bluuebunny/crossref_metadata_2025 huggingface dataset:
Next-Generation Sequencing: A Discovery Tool for Blood Disorders
Authors: Mardis Elaine R. , Ding Li , Westervelt Peter , Welch John S. , Klco Jeffery M. , DiPersio John F. , Wilson Richard K , Ley Timothy J.
Published: October 2019

Abstract SCI-10

The advent and evolution of next-generation massively parallel sequencing (MPS) has radically altered our approaches to studying the cancer genome and transcriptome. By using unbiased and comprehensive MPS in the context of clinically annotated samples from leukemia cases, rapid progress has resulted in our understanding of the mutational spectrum of hematopoietic malignancies, the heterogeneity of disease presentation, and the impact of chemotherapy on the cancer genome, among others. The complexity of the transcriptome, while daunting from an analytical standpoint, further reveals the nuances of gene expression changes in leukemia that often cannot be predicted simply by studying the genome. By applying the digital nature of MPS to explore tumor heterogeneity and tumor evolution, we have shown that de novo acute myeloid leukemia (AML) presents either as a mono- or multiclonal disease, and that the relapse presentation in the same patient is an evolved genetic derivation of the de novopresentation, often with novel driver mutations that have been acquired during the course of chemotherapy (1). New data from whole genome sequencing of hematopoietic stem cells in healthy volunteers indicates that somatic mutations largely are acquired during aging and occur randomly, carrying forward in the transformed cell. This baseline is important for the further comparison of AML subtypes, and provides a context for understanding tumor biology. Last, by studying 200 AML cases using whole-genome and exome sequencing, RNA-seq, miRNA-seq and array-based methylation, we have begun an integrated characterization of AML in an effort to inform tumor biology. These studies and the accompanying technologies set the stage for precision treatment of each AML patient according to the additional information provided by the person's integrated “omic” profile.

Disclosures:

No relevant conflicts of interest to declare.
von Willebrand Factor-ADAMTS13 Interactions
Authors: Zheng X. Long
Published: October 2019

Abstract SCI-18

ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) is a plasma metalloenzyme that cleaves von Willebrand factor (vWF), thereby limiting excessive platelet adhesion and aggregation and attenuating the rate of thrombus formation at the site of injury. Severe deficiency of plasma ADAMTS13 activity results in thrombotic thrombocytopenic purpura (TTP), a potentially fatal syndrome, while mild to moderate deficiency of plasma ADAMTS13 activity is associated with an increased risk for myocardial infarction and stroke. Adamts13 null mice in the ApoE null background exhibit a dramatically increased systemic inflammation and early atherosclerosis. Thus, ADAMTS13 plays a crucial role in pathogenesis of arterial thrombotic disorders such as TTP and chronic inflammatory diseases such as atherosclerosis. vWF is the only known substrate for ADAMTS13 to date. Structure-function analysis demonstrates that the N-terminal portion of ADAMTS13 (MDTCS) is sufficient for proteolytic cleavage of vWF, while the C-terminal portion of ADAMTS13 (T2–8 and CUB) inhibits shear-dependent platelet aggregation via free thiols (-SH). The proteolytic cleavage of vWF by ADAMTS13 is dramatically accelerated by binding of Factor VIII (FVIII) and/or platelets under shear stress. A B-domainless FVIII (FVIII-SQ) or a light chain of FVIII (FVIII-LC) appears to be sufficient for the enhanced proteolysis of vWF by ADAMTS13 in vitro and in vivo using a murine model. In contrast, a heavy chain of FVIII (FVIII-HC) or a light chain lacking an acidic region (a3) (FVIII-LC#x2610;¢a3) exhibits no rate enhancing effect under the same conditions. These results suggest that the light chain, particularly the acidic (a3) region that contains a high-affinity vWF binding site is required for the cofactor activity in accelerating vWF proteolysis. Consistent with this hypothesis, mutations in the D'D3 domain of vWF naturally occurring in patients with type 2N von Willebrand disease that reduces FVIII binding also impair the FVIII-dependent proteolysis by ADAMTS13 under fluid shear stress. To develop novel therapeutics, we have constructed, expressed, and screened for a panel of ADAMTS13 variants with a modification of the exosites (motif A and motif B) in the spacer domain. We identified several ADAMTS13 variants that have enhanced specific activity and are also resistant to inhibition by autoantibodies against ADAMTS13 in patients with acquired idiopathic TTP. These gain-of-function and autoantibody-resistant ADAMTS13 variants may be further developed as potential therapeutics for acquired TTP with inhibitors.

