Search

Introduction: The molecular diagnosis of the A1 blood group is based on the exclusion of ABO gene variants causing blood groups A2, B, or O. A specific genetic marker for the A1 blood group is still missing. Recently, long-read ABO sequencing revealed four sequence variations in intron 1 as promising markers for the ABO*A1 allele. Here, we evaluated the diagnostic values of the 4 variants in blood donors with regular and weak A phenotypes and genotypes. Methods: ABO phenotype data (A, B, AB, or O) were taken from the blood donor files. The ABO genotypes (low resolution) were known from a previous study and included the variants c.261delG, c.802G>A, c.803G>C, and c.1061delC. ABO variant alleles (ABO*AW.06,*AW.08,*AW.09,*AW.13, *AW.30, and *A3.02) were identified in weak A donors by sequencing the ABO exons before. For genotyping of the ABO intron 1 variants rs532436, rs1554760445, rs507666, and rs2519093, we applied TaqMan assays with endpoint fluorescence detection according to a standard protocol. Genotypes of the variants were compared with the ABO phenotype and genotype. Evaluation of diagnostic performance included sensitivity, specificity, positive (PPV), and negative predictive value (NPV). Results: In 1,330 blood donors with regular ABO phenotypes and genotypes, the intron 1 variants were significantly associated with the proposed A1 blood group. In 15 donors, we found discrepancies to the genotype of at least one of the 4 variants. For the diagnosis of the ABO*A1 allele, the variants showed 98.79–99.48% sensitivity, 99.66–99.81% specificity, 98.80–99.31% PPV, and 99.66–99.86% NPV. Regarding the A phenotype, the diagnostic values were 99.02–99.41% sensitivity, 99.63–99.76% specificity, 99.41–99.61% PPV, and 99.39–99.63% NPV. The *A1 marker allele of all intron 1 variants was also associated with the *AW.06, *AW.13, and *AW.30 variants. Samples with *AW.08, *AW.09, and *A3.02 variants lacked this association. Conclusion: The ABO intron 1 variants revealed significant association with the ABO*A1 allele and the A phenotype. However, the intron 1 genotype does not exclude variant alleles causing weak A phenotypes. With the introduction of reliable tag, single nucleotide variants for the A1, A2, B, and O blood groups and the genotyping instead of phenotyping of the ABO blood group are getting more feasible on a routine basis.

Coronavirus disease-2019 (COVID-19) is associated with increased thromboembolic complications. Long-term alteration in the coagulation system after acute COVID-19 infection is still a subject of research. Furthermore, the effect of sera from convalescent subjects on platelets is not known. In this study, we investigated platelet phenotype, coagulation, and fibrinolysis in COVID-19 convalescent plasma (CCP) donors and analyzed convalescent sera-induced effects on platelets. We investigated CCP donors who had a history of mild COVID-19 infection and donors who did not have COVID-19 were used as controls. We analyzed phosphatidylserine (PS) externalization, CD62p expression, and glycoprotein VI (GPVI) shedding both in platelet-rich plasma (PRP) and after incubation of washed healthy platelets with donors' sera using flow cytometry. Coagulation and fibrinolysis systems were assessed with thromboelastometry. Forty-seven CCP donors (22 males, 25 females; mean age (±SD): 41.4 ± 13.7 years) with a history of mild COVID-19 infection were included. Median duration after acute COVID-19 infection was 97 days (range, 34–401). We did not find an increased PS externalization, CD62p expression, or GPVI shedding in platelets from CCP donors. Sera from CCP donors did not induce PS externalization or GPVI shedding in healthy platelets. Sera-induced CD62p expression was slightly, albeit statistically significantly, lower in CCP donors than in plasma donors without a history of COVID-19. One patient showed increased maximum clot firmness and prolonged lysis time in thromboelastometry. Our findings suggest that procoagulant platelet phenotype is not present after mild COVID-19. Furthermore, CCP sera do not affect the activation status of platelets.

COVID-19 convalescent plasma (CCP) with high neutralizing antibodies has been suggested in preventing disease progression in COVID-19. In this study, we investigated the relationship between clinical donor characteristics and neutralizing anti-SARS-CoV-2 antibodies in CCP donors. COVID-19 convalescent plasma donors were included into the study. Clinical parameters were recorded and anti-SARS-CoV-2 antibody levels (Spike Trimer, Receptor Binding Domain (RBD), S1, S2 and nucleocapsid protein) as well as ACE2 binding inhibition were measured. An ACE2 binding inhibition < 20% was defined as an inadequate neutralization capacity. Univariate and multivariable logistic regression analysis was used to detect the predictors of inadequate neutralization capacity. Ninety-one CCP donors (56 female; 61%) were analyzed. A robust correlation between all SARS-CoV-2 IgG antibodies and ACE2 binding inhibition, as well as a positive correlation between donor age, body mass index, and a negative correlation between time since symptom onset and antibody levels were found. We identified time since symptom onset, normal body mass index (BMI), and the absence of high fever as independent predictors of inadequate neutralization capacity. Gender, duration of symptoms, and number of symptoms were not associated with SARS-CoV-2 IgG antibody levels or neutralization. Neutralizing capacity was correlated with SARS-CoV-2 IgG antibodies and associated with time since symptom onset, BMI, and fever. These clinical parameters can be easily incorporated into the preselection of CCP donors.

