bluuebunny/crossref_metadata_2025 huggingface dataset:
Is HLA-Haploidentical Hematopoietic Stem Cell Transplantation a Valid Alternative Option for Patients with Combined Paroxysmal Nocturnal Hemoglobinuria and Aplastic Anemia (AA/PNH Syndrome)?
Authors: Chen Feng , Depei Wu , Tang Xiaowen , Miao Miao , Fu Chengcheng , Qiu Huiying , Xue Shengli , Ma Xiao , Jin Zhengming
Published: October 2019

Background In the era of eculizumab, indentifying patients with PNH who may benefit from allogeneic stem cell transplantation(SCT) is challenging, especially for those who have no HLA-matched donors. Several recent studies have shown that HLA-haploidentical SCT for patients with hematological malignancy can achieved comparable outcomes with HLA-identical sibling transplantation. There are very few reports on the use of HLA-haploidentical SCT for AA/PNH. The aim of the present study was to assess the long-term clinical outcome of HLA-haploidentical SCT in patients with AA/PNH.

Methods Total of 9 AA/PNH patients received HLA-haploidentical SCT between Oct 2010 and Oct 2014 at our institution. The patients were aged 13 to 54(median 24 years). The median interval from the diagnosis to transplantation was 48 months (range 2-180). Of the 9 HLA-haploidentical donors, 3 were siblings, 2 fathers, 2 mothers, 1 son and 1 daughter. 8 patients received myeloablative conditioning regimen consisting of busulfan, cyclophosphamide and ATG (anti-thymocyte globulin), 1 patient who underwent salvage HLA-haploidentical SCT after the graft failure of double umbilical cord blood transplantation received conditioning including reduced-intensity total body irradiation, cyclophosphomide and ATG. G-CSF-mobilized bone marrow and peripheral blood stem cells were transplanted as graft. Prophylaxis for graft-versus-host disease(GVHD) consisted of cyclosporine or tacrolimus + mycophenolate mofetil + short-term methotrexate.

Results All 9 patients were engrafted successfully. The median time of neutrophils (ANC) reached to 0.5×109/L and platelets (PLT) reached to 20×109/L were 12 (range 11-26) days and 15 (range 11-120) days, respectively. 2 patients developed grade Ⅱ acute GVHD, 2 patients developed limited chronic GVHD. After a median follow-up time of 14.0 (range 4.0-30.8) months, the 2-year OS probability was 72.9±16.5%. 2 patients died of treatment-related mortality, including severe pulmonary infection (n=1) and transplant-associated thrombotic microangiopathy (n=1),respectively. No patients were documented to have a recurrence of a PNH clone after SCT.

Conclusion This study showed that long-term outcomes of HLA-haploidentical SCT in patients with AA/PNH were comparable to that of HLA-matched donor SCT ( the 2-year OS probability was 80.5±10.2%, P=0.02) at our institution. HLA-haploidentical SCT should be considered as a valid alternative therapeutic option for AA/PNH patients without HLA-matched donors.

Disclosures

No relevant conflicts of interest to declare.
A Highly Sensitive Assessment Tool for the Gene Editing Efficiency of Sickle Cell Disease Hemoglobin Beta Gene
Authors: Borjigin Mandula , Kmiec Eric Brian , Wu Rigumula
Published: October 2019

In sickle cell disease, a single point mutation in hemoglobin β gene (HBB) results in the substitution of valine for glutamic acid at position 6 of the β globin protein sequence, causing the deformation of red blood cells into a sickle (or crescent) shape. With the development of powerful gene editing tools, scientists are initiating the correction of the point mutation of HBB gene in CD34+ hematopoietic stem cells and induced pluripotent stem cells. Although the results are very exciting, the evaluation method of the gene editing is primitive. Currently, the modification at the mutation site is identified and quantified using Restriction Fragment Length Polymorphism (RFLP), which involves PCR amplification, restriction enzyme digestion and gel electrophoresis. The accuracy of the gene editing efficiency depends heavily on the quantification of the DNA bands in the gel images, which is inherently imprecise. We have developed a novel technique to quantify the correction efficiency of HBB gene editing using a fluorescence tagging of the edited DNA sequence. This method provides excellent sensitivity and accuracy, and saves time and labor, eliminating a process of gel electrophoresis. We demonstrate the assessment of gene editing in HBB of K562 cells, in which the wild type HBB (βA gene) is converted to mutant βs using the gene editing tools (i.e. Transcription Activator-Like Effector Nucleases (TALENs) and single-stranded oligo deoxynucleotides (ssODNs)). We present limited information here due to the sensitivity of the intellectual property, but will discuss in detail the experimental procedures and data at the American Society of Hematology meeting.

Disclosures

No relevant conflicts of interest to declare.
Subcellular Localization of Beta Hemoglobin Using pSNAPf fusion Protein
Authors: Jaffar Samia , Metzenberg Aida , Adams Gregor B.
Published: October 2019

Abstract: Much of what is known about hemoglobin relates to its structure and function. However, there is a great deal to learn about its assembly. The application of new technologies has allowed us to investigate the process of hemoglobin synthesis extensively. It has been shown that heme, synthesized in the mitochondria, is exported into the cytoplasm where it rapidly binds with apoglobin synthesized by polyribosomes. In this study,subcellular localization of newly synthesized alpha and beta subunits of human hemoglobin were examined using fluorescence microscopic imaging of cultured human erythroleukemic cells (cell line HEL 92.1.7). Experiments were used to determine the location of newly synthesized protein subunits. This paper will highlight the current knowledge of hemoglobin assembly, function and cellular interactions; and illustrate how cellular labels can be used to investigate this protein. It is hoped that future experiments will help to determine more details of the mechanism of globin assembly, which will be useful in the investigation of hemoglobinopathies.

