Nadine Odo

Regular Transfusion Lowers Plasma Free Hemoglobin in Children With Sickle-Cell Disease at Risk for Stroke

Background and Purpose— Intravascular hemolysis releases large amounts of free hemoglobin (PFH) in plasma of sickle- cell disease (SCD) patients. PFH has been associated with harmful endothelial actions including scavenging nitric oxide (NO). Whether PFH plays a role in stroke in SCD has not been examined. Methods— Serum levels of PFH, lactate dehydrogenase, and total bilirubin were measured in stored sera from children at risk for stroke treated in a randomized controlled trial of regular red cell transfusion (STOP study). Baseline and post-treatment (≈1 year of transfusion) were compared to determine whether treatment (which reduces stroke risk by 90%) was associated with reduction in markers of hemolysis. Results— Baseline serum PFH values did not differ between treatment groups. PFH declined with repeated transfusion from 78.7±8.2 mg/dL to 34.4±3.4 mg/dL (P<0.001). With only episodic or no transfusion the drop was smaller: 80.9±7.5 to 62.8±5.0 (P=0.019). The decrease was larger in those with regular transfusion (56% versus 22%; P<0.001). Reduction of lactate dehydrogenase and total bilirubin was observed only in those on regular transfusion. Conclusions— Regular transfusion which lowers stroke risk is associated with a significant reduction in PFH. A role for PFH in promoting stroke in SCD should be investigated.

Serum ferritin level changes in children with sickle cell disease on chronic blood transfusion are nonlinear and are associated with iron load and liver injury

Chronic blood transfusion is increasingly indicated in patients with sickle cell disease. Measuring resulting iron overload remains a challenge. Children without viral hepatitis enrolled in 2 trials for stroke prevention were examined for iron overload (STOP and STOP2; n = 271). Most received desferrioxamine chelation. Serum ferritin (SF) changes appeared nonlinear compared with prechelation estimated transfusion iron load (TIL) or with liver iron concentrations (LICs). Averaged correlation coefficient between SF and TIL (patients/observations, 26 of 164) was r = 0.70; between SF and LIC (patients/observations, 33 of 47) was r = 0.55. In mixed models, SF was associated with LIC (P = .006), alanine transaminase (P = .025), and weight (P = .026). Most patients with SF between 750 and 1500 ng/mL had a TIL between 25 and 100 mg/kg (72.8% +/- 5.9%; patients/observations, 24 of 50) or an LIC between 2.5 and 10 mg/g dry liver weight (75% +/- 0%; patients/observations, 8 of 9). Most patients with SF of 3000 ng/mL or greater had a TIL of 100 mg/kg or greater (95.3% +/- 6.7%; patients/observations, 7 of 16) or an LIC of 10 mg/g dry liver weight or greater (87.7% +/- 4.3%; patients/observations, 11 of 18). Although SF changes are nonlinear, levels less than 1500 ng/mL indicated mostly acceptable iron overload; levels of 3000 ng/mL or greater were specific for significant iron overload and were associated with liver injury. However, to determine accurately iron overload in patients with intermediately elevated SF levels, other methods are required. These trials are registered at www.clinicaltrials.gov as #NCT00000592 and #NCT00006182.

The proinflammatory cytokine GM-CSF downregulates fetal hemoglobin expression by attenuating the cAMP-dependent pathway in sickle cell disease