Background: Transfusion of red cell concentrates (RCCs) is an integral therapy after severe hemorrhage or trauma. Prehospital transfusion offers an immediate intervention in emergency cases. Air ambulance-based prehospital transfusion, already used in different countries, is currently established in Germany. Limited information is available for regulatory-compliant transport logistics of RCCs and their quality after repeated air rescue missions. Thus, the aim of this study was (i) to validate regulatory-compliant logistics and (ii) to assess product quality, analyzing biochemical parameters and RBC morphology. Study Design and Methods: Due to regulatory requirements, we adapted a rotation system of 1 day transport, 1 day quarantine storage and 1 day storage over the entire RCC shelf life. RCCs transported on air rescue missions (flight group) were compared against a control group, treated identically except for helicopter transport. RCCs were visually inspected, and their temperature was documented throughout the entire rotation cycles. RCCs at the end of shelf life (end point samples) were assessed for levels of hemoglobin, hematocrit, free hemoglobin, hemolysis, mean corpuscular volume, potassium and pH. In addition, morphological changes were assessed using flow morphometry. Results: In total 81 RCCs were assessed in the flight group and 50 in the control group. Within the flight group, 30 RCCs were transfused. RCCs were dispatched on average 11 times (7–13 times). The average flight time was 18.3 h (6.6–28.8 h). The rotation system ensured adherence to regulatory guidelines, especially compliance to storage conditions of +2 to +6°C of intermediate storage. Biochemical and morphological quality parameters did not exhibit any changes upon repeated air rescue missions. A correlation with respect to the flight time was not observed either. Discussion: The quality of RCCs after repeated air rescue missions is noninferior to control samples regarding biochemical and morphological parameters. The product quality is within German regulations for up to 42 days of storage. The logistics and maintenance of the thermal conditions are safe and feasible. Thus, a rotation system of RCCs offers a regulatory-compliant option to supply air rescue missions with RCCs to allow life-saving prehospital transfusions at the incident scene.

Background: The stem cell niche in human bone marrow provides scaffolds, cellular frameworks and essential soluble cues to support the stemness of hematopoietic stem and progenitor cells (HSPCs). To decipher this complex structure and the corresponding cellular interactions, a number of in vitro model systems have been developed. The cellular microenvironment is of key importance, and mesenchymal stromal cells (MSCs) represent one of the major cellular determinants of the niche. Regulation of the self-renewal and differentiation of HSPCs requires not only direct cellular contact and adhesion molecules, but also various cytokines and chemokines. The C-X-C chemokine receptor type 4/stromal cell-derived factor 1 axis plays a pivotal role in stem cell mobilization and homing. As we have learned in recent years, to realistically simulate the physiological in vivo situation, advanced model systems should be based on niche cells arranged in a three-dimensional (3D) structure. By providing a dynamic rather than static setup, microbioreactor systems offer a number of advantages. In addition, the role of low oxygen tension in the niche microenvironment and its impact on hematopoietic stem cells need to be taken into account and are discussed in this review. Summary: This review focuses on the role of MSCs as a part of the bone marrow niche, the interplay between MSCs and HSPCs and the most important regulatory factors that need to be considered when engineering artificial hematopoietic stem cell niche systems. Conclusion: Advanced 3D model systems using MSCs as niche cells and applying microbioreactor-based technology are capable of simulating the natural properties of the bone marrow niche more closely than ever before.

Background and Objectives This study aimed to describe motives as well as donation experiences and the intention to return for further donations of German whole blood donors who donated at the beginning of the COVID‐19 pandemic. Materials and Methods To describe motives and donor experiences, a retrospective survey was conducted among whole blood donors that had a donation appointment at the German Red Cross Blood Donation Service in the first 4 weeks of the pandemic. A donor questionnaire including 17 retrospective questions was sent to 7500 donors. Donor motivation and donor experiences were compared for different donor groups using chi‐square statistics. Finally, in an ordinal logistic regression model predictors for the intention to return were identified. Results More than half of the participating donors (56.9%) wanted to contribute to the fight against the pandemic by donating blood. Most of the donors were satisfied with their last donation experience and felt safe during the blood donor appointment. However, some donors would have liked more information on how to deal with the pandemic (20.3%). Intention to return for further donations was strongly associated with overall satisfaction (OR: 1.67, CI: 1.47–1.90) and the feeling of being safe during blood donation (OR: 1.33, CI: 1.05–1.68). Conclusion Donor satisfaction with the last donation was high and the vast majority of donors felt very safe. However, those donors who felt unsafe expressed a low intention to return and blood donation services should therefore carefully monitor donor satisfaction.

