Upper and/or Lower Respiratory Tract Infection Caused by Human Metapneumovirus After Allogeneic Hematopoietic Stem Cell Transplantation.

BACKGROUND: Human metapneumovirus (hMPV) epidemiology, clinical characteristics and risk factors for poor outcome after allogeneic stem cell transplantation (allo-HCT) remain a poorly investigated area. METHODS: This retrospective multicenter cohort study examined the epidemiology, clinical characteristics, and risk factors for poor outcomes associated with human metapneumovirus (hMPV) infections in recipients of allo-HCT. RESULTS: We included 428 allo-HCT recipients who developed 438 hMPV infection episodes between January 2012 and January 2019. Most recipients were adults (93%). hMPV infections were diagnosed at a median of 373 days after allo-HCT. The infections were categorized as upper respiratory tract disease (URTD) or lower respiratory tract disease (LRTD), with 60% and 40% of cases, respectively. Patients with hMPV LRTD experienced the infection earlier in the transplant course and had higher rates of lymphopenia, neutropenia, corticosteroid use, and ribavirin therapy. Multivariate analysis identified lymphopenia and corticosteroid use (>30 mg/d) as independent risk factors for LRTD occurrence. The overall mortality at day 30 after hMPV detection was 2% for URTD, 12% for possible LRTD, and 21% for proven LRTD. Lymphopenia was the only independent risk factor associated with day 30 mortality in LRTD cases. CONCLUSIONS: These findings highlight the significance of lymphopenia and corticosteroid use in the development and severity of hMPV infections after allo-HCT, with lymphopenia being a predictor of higher mortality in LRTD cases.

Impact of early cyclosporine A levels on acute graft-versus-host disease in allogeneic hematopoietic stem cell transplantation using in vivo T-cell depletion.

BACKGROUND AIMS: Cyclosporin A (CsA) remains a major component of immunosuppressive regimens applied in allogeneic hematopoietic stem cell transplantation (HSCT). The impact of CsA trough levels during the first weeks after HSCT has not yet been investigated specifically in anti-T-lymphocyte globulin (ATLG)-based HSCT from matched related and unrelated donors. METHODS: To address this issue, we have retrospectively examined 307 consecutive matched related (n = 145) and unrelated (n = 162) HSCTs, using peripheral blood stem cells or bone marrow. HSCTs for active, uncontrolled malignancies were excluded. The initial three weeks' average mean CsA trough levels were analyzed in landmark and multi-state models, using a cut-off of 200 ng/mL. RESULTS: CsA levels >200 ng/mL were associated with a reduced risk of acute graft-versus-host disease (GVHD) grade 3-4 at the first-week landmark (subdistribution hazard ratio [SHR] 0.59, P = 0.03) and the second-week landmark (SHR 0.48, P = 0.004), whereas there was no impact at the third-week landmark (HR 0.87, P = 0.69). This was supported by a multi-state model, in which week 1 (hazard ratio [HR] 0.53, P = 0.006) and week 2 (HR 0.48, P = 0.003), but not week 3 (HR 0.80, P = 0.44) CsA levels >200 ng/mL were associated with a reduced acute GVHD 3-4 risk. Relapse incidence was not significantly affected by week 1 through 3 CsA levels. Despite ATLG's inherent GVHD-preventive properties, week 1 CsA trough levels >200 ng/mL following ATLG-based HSCT (n = 220) were associated with a significantly reduced risk of non-relapse mortality (SHR 0.52, P = 0.02) and improved overall survival (HR 0.61, P = 0.02). CONCLUSIONS: Our findings emphasize the continuing importance of ensuring CsA levels ≥200 ng/mL immediately post-transplant in the setting of ATLG-based HSCT.

The SERCA residue Glu340 mediates interdomain communication that guides Ca2+ transport.

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that transports Ca2+ from the cytosol into the sarco(endo)plasmic reticulum (SR/ER) lumen, driven by ATP. This primary transport activity depends on tight coupling between movements of the transmembrane helices forming the two Ca2+-binding sites and the cytosolic headpiece mediating ATP hydrolysis. We have addressed the molecular basis for this intramolecular communication by analyzing the structure and functional properties of the SERCA mutant E340A. The mutated Glu340 residue is strictly conserved among the P-type ATPase family of membrane transporters and is located at a seemingly strategic position at the interface between the phosphorylation domain and the cytosolic ends of 5 of SERCA's 10 transmembrane helices. The mutant displays a marked slowing of the Ca2+-binding kinetics, and its crystal structure in the presence of Ca2+ and ATP analog reveals a rotated headpiece, altered connectivity between the cytosolic domains, and an altered hydrogen bonding pattern around residue 340. Supported by molecular dynamics simulations, we conclude that the E340A mutation causes a stabilization of the Ca2+ sites in a more occluded state, hence displaying slowed dynamics. This finding underpins a crucial role of Glu340 in interdomain communication between the headpiece and the Ca2+-binding transmembrane region.

