Search

Background/Purpose: In craniosynostosis, diagnostic imaging is crucial for identifying skull abnormalities, planning for surgical correction, and post-operative and follow-up evaluations. However, classifying postoperative corrected skull morphology is a highly subjective task for all craniofacial surgeons. Initially, many surgeons utilized the Whittaker categorization test as the post-operative outcome measure, which is based on the subjective esthetic appearance as determined by the surgeon, patient, and patient’s family. While the advancement of 3D imaging and anthropometric measurements techniques has allowed various research groups to produce normative skull models to serve as treatment guides, it is still difficult to determine the objective morphological standard. The purpose of this study is to review the current standards for classifying normal skull morphology. Methods: This paper is a review of the current surgical standards of normal cranial morphology. A systematic literature review following PRISMA guidelines was performed; all published studies over a 40-year period from 1980 to 2020 were reviewed that assessed cranial morphology of healthy pediatric samples. Results: A review of the strategies utilized by some of the major medical device manufacturers in craniosynostosis correction surgery is presented. Many of these companies have progressed to using 3D normative skull construction and 3D surface analysis to measure cranial morphology. However, many variations remain among industry standards. Conclusion: Normative head shapes and cohort data matched to similar population groups have a crucial function in the diagnosis, pre-operative planning, and post-operative evaluations in craniofacial surgery. However, industry standards for “normal head shape” utilized in surgical analysis and planning remain diverse and can be somewhat opaque to the craniofacial community. Despite the advancement of 3D imaging techniques, most reported outcomes following craniofacial surgical interventions are still subjectively defined.

To characterize how community mobility patterns across the United States correlate with local changes in retina visits during the pandemic.Outpatient retina clinic visits were linked to population mobility by combining multiple public data sets, including the Google Community Mobility Reports and data from the Centers for Disease Control and Infection. Percentage change from baseline in daily-average number of retina visits by county and mobility were measured by county.A total of 2,159,689 patient visits were examined across 332 counties. Daily-average retina visits decreased by 7.0%, 19.0%, 5.0%, and 4.0% from Quarter 1 to 4 of 2020. This decrease was negatively correlated with increased incident of COVID-19 deaths for Quarters 1 to 3 (r = -0.13, r = -0.16, and r = -0.15, respectively, P0.001) and increased incident cases for Quarters 1 and 2 (r = -0.18, r = -0.13, respectively, P0.001). Daily-average retina visits relative to baseline were significantly lower for metropolitan counties in Quarters 1 and 2, compared with urban and rural (P0.001). The decline in retina visits had greatest association with decline in workplace visits in Quarters 1 to 3 (r = 0.27, r = 0.09, r = 0.12, respectively, P0.001 for all).This study provides insight into how regional mobility patterns may help to explain and predict patient behaviors and retina outpatient visit responses during the COVID-19 pandemic.

Surgery is an important treatment for cancer; however, local recurrence following macroscopically-complete resection is common and a significant cause of morbidity and mortality. Systemic chemotherapy is often employed as an adjuvant therapy to prevent recurrence of residual disease, but has limited efficacy due to poor penetration and dose-limiting off-target toxicities. Selective delivery of chemotherapeutics to the surgical bed may eliminate residual tumor cells while avoiding systemic toxicity. While this is challenging for traditional drug delivery technologies, we utilized advances in 3D printing and drug delivery science to engineer a drug-loaded arrowhead array device (AAD) to overcome these challenges. We demonstrated that such a device can be designed, fabricated, and implanted intraoperatively and provide extended release of chemotherapeutics directly to the resection area. Using paclitaxel and cisplatin as model drugs and murine models of cancer, we showed AADs significantly decreased local recurrence post-surgery and improved survival. We further demonstrated the potential for fabricating personalized AADs for intraoperative application in the clinical setting.

Brachytherapy, which is the placement of radioactive seeds directly into tissue such as the prostate, is an important curative treatment for prostate cancer. By delivering a high dose of radiation from within the prostate gland, brachytherapy is an effective method of killing prostate cancer cells while limiting radiation dose to normal tissue. The main shortcomings of this treatment are: less efficacy against high grade tumor cells, acute urinary retention, and sub-acute urinary frequency and urgency. One strategy to improve brachytherapy is to incorporate therapeutics into brachytherapy. Drugs, such as docetaxel, can improve therapeutic efficacy, and dexamethasone is known to decrease urinary side effects. However, both therapeutics have high systemic side effects. To overcome this challenge, we hypothesized that we can incorporate therapeutics into the inert polymer spacers that are used to correctly space brachytherapy seeds during brachytherapy to enable local drug delivery. To accomplish this, we engineered 3D printed drug-loaded brachytherapy spacers using continuous liquid interface production (CLIP) with different surface patterns to control drug release. These devices have the same physical size as existing spacers, allowing them to easily replace commercial spacers. We examined these drug-loaded spacers using docetaxel and dexamethasone as model drugs in a murine model of prostate cancer. We found that drug-loaded spacers led to higher therapeutic efficacy for brachytherapy, and there was no discernable systemic toxicity from the drug-loaded spacers. STATEMENT OF SIGNIFICANCE: There has been high interest in the application of 3D printing to engineer novel medical devices. However, such efforts have been limited by the lack of technologies that can fabricate devices suitable for real world medical applications. In this study, we demonstrate a unique application for 3D printing to enhance brachytherapy for cancer treatment. We engineered drug-loaded brachytherapy spacers that can be fabricated using Continuous Liquid Interface Production (CLIP) 3D printing, allowing tunable printing of drug-loaded devices, and implanted intraoperatively with brachytherapy seeds. In combined chemotherapy and brachytherapy we are able to achieve greater therapeutic efficacy through local drug delivery and without systemic toxicities. We believe our work will facilitate further investigation in medical applications of 3D printing.

A male patient in his thirties presented to the hospital with meningismus and altered mental status. Cerebrospinal fluid (CSF) analysis confirmed meningitis, but no microorganisms were identified. Head imaging (MRI) revealed multiple brain abscesses. Surgically-collected brain specimens revealed Gram-positive cocci and subsequently grew Streptococcus intermedius. MRI of the thoracic spine subsequently also identified an intramedullary spinal abscess. While brain abscesses and spinal abscesses caused by S. intermedius have been previously reported, the extent of disseminated disease in the patient was noteworthy.

Human hemoglobin switching describes the highly regulated, sequential expression of the five β-like globin genes (HBE, HBG2, HBG1, HBD and HBB) of the human β-globin gene complex. The sequential activation of these β or β-like globin genes during human development from early embryonic through late fetal (‘adult’) stages, and during erythroid maturation, occurs in an order corresponding to their 5’ to 3’ location on chromosome 11. The β-hemoglobinopathies are the most common inherited diseases in humanity, and are diseases of mutated HBB or its altered regulation. Since the other β-like globin genes can potentially substitute for defective HBB, much translational research is directed toward understanding and manipulating sequential activation at the human β-globin gene complex to treat β-hemoglobinopathies. Non-human primates provide a vital contribution to such efforts because of their recapitulation of the developmental/maturational switch in hemoglobin production as observed in humans (mice do not model this switch). Valuable insights into druggable epigenetic forces that mediate the switch have been thereby gained. We review important lessons learned in non-human primates, complemented by other studies, and suggest rational next steps.