Disclosures

No relevant conflicts of interest to declare.
Actual Status of Inhibitor Hemophilia Management in Russia
Authors: Zozulya Nadezhda , Andreeva Tatiana , Vdovin Vladimir , Perina Farida , Parovichnikova Elena N. , Savchenko Valeri
Published: October 2019

Background: The number of inhibitor hemophilia patients in Russia is presently about 200 patients (4% of all hemophilia patients). Current medical community is moving towards increasing the efficacy and safety of treatment, as well as the standardization of services and improvement of standards of care in rare diseases area. Patient registries and databases are key tools for the improvement of patient care, healthcare resources planning as well as social, economical and quality of life outcomes. Due to the lack of data on clinical course of disease, treatment approaches and outcomes, National Hematology Society initiated continuously running prospective, multi-center, open-label, non-interventional, observational Registry of INhibitor Hemophilia patients (RING) in July 2014.

Aims: to document the natural history of hemophilia A or B disease in subjects with any type of inhibitors and to describe long-term treatment related outcomes in routine clinical practice.

Methods: Data of all inhibitor patients are collected by physicians from patients medical records documenting the routine patient's examination and reflecting the medical practice at the particular treatment center. All data are entered into e-database using on-line access secured by the Operator.

Results: In total 183 patients (101 adult and 82 children) from 47 centers were included in the Registry by June 2015. The majority of patients (90%) have severe hemophilia. The age of inhibitors diagnostics was: ≤10 years - 83%, 11-20 years - 3%, 21- 50 years - 5%, ≥51 years - 3% of patients. Chronic viral infections are diagnosed in 55 (30%) patients: 1 - HIV, 5- HBV and 49 - HCV. In 28% of patients annual bleeding rate (ABR) are ≥ 16, in 42% - 6-15, in 21% - ≤ 5. Approximately 25% of patients have zero joint ABR. Life threatening bleedings were registered in 25 patients (14%). Only 15 (8%) patients undergo immune tolerance induction (ITI); treatment with bypassing agents is administered in 151 (82%) patients. Due to the fact that activated prothrombin complex concentrate (aPCC) is not supplied by the State guarantees program 83% of patients receive rFVIIa (on-demand treatment - 50% and prophylaxis - 33%) and only 17% of patients have an access to aPCC treatment. The current data show that 34 (27%) patients with rFVIIa treatment need ≥3 injections to stop a bleeding episode thus they are identified as non-responders.

Conclusion: The clear view of current treatment situation based on available Registry data demonstrates the necessity of administering alternative treatment approaches for non-responding to rFVIIa inhibitor patients, which would optimize resources of the State guarantees program.

Disclosures

No relevant conflicts of interest to declare.
Lysosomal Newborn Screening in a Cohort in Mexican Population-
Authors: Navarrete Juana Ines
Published: October 2019

INTRODUCTION: The goal of newborn screening is an early detection of inborn erros of metabolism diseases. In Mexico we began newborn screening since 1977 with very few inborn errors of metabolism such as phenylketonuria, galactosemia, congenital hypothyroidism, sickle cell anemia and cytic fibrosis (1). Since that date we have been increasing our newborn screening our newborn screening slowly and now a days we screen in most states of the country 15 inborn errors of metabolism(2). In 2012 we started with some patients through out the country a wider neonatal screening that include 5 lysosomal storage diseases.

MATERIAL AND METHODS: Petróleos Mexicanos is a big governmental institution with approximately 10,000 workers and their families. Since 2005 a larger newborn screening has been done to all newborns in this institution through all the country. We test for most aminoacidopathies, including acidurias, hemoglobinopathies, G6PD deficiency, adrenal hyperplasia, cystic fibrosis and biotinidase deficiency; since August 2012 we included 6 lysosomal storage diseases; Gaucher disease, Fabry disease, Hurler disease, Pompe disease, Niemann-Pick type A and B disease and Krabbe disease.

RESULTS: Up to date we have screened 10,853 newborns, we have found 9 patients with lysosomal storage diseases. We found 4 newborns with mutations for Fabry disease, 4 newborns with Pompe disease, three were pseudodeficiencies and one was combined heterozygous for a late onset presentation and pseudodeficiencies and 1 patient with Hurler disease (Table 2). We present here our clinical correlation between genotype-phenotype in these patients. We found a frequency in our population of 1 in 2713 newborns for both Fabry and Pompe disease.

DISCUSSION: Newborn screening is a major public health achievement that has improve the morbidity and mortality of inborn errors of metabolism. The introduction of newborn screening for lysosomal storage diseases presents new challenges. This is the first latinamerican study of early detection of lysosomal storage diseases made by neonatal screening there are about 11 similar international studies. It is important point out that the most common lysosomal storage disease found in our study was Pompe diseases the pseudodeficiency type and Fabry disease type II with a frequency of 1 in 2713 newborns for both diseases. Spada et al; and Hwu et al; have reported frequencies of 1 in 1250 to 3100 male newborns. The mutation most commonly found was c.1088G>A, (p.R363H) for Fabry disease and c.1726G>A(p.G576S) for Pompe disease.

