GENETIC BEHAVIOUR, DISABILITY CAUSED DUE TO BLOOD DISORDER HAEMOPHILIA, THALASSEMIA, SICKLE CELL DISEASE

Sickle cell disease (SCD) is the monogenic hemoglobinopathy where mutated sickle hemoglobin molecules polymerize to form long fibers under deoxygenated state and deform red blood cells (RBCs) into predominantly sickle form. Sickled RBCs stick to the vascular bed and obstruct blood flow in extreme conditions, leading to acute painful vaso-occlusion crises (VOCs) – the leading cause of mortality in SCD. Being a blood disorder of deformed RBCs, SCD manifests a wide-range of organ-specific clinical complications of life (in addition to chronic pain) such as stroke, acute chest syndrome (ACS) and pulmonary hypertension in the lung, nephropathy, auto-splenectomy, and splenomegaly, hand-foot syndrome, leg ulcer, stress erythropoiesis, osteonecrosis and osteoporosis. The physiological inception for VOC was initially thought to be only a fluid flow problem in microvascular space originated from increased viscosity due to aggregates of sickled RBCs; however, over the last three decades, multiple molecular and cellular mechanisms have been identified that aid the VOC in vivo. Activation of adhesion molecules in vascular endothelium and on RBC membranes, activated neutrophils and platelets, increased viscosity of the blood, and fluid physics driving sickled and deformed RBCs to the vascular wall (known as margination of flow) – all of these come together to orchestrate VOC. Microfluidic technology in sickle research was primarily adopted to benefit from mimicking the microvascular network to observe RBC flow under low oxygen conditions as models of VOC. However, over the last decade, microfluidics has evolved as a valuable tool to extract biophysical characteristics of sickle red cells, measure deformability of sickle red cells under simulated oxygen gradient and shear, drug testing, in vitro models of intercellular interaction on endothelialized or adhesion molecule-functionalized channels to understand adhesion in sickle microenvironment, characterizing biomechanics and microrheology, biomarker identification, and last but not least, for developing point-of-care diagnostic technologies for low resource setting. Several of these platforms have already demonstrated true potential to be translated from bench to bedside. Emerging microfluidics-based technologies for studying heterotypic cell–cell interactions, organ-on-chip application and drug dosage screening can be employed to sickle research field due to their wide-ranging advantages.

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The gut microbiome in sickle cell disease: Characterization and potential implications

PLoS ONE ◽

10.1371/journal.pone.0255956 ◽

2021 ◽

Vol 16 (8) ◽

pp. e0255956

Author(s):

Hassan Brim ◽

James Taylor ◽

Muneer Abbas ◽

Kimberly Vilmenay ◽

Mohammad Daremipouran ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Gut Microbiome ◽

Tissue Injury ◽

Organ Damage ◽

Composition Analysis ◽

Cell Disease ◽

Blood Disorder ◽

Major Player ◽

Next Generation Sequencing Ngs

Background Sickle Cell Disease (SCD) is an inherited blood disorder that leads to hemolytic anemia, pain, organ damage and early mortality. It is characterized by polymerized deoxygenated hemoglobin, rigid sickle red blood cells and vaso-occlusive crises (VOC). Recurrent hypoxia-reperfusion injury in the gut of SCD patients could increase tissue injury, permeability, and bacterial translocation. In this context, the gut microbiome, a major player in health and disease, might have significant impact. This study sought to characterize the gut microbiome in SCD. Methods Stool and saliva samples were collected from healthy controls (n = 14) and SCD subjects (n = 14). Stool samples were also collected from humanized SCD murine models including Berk, Townes and corresponding control mice. Amplified 16S rDNA was used for bacterial composition analysis using Next Generation Sequencing (NGS). Pairwise group analyses established differential bacterial groups at many taxonomy levels. Bacterial group abundance and differentials were established using DeSeq software. Results A major dysbiosis was observed in SCD patients. The Firmicutes/Bacteroidetes ratio was lower in these patients. The following bacterial families were more abundant in SCD patients: Acetobacteraceae, Acidaminococcaceae, Candidatus Saccharibacteria, Peptostreptococcaceae, Bifidobacteriaceae, Veillonellaceae, Actinomycetaceae, Clostridiales, Bacteroidacbactereae and Fusobacteriaceae. This dysbiosis translated into 420 different operational taxonomic units (OTUs). Townes SCD mice also displayed gut microbiome dysbiosis as seen in human SCD. Conclusion A major dysbiosis was observed in SCD patients for bacteria that are known strong pro-inflammatory triggers. The Townes mouse showed dysbiosis as well and might serve as a good model to study gut microbiome modulation and its impact on SCD pathophysiology.