Sickle cell disease (SCD), an inherited blood disorder caused by a point mutation that renders hemoglobin susceptible to polymerization when deoxygenated, affects millions of people worldwide. Manifestations of SCD include chronic hemolytic anemia, inflammation, painful vaso-occlusive crises, multisystem organ damage, and reduced life expectancy. Part of SCD pathophysiology is the excessive formation of intracellular reactive oxygen species (ROS) in SCD red blood cells (RBCs), which accelerates their hemolysis. Normal RBC precursors eliminate their mitochondria during the terminal differentiation process. Strikingly, we observed an increased percentage of RBCs retaining mitochondria in SCD patient blood samples compared with healthy individuals. In addition, using an experimental SCD mouse model, we demonstrate that excessive levels of ROS in SCD are associated with this abnormal mitochondrial retention. Interestingly, the LSD1 inhibitor, RN-1, and the mitophagy-inducing agent mammalian target of rapamycin (mTOR) inhibitor, sirolimus, increased RBC lifespan and reduced ROS accumulation in parallel with reducing mitochondria-retaining RBCs in the SCD mouse model. Furthermore, gene expression analysis of SCD mice treated with RN-1 showed increased expression of mitophagy genes. Our findings suggest that reduction of mitochondria-retaining RBCs may provide a new therapeutic approach to preventing excessive ROS in SCD.

Pain is one of the most challenging and stressful conditions to patients with sickle cell disease (SCD) and their clinicians. Patients with SCD start experiencing pain as early as three months old and continue having it throughout their lives. Although many aspects of the disease are well understood, little progress has been made in understanding and treating pain in SCD. This study aimed to investigate the functional involvement of Ca2+/calmodulin-dependent protein kinase II (CaMKIIα) in the persistent and refractory pain associated with SCD. We found non-evoked ongoing pain as well as evoked hypersensitivity to mechanical and thermal stimuli were present in Berkeley sickle cell transgenic mice (BERK mice), but not non-sickle control littermates. Prominent activation of CaMKIIα was observed in the dorsal root ganglia and spinal cord dorsal horn region of BERK mice. Intrathecal administration of KN93, a selective inhibitor of CaMKII, significantly attenuated mechanical allodynia and heat hyperalgesia in BERK mice. Meanwhile, spinal inhibition of CaMKII elicited conditioned place preference in the BERK mice, indicating the contribution of CaMKII in the ongoing spontaneous pain of SCD. We further targeted CaMKIIα by siRNA knockdown. Both evoked pain and ongoing spontaneous pain were effectively attenuated in BERK mice. These findings elucidated, for the first time, an essential role of CaMKIIα as a cellular mechanism in the development and maintenance of spontaneous and evoked pain in SCD, which can potentially offer new targets for pharmacological intervention of pain in SCD.

Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease (SCD). More effective strategies to increase HbF are needed because the current standard of care, hydroxyurea, is not effective in a significant proportion of patients. Treatment of the millions of patients projected worldwide would best be accomplished with an orally administered drug therapy that increased HbF. LSD1 is a component of corepressor complexes that repress γ-globin gene expression and are a therapeutic target for HbF reactivation. We have shown that subcutaneous administration of RN-1, a pharmacological LSD1 inhibitor, increased γ-globin expression in SCD mice and baboons, which are widely acknowledged as the best animal model in which to test the activity of HbF-inducing drugs. The objective of this investigation was to test the effect of oral administration of a new LSD1 inhibitor, ORY-3001. Oral administration of ORY-3001 to SCD mice (n = 3 groups) increased γ-globin expression, Fetal Hemoglobin (HbF)-containing (F) cells, and F reticulocytes (retics). In normal baboons (n = 7 experiments) treated with ORY-3001, increased F retics, γ-globin chain synthesis, and γ-globin mRNA were observed. Experiments in anemic baboons (n = 2) showed that ORY-3001 increased F retics (PA8695, predose = 24%, postdose = 66.8%; PA8698: predose = 13%, postdose = 93.6%), γ-globin chain synthesis (PA8695: predose = 0.07 γ/γ+β, postdose = 0.20 γ/γ+β; PA8698: predose = 0.02 γ/γ+β, postdose = 0.44 γ/γ+β), and γ-globin mRNA (PA8695: predose = 0.06 γ/γ+β, postdose = 0.18 γ/γ+β; PA8698: predose = 0.03 γ/γ+β, postdose = 0.33 γ/γ+β). We conclude that oral administration of ORY-3001 increases F retics, γ-globin chain synthesis, and γ-globin mRNA in baboons and SCD mice, supporting further efforts toward the development of this drug for SCD therapy.

