Cytokine levels associated with favorable clinical outcome in the CAPSID randomized trial of convalescent plasma in patients with severe COVID-19.

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.

Upregulation of Checkpoint Ligand Programmed Death-Ligand 1 in Patients with Paroxysmal Nocturnal Hemoglobinuria Explained by Proximal Complement Activation.

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.

Translation of a standardized manufacturing protocol for mesenchymal stromal cells: A systematic comparison of validation and manufacturing data.

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.

Gender, cholinesterase, platelet count and red cell count are main predictors of peripheral blood stem cell mobilization in healthy donors.

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.

Immunosuppressive therapy for transplant-ineligible aplastic anemia patients.

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.

S100A4 and uric acid promote mesenchymal stromal cell induction of IL-10+/IDO+ lymphocytes.

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.

Mesenchymal stem cells remain of host origin even a long time after allogeneic peripheral blood stem cell or bone marrow transplantation.

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 in the arsenic trioxide (As2O3)-and anti-CD95-induced apoptosis in myeloid cells.

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 therapy in acute promyelocytic leukemia and beyond: from bench to bedside.

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.

Depolarization of mitochondria and activation of caspases are common features of arsenic(III)-induced apoptosis in myelogenic and lymphatic cell lines.

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.

Arsenic trioxide-induced apoptosis is independent of CD95 in lymphatic cell lines.

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 role of mitochondrial targeting in arsenic trioxide-induced apoptosis in myeloid cell lines.

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.

Progressive multifocal leukoencephalopathy with detection of JC virus in a patient with chronic lymphocytic leukemia parallel to onset of fludarabine therapy.

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.