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Introduction: COVID-19 convalescent plasma (CCP) is a possible treatment option for COVID-19. A comprehensive number of clinical trials on CCP efficacy have already been conducted. However, many aspects of CCP treatment still require investigations: in particular (1) Optimisation of the CCP product, (2) Identification of the patient population in need and most likely to benefit from this treatment approach, (3) Timing of administration and (4) CCP efficacy across viral variants in vivo. We aimed to test whether high-titre CCP, administered early, is efficacious in preventing hospitalisation or death in high-risk patients., Methods and Analysis: COVIC-19 is a multicentre, randomised, open-label, adaptive superiority phase III trial comparing CCP with very high neutralising antibody titre administered within 7 days of symptom onset plus standard of care versus standard of care alone. We will enrol patients in two cohorts of vulnerable patients [(1) elderly 70+ years, or younger with comorbidities; (2) immunocompromised patients]. Up to 1020 participants will be enrolled in each cohort (at least 340 with a sample size re-estimation after reaching 102 patients). The primary endpoint is the proportion of participants with (1) Hospitalisation due to progressive COVID-19, or (2) Who died by day 28 after randomisation. Principal analysis will follow the intention-to-treat principle., Ethics and Dissemination: Ethical approval has been granted by the University of Ulm ethics committee (#41/22) (lead ethics committee for Germany), Comité de protection des personnes Sud-Est I (CPP Sud-Est I) (#2022-A01307-36) (ethics committee for France), and ErasmusMC ethics committee (#MEC-2022-0365) (ethics committee for the Netherlands). Signed informed consent will be obtained from all included patients. The findings will be published in peer-reviewed journals and presented at relevant stakeholder conferences and meetings., Trial Registration: Clinical Trials.gov (NCT05271929), EudraCT (2021-006621-22)., Competing Interests: Competing interests: PT is an employee of Établissement Français du Sang, the blood establishment responsible for blood collection, qualification and supply in France. HS, SH, SK, TA and ES are employees of the German Red Cross Blood Transfusion Service Baden-Württemberg-Hessen (or its affiliates), the establishment responsible for blood collection, qualification and supply in Baden-Württemberg and Hesse, Germany. CEV, is an employee of Sanquin, the establishment responsible for blood collection, qualification and supply in the Netherlands. The authors declare no other competing interests., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Recent data on immune evasion of new SARS-CoV-2 variants raise concerns about the efficacy of antibody-based COVID-19 therapies. Therefore, in this study the in-vitro neutralization capacity against SARS-CoV-2 variant B.1 and the Omicron subvariants BA.1, BA.2 and BA.5 of sera from convalescent individuals with and without boost by vaccination was assessed., Methods and Findings: The study included 313 serum samples from 155 individuals with a history of SARS-CoV-2 infection, divided into subgroups without (n=25) and with SARS-CoV-2 vaccination (n=130). We measured anti-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 B.1, BA.1, BA.2 and BA.5 in a pseudovirus neutralization assay. Sera of the majority of unvaccinated convalescents did not effectively neutralize Omicron sublineages BA.1, BA.2 and BA.5 (51.7%, 24.1% and 51.7%, resp.). In contrast, 99.3% of the sera of superimmunized individuals (vaccinated convalescents) neutralized the Omicron subvariants BA.1 and BA.5 and 99.6% neutralized BA.2. Neutralizing titers against B.1, BA.1, BA.2 and BA.5 were significantly higher in vaccinated compared to unvaccinated convalescents (p<0.0001) with 52.7-, 210.7-, 141.3- and 105.4-fold higher geometric mean of 50% neutralizing titers (NT50) in vaccinated compared to unvaccinated convalescents. 91.4% of the superimmunized individuals showed neutralization of BA.1, 97.2% of BA.2 and 91.5% of BA.5 with a titer ≥ 640. The increase in neutralizing titers was already achieved by one vaccination dose. Neutralizing titers were highest in the first 3 months after the last immunization event. Concentrations of anti-S antibodies in the anti-SARS-CoV-2-QuantiVac-ELISA (IgG) and Elecsys Anti-SARS-CoV-2 S assays predicted neutralization capacity against B.1 and Omicron subvariants BA.1, BA.2 and BA.5., Conclusions: These findings confirm substantial immune evasion of the Omicron sublineages, 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., Competing Interests: SH, BJ, SK, CL, CV, RL, RM, PW, MS, HK, and HS are employees of the German Red Cross Blood Transfusion Service Baden-Württemberg-Hessen collecting plasma including convalescent plasma. The remaining 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 © 2023 Seidel, Hoffmann, Jahrsdörfer, Körper, Ludwig, Vieweg, Albers, von Maltitz, Müller, Lotfi, Wuchter, Klüter, Kirchhoff, Schmidt, Münch and Schrezenmeier.)

