Association of Apremilast With Vascular Inflammation and Cardiometabolic Function in Patients With Psoriasis: The VIP-A Phase 4, Open-label, Nonrandomized Clinical Trial.

Importance: Psoriasis is an inflammatory condition associated with metabolic and cardiovascular disease. Apremilast, a phosphodiesterase 4 inhibitor, is commonly used for psoriasis and can cause weight loss. Objective: To determine the association between apremilast and aortic vascular inflammation as assessed by 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT), cardiometabolic markers (primary outcomes at week 16), and abdominal fat composition. Design, Setting, and Participants: A single-arm, open-label, interventional, nonrandomized clinical trial in which the imaging and laboratory outcomes were measured by an investigator who was blinded to time was conducted between April 11, 2017, and August 17, 2021, at 7 dermatology sites in the United States. A total of 101 patients with moderate to severe psoriasis were screened, 70 enrolled, 60 completed week 16, and 39 completed week 52. Intervention: Apremilast, 30 mg, twice daily. Main Outcomes and Measures: Aortic vascular inflammation (measured by FDG-PET/CT), 68 cardiometabolic biomarkers, and abdominal fat composition (measured by CT) at week 16 and week 52 compared with baseline. Results: The mean (SD) age of the 70 patients was 47.5 (14.6) years, 54 were male (77.1%), 4 were Black (5.7%), and 58 were White (82.9%). There was no change in aortic vascular inflammation at week 16 (target to background ratio, -0.02; 95% CI, -0.08 to 0.05; P = .61) or week 52 (target to background ratio, -0.07; 95% CI, -0.15 to 0.01; P = .09) compared with baseline. At week 16, potentially beneficial decreases in interleukin 1b, valine, leucine, isoleucine, fetuin A, and branched-chain amino acids were observed. At week 52 compared with baseline, potentially beneficial decreases in ferritin, ß-hydroxybutyrate, acetone, and ketone bodies, with an increase in apolipoprotein A-1, were observed, but there was a reduction in cholesterol efflux. There was an approximately 5% to 6% reduction in subcutaneous and visceral adiposity at week 16 that was maintained at week 52. Conclusions and Relevance: The findings of this nonrandomized clinical trial suggest that apremilast has a neutral association with aortic vascular inflammation, variable but generally beneficial associations with a subset of cardiometabolic biomarkers, and associations with reductions in visceral and subcutaneous fat, indicating that the drug may have an overall benefit for patients with cardiometabolic disease and psoriasis. Trial Registration: ClinicalTrials.gov Identifier: NCT03082729.

An Imaging Flow Cytometry Method to Measure Citrullination of H4 Histone as a Read-out for Neutrophil Extracellular Traps Formation.

The formation of neutrophil extracellular traps (NETs) is thought to play a critical role in infections and propagating sterile inflammation. Histone citrullination is an essential and early step in NETs formation, detectable prior to the formation of the hallmark extracellular DNA-scaffolded strands. In addition to the classical microscopy method, new technologies are being developed for studies of NETs and their detection, both for research and clinical purposes. Classical microscopy studies of NETs are subjective, low throughput and semi-quantitative, and limited in their ability to capture the early steps. We have developed this novel Imaging Flow Cytometry (IFC) method that specifically identifies and quantifies citrullination of histone H4 as a NETs marker and its relationship with other alterations at nuclear and cellular level. These include nuclear decondensation and super-condensation, multi-lobulated nuclei versus 1-lobe nuclei and cell membrane damage. NETs markers can be quantified following variable periods of treatment with NETs inducers, prior to the formation of the specific extracellular DNA-scaffolded strands. Because these high throughput image-based cell analysis features can be performed with statistical rigor, this protocol is suited for both experimental and clinical applications as well as clinical evaluations of NETosis as a biomarker.

Detection and Quantification of Histone H4 Citrullination in Early NETosis With Image Flow Cytometry Version 4.