Disclosures:

No relevant conflicts of interest to declare.
Discovery Science and Integrating Molecular Diagnosis into Clinical Practice
Authors: Ebert Benjamin L.
Published: October 2019

Abstract SCI-31

Recent discoveries have yielded profound insights into the genetic basis of myeloid malignancies. A central challenge is now to translate these discoveries into clinical practice. Genetic lesions are powerfully associated with specific aspects of clinical phenotype, including hematologic parameters, particular morphologic abnormalities, response to therapy, and overall survival. Moreover, gene expression profiling, evaluation of microRNA expression, and epigenetic analyses add further biological, prognostic, and predictive information. Current research studies aim to integrate existing clinical prognostic scoring systems with molecular data to refine the taxonomy of myeloid malignancies, to improve the prediction of prognosis and response to specific therapies, and to develop novel therapeutic strategies based on insights into disease biology.

Disclosures:

Ebert: Celgene: Consultancy; Genoptix: Consultancy.
Induced Pluripotent Stem Cells and Erythrocyte Production
Authors: Slukvin Igor
Published: October 2019

Abstract SCI-38

Induced pluripotent stem cells (iPSCs) are somatic cells that have been turned into embryonic-like stem cells by forced expression of factors critical for establishing pluripotency. Because iPSCs can be differentiated into any type of cell in the human body, including hematopoietic cells, they are seen as a logical alternative source of red blood cells (RBCs) for transfusion. In addition, the unlimited expansion potential of iPSCs makes it easy to adopt iPSC technology for RBC biomanufacturing. iPSCs can be generated from any type of donor, including O/Rh-negative universal donors and donors with very rare blood phenotypes, which makes it possible to generate blood products to accommodate virtually all patient groups. We have developed an approach for generating large quantities of RBCs from iPSCs by inducing them to differentiate into CD34+CD43+ hematopoietic progenitors in coculture with OP9 stromal cells, followed by selective expansion of erythroid cells in serum-free media with erythropoiesis-supporting cytokines. Erythroid cultures produced by this approach consist of leukocyte-free populations of CD235a+ RBCs with robust expansion potential and long (up to 90 days) life spans. In these cultures, up to 1.8×105 RBCs can be generated from a single iPSC. Similar to embryonic stem cells, iPSC-derived RBCs express predominantly embryonic and fetal hemoglobin, with very little adult hemoglobin. It is already feasible to adopt iPSC technologies for producing cGMP-grade RBCs using defined animal-product-free differentiation conditions. However, the induction of the complete switch from embryonic to fetal and adult hemoglobin, as well as the terminal maturation and enucleation of iPSC-derived erythroid cells, remains a significant challenge. We recently identified at least three distinct waves of hematopoietic progenitors with erythroid potential in iPSC differentiation cultures. The characterization of erythroid cells produced from these waves of hematopoiesis may help to define populations with definitive erythroid potential and facilitate the production of erythrocytes from iPSCs. Additional critical steps toward translating iPSC-based RBC technologies to the clinic include the development of bioreactor-based-technology for further scaling-up of cell production, and evaluation of the therapeutic potential and safety of human pluripotent stem cell-derived blood cells in animal models. Overall, the manufacturing of RBCs provides several advantages. It can improve the continuity of the blood supply, minimize/eliminate the risk of infection transmission, reduce the incidence of hemolytic and nonhemolytic transfusion reactions, and provide an opportunity to generate RBCs that fit specific clinical needs by using genetically engineered iPSCs or iPSCs with rare blood groups.

