Flipped C-Terminal Ends of APOA1 Promote ABCA1-Dependent Cholesterol Efflux by Small HDLs.

BACKGROUND: Cholesterol efflux capacity (CEC) predicts cardiovascular disease independently of high-density lipoprotein (HDL) cholesterol levels. Isolated small HDL particles are potent promoters of macrophage CEC by the ABCA1 (ATP-binding cassette transporter A1) pathway, but the underlying mechanisms are unclear. METHODS: We used model system studies of reconstituted HDL and plasma from control and lecithin-cholesterol acyltransferase (LCAT)-deficient subjects to investigate the relationships among the sizes of HDL particles, the structure of APOA1 (apolipoprotein A1) in the different particles, and the CECs of plasma and isolated HDLs. RESULTS: We quantified macrophage and ABCA1 CEC of 4 distinct sizes of reconstituted HDL. CEC increased as particle size decreased. Tandem mass spectrometric analysis of chemically cross-linked peptides and molecular dynamics simulations of APOA1, the major protein of HDL, indicated that the mobility of C-terminus of that protein was markedly higher and flipped off the surface in the smallest particles. To explore the physiological relevance of the model system studies, we isolated HDL from LCAT-deficient subjects, whose small HDLs (like reconstituted HDLs) are discoidal and composed of APOA1, cholesterol, and phospholipid. Despite their very low plasma levels of HDL particles, these subjects had normal CEC. In both the LCAT-deficient subjects and control subjects, the CEC of isolated extra-small HDL (a mixture of extra-small and small HDL by calibrated ion mobility analysis) was 3- to 5-fold greater than that of the larger sizes of isolated HDL. Incubating LCAT-deficient plasma and control plasma with human LCAT converted extra-small and small HDL particles into larger particles, and it markedly inhibited CEC. CONCLUSIONS: We present a mechanism for the enhanced CEC of small HDLs. In smaller particles, the C-termini of the 2 antiparallel molecules of APOA1 are "flipped" off the lipid surface of HDL. This extended conformation allows them to engage with ABCA1. In contrast, the C-termini of larger HDLs are unable to interact productively with ABCA1 because they form a helical bundle that strongly adheres to the lipid on the particle. Enhanced CEC, as seen with the smaller particles, predicts decreased cardiovascular disease risk. Thus, extra-small and small HDLs may be key mediators and indicators of the cardioprotective effects of HDL.

Flipped C-Terminal Ends of APOA1 Promote ABCA1-dependent Cholesterol Efflux by Small HDLs.

Background: Cholesterol efflux capacity (CEC) predicts cardiovascular disease (CVD) independently of HDL cholesterol (HDL-C) levels. Isolated small HDL particles are potent promoters of macrophage CEC by the ABCA1 pathway, but the underlying mechanisms are unclear. Methods: We used model system studies of reconstituted HDL and plasma from control and lecithin-cholesterol acyltransferase (LCAT)-deficient subjects to investigate the relationships among the sizes of HDL particles, the structure of APOA1 in the different particles, and the CECs of plasma and isolated HDLs. Results: We quantified macrophage and ABCA1 CEC of four distinct sizes of reconstituted HDL (r-HDL). CEC increased as particle size decreased. MS/MS analysis of chemically crosslinked peptides and molecular dynamics simulations of APOA1 (HDL's major protein) indicated that the mobility of that protein's C-terminus was markedly higher and flipped off the surface in the smallest particles. To explore the physiological relevance of the model system studies, we isolated HDL from LCAT-deficient subjects, whose small HDLs-like r-HDLs-are discoidal and composed of APOA1, cholesterol, and phospholipid. Despite their very low plasma levels of HDL particles, these subjects had normal CEC. In both the LCAT-deficient subjects and control subjects, the CEC of isolated extra-small HDL (a mixture of extra-small and small HDL by calibrated ion mobility analysis) was 3-5-fold greater than that of the larger sizes of isolated HDL. Incubating LCAT-deficient plasma and control plasma with human LCAT converted extra-small and small HDL particles into larger particles, and it markedly inhibited CEC. Conclusions: We present a mechanism for the enhanced CEC of small HDLs. In smaller particles, the C-termini of the two antiparallel molecules of APOA1 are flipped off the lipid surface of HDL. This extended conformation allows them to engage with ABCA1. In contrast, the C-termini of larger HDLs are unable to interact productively with ABCA1 because they form a helical bundle that strongly adheres to the lipid on the particle. Enhanced CEC, as seen with the smaller particles, predicts decreased CVD risk. Thus, extra-small and small HDLs may be key mediators and indicators of HDL's cardioprotective effects.