Neutrophil extracellular traps (NETs) formation has been implicated in an increasing number of infectious and non-infectious pathologies. NETosis is a tightly regulated process; the end-stage and read-out is the formation of DNA strands extruded from the nuclei, and traditionally assessed by fluorescence microscopy. Since NETosis has emerged as a possible biomarker of the inflammatory process, there is a need for less time-consuming, consistent, and quantitative approaches to improve its application in clinical assessment of pro-inflammatory conditions. Imaging Flow Cytometry (IFC) combines features of conventional flow cytometry with qualitative power of fluorescence microscopy and has an added advantage of the capability of assessing the early processes leading up to extrusion of the DNA-scaffolded strands. We explored the optimal imaging-based tools that can be used to measure citrullination of H4 in early NETosis. IFC identified and quantified histone 4 citrullination (H4cit3) induced with several known NETosis stimuli (Ionophore, PMA, LPS, Hemin, and IL-8) following treatment periods ranging from 2 to 60 min. Its relationship with other alterations at nuclear and cellular level, such as nuclear decondensation and super-condensation, multi-lobulated nuclei vs. 1-lobe nuclei and cell membrane damage, were also quantified. We show that the early progress of the H4cit3 response in NETosis depends on the stimulus. Our method identifies fast (Ionophore and Hemin), intermediate and slow (PMA) inducers and shows that H4cit3 appears to have a limited contribution to both early LPS- and IL-8-induced NETosis. While this method is rapid and of a higher throughput compared to fluorescence microscopy, detection and quantification is limited to H4cit3-mediated nuclear events and is likely to be stimulus- and signaling pathway dependent.

Whole genome sequence-based haplotypes reveal a single origin of the 1393 bp HBB deletion.

BACKGROUND: Mutations of HBB give rise to two prevalent haemoglobin disorders-sickle cell disease (SCD) and ß-thalassaemia. While SCD is caused by a single base substitution, nearly 300 mutations that downregulate expression of HBB have been described. The vast majority of ß-thalassaemia alleles are point mutations or small insertion/deletions within the HBB gene; deletions causing ß-thalassaemia are very rare. We have identified three individuals with haemoglobin Sß0-thalassaemia in which the ß0-thalassaemia mutation is caused by a large deletion. OBJECTIVE: To use whole genome sequence data to determine whether these deletions arose from a single origin. METHODS: We used two approaches to confirm unrelatedness: pairwise comparison of SNPs and identity by descent analysis. Eagle, V.2.4, was used to generate phased haplotypes for the 683 individuals. The Neighbor-Net method implemented in SplitsTree V.4.13.1 was used to construct the network of haplotypes. RESULTS: All three deletions involved 1393 bp, encompassing the ß-promoter, exons 1 and 2, and part of intron 2, with identical breakpoints. The cases were confirmed to be unrelated. Haplotypes based on 29 SNPs in the HBB cluster showed that the three individuals harboured different ßS haplotypes. In contrast, the haplotype harbouring the 1393 bp deletion was the same in all three individuals. CONCLUSION: We suggest that all the reported cases of the 1393 bp HBB deletion, including the three cases here, are likely to be of the same ancestral origin.

Pro-inflammatory cytokines associate with NETosis during sickle cell vaso-occlusive crises.