ZusammenfassungPlasma von genesenen Spendern mit COVID-19 (COVID-19 Convalescent Plasma, CCP) wurde als Behandlungsoption für Patienten mit COVID-19 in Betracht gezogen. In der ersten Phase der Pandemie wurden mehrere Fallberichte und Fallkontrollstudien mit Hinweisen auf eine therapeutische Wirkung veröffentlicht. Inzwischen liegen die Ergebnisse zahlreicher randomisierter Studien vor. Die Studien unterscheiden sich in vielen Aspekten, u. a. in den Patientenpopulationen, die von ambulanten Patienten mit mildem COVID-19 bis zu kritisch Kranken reichten, wie auch den Endpunkten. Ebenso war der Behandlungsstandard innerhalb der klinischen Studien sehr unterschiedlich. Vor allem aber unterschied sich das Prüfpräparat CCP erheblich in Bezug auf das Behandlungsschema, das Volumen und den Gehalt an Antikörpern. Im Folgenden werden wir die Ergebnisse der bisher publizierten randomisierten Studien diskutieren. Aus den bisher veröffentlichten Ergebnissen lässt sich eine Wirksamkeit von CCP ableiten, sofern es sehr hohe Titer neutralisierender Antikörper enthält und früh im Krankheitsverlauf verabreicht wird. COVID-19-Rekonvaleszenten-Plasma ist noch keine Routinebehandlung und sollte möglichst weiter in klinischen Studien untersucht werden. Neu konzipierte Studien sollten sich auf die frühe Anwendung von CCP mit einem hohen Gehalt an neutralisierenden Antikörpern bei Patienten mit hohem Risiko für eine Progression zu einer schweren COVID-19-Erkrankung konzentrieren und wichtige Begleitmedikationen kontrollieren.

ZUSAMMENFASSUNGDer Anteil in Deutschland tätiger ausländischer Ärzte steigt an. In 2018 betrug der Zuwachs 3500 und insgesamt waren annähernd 55 000 Ärztinnen und Ärzte aus dem Ausland in deutschen Kliniken beschäftigt 1. Berücksichtigt man den demografischen Wandel und die zunehmende Zahl an altersbedingten Austritten in den Ruhestand wird zukünftig etwa jeder 4. bis 5. im Krankenhaus tätige Arzt aus dem Ausland stammen. In die ausländischen Ärzte muss zu Beginn ihrer Tätigkeit am neuen Arbeitsort in Deutschland auf vielfältige Weise zur beruflichen Qualifizierung investiert werden. Ein interkulturell ausgerichtetes Personalmanagement im Krankenhaus ist nötig, um die rekrutierten Kräfte in ihrer ärztlichen Rolle weiterzuentwickeln und zu binden.

Background Autologous lipofilling is an emerging procedure to treat and possibly reverse dermal scars and to reduce scar-related pain, but its efficacy and mechanisms are poorly understood. Objectives The aim of this study was to test the hypothesis that repeated lipografts reverse dermal scars by reinitiation of wound healing. Methods In a prospective, non-placebo-controlled clinical study, 27 adult patients with symptomatic scars were given 2 lipofilling treatments at 3-month intervals. As primary outcome, clinical effects were measured with the Patient and Observer Scar Assessment Scale (POSAS). Scar biopsies were taken before and after treatments to assess scar remodeling at a cellular level. Results Twenty patients completed the study. Patients’ scars improved after lipofilling. The total POSAS scores (combined patient and observer scores) decreased from 73.2 [14.7] points (mean [standard deviation]) pretreatment to 46.1 [14.0] and 32.3 [13.2] points after the first and second lipofilling treatment, respectively. Patient POSAS scores decreased from 37.3 [8.8] points to 27.2 [11.3] and 21.1 [11.4] points, whereas observer POSAS scores decreased from 35.9 [9.5] points to 18.9 [6.0] and 11.3 [4.5] points after the first and second treatment, respectively. After each lipofilling treatment, T lymphocytes, mast cells, and M2 macrophages had invaded scar tissue and were associated with increased vascularization. In addition, the scar-associated epidermis showed an increase in epidermal cell proliferation to levels similar to that normal in skin. Moreover, lipofilling treatment caused normalization of the extracellular matrix organization towards that of normal skin. Conclusions Autologous lipofilling improves the clinical outcome of dermal scars through the induction of a pro-regenerative immune response, increased vascularization, and epidermal proliferation and remodeling of scar tissue extracellular matrix. Level of Evidence: 4

Krise ist ein produktiver Zustand. Man muss ihm nur den Beigeschmack der Katastrophe nehmen. Max Frisch Sehr geehrte Leserinnen und Leser, liebe Kolleginnen und Kollegen,täglich erreichen uns Nachrichten über immer neue Krisenszenarien. Krisen scheinen die Welt in unserer Zeit zu bestimmen. Wie sind wir diesen Herausforderungen gewachsen?

