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Background: The excessive cardiovascular mortality of patients with chronic kidney disease (CKD) could be linked to mineral stress, the biological consequence of calcium-phosphate nanoparticle exposure. This study investigated whether zinc is associated with mineral stress markers in CKD. Methods: Zinc and T50 (serum calcification propensity) as well as hydrodynamic radius of secondary calciprotein particles (CPP2) were measured in blood donors and CKD patients with/out dialysis. Results: Serum zinc concentrations and T50 were reduced, while CPP2 radius was increased in CKD patients. Serum zinc levels positively correlated with T50 and inversely correlated with CPP2 radius. In a hierarchical linear regression model, T50 was associated with age, calcium, phosphate, magnesium and albumin. Addition of zinc significantly improved prediction of the model, confirming an additional contribution of zinc to T50. Similar observations were made for the association of zinc and CPP2 radius, but spiking experiments indicated that zinc may stronger modify T50 than CPP2 radius. Also, urinary zinc excretion was increased in patients with kidney disease and correlated to T50 and CPP2 radius. Serum zinc further correlated with markers of arterial stiffness in blood donors and CKD patients, but these associations did not remain significant in a multivariate linear regression model. Conclusions: Reduced serum zinc levels in CKD appear directly linked to lower T50 and associated with larger CPP2 radius. Further studies on the associations of zinc and mineral stress as well as putative therapeutic benefits of zinc supplementation are required.

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Medial vascular calcification in chronic kidney disease (CKD) involves pro-inflammatory pathways induced by hyperphosphatemia. Several interleukin 6 family members have been associated with pro-calcific effects in vascular smooth muscle cells (VSMCs) and are considered as therapeutic targets. Therefore, we investigated the role of leukemia inhibitory factor (LIF) during VSMC calcification. LIF expression was found to be increased following phosphate exposure of VSMCs. LIF supplementation aggravated, while silencing of endogenous LIF or LIF receptor (LIFR) ameliorated the pro-calcific effects of phosphate in VSMCs. The soluble LIFR mediated antagonistic effects towards LIF and reduced VSMC calcification. Mechanistically, LIF induced phosphorylation of the non-receptor tyrosine-protein kinase 2 (TYK2) and signal transducer and activator of transcription-3 (STAT3) in VSMCs. TYK2 inhibition by deucravacitinib, a selective, allosteric oral immunosuppressant used in psoriasis treatment, not only blunted the effects of LIF, but also interfered with the pro-calcific effects induced by phosphate. Conversely, TYK2 overexpression aggravated VSMC calcification. Ex vivo calcification of mouse aortic rings was ameliorated by Tyk2 pharmacological inhibition and genetic deficiency. Cholecalciferol-induced vascular calcification in mice was improved by Tyk2 inhibition and in the Tyk2-deficient mice. Similarly, calcification was ameliorated in Abcc6/Tyk2-deficient mice after adenine/high phosphorus-induced CKD. Thus, our observations indicate a role for LIF in CKD-associated vascular calcification. Hence, the effects of LIF identify a central pro-calcific role of TYK2 signaling, which may be a future target to reduce the burden of vascular calcification in CKD.

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BACKGROUND AND OBJECTIVES: Blood safety measures used by blood establishments to increase blood component safety can be validated using Transfusion-Relevant Bacterial Reference Strains (TRBRS). Ultra-cold storage conditions and manual preparation of the current TRBRS may restrict their practical use. To address this issue, the ISBT Transfusion-Transmitted Infectious Diseases Working Party's Bacterial Subgroup organized an international study to validate TRBRS in a user-friendly, lyophilised format. MATERIALS AND METHODS: Two bacterial strains Klebsiella pneumoniae PEI-B-P-08 and Staphylococcus aureus PEI-B-P-63 were manufactured as lyophilised material. The lyophilised bacteria were distributed to 11 different labs worldwide to assess the robustness for enumeration, identification and determination of growth kinetics in platelet concentrates (PCs). RESULTS: Production of lyophilised TRBRS had no impact on the growth properties compared with the traditional format. The new format allows a direct low-quantity spiking of approximately 30 bacteria in PCs for transfusion-relevant experiments. In addition, the lyophilised bacteria exhibit long-term stability across a broad temperature range and can even be directly rehydrated in PCs without losing viability. Interlaboratory comparative study demonstrated the robustness of the new format as 100% of spiked PC exhibited growth. CONCLUSION: Lyophilised TRBRS provide a user-friendly material for transfusion-related studies. TRBRS in the new format have improved features that may lead to a more frequent use in the quality control of transfusion-related safety measures in the future.