Recurring episodes of acute pain, also referred to as vaso-occlusive crises (VOC), are characteristic of sickle cell disease (SCD), during which pro-inflammatory cytokines, chemokines, adhesion markers and white cell count, some already elevated at steady state, increase further. Hydroxyurea (HU) is licensed by the FDA for reducing frequency of VOCs in SCD; increased fetal hemoglobin (HbF) together with reduction of the neutrophil count and circulating inflammatory markers, contribute to its clinical efficacy. Here, using paired plasma samples from HbSS patients (in steady-state and VOC) we determined that despite HU treatment, the SCD environment remained highly inflammatory and particularly at VOC, triggered neutrophil activity. While neutrophil extracellular traps (NETs) induction by the steady state plasmas were comparable to that of plasma from healthy donors, the NETs response triggered by crisis plasmas was significantly increased over that of the steady state (P = 0.0124*). Levels of IL-6 and IL-1α, IL-1ra/IL1F3 and adhesion molecule P-selectin were significantly increased in the VOC plasma when compared with steady state plasma. Higher levels of IL-6 and IL-1ra were also found in the crises samples that yielded an increased NETs response suggesting that increased NETs production associated with increased levels of the inflammatory products of the IL-6 family and regulators of IL-1 family of cytokines during sickle VOCs.

Neutrophils remain detrimentally active in hydroxyurea-treated patients with sickle cell disease.

Neutrophilia is a feature of sickle cell disease (SCD) that has been consistently correlated with clinical severity and has been shown to remain highly activated even at steady state. In addition to induction of fetal hemoglobin (HbF), hydroxyurea (HU) leads to reduction in neutrophil count and their adhesion properties, which contributes to the clinical efficacy of HU in SCD. Although HU reduces the frequency and severity of acute vaso-occlusive crises (VOCs) and chest syndrome, HU therapy does not abolish these acute clinical events. In this study we investigated whether neutrophils in SCD patients whilst on HU therapy retained features of detrimental pro-inflammatory activity. Freshly isolated neutrophils from SCD patients on HU therapy at steady state and from ethnic-matched healthy controls were evaluated ex vivo for their degranulation response and production of neutrophil extracellular traps (NETs). Unstimulated SCD patient neutrophils already produced NETs within 30 minutes, compared to none for healthy neutrophils, and by 4 hours, these neutrophils produced significantly more NETs than the control neutrophils (P = 0.0079**). Higher numbers of neutrophils from SCD patients also showed higher degree of degranulation-related intracellular features compared to healthy neutrophils, including rough-textured cellular membranes (P = 0.03*), double-positivity for F-Actin and CD63 (P = 0.02*) and re-located CD63 within cytoplasm more efficiently than their healthy counterparts (P = 0.02*). The neutrophils from SCD donors released more myeloperoxidase (P = 0.02*) in the absence of any trigger. Our data showed that neutrophils from patients with SCD at steady state remained active during hydroxyurea treatment and are likely to be able to contribute to the SCD pro-inflammatory environment.

The platelet NLRP3 inflammasome is upregulated in sickle cell disease via HMGB1/TLR4 and Bruton tyrosine kinase.

A key inflammatory mechanism recently identified in platelets involves the Nod-like receptor nucleotide-binding domain leucine-rich repeat containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK), which control activation of caspase-1 within inflammasome complexes. We investigated platelet caspase-1 activity in the context of sickle cell disease (SCD) directly in platelets isolated from SCD patients (n = 24) and indirectly by incubating platelets from healthy subjects with plasma obtained from SCD patients (n = 20), both in steady state and during an acute pain crisis (paired samples). The platelet NLRP3 inflammasome was upregulated in SCD patients under steady state conditions compared with healthy controls, and it was further upregulated when patients experienced an acute pain crisis. The results were consistent with indirect platelet assays, in which SCD plasma increased caspase-1 activity of platelets from healthy subjects in an NLRP3-dependent fashion. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) was elevated in plasma of SCD subjects compared with healthy controls and correlated with caspase-1 activity in platelets. Pharmacological or antibody-mediated inhibition of HMGB1, Toll-like receptor 4, and BTK interfered with sickle plasma-induced platelet caspase-1 activation. In Townes SCD mice, caspase-1 activity and aggregation of circulating platelets were elevated, which was suppressed by IV injection of an NLRP3 inhibitor and the BTK inhibitor ibrutinib. Activation of the platelet NLRP3 inflammasome in SCD may have diagnostic and therapeutic implications.

GlycA is not a useful biomarker of inflammation in sickle cell disease.

