Joseph DeSimone

Haemoglobinuria is associated with chronic kidney disease and its progression in patients with sickle cell anaemia.

To evaluate the association between haemoglobinuria and chronic kidney disease (CKD) in sickle cell anaemia (SCA), we analysed 356 adult haemoglobin SS or Sβo thalassaemia patients from the University of Illinois at Chicago (UIC) and 439 from the multi‐centre Walk‐Treatment of Pulmonary Hypertension and Sickle Cell Disease with Sildenafil Therapy (Walk‐PHaSST) cohort. CKD was classified according to National Kidney Foundation Kidney Disease Outcomes Quality Initiatives guidelines. Haemoglobinuria, defined as positive haem on urine dipstick with absent red blood cells on microscopy, was confirmed by enzyme‐linked immunosorbent assay in a subset of patients. The prevalence of CKD was 58% in the UIC cohort and 54% in the Walk‐PHaSST cohort, and haemoglobinuria was observed in 36% and 20% of the patients, respectively. Pathway analysis in both cohorts indicated an independent association of lactate dehydrogenase with haemoglobinuria and, in turn, independent associations of haemoglobinuria and age with CKD (P < 0·0001). After a median of 32 months of follow‐up in the UIC cohort, haemoglobinuria was associated with progression of CKD [halving of estimated glomerular filtration rate or requirement for dialysis; Hazard ratio (HR) 13·9, 95% confidence interval (CI) 1·7–113·2, P = 0·0012] and increasing albuminuria (HR 3·1, 95% CI: 1·3–7·7; logrank P = 0·0035). In conclusion haemoglobinuria is common in SCA and is associated with CKD, consistent with a role for intravascular haemolysis in the pathogenesis of renal dysfunction in SCA.

Clinical effectiveness of decitabine in severe sickle cell disease

precursors to the subsequent DLBCLs these patients develop. Several clonal B-cell populations may develop during the course of the disease, and these may be transient, with many disappearing subsequently. Hence, when a proportion of AITL patients subsequently develop a full-blown DLBCL or EBVcHL, one may or may not be able to establish clonal ancestry to ‘precursor’ cells seen in an earlier biopsy.

The LSD1 inhibitor RN-1 induces fetal hemoglobin synthesis and reduces disease pathology in sickle cell mice.

Inhibition of lysine-specific demethylase 1 (LSD1) has been shown to induce fetal hemoglobin (HbF) levels in cultured human erythroid cells in vitro. Here we report the in vivo effects of LSD1 inactivation by a selective and more potent inhibitor, RN-1, in a sickle cell disease (SCD) mouse model. Compared with untreated animals, RN-1 administration leads to induced HbF synthesis and to increased frequencies of HbF-positive cells and mature erythrocytes, as well as fewer reticulocytes and sickle cells, in the peripheral blood of treated SCD mice. In keeping with these observations, histologic analyses of the liver and spleen of treated SCD mice verified that they do not exhibit the necrotic lesions that are usually associated with SCD. These data indicate that RN-1 can effectively induce HbF levels in red blood cells and reduce disease pathology in SCD mice, and may therefore offer new therapeutic possibilities for treating SCD.

Detection of HB-papio B, A silent mutation of the baboon β chain, by high performance liquid chromatography. Improved procedures for the separation of globin chains by HPLC

A silent mutant of the β chain of the baboon (Papio cynocephalus) was detected by high performance liquid chromatography of the globin chains. The substitution is β13 Ala → Thr. The inclusion of nonylamine in the developers for HPLC results not only in an improved separation of baboon globin chains but also in the complete resolution of the human Gγ, Aγ, and AγT chains and some common human variant β chains.

DNA hypo-methylating agents and sickle cell disease.