Convalescent plasma was discussed as a therapeutic option early in the course of the COVID-19 pandemic. However, until the onset of the pandemic, only the results of mostly small single-arm studies in other infectious diseases were available, which did not prove efficacy. In the meantime, the results of more than 30 randomized trials of COVID-19 convalescent plasma (CCP) for treatment of COVID-19 are available 1. Despite the heterogeneity of the results, conclusions for an optimal use are possible., Competing Interests: Prof. Dr. Hubert Schrezenmeier, Prof. Dr. Dr. h.c. Erhard Seifried und Dr. Sixten Körper haben durch das BMG und BMBF geförderte klinische Prüfungen zum Einsatz von Rekonvalesztenplasma durchgeführt und sind bei Institutionen tätig, die Plasmapräparate herstellen., (Thieme. All rights reserved.)

COVID-19 convalescent plasma (CCP) has been explored as one of the treatment options for COVID-19. Results of many cohort studies and clinical trials have been recently published. At first glance, the results of the CCP studies appear to be inconsistent. However, it became clear that CCP is not beneficial if CCP with low anti-SARS-CoV-2 antibody concentrations is used, if it is administered late in advanced disease stages, and to patients who already mounted an antibody response against SARS-CoV-2 at the time of CCP transfusion. On the other hand, CCP may prevent progression to severe COVID-19 when very high-titer CCP is given early in vulnerable patients. Immune escape of new variants is a challenge for passive immunotherapy. While new variants of concern developed resistance to most clinically used monoclonal antibodies very rapidly, immune plasma from individuals immunized by both a natural SARS-CoV-2 infection and SARS-CoV-2 vaccination retained neutralizing activity against variants. This review briefly summarizes the evidence on CCP treatment to date and identifies further research needs. Ongoing research on passive immunotherapy is not only relevant for improving care for vulnerable patients in the ongoing SARS-CoV-2 pandemic, but even more as a model for passive immunotherapy in case of future pandemics with a newly evolving pathogen. Compared to other drugs, which must be newly developed in a pandemic (e.g., monoclonal antibodies, antiviral drugs), convalescent plasma is rapidly available, inexpensive to produce, and can be adaptive to viral evolution by selection of contemporary convalescent donors., Competing Interests: None declared., (Thieme. All rights reserved.)

BACKGROUNDResults of many randomized trials on COVID-19 convalescent plasma (CCP) have been reported, but information on long-term outcome after CCP treatment is limited. The objectives of this extended observation of the randomized CAPSID trial are to assess long-term outcome and disease burden in patients initially treated with or without CCP.METHODSOf 105 randomized patients, 50 participated in the extended observation. Quality of life (QoL) was assessed by questionnaires and a structured interview. CCP donors (n = 113) with asymptomatic to moderate COVID-19 were included as a reference group.RESULTSThe median follow-up of patients was 396 days, and the estimated 1-year survival was 78.7% in the CCP group and 60.2% in the control (P = 0.08). The subgroup treated with a higher cumulative amount of neutralizing antibodies showed a better 1-year survival compared with the control group (91.5% versus 60.2%, P = 0.01). Medical events and QoL assessments showed a consistent trend for better results in the CCP group without reaching statistical significance. There was no difference in the increase in neutralizing antibodies after vaccination between the CCP and control groups.CONCLUSIONThe trial demonstrated a trend toward better outcome in the CCP group without reaching statistical significance. A predefined subgroup analysis showed a significantly better outcome (long-term survival, time to discharge from ICU, and time to hospital discharge) among those who received a higher amount of neutralizing antibodies compared with the control group. A substantial long-term disease burden remains after severe COVID-19.Trial registrationEudraCT 2020-001310-38 and ClinicalTrials.gov NCT04433910.FundingBundesministerium für Gesundheit (German Federal Ministry of Health).