INTRODUCTION: Sickle cell disease (SCD) is a multisystemic disorder, the pathology being driven by recurrent inflammation particularly during a vaso-occlusive crisis. GlycA, a composite measure of protein glycation, is a sensitive biomarker for disorders associated with vascular inflammation. We determined the utility of GlycA as a biomarker of inflammation in SCD. METHODS: Stored plasma samples from patients with SCD recruited to two clinical studies were analyzed. One study encompasses 488 patient samples with SCD (HbSS, HbSß0 and HbSC) at steady state and 52 race-matched, healthy controls. The other study included paired plasma samples during steady state and acute pain crisis from (HbSS) patients with SCD. Plasma GlycA was measured using a proton NMR on the Vantera® Clinical Analyzer. We performed analysis comparing patients with SCD, healthy controls, and paired samples analysis. RESULTS: The mean plasma GlycA level was lower in SCD compared with healthy controls (324.6 ± 70.4 µmol/L vs. 386.3 ± 74.6 µmol/L, P < 0.0001). Within the same patient, mean plasma GlycA during acute pain crisis was lower than steady state, although the difference was not significant (300.5 ± 36.3 µmol/L vs 314.2 ± 34.8 µmol/L, P = 0.020). Plasma GlycA correlated inversely with serum LDH (P = 0.009). CONCLUSION: GlycA is not a suitable biomarker of inflammation in SCD. We surmise that its signal is confounded by hemolysis leading to a depletion of haptoglobin, one of the major plasma proteins included in the composite NMR signal. Hemolysis is further exacerbated during an acute pain crisis, hence the lower GlycA levels in crisis compared to steady state.

Reversible binding of hemoglobin to band 3 constitutes the molecular switch that mediates O2 regulation of erythrocyte properties.

Functional studies have shown that the oxygenation state of the erythrocyte regulates many important pathways, including glucose metabolism, membrane mechanical stability, and cellular adenosine triphosphate (ATP) release. Deoxyhemoglobin (deoxyHb), but not oxyhemoglobin, binds avidly and reversibly to band 3, the major erythrocyte membrane protein. Because band 3 associates with multiple metabolic, solute transport, signal transduction, and structural proteins, the hypothesis naturally arises that the O2-dependent regulation of erythrocyte properties might be mediated by the reversible association of deoxyHb with band 3. To explore whether the band 3-deoxyHb interaction constitutes a "molecular switch" for regulating erythrocyte biology, we have generated transgenic mice with mutations in the deoxyHb-binding domain of band 3. One strain of mouse contains a "humanized" band 3 in which the N-terminal 45 residues of mouse band 3 are replaced by the homologous sequence from human band 3, including the normal human band 3 deoxyHb-binding site. The second mouse contains the same substitution as the first, except the deoxyHb site on band 3 (residues 12-23) has been deleted. Comparison of these animals with wild-type mice demonstrates that the following erythrocyte properties are controlled by the O2-dependent association of hemoglobin with band 3: (1) assembly of a glycolytic enzyme complex on the erythrocyte membrane which is associated with a shift in glucose metabolism between the pentose phosphate pathway and glycolysis, (2) interaction of ankyrin with band 3 and the concomitant regulation of erythrocyte membrane stability, and (3) release of ATP from the red cell which has been linked to vasodilation.

Sickle Cell Imaging Flow Cytometry Assay (SIFCA).

Hemoglobin S polymerization under hypoxic conditions in sickle cell disorders causes characteristic shape changes to human red blood cells. Previous sickling assays used to investigate the efficacy of novel agents to treat these disorders are laborious and observer dependent. Here, we describe a partially automated, high-throughput sickling assay using imaging flow cytometry.

Production of Gene-Corrected Adult Beta Globin Protein in Human Erythrocytes Differentiated from Patient iPSCs After Genome Editing of the Sickle Point Mutation.