Association of uric acid levels before start of conditioning with mortality after allogeneic hematopoietic stem cell transplantation - a prospective, non-interventional study of the EBMT Transplant Complication Working Party.

Uric acid is a danger signal contributing to inflammation. Its relevance to allogeneic stem cell transplantation (alloSCT) derives from preclinical models where the depletion of uric acid led to improved survival and reduced graft-versus-host disease (GvHD). In a clinical pilot trial, peri-transplant uric acid depletion reduced acute GvHD incidence. This prospective international multicenter study aimed to investigate the association of uric acid serum levels before start of conditioning with alloSCT outcome. We included patients with acute leukemia, lymphoma or myelodysplastic syndrome receiving a first matched sibling alloSCT from peripheral blood, regardless of conditioning. We compared outcomes between patients with high and low uric acid levels with univariate- and multivariate analysis using a cause-specific Cox model. Twenty centers from 10 countries reported data on 366 alloSCT recipients. There were no significant differences in terms of baseline comorbidity and disease stage between the high- and low uric acid group. Patients with uric acid levels above median measured before start of conditioning did not significantly differ from the remaining in terms of acute GvHD grades II-IV incidence (Hazard ratio [HR] 1.5, 95% Confidence interval [CI]: 1.0-2.4, P=0.08). However, they had significantly shorter overall survival (HR 2.8, 95% CI: 1.7-4.7, P<0.0001) and progression free survival (HR 1.6, 95% CI: 1.1-2.4, P=0.025). Non-relapse mortality was significantly increased in alloSCT recipients with high uric acid levels (HR 2.7, 95% CI: 1.4-5.0, P=0.003). Finally, the incidence of relapse after alloSCT was increased in patients with higher uric acid levels (HR 1.6, 95% CI: 1.0-2.5, P=0.04). We conclude that high uric acid levels before the start of conditioning correlate with increased mortality after alloSCT.

Novel Zinc-Attenuating Compounds as Potent Broad-Spectrum Antifungal Agents with In Vitro and In Vivo Efficacy.

An increase in the incidence of rare but hard-to-treat invasive fungal pathogens as well as resistance to the currently available antifungal drugs calls for new broad-spectrum antifungals with a novel mechanism of action. Here we report the identification and characterization of two novel zinc-attenuating compounds, ZAC307 and ZAC989, which exhibit broad-spectrum in vitro antifungal activity and in vivo efficacy in a fungal kidney burden candidiasis model. The compounds were identified serendipitously as part of a drug discovery process aimed at finding novel inhibitors of the fungal plasma membrane proton ATPase Pma1. Based on their structure, we hypothesized that they might act as zinc chelators. Indeed, both fluorescence-based affinity determination and potentiometric assays revealed these compounds, subsequently termed zinc-attenuating compounds (ZACs), to have strong affinity for zinc, and their growth inhibitory effects on Candida albicans and Aspergillus fumigatus could be inactivated by the addition of exogenous zinc to fungal growth media. We determined the ZACs to be fungistatic, with a low propensity for resistance development. Gene expression analysis suggested that the ZACs interfere negatively with the expression of genes encoding the major components of the A. fumigatus zinc uptake system, thus supporting perturbance of zinc homeostasis as the likely mode of action. With demonstrated in vitro and in vivo antifungal activity, low propensity for resistance development, and a novel mode of action, the ZACs represent a promising new class of antifungal compounds, and their advancement in a drug development program is therefore warranted.

SERCA mutant E309Q binds two Ca(2+) ions but adopts a catalytically incompetent conformation.

The sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) couples ATP hydrolysis to transport of Ca(2+). This directed energy transfer requires cross-talk between the two Ca(2+) sites and the phosphorylation site over 50 Å distance. We have addressed the mechano-structural basis for this intramolecular signal by analysing the structure and the functional properties of SERCA mutant E309Q. Glu(309) contributes to Ca(2+) coordination at site II, and a consensus has been that E309Q only binds Ca(2+) at site I. The crystal structure of E309Q in the presence of Ca(2+) and an ATP analogue, however, reveals two occupied Ca(2+) sites of a non-catalytic Ca2E1 state. Ca(2+) is bound with micromolar affinity by both Ca(2+) sites in E309Q, but without cooperativity. The Ca(2+)-bound mutant does phosphorylate from ATP, but at a very low maximal rate. Phosphorylation depends on the correct positioning of the A-domain, requiring a shift of transmembrane segment M1 into an 'up and kinked position'. This transition is impaired in the E309Q mutant, most likely due to a lack of charge neutralization and altered hydrogen binding capacities at Ca(2+) site II.

Elucidation of antimicrobial activity and mechanism of action by N-substituted carbazole derivatives.

Compounds belonging to a carbazole series have been identified as potent fungal plasma membrane proton adenosine triphophatase (H+-ATPase) inhibitors with a broad spectrum of antifungal activity. The carbazole compounds inhibit the adenosine triphosphate (ATP) hydrolysis activity of the essential fungal H+-ATPase, thereby functionally inhibiting the extrusion of protons and extracellular acidification, processes that are responsible for maintaining high plasma membrane potential. The compound class binds to and inhibits the H+-ATPase within minutes, leading to fungal death after 1-3h of compound exposure in vitro. The tested compounds are not selective for the fungal H+-ATPase, exhibiting an overlap of inhibitory activity with the mammalian protein family of P-type ATPases; the sarco(endo)plasmic reticulum calcium ATPase (Ca2+-ATPase) and the sodium potassium ATPase (Na+,K+-ATPase). The ion transport in the P-type ATPases is energized by the conversion of ATP to adenosine diphosphate (ADP) and phosphate and a general inhibitory mechanism mediated by the carbazole derivative could therefore be blocking of the active site. However, biochemical studies show that increased concentrations of ATP do not change the inhibitory activity of the carbazoles suggesting they act as allosteric inhibitors. Furthermore decreased levels of intracellular ATP would suggest that the compounds inhibit the H+-ATPase indirectly, but Candida albicans cells exposed to potent H+-ATPase-inhibitory carbazoles result in increased levels of intracellular ATP, indicating direct inhibition of H+-ATPase.

Critical roles of interdomain interactions for modulatory ATP binding to sarcoplasmic reticulum Ca2+-ATPase.

ATP has dual roles in the reaction cycle of sarcoplasmic reticulum Ca(2+)-ATPase. Upon binding to the Ca2E1 state, ATP phosphorylates the enzyme, and by binding to other conformational states in a non-phosphorylating modulatory mode ATP stimulates the dephosphorylation and other partial reaction steps of the cycle, thereby ensuring a high rate of Ca(2+) transport under physiological conditions. The present study elucidates the mechanism underlying the modulatory effect on dephosphorylation. In the intermediate states of dephosphorylation the A-domain residues Ser(186) and Asp(203) interact with Glu(439) (N-domain) and Arg(678) (P-domain), respectively. Single mutations to these residues abolish the stimulation of dephosphorylation by ATP. The double mutation swapping Asp(203) and Arg(678) rescues ATP stimulation, whereas this is not the case for the double mutation swapping Ser(186) and Glu(439). By taking advantage of the ability of wild type and mutant Ca(2+)-ATPases to form stable complexes with aluminum fluoride (E2·AlF) and beryllium fluoride (E2·BeF) as analogs of the E2·P phosphoryl transition state and E2P ground state, respectively, of the dephosphorylation reaction, the mutational effects on ATP binding to these intermediates are demonstrated. In the wild type Ca(2+)-ATPase, the ATP affinity of the E2·P phosphoryl transition state is higher than that of the E2P ground state, thus explaining the stimulation of dephosphorylation by nucleotide-induced transition state stabilization. We find that the Asp(203)-Arg(678) and Ser(186)-Glu(439) interdomain bonds are critical, because they tighten the interaction with ATP in the E2·P phosphoryl transition state. Moreover, ATP binding and the Ser(186)-Glu(439) bond are mutually exclusive in the E2P ground state.

Distinct roles of the C-terminal 11th transmembrane helix and luminal extension in the partial reactions determining the high Ca2+ affinity of sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2b (SERCA2b).