Introduction: Humoral immunity after SARS-CoV-2 vaccination has been extensively investigated in blood. Aim of this study was to develop an ELISA method in order to determine the prevalence of IgG and IgA SARS-CoV-2 domain 1 spike-protein (S) specific antibodies (Abs) in buccal and nasal mucosal surfaces of vaccinees., Methods: To this end, we analyzed 69 individuals who received their first vaccine dose between February and July 2021. Vaccines administered were BNT162b2, mRNA-1273 or ChAdOx1-nCoV-19. Detection of IgG and IgA Abs was performed using commercial ELISA kits for both blood and swab samples after protocol modification for the latter., Results: Anti-spike IgG and IgA Abs in the buccal and/or nasal swabs were detectable in >81% of the study subjects after the second dose. The IgG measurements in buccal swabs appeared to correlate in a more consistent way with the respective measurements in blood with a correlation coefficient of r=0.74. It is of note that IgA Abs appeared to be significantly more prevalent in the nasal compared to the buccal mucosa. Optimal selection of the assay cut-off for the IgG antibody detection in buccal swabs conferred a sensitivity of 91.8% and a specificity of 100%. Last, individuals vaccinated with mRNA-based vaccines exhibited higher antibody levels in both blood and mucosal surfaces compared to those receiving ChAdOx1-nCoV-19 confirming previously reported results., Conclusion: In conclusion, our findings show a differential prevalence of anti-S Abs on mucosal surfaces after vaccination for SARS-CoV-2, while they also set the basis for potential future use of IgG antibody detection in buccal swabs for extended immunity screening in large populations., 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 Tsamadou, Ludwig, Scholz, Proffen, Hägele, Rode, Körper, Fabricius, Jahrsdörfer, Neuchel, Amann, Schrezenmeier and Fürst.)

Objectives: To determine the profile of cytokines in patients with severe COVID-19 who were enrolled in a trial of COVID-19 convalescent plasma (CCP)., Methods: Patients were randomized to receive standard treatment and 3 CCP units or standard treatment alone (CAPSID trial, ClinicalTrials.gov NCT04433910). The primary outcome was a dichotomous composite outcome (survival and no longer severe COVID-19 on day 21). Time to clinical improvement was a key secondary endpoint. The concentrations of 27 cytokines were measured (baseline, day 7). We analyzed the change and the correlation between serum cytokine levels over time in different subgroups and the prediction of outcome in receiver operating characteristics (ROC) analyses and in multivariate models., Results: The majority of cytokines showed significant changes from baseline to day 7. Some were strongly correlated amongst each other (at baseline the cluster IL-1ß, IL-2, IL-6, IL-8, G-CSF, MIP-1α, the cluster PDGF-BB, RANTES or the cluster IL-4, IL-17, Eotaxin, bFGF, TNF-α). The correlation matrix substantially changed from baseline to day 7. The heatmaps of the absolute values of the correlation matrix indicated an association of CCP treatment and clinical outcome with the cytokine pattern. Low levels of IP-10, IFN-γ, MCP-1 and IL-1ß on day 0 were predictive of treatment success in a ROC analysis. In multivariate models, low levels of IL-1ß, IFN-γ and MCP-1 on day 0 were significantly associated with both treatment success and shorter time to clinical improvement. Low levels of IP-10, IL-1RA, IL-6, MCP-1 and IFN-γ on day 7 and high levels of IL-9, PDGF and RANTES on day 7 were predictive of treatment success in ROC analyses. Low levels of IP-10, MCP-1 and high levels of RANTES, on day 7 were associated with both treatment success and shorter time to clinical improvement in multivariate models., Conclusion: This analysis demonstrates a considerable dynamic of cytokines over time, which is influenced by both treatment and clinical course of COVID-19. Levels of IL-1ß and MCP-1 at baseline and MCP-1, IP-10 and RANTES on day 7 were associated with a favorable outcome across several endpoints. These cytokines should be included in future trials for further evaluation as predictive factors., 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 Körper, Schrezenmeier, Rincon-Arevalo, Grüner, Zickler, Weiss, Wiesmann, Zacharowski, Kalbhenn, Bentz, Dollinger, Paul, Lepper, Ernst, Wulf, Zinn, Appl, Jahrsdörfer, Rojewski, Lotfi, Dörner, Jungwirth, Seifried, Fürst and Schrezenmeier.)

In this prospective observational cohort study we analyzed cellular and serological immune response parameters against SARS-CoV-2 and current variants of concern (VOC) in 147 COVID-19-convalescent and 39 COVID-19-naïve individuals before and after BNT162b2 booster vaccination. No significant differences regarding immunological response parameters were observed between younger and older individuals. Booster vaccination induced full recovery of both cellular and serological response parameters including IFN-γ secretion and anti-spike antibody titers with strong neutralization capacities against wild type SARS-COV-2 and Delta. Surprisingly, even serological neutralization capacity against Omicron was detectable one month after second vaccination and four months before it had been first observed in South Africa. As a result, more than 90% of convalescent individuals exhibited detectable and 75% strong Omicron neutralization capacity after booster vaccination, compared with 72% and 46% of COVID-19-naïve individuals. Our results support the notion that broad and cross-reactive immune memory against SARS-CoV-2 including currently known VOCs can be established by booster vaccination with spike-based mRNA vaccines like BNT162b2, particularly in COVID-19-convalescent individuals of all ages. Nevertheless, especially in COVID-19-naïve individuals future variants escaping the memory immune response may require vaccine approaches such as inactivated whole virus vaccines , which include all antigenic components of the virus., 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 Jahrsdörfer, Proffen, Scholz, Hägele, Ludwig, Vieweg, Grempels, Fabricius, Lotfi, Körper, Adler and Schrezenmeier.)

