Endothelial injury and thrombotic microangiopathy in COVID-19: Treatment with the lectin-pathway inhibitor narsoplimab.

In COVID-19, acute respiratory distress syndrome (ARDS) and thrombotic events are frequent, life-threatening complications. Autopsies commonly show arterial thrombosis and severe endothelial damage. Endothelial damage, which can play an early and central pathogenic role in ARDS and thrombosis, activates the lectin pathway of complement. Mannan-binding lectin-associated serine protease-2 (MASP-2), the lectin pathway's effector enzyme, binds the nucleocapsid protein of severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2), resulting in complement activation and lung injury. Narsoplimab, a fully human immunoglobulin gamma 4 (IgG4) monoclonal antibody against MASP-2, inhibits lectin pathway activation and has anticoagulant effects. In this study, the first time a lectin-pathway inhibitor was used to treat COVID-19, six COVID-19 patients with ARDS requiring continuous positive airway pressure (CPAP) or intubation received narsoplimab under compassionate use. At baseline and during treatment, circulating endothelial cell (CEC) counts and serum levels of interleukin-6 (IL-6), interleukin-8 (IL-8), C-reactive protein (CRP) and lactate dehydrogenase (LDH) were assessed. Narsoplimab treatment was associated with rapid and sustained reduction of CEC and concurrent reduction of serum IL-6, IL-8, CRP and LDH. Narsoplimab was well tolerated; no adverse drug reactions were reported. Two control groups were used for retrospective comparison, both showing significantly higher mortality than the narsoplimab-treated group. All narsoplimab-treated patients recovered and survived. Narsoplimab may be an effective treatment for COVID-19 by reducing COVID-19-related endothelial cell damage and the resultant inflammation and thrombotic risk.

Renal function in HIV/HBV co-infected and HBV mono-infected patients on a long-term treatment with tenofovir in real life setting.

The human immunodeficiency virus (HIV)/hepatitis B virus (HBV) co-infection is likely to be associated with an increased risk of kidney disease, due to the additional factors that may affect renal function in the HIV population. We aimed to evaluate renal toxicity in HIV/HBV and HBV mono-infected patients on long-term therapy with tenofovir (TDF) and to explore the association of polymorphisms in ATP-binding cassette (ABCC)2, ABCC4, ABCC10 with the development of renal dysfunction. From September 2006 to November 2014, 44 HIV/HBV co-infected and 34 HBV mono-infected patients were commenced on TDF. Data of renal safety were retrospectively collected and analyzed. ABCC2, ABCC4 and ABCC10 genotypes were identified by real-time PCR. Over 60 months of observation, there was a significant increase in mean creatinine levels from baseline (P<.01) that was not significantly different between the two study groups. Moreover, a significant decline in estimated glomerular filtration rate (eGFR) was observed from baseline (P<.01), and it was significantly greater in HBV mono-infected than co-infected patients (P=.03). The distribution of ABCC2, ABCC4 and ABCC10 genotypes among a subgroup of 34 patients did not show significant association with eGFR decline <90 mL/min per 1.73 m2 . Although our findings showed a statistically significant decrease in eGFR with long-term use of TDF, its clinical impact seems to be modest. The role of genetic factors to identify patients at greater risk for developing tenofovir-induced renal toxicity needs to be further investigated.

Detection of erythroblast antibodies in mitogen-stimulated bone marrow cultures from patients with myelodysplastic syndromes.