The molecular mechanism underlying the characteristic high apparent Ca(2+) affinity of SERCA2b relative to SERCA1a and SERCA2a isoforms was studied. The C-terminal tail of SERCA2b consists of an 11th transmembrane helix (TM11) with an associated 11-amino acid luminal extension (LE). The effects of each of these parts and their interactions with the SERCA environment were examined by transient kinetic analysis of the partial reaction steps in the Ca(2+) transport cycle in mutant and chimeric Ca(2+)-ATPase constructs. Manipulations to the LE of SERCA2b markedly increased the rate of Ca(2+) dissociation from Ca(2)E1. Addition of the SERCA2b tail to SERCA1a slowed Ca(2+) dissociation, but only when the luminal L7/8 loop of SERCA1 was simultaneously replaced with that of SERCA2, thus suggesting that the LE interacts with L7/8 in Ca(2)E1. The interaction of LE with L7/8 is also important for the low rate of the Ca(2)E1P → E2P conformational transition. These findings can be rationalized in terms of stabilization of the Ca(2)E1 and Ca(2)E1P forms by docking of the LE near L7/8. By contrast, low rates of E2P dephosphorylation and E2 → E1 transition in SERCA2b depend critically on TM11, particularly in a SERCA2 environment, but do not at all depend on the LE or L7/8. This indicates that interaction of TM11 with SERCA2-specific sequence element(s) elsewhere in the structure is critical in the Ca(2+)-free E2/E2P states. Collectively these properties ensure a higher Ca(2+) affinity of SERCA2b relative to other SERCA isoforms, not only on the cytosolic side, but also on the luminal side.

Chimerism in DNA of buccal swabs from recipients after allogeneic hematopoietic stem cell transplantations: implications for forensic DNA testing.

We attempted to quantitatively determine the chimeric state in a total of 162 buccal swabs from 77 adult recipients aged 19-74 (median 50 years) after allogeneic hematopoietic cell transplantation by estimating the chimeric recipient/donor DNA ratios through analysis of 15 autosomal short tandem repeat markers. From each individual between one and nine, buccal swabs were taken at known time intervals after transplantation, ranging from 17 to 3,361 days (median 394 days). In buccal cells, the determined recipient/donor DNA ratios turned out to be highly variable between individuals and also within an individual. Relative donor chimerism levels (%Ch) between 0 and 100 % were detected with maximal frequencies between 10 and 30 %. Blood was always found to show the donor's genotype while hair samples in all cases gave the recipient's genotype. We examine chimerism levels with respect to age, gender, and posttransplantation period and discuss the results in the context of forensic identity testing.

Impact of the Recipient's Pre-Treatment Blood Lymphocyte Count on Intended and Unintended Effects of Anti-T-Lymphocyte Globulin in Allogeneic Hematopoietic Stem Cell Transplantation.

Background: In allogeneic hematopoietic stem cell transplantation (HSCT), Anti-T-Lymphocyte Globulin (ATLG) may be used for the prevention of severe graft-versus-host disease (GVHD). ATLG targets both the recipient's lymphocytes and those transferred with the graft. Assuming an inverse relation between the recipient's absolute lymphocyte count (ALC) and exposure of remaining ATLG to the graft, we aim to evaluate the impact of the recipient's ALC before the first ATLG administration on the benefits (prevention of GVHD and GVHD-associated mortality) and potential risks (increased relapse incidence) associated with ATLG. Methods: In recipients of HLA-matched, ATLG-based HSCT (n = 311), we assessed the incidence of acute GVHD, GVHD-related mortality and relapse, as well as other transplant-related outcomes, in relation to the respective ALC (divided into tertiles) before ATLG. Results: The top-tertile ALC group had a significantly increased risk of aGVHD (subhazard ratio (sHR) 1.81; [CI 95%; 1.14-2.88]; p = 0.01) and aGVHD-associated mortality (sHR 1.81; [CI 95%; 1.03-3.19]; p = 0.04). At the highest ATLG dose level (≥45 mg/kg), recipients with lowest-tertile ALC had a trend towards increased relapse incidence (sHR 4.19; [CI 95%; 0.99-17.7]; p = 0.05, n = 32). Conclusions: ATLG dosing based on the recipient's ALC may be required for an optimal balance between GVHD suppression and relapse prevention.

Allogeneic Stem Cell Transplantation in Multiple Myeloma: Risk Factors and Outcomes in the Era of New Therapeutic Options-A Single-Center Experience.