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Myelodysplastic syndromes (MDS) are myeloid neoplasms presenting with dysplasia in the bone marrow (BM) and peripheral cytopenia. In most patients anemia develops. We screened for genes that are expressed abnormally in erythroid progenitor cells (EP) and contribute to the pathogenesis of MDS. We found that the Coxsackie-Adenovirus receptor (CAR = CXADR) is markedly downregulated in CD45low/CD105+ EP in MDS patients compared to control EP. Correspondingly, the erythroblast cell lines HEL, K562, and KU812 stained negative for CAR. Lentiviral transduction of the full-length CXADR gene into these cells resulted in an increased expression of early erythroid antigens, including CD36, CD71, and glycophorin A. In addition, CXADR-transduction resulted in an increased migration against a serum protein gradient, whereas truncated CXADR variants did not induce expression of erythroid antigens or migration. Furthermore, conditional knock-out of Cxadr in C57BL/6 mice resulted in anemia and erythroid dysplasia. Finally, decreased CAR expression on EP was found to correlate with high-risk MDS and decreased survival. Together, CAR is a functionally relevant marker that is down-regulated on EP in MDS and is of prognostic significance. Decreased CAR expression may contribute to the maturation defect and altered migration of EP and thus their pathologic accumulation in the BM in MDS.

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Acting as the largest energy reservoir in the body, adipose tissue is involved in longevity and progression of age-related metabolic dysfunction. Here, cellular senescence plays a central role in the generation of a pro-inflammatory environment and in the evolution of chronic diseases. Within the complexity of a tissue, identification and targeting of senescent cells is hampered by their heterogeneity. In this study, we generated stress-induced premature senescence 2D and 3D in vitro models for the stromal vascular niche of human adipose tissue. We established treatment conditions for senescence induction using Doxorubicin (Dox), starting from adipose-derived stromal/stem cells (ASCs), which we adapted to freshly isolated microtissue-stromal vascular fraction (MT-SVF), where cells are embedded within their native extracellular matrix. Senescence hallmarks for the established in vitro models were verified on different cellular levels, including morphology, cell cycle arrest, senescence-associated ß-galactosidase activity (SA-ßgal) and gene expression. Two subsequent exposures with 200 nM Dox for six days were suitable to induce senescence in our in vitro models. We demonstrated induction of senescence in the 2D in vitro models through SA-ßgal activity, at the mRNA level (LMNB1, CDK1, p21) and additionally by G2/M phase cell cycle arrest in ASCs. Significant differences in Lamin B1 and p21 protein expression confirmed senescence in our MT-SVF 3D model. MT-SVF 3D cultures were composed of multiple cell types, including CD31, CD34 and CD68 positive cells, while cell death remained unaltered upon senescence induction. As heterogeneity and complexity of adipose tissue senescence is given by multiple cell types, our established senescence models that represent the perivascular niche embedded within its native extracellular matrix are highly relevant for future clinical studies.