Disclosures:

Slukvin: CDI: Consultancy, Equity Ownership; Cynata: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Inflammation, Obesity, and Thrombosis
Authors: Samad Fahumiya , Badeanlou Leylla , Furlan-Freguia Christian , Yang Guang , Ruf Wolfram
Published: October 2019

Abstract SCI-45

Clinical and epidemiological studies have long established a connection between obesity and thrombosis leading to increased cardiovascular complications, in part attributed to elevated expression of the prothrombotic molecules plasminogen activator inhibitor 1 and circulating tissue factor (TF). Obesity also increases the risk for metabolic dysfunction, and emerging evidence indicates that coagulation proteases offer a range of signaling pathways that control multiple aspects of the metabolic syndrome, including weight gain, insulin resistance, type 2 diabetes, and hepatic steatosis. While adipose inflammation due to recruitment and activation of macrophages and secretion of inflammatory cytokines promotes systemic hypercoagulability and insulin resistance, pathways that sustain adipose macrophage inflammation in obesity are unclear. Our studies suggest that TF signaling via the G-protein-coupled receptor, protease activated receptor 2 (PAR2), represents a novel link between obesity, coagulation, and associated adipose inflammation and insulin resistance. Mice lacking the cytoplasmic domain of TF or PAR2 were protected from high fat diet-induced weight gain, adipose inflammation, and insulin resistance. Genetic loss of TF cytoplasmic domain and PAR2 in myeloid cells attenuated adipose macrophage inflammation and increased insulin sensitivity, an effect that was also achieved by acute pharmacologic inhibition of TF-PAR2 signaling in macrophages. In contrast, TF signaling in nonhematopoietic cells specifically promoted obesity via its effects on energy metabolism. In adipocytes, TF-VIIa signaling suppressed basal and insulin-mediated AKT phosphorylation, with concordant transcriptional changes in genes regulating lipid and glucose metabolism. Thus, the procoagulant state induced by obesity can in fact contribute to the metabolic syndrome, and adipose TF signaling may be at the intersection between obesity, inflammation, and thrombosis.

Disclosures:

No relevant conflicts of interest to declare.
Intestinal Microbiota in Bone Marrow Transplantation
Authors: Jenq Robert R.
Published: October 2019

Abstract SCI-51

Regulators of the intestinal flora include diet, antibiotics, and importantly, intestinal inflammation. As a result, the cause-effect relationships between intestinal inflammation and changes in microbiota have been difficult to define. The success of allogeneic bone marrow transplantation (allo BMT), a standard therapy for conditions including hematopoietic malignancies and inherited hematopoietic disorders, is limited by graft-versus-host disease (GVHD) morbidity and mortality. With GVHD, vigorous activation of donor immune cells, most importantly T cells, leads to damage of the skin, liver, hematopoietic system, and gut. The major sources of immune activation are histocompatibility complex differences between donor and recipient. Combinations of chemotherapy and radiation also contribute, as damage to the intestinal epithelium results in systemic exposure to microbial products normally sequestered in the intestinal lumen. We have demonstrated in murine and human recipients of allo BMT that intestinal inflammation secondary to GVHD is associated with major shifts in the composition of the intestinal microbiota. The microbiota, in turn, can modulate the severity of intestinal inflammation. In mouse models of GVHD we observed loss of overall diversity and expansion of Lactobacillales and loss of Clostridiales. Eliminating Lactobacillales from the flora of mice prior to BMT aggravated GVHD, while reintroducing the predominant species of Lactobacillus mediated significant protection against GVHD. We then characterized the gut flora of patients during onset of intestinal inflammation due to GVHD and found patterns mirroring those in mice. We also identified increased microbial chaos soon after allo BMT as a potential risk factor for subsequent GVHD. Together, these data demonstrate regulation of flora by intestinal inflammation, and suggest that flora manipulation may reduce intestinal inflammation and improve outcomes for allo BMT recipients.

Disclosures:

No relevant conflicts of interest to declare.
Flow Cytometry-Based Maturity Score As a Novel Prognostic Parameter in AML
Authors: Flörcken Anne , Tanja Schneider , Singh Anju , Türkmen Seval , Thomas Burmeister , Anagnostopoulos Joannis , Pezzutto Antonio , Dörken Bernd , Westermann Jörg
Published: October 2019

Background: Based on cytogenetic and molecular phenotypes, the European LeukemiaNet (ELN) classification is widely accepted for risk stratification of patients with acute myeloid leukemia (AML). Single leukemic blast cell antigens were also shown to have prognostic value, but the influence of AML blast maturity within the different ELN risk groups is largely unknown.