Efficacy and safety of topically applied therapeutic ammonia oxidising bacteria in adults with mild-to-moderate atopic dermatitis and moderate-to-severe pruritus: a randomised, double-blind, placebo-controlled, dose-ranging, phase 2b trial.

Background: Topical anti-inflammatory therapy is a cornerstone of treatment for atopic dermatitis (AD). However, many unmet needs remain with existing therapies. B244 is a live topical biotherapeutic being tested for the reduction of pruritus and improvement of eczema signs in patients with AD. We aimed to assess the safety and efficacy of B244, compared to vehicle, for patients with mild-to-moderate AD and moderate-to-severe pruritus. Methods: In this randomised, placebo-controlled, double-blind phase 2b trial, adults aged 18-65 years with mild-to-moderate AD and moderate-to-severe pruritus were enrolled across 56 sites in the USA. Patients were randomised 1:1:1 into a low-dose (optical density at 600 nm [OD] 5.0), high-dose (OD 20.0), or vehicle group for the 4-week treatment period and a 4 week follow-up period. Patients were instructed to apply the topical spray twice daily throughout the treatment period. Randomisation was centrally based (random alternating blocks of 6 and 3) and stratified by site. All participants, investigators, and those assessing outcomes were blinded to the treatment group assignments. The primary endpoint was the mean change in pruritus as measured by the Worst Itch Numeric Rating Scale (WI-NRS) at 4 weeks. Safety was tracked throughout the study. Primary efficacy analyses included the modified intent-to-treat (mITT) population, encompassing those who received at least one dose of study drug and attended at least one post-baseline visit. The safety population included all participants who received at least one does of study drug. This study is registered with ClinicalTrials.gov, NCT04490109. Findings: Between June 4, 2020 and October 22, 2021, 547 eligible patients were enrolled. All study endpoints were meaningfully improved with B244 compared to vehicle. The WI-NRS score was reduced by 34% (-2.8 B244 vs -2.1 placebo, p = 0.014 and p = 0.015 for OD 20.0 and OD 5.0), from a baseline score of >8. B244 was well tolerated with no serious adverse events (SAEs); treatment-emergent adverse events (TEAEs) and treatment related TEAEs were low in incidence, mild in severity, and transient. 33 (18%) of 180 patients given B244 OD 5.0, 29 (16%) of 180 patients given B244 OD 20.0, and 17 (9%) of 186 patients given placebo reported treatment-emergent adverse events; headache was the most frequent (3%, 2%, and 1%, respectively). Interpretation: B244 was well tolerated and demonstrated improved efficacy compared to vehicle in all primary, secondary, and exploratory endpoints and should be further developed as a novel, natural, fast-acting topical spray treatment option for AD and associated pruritus. Funding: AOBiome Therapeutics.

ABCA1 is an extracellular phospholipid translocase.

Production of high density lipoprotein (HDL) requires ATP-binding cassette transporter A1 (ABCA1) to drive phospholipid (PL) from the plasma membrane into extracellular apolipoprotein A-I. Here, we use simulations to show that domains of ABCA1 within the plasma membrane remove PL from the membrane's outer leaflet. In our simulations, after the lipid diffuses into the interior of ABCA1's outward-open cavity, PL extracted by the gateway passes through a ring-shaped domain, the annulus orifice, which forms the base of an elongated hydrophobic tunnel in the transporter's extracellular domain. Engineered mutations in the gateway and annulus strongly inhibit lipid export by ABCA1 without affecting cell-surface expression levels. Our finding that ABCA1 extracts lipid from the outer face of the plasma membrane and forces it through its gateway and annulus into an elongated hydrophobic tunnel contrasts with the alternating access model, which proposes that ABCA1 flops PL substrate from the inner leaflet to the outer leaflet of the membrane. Consistent with our model, ABCA1 lacks the charged amino acid residues in the transmembrane domain found in the floppase members of the ABC transporter family.

Direct observation of large electron-phonon interaction effect on phonon heat transport.