Although reduction in leukocyte counts following hydroxyurea therapy in sickle cell disease (SCD) predicts fetal hemoglobin (HbF) response, the underlying mechanism remains unknown. We previously reported that leukocyte counts are regulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) in SCD patients. Here we examined the roles of GM-CSF in the regulation of HbF expression in SCD. Upon the analysis of retrospective data in 372 patients, HbF levels were inversely correlated with leukocyte counts and GM-CSF levels in SCD patients without hydroxyurea therapy, while HbF increments after hydroxyurea therapy correlated with a reduction in leukocyte counts, suggesting a negative effect of GM-CSF on HbF expression. Consistently, in vitro studies using primary erythroblasts showed that the addition of GM-CSF to erythroid cells decreased HbF expression. We next examined the intracellular signaling pathway through which GM-CSF reduced HbF expression. Treatment of erythroid cells with GM-CSF resulted in the reduction of intracellular cAMP levels and abrogated phosphorylation of cAMP response-element-binding-protein, suggesting attenuation of the cAMP-dependent pathway, while the phosphorylation levels of mitogen-activated protein kinases were not affected. This is compatible with our studies showing a role for the cAMP-dependent pathway in HbF expression. Together, these results demonstrate that GM-CSF plays a role in regulating both leukocyte count and HbF expression in SCD. Reduction in GM-CSF levels upon hydroxyurea therapy may be critical for efficient HbF induction. The results showing the involvement of GM-CSF in HbF expression may suggest possible mechanisms for hydroxyurea resistance in SCD.

Glycosylation Inhibitors Efficiently Inhibit P-Selectin-Mediated Cell Adhesion to Endothelial Cells

Adhesion molecules play a critical role in the adhesive interactions of multiple cell types in sickle cell disease (SCD). We previously showed that anti-P-selectin aptamer efficiently inhibits cell adhesion to endothelial cells (ECs) and permits SCD mice to survive hypoxic stress. In an effort to discover new mechanisms with which to inhibit P-selectin, we examined the role of glycosylation. P-selectin is a 90 kDa protein but was found to migrate as 90 and 140 kDa bands on gel electrophoresis. When P-selectin isolated from ECs was digested with peptide N-glycosidase F, but not O-glycosidase, the 140 kDa band was lost and the 90 kDa band was enhanced. Treatment of ECs with tunicamycin, an N-glycosylation inhibitor, suppressed CD62P (P-selectin) expression on the cell surface as well as the 140 kDa form in the cytoplasm. These results indicate that the 140 kDa band is N-glycosylated and glycosylation is critical for cell surface expression of P-selectin in ECs. Thrombin, which stimulates P-selectin expression on ECs, induced AKT phosphorylation, whereas tunicamycin inhibited AKT phosphorylation, suggesting that AKT signaling is involved in the tunicamycin-mediated inhibition of P-selectin expression. Importantly, the adhesion of sickle red blood cells (sRBCs) and leukocytes to ECs induced by thrombin or hypoxia was markedly inhibited by two structurally distinct glycosylation inhibitors; the levels of which were comparable to that of a P-selectin monoclonal antibody which most strongly inhibited cell adhesion in vivo. Knockdown studies of P-selectin using short-hairpin RNAs in ECs suppressed sRBC adhesion, indicating a legitimate role for P-selectin in sRBC adhesion. Together, these results demonstrate that P-selectin expression on ECs is regulated in part by glycosylation mechanisms and that glycosylation inhibitors efficiently reduce the adhesion of sRBCs and leukocytes to ECs. Glycosylation inhibitors may lead to a novel therapy which inhibits cell adhesion in SCD.

A common signaling pathway is activated in erythroid cells expressing high levels of fetal hemoglobin: a potential role for cAMP-elevating agents in β-globin disorders

Background Although erythroid cells prepared from fetal liver, cord blood, or blood from β-thalassemia patients are known to express fetal hemoglobin at high levels, the underlying mechanisms remain elusive. We previously showed that cyclic nucleotides such as cAMP and cGMP induce fetal hemoglobin expression in primary erythroid cells. Here we report that cAMP signaling contributes to high-level fetal hemoglobin expression in erythroid cells prepared from cord blood and β-thalassemia. Methods The status of the cAMP signaling pathway was investigated using primary erythroid cells prepared from cord blood and the mononuclear cells of patients with β-thalassemia; erythroid cells from adult bone marrow mononuclear cells served as the control. Results We found that intracellular cAMP levels were higher in erythroid cells from cord blood and β-thalassemia than from adult bone marrow. Protein kinase A activity levels and cAMP-response element binding protein phosphorylation were higher in erythroid cells from cord blood or β-thalassemia than in adult bone marrow progenitors. Mitogen-activated protein kinase pathways, which play a role in fetal hemoglobin expression, were not consistently activated in cord blood or β-thalassemia erythroid cells. When cAMP signaling was activated in adult erythroid cells, fetal hemoglobin was induced at high levels and associated with reduced expression of BCL11A, a silencer of the β-globin gene. Conclusion These results suggest that activated cAMP signaling may be a common mechanism among erythroid cells with high fetal hemoglobin levels, in part because of downregulation of BCL11A. Activation of the cAMP signaling pathway with cAMP-elevating agents may prove to be an important signaling mechanism to reactivate fetal hemoglobin expression in erythroid cells.