Methods: Our study includes 300 AML patients, who were diagnosed at our institution between 01/2003 and 04/2012 and in whom morphologic, immunophenotypic, cytogenetic and molecular data from primary diagnosis were available. A flow cytometric maturity score was developed in order to distinguish mature AML (AML-ma) from immature AML (AML-im) by means of a quantitative analysis of the expression of early progenitor cell antigens (CD34, CD117, and TdT). Clinical outcome analysis was performed both in the whole cohort and in the different ELN risk groups using Chi Square and Fisher's Exact Test (CR/CRi) and Logrank Test (RFS, OS).

Results: A mature AML blast phenotype (AML-ma) was associated with a significantly longer RFS (7.5 vs. 4.3 years) and OS (5.3 vs. 2.7 years) than an immature phenotype (AML-im) (p < 0.001). Low-risk phenotypes according to ELN (NPM1, PML-RARA) significantly accumulated in the AML-ma group, whereas high-risk aberrations (monosomal and complex aberrant karyotypes, -5 or del(5q), -7) had a higher frequency in AML-im.

Interestingly, the differences in RFS and OS were maintained in a subgroup analysis within the different ELN risk groups. Statistical significance was obtained in the intermediate risk group with AML-ma being superior to AML-im (RFS: 7.0 years (AML-ma) vs. 3.3 years (AML-im); p = 0.002; OS: 5.1 years (AML-ma) vs. 3.0 years (AML-im); p = 0.022). In contrast, there was no correlation between AML blast maturity and CR/CRi rates.

Conclusions: Our novel flow cytometric score easily determines AML blast maturity and can predict clinical outcome, even within the different ELN risk groups. Particularly for intermediate risk patients according to ELN, the score may be useful in post remission therapy decision-making. It remains to be clarified whether these results reflect differences in genetic instability and/or a different frequency of leukemia stem cells in AML-ma and AML-im.

Figure 1 Figure 1. Figure 2 Figure 2.

RFS (A) and OS (B) of AML patients with mature (AML-ma) and immature (AML-im) blast phenotype within the ELN intermediate risk group. AML-ma show a significantly longer RFS and OS compared to AML-im.

Disclosures

No relevant conflicts of interest to declare.
Single Cell Whole Exome Sequencing in NPM1/Flt3 Positive Pediatric Acute Myeloid Leukemia
Authors: Walter Christiane , Hofmann Winfried , Reinhardt Katarina , Reinhardt Dirk , von Neuhoff Nils
Published: October 2019

Introduction:

Acute myeloid leukemia (AML) is one of the most frequent forms of leukemia in children younger than 15 years. The detection of several mutations in a blast population of pediatric AML (pAML) is supposed to be caused by a clonal evolution from a leukemic stem cell (LSC) to leukemic blasts. LSC are believed to be more resistant to chemotherapy, to be able to survive during treatment and to be responsible for the emergence of a relapse due to the persistence in the bone marrow (BM) niche. Since LSC and potential leukemic subclones are only present in small subpopulations, it has been a major technical challenge to particular analyse only the specific population. To acquire a better understanding of the underlying mechanisms of mutagenesis, clonal evolution and leukemogenesis, the aim of this study was to establish methods that allow the analysis and detection of mutations in single cells of a subpopulation known to contain HSC as well as LSC (CD34+CD38-). We especially focused on a pAML subgroup with mutations in Nucleophosmin (NPM1) and/or fms related tyrosine kinase 3 (Flt3).