As a foundational concept in many-body physics, electron-phonon interaction is essential to understanding and manipulating charge and energy flow in various electronic, photonic, and energy conversion devices. While much progress has been made in uncovering how phonons affect electron dynamics, it remains a challenge to directly observe the impact of electrons on phonon transport, especially at environmental temperatures. Here, we probe the effect of charge carriers on phonon heat transport at room temperature, using a modified transient thermal grating technique. By optically exciting electron-hole pairs in a crystalline silicon membrane, we single out the effect of the phonon-carrier interaction. The enhanced phonon scattering by photoexcited free carriers results in a substantial reduction in thermal conductivity on a nanosecond timescale. Our study provides direct experimental evidence of the elusive role of electron-phonon interaction in phonon heat transport, which is important for understanding heat conduction in doped semiconductors. We also highlight the possibility of using light to dynamically control thermal transport via electron-phonon coupling.

Diabetes Impairs Cellular Cholesterol Efflux From ABCA1 to Small HDL Particles.

RATIONALE: HDL (high-density lipoprotein) may be cardioprotective because it accepts cholesterol from macrophages via the cholesterol transport proteins ABCA1 (ATP-binding cassette transporter A1) and ABCG1 (ATP-binding cassette transporter G1). The ABCA1-specific cellular cholesterol efflux capacity (ABCA1 CEC) of HDL strongly and negatively associates with cardiovascular disease risk, but how diabetes mellitus impacts that step is unclear. OBJECTIVE: To test the hypothesis that HDL's cholesterol efflux capacity is impaired in subjects with type 2 diabetes mellitus. METHODS AND RESULTS: We performed a case-control study with 19 subjects with type 2 diabetes mellitus and 20 control subjects. Three sizes of HDL particles, small HDL, medium HDL, and large HDL, were isolated by high-resolution size exclusion chromatography from study subjects. Then we assessed the ABCA1 CEC of equimolar concentrations of particles. Small HDL accounted for almost all of ABCA1 CEC activity of HDL. ABCA1 CEC-but not ABCG1 CEC-of small HDL was lower in the subjects with type 2 diabetes mellitus than the control subjects. Isotope dilution tandem mass spectrometry demonstrated that the concentration of SERPINA1 (serpin family A member 1) in small HDL was also lower in subjects with diabetes mellitus. Enriching small HDL with SERPINA1 enhanced ABCA1 CEC. Structural analysis of SERPINA1 identified 3 amphipathic α-helices clustered in the N-terminal domain of the protein; biochemical analyses demonstrated that SERPINA1 binds phospholipid vesicles. CONCLUSIONS: The ABCA1 CEC of small HDL is selectively impaired in type 2 diabetes mellitus, likely because of lower levels of SERPINA1. SERPINA1 contains a cluster of amphipathic α-helices that enable apolipoproteins to bind phospholipid and promote ABCA1 activity. Thus, impaired ABCA1 activity of small HDL particles deficient in SERPINA1 could increase cardiovascular disease risk in subjects with diabetes mellitus.

Association of plasma fibrinogen concentrations and blood eosinophil counts with clinical phenotypes of COPD.

BACKGROUND: In patients with chronic obstructive pulmonary disease (COPD), the plasma fibrinogen level is associated with a decline in lung function and exacerbation of COPD. High blood eosinophil count is also associated with exacerbation of COPD in some studies but not others.OBJECTIVE: To investigate the associations between clinical phenotypes and plasma fibrinogen levels and blood eosinophil counts in patients with COPD.METHODS: Outpatients with COPD, in whom plasma fibrinogen level and blood eosinophil count were measured at least once simultaneously, were analysed retrospectively. Patients were classified into four groups, based on plasma fibrinogen level (threshold, 350 mg/dl) and blood eosinophil percentage (threshold, 2%). Clinical characteristics, comorbidities, laboratory data, COPD severity and exacerbations were compared in the four groups.RESULTS: Of 370 patients with COPD, the group with both high fibrinogen levels and eosinophil counts had more severe airflow limitation, more comorbidities and higher COPD severity indexes than the groups with low plasma fibrinogen. The annual rates of severe (0.29/year) and total (0.42/year) exacerbations were significantly higher in patients with both high fibrinogen and eosinophils than in the other three groups.CONCLUSION: Plasma fibrinogen levels and blood eosinophil counts may predict the clinical phenotype and frequency of exacerbations of COPD.

Glutamine-451 Confers Sensitivity to Oxidative Inhibition and Heme-Thiolate Sulfenylation of Cytochrome P450 4B1.