References: 1. Nakamura K, Am J Med Genet Part C, 2011; 157, 63-71. 2. Zhou et al, J. Pediatr 2011 159 1 7-13. 3. Alterescu GM, Clin. Genet 2001:60:46-51. 2001. 4. Desnick R. J.: Enzyme Replacement Therapy and Enhancement therapies for Lysosomal Storage Diseases. J. Inher Metab Dis 2004; 27:385-4013.

Disclosures

No relevant conflicts of interest to declare.
Acute Promyelocytic Leukemia, Are US Doctors and Hospitals Prepared? Tools and Know How, Yes; But, What about Readiness?
Authors: Morris Daniel
Published: October 2019

Acute Promyelocytic Leukemia (APL) is a hematologic emergency or urgency. It is a potential disaster that is also potentially curable. My research attempts to ascertain the readiness to care for patients who present with this (suspected) diagnosis and require urgent therapy.

I polled a sample of approximately 140 (United States) hematologist-oncologists as to the availability of Tretinoin/all-trans retinoid acid (ATRA) on a stat basis at the hospitals at which they practice and 18 responded. In other words, is the medicine on the shelf and can be given when ordered.

Available on the shelf and ready to give ATRA at all hospitals in which practiced: 2/18

Do not know: 3/18

Not available at all hospitals practiced: 13/18

Amazingly, one of the physicians stated that the family is given a prescription to get ATRA filled, and another responded that he had to "beg" to get the hospital to obtain it.

I posted two questions on a social media site in the Emergency Medicine category; This social media web site states that the total physician membership is 343,000 (verified). The responses are as follows:

Is ATRA on the shelf and available at the hospital they practice (17 responses to this question)

13/17 Do not know

2/17 On the shelf and available

2/17 Not on the shelf and would need to be ordered

Is APL a possible question for board exam in Emergency Medicine? (16 responses to this question)

9/16 No

4/16 Not sure

3/16 Yes

The price for ATRA according to www.uptodate.com is (Tretinoin Oral) 10 mg (100): $2988.16. Issues related to this topic include the cost, the supply (sold in quantities of 100), shelf life/expiration date, the agggregate cost to maintain it at every hospital based ER , and consideration that the patients are not all acutely ill and sometimes can wait a day before starting therapy. Even if this approach of not having ATRA in stock is not optimal, this approach is occurring.

Whether or not Joint Commission or the more voluntary Commission on Cancer should require that ATRA be on the shelf at a hospital, or available within a certain amount of time may merit further discussion.

Finding out how prepared we are to treat APL is not an easy undertaking and my effort seems to indicate that we probably do not have the wherewithal in many cases. Obtaining more data on the scope of the problem and perhaps devising a "Practice Guideline" by experts in the APL area dealing with the timeliness of availability of ATRA would seem to be in order. The lifesaving nature of the intervention would seem to justify the consideration of having hospitals maintaining a supply at all times. If the manufacturer would be willing to sell a supply of #30 (currently available in bottles of 100 per Facts and Comparison 2015 edition), this might be useful in implementing wider availability.

Disclosures

No relevant conflicts of interest to declare.
Exposure to New Therapies and Treatment Delay in Multiple Myeloma: Experience from a Safety Net Hospital
Authors: O'Brien Timothy , Ruda Barbara , Cluckey Andrew
Published: October 2019

Introduction:

Treatments for myeloma have evolved over the past several years with resultant improvements in survival. Two new medications, bortezomib (B) and lenalidomide (L), have played a major role in this development. Each has received FDA approval in the past 10 years. Both medications are relatively expensive and, due to its teratogenic effects, lenalidomide requires a pt training and education program before pharmacies may fill the prescription. In addition, specialty pharmacies need to be identified and prior approval is required from the insurance carrier. This study addresses whether or not a relatively underinsured, indigent population was able to receive these medications and in a timely fashion.

Methods:

Electronic medical records of all pts diagnosed with myeloma from 1/2005-12/2014 at MetroHealth Medical Center (a large safety net institution in Cleveland, OH) were reviewed.

88 pts were identified. Med age was 59 (35 -87); 50% were African American (AA), 43% caucasian (C), 1% Hispanic and 6% unknown. Health insurance status: 26% private; 35% medicare; 17% medicaid; and 22% were uninsured.

Results:

77% AA received either B or L or both vs 82% C pts (NS).

Overall, 17/88 (19%) never received either L or B. This included: 5 due to pt preference or co-morbidities which precluded treatment,; 2 who died before they could receive any therapy; 3 lost to follow-up before treatment could be started; 6 who received thalidomide and then either transferred care elsewhere, died or did not require another treatment; and 1 who transferred to another facility for stem cell transplant evaluation before treatment was initiated. Therefore, all 71 pts who were candidates and w/treatment data available received B or L. Of the 17 who did not get B or L: 6 were uninsured, 5 private insurance, 4 medicare, and 2 medicaid.

Overall, 66 pts received B; 31 received L.

Ave time from prescription to pt start of medication: 19.1 days for L (med 14.5d, range 0-65d) vs 1.56d for B (med 0d, range 0-20d), p<0.00001. The pts treated with L were not overrepresented from any particular insurance status (29% were uninsured; 19% medicaid; 29% medicare; 23% private). This payor mix was similar for those who received B (21% uninsured; 13% medicaid, 36% medicare and 30% private).

Conclusions:

In this underinsured, indigent population, all appropriate pts with myeloma received at least one of the 2 novel drugs. There was a statistically significant delay in treatment for those pts who were prescribed lenalidomide as compared to bortezomib (19.1 days L vs 1.56 days for B). It is not clear that this is a clinically significant difference but suggests that bortezomib containing regimens may be a better option in cases where urgent therapy is needed, particularly in this population.