Methods and Results:

We established methods to perform single cell sorting, whole genome amplification (WGA) using multiple displacement amplification (MDA) technology (Qiagen) and subsequent whole exome sequencing. The sorting efficiency was checked as Hoechst stained cells were sorted onto glas slides with 48 defined spots and the presence of single cells was checked under an inverse fluorescent microscope. Subsequently, single CD34+CD38- patient derived cells were sorted into 0,5ml low binding tubes containing 4µl PBS followed by WGA and whole exome sequencing. The mutational status of the sorted single cells from three patients suffering from pAML was analysed and compared to mutations detected at initial diagnosis in DNA from a bulk of BM cells. WGA from single CD34+CD38-PI- cells resulted in an amount of 29 to 31.7µg DNA from each of five single cells. The quality of the amplified DNA was sufficient for whole exome sequencing. A 4bp insertion in exon 12 of NPM1 reflecting a common NPM1 mutation (MutA) initially detected from a bulk of cells was identified in amplified DNA from single cells using whole exome sequencing in 2/3 patients. Internal tandem duplications in Flt3 indicated by mismatches in the alignment could be detected in amplified DNA from single cells of two patients. The detected ITD resemble those initially detected in DNA from a bulk of BM cells.

Discussion and Conclusion:

Single cell sequencing provides a useful tool to amend the detection of genetic aberrations from a bulk of cells and to confirm the presence of specific mutations in single cells from small subpopulations. It therefore helps to get further insights into the clonal evolution in pAML.

Disclosures

No relevant conflicts of interest to declare.
Identification of Cytogenetically Normal Human CD34+CD38+ Hematopoietic Stem/Progenitor Cells from Inv(16)+ Leukemic Bone Marrow
Authors: LeGrand Jason N. , Heidemann Stephanie C. , Swindle C. Scott , Klug Christopher A.
Published: October 2019

For many subtypes of AML including cases with the inv(16), mutations that give rise to the leukemic phenotype occur, at least in part, in the hematopoietic stem/progenitor (HSPC) cell subset, as suggested by studies showing that primitive CD34+ CD38- bone marrow cells can function as leukemia-initiating cells (LIC) when transferred into immunodeficient mice. A significant challenge has been that LIC share many of the same cell-surface markers as their normal HSPC counterparts, thus making it difficult to purify and functionally characterize either subset from the bulk bone marrow of leukemia patients. Here we report the FACS analysis of several previously reported human LIC markers on bone marrow samples from inv(16) AML patients and show that a combination of TIM3, CLL1, and CD33 can significantly enrich for a rare population of CD34+ CD38- cells that lack the inv(16) fusion mRNA when tested by nested RT-PCR. Heterogeneous expression of these markers among different patient samples often causes incomplete elimination of the fusion mRNA when FACS-sorting the CD34+ CD38- population as single TIM3-, CLL1-, or CD33- subsets. The combination of TIM3 with CLL1 and/or CD33 leads to a more consistent elimination of the fusion mRNA from the FACS-sorted CD34+ CD38- subsets. Results from methylcellulose assays showed that the TIM3- CLL1- CD33- subset of CD34+CD38- cells could form multiple colony types, including CFU-GEMM, that were all negative for the fusion mRNA by RT-PCR. In contrast, colonies derived from bulk bone marrow were all positive for the fusion mRNA. The TIM3- CLL1- CD33- subset of CD34+CD38- cells displayed greater than 600-fold enrichment for progenitor activity compared to bulk bone marrow but did not form additional colonies upon serial re-plating. These results have important implications for the therapeutic targeting of inv(16)+ hematopoietic stem/progenitor cells in patients with relapsed and refractory disease and for purification of normal HSPC from leukemic bone marrow samples.

Disclosures

No relevant conflicts of interest to declare.
Infused Autograft Lymphocyte to Monocyte Ratio and Survival in Classical Hodgkin Lymphoma
Authors: Porrata Luis F. , Inwards David J. , Ansell Stephen M. , Micallef Ivana N. , Johnston Patrick B. , Hogan William , Markovic Svetomir N.
Published: October 2019