BACKGROUND: Low-risk myelodysplastic syndromes (MDS) show several immunologic abnormalities, including increased frequency of autoimmune manifestations and/or overt autoimmune diseases, whose prognostic significance still remains controversial. STUDY DESIGN AND METHODS: We studied the presence of erythroblast antibodies in mitogen-stimulated bone marrow (BM) cultures of 70 patients with early-stage MDS (refractory anemia and refractory anemia with ringed sideroblasts). RESULTS: Sixty-six percent of patients showed positive erythroblast antibodies, along with BM erythroid hyperplasia and a hemolytic picture in the peripheral blood. Supernatants from positive cultures induced an increase of overall cellularity, the appearance of erythroblastic clustering, and dyserythropoietic signs in normal BM. We identified CD45(dim) Gly-A(dim) CD71(bright) cells (red blood cell precursors at different maturation stage) as the target of the antibodies. Erythropoietin (EPO) levels were reduced and EPO receptors (EPO-R) increased in BM culture supernatants from positive patients. However, flow cytometric analysis showed that neither EPO nor EPO-R was involved in an abnormal stimulation driven by these autoantibodies. Values of the proapoptotic protein Bax were increased in positive patients and Bcl-2 levels were decreased, although not significantly. CONCLUSION: MDS patients with anti-erythroblast autoimmunity showed increased BM apoptosis, suggesting that the autoimmune reaction may contribute to an unfavorable BM microenvironment for optimal erythropoiesis.

Reduced 25-OH vitamin D in patients with autoimmune cytopenias, clinical correlations and literature review.

Vitamin D deficiency is widespread in Western Countries and has been found related to autoimmune and hematologic disease incidence and clinical course. We evaluated vitamin D levels, vitamin D receptor (VDR) and T helper (Th)1, Th2 and Th17 immunomodulatory cytokines in patients with immune thrombocytopenic purpura (ITP, N=44), primary autoimmune hemolytic anemia (AIHA, n=35), Evans' syndrome (n=5) and chronic idiopathic neutropenia (CIN, n=19) and also tested vitamin D effect on the in vitro production of anti-erythrocyte autoantibodies. 25-OH-vitamin D levels were significantly lower and vitamin D receptor higher in patients than in controls. Among ITP cases, those with very low vitamin D levels displayed reduced platelet counts, irrespective of the bleeding history. In AIHA patients, LDH values negatively correlated with vitamin D levels in mixed forms, and reticulocyte counts were positively related with vitamin D. Considering treatment, AIHA patients who had been treated with 2 therapy lines or more showed lower mean 25-OH-vitamin D levels than those untreated or treated with one line of therapy only. IL-6, IL-10, IL-17 and IFN-γ levels were higher in patients versus controls, whereas TNF-α was significantly reduced. Finally, vitamin D at concentrations of 10, 20, and 40ng/mL reduced the in vitro production of anti-erythrocyte autoantibodies both in pokeweed-stimulated and unstimulated cultures. In conclusion, vitamin D is reduced in autoimmune cytopenias and correlate with disease severity, supporting its possible protective role against the development of autoimmunity. Literature review showed vitamin D deficiency reports both in onco- and in non onco-hematologic diseases with a relationship with disease severity/activity in myeloid and lymphoid neoplasms, as well as in sickle cell disease. Supplementation has produced weak results in autoimmune and hematologic diseases, and further studies are needed.

Low-dose alemtuzumab-associated immune thrombocytopenia in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is frequently complicated during its course by autoimmune disorders (from 2 to 12% of cases), such as autoimmune hemolytic anemia (AIHA) and immune thrombocytopenic purpura (ITP). In particular, ITP has been reported in about 2­5% of CLL population. Recently, Cuker et al. reported the occurrence of ITP in 6/216 patients with relapsing-remitting multiple sclerosis in a phase 2 clinical trial of annual alemtuzumab. Alemtuzumab is an anti-CD52 monoclonal antibody used in CLL both as first-line treatment and in relapsed/refractory patients. We evaluated a cohort of 64 consecutive patients affected by relapsed-refractory CLL treated with low-dose alemtuzumab and we observed a incidence of ITP higher than predicted. Our data, associated with the report of Cuker et al., seem to suggest an important role of alemtuzumab in the pathogenesis of ITP which could be related to its induced dysregulation of T-lymphocyte activity.

The relation between charge movement and transport-associated currents in the rat GABA cotransporter rGAT1.