Disclosures

No relevant conflicts of interest to declare.
COL4A1 is a Novel Causative Gene Responsible for Congenital Hemolytic Anemia, Representing Characteristic Clinical Course in Infants
Authors: Ogura Hiromi , Ohga Shouichi , Aoki Takako , Utsugisawa Taiju , Takahashi Hidehiro , Iwai Asayuki , Watanabe Kenichiro , Okuno Yusuke , Yoshida Kenichi , Miyano Satoru , Ogawa Seishi , Kojima Seiji , Kanno Hitoshi
Published: October 2019

We have been working on the differential diagnosis of congenital hemolytic anemia, but even with extensive analysis of hemoglobin, red cell membrane and enzymes, approximately 40% of patients remained to be diagnosed. In this study, we analyzed 17 undiagnosed hemolytic anemia subjects under the age of 1 by whole-exome sequencing, and identified COL4A1 gene mutations in 5 cases (29.4%). All patients were de novo cases without family histories and exhibited moderate to severe neonatal hemolytic anemia: Hgb, 5.2-9.3 g/dl; MCV, 90.0-126.9; MCHC, 29.9-32.7; and reticulocyte count, 9.2-33.0%. Either schizocytes or poikilocytes were observed in peripheral blood smears of 3 cases, suggesting that the microangiopathy was attributable to hemolysis. Previous reports showed that mutation of COL4A1 accounts for brain small-vessel disease characterized by stroke and eye abnormalities and the most characteristic complications of the present cases were congenital anomaly in the central nervous system, such as porencephaly, schizencephaly, congenital hydrocephalus, cataracts or paraventricular calcification, as reported previously. Hemolytic anemia became less severe within 2 months after birth, and all cases no longer required red cell transfusion after Day 50. COL4A1 encodes subtype 1 of type IV collagen, which is most abundantly expressed in basement membranes, including the vasculature. The COL4A1 gene mutations identified in the cases were all novel missense mutations except one, located in exons 26, 27, 37, 38 and 51. Although the pathophysiological significance of the mutations remains unclear, COL4A1 is the first identified causative gene responsible for congenital hemolytic anemia without intrinsic defects of red blood cells, and mutation of COL4A1 is the most prevalent cause of neonatal hemolytic anemia.

Disclosures

No relevant conflicts of interest to declare.
Deep Proteomic Analysis of Human Erythropoiesis
Authors: Ducamp Sarah , Gautier Emilie-Fleur , Leduc Marjorie , Salnot Virginie , Dussiot Michael , Guillonneau François , Giarratana Marie-Catherine , Douay Luc , Lacombe Catherine , Verdier Frederique , Zermati Yael , Mayeux Patrick
Published: October 2019

*the first two authors are co-first authors

Introduction. Erythropoiesis is a complex process starting from pluripotent medullary progenitors and leading to the production of highly specialized and enucleated erythrocytes. Two successive phases are generally distinguished: an amplification phase with intense proliferation of morphologically similar progenitors and a terminal differentiation phase with few cell divisions and strong cellular modifications. Although erythropoiesis is a continuous process, these modifications allow the identification of specific maturation stages and the passage from one stage to the following one seems to correlate with a cell division. Several transcriptomic analyses of erythroid differentiation have been published but only few and very limited proteomic studies have been reported. Since post transcriptomic modifications are responsible for a large part of the proteome variations, a direct proteomic analysis of the erythroid differentiation is required to accurately assess the modifications that occur during this process.

Results. For this study, we used CD34+ cord blood progenitors and an optimized three step cell culture method allowing the production of highly synchronized cell populations of erythroid cells at various differentiation stages. Several cellular populations from erythroid progenitors up to reticulocytes were analyzed by a label-free analysis and mass spectrometry that led to the absolute quantification of more than 6000 proteins with a false discovery rate of less than 1% (n=3). Moreover, the relative expression of well-known stage-specific erythroid markers such as TFRC, BAND3 or GLUT1, transcription factors, heme biosynthesis enzymes followed the expected pattern.

To complete this study, we performed a quantitative analysis of the repartition of proteins between the generated reticulocytes and the expelled nucleus (pyrenocyte). To do that, pyrenocytes and reticulocytes were sorted by FACS according to size, Hoechst 33342 and glycophorin A labelling. Equal numbers of reticulocytes and pyrenocytes were used to prepare peptides that were analyzed by mass spectrometry after iTRAQ labelling. These experiments allowed the quantitative repartition of 1153 proteins including most erythrocyte-specific membrane proteins.

Conclusion. All these results significantly increase our knowledge of the protein expression pattern during erythropoiesis and should constitute a valuable data base for subsequent studies regarding both physiological and disordered erythropoiesis.