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hemolytic disease driven by impaired complement regulation. Mutations in genes encoding the enzymes that build the GPI anchors are causative, with somatic mutations in the PIG-A gene occurring most frequently. As a result, the important membrane-bound complement regulators CD55 and CD59 are missing on the affected hematopoietic stem cells and their progeny, rendering those cells vulnerable to complement attack. Immune escape mechanisms sparing affected PNH stem cells from removal are suspected in the PNH pathogenesis, but molecular mechanisms have not been elucidated. We hypothesized that exuberant complement activity in PNH results in enhanced immune checkpoint interactions, providing a molecular basis for the potential immune escape in PNH. In a series of PNH patients, we found increased expression levels of the checkpoint ligand programmed death-ligand 1 (PD-L1) on granulocytes and monocytes, as well as in the plasma of PNH patients. Mechanistically, we demonstrate that complement activation leading to the decoration of particles/cells with C3- and/or C4-opsonins increased PD-L1 expression on neutrophils and monocytes as shown for different in vitro models of classical or alternative pathway activation. We further establish in vitro that complement inhibition at the level of C3, but not C5, inhibits the alternative pathway-mediated upregulation of PD-L1 and show by means of soluble PD-L1 that this observation translates into the clinical situation when PNH patients are treated with either C3 or C5 inhibitors. Together, the presented data show that the checkpoint ligand PD-L1 is increased in PNH patients, which correlates with proximal complement activation., (Copyright © 2022 by The American Association of Immunologists, Inc.)

The interferon pathway, a key antiviral defense mechanism, is being considered as a therapeutic target in COVID-19. Both, substitution of interferon and JAK/STAT inhibition to limit cytokine storms have been proposed. However, little is known about possible abnormalities in STAT signaling in immune cells during SARS-CoV-2 infection. We investigated downstream targets of interferon signaling, including STAT1, STAT2, pSTAT1 and 2, and IRF1, 7 and 9 by flow cytometry in 30 patients with COVID-19, 17 with mild, and 13 with severe infection. We report upregulation of STAT1 and IRF9 in mild and severe COVID-19 cases, which correlated with the IFN-signature assessed by Siglec-1 (CD169) expression on peripheral monocytes. Interestingly, Siglec-1 and STAT1 in CD14+ monocytes and plasmablasts showed lower expression among severe cases compared to mild cases. Contrary to the baseline STAT1 expression, the phosphorylation of STAT1 was enhanced in severe COVID-19 cases, indicating a dysbalanced JAK/STAT signaling that fails to induce transcription of interferon stimulated response elements (ISRE). This abnormality persisted after IFN-α and IFN-γ stimulation of PBMCs from patients with severe COVID-19. Data suggest impaired STAT1 transcriptional upregulation among severely infected patients may represent a potential predictive biomarker and would allow stratification of patients for certain interferon-pathway targeted treatments., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

BACKGROUNDCOVID-19 convalescent plasma (CCP) has been considered a treatment option for COVID-19. This trial assessed the efficacy of a neutralizing antibody containing high-dose CCP in hospitalized adults with COVID-19 requiring respiratory support or intensive care treatment.METHODSPatients (n = 105) were randomized 1:1 to either receive standard treatment and 3 units of CCP or standard treatment alone. Control group patients with progress on day 14 could cross over to the CCP group. The primary outcome was a dichotomous composite outcome of survival and no longer fulfilling criteria for severe COVID-19 on day 21.ResultsThe primary outcome occurred in 43.4% of patients in the CCP group and 32.7% in the control group (P = 0.32). The median time to clinical improvement was 26 days in the CCP group and 66 days in the control group (P = 0.27). The median time to discharge from the hospital was 31 days in the CCP group and 51 days in the control group (P = 0.24). In the subgroup that received a higher cumulative amount of neutralizing antibodies, the primary outcome occurred in 56.0% of the patients (vs. 32.1%), with significantly shorter intervals to clinical improvement (20 vs. 66 days, P < 0.05) and to hospital discharge (21 vs. 51 days, P = 0.03) and better survival (day-60 probability of survival 91.6% vs. 68.1%, P = 0.02) in comparison with the control group.ConclusionCCP added to standard treatment was not associated with a significant improvement in the primary and secondary outcomes. A predefined subgroup analysis showed a significant benefit of CCP among patients who received a larger amount of neutralizing antibodies.Trial registrationClinicalTrials.gov NCT04433910.FundingBundesministerium für Gesundheit (German Federal Ministry of Health): ZMVI1-2520COR802.