Background Convalescent plasma is one of the treatment options for COVID-19 which is currently being investigated in many clinical trials. Understanding of donor and product characteristics is important for optimization of convalescent plasma. Methods Patients who had recovered from CO­VID-19 were recruited as donors for COVID-19 convalescent plasma (CCP) for a randomized clinical trial of CCP for treatment of severe COVID-19 (CAPSID Trial). Titers of neutralizing antibodies were measured by a plaque-reduction neutralization test (PRNT). Correlation of antibody titers with host factors and evolution of neutralizing antibody titers over time in repeat donors were analysed. Results A series of 144 donors (41% females, 59% males; median age 40 years) underwent 319 plasmapheresis procedures providing a median collection volume of 850 mL and a mean number of 2.7 therapeutic units per plasmapheresis. The majority of donors had a mild or moderate course of COVID-19. The titers of neutralizing antibodies varied greatly between CCP donors (from 1:640). Donor factors (gender, age, ABO type, body weight) did not correlate significantly with the titer of neutralizing antibodies. We observed a significant positive correlation of neutralization titers with the number of reported COVID-19 symptoms and with the time from SARS-CoV-2 diagnosis to plasmapheresis. Neutralizing antibody levels were stable or increased over time in 58% of repeat CCP donors. Mean titers of neutralizing antibodies of first donation and last donation of repeat CCP donors did not differ significantly (1:86 at first compared to 1:87 at the last donation). There was a significant correlation of neutralizing antibodies measured by PRNT and anti-SARS-CoV-2 IgG and IgA antibodies which were measured by ELISA. CCP donations with an anti-SARS-CoV-2 IgG antibody content above the 25th percentile were substantially enriched for CCP donations with higher neutralizing antibody levels. Conclusion We demonstrate the feasibility of collection of a large number of CCP products under a harmonized protocol for a randomized clinical trial. Titers of neutralizing antibodies were stable or increased over time in a subgroup of repeat donors. A history of higher number of COVID-19 symptoms and higher levels of anti-SARS-CoV-2 IgG and IgA antibodies in immunoassays can preselect donations with higher neutralizing capacity.

The coronavirus disease-2019 (COVID-19) is a systemic disease with severe implications on the vascular and coagulation system. A procoagulant platelet phenotype has been reported at least in the acute disease phase. Soluble P-selectin (sP-sel) in the plasma is a surrogate biomarker of platelet activation. Increased plasma levels of sP-sel have been reported in hospitalized COVID-19 patients associated with disease severity. Here, we evaluated in a longitudinal study the sP-sel plasma concentration in blood donors who previously suffered from moderate COVID-19.154 COVID-19 convalescent and 111 non-infected control donors were recruited for plasma donation and for participation in the CORE research trial. First donation (T1) was performed 43-378 days after COVID-19 diagnosis. From most of the donors the second (T2) plasma donation including blood sampling was obtained after a time period of 21-74 days and the third (T3) donation after additional 22-78 days. Baseline characteristics including COVID-19 symptoms of the donors were recorded based on a questionnaire. Platelet function was measured at T1 by flow cytometry and light transmission aggregometry in a representative subgroup of 25 COVID-19 convalescent and 28 control donors. The sP-sel plasma concentration was determined in a total of 704 samples by using a commercial ELISA.In vitro platelet function was comparable in COVID-19 convalescent and control donors at T1. Plasma samples from COVID-19 convalescent donors revealed a significantly higher sP-sel level compared to controls at T1 (1.05 ± 0.42 ng/mL vs. 0.81 ± 0.30 ng/mL; p 0.0001) and T2 (0.96 ± 0.39 ng/mL vs. 0.83 ± 0.38 ng/mL; p = 0.0098). At T3 the sP-sel plasma level was comparable in both study groups. Most of the COVID-19 convalescent donors showed a continuous decrease of sP-sel from T1 to T3.Increased sP-sel plasma concentration as a marker for platelet or endothelial activation could be demonstrated even weeks after moderate COVID-19, whereas, in vitro platelet function was comparable with non-infected controls. We conclude that COVID-19 and additional individual factors could lead to an increase of the sP-sel plasma level.

Binding of podoplanin to the C-type lectin-like receptor 2 (CLEC-2) promotes platelet activation and soluble CLEC-2 (sCLEC-2) is shed from activated platelets. The role of sCLEC-2 in the plasma is ...

ZusammenfassungRekonvaleszentenplasma wurde bei Patienten mit akuten, viralen respiratorischen Erkrankungen mit schwerem Verlauf eingesetzt (SARS- oder MERS-Coronavirus, Influenza H1N1, H5N1). Diese Studien erbrachten Hinweise auf eine Reduktion der Viruslast, einer Verkürzung des Krankenhausaufenthalts und eine Reduktion der Mortalität durch Rekonvaleszentenplasma. Die Wirkung konnte jedoch nicht abschließend belegt werden, da es sich nicht um randomisierte Studien handelte. Nach einer SARS-CoV-2-Infektion kommt es bei den meisten Patienten zu einer raschen Bildung von neutralisierenden Antikörpern. Wegen der pandemischen Entwicklung gibt es eine wachsende Zahl Genesener mit neutralisierenden Antikörpern, die als potenzielle Spender in Betracht kommen. Wir konzipierten eine prospektive, randomisierte Studie, um die Wirksamkeit und Sicherheit von Rekonvaleszentenplasma zur Therapie von schwerem COVID-19 und dessen Wirkmechanismus zu untersuchen (CAPSID-Studie). In diesem Beitrag werden die Rationale und das Design der Studie vorgestellt.