BACKGROUND: Despite major treatment advances, multiple myeloma remains incurable. The outcome of patients who are refractory to immunomodulatory agents, proteasome inhibitors, and anti-CD38 monoclonal antibodies is poor, and improved treatment strategies for this difficult-to-treat patient population are an unmet medical need. METHODS: This retrospective, unicentric analysis included 38 patients with relapsed/refractory multiple myeloma or plasma cell leukemia who underwent allogeneic stem cell transplantation (allo-HSCT) between 2013 and 2022. Survival outcomes, relapse incidence, and non-relapse mortality were calculated according to remission status, date of allo-HSCT, cytogenetic risk status, timing, and number of previous autologous HSCTs. RESULTS: The median PFS was 13.6 months (95% CI, 7.7-30.4) and the median OS was 51.4 months (95% CI, 23.5-NA) in the overall cohort. The cumulative incidence of relapse at 3 years was 57%, and non-relapse mortality was 16%. The median PFS and OS were significantly longer in patients with very good partial remission (VGPR) or better compared to patients with less than VGPR at the time of allo-HSCT (mPFS 29.7 months (95% CI, 13.7-NA) vs. 6.5 months (95% CI, 2.6-17.0); p = 0.009 and mOS not reached vs. 18.6 months (95% CI, 7.0-NA); p = 0.006). CONCLUSION: For selected patients, allo-HSCT may result in favorable overall survival, in part by providing an appropriate hemato-immunological basis for subsequent therapies.

Successful SARS-CoV-2 mRNA Vaccination Program in Allogeneic Hematopoietic Stem Cell Transplant Recipients-A Retrospective Single-Center Analysis.

(1) Background: mRNA COVID-19 vaccines are effective but show varied efficacy in immunocompromised patients, including allogeneic hematopoietic stem cell transplant (HSCT) recipients. (2) Methods: A retrospective study on 167 HSCT recipients assessed humoral response to two mRNA vaccine doses, using the manufacturer cut-off of ≥7.1 BAU/mL, and examined factors affecting non-response. (3) Results: Twenty-two percent of HSCT recipients failed humoral response. Non-responders received the first vaccine a median of 10.2 (2.5-88.9) months post-HSCT versus 35.3 (3.0-215.0) months for responders (p < 0.001). Higher CD19 (B cell) counts favored vaccination response (adjusted odds ratio (aOR) 3.3 per 100 B-cells/microliters, p < 0.001), while ongoing mycophenolate mofetil (MMF) immunosuppression hindered it (aOR 0.04, p < 0.001). By multivariable analysis, the time from transplant to first vaccine did not remain a significant risk factor. A total of 92% of non-responders received a third mRNA dose, achieving additional 77% seroconversion. Non-converters mostly received a fourth dose, with an additional 50% success. Overall, a cumulative seroconversion rate of 93% was achieved after up to four doses. (4) Conclusion: mRNA vaccines are promising for HSCT recipients as early as 3 months post-HSCT. A majority seroconverted after four doses. MMF usage and low B cell counts are risk factors for non-response.

Modulatory ATP binding affinity in intermediate states of E2P dephosphorylation of sarcoplasmic reticulum Ca2+-ATPase.

The mechanism of ATP modulation of E2P dephosphorylation of sarcoplasmic reticulum Ca(2+)-ATPase wild type and mutant forms was examined in nucleotide binding studies of states analogous to the various intermediates of the dephosphorylation reaction, obtained by binding of metal fluorides, vanadate, or thapsigargin. Wild type Ca(2+)-ATPase displays an ATP affinity of 4 µM for the E2P ground state analog, 1 µM for the E2P transition state and product state analogs, and 11 µM for the E2 dephosphoenzyme. Hence, ATP binding stabilizes the transition and product states relative to the ground state, thereby explaining the accelerating effect of ATP on dephosphorylation. Replacement of Phe(487) (N-domain) with serine, Arg(560) (N-domain) with leucine, or Arg(174) (A-domain) with alanine or glutamate reduces ATP affinity in all E2/E2P intermediate states. Alanine substitution of Ile(188) (A-domain) increases the ATP affinity, although ATP acceleration of dephosphorylation is disrupted, thus indicating that the critical role of Ile(188) in ATP modulation is mechanistically based rather than being associated with the binding of nucleotide. Mutants with alanine replacement of Lys(205) (A-domain) or Glu(439) (N-domain) exhibit an anomalous inhibition by ATP of E2P dephosphorylation, due to ATP binding increasing the stability of the E2P ground state relative to the transition state. The ATP affinity of Ca(2)E2P, stabilized by inserting four glycines in the A-M1 linker, is similar to that of the E2P ground state, but the Ca(2+)-free E1 state of this mutant exhibits 3 orders of magnitude reduction of ATP affinity.