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The prevalence of leukocyte abnormalities among Sudanese patients with sickle cell disease

World Journal of Advanced Research and Reviews ◽

10.30574/wjarr.2021.9.1.0026 ◽

2021 ◽

Vol 9 (1) ◽

pp. 262-267

Author(s):

Tarig Osman Khalafallah Ahmed ◽

Ekhlas Alrasheid Abu Elfadul ◽

Ahmed A. Agab Eldour ◽

Omer Ibrahim Abdallah Mohammed

Keyword(s):

Sickle Cell Disease ◽

Cell Volume ◽

Sickle Cell ◽

Blood Cells ◽

White Blood Cells ◽

Blood Film ◽

Cell Disease ◽

Mean Cell Volume ◽

Blood Disorder ◽

Age Range

Sickle cell disease (SCD) is an inherited blood disorder that affects red blood cells. The study was conducted in Elobied town during the period May 2011 to September 2011. The aim of this study is to detect the abnormalities of leucocytes among sickle cell anemic patients. 40 sickle cell anemic patients; age range between 8 months to 23 years. Blood sample was taken for all patients and the laboratory investigation were performed using automated estimation for: hemoglobin (Hb), Packed cell volume (PCV), red cell count (RBCs), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell concentration (MCHC), and total white blood cells, comment on blood film using manual methods. The conclusion of this study there is increase in total white blood cells with shift to left in neutrophil precursor in sickle cell patients with complications ,the most immature cells are band form, myelocytes and metamyelocytes, and there also lymphocytosis and neutrophilia which has been increases in response to infections.

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Review of Sickle Cell Disease and Spinal Pathology

Global Spine Journal ◽

10.1177/2192568218799074 ◽

2018 ◽

Vol 9 (7) ◽

pp. 761-766 ◽

Cited By ~ 1

Author(s):

Hayeem L. Rudy ◽

David Yang ◽

Andrew D. Nam ◽

Woojin Cho

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Vertebral Osteomyelitis ◽

Compression Fracture ◽

Symptomatic Therapy ◽

Cell Disease ◽

Blood Disorder ◽

Increased Risk ◽

Adequate Hydration ◽

Surgical Treatments

Study Design:Sickle cell disease (SCD) is a relatively common blood disorder that has profound implications on the musculoskeletal system and particularly the spine; however, there is a paucity of data in the literature discussing this important topic.Objectives:(1) To elucidate common spinal pathologies affecting patients with SCD, as well as the medical and surgical treatments available for these patients. (2) To discuss indications for surgical management of spinal complications of SCD and important for orthopedic surgeons when taking patients with SCD to the operating room.Methods:A narrative review of the literature was performed.Results:Patients with SCD have a significantly higher risk of developing spinal pathologies including vertebral osteomyelitis, compression fracture, vertebral vaso-occlusive crises, and osteoporosis, among others. In addition, patients with sickle cell disease are particularly susceptible to developing perioperative and post-operative complications including surgical site infection, implant malfunction, and vertebral body compression fracture. Postoperatively patients with SCD are prone to developing complications and adequate hydration is necessary in order to reduce complications of SCD.Conclusions:Several spinal pathologies may arise secondary to SCD and distinguishing these pathologies from one another may be challenging due to similarities in symptoms and inflammatory markers. Although most patients with SCD can benefit from conservative treatment involving rest, symptomatic therapy, antibiotic therapy, and/or orthosis, surgical intervention may be indicated in certain cases. It is preferable to avoid surgery in patients with SCD due to an increased risk of complications such as wound infection and vaso-occlusive crisis.