Infused autograft absolute lymphocyte count (A-ALC) is a prognostic factor for survival post-autologous peripheral hematopoietic stem cell transplantation (APHSCT) for lymphoma. Myeloid-derived suppressor cells (MDSCs) affect tumor growth by suppression of host anti-tumor immunity. Thus, we set out to investigate if the infused autograft lymphocyte /monocyte ratio (A-LMR), as a biomarker of host immunity (i.e., lymphocytes) and MDSCs (i.e., monocytes), affects survival post-APHSCT in patients with classical Hodgkin lymphoma (cHL). Only patients that collected their stem cells through the peripheral blood were included in the study. From 1994 to 2012, 183 cHL patients qualified for the study. The 183 patients were randomly divided into a training set (n = 122) and a validation set (n = 61). Receiver operating characteristic and area under the curve (AUC) identified an A-LMR ≥ 1 as the best cut-off value (AUC = 0.8, p < 0.01) and validated by the k-fold cross-validation (AUC = 0.8) in the training set. Multivariate analysis showed A-LMR to be an independent prognostic factor for survival in the training set. Patients with an A-LMR ≥ 1.0 experienced a superior overall survival (OS) versus patients with an A-LMR < 1.0 (median OS was not reached vs 40.4 months, 5-year OS rates of 86% (95 CI, 72%-93%) vs 43% (95 CI, 28-58 %), p < 0.0001, respectively) in the training set. In the validation set, an A-LMR ≥ 1 showed a median OS of not reached vs 41.8 months for an A-LMR < 1, 5 year OS rates of 90% (95 CI, 73%-97%) vs 48% (95 CI, 29-68%), p < 0.0001, respectively. A-LMR provides a platform to engineer an immunocompetent autograft to improve clinical outcomes in cHL patients undergoing APHSCT.

Disclosures

No relevant conflicts of interest to declare.
Using the Rothman Index to Predict Discharge and Readmission Rates in an Inpatient Hematology Unit
Authors: Kim Roger Y. , Yao Xiaopan , Longley Peter , Rimar Joan , Kournioti Chryssanthi , Lee Alfred Ian
Published: October 2019

BACKGROUND: Limited data exists regarding predictors of discharge (DC) disposition and readmission (RA) rates for hematology-oncology patients who are hospitalized. The Rothman Index (RI) is a single numerical score ranging from -91 to 100 derived from 26 data elements including vital signs, laboratory results, and nursing assessments. It is automatically calculated and displayed in real-time in the electronic medical record by a commercial software system. A previous analysis at our institution revealed a significant association between RI and DC disposition, and between RI and RA rates, for patients with solid tumors who were hospitalized in a dedicated inpatient oncology unit. We sought to determine the effectiveness of RI in predicting DC disposition and RA rates for hospitalized patients with hematologic malignancies.

METHODS: Medical records of patients with hematologic malignancies admitted to Yale Cancer Center between 3/1/13 and 2/28/14 were reviewed. Admissions for elective chemotherapy were excluded. DC disposition was categorized as home (Hm), nursing home (NH), hospice (Hp), or death (D). RI at DC was examined as a predictor of DC disposition, overall RA rate, and RA rate of patients discharged to Hm. As an individual patient might have several admissions, generalized estimating equation models were used to assess the effect of RI at DC on DC disposition and the possibility of RA by controlling the within subject correlation.

RESULTS: A total of 281 patients comprising 520 admissions were included in the final analysis. The median age was 59 (range: 19-93; age 70 or older: 21%), and 53% of patients were male. The most common hematologic malignancies were non-Hodgkin lymphoma (33%), acute myeloid leukemia (25%), and multiple myeloma (19%). Among all admissions, DC disposition was as follows: Hm, 439 (84%); NH, 53 (10%); Hp, 18 (4%); and D, 10 (2%). RI at DC was a significant predictor of DC disposition (p = 0.0001), with median RI at DC as follows: Hm, 82.1; NH, 61.4; Hp, 38.7; and D, -1.3. Patients with lower RI at DC were significantly more likely to be readmitted (p = 0.0006), even when the analysis was restricted to patients who had been discharged to Hm (p = 0.0131). Among Hm patients, RA rate was 61% for those whose RI at DC was < 60 and 46% for RI at DC > 60.

CONCLUSIONS: The RI at DC significantly correlated with patient DC disposition and RA rates and may assist health care providers in DC planning with the goal of reducing hospital RA rates and ultimately health care costs.