To identify the most efficient methods of immunological protection against SARS-CoV-2, including the currently most widespread variants of concern (VOCs)-B.1.1.7, B.1.351 and P.1-a simultaneous side-by-side-comparison of available vaccination regimes is required. In this observational cohort study, we compared immunological responses in 144 individuals vaccinated with the mRNA vaccines BNT162b2 or mRNA-1273 and the vector vaccine ChAdOx1-nCoV-19, either alone, in combination, or in the context of COVID-19-convalescence. Unvaccinated COVID-19-convalescent subjects served as a reference. We found that cellular and serological immune responses, including neutralizing capacity against VOCs, were significantly stronger with mRNA vaccines as compared with COVID-19-convalescent individuals or vaccinated individuals receiving the vector vaccine ChAdOx1-nCoV-19. Booster immunizations with mRNA vaccines triggered strong and broadly neutralizing antibody and IFN-γ responses in 100% of vaccinated individuals investigated. This effect was particularly strong in COVID-19-convalescent and ChAdOx1-nCoV-19-primed individuals, who were characterized by comparably moderate cellular and neutralizing antibody responses before mRNA vaccine booster. Heterologous vaccination regimes and convalescent booster regimes using mRNA vaccines may allow enhanced protection against SARS-CoV-2, including current VOCs. Furthermore, such regimes may facilitate rapid (re-)qualification of convalescent plasma donors with high titers of broadly neutralizing antibodies.

The current SARS-CoV-2 pandemic has triggered the development of various SARS-CoV-2 neutralization tests. A wild-type virus (using African green monkey VeroE6 cells), a pseudovirus (using human Caco-2 cells), and a surrogate neutralization test platform were applied to characterize the SARS-CoV-2 neutralization potential of a cohort of 111 convalescent plasma donors over a period of seven months after diagnosis. This allowed an in-depth validation and assay performance analysis of these platforms. More importantly, we found that SARS-CoV-2 neutralization titers were stable or even increased within the observation period, which contradicts earlier studies reporting a rapid waning of Ab titers after three to four months. Moreover, we observed a positive correlation of neutralization titers with increasing age, number of symptoms reported, and the presence of the Rhesus Ag RhD. Validation of the platforms revealed that highest assay performances were obtained with the wild-type virus and the surrogate neutralization platforms. However, our data also suggested that selection of cutoff titers had a strong impact on the evaluation of neutralization potency. When taking strong neutralization potency, as demonstrated by the wild-type virus platform as the gold standard, up to 55% of plasma products had low neutralization titers. However, a significant portion of these products were overrated in their potency when using the surrogate assay with the recommended cutoff titer. In summary, our study demonstrates that SARS-CoV-2 neutralization titers are stable for at least seven months after diagnosis and offers a testing strategy for rapid selection of high-titer convalescent plasma products in a biosafety level 1 environment., (Copyright © 2021 by The American Association of Immunologists, Inc.)

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 CO-VID-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 <1:20 to >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., Competing Interests: Dr. Victor M. Corman is named together with Euroimmun on a patent application filed recently regarding the diagnostic of SARS-CoV-2 by antibody testing. All other authors have no conflicts of interest to declare., (Copyright © 2021 by S. Karger AG, Basel.)