Invasive fungal breakthrough infections, fungal colonization and emergence of resistant strains in high-risk patients receiving antifungal prophylaxis with posaconazole: real-life data from a single-centre institutional retrospective observational study.

OBJECTIVES: The broad-spectrum triazole posaconazole showed promising results in preventing invasive fungal infections (IFIs) in high-risk patients. Concerns rise over the relevance of breakthrough IFIs (bIFIs) and the emergence of azole-resistant strains. The current retrospective analysis was undertaken to evaluate the incidence of bIFIs and to study fungal colonization and resistance following posaconazole exposure. METHODS: Ninety-five patients who underwent 202 courses of primary antifungal prophylaxis with 200 mg of posaconazole three times daily during neutropenia after chemotherapy/haematopoietic stem cell transplantation between September 2008 and September 2010 were evaluated. An IFI was considered to be a bIFI if its occurrence was detected ≥4 days after initiation of preventative posaconazole prophylaxis. RESULTS: The incidence of bIFIs was 13% (27/202), with 11/27 (41%) proven and 16/27 (59%) probable bIFIs. Proven infections were mainly localized in the lungs (85%). Species diagnosis exclusively revealed non-Aspergillus species, i.e. mucormycetes in 55% and yeasts in 45%. The median overall survival for patients with bIFIs was 5.2 months. Sixteen of 27 patients with bIFIs (proven and probable) succumbed. Regarding only proven cases, 8/11 patients died, whereas only 1/16 deaths was caused by fungal disease. Prospective screening confirmed colonization with yeasts in 42/202 (21%) courses; moulds were not identified. The spectrum of colonizing yeasts changed slightly over time, shifting to more rare yeasts. There were no deaths due to invasive yeast infections. CONCLUSIONS: A significant proportion of bIFIs, compared with historical data, with a shift to non-Aspergillus spp. and in particular to mucormycetes was observed in patients at high risk for IFI during posaconazole prophylaxis.

Efficacy and safety of itacitinib versus placebo in combination with corticosteroids for initial treatment of acute graft-versus-host disease (GRAVITAS-301): a randomised, multicentre, double-blind, phase 3 trial.

BACKGROUND: Acute graft-versus-host disease (GVHD) is a common and life-threatening complication of allogeneic haematopoietic stem cell transplantation (HSCT); there is an urgent unmet need for effective therapies. We aimed to evaluate the Janus kinase 1 inhibitor itacitinib versus placebo, both in combination with corticosteroids, for initial treatment of acute GVHD. METHODS: GRAVITAS-301 was an international, double-blind, adaptive (group sequential design) phase 3 study conducted at 129 hospitals and community practices in 19 countries. Eligible patients were aged 18 years or older, had previously received allogeneic HSCT for a haematological malignancy, developed grades II-IV acute GVHD, and received up to 2 days of systemic corticosteroids. Patients were stratified by clinical standard-risk or high-risk acute GVHD and randomly assigned (1:1), via a centralised interactive voice response system, to receive either oral itacitinib (200 mg) or placebo once daily, both in addition to corticosteroids. The primary endpoint was overall response rate (ORR) at day 28 (defined as the proportion of patients with complete response, very good partial response, or partial response 28 days after the start of treatment). For sample size determination, an absolute improvement in ORR at day 28 over standard therapy of 16% was considered clinically meaningful. Efficacy analyses were performed in the intention-to-treat population; safety analyses included patients who received at least one dose of study drug. GRAVITAS-301 is registered with ClinicalTrials.gov (NCT03139604) and is complete. FINDINGS: Between July 19, 2017, and Oct 3, 2019, 439 patients were randomly assigned to receive either itacitinib plus corticosteroids (n=219; itacitinib group) or placebo plus corticosteroids (n=220; placebo group). 173 (39%) patients were female and 390 (89%) were White. At baseline, 107 (24%) of 439 patients (itacitinib 51 [23%] of 219; placebo 56 [25%] of 220) had clinical high-risk acute GVHD. The ORR at day 28 was 74% (95% CI 67·6-79·7; 162 of 219; complete response 53% [116 of 219]) for itacitinib and 66% (59·7-72·6; 146 of 220; complete response, 40% [89 of 220]) for placebo (odds ratio for ORR 1·45, 95% CI 0·96-2·20; two-sided p=0·078). Grade 3 or worse adverse events occurred in 185 (86%) of 215 itacitinib recipients and 178 (82%) of 216 placebo recipients, and most commonly included thrombocytopenia or platelet count decreased (78 [36%] vs 68 [31%]), neutropenia or neutrophil count decreased (49 [23%] vs 45 [21%]), anaemia (42 [20%] vs 26 [12%]), and hyperglycaemia (26 [12%] vs 28 [13%]). Treatment-related deaths occurred in three of 215 patients (1%) in the itacitinib group and four of 216 (2%) in the placebo group. INTERPRETATION: The observed improvement in ORR at day 28 with the addition of itacitinib versus placebo to corticosteroids did not reach the prespecified significance level. Further studies might provide additional insight into the utility of selective JAK1 inhibition for the treatment of acute GVHD. FUNDING: Incyte.