Fetal haemoglobin (HbF, α2) decreases polymerization of sickle haemoglobin (HbS) and high levels correlate with decreased morbidity and mortality in sickle cell disease (SSD). Therefore, a therapeutic goal in SSD is the pharmacologic reactivation of HbF. Silencing of the globin (HbF) gene is associated with DNA methylation. The cytosine analogues 5‐azacytidine and 5‐aza‐2′‐deoxycytidine (decitabine) hypomethylate DNA by inhibiting DNA methyl‐transferase. In clinical trials, 5‐azacytidine and decitabine have demonstrated the greatest efficacy in HbF reactivation. Clinical development of these drugs has been delayed by concerns regarding the carcinogenic potential of 5‐azacytidine. Furthermore, controversy regarding DNA hypomethylation versus more generic cytotoxic effects as the mechanism of action suggested that other cytotoxic/cytostatic agents might be as effective. Additional preclinical data and clinical studies of decitabine have tempered many safety concerns and have confirmed that DNA hypomethylation is the mechanism of action. Pharmacologic reactivation of HbF through DNA hypomethylation holds promise as an effective disease modifying intervention for patients with SSD. Larger studies are required to confirm safety and effectiveness with chronic use.

Pain, coping and health care utilization in younger and older adults with sickle cell disease.

Sickle cell disease is characterized by acute pain crises. Pain, chronic medical problems, utilization and coping were compared in younger vs older patients using questionnaires and medical record review. Groups reported similar pain intensity and medical conditions. The pattern of utilization differed such that older patients attended outpatient clinic, and younger patients went to the Emergency Department. Younger patients were more likely to cope by ignoring pain, or by using heat, cold or massage. Older patients were more likely to pray and hope. We conclude that age plays an important role in the utilization and coping of sickle cell patients.

Effects of hydroxurea, stem cell factor, and erythropoietin in combination on fetal hemoglobin in the baboon.

OBJECTIVE Augmentation of the level of fetal hemoglobin (HbF) is considered therapeutic for patients with sickle cell disease. The objective of this study was to determine the effect of treatment with a combination of erythropoietin (Epo), stem cell factor (SCF), and hydroxyurea (HU) on HbF levels. MATERIALS AND METHODS The effect of treatment with a combination of Epo, SCF, and HU on HbF, F-cell numbers, and globin chain synthesis was evaluated in a baboon model. RESULTS Treatment with a combination of SCF+Epo resulted in a two-fold increase in HbF, F-cells, and F-reticulocytes compared to Epo alone. The combination of SCF+Epo+HU resulted in an additional two-fold increase in HbF, whereas F-cells and F-reticulocytes increased only 25% compared to the SCF+Epo regimen. Measurement of differential globin chain synthesis indicated that the SCF+Epo+HU treatment also increased the I gamma/V gamma (homologous to human G gamma and A gamma) synthetic ratio toward the fetal ratio. CONCLUSIONS HU can effectively augment growth factor-induced HbF synthesis in vivo. Because I gamma/V gamma ratios are unaffected by erythropoietic stress and similar increases in this ratio have only been observed following administration of 5-azacytidine, we suggest that these two agents may share a common mechanism of action involving the recruitment of a similar target cell population to terminal erythroid differentiation.

Transcriptional activation of the γ-globin gene in baboons treated with decitabine and in cultured erythroid progenitor cells involves different mechanisms