Nitric oxide-cGMP signaling stimulates erythropoiesis through multiple lineage-specific transcription factors: Clinical implications and a novel target for erythropoiesis

Much attention has been directed to the physiological effects of nitric oxide (NO)-cGMP signaling, but virtually nothing is known about its hematologic effects. We reported for the first time that cGMP signaling induces human γ-globin gene expression. Aiming at developing novel therapeutics for anemia, we examined here the hematologic effects of NO-cGMP signaling in vivo and in vitro. We treated wild-type mice with NO to activate soluble guanylate cyclase (sGC), a key enzyme of cGMP signaling. Compared to untreated mice, NO-treated mice had higher red blood cell counts and total hemoglobin but reduced leukocyte counts, demonstrating that when activated, NO-cGMP signaling exerts hematopoietic effects on multiple types of blood cells in vivo. We next generated mice which overexpressed rat sGC in erythroid and myeloid cells. The forced expression of sGCs activated cGMP signaling in both lineage cells. Compared with non-transgenic littermates, sGC mice exhibited hematologic changes similar to those of NO-treated mice. Consistently, a membrane-permeable cGMP enhanced the differentiation of hematopoietic progenitors toward erythroid-lineage cells but inhibited them toward myeloid-lineage cells by controlling multiple lineage-specific transcription factors. Human γ-globin gene expression was induced at low but appreciable levels in sGC mice carrying the human β-globin locus. Together, these results demonstrate that NO-cGMP signaling is capable of stimulating erythropoiesis in both in vitro and vivo settings by controlling the expression of multiple lineage-specific transcription factors, suggesting that cGMP signaling upregulates erythropoiesis at the level of gene transcription. The NO-cGMP signaling axis may constitute a novel target to stimulate erythropoiesis in vivo.

Serum of sickle cell disease patients contains fetal hemoglobin silencing factors secreted from leukocytes

Background The mechanisms that regulate fetal hemoglobin (HbF) expression in sickle cell disease (SCD) remain elusive. We previously showed that steady-state SCD patients with high HbF levels due to a γ-globin gene mutation demonstrate strong inverse correlations between HbF levels and leukocyte counts, suggesting that leukocytes play a role in regulating HbF in SCD. Materials and methods To further investigate the role of leukocytes in HbF expression in SCD, we examined the presence of HbF silencing factors in the serum of 82 SCD patients who received hydroxyurea (HU) therapy. Results HU-mediated HbF induction was associated with elevated total hemoglobin levels and improved red blood cell parameters, but there was no correlation with reticulocyte or platelet counts. Importantly, we again found that HU-induced HbF levels correlated with reductions in both neutrophils and lymphocytes/monocytes, indicating that these cell lineages may have a role in regulating HU-mediated HbF expression. Our in vitro studies using CD34+-derived primary erythroblasts found that patient serum preparations include HbF silencing factors that are distinct from granulocyte-macrophage colony-stimulating factor, and the activity of such factors decreases upon HU therapy. Conclusion Together, these results demonstrate the importance of leukocyte numbers in the regulation of HbF levels for SCD patients both in steady state and under HU therapy, and that leukocytes secrete HbF silencing factors that negatively affect HbF expression in erythroid-lineage cells in SCD.

Iatrogenic atrio-esophageal fistula following a video-assisted thoracoscopic maze procedure: Is esophageal instrumentation justified even when the diagnosis is equivocal?