Human cytochrome P450 (P450) family 4 enzymes are involved in the metabolism of fatty acids and the bioactivation of carcinogenic arylamines and toxic natural products, e.g., 4-ipomeanol. These and other drug-metabolizing P450s are redox sensitive, showing a loss of activity resulting from preincubation with H2O2 and recovery with mild reducing agents [Albertolle, M. W., et al. (2017) J. Biol. Chem. 292, 11230-11242]. The inhibition is due to sulfenylation of the heme-thiolate ligand, as determined by chemopreoteomics and spectroscopy. This phenomenon may have implications for chemical toxicity and observed disease-drug interactions, in which the decreased metabolism of P450 substrates occurs in patients with inflammatory diseases (e.g., influenza and autoimmunity). Human P450 1A2 was determined to be redox insensitive. To determine the mechanism underlying the differential redox sensitivity, molecular dynamics (MD) simulations were employed using the crystal structure of rabbit P450 4B1 (Protein Data Bank entry 5T6Q ). In simulating either the thiolate (Cys-S-) or the sulfenic acid (Cys-SOH) at the heme ligation site, MD revealed Gln-451 in either an "open" or "closed" conformation, respectively, between the cytosol and heme-thiolate cysteine. Mutation to either an isosteric leucine (Q451L) or glutamate (Q451E) abrogated the redox sensitivity, suggesting that this "open" conformation allows for reduction of the sulfenic acid and religation of the thiolate to the heme iron. In summary, MD simulations suggest that Gln-451 in P450 4B1 adopts conformations that may stabilize and protect the heme-thiolate sulfenic acid; mutating this residue destabilizes the interaction, producing a redox insensitive enzyme.

Apolipoprotein A1 Forms 5/5 and 5/4 Antiparallel Dimers in Human High-density Lipoprotein.

Apolipoprotein A1 (APOA1), the major protein of high-density lipoprotein (HDL), contains 10 helical repeats that play key roles in protein-protein and protein-lipid interactions. The current structural model for HDL proposes that APOA1 forms an antiparallel dimer in which helix 5 in monomer 1 associates with helix 5 in monomer 2 along a left-left (LL5/5) interface, forming a protein complex with a 2-fold axis of symmetry centered on helix 5. However, computational studies suggest that other orientations are possible. To test this idea, we used a zero-length chemical cross-linking reagent that forms covalent bonds between closely apposed basic and acidic residues. Using proteolytic digestion and tandem mass spectrometry, we identified amino acids in the central region of the antiparallel APOA1 dimer of HDL that were in close contact. As predicted by the current model, we found six intermolecular cross-links that were consistent with the antiparallel LL5/5 registry. However, we also identified three intermolecular cross-links that were consistent with the antiparallel LL5/4 registry. The LL5/5 is the major structural conformation of the two complexes in both reconstituted discoidal HDL particles and in spherical HDL from human plasma. Molecular dynamic simulations suggest that that LL5/5 and LL5/4 APOA1 dimers possess similar free energies of dimerization, with LL5/5 having the lowest free energy. Our observations indicate that phospholipidated APOA1 in HDL forms different antiparallel dimers that could play distinct roles in enzyme regulation, assembly of specific protein complexes, and the functional properties of HDL in humans.

Tertiary structure of apolipoprotein A-I in nascent high-density lipoproteins.

Understanding the function of high-density lipoprotein (HDL) requires detailed knowledge of the structure of its primary protein, apolipoprotein A-I (APOA1). However, APOA1 flexibility and HDL heterogeneity have confounded decades of efforts to determine high-resolution structures and consistent models. Here, molecular dynamics simulations totaling 30 µs on two nascent HDLs, each with 2 APOA1 and either 160 phospholipids and 24 cholesterols or 200 phospholipids and 20 cholesterols, show that residues 1-21 of the N-terminal domains of APOA1 interact via strong salt bridges. Residues 26-43 of one APOA1 in the smaller particle form a hinge on the disc edge, which displaces the C-terminal domain of the other APOA1 to the phospholipid surface. The proposed structures are supported by chemical cross-linking, Rosetta modeling of the N-terminal domain, and analysis of the lipid-free ∆185APOA1 crystal structure. These structures provide a framework for understanding HDL maturation and revise all previous models of nascent HDL.

A consensus model of human apolipoprotein A-I in its monomeric and lipid-free state.