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Management of chronic respiratory complications in children and adolescents with sickle cell disease

European Respiratory Review ◽

10.1183/16000617.0054-2020 ◽

2020 ◽

Vol 29 (157) ◽

pp. 200054

Author(s):

Michele Arigliani ◽

Atul Gupta

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Current Knowledge ◽

Sub Saharan Africa ◽

Cell Disease ◽

Respiratory Complications ◽

Progressive Decline ◽

Blood Disorder ◽

Life Threatening ◽

Sub Saharan

Sickle cell disease (SCD) is a life-threatening hereditary blood disorder that affects millions of people worldwide, especially in sub-Saharan Africa. This condition has a multi-organ involvement and highly vascularised organs, such as the lungs, are particularly affected. Chronic respiratory complications of SCD involve pulmonary vascular, parenchymal and airways alterations. A progressive decline of lung function often begins in childhood. Asthma, sleep-disordered breathing and chronic hypoxaemia are common and associated with increased morbidity. Pulmonary hypertension is a serious complication, more common in adults than in children. Although there is a growing attention towards respiratory care of patients with SCD, evidence regarding the prognostic meaning and optimal management of pulmonary issues in children with this condition is limited.This narrative review presents state-of-the-art evidence regarding the epidemiology, pathophysiology and therapeutic options for chronic respiratory complications commonly seen in paediatric patients with SCD. Furthermore, it highlights the gaps in the current knowledge and indicates future directions for studies that aim to improve our understanding of chronic respiratory complications in children with SCD.

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Building access to care in adult sickle cell disease: defining models of care, essential components, and economic aspects

Blood Advances ◽

10.1182/bloodadvances.2020001743 ◽

2020 ◽

Vol 4 (16) ◽

pp. 3804-3813

Author(s):

Julie Kanter ◽

Wally R. Smith ◽

Payal C. Desai ◽

Marsha Treadwell ◽

Biree Andemariam ◽

...

Keyword(s):

United States ◽

Sickle Cell Disease ◽

Access To Care ◽

Sickle Cell ◽

The United States ◽

Models Of Care ◽

System Level ◽

Cell Disease ◽

Blood Disorder ◽

Essential Components

Abstract Sickle cell disease (SCD) is the most common inherited blood disorder in the United States. It is a medically and socially complex, multisystem illness that affects individuals throughout the lifespan. Given improvements in care, most children with SCD survive into adulthood. However, access to adult sickle cell care is poor in many parts of the United States, resulting in increased acute care utilization, disjointed care delivery, and early mortality for patients. A dearth of nonmalignant hematology providers, the lack of a national SCD registry, and the absence of a centralized infrastructure to facilitate comparative quality assessment compounds these issues. As part of a workshop designed to train health care professionals in the skills necessary to establish clinical centers focused on the management of adults living with SCD, we defined an SCD center, elucidated required elements of a comprehensive adult SCD center, and discussed different models of care. There are also important economic impacts of these centers at an institutional and health system level. As more clinicians are trained in providing adult-focused SCD care, center designation will enhance the ability to undertake quality improvement and compare outcomes between SCD centers. Activities will include an assessment of the clinical effectiveness of expanded access to care, the implementation of SCD guidelines, and the efficacy of newly approved targeted medications. Details of this effort are provided.

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Comorbidity of Glucose-6-Phosphate Dehydrogenase Deficiency and Sickle Cell Disease Exert Significant Effect on RBC Indices

Anemia ◽

10.1155/2019/3179173 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9 ◽

Cited By ~ 3

Author(s):

Samuel Antwi-Baffour ◽

Jonathan Kofi Adjei ◽

Peter Owadee Forson ◽

Stephen Akakpo ◽

Ransford Kyeremeh ◽

...

Keyword(s):

Sickle Cell Disease ◽

Red Blood Cells ◽

Sickle Cell ◽

Blood Cells ◽

G6pd Deficiency ◽

Pentose Phosphate ◽

Full Blood Count ◽

Cell Disease ◽

Blood Disorder ◽

Glucose 6 Phosphate Dehydrogenase

Background. Glucose-6-phosphate dehydrogenase (G6PD) converts glucose-6-phosphate into 6-phosphogluconate in the pentose phosphate pathway and protects red blood cells (RBCs) from oxidative damage. Their deficiency therefore makes RBCs prone to haemolysis. Sickle cell disease (SCD) on the other hand is a hereditary blood disorder in which there is a single nucleotide substitution in the codon for amino acid 6 substituting glutamic acid with valine. SCD patients are prone to haemolysis due to the shape of their red blood cells and if they are deficient in G6PD, the haemolysis may escalate. Reported studies have indicated variations in the prevalence of G6PD deficiency in SCD patients and as such further work is required. The aim of this study was therefore to estimate the incidence of G-6-PD deficiency among SCD patients and to determine its impact on their RBC parameters as a measure of incidence of anaemia.Methods. A total of 120 clinically diagnosed SCD patients of genotypes HbSS and HbSC were recruited into the study. About 5ml of blood was collected via venipuncture from each patient and used to run G6PD, full blood count, and haemoglobin (Hb) electrophoresis tests. The data were analyzed using SPSS version 20 and Graphpad prism.Result. G6PD deficiency was detected in 43 (35.83%) of the participants made up of 16 (13.33%) males and 27 (22.50%) females of whom 17 (14.17%) had partial deficiency and 10 (8.33%) full deficiency. Statiscally significant differences p=0.036 and p=0.038 were established between the Hb concentration of the participants having a G6PD deficiency and those with normal G6PD activity for males and females, respectively.Conclusion. From the results obtained, it implies that G6PD deficiency may increase the severity of anaemia in SCD patients. There is therefore the need to screen all SCD patients for G6PD deficiency to ensure that their condition is not exacerbated during treatment.