A 74-year-old female underwent an uneventful bilateral thoracoscopic maze procedure for persistent atrial fibrillation with continuous transesophageal echocardiographic (TEE) guidance. She presented six weeks later with persistent fever and focal neurological signs. Computed tomography of the thorax revealed air in the posterior LA, raising suspicion for an abscess versus an atrioesophageal fistula (AEF). Before undergoing an exploratory median sternotomy, an esophagogastroduodenoscopy (EGD) was performed by the surgeon to check for any esophageal pathology. This however, resulted in sudden hemodynamic compromise that required intensive treatment with vasopressors and inotropes. In this case-report, we review the various intraoperative risk factors associated with the development of AEF during cardiac ablation procedures as well as the potential hazards of esophageal instrumentation with TEE, naso- or oro- gastric devices, and/or an EGD when an AEF is suspected.

Cervical Deformity and Potential Difficult Airway Management in Klippel–Feil Syndrome

1 XXX 2017 K LIPPEL–FEIL syndrome presents with a short neck and severe restriction of cervical motion.1 The accompanying magnetic resonance image demonstrates this neck deformity due to the fusion of cervical vertebrae (image A, arrow A) and severe central canal stenosis (image A, arrow B). A lateral cervical spine X-ray (image B) demonstrates cervical fusion (arrow A’ ) but not the extent of spinal canal narrowing, and thus magnetic resonance imaging or computed tomography better informs preoperative airway assessment. Preoperative review of the imaging is instrumental to objective airway assessment, risk stratification, and development of an airway management plan. During laryngoscopy, the neck should be maintained in a neutral position to prevent hyperextension, which could cause severe neurologic injury.2 In patients with symptomatic cord compression, an awake fiberoptic intubation is often a better option due to severely limited neck range of motion, atlanto-occipital abnormalities,1,2 and possible impingement on the spine. The endotracheal tube should be meticulously secured and inline stabilization maintained, especially during procedures that require prone positioning. A laryngeal mask airway can be used to manage a difficult airway; however, it has a limited role in prone positioning. Due to skeletal abnormalities, protective padding should be used to prevent perioperative nerve injury and pressure ulcers. On emergence from anesthesia, a neurologic examination must be performed to ensure integrity of the spinal cord. The presence of congenital cardiac, renal, and other skeletal abnormalities can also affect anesthetic management.3 Of particular note, kyphoscoliosis may lead to restrictive respiratory mechanics and can cause difficulties and complications when placing spinal or epidural anesthesia.

“Golf Ball” in the Left Ventricular Outflow Tract?

1 XXX 2017 A 13 × 20 mm mobile vegetation (Panel A) resembling a golf ball, and causing dynamic left ventricular outflow tract (LVOT) obstruction, was found to be attached to the anterior mitral valve leaflet (AMVL) on the ventricular side by transesophageal echocardiography in the midesophageal long-axis view, sparing the posterior mitral valve leaflet (PMVL). Most vegetations on the mitral valve attach to the atrial (low flow, left atrium [LA]) side of the leaflet, but on rare occasions are on the ventricular (high flow) side.1 Vegetation location, regurgitation mechanism, and severity of the LVOT obstruction were assessed by color flow Doppler. The presence of the mass on the leaflet tip prevented leaflet coaptation, resulting in central mitral regurgitation (MR) in systole.2 There was flow convergence within the left ventricle (LV) proximal to the mass as well as flow acceleration that began at the mass proximal to the aortic valve, causing a dynamic LVOT obstruction (Panel B). This large (greater than 10 mm) mass posed a high risk for systemic embolization.3 Hemodynamic management can be challenging in the presence of MR, low systemic vascular resistance in the presence of sepsis, general anesthesia, and dynamic LVOT obstruction. Ensuring adequate coronary and systemic perfusion by optimizing preload and maintaining sinus rhythm with a moderate rate should be the goal. Continuous intraoperative transesophageal echocardiography confirms vegetation size and location; defines the degree of valve involvement and damage to surrounding tissue; facilitates hemodynamic monitoring; and alerts the anesthesiologist to systemic embolization.2