Human induced pluripotent stem cells (iPSCs) and genome editing provide a precise way to generate gene-corrected cells for disease modeling and cell therapies. Human iPSCs generated from sickle cell disease (SCD) patients have a homozygous missense point mutation in the HBB gene encoding adult ß-globin proteins, and are used as a model system to improve strategies of human gene therapy. We demonstrate that the CRISPR/Cas9 system designer nuclease is much more efficient in stimulating gene targeting of the endogenous HBB locus near the SCD point mutation in human iPSCs than zinc finger nucleases and TALENs. Using a specific guide RNA and Cas9, we readily corrected one allele of the SCD HBB gene in human iPSCs by homologous recombination with a donor DNA template containing the wild-type HBB DNA and a selection cassette that was subsequently removed to avoid possible interference of HBB transcription and translation. We chose targeted iPSC clones that have one corrected and one disrupted SCD allele for erythroid differentiation assays, using an improved xeno-free and feeder-free culture condition we recently established. Erythrocytes from either the corrected or its parental (uncorrected) iPSC line were generated with similar efficiencies. Currently ∼6%-10% of these differentiated erythrocytes indeed lacked nuclei, characteristic of further matured erythrocytes called reticulocytes. We also detected the 16-kDa ß-globin protein expressed from the corrected HBB allele in the erythrocytes differentiated from genome-edited iPSCs. Our results represent a significant step toward the clinical applications of genome editing using patient-derived iPSCs to generate disease-free cells for cell and gene therapies. Stem Cells 2015;33:1470-1479.

Iron, inflammation, and early death in adults with sickle cell disease.

RATIONALE: Patients with sickle cell disease (SCD) have markers of chronic inflammation, but the mechanism of inflammation and its relevance to patient survival are unknown. OBJECTIVE: To assess the relationship between iron, inflammation, and early death in SCD. METHODS AND RESULTS: Using peripheral blood mononuclear cell transcriptome profile hierarchical clustering, we classified 24 patients and 10 controls in clusters with significantly different expression of genes known to be regulated by iron. Subsequent gene set enrichment analysis showed that many genes associated with the high iron cluster were involved in the toll-like receptor system (TLR4, TLR7, and TLR8) and inflammasome complex pathway (NLRP3, NLRC4, and CASP1). Quantitative PCR confirmed this classification and showed that ferritin light chain, TLR4, and interleukin-6 expression were >100-fold higher in patients than in controls (P<0.001). Further linking intracellular iron and inflammation, 14 SCD patients with a ferroportin Q248H variant that causes intracellular iron accumulation had significantly higher levels of interleukin-6 and C-reactive protein compared with 14 matched SCD patients with the wild-type allele (P<0.05). Finally, in a cohort of 412 patients followed for a median period of 47 months (interquartile range, 24-82), C-reactive protein was strongly and independently associated with early death (hazard ratio, 3.0; 95% confidence interval, 1.7-5.2; P<0.001). CONCLUSIONS: Gene expression markers of high intracellular iron in patients with SCD are associated with markers of inflammation and mortality. The results support a model in which intracellular iron promotes inflammatory pathways, such as the TLR system and the inflammasome, identifying important new pathways for additional investigation.

Heme-bound iron activates placenta growth factor in erythroid cells via erythroid Krüppel-like factor.

