What happens after graft loss? A large,long-term,single-center observation

The number of patients returning to dialysis after graft failure increases. Surprisingly, little is known about the clinical and immunological outcomes of this cohort. We retrospectively analyzed 254 patients after kidney allograft loss between 1997 and 2017 and report clinical outcomes such as mortality, relisting, retransplantations, transplant nephrectomies, and immunization status. Of the 254 patients, 49% had died 5 years after graft loss, while 27% were relisted, 14% were on dialysis and not relisted, and only 11% were retransplanted 5 years after graft loss. In the complete observational period, 111/254 (43.7%) patients were relisted. Of these, 72.1% of patients were under 55 years of age at time of graft loss and only 13.5% of patients were ≥65 years. Age at graft loss was associated with relisting in a logistic regression analysis. In the complete observational period, 42 patients (16.5%) were retransplanted. Only 4 of those (9.5%) were ≥65 years at time of graft loss. Nephrectomy had no impact on survival, relisting, or development of dnDSA. Patients after allograft loss have a high overall mortality. Immunization contributes to long waiting times. Only a very limited number of patients are retransplanted especially when ≥65 years at time of graft loss.     

The tyrosine kinase NPM-ALK, associated with anaplastic large cell lymphoma, binds the intracellular domain of the surface receptor CD30 but is not activated by CD30 stimulation

The heterogenous group of anaplastic large cell lymphomas (ALCLs) is characterized by expression of the Ki-1/CD30 antigen, a member of the tumor necrosis factor receptor superfamily. About 40 to 50% of cases diagnosed as ALCL contain a specific chromosomal rearrangement, t(2;5)(p23;q35), resulting in expression of the chimeric tyrosine kinase NPM-ALK. As NPM-ALK-positive lymphomas define a distinct subtype within the group of ALCL, the chimeric protein might be responsible for certain pathogenetic and clinicopathologic characteristics. To better elucidate the function of NPM-ALK, we investigated a possible mechanism for regulation of its activity. We demonstrate that NPM-ALK specifically binds to the intracellular domain of the cytokine receptor CD30. In vitro binding assays revealed that the ALK portion of NPM-ALK mediates interaction of the two proteins. Stimulation of the CD30 receptor by cross-linking with immobilized anti-CD30 antibody results in complete growth inhibition of Karpas 299, an NPM-ALK-positive ALCL cell line, but does not alter proliferation of HDLM-2, a Hodgkin's lymphoma-derived cell line lacking t(2;5). Western blot analysis of coimmunoprecipitated CD30 and NPM-ALK proteins from stimulated Karpas 299 cells showed that the interaction of the proteins is not modified by stimulation. Activation of CD30 neither enhanced NPM-ALK activity measured by autophosphorylation of the chimeric tyrosine kinase nor phosphorylation of phospholipase C-gamma, an NPM-ALK substrate. We conclude that NPM-ALK is not stimulated by CD30 activation, but exists as a constitutively hyperactivated protein. Interaction with CD30 may extend the subcellular localization of NPM-ALK to the microenvironment of membrane-associated proteins.     

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.     

Donors for SARS-CoV-2 Convalescent Plasma for a Controlled Clinical Trial: Donor Characteristics,Content and Time Course of SARS-CoV-2 Neutralizing Antibodies

BackgroundConvalescent 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.MethodsPatients 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.ResultsA 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.ConclusionWe 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 K+ channel openers diazoxide and NS1619 induce depolarization of mitochondria and have differential effects on cell Ca2+ in CD34+ cell line KG-1a

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.     

Paroxysmal nocturnal haemoglobinuria treatment with eculizumab is associated with a positive direct antiglobulin test

Background/Objectives Paroxysmal nocturnal haemoglobinuria (PNH) is characterized by intravascular haemolysis with a negative direct antiglobulin test (DAT). Eculizumab is a humanized monoclonal antibody that inhibits complement component C5 and is approved for PNH treatment. Recent publications demonstrated that some patients with PNH develop a positive DAT during eculizumab treatment. These published clinical trials investigated a highly selected patient population. Therefore, it seems important to study this topic in a general PNH patient population with a longer follow‐up. Materials and Methods We analysed haemolytic activity, RBC transfusion requirement, effect on DAT and ferritin levels in 41 patients with PNH before and during eculizumab therapy with a median follow‐up of 24 months (range 1–63 months). Results During eculizumab therapy, median LDH decreased (1657–258 U/l; P < 0·0001), while median haemoglobin increased (9·2–10·3 g/dl). Eighteen of 32 pts (56%) who previously required regular transfusions became transfusion independent. DAT was positive for C3d in 72·4% of 21 eculizumab‐treated pts with available DAT. Ferritin levels increased (69–348 ng/ml, P < 0·0001). This increase was more pronounced in pts with ongoing transfusion dependency during eculizumab therapy. Conclusion Eculizumab therapy for PNH should be added to the list of possible causes for a positive DAT. Intravascular haemolysis was inhibited by eculizumab, but signs of extravascular haemolysis should be monitored. Because renal iron loss was stopped, eculizumab‐treated pts can be prone to iron overload and therefore ferritin concentrations should be monitored closely.     

