble factors from PTC induce signal transduction and differentiation of ASC., Conclusions: Our study shows that conditioned medium from renal tubular epithelial cells provides a convenient source of inductive signals to initiate differentiation of ASC towards epithelial lineage. We deduce that these interactions may play an important role during renal repair mechanisms.

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 IgG(Mite) and IgG4(Mite), 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 IgG(Mite) (p < 0.001) and specific IgG4(Mite) (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., ((c) 2008 S. Karger AG, Basel.)

The chemokine CXCL16 plays an important role in the recruitment of leukocytes to sites of inflammation influencing the course of experimental glomerulonephritis. Here we show that human kidneys highly express CXCL16 in the distal tubule, connecting tubule and principal cells of the collecting duct with weak expression in the thick ascending limb of Henle. Beside the membrane localization, a soluble form of CXCL16 can be proteolytically released which acts as a chemotactic factor. In human renal tissue the expression pattern of the disintegrin-like metalloproteinase ADAM10 is similar to that of CXCL16, suggesting ADAM10 can potentially cleave CXCL16 in vivo. When we tested this in primary tubular cells we found that blockade of ADAM10 activity inhibited the IFN-gamma induced release of soluble CXCL16. Acute tubular damage in renal allografts was associated with elevated urinary CXCL16 and this correlated with focally increased apical CXCL16 expression in the distal tubules and collecting ducts. Renal allograft biopsies, with a histopathological diagnosis of acute interstitial rejection, showed increased basolateral ADAM10 expression together with high numbers of infiltrating T cells. Our results suggest that CXCL16 and ADAM10 are involved in the recruitment of T cells to the kidney and play an important role in inflammatory kidney diseases.

Aim: Human renal tubular cells of well-defined nephron origin are an important basis in the research of various physiological and pathophysiological mechanisms in the kidney. Whereas an exceeding amount of data has been obtained on proximal tubular cells, only limited data of cells of the human thick ascending limb and the early distal tubule (TALDC) are available., Methods: TALDC have been isolated immunomagnetically according to their specific antigen expression of Tamm-Horsfall glycoprotein (THG). Cells were either directly processed for analysis or cultured under normal cell culture conditions. Differentiation of primary isolates and cultured cells was assessed by reverse transcription polymerase chain reaction using characteristic markers. As controls, we used RNA from whole human kidney and cultured HK-2 cells. Additional characterizations were made by morphological analysis and western blotting., Results: Primary isolated TALDC express the characteristic markers epidermal growth factor receptor, Na-K-2Cl transporter 2, epithelial calcium canal, and THG but were negative for Pax-2, aquaporin-2 and -3. Cultured TALDC were positive for epidermal growth factor receptor and Na-K-2Cl transporter 2 but have lost their epithelial calcium canal and THG expression and started to express Pax-2. All probes were positive for the specific markers kidney-specific cadherin and cytokeratin-8. Furthermore, differentiation of cultured TALDC was shown by cell morphology and their characteristic protein expression pattern., Conclusion: Our results highlight the purity of primary isolates and the differentiation of cultured TALDC, and show that they can be used as an in vitro system studies of the human thick ascending limb of Henle's loop and early distal tubule.

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: alpha-linolenic acid, 240 mg/d; eicosapentaenoic acid, 120 mg/d; stearidonic acid, 49 mg/d; and gamma-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-alpha, 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 alpha-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-alpha 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.

Aims: C-reactive protein (CRP) is a component of the acute-phase reaction to inflammation, severe tissue injury, and infection. Investigations have shown that CRP concentration is highly increased in the urine during acute renal graft dysfunction and, therefore, may affect tubular cell metabolism. Nevertheless, no data about the effects of CRP on human renal tubular epithelial cells are available., Methods: Human renal distal tubular cells (DTC) were isolated immunomagnetically and cultured. Cells were stimulated with affinity chromatography pure native CRP from human ascites (10 - 0.001 microg/ml). Phosphorylation of MAP-K was assessed by Westernblot analysis. Release of RANTES and interleukin-6 was evaluated with an enzyme immunoassay. Cytotoxic effects of CRP were determined by a commercially available Live/Dead assay and MTT assay. Effects on cell proliferation were analyzed by a fluorimetric assay., Results: Westernblot analysis clearly showed that CRP activates the MAP-K pathway of DTC. CRP upregulated RANTES expression of DTC in a significant and dose-dependent manner. CRP (10 microg/ml) induced a 12.3-fold upregulation, CRP 1 or 0.1 microg/ml induced a 6.3-/2.8-fold RANTES upregulation, respectively. Interleukin-6 synthesis was not influenced. Cytotoxic, proliferative or apoptotic effects were not observed at the concentrations used., Conclusions: We demonstrated an activating effect of CRP on DTC in vitro. In vivo, this effect of CRP might be part of the immune activation cascade during episodes of renal graft rejection or bacterial infections.