Apolipoprotein (apo)A-I is an organizing scaffold protein that is critical to high-density lipoprotein (HDL) structure and metabolism, probably mediating many of its cardioprotective properties. However, HDL biogenesis is poorly understood, as lipid-free apoA-I has been notoriously resistant to high-resolution structural study. Published models from low-resolution techniques share certain features but vary considerably in shape and secondary structure. To tackle this central issue in lipoprotein biology, we assembled a team of structural biologists specializing in apolipoproteins and set out to build a consensus model of monomeric lipid-free human apoA-I. Combining novel and published cross-link constraints, small-angle X-ray scattering (SAXS), hydrogen-deuterium exchange (HDX) and crystallography data, we propose a time-averaged model consistent with much of the experimental data published over the last 40 years. The model provides a long-sought platform for understanding and testing details of HDL biogenesis, structure and function.

Impact of pre-operative body mass index in head and neck cancer patients undergoing microvascular reconstruction.

OBJECTIVES: To analyse the relationship of pre-operative body mass index with surgical complications and oncological outcomes in patients undergoing microvascular reconstruction for head and neck squamous cell cancer. METHOD: A retrospective review was conducted of 259 patients who underwent microvascular free flap reconstruction after head and neck ablative surgery. RESULTS: Mean body mass index was 22.48 kg/m2. There were no correlations between body mass index and: flap failure (p = 0.739), flap ischaemia (p = 0.644), pharyngocutaneous fistula (p = 0.141) or wound infection (p = 0.224). The five-year disease-specific survival rate was 63 per cent. On univariate analysis, the five-year disease-specific survival rate was significantly correlated with pre-operative body mass index, based on Kaplan-Meier survival curves (p = 0.028). The five-year disease-specific survival rates in underweight, normal weight, overweight and obese groups were 47 per cent, 55 per cent, 65 per cent and 80 per cent, respectively. CONCLUSION: Pre-operative body mass index was a useful predictor for recurrence and survival in patients who underwent microvascular reconstruction for head and neck squamous cell cancer.

Time-domain Brillouin scattering for the determination of laser-induced temperature gradients in liquids.

We present an optical technique based on ultrafast photoacoustics to determine the local temperature distribution profile in liquid samples in contact with a laser heated optical transducer. This ultrafast pump-probe experiment uses time-domain Brillouin scattering (TDBS) to locally determine the light scattering frequency shift. As the temperature influences the Brillouin scattering frequency, the TDBS signal probes the local laser-induced temperature distribution in the liquid. We demonstrate the relevance and the sensitivity of this technique for the measurement of the absolute laser-induced temperature gradient of a glass forming liquid prototype, glycerol, at different laser pump powers-i.e., different steady state background temperatures. Complementarily, our experiments illustrate how this TDBS technique can be applied to measure thermal diffusion in complex multilayer systems in contact with a surrounding liquid.

Sarcomatoid carcinoma in the trachea: A case report and literature review.

Tracheal sarcomatoid carcinoma is an extremely infrequent neoplasm with unclear pathogenesis and clinical outcomes. To date, only two cases have been described in English literature. We report a case of a 37-year-old patient complaining of hemoptysis, dyspnea, and cough. An intraluminal polypoid mass in the trachea was found and ultimately diagnosed as tracheal sarcomatoid carcinoma in the cervical trachea with both carcinomatous and sarcomatoid morphology. The patient is alive without recurrence after segmental resection of the trachea. We also present a comparative analysis of our case with a prior tracheal sarcomatoid carcinoma case.

Referral Patterns and Clinical Outcomes for Transplant-Eligible Lymphoma and Myeloma Patients Evaluated at an Urban County Hospital.

Disparities in clinical care have been described for patients with limited insurance coverage or social support. We hypothesized that patients with relapsed Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), or multiple myeloma (MM) treated at an urban county hospital serving indigent and under-insured patients would face barriers for referral to a private academic transplant center for autologous stem cell transplantation (ASCT). Charts of patients with HL, NHL, or MM treated at Grady Memorial Hospital between 2007 and 2013 were reviewed, and 215 patients with diagnosis of HD (n=40), NHL (n=96), and MM (n=79). 55 patients were referred for ASCT consults and 160 patients were not referred. Reasons for transplant non-referral included established clinical criteria (64% of cases), poor performance status (13%), refusal (4%), moved/lost-to-follow-up (4%), medical non-compliance (3%), death (3%), or referral to another hospital (1%). Non-referral based upon socio-economic criteria included: lack of legal immigration status/insurance (2%), and lack of social support/substance abuse (2%). Among the 55 referred patients, 27 patients (49%) underwent ASCT. Median follow-up for all referred patients from the time of diagnosis was 3.9 [0.7-22.7] years. 5-year survival from the date of diagnosis for patients who received ASCT was 80.2% versus 65.7% for non-transplanted patients (log-rank test, p-value=0.11). While the referral process did not demonstrate significant barriers based upon insurance or social status, further evaluation is needed to identify modifiable factors that can improve referral and assess the impact of the Affordable Care Act on access to ASCT.