The ADP receptor P2Y12, the thromboxane A2 receptor (TXA2R) and the C-type lectin-like receptor 2 (CLEC-2) mediate platelet activation by different mechanisms. Only little is known about the expres...

Introduction: The aim was to evaluate the impact of perioperative blood transfusions (PBTs) on renal function after surgery for renal cell carcinoma (RCC). Methods: Consecutive patients with RCC who underwent partial nephrectomy or radical nephrectomy between 2005 and 2015 at a tertiary care center were included. Minimum follow-up period was 6 months. A PBT was defined as the transfusion of packed erythrocyte concentrate (EC) within 7 days before until 30 days after surgery. The multivariable analyses were carried out by Cox regression. Results: The overall cohort included 851 patients, of whom 93 (10.9%) received a PBT. The median follow-up was 46 months (range 28–72). In case of a PBT, a median of 2 EC was transfused. PBT patients were older and had a poorer performance status and more comorbidities as well as locally more advanced or metastatic tumors. In the multivariable analyses, PBT was an independent prognostic factor for acute as well as chronic renal impairment (hazard ratio (HR) 2.72, 95% confidence interval (95% CI) 1.45–5.10, p = 0.002 and HR 2.23, 95% CI 1.26–3.70, p = 0.007). Conclusion: PBT is associated with acute and chronic deterioration of kidney function after surgery for RCC. Thus, it may be used to identify patients requiring close nephrological monitoring.

BackgroundRecent data on immune evasion of new SARS-CoV-2 variants raise concerns about antibody-based COVID-19 therapies. Therefore in this study the in-vitro neutralization capacity against SARS-CoV-2 variants Wuhan D614G, Delta and Omicron in sera of convalescent individuals with and without boost by vaccination was assessed.Methods and FindingsThis in-vitro study included 66 individuals with a history of SARS-CoV-2 infection, divided into subgroups without (n=29) and with SARS-CoV-2 vaccination (n=37). We measured SARS-CoV-2 antibody concentrations by serological assays (anti-SARS-CoV-2-QuantiVac-ELISA (IgG) and Elecsys Anti-SARS-CoV-2 S) and neutralizing titers against Wuhan D614G, Delta and Omicron in a pseudovirus neutralization assay.Sera of the majority of unvaccinated convalescents did not effectively neutralize Delta and Omicron (4/29, 13.8% and 19/29, 65.5%, resp.). Neutralizing titers against Wuhan D614G, Delta and Omicron were significantly higher in vaccinated compared to unvaccinated convalescents (pin-vitro by Bamlanivimab, Casirivimab and Imdevimab, Omicron was resistant to these monoclonal antibodies.ConclusionsThese findings confirm substantial immune evasion of Delta and Omicron which can be overcome by vaccination of convalescents. This informs strategies for choosing of plasma donors in COVID-19 convalescent plasma programs that shall select specifically vaccinated convalescents with very high titers of anti-S antibodies.

This paper investigates the impact of monetary incentives on whole blood donations. We take advantage of a quasi-natural experiment in Germany, in which one blood donation site changes its payment scheme from remunerated to non-remunerated. All other donation sites maintain their payment schemes. We show that donation volumes drop significantly after the pay drop and do not recuperate. At the same time, donation volumes increase at other paid donation sites, which is partly due to donor migration to these sites. We do not find any impact of the changed payment scheme on blood quality. Our results offer additional insight into the complex question whether it is efficient to ensure blood supply by paying donors a direct monetary compensation.

Mesenchymal stromal cells (MSC) exert their immunomodulatory potential on several cell types of the immune system, affecting and influencing the immune response. MSC efficiently inhibit T cell proliferation, reduce the secretion of pro-inflammatory cytokines, limit the differentiation of pro-inflammatory Th subtypes and promote the induction of regulatory T cells (Treg). In this study, we analyzed the immunomodulatory potential of human adipose tissue-derived MSC (ASC), on CD4+ T cells, addressing potential cell-contact dependency in relation to T cell receptor stimulation of whole human peripheral blood mononuclear cells (PBMC). ASC were cultured with not stimulated or anti-CD3/CD28-stimulated PBMC in direct and transwell cocultures, PBMC alone were used as controls. After 7 days, cocultures were harvested and we analyzed: (1) the inhibitory potential of ASC on CD4+ cell proliferation and (2) phenotypic changes in CD4+ cells in respect of Treg marker (CD25, CD127 and FoxP3) expression. We confirmed the inhibitory potential of ASC on CD4+ cell proliferation, which occurs upon PBMC stimulation and is mediated by indoleamine 2,3-dioxygenase. Importantly, ASC reduce both pro- and anti-inflammatory cytokine secretion, without indications on specific Th differentiation. We found that stimulation induces CD25 expression on CD4+ cells and that, despite inhibiting overall CD4+ cell proliferation, ASC can specifically induce the proliferation of CD4+CD25+ cells. We observed that ASC induce Treg (CD4+CD25+CD127-FoxP3+) only in not stimulated cocultures and that ASC increase the ratio of CD4+CD25+CD127+FoxP3- cells at the expense of CD4+CD25+CD127-FoxP3- cells. Our study provides new insights on the interplay between ASC and CD4+ T cells, proposing that ASC-dependent induction of Treg depends on PBMC activation which affects the balance between the different subpopulations of CD4+CD25+ cells expressing CD127 and/or FoxP3.