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BACKGROUND AND OBJECTIVES: Fainting is a well-known side effect of blood donation. Such adverse experiences can diminish the return rate for further blood donations. Identifying factors associated with fainting could help prevent adverse incidents during blood donation. MATERIALS AND METHODS: Data of 85,040 blood donations from whole blood and apheresis donors within four consecutive years were included in this retrospective study. Seven different machine learning models (random forests, artificial neural networks, XGradient Boosting, AdaBoost, logistic regression, K nearest neighbors, and support vector machines) for predicting fainting during blood donation were established. The used features derived from the data obtained from the questionnaire every donor has to fill in before the donation and weather data of the day of the donation. RESULTS: One thousand seven hundred fifteen fainting reactions were observed in 228 846 blood donations from 88,003 donors over a study period of 48 months. Similar values for all machine learning algorithms investigated for NPV, PPV, AUC, and F1-score were obtained. In general, NPV was above 0.996, whereas PPV was below 0.03. AUC and F1-score were close to 0.9 for all models. Essential features predicting fainting during blood donation were systolic and diastolic blood pressure and ambient temperature, humidity, and barometric pressure. CONCLUSION: Machine-learning algorithms can establish prediction models of fainting in blood donors. These new tools can reduce adverse reactions during blood donation and improve donor safety and minimize negative associations relating to blood donation.

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BACKGROUND: Viral reduction and inactivation of cell-derived biologicals is paramount for patients' safety and so viral reduction needs to be demonstrated to regulatory bodies in order to obtain marketing authorisation. Allogeneic human blood-derived biological medicinal products require special attention. APOSECTM, the secretome harvested from selected human blood cells, is a new biological with promising regenerative capabilities. We evaluated the effectiveness of inactivation of model viruses by methylene blue/light treatment, lyophilisation, and gamma irradiation during the manufacturing process of APOSECTM. MATERIALS AND METHODS: Samples of intermediates of APOSECTM were acquired during the manufacturing process and spiked with bovine viral diarrhoea virus (BVDV), human immunodeficiency virus type 1 (HIV-1), pseudorabies virus (PRV), hepatitis A virus (HAV), and porcine parvovirus (PPV). Viral titres were assessed with suitable cell lines. RESULTS: Methylene blue-assisted viral reduction is mainly effective against enveloped viruses: the minimum log10 reduction factors for BVDV, HIV-1, and PRV were ≥6.42, ≥6.88, and ≥6.18, respectively, with no observed residual infectivity. Viral titres of both HAV and PPV were not significantly reduced, indicating minor inactivation of non-enveloped viruses. Lyophilisation had minor effects on the viability of several enveloped model viruses. Gamma irradiation contributes to the viral safety by reduction of enveloped viruses (BVDV: ≥2.42; HIV-1: 4.53; PRV: ≥4.61) and to some degree of non-enveloped viruses as seen for HAV with a minimum log10 reduction factor of 2.92. No significant reduction could be measured for the non-enveloped virus PPV (2.60). DISCUSSION: Three manufacturing steps of APOSECTM were evaluated under Good Laboratory Practice conditions for their efficacy at reducing and inactivating potentially present viruses. It could be demonstrated that all three steps contribute to the viral safety of APOSECTM.

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A cell-free approach using secretomes derived from stem cells or peripheral blood mononuclear cells is an active area of regenerative medicine that holds promise for therapies. Regulatory authorities classify these secretomes as biological medicinal products, and non- clinical safety assessment thus falls under the scope of ICH S6. A secretome of stressed peripheral blood mononuclear cells (APOSEC) was successfully tested in a toxicology program, supporting clinical use of the new drug candidate. Here, to allow for topical, dermal treatment of patients with diabetic foot ulcer, several non-clinical safety studies were performed. Acute toxicity (single dose) and neuropharmacological screening were tested intravenously in a rat model. Risk for skin sensitisation was tested in mice. A 4-week intravenous toxicity study in mice and a 4-week subcutaneous toxicity study in minipigs were conducted to cover the clinical setting and application in a rodent and a non-rodent model. Acute and repeated-dose toxicity studies show that APOSEC administered intravenously and subcutaneously does not involve major toxicities or signs of local intolerance at levels above the intended total human maximal dose of 3.3 U/kg/treatment, 200 U/wound/treatment, and 100 U/cm2/treatment. The non-clinical data support the safe topical use of APOSEC in skin diseases related to deficient wound healing.