Disclosures

No relevant conflicts of interest to declare.
Induction of Fetal Hemoglobin and Reduction of Disease Pathology in Sickle Cell Mice By a Synthetic Zinc Finger Gamma-Globin Activator
Authors: Peterson Kenneth R , Makala Levi C , Takezaki Mayuko , Barbas Carlos F , Pace Betty
Published: October 2019

A plethora of research has established that the most effective treatment for sickle cell disease (SCD) is increased fetal hemoglobin (HbF). Fetal hemoglobin normally accounts for less than 0.5% of total hemoglobin in adults; increasing levels to approximately 10% alleviates much of the pathophysiology associated with SCD. Hydroxyurea is the only FDA-approved treatment for SCD that results in enhanced HbF production, but this drug is highly pleiotropic in its action and does not exclusively modulate γ-globin gene expression. Thus, research has focused on identifying agents that specifically reactivate γ-globin gene expression during adult definitive erythropoiesis, with minimal off-target effects. Artificial transcription factors (ATFs) are synthetic proteins designed to bind at a specific DNA sequence and modulate gene expression. The artificial zinc finger gg1-VP64 was designed to target the -117 region of the A γ-globin gene proximal promoter and activate expression of this gene. Previous studies demonstrated that HbF levels were increased in K562 cells, murine chemical inducer of dimerization (CID)-dependent bone marrow cells carrying a human β-globin locus yeast artificial chromosome (β-YAC) transgene, in CD34+ erythroid progenitor cells from normal donors and β-thalassemia patients, and in vivo, in gg1-VP64 β-YAC double transgenic (bigenic) mice. Transgenic mice with enforced expression of the gg1-VP64 fusion protein only in the erythroid-megakaryocytic compartment were crossed into the Townes sickle cell knock-in mouse (Jackson Laboratory) background. Compared with control sickle cell (HbSS) mice, gg1-VP64 ATF sickle cell (gg1-HbSS) mice had hematological values at levels found in wild-type homozygous or heterozygous adult hemoglobin (HbAA or HbAS, respectively) mice. For example, average RBC (106/mm3) was 11.7 for wild-type mice and 12.9 for gg1 HbSS, compared to 8.2 for HbSS mice. Average HGB (g/dl) was 15.1 for wild-type mice and gg1 HbSS mice, versus 10.0 for HbSS mice. Average HCT was 52.5% for wild-type mice, 53.7% for gg1 HbSS mice, but only 41.5% for HbSS mice. Finally, average WBC (103/mm3) was 9.4 for wild-type mice, 9.0 for gg1 HbSS mice and 91.0 for HbSS mice. HPLC and Western blot analysis to determine the effect of gg1-VP64 on HbF synthesis are underway. In addition, we are examining mice for numbers of HbF-positive cells, mature cells, and reticulocytes, as well as looking at organ damage. Our results demonstrate that the ATF class of reagent may be an effective gene therapy for treatment of SCD.

Disclosures

Makala: Georgia Regents University: Employment.
Sustained Elevation of Adenosine-ADORA2B Signaling Promotes Chronic Pain through Neuro-Immune Interaction in Sickle Cell Disease
Authors: Adebiyi Morayo G. , Hu Xia , Luo Jialie , Sun Kaiqi , Le Thanh-Thuy T. , Zhang Yujin , Wu Hongyu , Liu Hong , Huang Aji , Kellems Rodney E. , Blackburn Michael R. , Walters Edgar T. , Hu Hongzhen , Xia Yang
Published: October 2019

Sickle cell disease (SCD) is a common inherited hemolytic disorder that can cause severe health complications. One of the main complications of SCD is chronic pain. The molecular mechanism behind SCD chronic pain is poorly understood and effective mechanism-based treatments for SCD chronic pain are extremely limited. Recent studies show that an accumulated adenosine level induces sickling and disease progression by activation of adenosine A2B receptor (ADORA2B) (Zhang et al. 2011, Nature Medicine). However, the functional role of persistently elevated adenosine signaling in SCD chronic pain is undetermined. Here we report that SCD mice have persistent mechanical and thermal hypersensitivity due to sustained elevated circulating adenosine by activation of ADORA2B. Extending from SCD mice, we further demonstrated that elevated circulating adenosine contributes to chronic pain behavior in two additional animal models independent of sickling, mice lacking adenosine deaminase (ADA), an enzyme necessary for the breakdown of adenosine, and mice with Complete Freund's Adjuvant pawn injection, a well-accepted inflammatory chronic pain model, by activation of ADORA2B. Mechanistically, we revealed that adenosine does not directly activates neuronal cells. Instead, elevated adenosine activating ADORA2B on myeloid-derived immune cells causes nociceptor hyperexcitability and promotes chronic pain via IL-6-soluble IL-6 receptor trans-signaling cascade. Overall, our findings have identified that prolonged elevated adenosine promotes the cross talk between immune and neural cells and contributes to chronic pain in three independent animal models. These findings add significant new insight to molecular basis for chronic pain and immediately suggest novel therapeutic possibilities for anti-chronic pain.

Disclosures

No relevant conflicts of interest to declare.
Heterogeneity in the Making of Blood
Authors: Scadden David T.
Published: October 2019

It is increasingly clear that the bone marrow is comprised of a heterogeneous complex of niches for hematopoietic cells, some for stem cells in the perivascular space and some for progenitors. We have used two approaches to define the role of specific cells in the marrow. First, single cell selection and characterization based on in vivo proximity to HSPC. This method has defined a subset of endosteal lining cells that can be immunophenotypically defined and isolated and reveals IL-18 as a regulator of hematopoietic progenitor quiescence. Second, candidate cell depletion that revealed mature osteolineage cells expressing osteocalcin as regulating the production of thymic emigrants through the expression of Dll4. Deletion of these cells reduces the number and function of T-biased lymphoid progenitors in the marrow space as well as thymic populations and mature T cells in the blood. These data suggest that specific niche subsets can be defined and through them, novel molecular regulators of HSPC function. The bone marrow niche is a heterogeneous composite of distinctive niches.