Binding of podoplanin to the C-type lectin-like receptor 2 (CLEC-2) promotes platelet activation and soluble CLEC-2 (sCLEC-2) is shed from activated platelets. The role of sCLEC-2 in the plasma is unknown. The expression level and plasma concentration of sCLEC-2 could be affected by variants of the corresponding gene

Over recent years, mesenchymal stromal cells (MSC) have gained immense attraction in immunotherapy, regenerative medicine and tissue engineering. MSC microenvironment modulation occurs through synergy of direct cell&ndash, cell contact, and secreted soluble factors and extracellular vesicles (EV). MSC-derived EV have been suggested as cell-free immunomodulatory alternative to MSC, however, previous findings have challenged this. Furthermore, recent data suggest that evaluating the mechanism of action of human MSC (hMSC) in animal models might promote adverse immune reactions or lack of functionality due to xeno-incompatibilities. In this study, we first assessed the immunomodulatory strength of different human MSC sources on in vitro stimulated T cells and compared this to interferon-gamma (IFN&gamma, ) primed MSC conditioned medium (CM) and EV. Second, we addressed the main molecular mechanisms, and third, we assessed the MSC in vitro immunosuppressive effect across interspecies barriers. We identified human adipose tissue-derived stromal cells (ASC) with strongest immunomodulatory strength, followed by bone marrow (BM) and cord blood-derived MSC (CB). Whilst CM from primed ASC managed to exert analogous effects as their cellular counterpart, EV derived thereof did not, reproducing previous findings. IFN&gamma, induced indoleamine 2,3-dioxygenase (IDO) activity was identified as key mechanism to suppress human lymphocyte proliferation, as in the presence of the IDO inhibitor epacadostat (Epac) a stimulation of proliferation was seen. In addition, we revealed MSC immunosuppressive effects to be species-specific, because human cells failed to suppress murine lymphocyte proliferation. In summary, ASC were the strongest immunomodulators with the IDO-kynurenine pathway being key within the human system. Importantly, the in vitro lack of interspecies immunomodulatory strength suggests that preclinical data need to be carefully interpreted especially when considering a possible translation to clinical field.

The ADP receptor P2Y

Introduction: Recently, we identified a huge discrepancy between the collection practice and the actual utilization of cryopreserved peripheral blood stem cells (PBSCs) for high-dose chemotherapy (HDCT) and autologous blood stem cell transplantation (ABSCT). Specifically, patients with Burkitt lymphoma, acute leukemia, and myeloproliferative neoplasms (MPN) were frequently not referred for ABSCT after successful PBSC collection. Objective: The aim of this study was to identify variables that are associated with the non-utilization of PBSC grafts. Methods: We retrospectively analyzed the collection, storage, and disposal of PBSC grafts in Burkitt lymphoma (n = 18), acute lymphoblastic leukemia (ALL, n = 22), MPN (n = 18), and acute myeloid leukemia (AML, n = 71) patients. Patients who underwent autologous PBSC collection at 2 collection and transplantation centers between 2001 and 2012 were included and followed up until 2016. Results: None of the Burkitt lymphoma patients were referred for ABSCT. Only in 1 (6%) patient, the graft was discarded after the patient’s death. In all other patients (n = 17, 94%), the grafts were stored independently of the patient’s status (death, n = 4, 22%; no follow-up, n = 6, 33%; no indication for ABSCT given, n = 7, 39%). In ALL patients, 4 (18%) patients underwent ABSCT after a median follow-up of 74 (1–182) months. In the remaining patients, PBSC grafts were either discarded (8 patients, 36%) or stored until the reference date (10 patients, 45%). Seven of 18 MPN patients (39%) underwent ABSCT. ABSCT was performed in 24 (34%) AML patients. In 20 (28%) patients who were not referred to ABSCT, an allogeneic transplantation (TPL) was performed. Fifteen (21%) patients received palliative care or deceased, and their grafts were discarded in all but 1 patient. Additional grafts were discarded in 21 (31%) patients and stored in 9 (13%) patients who underwent ABSCT or allogeneic TPL (n = 44). Conclusions: As the role and efficacy of autologous HDCT/ABSCT are not established in the analyzed entities, the indication for PBSC collection should be reanalyzed in regular intervals. Moreover, PBSC grafts from patients who have deceased, have insufficient grafts, or have already undergone an allogeneic TPL should be considered for disposal or (if applicable) for research use, to economize storage costs on a rational basis.