Background Sickle cell disease (SCD), a congenital hemolytic anemia that exacts terrible global morbidity and mortality, is driven by polymerization of mutated sickle hemoglobin (HbS) in red blood cells (RBCs). Fetal hemoglobin (HbF) interferes with this polymerization, but HbF is epigenetically silenced from infancy onward by DNA methyltransferase 1 (DNMT1). Methods and findings To pharmacologically re-induce HbF by DNMT1 inhibition, this first-in-human clinical trial (NCT01685515) combined 2 small molecules—decitabine to deplete DNMT1 and tetrahydrouridine (THU) to inhibit cytidine deaminase (CDA), the enzyme that otherwise rapidly deaminates/inactivates decitabine, severely limiting its half-life, tissue distribution, and oral bioavailability. Oral decitabine doses, administered after oral THU 10 mg/kg, were escalated from a very low starting level (0.01, 0.02, 0.04, 0.08, or 0.16 mg/kg) to identify minimal doses active in depleting DNMT1 without cytotoxicity. Patients were SCD adults at risk of early death despite standard-of-care, randomized 3:2 to THU–decitabine versus placebo in 5 cohorts of 5 patients treated 2X/week for 8 weeks, with 4 weeks of follow-up. The primary endpoint was ≥ grade 3 non-hematologic toxicity. This endpoint was not triggered, and adverse events (AEs) were not significantly different in THU-decitabine—versus placebo-treated patients. At the decitabine 0.16 mg/kg dose, plasma concentrations peaked at approximately 50 nM (Cmax) and remained elevated for several hours. This dose decreased DNMT1 protein in peripheral blood mononuclear cells by >75% and repetitive element CpG methylation by approximately 10%, and increased HbF by 4%–9% (P < 0.001), doubling fetal hemoglobin-enriched red blood cells (F-cells) up to approximately 80% of total RBCs. Total hemoglobin increased by 1.2–1.9 g/dL (P = 0.01) as reticulocytes simultaneously decreased; that is, better quality and efficiency of HbF-enriched erythropoiesis elevated hemoglobin using fewer reticulocytes. Also indicating better RBC quality, biomarkers of hemolysis, thrombophilia, and inflammation (LDH, bilirubin, D-dimer, C-reactive protein [CRP]) improved. As expected with non-cytotoxic DNMT1-depletion, platelets increased and neutrophils concurrently decreased, but not to an extent requiring treatment holds. As an early phase study, limitations include small patient numbers at each dose level and narrow capacity to evaluate clinical benefits. Conclusion Administration of oral THU-decitabine to patients with SCD was safe in this study and, by targeting DNMT1, upregulated HbF in RBCs. Further studies should investigate clinical benefits and potential harms not identified to date. Trial registration ClinicalTrials.gov, NCT01685515, In a clinical trial, Yogen Saunthararajah and colleagues target DNA methyltransferase with small molecules to reactivate fetal hemoglobin which inhibits polymerisation of mutated sickle cell hemoglobin., Author summary Why was this study done? Sickle cell disease, one of the most frequent inherited diseases in humans, is driven by aggregation and precipitation of less soluble sickle cell hemoglobin in red blood cells—this destroys red blood cells and blocks blood vessels, causing organ damage, suffering, and early death. Fetal hemoglobin, expressed in red blood cells until infancy, mixes with sickle cell hemoglobin and promotes its dissolution. Individuals who naturally express high levels of fetal hemoglobin beyond infancy thus receive some protection from sickle complications. To mimic this natural state using drugs, one relevant observation was that fetal hemoglobin is increased during recovery of bone marrow from extreme stress. This led to evaluation and approval of the cytotoxic (cell killing) drug hydroxyurea, the only drug approved to treat sickle cell disease. This approach to fetal hemoglobin induction, however, is limited in potency and sustainability. Our goal was to increase fetal hemoglobin by inhibiting an enzyme, DNA methyltransferase 1 (DNMT1), involved in shutting off the fetal hemoglobin gene from infancy onward. What did the researchers do and find? We used the drug decitabine to inhibit DNMT1. However, decitabine is very rapidly inactivated in the body by another enzyme, cytidine deaminase (CDA). We therefore combined it with a CDA inhibitor, tetrahydrouridine (THU). Oral THU and decitabine were given to patients with severe, symptomatic sickle cell disease who had not derived benefit from the standard treatment of hydroxyurea. Oral THU-decitabine was safe and well-tolerated by the patients. Measurement of decitabine levels in the blood confirmed that THU enabled oral absorption of very small doses of oral decitabine. Measurements of DNMT1 protein levels and DNA methylation confirmed that this decitabine exposure was sufficient to deplete DNMT1 from cells. DNMT1 depletion by decitabine produced large increases in fetal hemoglobin and increased numbers of healthy red blood cells. What do these findings mean? Oral THU-decitabine to deplete DNMT1 safely increased fetal hemoglobin and numbers of healthy red blood cells. Since this was a first-in-human study, only a small number of patients were treated and for a fairly short time. Further clinical studies are therefore needed.

Background Varicella-zoster virus (VZV) infection of the central nervous system (CNS) is relatively uncommon. Diagnostic tests historically utilized culture, serologies, and targeted PCR methods. In April 2016, our institution began using the BioFire FilmArray meningitis/encephalitis (BFME) panel for cerebrospinal fluid (CSF) specimen analysis. We hypothesized that the diagnosis of VZV CNS infection increased at our institution with the implementation of this diagnostic panel. Methods We conducted chart reviews of patients from 2 time periods. In the first period, April 2013–March 2016, BFME was not available for CSF analysis (pre-BFME period). We reviewed all positive CSF VZV PCR results during this period. Medical charts of these patients were reviewed for epidemiology, clinical presentation, treatment course, and outcome. In the second period, April 2016–March 2018, BFME was performed on all CSF specimens obtained by lumbar puncture (BFME period). Patients with a positive VZV result on BFME underwent similar chart review. Results In the 3-year pre-BFME period, 292 VZV PCR tests were performed. Six patients were diagnosed with VZV CNS infection; median age 61 years. Five of the 6 patients (83%) had cutaneous zoster. All 6 patients received antiviral therapy. Five of the 6 patients clinically improved; 1 patient with VZV encephalitis died. In the 2-year BFME period, 1,113 CSF samples were evaluated, and 18 of these were positive for VZV (1.6%); median age 55 years. Only 7 of the 18 (39%) had cutaneous zoster at the time of hospitalization. All 18 received antiviral therapy with clinical improvement. Conclusion Prior to implementation of the BFME panel at our institution, VZV CNS infection was rarely diagnosed. Diagnosis at that time relied on physicians’ requests for a targeted CSF VZV PCR. The majority of the patients during that period had a concurrent zoster rash. In a shorter period utilizing syndromic testing (BFME) on CSF specimens, we diagnosed 3 times as many cases of VZV CNS disease. Only a minority of these patients presented with a concurrent zoster rash. The use of syndromic testing of CSF will likely identify more cases of VZV CNS disease. Disclosures All authors: No reported disclosures.

To evaluate the association between haemoglobinuria and chronic kidney disease (CKD) in sickle cell anaemia (SCA), we analysed 356 adult haemoglobin SS or Sβ(o) 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.

To the editor: Elevated levels of fetal hemoglobin (HbF; α2γ2) lessen the severity of symptoms and increase the life span of patients with sickle cell disease (SCD).[1][1],[2][2] Hydroxyurea, the only approved drug for the treatment of SCD, is ineffective in a large proportion of patients, and

Summary In sickle cell disease (SCD), vigorous reticulocytosis is required to partially compensate for chronic hemolytic anaemia. Consequently, early renal damage, insufficient to cause azotemia but sufficient to cause erythropoietin deficiency and chronic relative reticulocytopenia (chRR), could have severe clinical consequences. chRR was defined as reticulocytes