Highly sensitive and specific platforms for the detection of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies are becoming increasingly important for evaluating potential SARS-CoV-2 convalescent plasma donors, studying the spread of SARS-CoV-2 infections, and identifying individuals with seroconversion. This study provides a comparative validation of 4 anti-SARS-CoV-2 platforms. A unique feature of the study is the use of a representative cohort of convalescent patients with coronavirus disease 2019 and a mild to moderate disease course. All platforms showed significant correlations with a SARS-CoV-2 plaque reduction neutralization test, with highest sensitivities for the Euroimmun and the Roche platforms, suggesting their preferential use for screening persons at increased risk of SARS-CoV-2 infections., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Many data are available on expansion protocols for mesenchymal stromal cells (MSCs) for both experimental settings and manufacturing for clinical trials. However, there is a lack of information on translation of established protocols for Good Manufacturing Practice (GMP) from validation to manufacturing for clinical application. We present the validation and translation of a standardized pre-clinical protocol for isolation and expansion of MSCs for a clinical trial for reconstitution of alveolar bone., Methods: Key parameters of 22 large-scale expansions of MSCs from bone marrow (BM) for validation were compared with 11 expansions manufactured for the clinical trial "Jaw bone reconstruction using a combination of autologous mesenchymal stromal cells and biomaterial prior to dental implant placement (MAXILLO1)" aimed at reconstruction of alveolar bone., Results: Despite variations of the starting material, the robust protocol led to stable performance characteristics of expanded MSCs. Manufacturing of the autologous advanced therapy medicinal product MAXILLO-1-MSC was possible, requiring 21 days for each product. Transport of BM aspirates and MSCs within 24 h was guaranteed. MSCs fulfilled quality criteria requested by the national competent authority. In one case, the delivered MSCs developed a mosaic in chromosomal finding, showing no abnormality in differentiation capacity, growth behavior or surface marker expression during long-term culture. The proportion of cells with the mosaic decreased in long-term culture and cells stopped growth after 38.4 population doublings., Conclusions: Clinical use of freshly prepared MSCs, manufactured according to a standardized and validated protocol, is feasible for bone regeneration, even if there was a long local distance between manufacturing center and clinical site. Several parameters, such as colony forming units fibroblasts (CFU-F), percentage of CD34+ cells, cell count of mononuclear cells (MNCs) and white blood cells (WBCs), of the BM may serve as a predictive tool for the yield of MSCs and may help to avoid unnecessary costs for MSC manufacturing due to insufficient cell expansion rates., (Copyright © 2019 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Background and Objectives: Mobilization of CD34 + cells by stimulation with G-CSF shows considerable variation across stem cell donors. Upfront prediction of CD34 + cell counts in peripheral blood based on easily available steady-state parameters would be helpful for the planning of apheresis and stem cell transplantation. Commonly accepted steady-state predictors for the mobilization are gender, body mass index and platelet count. The aim of the study was the identification of novel predictors that might influence mobilization efficacy and to create a model for the prediction of stem cell mobilization., Methods: A total of 333 healthy stem cell donors who donated peripheral stem cells in our institution were retrospectively analysed. All available data before stem cell mobilization with G-CSF were included in the database. Primary end-point was CD34 + cell count before the first apheresis., Results: In this cohort cholinesterase, differential blood cell counts including platelets, gender and body mass index were significantly correlated with CD34 + cell count. G-CSF dose per lean body weight showed a significant correlation with mobilization efficacy in women but not in men. A multivariate analysis identified gender, cholinesterase and platelet and red cell count as main predictors of mobilization. Red cell count showed a strong gender dependence, with higher predictive value in females., Conclusion: The counts of eosinophils, platelets, red cells, cholinesterase and gender are the most important predictors of CD34 + cell mobilization in our deduced models. The red cell count as a predictor for mobilization showed a differential gender dependence., (© 2019 International Society of Blood Transfusion.)

Aplastic anemia is a rare life-threatening bone marrow failure that is characterized by bicytopenia or pancytopenia in the peripheral blood and a hypoplastic or aplastic bone marrow. The patients are at risk of infection and hemorrhage due to neutropenia and thrombocytopenia and suffer from symptoms of anemia. The main treatment approaches are allogeneic stem cell transplantation and immunosuppression. Here, we review current standard immunosuppression and the attempts that have been made in the past two decades to improve results: review of recent developments also reveals that sometimes not only the advent of new drugs, good ideas and well-designed clinical trials decide the progress in the field but also marketing considerations of pharmaceutical companies. Aplastic anemia experts unfortunately had to face the situation that efficient drugs were withdrawn simply for marketing considerations. We will discuss the current options and challenges in first-line treatment and management of relapsing and refractory patients with an emphasis on adult patients. Some promising new approaches are currently under investigation in prospective, randomized trials.