Most cotransporters characteristically display two main kinds of electrical activity: in the absence of organic substrate, transient presteady-state currents (I(pre)) are generated by charge relocation during voltage steps; in the presence of substrate, sustained, transport-associated currents (I(tr)) are recorded. Quantitative comparison of these two currents, in Xenopus oocytes expressing the neural GABA cotransporter rGAT1, revealed several unforeseen consistencies between I(pre) and I(tr), in terms of magnitude and kinetic parameters. The decay rate constant (r) of I(pre) and the quantity of charge displaced to an inner position in the transporter (Q(in)(0)) depended on voltage and ionic conditions. Saturating GABA concentrations, applied under the same conditions, suppressed I(pre) (i.e. Q(in)( infinity ) = 0) and produced a transport-associated current with amplitude I(tr) = Q(in)(0)r. At non-saturating levels of GABA, changes of I(tr) were compensated by corresponding variations in Q(in), such that I(pre) and I(tr) complemented each other, according to the relation: I(tr) = (Q(in)(0) - Q(in)) r. Complementarity of magnitude, superimposable kinetic properties and equal dependence on voltage and [Na(+)](o) point to the uniqueness of the charge carrier for both processes, suggesting that transport and charge migration arise from the same molecular mechanism. The observed experimental relations were correctly predicted by a simple three-state kinetic model, in which GABA binding takes place after charge binding and inward migration have occurred. The model also predicts the observed voltage dependence of the apparent affinity of the transporter for GABA, and suggests a voltage-independent GABA binding rate with a value around 0.64 microM(-1) s(-1).

Role of anion-cation interactions on the pre-steady-state currents of the rat Na(+)-Cl(-)-dependent GABA cotransporter rGAT1.

The effects of sodium and chloride on the properties of the sodium-dependent component of the 'pre-steady-state' currents of rGAT1, a GABA cotransporter of the Na(+)-Cl(-)-dependent family, were studied using heterologous oocyte expression and voltage clamp. Reductions in either extracellular sodium or chloride shifted the charge-voltage (Q-V) and time constant-voltage (tau-V) characteristics of the process towards more negative potentials. The shift induced by sodium (TMA(+), tetramethylammonium substitution) was stronger than that induced by chloride (acetate substitution), and the shift of tau was accompanied by a decrease in its maximum value. Increasing extracellular Ca(2+) did not produce significant shifts in Q-V and tau-V curves. The negative shift of the Q-V curve upon chloride reduction and the decrease in the value of the relaxation time constant, tau, when either sodium or chloride were lowered, contrasted with the prediction of the Hill-Boltzmann interpretation of the process. Analysis of the unidirectional rate constants under different conditions revealed that both sodium and chloride shifted the outward rate more than the inward rate; furthermore, the shifts induced by sodium were larger than those induced by chloride. These observations are qualitatively compatible with the existence of a selective vestibule at the mouth of the transporters, acting similarly to a Donnan system.

Temperature effects on the presteady-state and transport-associated currents of GABA cotransporter rGAT1.

The effects of temperature on the gamma-aminobutyric acid (GABA) uptake and on the presteady-state and transport-associated currents of the GABA cotransporter, rat gamma-aminobutyric acid transporter 1 (rGAT1), have been studied using heterologous oocyte expression and voltage-clamp. Increasing temperature from 15 to 30 degrees C increased GABA uptake, diminished the maximal value of the relaxation time constant of the presteady-state currents and increased the amplitude of the current associated with the transport of GABA. The curve of the presteady-state charge versus voltage was shifted toward negative potentials by increasing the temperature, while the maximal amount of charge (Q(max)) remained constant; the tau versus V curve was also negatively shifted by increasing temperatures. Analysis of the outward (alpha) and inward (beta) rate constants as functions of temperature showed that they are affected differently, with a Q(10)=3.4 for alpha and Q(10)=1.5 for beta. The different temperature coefficients of the rate constants account for the observed shifts. These observations are consistent with a charge moving mechanism based on a conformational change of the protein; the weaker temperature sensitivity of the inward rate constant suggests a rate-limiting diffusional component on this process.