Ultra-low-field NMR relaxation and diffusion measurements using an optical magnetometer.

Nuclear magnetic resonance (NMR) relaxometry and diffusometry are important tools for the characterization of heterogeneous materials and porous media, with applications including medical imaging, food characterization and oil-well logging. These methods can be extremely effective in applications where high-resolution NMR is either unnecessary, impractical, or both, as is the case in the emerging field of portable chemical characterization. Here, we present a proof-of-concept experiment demonstrating the use of high-sensitivity optical magnetometers as detectors for ultra-low-field NMR relaxation and diffusion measurements.

Asthma and proinflammatory conditions: a population-based retrospective matched cohort study.

OBJECTIVE: To determine the association between asthma and proinflammatory conditions. PARTICIPANTS AND METHODS: This population-based retrospective matched cohort study enrolled all asthmatic patients among Rochester, Minnesota, residents between January 1, 1964, and December 31, 1983. For each asthmatic patient, 2 age-and sex-matched nonasthmatic individuals were drawn from the same population. The asthmatic and nonasthmatic cohorts were followed forward in the Rochester Epidemiology Project diagnostic index for inflammatory bowel disease (IBD), rheumatoid arthritis (RA), diabetes mellitus (DM), and coronary heart disease (CHD) as outcome events. Data were fitted to Cox proportional hazards models. RESULTS: We identified 2392 asthmatic patients and 4784 nonasthmatic controls. Of the asthmatic patients, 1356 (57%) were male, and mean age at asthma onset was 15.1 years. Incidence rates of IBD, RA, DM, and CHD in nonasthmatic controls were 32.8, 175.9, 132.0, and 389.7 per 100,000 person-years, respectively; those for asthmatic patients were 41.4, 227.9, 282.6, and 563.7 per 100,000 person-years, respectively. Asthma was associated with increased risks of DM (hazard ratio, 2.11; 95% confidence interval, 1.43-3.13; P<.001) and CHD (hazard ratio, 1.47; 95% confidence interval, 1.05-2.06; P=.02) but not with increased risks of IBD or RA. CONCLUSION: Although asthma is a helper T cell type 2-predominant condition, it may increase the risks of helper T cell type 1-polarized proinflammatory conditions, such as CHD and DM. Physicians who care for asthmatic patients need to address these unrecognized risks in asthmatic patients.

Phase 1 dose-escalation trial of clofarabine followed by escalating dose of fractionated cyclophosphamide in adults with relapsed or refractory acute leukaemias.

The prognosis of patients with relapsed and refractory acute leukaemia (RRAL) is very poor. Forty patients with RRAL were enroled [28 acute myeloid leukaemia (AML), 12 acute lymphoblastic leukaemia (ALL)] in this Phase 1 dose-escalation trial of daily-infused clofarabine (CLO) followed by cyclophosphamide (CY) for four consecutive days (CLO-CYx4). The median age was 48·5 years. The median number of prior regimens was 2 (range 1-5), and 6/40 patients (15%) had prior allogeneic haematopoietic stem cell transplant. 28/40 patients (70%) had adverse genetic features. 6/40 patients (15%) died within 60 d of induction (two infections, four progressive disease). The average time to neutrophil recovery (absolute neutrophil count ≥0·5 × 10(9) /l was 34 d, (range, 17-78). The overall response rate (ORR) was 33% (13/40), with seven complete remissions (18%), four complete remissions with incomplete recovery of blood counts (10%), and two partial remissions (5%). ORR was 25% (7/28), and 50% (6/12), for AML and ALL respectively. Notably, the clinical responses were independent of dose level. 7/17 patients (41%) exhibited CLO-mediated enhancement of CY-induced DNA, which was associated with, but not necessary for, improved clinical outcomes. In summary, the CLO-CYx4 regimen was well tolerated and had activity in patients with RRAL, especially relapsed ALL. Therefore, CLO-CYx4 can be considered a salvage therapy for adults with RRALs, and warrants further investigations.