Simple stress or necrotic cell death with subsequent release of damage-associated molecular patterns (DAMPs) is a characteristic feature of most advanced tumors. DAMPs within the tumor microenvironment stimulate tumor-associated cells, including dendritic cells and mesenchymal stromal cells (MSCs). The presence of tumor-infiltrating MSCs is associated with tumor progression and metastasis. Oxidized necrotic material loses its stimulatory capacity for MSCs. As a DAMP, S100A4 is sensitive to oxidation whereas uric acid (UA) acts primarily as an antioxidant. We tested these two biologic moieties separately and in combination for their activity on MSCs. Similar to necrotic tumor material, S100A4 and UA both dose-dependently induced chemotaxis of MSCs with synergistic effects when combined. Substituting for UA, alternative antioxidants (vitamin C, DTT, and N-acetylcysteine) also enhanced the chemotactic activity of S100A4 in a synergistic manner. This emphasizes the reducing potential of UA being, at least in part, responsible for the observed synergy. With regard to MSC proliferation, both S100A4 and UA inhibited MSCs without altering survival or inducing differentiation toward adipo-, osteo-, or chondrocytes. In the presence of S100A4 or UA, MSCs gained an immunosuppressive capability and stably induced IL-10- and IDO-expressing lymphocytes that maintained their phenotype following proliferation. We have thus demonstrated that both S100A4 and UA act as DAMPs and, as such, may play a critical role in promoting some aspects of MSC-associated immunoregulation. Our findings have implications for therapeutic approaches targeting the tumor microenvironment and addressing the immunosuppressive nature of unscheduled cell death within the tumor microenvironment., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Objective: Plasticity of hematopoietic stem cells (HSC) has gained major interest in stem cell research. In order to investigate whether HSC may differentiate into mesenchymal stem cells (MSC), we assessed chimerism in peripheral blood (PB), mononuclear cell fractions (MNC) of bone marrow, and MSC derived from bone marrow (BM) from 27 up to 4225 days after allogeneic transplantation., Patients and Methods: We applied fluorescence in situ hybridization using X/Y gene probes in sex-mismatched and STR-PCR in sex-matched patients. MSC could have been generated in 27 of 55 bone marrow samples derived from 20 patients. Fifteen patients received peripheral blood stem cell transplants (PBSCT), including CD34-selected PBSCT in two. Five patients received bone marrow., Results: While all patients had chimerism in PB and MNC of the BM, in all but one patient BM-derived MSC were of recipient origin. This single patient showed reproducibly MSC of donor origin in a frequency of 1% after having received a CD34-selected PBSCT. Looking at graft collections, MSCs were easily generated from BM specimens, while no MSC could be derived from PBSC samples., Conclusion: Even though HSC have been found to differentiate into a variety of nonhematological cell types, they usually do not differentiate into MSC after allogeneic transplantation.

Depolarisation of the plasma membrane has been shown to be actively regulated during lymphocyte-apoptosis. Here, we present data about anti-Fas and As2O3 induced depolarisation of myeloid U-937 cells. Anti-Fas but not As2O3-induced depolarisation was significantly dependent on caspase-activation. Na+-fluxes contributed to the depolarisation in early stages of As2O3-induced apoptosis, whereas the membrane potential in late stages depended on Cl- -fluxes. Cl- -channels also played an important role in the induction of cell shrinkage in As2O3-induced apoptosis. However, none of these ions contributed significantly to anti-Fas induced depolarisation. This indicates the existence of different mechanisms for apoptotic plasma membrane depolarisation within one cell type.

Arsenic trioxide (As2O3) has a long history of use in medicine. However, it was almost forgotten in Western medicine in the recent centuries. Prompted by reports from China about successful treatment of acute promyelocytic leukemia (APL) with As2O3, there was again increasing interest in this drug in the 1990s. This review summarizes the considerable knowledge about the mechanisms of action of As2O3 that was gained during the last 5-10 years. It is focused in particular on the effects of As2O3 in non-APL cells. Since As2O3 seems to induce apoptosis and inhibits growth in a large variety of cellular targets, it might become an alternative or adjunct drug to conventional chemotherapy. As2O3 can even be effective in cells resistant to conventional cytostatic agents. Insight into the cellular mechanisms, in particular the impact of the redox state on sensitivity towards As2O3 opens the possibility to enhance As2O3 effects by appropriate combination therapies.

The potency of arsenic trioxide (As2O3) as chemotherapeutic agent is under investigation in various clinical trials. As2O3 was shown to be a potent inductor of apoptosis, and several publications describe the involvement of caspases, reduction of mitochondrial membrane potential and modulation of intracellular glutathione level. However, little is known about the involvement of membrane bound cell death receptors. We investigated the role of CD95 and CD95L in As2O3 mediated apoptosis in various lympho-haematopoietic cell lines. Basal CD95-expression did not correlate with sensitivity to As2O3 and incubation with As2O3 did not alter CD95-expression. We therefore chose two CD95 positive cell lines (CCRF-CEM and Jurkat) to analyse a potential activation of this pathway. We were able to induce apoptosis in these CD95 positive cell lines with activating anti-CD95 antibodies and could block induction of apoptosis by inhibitory anti-CD95 antibodies. In contrast we were not able to block As2O3-induced apoptosis by inhibitory anti-CD95 antibodies. We could block additive effects of arsenic trioxide and an apoptosis-inducing anti-CD95 antibody against CD95 to levels of arsenic trioxide alone using an inhibitory anti-CD95 antibody. Thus, our data provide no evidence for a role of the CD95L/CD95 pathway in As2O3-induced apoptosis.