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Developing effective therapies against chronic wound healing deficiencies is a global priority. Thus we evaluated the safety of two different doses of topically administered autologous APOSEC, the secretome of apoptotic peripheral blood mononuclear cells (PBMCs), in healthy male volunteers with artificial dermal wounds. Ten healthy men were enrolled in a single-center, randomized, double-blinded, placebo-controlled phase 1 trial. Two artificial wounds at the upper arm were generated using a 4-mm punch biopsy. Each participant was treated with both topically applied APOSEC and placebo in NuGel for 7 consecutive days. The volunteers were randomized into two groups: a low-dose group (A) receiving the supernatant of 12.5 × 106 PBMCs and a high-dose group (B) receiving an equivalent of 25 × 106 PBMCs resuspended in NuGel Hydrogel. Irradiated medium served as placebo. The primary outcome was the tolerability of the topical application of APOSEC. All adverse events were recorded until 17 days after the biopsy. Local tolerability assessment was measured on a 4-point scale. Secondary outcomes were wound closure and epithelization at day 7. No therapy-related serious adverse events occurred in any of the participants, and both low- and high-dose treatments were well tolerated. Wound closure was not affected by APOSEC therapy.

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BACKGROUND: Cell-based therapies with autologous adipose tissue-derived cells have shown great potential in several clinical studies in the last decades. The majority of these studies have been using the stromal vascular fraction (SVF), a heterogeneous mixture of fibroblasts, lymphocytes, monocytes/macrophages, endothelial cells, endothelial progenitor cells, pericytes and adipose-derived stromal/stem cells (ASC) among others. Although possible clinical applications of autologous adipose tissue-derived cells are manifold, they are limited by insufficient uniformity in cell identity and regenerative potency. METHODS: In our experimental set-up, low-energy extracorporeal shock wave therapy (ESWT) was performed on freshly obtained human adipose tissue and isolated adipose tissue SVF cells aiming to equalize and enhance stem cell properties and functionality. RESULTS: After ESWT on adipose tissue we could achieve higher cellular adenosine triphosphate (ATP) levels compared with ESWT on the isolated SVF as well as the control. ESWT on adipose tissue resulted in a significantly higher expression of single mesenchymal and vascular marker compared with untreated control. Analysis of SVF protein secretome revealed a significant enhancement in insulin-like growth factor (IGF)-1 and placental growth factor (PLGF) after ESWT on adipose tissue. DISCUSSION: Summarizing we could show that ESWT on adipose tissue enhanced the cellular ATP content and modified the expression of single mesenchymal and vascular marker, and thus potentially provides a more regenerative cell population. Because the effectiveness of autologous cell therapy is dependent on the therapeutic potency of the patient's cells, this technology might raise the number of patients eligible for autologous cell transplantation.

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BACKGROUND: The Rhesus (Rh) complex consists of a core comprising the Rh proteins (RhD/RhCE) and the Rh-associated glycoprotein (RhAG) with accessory chains (GPB, LW, CD47). Molecular defects of the RHAG gene may cause a regulator Rhnull phenotype without Rh antigen expression or a Rhmod phenotype with decreased Rh antigen expression. STUDY DESIGN AND METHODS: Blood samples of a donor with strongly diminished Rh antigens and five family members were analyzed by serological phenotyping, flow cytometry, molecular testing, and gene expression analysis of Rh complex candidate genes. RESULTS: RHAG sequencing identified a missense mutation, c.241G>C (p.Gly81Arg) and a splice site mutation, c.640 + 3del14, among the cohort. Compound heterozygosity of these novel alleles identified in the propositus and two siblings gave rise to a strongly diminished expression of RhAG, Rh, and CD47 antigens on the RBC surface. CONCLUSION: The Rhmod phenotype was caused by a novel RHAG splice site mutation in association with a non-functional allele. The primary depression of RhAG is most likely due to posttranslational events that affect the interaction and processing of the RhAG glycoprotein and gave rise to a secondary depression of RhD, RhCE, and CD47, the major members of the Rh complex.