The HbF to HbA developmental globin switch is recapitulated during adult erythroid differentiation and involves the acquisition of repressive epigenetic marks at the γ-globin promoter catalyzed by "druggable" enzymes such as histone deacetylases (HDACs), DNA methyltransferase (DNMT1), LSD1 (KDM1A), and G9A (EHMT2) that are functional components of multiprotein co-repressors recruited to the γ-globin gene promoter by the trans-acting repressors TR2/TR4, BCL11A, and ZBTB7A. A logical approach to increase HbF that has been successfully pursued by our laboratory is to intervene with the epigenetic repression mechanism that executes the switch from HbF to HbA using pharmacological inhibitors of these enzymes. Simian primates such as the baboon are widely acknowledged as the best animal models for testing the ability of new drugs to increase γ-globin expression because results in baboons are predictive of effects in man due to conservation of the structure and developmental stage-specific regulation of the β-like globin genes in simian primates.Our laboratory has developed and utilized an in vivo baboon model for over thirty years to investigate globin gene regulation and the ability of pharmacological inhibitors of enzymes that catalyze repressive epigenetic modifications to increase HbF, generating results with DNMT inhibitors that were extended and confirmed in a number of clinical studies in patients with SCD and β-thalassemia. Following studies that identified LSD1 as an additional therapeutic target (Shi et al, Nat Med 19:291, 2013), we showed that the LSD1 inhibitor RN-1 dramatically increased HbF, F cells and F retics in baboons (Rivers et al, Haematol 101:698, 2015) and that these effects were sustained upon long-term treatment (>265d; Ibanez et al, Blood 129:260, 2017). ChIP analysis showed increased levels of Histone H3 di and tri-methyl K4 at the γ-globin gene, consistent with LSD1 inhibition. This current investigation seeks to identify the stage in the erythroid differentiation pathway targeted by LSD1 inhibitors to increase HbF and also to characterize additional effects of these drugs on erythroid differentiation that potentially impact mechanism of action. Subpopulations of bone marrow (BM) cells highly enriched in BFUe (CD105+CD34+CD117+bRBC-), CFUe (CD105+CD34+CD117+bRBC+), proerythroblasts (CD105+CD34-CD117+bRBC+) were purified from RN-1 treated (0.25mg/kg/d; 3d) and untreated baboons by immunomagnetic column separation combined with FACS. RT-PCR analysis of γ-globin expression within purified BM erythroid subpopulations showed that relative levels of γ-globin mRNA (γ/γ+β) in untreated baboons were 4 fold higher in BFUe than in CFUe and 11 fold higher in BFUe than in proerythroblasts consistent with repression of the γ-globin gene during the BFUe to CFUe transition. RN-1 treatment increased relative γ-globin expression (γ/γ+β) 4 fold in BFUe, 25 fold in CFUe, and >100 fold in proerythroblasts compared to untreated controls and thus partially prevented repression of the γ-globin expression at early stages of differentiation. Additional effects of the LSD1 inhibitor RN-1 on erythroid differentiation were measured by flow cytometry in baboon BM aspirates following RN-1 administration. RN-1 treatment (0.25mg/kg/d; 3d) increased the proportion of CD105+CD117+bRBC+BM cells (proerythroblasts) (24.2% pre-treatment; 48.4% post-treatment; p265d) identified a common set of 41 genes whose expression was increased while the expression of 6 genes was decreased. RT-PCR assays confirmed increased expression of γ-globin, GATA-2, GFi-1B, DAP, IFI6, and Lyn in proerythroblasts of RN-1-treated baboons. We conclude that the LSD1 inhibitor RN-1 administered to baboons 1) restrains erythroid differentiation by modulating the expression of genes such as GATA-1 and GFi-1B that regulate erythroid differentiation, and 2) targets an early event responsible for γ-globin repression to increase γ-globin expression. Disclosures Saunthararajah: Novo Nordisk, A/S: Patents & Royalties; EpiDestiny, LLC: Patents & Royalties. Lavelle:Global Blood therapeutics: Research Funding.

Pain is one of the most dreadful symptoms in sickle cell disease (SCD) and is often refractory to currently available analgesics. Besides acute painful vaso-occlusive crises, SCD is also accompanied by intractable chronic pain. This persistent, and often unrelieved, pain starts early in childhood and continues throughout life. The neurobiological mechanisms of chronic pain in SCD remain unclear, which markedly limits effective pain management and the quality of life in patients with SCD. Taking advantage of two humanized mouse models of SCD, this study aimed to investigate protein phosphorylation mechanisms for chronic pain in SCD. We characterized pain in two transgenic mice models of SCD that exclusively express human alleles encoding normal α- and sickle β-globin. Berkeley SCD mice (BERK mice) and Townes' SCD mice (TOW mice) exhibited ongoing spontaneous pain behavior and increased sensitivity to evoked pain stimuli compared with littermate control mice expressing normal human hemoglobins. To investigate the underlying protein phosphorylation mechanisms of chronic pain in SCD, we examined PKC isoform mediated nociceptive signaling. Prominent activation of multiple PKC isoforms were observed in the superficial laminae of the spinal cord dorsal horn in BERK and TOW mice. Functional inhibition and silencing of specific PKC isoforms attenuated spontaneous pain, mechanical allodynia, and heat hyperalgesia in both transgenic SCD mice. Furthermore, employing hematopoietic stem cell transplantation approach, we were able to generate a sickle cell anemia model in PKC-null mice, allowing us to specifically target neuronal PKC in SCD. Neither spontaneous pain nor evoked pain was detected in the mice lacking specific PKC isoform despite the full establishment of SCD phenotypes. In summary, this study is the first to identify the presence of ongoing spontaneous pain in preclinical sickle cell models, which closely mimic the most prevalent manifestation in patients with SCD. Moreover, we found that spinal PKC is a critical mechanism for the generation and maintenance of ongoing and evoked pain in SCD. These findings offer insights into sickle cell pain mechanisms, which may become a potential target for pharmacological interventions. Disclosures No relevant conflicts of interest to declare.

Objective To determine whether the difference in γ-globin gene promoter methylation in terminal erythroblasts at the fetal and adult stages of development is a result of fetal stage-specific demethylation or adult stage-specific de novo methylation during erythropoiesis. Materials and Methods Fetal liver- (FL, n=2) and adult bone marrow- (ABM, n=3) derived hematopoietic stem/progenitor cells and mature erythroblasts were purified by passage through a Miltenyi Magnetic Column followed by fluorescein-activated cell sorting (FACS) into subpopulations, defined by expression of CD34 and CD36 antigens. CD34 + CD36 − , CD34 + CD36 + , and CD34 − CD36 + subpopulations were purified by FACS and their degree of differentiation verified using the colony-forming cell assay. The methylation pattern of 5 CpG sites in the γ-globin promoter region of these purified cell populations was determined using bisulfite sequencing. Results The γ-globin promoter was highly methylated in the earliest stage of hematopoietic stem progenitor cells (CD34 + CD36 − ) and methylation progressively decreased as erythroid differentiation progressed in FL and appears so in ABM as well. Conclusions These data support a model in which differences in the methylation pattern of the γ-globin gene in differentiating erythroblasts at different stages of development is the result of fetal stage-specific demethylation associated with transcriptional activation, rather than de novo methylation in the adults. The difference in the extent of γ-globin gene demethylation in FL and ABM is correlated with the difference in γ-globin expression at these developmental stages.

The silencing of tumor suppressor genes associated with increased DNA methylation of the promoter regions is a frequent observation in many forms of cancer. Reactivation of these genes using pharmacological inhibitors of DNA methyltransferase such as 5-aza-2'-deoxycytidine (decitabine) is a worthwhile therapeutic goal. The effectiveness and tolerability of low-dose intravenous and subcutaneous decitabine regimens to demethylate and reactivate expression of the methylated gamma-globin gene in baboons and in patients with sickle cell disease led to successful trials of low-dose regimens of this drug in patients with myelodysplastic syndrome. Since these low-dose regimens are well-tolerated with minimal toxicity, they are suitable for chronic dosing to maintain promoter hypomethylation and expression of target genes. The development of an orally administered therapy using DNA methyltransferase inhibitors would facilitate such chronic approaches to therapy. We tested the ability of decitabine and a new salt derivative, decitabine mesylate, to reactivate the methylated gamma-globin gene in baboons when administered orally. Our results demonstrate that oral administration of these drugs at doses 17-34 times optimal subcutaneous doses of decitabine reactivates fetal hemoglobin, demethylates the epsilon- and gamma-globin gene promoters, and increases histone acetylation of these promoters in baboons (Papio anubis).