Disclosures

No relevant conflicts of interest to declare.
Role of Red Cells in Thrombosis and Hemostasis
Authors: Weisel John W.
Published: October 2019

Until recently it has been assumed that red blood cells (RBCs) play mainly a passive role in hemostasis and thrombosis, but now some studies have uncovered several novel roles for them in both of these processes. The best-known effects of RBCs in clotting in vivo are rheological, involving laminar shearing plus aggregation and deformability of RBCs. In addition, RBCs have specific receptors for platelets and fibrin(ogen) that may be involved in thrombosis. Furthermore, in the contraction of blood clots, the trapping of RBCs in the platelet-fibrin network and subsequent physical changes may be important for hemostasis and thrombosis. Contracted blood clots develop a remarkable structure, with a meshwork of fibrin and platelet aggregates on the exterior of the clot and a close-packed, tessellated array of compressed polyhedral RBCs within. The structure and properties of contracted clots vary depending on the relative amounts of platelets, fibrinogen and RBCs and the conditions of clotting. Such close-packed arrays of polyhedral erythrocytes, or polyhedrocytes, have also been observed in human arterial and especially venous thrombi taken from patients. The mechanical nature of this RBC shape change was confirmed by polyhedrocyte formation from the forces of centrifugation of blood without clotting. Many experiments suggest that polyhedrocytes are more prevalent under conditions associated with thrombosis and could be a marker of prothrombotic conditions. Consistent with this hypothesis, RBC membranes have been found to be a major source of exposure of phosphatidylserine, which forms a procoagulant surface to generate thrombin. Platelets (with their cytoskeletal motility proteins) and fibrin(ogen) (as the substrate bridging platelets for contraction) are required to generate the forces necessary to segregate platelets/fibrin from RBCs and to compress these cells into a tightly packed array. Clots formed after puncture of the mouse saphenous vein are composed primarily of polyhedrocytes, suggesting that they may be important in venous hemostasis. Moreover, the permeability of contracted clots is nearly the same as that of the endothelial cell lining, consistent with their functional significance. These results demonstrate how contracted clots form an impermeable barrier important for hemostasis and wound healing and also to restore flow past obstructive thrombi. On the other hand, they also help explain how fibrinolysis is greatly retarded after clots contract, as a result of these striking structural changes. In summary, RBCs may perform a dual role, both helping to stem bleeding but at the same time contributing to thrombosis in a variety of ways, including when the tightly packed array of RBCs in a contracted clot blocks blood flow and resists dissolution.

Disclosures

No relevant conflicts of interest to declare.
Therapeutic Strategies for the Treatment of Telomere-Associated Diseases
Authors: Blasco Maria A.
Published: October 2019

Aplastic anemia is a rare but life-threatening disorder often accompanied by the presence of short telomeres in the hematopoietic compartment in both the acquired and the inherited form. We recently generated a mouse model that faithfully recapitulates the bone marrow phenotype observed in aplastic anemia patients. In this model, incomplete conditional Trf1 gene deletion specifically in the hematopoietic system causes acute telomere uncapping and persistent activation of a DNA damage response at telomeres, leading to a fast elimination of those HSCs lacking Trf1 and a compensatory hyperproliferation along with rapid telomere shortening in those which retain intact Trf1. Thus, this model recapitulates the high turnover and hyperproliferation observed in aplastic anemia patients of autoimmune origin, as well as presence of very short telomeres owing to mutations in telomere maintenance genes. We are currently employing this model to test two different therapeutic strategies for the reactivation of telomerase in the hematopoietic system, therefore counteracting telomere attrition and alleviating the aplastic anemia phenotype owed to the presence of short telomeres. First, we explored the possibility that the recently described regulation of telomerase activity by sex hormones may be the responsible mechanism for the frequently observed induction of remission in aplastic anemia patients following hormone therapy. We show that androgen therapy results in telomerase up-regulation, improved blood counts, and a significant extension of life-span in our mouse model. Importantly, longitudinal follow-up studies revealed longer telomeres in peripheral blood in mice subjected to hormone treatment. In addition to this chemical approach for telomerase activation we are also testing the efficacy of increasing telomerase gene dosage by virtue of AAV9 gene therapy. Our preliminary data indicate that AAV9-Tert treatment leads to a significantly reduces aplastic anemia related morbidity which coincides with telomere elongation in blood and bone marrow cell compared to empty vector treated mice. In summary, we used a mouse model of aplastic anemia produced by short telomeres and demonstrate two different strategies of telomerase activation which are effective in treating the disease. Thus this work may pave the way for the development of novel treatment options for aplastic anemia in humans.

Disclosures

No relevant conflicts of interest to declare.
Clonal Heterogeneity and Genetic Instability of Multiple Myeloma and Impact on Treatment
Authors: Keats Jonathan J
Published: October 2019

Multiple myeloma is a pathological definition for a series of distinct genetic entities with similar phenotypic and clinical characteristics. Multiple studies have now identified distinct subtypes of the disease, which are associated with different clinical outcomes. The genetic complexity underlying these different subtypes is very diverse. Some subtypes like those characterized by immunoglobulin translocations targeting cyclin D1 are associated with very minimal changes or no copy number changes. Conversely, subtypes defined by translocations targeting WHSC1/MMSET or the MAF and MAFB transcription factors often have highly complex and diverse copy number changes. With the substantial advances in DNA sequencing technology we now know there is a diverse array of somatic mutations in multiple myeloma tumors. Through the integration of copy number changes and somatic mutations multiple groups have now shown the existence of multiple co-existing subclones within individual tumors. Additional studies following patients through their individual disease courses have shown these subclones can ebb and flow with time through multiple rounds of therapeutic selection. This session will highlight our current understanding of how the interplay between tumor evolution and clonal heterogeneity should influence our treatment decisions, particularly when applying a personalized medicine approach.