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Paper-Based Assay for Quantification of HbS in Blood of Sickle Cell Disease Patients

Blood ◽

10.1182/blood.v124.21.1371.1371 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1371-1371

Author(s):

Nathaniel Z. Piety ◽

Xiaoxi Yang ◽

Bogdan R. Dinu ◽

Alex George ◽

Sergey S. Shevkoplyas

Keyword(s):

Image Analysis ◽

Sickle Cell Disease ◽

Real Time ◽

Sickle Cell ◽

Cell Disease ◽

Transfusion Therapy ◽

Blood Samples ◽

Blood Disorder ◽

Peripheral Ring ◽

The Difference

Abstract Introduction: Sickle cell disease (SCD) is a common inherited blood disorder caused by sickle hemoglobin (HbS) which, unlike normal adult hemoglobin (HbA), becomes insoluble and polymerizes under hypoxic conditions. Patients with SCD experience chronic hemolytic anemia, episodic pain crises and abnormal blood flow to critical organs that cumulatively result in significant illness and shortened lifespans for many. The severity of SCD varies significantly between patients, but for individuals the rate of adverse events is strongly correlated with intraerythrocytic concentration of HbS (%HbS). High per test costs and long turnaround times make conventional laboratory methods (e.g. Hb electrophoresis, HPLC, IEF) impractical for quantifying %HbS in real-time (e.g. during transfusion therapy). The objective of this study was to demonstrate that %HbS in blood could be quantified using our recently developed rapid, low-cost paper-based SCD assay [1]. Methods: Blood samples were obtained from SCD and sickle cell trait (SCT) patients at the Texas Children’s Hematology Center (Houston, TX). To perform the SCD assay a 20μL droplet of blood mixed with Hb solubility buffer (1:10 by volume) was dropped on chromatography paper. The resulting blood stain was digitized with a flatbed scanner (Canon USA Inc, Melville, NY) and analyzed using a custom image analysis code (The MathWorks Inc, Natick, MA). Conventional Hb electrophoresis was performed with the semi-automated Sebia Hydrasys 2 Scan system (Sebia Inc, Norcross, GA). Results: The difference in transport of Hb through the paper produced a blood stain with two parts: the area of the initial drop where polymerized HbS is retained (center spot) and the area where soluble Hb is wicked laterally (peripheral ring). The relative color intensity of the center spot and peripheral ring is related to the blood sample %HbS (Fig. 1). The image analysis code automatically isolates and calculates the ratio of the average color intensities of each area (S-index). A series of reconstituted blood samples with artificially adjusted %HbS from 0 to 60% was used to calibrate the assay so that %HbS could be estimated based on blood stain color intensities (Fig. 2a). The values of %HbS estimated for patient samples using our paper-based SCD assay and actual values measured using conventional Hb electrophoresis were highly correlated with R2 = 0.898 (Fig. 2b). The estimated and actual %HbS values also showed strong agreement with the standard deviation of the difference between the two measurements = 5.5 %HbS (Fig. 2c). The majority of the differences between actual and estimated %HbS (96.67%) are within 2 standard deviations of the mean of the differences. The assay could be performed in under 35 minutes and multiple assays could be performed and analyzed in parallel. The cost of consumable materials and reagents for the paper-based SCD assay is less than $0.03. Conclusions: This study demonstrates the feasibility of using our recently developed paper-based assay to quantify %HbS in blood samples in real-time. The ability to rapidly, inexpensively measure %HbS will be particularly useful for monitoring the effectiveness of chronic transfusion or hydroxyurea therapy for long-term control of HbS content in blood of SCD patients. The ability to measure %HbS in real-time could also potentially facilitate more aggressive prophylactic therapy to intervene rapidly and significantly reduce the rate of life-threatening complications in SCD patients, including stroke. Figure 1: Figure 1:. Figure 2: Figure 2:. Acknowledgments: This work was supported in part by a 2012 NIH Director's Transformative Research Award (NHLBI R01HL117329, PI: SSS). References: [1] Yang X, et al. Lab Chip, 2013, 13, 1464-1467. Disclosures Piety: Tulane University: PCT/US2012/064856 Patents & Royalties. Yang:Tulane University: PCT/US2012/064856 Patents & Royalties. Shevkoplyas:Tulane University: PCT/US2012/064856 Patents & Royalties; Halcyon Biomedical Incorporated: Equity Ownership.