Pain is a life-long symptom in sickle cell disease (SCD) and a predictor of disease progression and mortality, but little is known about its molecular mechanisms. Here, we characterized pain in a targeted knockin mouse model of SCD (TOW mouse) that exclusively expresses human alleles encoding normal α- and sickle β-globin. TOW mice exhibited ongoing spontaneous pain behavior and increased sensitivity to evoked pain compared with littermate control mice expressing normal human hemoglobins. PKCδ activation was elevated in the superficial laminae of the spinal cord dorsal horn in TOW mice, specifically in GABAergic inhibitory neurons. Functional inhibition and neuron-specific silencing of PKCδ attenuated spontaneous pain, mechanical allodynia, and heat hyperalgesia in TOW mice. Furthermore, we took a hematopoietic stem cell transplantation approach to generating a SCD model in PKCδ-deficient mice. Neither spontaneous pain nor evoked pain was detected in the mice lacking PKCδ despite full establishment of SCD phenotypes. These findings support a critical role of spinal PKCδ in the development of chronic pain in SCD, which may become a potential target for pharmacological interventions.

Fetal haemoglobin (HbF, alpha2) 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.

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.

We have previously demonstrated that 5-aza-2'-deoxycytidine (decitabine) augments fetal hemoglobin (HbF) levels in patients with sickle cell anemia (SS) who did not respond to hydroxyurea (HU). The present study was designed to determine the effect of repeated decitabine dosing on HbF levels and hematologic toxicity over a 9-month treatment period. Seven patients (5 HU nonresponders) were entered. One patient had alpha-thalassemia sickle cell anemia. Decitabine was administered by intravenous infusion at a starting dose of 0.3 mg/kg per day, 5 days a week for 2 weeks, followed by a 4-week observation period. If the absolute neutrophil count dropped below 1000, the dose was reduced by 0.05 mg/kg per day in the next cycle. A drug dose was obtained for each patient, and it resulted in an elevated HbF without neutropenia (absolute neutrophil count nadir greater than 1500) or evidence of cumulative toxicity. Average HbF and average maximal HbF levels attained during the last 20 weeks of treatment for the 6 SS patients increased to 13.93% +/- 2.75% and 18.35% +/- 4.46%, respectively, from a pretreatment mean of 3.12% +/- 2.75%. Mean and mean maximal hemoglobin (Hb) levels increased from 7.23 +/- 2.35 g/dL to 8.81 +/- 0.42 g/dL and 9.73 +/- 0.53 g/dL, respectively. Individual maximal F-cell number observed during the trial was 69% +/- 10.12%. The absence of cumulative toxicity may allow shorter intervals between drug treatments, which may lead to higher hemoglobin and HbF levels after several treatment cycles and, therefore, to greater clinical improvement.

Histone deacetylase (HDAC) inhibitors cause growth arrest and apoptosis of cancer cells by both p21-dependent and independent mechanisms. Decreased expression of growth factor receptors may be a key factor in the p21-independent mechanism, although this has not been directly tested. We have tested the effects of sodium butyrate and trichostatin A on human myeloma cell lines and have observed G1 arrest and apoptosis associated with increased expression of p21WAF1, Bax, Rb dephosphorylation, and decreased IL-6 receptor (IL-6R) expression. Experiments to determine the role of disruption of IL-6 signaling as a result of decreased IL-6 receptor expression in mediating these effects were conducted using a stable transfectant of the OPM-2 line which constitutively expressed the IL-6 receptor. Our results indicated that decreased IL-6R expression was not required for induction of p21WAF1 or apoptosis. Thus, HDAC inhibitors appear to activate multiple cellular pathways, leading to growth arrest and apoptosis, and their use in the treatment of myeloma, particularly in combination with other agents, warrants further investigation. Am. J. Hematol. 68:170–178, 2001. © 2001 Wiley-Liss, Inc.

Retinoic acid and dexamethasone, in combination, inhibit the growth of human myeloma cell lines in a synergistic manner. Previously, we observed that all-trans retinoic acid (ATR4) caused G1 arrest and inhibited clonogenic growth of the OPM-2 human myeloma cell line. This was associated with downregulation of the IL-6 receptor (IL-6R) gp80 protein, while autocrine IL-6 production and gp130 were not affected. Growth inhibition was not reversed by the addition of exogenous IL-6 or forced, constitutive expression of the IL-6 receptor gp80 protein, suggesting that the mechanism of action of ATRA may be due to effects on the post-receptor pathway. Therefore, in this study we have investigated whether growth arrest was associated with changes in the level of phosphorylation of the RE3 protein. ATRA decreased the level of phosphorylation of the RB protein at doses > 5 × 10−9 M and also induced a five fold increase in p2lWAF1, while levels of p27KIPI and CDK2 were unchanged. The ATRA-mediated increase in p21 prec...