In adults with sickle cell disease (SCD), markers of iron burden are associated with excessive production of the angiogenic protein placenta growth factor (PlGF) and high estimated pulmonary artery pressure. Enforced PlGF expression in mice stimulates production of the potent vasoconstrictor endothelin-1, producing pulmonary hypertension. We now demonstrate heme-bound iron (hemin) induces PlGF mRNA >200-fold in a dose- and time-dependent fashion. In murine and human erythroid cells, expression of erythroid Krüppel-like factor (EKLF) precedes PlGF, and its enforced expression in human erythroid progenitor cells induces PlGF mRNA. Hemin-induced expression of PlGF is abolished in EKLF-deficient murine erythroid cells but rescued by conditional expression of EKLF. Chromatin immunoprecipitation reveals that EKLF binds to the PlGF promoter region. SCD patients show higher level expression of both EKLF and PlGF mRNA in circulating blood cells, and markers of iron overload are associated with high PlGF and early mortality. Finally, PlGF association with iron burden generalizes to other human diseases of iron overload. Our results demonstrate a specific mechanistic pathway induced by excess iron that is linked in humans with SCD and in mice to markers of vasculopathy and pulmonary hypertension. These trials were registered at www.clinicaltrials.gov as #NCT00007150, #NCT00023296, #NCT00081523, and #NCT00352430.

Microvascular oxygen consumption during sickle cell pain crisis.

Sickle cell disease is an inherited blood disorder characterized by chronic hemolytic anemia and episodic vaso-occlusive pain crises. Vaso-occlusion occurs when deoxygenated hemoglobin S polymerizes and erythrocytes sickle and adhere in the microvasculature, a process dependent on the concentration of hemoglobin S and the rate of deoxygenation, among other factors. We measured oxygen consumption in the thenar eminence during brachial artery occlusion in sickle cell patients and healthy individuals. Microvascular oxygen consumption was greater in sickle cell patients than in healthy individuals (median [interquartile range]; sickle cell: 0.91 [0.75-1.07] vs healthy: 0.75 [0.62-0.94] -ΔHbO2/min, P < .05) and was elevated further during acute pain crisis (crisis: 1.10 [0.78-1.30] vs recovered: 0.88 [0.76-1.03] -ΔHbO2/min, P < .05). Increased microvascular oxygen consumption during pain crisis could affect the local oxygen saturation of hemoglobin when oxygen delivery is limiting. Identifying the mechanisms of elevated oxygen consumption during pain crisis might lead to the development of new therapeutic interventions. This trial was registered at www.clinicaltrials.gov as #NCT01568710.

Imaging flow cytometry for automated detection of hypoxia-induced erythrocyte shape change in sickle cell disease.

In preclinical and early phase pharmacologic trials in sickle cell disease, the percentage of sickled erythrocytes after deoxygenation, an ex vivo functional sickling assay, has been used as a measure of a patient's disease outcome. We developed a new sickle imaging flow cytometry assay (SIFCA) and investigated its application. To perform the SIFCA, peripheral blood was diluted, deoxygenated (2% oxygen) for 2 hr, fixed, and analyzed using imaging flow cytometry. We developed a software algorithm that correctly classified investigator tagged "sickled" and "normal" erythrocyte morphology with a sensitivity of 100% and a specificity of 99.1%. The percentage of sickled cells as measured by SIFCA correlated strongly with the percentage of sickle cell anemia blood in experimentally admixed samples (R = 0.98, P ≤ 0.001), negatively with fetal hemoglobin (HbF) levels (R = -0.558, P = 0.027), negatively with pH (R = -0.688, P = 0.026), negatively with pretreatment with the antisickling agent, Aes-103 (5-hydroxymethyl-2-furfural) (R = -0.766, P = 0.002), and positively with the presence of long intracellular fibers as visualized by transmission electron microscopy (R = 0.799, P = 0.002). This study shows proof of principle that the automated, operator-independent SIFCA is associated with predictable physiologic and clinical parameters and is altered by the putative antisickling agent, Aes-103. SIFCA is a new method that may be useful in sickle cell drug development.

Extensive ex vivo expansion of functional human erythroid precursors established from umbilical cord blood cells by defined factors.