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Recent advances in high-throughput sequencing allow for the competitive analysis of the human B and T cell immune repertoire. In this study we compared Immunoglobulin and T cell receptor repertoires of lymphocytes found in kidney and blood samples of 10 patients with various renal diseases based on next-generation sequencing data. We used Biomed-2 primer panels and ImmunExplorer software to sequence, analyze and compare complementarity determining regions and V-(D)-J elements. While generally an individual's renal receptor repertoire is different from the repertoire present in blood, 94% (30/32) of the lymphocytes with clonal expansion in kidney can also be traced in blood however, not all of these clonotypes are equally abundant. Summarizing the data of all analyzed patients, 68% of highly expanded T cell clonotypes and 30% of the highly expanded B cell clonotypes that have infiltrated the kidney can be found amongst the five most abundant clonotypes in blood. In addition, complementarity determining region 3 sequences of the immunoglobulin heavy chains are on average more diverse than T cell receptor beta chains. Immune repertoire analysis of tissue infiltrating B and T cells adds new approaches to the assessment of adaptive immune response in kidney diseases. Our data suggest that expanded clonotypes in the tissues might be traceable in blood samples in the course of treatment or the natural history of the disease.

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Loss of the Y-chromosome (LOY) is described as both a normal age-related event and a marker of a neoplastic clone in hematologic diseases. To assess the significance of LOY in myelodysplastic syndromes (MDS), we determined the percentage of LOY in clonal CD34+ peripheral blood cells in comparison to normal CD3+ T-cells of 27 MDS patients using fluorescence in situ hybridization (FISH) analysis. Results were compared with the percentage of LOY in CD34+ and CD3+ cells of 32 elderly men without hematologic diseases and in 25 young blood donors. While LOY could not be detected in CD3+ cells of young men, it was observed in CD3+ cells of elderly men without hematologic diseases (2.5% LOY) as well as in CD3+ cells of elderly MDS patients (5.8% LOY). The percentage of CD34+ cells affected by LOY was significantly higher in MDS patients compared to elderly men without hematologic diseases (43.3% vs. 13.2%, P = 0.005), indicating that LOY has an age-related basis but is also associated with MDS. Furthermore, we aimed to define a threshold between age- and disease-associated LOY in MDS. Statistical analysis revealed that a value of 21.5% LOY in CD34+ peripheral blood cells provided the best threshold to discriminate between these two conditions in MDS. We conclude that LOY is clonal in a substantial number of MDS based on an age-related predisposition.

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According to the European Pharmacopoeia sterility testing of products includes an incubation time of 14 days in thioglycollate medium and soya-bean casein medium. In this case a large period of time is needed for product testing. So we designed a study to evaluate an alternative method for sterility testing. The aim of this study was to reduce the incubation time for the routinely produced products in our tissue bank (cornea and amnion grafts) by obtaining the same detection limit, accurateness and recovery rates as the reference method described in the European Pharmacopoeia. The study included two steps of validation. Primary validation compared the reference method with the alternative method. Therefore eight bacterial and two fungi test strains were tested at their preferred milieu. A geometric dilution series from 10 to 0.625 colony forming unit per 10 ml culture media was used. Subsequent to the evaluation the second part of the study started including the validation of the fertility of the culture media and the parallel testing of the two methods by investigating products. For this purpose two product batches were tested in three independent runs. Concerning the validation we could not find any aberration between the alternative and the reference method. In addition, the recovery rate of each microorganism was between 83.33 and 100 %. The alternative method showed non-inferiority regarding accuracy to the reference method. Due to this study we reduced the sterility testing for cornea and amniotic grafts to 9 days.

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