Fetal hemoglobin (HbF) potently corrects red cell pathology caused by β-globin (HBB) mutations in β-hemoglobinopathies (e.g., sickle cell disease, SCD). DNA methyltransferase (DNMT1) is a central component of the chromatin-modifying enzyme network that represses the gene for HbF, ƴ-globin (HBG), in adult erythropoiesis. Thus, DNMT1 has been scientifically validated as a molecular target to increase HbF. The generic deoxycytidine analogue decitabine (Dec) can incorporate into DNA without terminating chain elongation (the sugar in Dec is natural) and deplete DNMT1 without cytotoxicity. Unfortunately, Dec is very rapidly metabolized by cytidine deaminase (CDA) in vivo into uridine counterparts that do not deplete DNMT1. Moreover, the uridine degradation products can mis-incorporate into DNA and cause cytotoxicity. Also, solid tissues, e.g., intestine/liver, highly express CDA, severely limiting Dec tissue distribution, oral bioavailability and exposure time. To address these barriers to clinical translation, which have as a common cause CDA, we evaluated the combination of oral Dec with a CDA-inhibitor, tetrahydrouridine (THU). Pre-clinical in vivo studies indicated improved distribution and oral bioavailability (~10-fold increase) with the low Cmax but extended Tmax desired for non-cytotoxic DNMT1-depletion. Here we report the first-in-man clinical translation (NCT01685515). Patients: This Phase 1 clinical trial enrolled adult SS or S-b-thal patients with multiple, clinically significant complications of SCD and risk of early death despite standard of care hydroxyurea. The primary objective was to identify the dose of oral Dec that can be safely co-administered 2X/week with oral THU. Treatment: Fixed dose oral THU 10mg/kg was ingested 60 minutes before oral Dec at 0.01, 0.02, 0.04, 0.08 or 0.16 mg/kg (mpk) in 5 cohorts of 5 pts, randomized 3/2 THU-Dec/placebo (Fig.1). Repeat dose administration 2X/wk for 8 wks was used to assess safety, increasing the likelihood that the identified dose would be safe for the intended application of chronic disease modification. Pharmacokinetics: Samples were obtained at 0, 2, 4 and 24 hrs from 12 of 15 THU-Dec subjects (dictated by venous access). Dec was detected (standard LCMS/MS method) at the lowest dose 0.01 mpk, and a dose-dependent increase was observed. Dec 0.08 and 0.16 mpk produced Cmax 9-21 and 39-54 nM respectively, levels that produced clinical pharmacodynamic effects (described below). Adverse events (AE):AE were similar in placebo and THU-Dec cohorts. No significant AE, including no significant gastro-intestinal AE, were attributed to study drug. There were no Grade 4 AE. No subjects discontinued placebo or THU-Dec. Hematology: HbF was measured by HPLC and in individual red cells by flow cytometry (F-cells). Dec 0.08 and 0.16 mpk substantially increased HbF (~2-fold increase in F-cells at 0.16 mpk) (Fig.1). In parallel, total hemoglobin (hb) increased (e.g., by 1.9, 1.8 and 1.2 g/dL at 0.16mpk) while LDH, t.bilirubin and D-dimers decreased (improvement in hemolysis and thrombophilia biomarkers) (Fig.1). Non-cytotoxic DNMT1-depletion alters hematopoietic differentiation and is expected to simultaneously increase platelet counts and decrease absolute neutrophil counts. This was seen at Dec 0.08 and 0.16 mpk. The HbF and Hb increases and biomarker improvements were produced within boundaries of platelet and neutrophil counts that did not require treatment holds. Discussion: Combining oral Dec with oral THU to inhibit CDA thus produced the Dec tissue distribution and wide concentration-time profile that is a basic requirement for clinical translation of observations regarding the safety and value of DNMT1 as a molecular target for therapy, moreover with the accessibility of oral administration. Platelet count increases by non-cytotoxic DNMT1-depletion were not linked with increases in thrombophilia biomarkers here or in previous trials of Dec in SCD. This impression is consistent also with clinical experience after standard of care splenectomy, which increases platelet counts even more drastically. Multi-organ damage in SCD incorporates the bone marrow, causing a decline in reticulocytosis/hb that contributes to early death. This underscores the import in further evaluation of this non-cytotoxic, normal stem cell sparing, rationally targeted approach to disease modification of SCD. Figure 1 Figure 1. Disclosures Lavelle: Acetylon: Research Funding. Hsu:Gerson Lehman Group: Consultancy; Mast Therapeutics: Research Funding; Eli Lilly: Research Funding; Astra Zeneca: Consultancy, Research Funding; Centers for Medicare and Medicaid Innovation: Research Funding; EMMI Solutions: Consultancy; Purdue Pharma: Research Funding; Hilton Publishing: Consultancy, Research Funding; Sancilio: Research Funding; Pfizer: Consultancy, Research Funding. DeSimone:EpiDestiny: Consultancy, Other: patents around decitabine and tetrahydrouridine. Saunthararajah:EpiDestiny: Consultancy, Other: patents around decitabine and tetrahydrouridine.

Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease. New and more effective strategies to increase HbF are needed because current therapies are ineffective in a significant proportion of patients. The lysine-specific demethylase, LSD1, an enzyme that demethylates Histone H3 mono- and dimethyl- K4 and K9 residues, is a component of the DRED co-repressor complex that represses the γ-globin gene (Cui et al, Mol Cell Biol 31:3298, 2011). Our laboratory has recently shown that RN-1, a pharmacological inhibitor of LSD1, increases γ-globin expression in sickle cell disease (SCD) transgenic mice (Rivers et al, Exp Hematol 43:546, 2015; Cui et al, Blood 126:386, 2015) and in baboons (Rivers et al, Haematol 101:688, 2016), widely acknowledge as the best animal model to test the activity of HbF-inducing drugs. To evaluate the safety and efficacy of RN-1, two normal, non-anemic baboons were treated for an extended period with RN-1 (PA8695, 264d; PA8698, 278d; 0.25mg/kg/d; 5d/wk, sc) and the effect on HbF, F cells, and hematological parameters evaluated weekly. Both animals exhibited weight gain (PA8695: 14.4%; PA8698: 20%) during the course of the study. F retics were increased 8-10 fold by the second week (d9) and were maintained at high levels throughout the course of the study (PA8695: 55.7±17.7% (mean±SD), median (M)=59.0%; PA8698: 56.4±14.5%, M=56.9%). F cell levels increased until d169 and were maintained thereafter at levels 18-25 times greater than pre-treatment (PA8695: 54.5±3.3%, M=54.2%; PA8698: 51.7±3.2%, M=51.4%). HbF levels also increased until d169 and were also maintained thereafter at levels 10-12 times greater than pre-treatment (PA8695: 12.8±1.7%, M=12.1%; PA8698: 11.4±1.4%, M=11.5%). Elevated levels of γ-globin chain synthesis were observed in the peripheral blood on multiple days (d162, d190, d267) of treatment (PA8695, 0.28±0.08 γ/γ+β;PA8698, 0.26±0.08 γ/γ+β). RT-PCR analysis showed that γ-globin mRNA levels were increased 9-22 fold (p Disclosures Rivers: Acetylon: Research Funding. DeSimone:EpiDestiny: Consultancy, Other: patents around decitabine and tetrahydrouridine. Lavelle:Acetylon: Research Funding.

Sickle cell disease (SCD) is an inherited blood disorder that affects millions of people worldwide. A single point mutation of the sixth amino acid of β-globin causes glutamic acid to be replaced by valine, rendering the hemoglobin susceptible to polymerization when deoxygenated. SCD patients suffer from the wide variety of disease manifestations including chronic hemolytic anemia, inflammation, painful vaso-occlusive crises, multisystem organ damage, and reduced life expectancy. In addition to the HbS polymerization-mediated rigid and fragile sickle-shaped red blood cell (RBC) formation, an excessive formation of intracellular reactive oxygen species (ROS) occurs in SCD red blood cells, which accelerates their hemolysis. This causes the release of ROS, free extracellular hemoglobin, hemin, and inflammatory cytokines that trigger disease progression. We analyzed levels of ROS in SCD patient RBCs and observed a higher fraction of intracellular ROS positive RBC in SCD (HbSS) compared to control (HbAA) RBC of adults [Control (HbAA): 7.1%± 1.4 %, n=11; SCD (HbSS): 25.3 % ± 4.3%, n=9; p Disclosures Jagadeeswaran: Acetylon: Research Funding. DeSimone:EpiDestiny: Consultancy, Other: patents around decitabine and tetrahydrouridine. Lavelle:Acetylon: Research Funding. Rivers:Acetylon: Research Funding.

This study was performed to investigate the hypothesis that the erythroid micro-environment plays a role in regulation of globin gene expression during adult erythroid differentiation. Adult baboon bone marrow and human cord blood CD34+ progenitors were grown in methylcellulose, liquid media, and in co-culture with stromal cell lines derived from different developmental stages in identical media supporting erythroid differentiation to examine the effect of the micro-environment on globin gene expression. Adult progenitors express high levels of γ-globin in liquid and methylcellulose media but low, physiological levels in stromal cell co-cultures. In contrast, γ-globin expression remained high in cord blood progenitors in stromal cell line co-cultures. Differences in γ-globin gene expression between adult progenitors in stromal cell line co-cultures and liquid media required cell-cell contact and were associated with differences in rate of differentiation and γ-globin promoter DNA methylation. We conclude that γ-globin expression in adult-derived erythroid cells can be influenced by the micro-environment, suggesting new potential targets for HbF induction.