There is a constant shortage of red blood cells (RBCs) from sufficiently matched donors for patients who need chronic transfusion. Ex vivo expansion and maturation of human erythroid precursors (erythroblasts) from the patients or optimally matched donors could represent a potential solution. Proliferating erythroblasts can be expanded from umbilical cord blood mononuclear cells (CB MNCs) ex vivo for 10(6)-10(7)-fold (in ~50 days) before proliferation arrest and reaching sufficient number for broad application. Here, we report that ectopic expression of three genetic factors (Sox2, c-Myc, and an shRNA against TP53 gene) associated with iPSC derivation enables CB-derived erythroblasts to undergo extended expansion (~10(68)-fold in ~12 months) in a serum-free culture condition without change of cell identity or function. These expanding erythroblasts maintain immature erythroblast phenotypes and morphology, a normal diploid karyotype and dependence on a specific combination of growth factors for proliferation throughout expansion period. When being switched to a terminal differentiation condition, these immortalized erythroblasts gradually exit cell cycle, decrease cell size, accumulate hemoglobin, condense nuclei and eventually give rise to enucleated hemoglobin-containing erythrocytes that can bind and release oxygen. Our result may ultimately lead to an alternative approach to generate unlimited numbers of RBCs for personalized transfusion medicine.

Topical sodium nitrite for chronic leg ulcers in patients with sickle cell anaemia: a phase 1 dose-finding safety and tolerability trial.

BACKGROUND: Well-tolerated and effective treatments are needed for chronic leg ulcers in sickle cell anaemia. Topical sodium nitrite, a known nitric oxide donor, enhances blood flow in ulcers and has known bacteriostatic effects. We aimed to assess the safety, tolerability, and pharmacokinetics of topical sodium nitrite in patients with sickle cell disease and chronic leg ulcers. METHODS: We enrolled adult patients from an ambulatory clinic at the National Institutes of Health (Bethesda, MD, USA) with sickle cell anaemia with leg ulcers (with a surface area of 2.5-100 cm2) persisting for at least 4 weeks into a safety and tolerability phase 1 dose-escalation trial of topical sodium nitrite. Increasing concentrations of sodium nitrite cream were applied twice weekly for 4 weeks to one ulcer per patient at five dose levels (0.5%, 1%, 1.5%, 1.8%, and 2%). The primary endpoints were safety and tolerability, with secondary endpoints of pharmacokinetics, blood flow, and wound healing. Pain relief was analysed post hoc. Endpoints were analysed over time for the whole study population and according to dose level. This study is registered with ClinicalTrials.gov, number NCT01316796. FINDINGS: Between April 4, 2011, and March 19, 2013, we enrolled 18 adult patients with sickle cell anaemia and leg ulcers into our trial. We assigned three patients into each cohort, and each cohort was treated with a different concentration of sodium nitrite cream (cohort 1: 0.5%, cohort 2: 1.0%, cohort 3: 1.5%, and cohort 4: 2.0%). Patients were not enrolled into the next cohort dose until we were able to establish that no dose-limiting toxicities were observed. An additional six patients were enrolled to cohort 3a: 1.8%, after two patients in cohort 4 had asymptomatic drops in diastolic blood pressure. No grade 3-4 adverse events were observed, and there were no serious adverse events or dose-limiting side-effects. Pharmacokinetic analysis showed that systemic absorption of sodium nitrite was very low. Application of topical sodium nitrite was associated with a significant increase in peri-wound cutaneous blood flow measured by laser speckle contrast imaging (p=0.0002), corroborated by increased peri-wound skin temperature by infrared thermography (p=0.0119). We recorded a dose-dependent decrease in leg ulcer size (p=0.0012) and pain (p<0.0001). Ulcers healed completely in three patients who received the highest concentrations of topical sodium nitrite (the 1.8% and 2% cream). In our post-hoc analysis of pain, brief pain inventory scores improved in pain severity (p=0.0048) and pain interference (p=0.0013). INTERPRETATION: Our results indicate that topical sodium nitrite 2% cream is suitable for additional clinical trials in adults with sickle cell anaemia to promote healing of leg ulcers. FUNDING: National Heart, Lung and Blood Institute Division of Intramural Research (National Institutes of Health).