Hyperglycemia, a hallmark of diabetes, can induce inflammatory programming of macrophages. The macrophage scavenger receptor CD163 internalizes and degrades hemoglobin-haptoglobin (Hb-Hp) complexes built due to intravascular hemolysis. Clinical studies have demonstrated a correlation between impaired scavenging of Hb-Hp complexes via CD163 and diabetic vascular complications. Our aim was to identify whether hyperglycemia is able to amplify inflammation via Hb-Hp complex interactions with the immune system. M(IFNγ), M(IL-4), and control M0 macrophages were differentiated out of primary human monocytes in normo- (5 mM) and hyperglycemic (25 mM) conditions. CD163 gene expression was decreased 5.53 times in M(IFNγ) with a further decrease of 1.99 times in hyperglycemia. Hyperglycemia suppressed CD163 surface expression in M(IFNγ) (1.43 times). Flow cytometry demonstrated no impairment of Hb-Hp uptake in hyperglycemia. However, hyperglycemia induced an inflammatory response of M(IFNγ) to Hb-Hp1-1 and Hb-Hp2-2 uptake with different dynamics. Hb-Hp1-1 uptake stimulated IL-6 release (3.03 times) after 6 h but suppressed secretion (5.78 times) after 24 h. Contrarily, Hb-Hp2-2 uptake did not affect IL-6 release after 6h but increased secretion after 24 h (3.06 times). Our data show that hyperglycemia induces an inflammatory response of innate immune cells to Hb-Hp1-1 and Hb-Hp2-2 uptake, converting the silent Hb-Hp complex clearance that prevents vascular damage into an inflammatory process, hereby increasing the susceptibility of diabetic patients to vascular complications.

Background: The provision of compatible blood products to patients is the most essential task of transfusion medicine. Besides ABO and Rh, a number of additional blood group antigens often have to be considered for the blood supply of immunized or chronically transfused patients. It also applies for platelet antigens (HPA) and neutrophil antigens (HNA) for patients receiving platelet or granulocyte concentrates. Here, we describe the molecular screening for a number of blood group, HPA, and HNA alleles. Based on the screening results we are building up a regional blood donor registry to provide extended matched blood products on demand. Methods: We developed and validated TaqMan™ PCR and PCR-SSP methods for genetic markers defining 37 clinically relevant blood group antigens (beyond ABO and Rh), 10 HPA, and 11 HNA. Furthermore, we describe a feasible method for fast molecular screening of the HNA-2null phenotype. All data were statistically evaluated regarding genotype distribution. Allele frequencies were compared to ExAC data from non-Finnish Europeans. Results: Up to now more than 2,000 non-selected regular blood donors in south-west Germany have been screened for blood group, HPA, and HNA alleles. The screening results were confirmed by serology and PCR-SSP methods for selected numbers of samples. The allele frequencies were similar to non-finnish Europeans in the ExAC database except for the alleles encoding the S, HPA-3b and HNA-4b antigens, with significantly lower prevalence in our cohort, as well as the LU14 and the HNA-3b antigens, with a higher frequency compared to the ExAC data. We identified 71 donors with rare blood groups such as Lu(a+b-), Kp(a+b-), Fy(a-b-) and Vel-, and 169 donors with less prevalent HPA or HNA types. Conclusion: Molecular screening for blood group alleles by using TaqMan™ PCR is an effective and reliable high-throughput method for establishing a rare donor registry.

Bioactive polylactic acid based (PLA) scaffolds with hyaluronic acid immobilized on their surface by atmospheric pressure plasma assisted modification method were developed. By using X-ray photoelectron spectroscopy and wettability measurements it was shown that atmospheric pressure plasma treatment leads to the changes in surface chemical composition of the PLA-based scaffolds that resulted in an increased long-term hydrophilicity of the scaffolds surface. Scanning electron microscopy and mechanical studies revealed that the use of plasma for surface activation allows for the non-destructive immobilization of bioactive compounds like hyaluronic acid. The modified PLA-based scaffolds effect on the release of cytokines and matrix metalloproteinases by primary human monocyte-derived macrophages was investigated. The macrophages reaction to the scaffolds was donor-specific, however, the two best materials from immunological point of view were identified - plasma treated PLA-based scaffold and PLA-based scaffold with the least amount of immobilized hyaluronic acid. Both hyaluronic acid attachment and atmospheric pressure plasma treatment enhance PLA-based scaffolds biocompatibility. It was found that supernatants collected after the macrophages coculture with modified PLA-based scaffolds stimulate HUVECs' tube formation. The modified PLA-based scaffolds possess pro-angiogenic activity. Thus, our research offers a high-performing method for the creation of polymer-based tissue engineering scaffolds with modified bioactive surface. Keywords: Polylactic acid (PLA), Hyaluronic acid (HA), Tissue engineering scaffold (TES), Plasma, Macrophages, Cytokines