Glutamate 90 at the luminal ion gate of sarcoplasmic reticulum Ca2+-ATPase is critical for Ca(2+) binding on both sides of the membrane.

The roles of Ser(72), Glu(90), and Lys(297) at the luminal ends of transmembrane helices M1, M2, and M4 of sarcoplasmic reticulum Ca(2+)-ATPase were examined by transient and steady-state kinetic analysis of mutants. The dependence on the luminal Ca(2+) concentration of phosphorylation by P(i) ("Ca(2+) gradient-dependent E2P formation") showed a reduction of the apparent affinity for luminal Ca(2+) in mutants with alanine or leucine replacement of Glu(90), whereas arginine replacement of Glu(90) or Ser(72) allowed E2P formation from P(i) even at luminal Ca(2+) concentrations much too small to support phosphorylation in wild type. The latter mutants further displayed a blocked dephosphorylation of E2P and an increased rate of conversion of the ADP-sensitive E1P phosphoenzyme intermediate to ADP-insensitive E2P as well as insensitivity of the E2.BeF(3)(-) complex to luminal Ca(2+). Altogether, these findings, supported by structural modeling, indicate that the E2P intermediate is stabilized in the mutants with arginine replacement of Glu(90) or Ser(72), because the positive charge of the arginine side chain mimics Ca(2+) occupying a luminally exposed low affinity Ca(2+) site of E2P, thus identifying an essential locus (a "leaving site") on the luminal Ca(2+) exit pathway. Mutants with alanine or leucine replacement of Glu(90) further displayed a marked slowing of the Ca(2+) binding transition as well as slowing of the dissociation of Ca(2+) from Ca(2)E1 back toward the cytoplasm, thus demonstrating that Glu(90) is also critical for the function of the cytoplasmically exposed Ca(2+) sites on the opposite side of the membrane relative to where Glu(90) is located.

Purified E255L mutant SERCA1a and purified PfATP6 are sensitive to SERCA-type inhibitors but insensitive to artemisinins.

The antimalarial drugs artemisinins have been described as inhibiting Ca(2+)-ATPase activity of PfATP6 (Plasmodium falciparum ATP6) after expression in Xenopus oocytes. Mutation of an amino acid residue in mammalian SERCA1 (Glu(255)) to the equivalent one predicted in PfATP6 (Leu) was reported to induce sensitivity to artemisinin in the oocyte system. However, in the present experiments, we found that artemisinin did not inhibit mammalian SERCA1a E255L either when expressed in COS cells or after purification of the mutant expressed in Saccharomyces cerevisiae. Moreover, we found that PfATP6 after expression and purification from S. cerevisiae was insensitive to artemisinin and significantly less sensitive to thapsigargin and 2,5-di(tert-butyl)-1,4-benzohydroquinone than rabbit SERCA1 but retained higher sensitivity to cyclopiazonic acid, another type of SERCA1 inhibitor. Although mammalian SERCA and purified PfATP6 appear to have different pharmacological profiles, their insensitivity to artemisinins suggests that the mechanism of action of this class of drugs on the calcium metabolism in the intact cell is complex and cannot be ascribed to direct inhibition of PfATP6. Furthermore, the successful purification of PfATP6 affords the opportunity to develop new antimalarials by screening for inhibitors against PfATP6.