Emergence of CD52-, phosphatidylinositolglycan-anchor-deficient T lymphocytes after in vivo application of Campath-1H for refractory B- cell non-Hodgkin lymphoma

CD52 is a phosphatidylinositolglycan (PIG)-anchored glycoprotein (PIG- AP) expressed on normal T and B lymphocytes, monocytes, and the majority of B-cell non-Hodgkin lymphomas. We observed the emergence of CD52- T cells in 3 patients after intravenous treatment with the humanized anti-CD52 monoclonal antibody Campath-1H for refractory B- cell lymphoma and could identify the underlaying mechanism. In addition to the absence of CD52, the PIG-AP CD48 and CD59 were not detectable on the CD52- T cells in 2 patients. PIG-AP-deficient T-cell clones from both patients were established. Analysis of the mRNA of the PIG-A gene showed an abnormal size in the T-cell clones from 1 of these patients, suggesting that a mutation in the PIG-A gene was the cause of the expression defect of PIG-AP. An escape from an immune attack directed against PIG-AP+ hematopoiesis has been hypothesized as the cause of the occurrence of PIG-AP-deficient cells in paroxysmal nocturnal hemoglobinuria (PNH) and aplastic anemia. Our results support the hypothesis that an attack against the PIG-AP CD52 might lead to the expansion of a PIG-anchor-deficient cell population with the phenotypic and molecular characteristics of PNH cells.     

Outcome of aplastic anemia in adolescence: a survey of the Severe Aplastic Anemia Working Party of the European Group for Blood and Marrow Transplantation

We analyzed the outcome of 537 adolescents (age 12–18 years) with idiopathic aplastic anemia included in the database of the Severe Aplastic Anemia Working Party of the European Group for Blood and Marrow Transplantation comparing: i) matched family donor hematopoietic stem cell transplantation performed as first-line treatment with ii) front-line immunosuppressive therapy not followed by subsequent transplant given for failure and with iii) hematopoietic stem cell transplantation performed after failed front-line immunosuppressive therapy. Overall survival was 86% in the matched family donor hematopoietic stem cell transplantation group, 90% in patients given front-line immunosuppressive alone (those who did not fail this treatment and who did not receive subsequent rescue with hematopoietic stem cell transplantation) and 78% in subjects who underwent hematopoietic stem cell transplantation post failed front-line immunosuppressive therapy (P=0.14). Event-free survival in the same groups was respectively 83%, 64% and 71% (P=0.04). Cumulative incidence of rejection was 8% in matched family donor hematopoietic stem cell transplantation and 9% in transplants post failed front-line immunosuppression (P=0.62). Cumulative incidence of acute graft-versus-host disease was 12% in matched family donor transplants and 18% in transplants post failed immunosuppression (P=0.18). Chronic graft-versus-host disease was higher in matched family donor hematopoietic stem cell transplantation (8%) than in transplants post failed immunosuppressive therapy (20%) (P=0.0009). Cumulative incidence of post-therapy malignancies was 0.7% in matched family donor transplantations, 7% in transplantations post failed immunosuppression and 21% after front-line immunosuppression (P=0.0017). In the whole cohort, under multivariate analysis, the diagnosis to treatment interval of two months or under positively affected overall survival whereas up-front immunosuppression alone (with no subsequent rescue transplants) negatively affected event-free survival. In transplanted patients an interval from diagnosis to treatment of 2 months or under, bone marrow as source of cells and first-line matched family donor transplants provided a significant advantage in overall and event-free survival. Aplastic anemia in adolescents has a very good outcome. If a matched family donor is available, hematopoietic stem cell transplantation using bone marrow cells is the first choice treatment. If such a donor is not available, immunosuppressive treatment may still be an acceptable second choice, also because, in case of failure, hematopoietic stem cell transplantation is a very good rescue option.