Disclosures

No relevant conflicts of interest to declare.
Checkpoint Blockade with Neoantigen Cancer Vaccines for Personalized Immunotherapy
Authors: Wu Catherine J.
Published: October 2019

Methods for harnessing the exquisite specificity of the immune system to eliminate tumors have been under development since the start of the 20th century, with numerous studies in animal models and trials in humans. Despite accumulating evidence, there has been widespread skepticism in the cancer community about whether tumors could be controlled by spontaneous immunity, and whether effective immunotherapy would be feasible to develop. However, the field has now passed an inflection point as new results have quelled these doubts, including: (i) several large clinical studies that have identified infiltrating CD8+ T cells as one of the best predictors of overall survival in human cancers, with strong validation in a large study of colorectal cancers; (ii) adoptive T cell therapy that effectively eradicates some types of B cell leukemias and (iii) novel checkpoint blockade (CPB) therapies inducing unprecedented long-term cures for patients with solid tumors, and more recently with blood malignancies. With the availability of these novel immunologic agents, the priority has shifted to understanding the mechanisms of and predicting responses to each treatment. Fundamental to this endeavor is the understanding of the crosstalk between tumor cells and immune cell populations within the tumor microenvironment and the selective pressure they mutually exert on each other. At the heart of cancer and host immune cell coevolution is the tumor antigen and host antigen-specific T cell interaction. The cytotoxic T cell-cognate antigen interaction forms the mechanistic basis for immune-mediated recognition and the killing of malignant cells, and provides selective pressure on the cancer cell to evolve. While the search for immunogenic tumor antigens has been the subject of decades-long studies, multiple lines of evidence have convincingly demonstrated tumor neoantigens as an important class of immunogenic tumor antigens. Neoantigens arise from amino acid changes encoded by somatic mutations in the tumor cell and have the potential to bind to and be presented by personal HLA molecules. With the recent availability of next-generation sequencing, systematic approaches to identify mutations leading to amino acid changes have become feasible. This development, together with the improved sensitivity of well-validated neural network/machine learning-based algorithms that predict binding to class I MHC, has enabled the implementation of neoantigen discovery pipelines. Recent studies by us and others in humans and mice have leveraged these tools to demonstrate evidence of protective immunity when targeting tumor neoantigens. We have further incorporated this approach to discover antigen targets to generate a multi-epitope personalized therapeutic cancer vaccine, that we are now testing in phase I clinical trials. Altogether, effective targeting of tumor neoantigens occurs spontaneously and during CPB, and offers prospects of a powerful, new immunotherapy.

Disclosures

No relevant conflicts of interest to declare.
High Resolution Mapping of Human Lymphopoiesis Reveals a Common Lymphoid Progenitor (CLP) Population
Authors: Saxena Nivedita Shankar , Davis Kara L. , Inlay Matthew Arthur , Junger Astraea , Yang Yujun , Krampf Mark L. , Dejene Beruh , Nolan Garry P. , Weissman Irving L , Weinberg Kenneth I.
Published: October 2019

In vivo reconstitution studies have established the phenotype of murine CLP, a progenitor population with restricted developmental potential for T, NK, B lymphoid and plasmacytoid dendritic cell (pDC) lineages. Co-transplantation of murine CLP with purified HSC more rapidly restores lymphopoiesis, immune function and resistance to viral challenge in experimental models of HSC transplantation. Thus, the identification of human CLP would have translational impact in clinical HSCT. Based on the expression of IL-7Rα (CD127) as a key distinguishing marker of murine CLP, we studied the CD127+ subset of immature CD34+ Lin- CD38+ cells for lymphoid committed progenitors, comprising approximately 0.1-0.2% of total marrow. Like murine CLP, the CD34+ Lin- CD38+ CD127+ cells also expressed Kit and Flt3. OP9 stromal cells were transduced with a vector expressing a doxycycline inducible delta-like ligand 1 (dll1) gene to test the differentiation potential in a single-well switch culture. Progenitors co-cultured with uninduced OP9Δdll1 cells can differentiate into B and myeloid cells; after induction of dll1, the culture is permissive for T and NK differentiation in the same well. In the presence of Kit and Flt3 ligands, erythromyeloid growth factors (IL-6, IL-3, GM-CSF, EPO, TPO) and lymphoid factors (IL-7, IL-15), CD34+ Lin- CD38- CD127- HSC generated erythroid (E), myelomonocytic (MM), myeloid DC (mDC), T, NK, and B lymphocytes and pDC. In contrast, CD34+ Lin- CD38+ CD127+ cells generated T/NK/B/pDC progeny, distributed in varying proportions, but did not generate E/MM/mDC. Singly plated CD34+ Lin- CD38+ CD127+ cells generate T/NK/B cells in the same well, consistent with these cells being human CLP. CD34+ Lin- CD38+ CD127- "non-CLP" generated E/MM/mDC, but not T/NK/B/pDC progeny. Limiting dilution analyses under lymphoid conditions demonstrated that the CD34+ Lin- CD38+ CD127+ CLP had approximately 2-3-fold greater efficiency of lymphocyte generation than HSC under the same conditions. Transcriptional profiling of the human CLP showed patterns of gene expression distinct from that of HSC, CD34+ Lin- CD38+ CD127- progenitors and immature CD3-CD4-CD8- thymocytes. Approximately 50% of the CD34+ Lin- CD38+ CD127+ CLP also express CD10, a subpopulation which CyTOF analyses revealed to have other markers of B-cell commitment and/or differentiation, e.g., CD179a. Human CD34+ Lin- CD38+ CD127+ marrow cells are a unique multipotent, lymphoid-restricted progenitor population for T/NK/B/pDC cell development, and within which subsets of cells biased for individual lymphoid lineages may exist. Identification of human CLP will permit further analyses of lymphoid specification, commitment to lymphoid lineages, and potential adoptive transfer as a short-term source of polyclonal lymphocytes.