We have studied the effect of site-specific cytosine methylation on the binding of nuclear proteins to the γ-globin promoter region from −71 to −34. This sequence was recently shown to contain a stage selector element responsible for increased transcription from the γ compared to the β promoter when both are linked to Locus Control Region enhancer sequences. We observed that Sp1 binding to this sequence is increased upon site-specific cytosine methylation, such that only methylation of the cytosine in the −50 CpG dinucleotide effects Sp1 binding. Methylation of the distal C of the Hpall site has no effect. The possible functional role of Sp1 as a repressor of γ-globin synthesis is discussed. © 1994 Wiley-Liss, Inc.

Developmental regulation of globin gene expression may be controlled by developmental stage-specific nuclear proteins that influence interactions between the locus control region and local regulatory sequences near individual globin genes. We previously isolated an 87-kD nuclear protein from K562 cells that bound to DNA sequences in the beta- globin locus control region, gamma-globin promoter, and A gamma-globin enhancer. The presence of this protein in fetal globin-expressing cells and its absence in adult globin-expressing cells suggested that it may be a developmental stage-specific factor. A lambda gt11 K562 cDNA clone encoding a portion of the HOXB2 (formerly HOX2H) homeobox gene was isolated on the basis of the ability of its beta-galactosidase fusion protein to bind to the same DNA sequences as the 87-kD K562 protein. Because no other relationship had been established between the 87-kD K562 protein and the HOXB2 protein other than their ability to bind ot the same DNA sequences, we have investigated whether the two proteins are related antigenically. Our data show that antisera produced against the HOXB2-beta-gal fusion protein and a synthetic HOXB2 decapeptide react specifically with an 87-kD protein from K562 nuclear extract, showing that the 87-kD K562 nuclear protein is a product of the HOXB2 locus, and is the first demonstration of cellular HOXB2 protein.

The deoxycytidine analog decitabine (DAC) can deplete DNA methyl-transferase 1 (DNMT1) and thereby modify cellular epigenetics, gene expression, and differentiation. However, a barrier to efficacious and accessible DNMT1-targeted therapy is cytidine deaminase, an enzyme highly expressed in the intestine and liver that rapidly metabolizes DAC into inactive uridine counterparts, severely limiting exposure time and oral bioavailability. In the present study, the effects of tetrahydrouridine (THU), a competitive inhibitor of cytidine deaminase, on the pharmacokinetics and pharmacodynamics of oral DAC were evaluated in mice and nonhuman primates. Oral administration of THU before oral DAC extended DAC absorption time and widened the concentration-time profile, increasing the exposure time for S-phase-specific depletion of DNMT1 without the high peak DAC levels that can cause DNA damage and cytotoxicity. THU also decreased interindividual variability in pharmacokinetics seen with DAC alone. One potential clinical application of DNMT1-targeted therapy is to increase fetal hemoglobin and treat hemoglobinopathy. Oral THU-DAC at a dose that would produce peak DAC concentrations of less than 0.2μM administered 2x/wk for 8 weeks to nonhuman primates was not myelotoxic, hypomethylated DNA in the γ-globin gene promoter, and produced large cumulative increases in fetal hemoglobin. Combining oral THU with oral DAC changes DAC pharmacology in a manner that may facilitate accessible noncytotoxic DNMT1-targeted therapy. Fil: Lavelle, Donald. University of Illinois at Chicago; Estados Unidos. Jesse Brown VA Medical Center; Estados Unidos Fil: Vaitkus, Kestis. University of Illinois at Chicago; Estados Unidos. Jesse Brown VA Medical Center; Estados Unidos Fil: Ling, Yonghua. Ohio State University; Estados Unidos Fil: Ruiz, Maria A.. University of Illinois at Chicago; Estados Unidos Fil: Mahfouz, Reda. Cleveland Clinic; Estados Unidos Fil: Peng Ng, Kwok. Cleveland Clinic; Estados Unidos Fil: Negrotto, Soledad. Cleveland Clinic; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Smith, Nicola. National Cancer Institute; Estados Unidos Fil: Terse, Pramod. National Cancer Institute; Estados Unidos Fil: Engelke, Kory J.. Avanza Laboratories; Estados Unidos Fil: Covey, Joseph. National Cancer Institute; Estados Unidos Fil: Chan, Kenneth K.. Ohio State University; Estados Unidos Fil: DeSimone, Joseph. University of Illinois at Chicago; Estados Unidos. Jesse Brown VA Medical Center; Estados Unidos Fil: Saunthararajah, Yogen. University of Illinois at Chicago; Estados Unidos. Cleveland Clinic; Estados Unidos

In sickle cell disease (SCD), vigorous reticulocytosis is required to partially compensate for chronic hemolytic anaemia. Consequently, early renal damage, insufficient to cause azotemia but sufficient to cause erythropoietin deficiency and chronic relative reticulocytopenia (chRR), could have severe clinical consequences. chRR was defined as reticulocytes250×10(9) /l despite haemoglobin9 g/dl on ≥ two occasions ≥4 weeks apart. The influence of multiple variables including chRR on time from first clinic visit to death was evaluated in 306 SCD patients. In univariate analyses, fetal haemoglobin, indices of renal damage (serum creatinine, proteinuria), chRR and age, were associated with rate of death. In multivariate analysis, only age and chRR (Hazard ratio 3·6, 95% CI 2·049-6·327, P0·0001) were significant, underlining that chRR could be an early and important clinical consequence of renal damage. Even in chRR patients with normal serum creatinine levels, low haemoglobin and low reticulocyte counts were associated with low erythropoietin levels. In the general population, evaluation of erythropoietin levels is prompted by the combination of anaemia and abnormal serum creatinine. In SCD patients, this standard approach can miss a substantial risk factor for early death. chRR could be a practical and important criterion for diagnosis of erythropoietin deficiency in SCD.

Present concepts of the mechanism of reactivation of synthesis of fetal hemoglobin (HbF) in the adult under conditions of erythropoietic stress are briefly reviewed. Since HbF can be considered an effective natural antisickling agent, the reactivation of its synthesis in patients with sickle cell anemia as a desirable therapeutic goal has been extensively explored since the discovery in 1982 that 5-azacytidine increases HbF levels in the baboon. Hydroxyurea (HU) has become the most widely used agent, although its effectiveness in increasing HbF levels and the number of F cells is highly variable. Recent investigations are cited showing that other agents such as butyrate, and the addition of recombinant hemopoietic growth factors, such as erythropoietin and stem cell factor, especially in combination with HU, offer important therapeutic possibilities. Transacting nuclear proteins are briefly discussed as possibly having a future role in the efforts of stimulating γ-chain synthesis. © 1993 Wiley-Liss, Inc.