The clinical efficacy of arsenic(III) oxide (As(2)O(3)) has been shown in patients with relapsed acute promyelocytic leukemia (APL). To identify potential common primary targets of action of As(2)O(3) in myelogenic and lymphatic cell lines, we analyzed As(2)O(3) effects on caspases and on the mitochondrial membrane potential (Psi(M)) under uniform conditions. As(2)O(3) induced breakdown of Psi(M) and activated caspases in cell lines with different sensitivities for As(2)O(3), including cell lines resistant to mitoxantron or camptothecin but sensitive to As(2)O(3). Caspase inhibitors could not prevent breakdown of Psi(M) in lymphoid cell lines, whereas activation of caspases and apoptosis could be inhibited. Activation of caspases seems to be a downstream effect occurring after breakdown of Psi(M). We could show that all of these effects are independent of MDR-1 expression. There was no difference in the mode of action of As(2)O(3) in cell lines sensitive or resistant to camptothecin, mitoxantrone, or doxorubicin. As(2)O(3) deserves further evaluation as an adjunct or alternative to other cytostatic drugs.

Data regarding the role of mitochondria in arsenic trioxide (As2O3)-induced apoptosis are controversial. We investigated the contribution of caspases and mitochondrial depolarization to As2O3-induced apoptosis in the myeloid cell lines NB-4, HL-60 and U-937. Caspase inhibition reduced the amount of cells with As2O3 (20 micromol/l)-induced mitochondrial depolarization by about 50% in all cell lines. As2O3 also induced dose-dependent phosphatidylserine exposure in cells without depolarized mitochondria. We conclude that caspase activation is of similar importance in As2O3-induced apoptosis in myeloid cell lines as direct mitochondrial targeting and mitochondria are not necessary for caspase activation downstream of mitochondria.

Along with the establishment of more intense chemotherapeutic regimens including fludarabine for the treatment of indolent lymphoproliferative diseases like chronic lymphocytic leukemia (CLL), an increasing amount of cases with progressive multifocal leukoencephalopathy (PML) due to JC virus have been observed. We report a patient with CLL who developed PML parallel to the onset of fludarabine therapy. Spinal fluid was tested positive for JC virus. Despite virostatic treatment with cidofovir, neurologic symptoms were progressive and the disease ultimately fatal. The present case suggests that immunosuppression caused by chronic lymphoproliferative malignancies alone may be a factor in the development of PML. Chemotherapy with fludarabine may act as an additional trigger. The question remains whether serologic screening for JC virus in patients with chronic lymphoproliferative disease undergoing intense chemotherapy might be valuable once sufficient antiviral treatment has been established.

Objective: Mitochondrial membrane potential (deltaPsim) and intracellular Ca2+ play a crucial role in growth and differentiation in hemopoiesis. Some potassium channel openers such as diazoxide have the capacity to elevate cytosolic Ca2+ and depolarize mitochondria in cardiomyocytes. To clarify if such substances have effects on hemopoietic cells we investigated the commonly used opener of the mitoK(ATP) channel, diazoxide, and the opener of BK channels, NS1619, for their potential to depolarize mitochondria, elevate cytosolic Ca2+, and induce apoptosis in the hemopoietic CD34+ cell line KG-1a., Methods: Fluorescent probes were used to investigate deltaPsim, free Ca2+, and apoptosis (JC-1, fluo-3-AM and annexin V-FITC) by flow cytometry. To measure deltaPsim with JC-1 in glycoprotein P+ cells we used an improved dye loading technique with verapamil., Results: NS1619 induced stronger dose-dependent mitochondrial depolarizations than diazoxide. Depolarization was independent from caspase activation and could also be induced when the driving force for K+ out of cells was near 0 mV. In Ca2+ free solutions NS1619 induced stronger Ca2+ elevations than diazoxide and elevated Ca2+ also after Ca2+ depletion of the endoplasmatic reticulum with caffeine. NS1619 did not enhance the Ca2+ elevation induced by ionophores (CCCP, valinomycin) that depolarize mitochondria. Both agents were weak inducers of apoptosis., Conclusion: Diazoxide has similar effects in CD34+ cells as described for muscle or nerve cells. In accordance to the single channel conductance of mitoK(ATP) and BK channels, NS1619 is a more potent inducer of mitochondrial depolarization than diazoxide. NS1619 releases Ca2+ from an intracellular pool that is insensitive to caffeine but depends strongly on deltaPsim.

The effects of the alkaloids ajmaline, lupanine, sparteine, serpentine, strychnine, and yohimbine were studied with the loose patch clamp technique on sodium currents of isolated single skeletal muscle fibers. The IC50 values for half-maximal blocking of the sodium currents were 6.6 microM for ajmaline, 55.7 microM for quinidine, 168.8 microM for sparteine, and 1.2 mM for lupanine. The observed Na+ channel inhibition is in accordance with the use of ajmaline, quinidine and sparteine as antiarrhythmic drugs. The interference of alkaloids with Na+ channels can also be interpreted as a means to strongly interfere with neuronal transmission in herbivores. Alkaloids thus serve as chemical defense compounds for the plants producing them.