We present a novel automated system for morphology analysis of red blood cells (RBC) under flow. RBC concentrates collected by blood banks for transfusions are stored for periods of up to several weeks, during which time a number of changes occur, collectively termed the storage lesion. Typically the extent of hemolysis is the defining criterion to determine the acceptability of the RBCs for transfusions. Morphological changes are related with biochemical alteration during the storage of RBCs. The typical blood smear procedure for determining such changes is a labor-intensive and potentially biased manual process. The advantage of the flow morphometry system presented here is that it provides fully automated morphological classification of RBCs with large sample numbers in a short time. Our system uses a commercially available flow cell and flow conditions that prevent adhesion of RBCs, thus eliminating the need for blocking agents such as albumin that affect the distribution of cell shapes. Our morphometry results are validated by comparison with standard biochemical assays (hemolysis, ATP) for blood from 17 donors stored under blood bank conditions for 13 weeks. We show that the percentage of spherocytes present can be used to estimate the status of RBC concentrates. © 2017 International Society for Advancement of Cytometry.

Background: In the recent past, the discrepancy between blood supply and future demand may have been overestimated. As medical progress develops rapidly, it will be essential to monitor ongoing demographic changes in the donor population regularly and to re-evaluate retention and recruiting strategies. The aim of the current study was to compare first-time donor (FTD) characteristics and their return rates. We therefore compared whole blood (WB) donations in total and the annual donation frequencies in 2010 and in 2015/2016. Furthermore, we evaluated whether over the same observation period, medical reasons for deferral underwent a change (2010 vs. 2015). Methods: The return probability of FTD within 12 months was analysed in 2010 and 2015 with respect to successful donation versus deferral and with regard to age. The total number of WB donations was investigated, and age distribution was compared between 2010, 2013 and 2016. WB donation frequencies were calculated with respect to age and gender in 2010 and 2016. In a second analysis, medical reasons for deferral were differentiated into 14 categories and a possible impact of time (2010 vs. 2015) on the respective percentage was studied. Results: We observed a significant decline of the FTD return rate from 42.5% to 38.8% in donors that successfully donated WB while the rate remained unchanged in deferred FTD. At the same time the mean FTD age decreased from 29.1 ± 11.6 to 28.5 ± 11.7 years in 2016. Analysis of total WB donations revealed an increase of all donations from donors ≥60 years, a constant percentage from donors Conclusion: There is ongoing demographic change in our WB donor population. Our data prove a need for a re-evaluation of retention and recruitment strategies since previous marketing campaigns seem to have neglected the age group 30-59 years. This must be addressed in further studies as this age group will be highly relevant for assuring future blood supplies since donor recruitment from adolescents will be limited due to declining birth rates. Furthermore, deferral due to travel history is increasing significantly. Thus we will require further studies on the possible impact on donor retention.

The number of diabetic patients in Europe and world-wide is growing. Diabetes confers a 2-fold higher risk for vascular disease. Lack of insulin production (Type 1 diabetes, T1D) or lack of insulin responsiveness (Type 2 diabetes, T2D) causes systemic metabolic changes such as hyperglycemia (HG) which contribute to the pathology of diabetes. Monocytes and macrophages are key innate immune cells that control inflammatory reactions associated with diabetic vascular complications. Inflammatory programming of macrophages is regulated and maintained by epigenetic mechanisms, in particular histone modifications. The aim of our study was to identify the epigenetic mechanisms involved in the hyperglycemia-mediated macrophage activation. Using Affymetrix microarray profiling and RT-qPCR we identified that hyperglycemia increased the expression of S100A9 and S100A12 in primary human macrophages. Expression of S100A12 was sustained after glucose levels were normalized. Glucose augmented the response of macrophages to Toll-like receptor (TLR)-ligands Palmatic acid (PA) and Lipopolysaccharide (LPS) i.e., pro-inflammatory stimulation. The abundance of activating histone Histone 3 Lysine 4 methylation marks (H3K4me1, H3K4me3) and general acetylation on histone 3 (AceH3) with the promoters of these genes was analyzed by chromatin immunoprecipitation. Hyperglycemia increased acetylation of histones bound to the promoters of S100A9 and S100A12 in M1 macrophages. In contrast, hyperglycemia caused a reduction in total H3 which correlated with the increased expression of both S100 genes. The inhibition of histone methyltransferases SET domain-containing protein (SET)7/9 and SET and MYND domain-containing protein (SMYD)3 showed that these specifically regulated S100A12 expression. We conclude that hyperglycemia upregulates expression of S100A9, S100A12 via epigenetic regulation and induces an activating histone code on the respective gene promoters in M1 macrophages. Mechanistically, this regulation relies on action of histone methyltransferases SMYD3 and SET7/9. The results define an important role for epigenetic regulation in macrophage mediated inflammation in diabetic conditions.