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Mitophagy Induction Is a Potential Therapeutic Approach for Sickle Cell Disease

Blood ◽

10.1182/blood.v128.22.267.267 ◽

2016 ◽

Vol 128 (22) ◽

pp. 267-267

Author(s):

Ramasamy Jagadeeswaran ◽

Benjamin Alejandro Vazquez ◽

Vinzon Ibanez ◽

Maria A Ruiz ◽

Robert E Molokie ◽

...

Keyword(s):

Flow Cytometry ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Therapeutic Approach ◽

Research Funding ◽

Single Point Mutation ◽

Cell Disease ◽

Blood Disorder ◽

In Vivo Treatment

Abstract 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<0.0004]. We also made the novel observation that mature RBCs from SCD patients abnormally contain mitochondria as evidenced by flow cytometry analysis of blood samples of 36 SCD patients and 14 normal human control subjects.[Control (HbAA):0.4 % ± 0.04%, n=14; SCD (HbSS): 7.8%± 0.9%, n=30; p<0.0001]. Further subset analysis from SCD patients with HbSC showed mitochondrial retention in their mature RBCs [HbSC: 2.2%± 0.6%,n=6 p<0.01], however to a lesser degree than patients with HbSS. Transmission electron microscopy confirmed the presence of mitochondria in mature RBC of patients with SCD. ROS analysis between mitochondria positive vs. negative fractions showed that mitochondria-positive (TMRM+) RBC fractions have higher levels of ROS compared to mitochondria-negative (TMRM-) RBC fractions. This data strongly suggests that retained mitochondria significantly contribute to the production of ROS in SCD RBCs. Similar to humans, a higher fraction of RBCs of SCD mice (B6;129-Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB*)Tow/J) retain mitochondria compared to control mice RBC [Control (HbAA): 0.29% ± 0.18%; SCD (HbSS): 16.68%± 1.9%, p<0.0001]. While investigating RN-1, a lysine specific demethylase-1 (LSD-1) inhibitor, as a HbF inducing agent, we observed that SCD mice treated with RN-1 showed a reduction in the fraction of RBCs which retain mitochondria. Therefore, we investigated mitophagy-inducing drugs as a possible useful therapeutic approach for SCD by administering mitophagy-inducing agent Sirolimus. SCD mice treated with RN-1 (5mg), or Sirolimus (5mg) had a significant decrease in the fraction of mitochondria containing RBCs (RN1: 4.96± 1.0%, p<0.0005; Sirolimus: 6.4% ± 1.8%, p<0.002). We observed a reduction of ROS in mature RBCs coupled with decreased mitochondrial retention in RBCs after in vivo treatment with RN1 or Sirolimus as measured by co-staining of TMRM, APC-conjugated CD71antibody, and CM-H2DCFDA. We also observed a significant improvement in RBC survival after the in vivo treatment with Sirolimus or RN-1. RBC survival was measured by flow cytometry and calculated biotin positive circulating RBCs after 2 days of in vivo labeling [SCD treated with vehicle control: 40 %± 2.6%; SCD treated with RN1 (2.5mg): 69.9 ± 2.6%, p<0.004; Sirolimus (5mg): 67.5% ± 6.1%, p<0.04]. Based on this data, mitophagy-inducing drugs have the potential to be a novel therapeutic approach for the treatment of SCD patients. Disclosures Jagadeeswaran: Acetylon: Research Funding. DeSimone:EpiDestiny: Consultancy, Other: patents around decitabine and tetrahydrouridine. Lavelle:Acetylon: Research Funding. Rivers:Acetylon: Research Funding.