Allogeneic bone marrow vs. peripheral blood stem cell transplantation: a long-term retrospective single-center analysis in 329 patients.

OBJECTIVES: Granulocyte colony-stimulating factor-mobilized peripheral blood hematopoietic stem cell transplantation (HSCT) provides a valuable and increasingly used alternative to bone marrow transplantation (BMT). This retrospective study aimed at determining whether the stem cell source is predictive for outcome, relapse incidence, non-relapse mortality, and severity and incidence of both, acute and chronic graft-versus-host disease (GVHD) in patients undergoing allogeneic HSCT. PATIENTS AND METHODS: Between 1983 and 2007, 329 adult patients (median age 40, range 18-76) received a first allogeneic HSCT from either sibling (n = 203) or volunteer unrelated donors (n = 126) at our institution. The source of stem cells was bone marrow in 177 (54%) and peripheral blood in the remaining 152 (46%) patients. RESULTS: Overall survival was 37% (31-43%, 95% confidence interval, CI), the relapse incidence was 30% (25-36%, 95% CI), and the non-relapse mortality was 43% (38-49%, 95% CI) for the entire cohort with no significant differences between peripheral blood stem cell or BMT. In patients receiving myeloablative conditioning, peripheral blood stem cell transplantation (PBSCT) was associated with a significantly lower non-relapse mortality (32% vs. 46%, P = 0.05), which, however, was restricted to standard-risk disease (23% vs. 42%, P = 0.02). The overall cumulative incidences of acute GVHD II-IV were 51% and 54% following bone marrow and PBSCT, respectively. Severe acute GVHD III-IV was significantly more frequent after BMT (24% vs. 14%, P = 0.04), whereas chronic GVHD was significantly more frequent following PBSCT (48% vs. 24%, P = 0.0001). By multivariate analysis, PBSCT was only predictive for chronic GVHD (RR 2.29, P = 0.02). CONCLUSION: Although we failed to demonstrate any advantage of PBSCT over conventional BMT with regard to overall survival, relapse incidence and non-relapse mortality PBSCT were associated with a significantly higher incidence of chronic graft-versus-host disease. Therefore, and by virtue of observations, that some patient groups might benefit from either stem cell source, there is still need for prospective randomized trials with special emphasize on quality of life in long-term survivors.

Relapse Protection Following Early Cytomegalovirus Reactivation after Hematopoietic Stem Cell Transplantation Is Limited to HLA-C Killer Cell Immunoglobulin-Like Receptor Ligand Homozygous Recipients.

Although the risk for nonrelapse mortality (NRM) associated with early cytomegalovirus (CMV) reactivation (CMVR) after allogeneic hematopoietic stem cell transplantation (HSCT) is well established, debate is ongoing on whether CMVR may reduce the risk of primary disease relapse. The aim of this study was to evaluate relapse protection following early CMV reactivation after HSCT in the context of the recipient HLA-C killer cell immunoglobulin-like receptor ligands (KIRLs). In this retrospective bicentric study, 406 matched related or unrelated donor transplantations for acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS) were stratified by HLA-C KIRL group (homozygous versus heterozygous) and analyzed separately for the impact of early CMVR on the cumulative incidences of relapse, NRM, and acute and chronic graft-versus-host-disease (GVHD) using landmark and multistate analyses. By landmark analysis of patients alive and relapse-free at 45 days post-HSCT, HLA-C KIRL homozygous recipients (C1/1 or C2/2) had a lower risk of subsequent relapse if CMVR occurred before this landmark (subhazard ratio [sHR], 0.36; P = .002). In contrast, in HLA-C KIRL heterozygous (C1/2) recipients, early CMVR had no impact on subsequent relapse (sHR, 0.88; P = .63). NRM (sHR, 3.31; P < .001) and grade III-IV acute GVHD (sHR, 2.60; P = .04) were significantly increased after early CMVR in the homozygous cohort, but not in the heterozygous cohort (NRM: sHR, 1.23; P = .53; grade III-IV acute GVHD: sHR, 1.40; P = .50). Multivariable landmark analyses and a multistate model confirmed the limitation of the relapse-protective effect of early CMVR to the homozygous cohort. Chronic GVHD and overall survival were not influenced in neither cohort. An antileukemic effect of early CMVR after HSCT for AML/MDS was significant but strictly limited to recipients homozygous for HLA-C KIRL. However, particularly in this cohort, CMVR had an adverse impact on aGVHD and NRM.