Disclosures

Nolan: Fluidigm: Consultancy.
Dual-Method Clone Tracking in Nonhuman Primates Confirms Long-Term Hematopoietic Reconstitution Initiated By Early Engrafting Clones
Authors: Adair Jennifer E , Norgaard Zachary K. , Haworth Kevin G , Schefter Lauren E , Tam Kenric , Porteus Matthew , Kiem Hans-Peter
Published: October 2019

Summary:

Several models of hematopoietic reconstitution after transplant have been proposed with multiple methods of tracking individual clones in both animal models and patients. Lentivirus-mediated gene therapy is the most widely applied strategy for marking individual cells, tracked by either provirus integration site analysis (ISA) or by proviral DNA barcode sequencing (DBS). Two previous studies have applied one of each of these methods to track hematopoiesis in a nonhuman primate model of myeloablative autologous transplantation. The report using ISA described long-term, multi-lineage hematopoietic clones emerging at 1 year after transplantation [Kim S. et al., Cell Stem Cell, 2014]. The report using DBS described only early engraftment up to 9 months after transplant, precluding long term analysis [Wu C. et al., Cell Stem Cell, 2014]. We applied a combination of ISA and DBS methods to follow longitudinal hematopoiesis in a total of six nonhuman primates following autologous transplant in the myeloablative setting, tracking >120,000 individual clones for up to 10 years. We measured hematopoietic lineage reconstitution, as well as spatial and temporal contributions of individual clones within the bone marrow niche and peripheral blood. Here we applied a stringent definition of the hematopoietic stem cell (HSC) phenotype in vivo (i.e. shared clonal identity between short-lived granulocytes and long-lived B cells with multiple time points of detection), to assess HSC contributions after engraftment. Strikingly, we demonstrate sustained contribution of HSC clones occurs as early as 1 month after transplant and that minor clonal waves continuously emerge when the measureable clonal pool is small in size, but not when the initial engrafting clonal pool is robust. Nearly one half of all HSC clones identified over the length of follow-up were detectable in the periphery within a few months after transplant by both ISA and DBS methods. However, we also identify technical differences between these clone tracking methods that can obscure biological interpretation. This study demonstrates an early and robust phase of multi-lineage hematopoietic reconstitution after myeloablative transplantation in the nonhuman primate, suggesting a single subpopulation of CD34+ cells which maintains clonal stability over the lifetime of the recipient in the autologous setting.

Disclosures

Adair: Rocket Pharmaceuticals: Consultancy, Equity Ownership. Porteus:CRISPR Therapeutics: Consultancy, Equity Ownership. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Research Funding.
GATA Factor Switching during Erythroid Differentiation Is Facilitated By FBW7 Mediated Clearance of GATA2
Authors: Ghamari Alireza , Pregerning Gabriela , Fraenkel Ernest , Cantor Alan B.
Published: October 2019

Erythroid differentiation is controlled by the dynamic exchange of GATA family transcription factors. During early erythroid maturation, high GATA2 levels activate progenitor genes such as c-kit-, c-myc, and GATA2 itself. In contrast, GATA1 levels are low in early progenitor cells, but rise during terminal maturation. During this process GATA1 turns off GATA2 controlled early progenitor genes and activates terminal maturation genes, such as globin genes, heme biosynthesis enzymes, and iron transporters. This involves the exchange of GATA1 for GATA2 at key chromatin sites, the so-called "GATA factor switch". GATA factor switching is facilitated by the much shorter half-life of GATA2 (~30-60 min) compared to GATA1 (>4-6 hrs). We and others recently demonstrated that the E3 ubiquitin ligase adaptor protein FBW7 contributes to GATA2's relative instability. This prompted us to dissect the role of FBW7 during GATA switching and erythroid differentiation. We deleted the Fbw7 gene using CRISPR/Cas9 gene editing in the inducible G1-ER murine erythroid cell line. This resulted in the delayed clearance of GATA2 during differentiation. RNA-seq analysis at an early time points (7 hr) demonstrated impaired repression of GATA2 regulated genes and reduced activation of GATA1 target genes. Globally, altered gene expression was enriched for GATA factor switch genes. This ultimately resulted in delayed erythroid maturation. We also found that Fbw7 mRNA transcript levels increase during erythroid maturation in wild type cells. We identified a site ~40kb upstream of the Fbw7 gene transcriptional start site, which is itself a GATA factor switch site. We propose that FBW7 facilitates GATA factor switching by promoting the clearance of GATA2 from GATA factor switch sites. Moreover, we suggest that GATA factor switching at the Fbw7 locus itself reinforces the commitment of erythroid cells to terminal maturation, by enhancing the clearance of GATA2 and other Fbw7 progenitor target gene proteins such as c-Myc and c-Myb. As Fbw7 recognition of GATA2 requires phosphorylation of GATA2's degron motif, this suggests that signaling pathways, acting through Fbw7, may modulate erythroid maturation kinetics.

Disclosures

No relevant conflicts of interest to declare.