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Cathepsin B, a Negative Regulator of Autophagy, Identified As a Novel Therapeutic Drug Target in Sickle Cell Disease

Blood ◽

10.1182/blood-2020-140119 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 32-32

Author(s):

Jagadeesh Ramasamy ◽

Vinzon Ibanez ◽

Kareem Al-Qadi ◽

Helen Zhang ◽

Jennifer Afranie-Sakyi ◽

...

Keyword(s):

Sickle Cell Disease ◽

Western Blot ◽

Sickle Cell ◽

Cathepsin B ◽

Negative Regulator ◽

Differentially Expressed ◽

Therapeutic Drug ◽

Cell Disease ◽

Blood Disorder ◽

Autophagy Pathway

Sickle cell disease (SCD) is an inherited blood disorder that affects millions of people worldwide. The disease is caused by a mutation of the beta-globin gene that results in polymerization of the sickle hemoglobin (HbS) when deoxygenated. Reactive oxygen species (ROS) induced hemolysis is a major critical event in SCD. We have previously shown that abnormal retention of mitochondria in the erythrocytes of both SCD patients and SCD mice is associated with elevated ROS levels and hemolysis (Exp. Hem.2017;50:46-52). The mechanism responsible for mitochondrial retention in SCD is unknown. Autophagy is one of the processes responsible for the elimination of mitochondria during erythroid differentiation. Autophagy is a conserved physiological process that promotes cellular homeostasis through the recycling of proteins, protein aggregates, and removal of damaged organelles. Hypothesis: In this study, we have investigated the hypothesis that autophagy pathway dysregulation is responsible for abnormal mitochondrial retention in SCD erythrocytes. We propose that pharmacological methods that address the abnormal activity of these genes may be therapeutic for SCD. Methods: The expression of autophagy pathway genes in reticulocytes of SCD patients and experimental SCD mice was compared with their respective controls using the RT2 Autophagy Profiler PCR Array. The expression of autophagy proteins was measured by Western blot. Cathepsin B (CTSB) activity was measured by a fluorometric assay using the substrate RR-AFC (amino-4-trifluoromethyl coumarin). Results: Six autophagy pathway genes were differentially expressed in SCD patients compared to control subjects. Expression of the autophagy pathway genes was also analyzed in SCD mice, normal AA mice, and phlebotomized anemic AA mice as a control for increased reticulocytosis. Eighteen genes were differentially expressed in SCD mice compared with control AA mice (p<0.05). Four genes were differentially expressed in SCD mice compared with control phlebotomized anemic AA mice (p<0.05). CTSB, a known negative regulator of autophagy (J Exp Med. 2016;213(10):2081-97), was over-expressed in the reticulocytes of both SCD patients and SCD mice. Expression of CTSB was 4.5 fold (p<0.007) higher in SCD mice compared to normal control AA mice and 8.6 fold (p<0.001) higher compared to phlebotomized AA anemic mice. We therefore focused our further analysis on CTSB. Western blot data confirmed that CTSB protein was overexpressed in red blood cells of SCD patients and experimental SCD mice (p<0.01) compared to respective controls. The level of CTSB enzyme activity was 3.3 fold higher in SCD mice compared to the control mice(p<0.01). To evaluate the functional significance of these differences in CTSB expression, CTSB inhibitor E64d was investigated in SCD mice. SCD mice treated with E64d (40mg/kg b.wt.) for two weeks showed a reduction in CTSB activity (SCD vehicle: 11.8±1.1 RFU/µg, n=3 vs SCD E64d treatment: 5 ± 0.1 RFU/µg, n=3, p<0.008) and in the proportion of reticulocytes that retained mitochondria (SCD vehicle: 45.2%±3%, n=3 vs. SCD E64d treatment: 32 % ± 1.9%, n=3, p<0.003). Reticulocytosis was also significantly reduced in SCD mice treated with the CTSB inhibitor (SCD vehicle: 47.2%±2.8%, n=3 vs. SCD E64d treatment: 35.9 % ± 3.9%, n=3, p<0.01). Conclusion: Our data shows that the multiple autophagy pathway gene expression is dysregulated in SCD. Among these genes, CTSB, a key negative regulator of autophagy, is overexpressed in the reticulocytes of both SCD patients and SCD mice. Pharmacological inhibition of CTSB activity partially reverses mitochondrial retention and decreases reticulocytosis in SCD mice, confirming that overexpression of CTSB contributes to SCD pathogenesis and is a potential therapeutic target. Disclosures No relevant conflicts of interest to declare.

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