The Carolina Center of Cancer Nanotechnology Excellence (C-CCNE) is funded by the National Cancer Institute and is based at the University of North Carolina. The C-CCNE features interactions between physical and biological scientists in a series of projects and cores that work together to quickly harness innovations in nanotechnology for the early diagnosis and treatment of cancer. Two key focus areas of the C-CCNE are, first, the selective delivery of drugs and imaging agents utilizing advanced nanoparticle technology, and second, novel approaches to imaging and radiotherapy utilizing carbon nanotube-based x-ray sources.

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.

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.

Objective To investigate the mechanism(s) responsible for increased γ-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 γ-globin gene complex by chromatin immunoprecipitation. DNA methylation of the γ-globin promoter was determined by bisulfite sequencing. Expression of the baboon Iγ- and Vγ-globin chains was determined by high performance liquid chromatography (HPLC). Expression of BCL11A, a recently identified repressor of γ-globin expression, was analyzed by Western blot. Results Increased γ-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 γ-globin gene consistent with a transcriptional activation mechanism. Cultured EPC expressed the Iγ- and Vγ-globin chains in a pattern characteristic of fetal development. The level of DNA methylation of the γ-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 γ-globin expression in cultured EPC was unexpectedly not associated with increased DNA hypomethylation of the γ-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 Iγ/Vγ chain ratio and a difference in the size of the BCL11A protein compared to normal BM erythroid cells.

Case Report A 30 year old male with a remote history of intravenous drug abuse (IVDA) and Hepatitis C was admitted in July 2008 with changes in mental status and a new right sided paresis. According to the family, the patient was in his usual state of health until approximately 2-3 weeks prior to admission when he developed headaches, generalized malaise, and fever to 101 degrees Fahrenheit after swimming in a lake. He was seen in an outside hospital emergency department, diagnosed with otitis media and subsequently discharged on prednisone and ciprofloxacin. The morning of admission, the patient was found naked, nonverbal, confused and was subsequently taken again to an outside hospital emergency department where he was found to have right sided weakness, diagnosed with a new cerebrovascular accident (CVA) and transferred to TJUH for further management.

Developmental regulation of gamma-globin gene expression probably occurs through developmental-stage-specific trans-acting factors able to promote the interaction of enhancer elements located in the far upstream locus control region with regulatory elements in the gamma gene promoters and 3' A gamma enhancer located in close proximity to the genes. We have detected a nuclear protein in K562 and baboon fetal bone marrow nuclear extracts capable of binding to A+T-rich sequences in the locus control region, gamma gene promoter, and 3' A gamma enhancer. SDS/polyacrylamide gel analysis of the purified K562 binding activity revealed a single protein of 87 kDa. A K562 cDNA clone was isolated encoding a beta-galactosidase fusion protein with a DNA binding specificity identical to that of the K562/fetal bone marrow nuclear protein. The cDNA clone encodes a homeodomain homologous to the Drosophila antennapedia protein.

Background: Infection is a potentially life-threatening complication of cardiac device implantation. Lead-associated endocarditis (LAE) may be the most serious complication since it is associated with a high mortality. Methods: The medical records of patients referred to our institution for the treatment of LAE between 1999 and 2007 were reviewed. Results: A total of 51 of 107 patients referred for device-related infections met the criteria for LAE. Of these, 19 occurred within 6 months of their most recent procedure (early), while the remaining 32 occurred more than 6 months later (mean = 31.9 months post procedure). Devices included pacemakers in 33 patients and ICDs in 18 patients. The most common organism responsible for infection was Staphylococcus aureus (S. aureus) followed by coagulase-negative staphylocci (22%) and streptococci (12%). Methicillin-resistant S. aureus (MRSA) accounted for 67% of the S. aureus infections. Coagulase-negative staphylococci were responsible for only 26% of early and 19% of late cases. A distant site of infection was common (26/51 = 51%), particularly in patients with MRSA LAE. The device and leads were removed percutaneously in all patients. Only one patient failed to respond to intravenous antibiotics. Conclusions: Our data suggest that methicillin-resistant S. aureus is an important pathogen in LAE. Since many infections occur months after the last device procedure, hematogenous spread of organisms from a distant site may be an important contributing factor. These data suggest that strategies to prevent hematogenous infection, particularly with S. aureus, are critical in patients with implantable cardiac devices.

Sickle cell disease (SCD) results from the substitution of glutamic acid for valine at the sixth amino acid of β-globin protein, leading to the formation of the abnormal hemoglobin S (HbS). Following deoxygenation in red blood cells (RBCs), HbS forms polymers causing the RBCs to become deformed and adherent leading to vaso-occlusive events resulting in splenic infarction, kidney failure, stroke, painful crises, chronic anemia, inflammation and decreased survival. Hydroxyurea, the only drug currently FDA approved for SCD, is not effective in some patients and, therefore, new combination regimens are needed. RN-1, a new drug targeting the histone demethylase LSD-1, is a more effective inducer of γ-globin expression in sickle cell mice and HbF in baboons (P. anubis). Decitabine(DAC), a DNMT1 inhibitor, is well known to induce high levels of HbF in non-human primates and SCD patients and is currently in clinical trials. We investigated the effect of two combination regimens: RN-1 in combination with hydroxyurea vs. RN-1 in combination with DAC. The knock-in humanized sickle cell mice (B6;129-Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB*)Tow/J) were treated with 1) DMSO (vehicle control), 2) RN-1 (5mg/kg), 3) DAC(0.25mg/kg) and 4) RN-1(5mg/kg) + DAC(0.5mg/kg) for 10 days. Analysis of F-retics and γ-globin mRNA performed on d10 showed F-retics were increased two fold (p=0.01) in mice treated with RN-1(5mg/kg) or DAC(0.25mg/kg) compared to DMSO-treated. γ-globin mRNA levels (γ/γ+β) were increased three fold in RN-1(5mg/kg) or DAC(0.25mg/kg) compared to DMSO. The combination of RN-1(5mg/kg) and DAC(0.25mg/kg) did increase F-retics and mRNA levels compared to mice treated with RN-1 alone(p=0.02). Also, the RN-1 and DAC combination led to neutropenia and thrombocytopenia. We next examined the effect of RN-1(5mg/kg) and HU (100mg/kg) in combination. Knock-in humanized sickle cell mice (B6;129-Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB)Tow/J) were treated with 1) DMSO (vehicle) 2) RN-1(5mg/kg) and 3) RN-1(5mg/kg) + HU (100mg/kg). While RN-1 in combination with HU did not significantly increase F-cells or F-retics, there was a significant decrease in spleen weight [(DMSO: 1.17±0..06g; RN1:0.71± 0.1g; RN1+HU: 0.46±0.05g)(p=0.004:p=0.02)] and percentage of sickle cells in a peripheral blood smear.[(DMSO: 8.4 ±1.3%; RN-1:4.1± 0.2%; RN1+HU: 1.57±0.6%)(p=0.006:p-0.014)]. We conclude that further studies to determine the effect of RN-1 in combination with HU or DAC in the sickle cell mouse model and cultures from sickle cell CD34+ cells are needed to evaluate its potential use in the treatment of sickle cell disease. These results suggest that RN-1 in combination with hydroxyurea may be a useful therapeutic regimen. Disclosures No relevant conflicts of interest to declare.