OBJECTIVE To investigate the mechanism(s) responsible for increased gamma-globin expression in vivo in decitabine-treated baboons and in vitro in cultured erythroid progenitor cells (EPC) from adult baboon bone marrow (BM). MATERIALS AND METHODS Fetal liver, adult BM erythroid cells pre- and post-decitabine, and cultured EPCs were analyzed for distribution of RNA polymerase II, histone acetylation, and histone H3 (lys4) trimethyl throughout the gamma-globin gene complex by chromatin immunoprecipitation. DNA methylation of the gamma-globin promoter was determined by bisulfite sequencing. Expression of the baboon Igamma- and Vgamma-globin chains was determined by high performance liquid chromatography (HPLC). Expression of BCL11A, a recently identified repressor of gamma-globin expression, was analyzed by Western blot. RESULTS Increased gamma-globin expression in decitabine-treated baboons and cultured EPC correlated with increased levels of RNA polymerase II, histone acetylation, and histone H3 (lys4) trimethyl associated with the gamma-globin gene consistent with a transcriptional activation mechanism. Cultured EPC expressed the Igamma- and Vgamma-globin chains in a pattern characteristic of fetal development. The level of DNA methylation of the gamma-globin gene promoter in EPC cultures was similar to BM erythroid cells from normal adult baboons. Different BCL11A isoforms were observed in BM erythroid cells and cultured EPC. CONCLUSION The mechanism responsible for increased gamma-globin expression in cultured EPC was unexpectedly not associated with increased DNA hypomethylation of the gamma-globin gene promoter compared to normal BM erythroid cells, in contrast to BM erythroid cells of decitabine-treated baboons. Rather, increased fetal hemoglobin in EPC cultures was associated with a fetal Igamma/Vgamma chain ratio and a difference in the size of the BCL11A protein compared to normal BM erythroid cells.

RN-1, a potent and selective lysine-specific demethylase 1 inhibitor, increases γ-globin expression, F reticulocytes, and F cells in a sickle cell disease mouse model

Increased levels of fetal hemoglobin are associated with decreased symptoms and increased lifespan in patients with sickle cell disease (SCD). Hydroxyurea, the only drug currently approved for SCD, is not effective in a large fraction of patients, and therefore, new agents are urgently needed. Recently it was found that lysine demethylase 1, an enzyme that removes monomethyl and dimethyl residues from the lysine 4 residue of histone H3, is a repressor of γ-globin gene expression. In this article, we have compared the ability of tranylcypromine (TCP) and a more potent TCP derivative, RN-1, to increase γ-globin expression in cultured baboon erythroid progenitor cells and in the SCD mouse model. The results indicate that the ability of RN-1 to induce F cells and γ-globin mRNA in SCD mice is similar to that of decitabine, the most powerful fetal hemoglobin-inducing drug known, and greater than that of either TCP or hydroxyurea. We conclude that RN-1 and other lysine demethylase 1 inhibitors may be promising new γ-globin-inducing agents for the treatment of SCD that warrant further studies in other preclinical models, such as nonhuman primates.

siDNMT1 increases γ-globin expression in chemical inducer of dimerization (CID)−dependent mouse βYAC bone marrow cells and in baboon erythroid progenitor cell cultures

OBJECTIVE These studies were performed to test the hypothesis that DNMT1 is required for maintenance of DNA methylation and repression of the γ-globin gene in adult-stage erythroid cells. MATERIALS AND METHODS DNMT1 levels were reduced by nucleofection of small interfering RNA targeting DNMT1 in chemical inducer of dimerization-dependent multipotential mouse bone marrow cells containing the human β-globin gene locus in the context of a yeast artificial chromosome and in primary cultures of erythroid progenitor cells derived from CD34(+) baboon bone marrow cells. The effect of reduced DNMT1 levels on globin gene expression was measured by real-time polymerase chain reaction and the effect on globin chain synthesis in primary erythroid progenitor cell cultures was determined by biosynthetic radiolabeling of globin chains followed by high-performance liquid chromatography analysis. The effect on DNA methylation was determined by bisulfite sequence analysis. RESULTS Reduced DNMT1 levels in cells treated with siDNMT1 were associated with increased expression of γ-globin messenger RNA, an increased γ/γ+β chain ratio in cultured erythroid progenitors, and decreased DNA methylation of the γ-globin promoter. Similar effects were observed in cells treated with decitabine, a pharmacological inhibitor of DNA methyltransferase inhibitor. CONCLUSIONS DNMT1 is required to maintain DNA methylation of the γ-globin gene promoter and repress γ-globin gene expression in adult-stage erythroid cells.