Disclosures

Lee: Pfizer: Consultancy.
Hyperexpression of Inflammatory Genes in Placental Tissue in Patients with Hemoglobin SC Disease
Authors: Baptista Letí­cia Carvalho , Fertrin Kleber Yotsumoto , Ferreira Regiane , Lanaro Carolina , Albuquerque Dulcinéia Martins , Costa Fernando Ferreira , Costa Maria Laura , Melo Monica Barbosa
Published: October 2019

Introduction: Pregnant women with sickle cell disease (SCD) are at higher risk of obstetrical complications, such as preeclampsia and preterm delivery. The placental circulation is susceptible to vaso-occlusion, which may contribute to fibrosis, necrosis and infarction of the placental villi. Restricted uteroplacental circulation is associated with low birth weight and fetal growth restriction. The mechanisms by which SCD affects placental physiology are largely unknown, and the chronic inflammatory state may be involved in this pathological process. The aim of this study was to evaluate the expression of mediators of inflammatory response in the placenta of pregnant women with SCD.

Methods: This is an ongoing case control study that has recruited 4 pregnant patients with hemoglobin SC disease and 4 healthy control pregnant women paired for gestational age and route of delivery. Placental tissue was collected for total RNA extraction and synthesis of cDNA. Samples were run on a qPCR Array (Inflammatory Response and Autoimmunity RT2 Profiler, Qiagen), which includes 84 genes. Gene expression levels were normalized to housekeeping genes (ACTB, B2M, and RPLP0), and will be further validated by RT-qPCR.

Results: Two patients underwent cesarean delivery and one patient delivered preterm. The analysis of placental gene expression showed that 16 genes were upregulated. The top 5 most upregulated genes were CCL24 (18.1 fold), TLR9 (8.6 fold), inducible nitric oxide synthase NOS2 (6.8 fold), IL17A (5.4 fold), and CCL16 (4.8 fold). Upregulated genes also included other chemokines and their receptors (CCL21, CCL22, CXCL3, and CCR7), interleukins (IL1RAP, IL5, and IL9), another toll-like receptor (TLR3), transcription factor BCL6, CD40 ligand (CD40LG), and kininogen 1 (KNG1). Interestingly, the two genes found to be downregulated were CRP (C reactive protein) and IFNG (interferon gamma).

Conclusion: This is the first study of differential gene expression in placenta of patients with SCD. Despite patients with hemoglobin SC disease being clinically less severe than homozygous sickle cell anemia, we have found that placental tissue in this subset of patients displays increased expression of several genes involved in inflammatory response. Our data support local inflammatory changes in the placenta of these patients that might explain the development of complications in SCD pregnancies. Although the association between obstetric complications and SCD has been long described, our data should contribute to better define the mechanisms affecting the placental physiology that lead to complications, such as preeclampsia.

Disclosures

No relevant conflicts of interest to declare.
Evidence for Transient Acute Liver Injury in Mouse Models of Sickle Cell Disease during Steady State Health
Authors: Wandersee Nancy J. , Holzhauer Sandra L. , Retherford Dawn M. , Foster Thomas D. , Hillery Cheryl A.
Published: October 2019

Individuals with sickle cell disease experience episodes of acute vaso-occlusive complications interspersed by periods of feeling well and apparent health during “steady state”. It is likely that episodic and subclinical sickling and vaso-occlusion occur on an ongoing basis even during periods of steady state. In order to determine whether mice with sickle disease have acute vaso-occlusion sufficient for organ injury at apparent steady state health, we chose to study 2 mouse models of severe sickle cell disease with serial blood draws over time. Since we have shown in previous studies that the liver is highly susceptible to acute sickling/vaso-occlusion induced by hypoxia, we first examined serial ALT measurements in a total of 13 Berkeley sickle cell (BERK-SS) mice, initially from week-to-week and month-to-month, and then day-to-day time frames. Individual BERK-SS mice showed week-to-week and month-to-month variations of up to 5-fold over baseline ALT levels, followed by return to baseline levels in most mice. This suggests that BERK-SS mice, during steady state health, experience acute, yet transient, liver injury. Surprisingly, we also found day-to-day variations of up to 3-fold in ALT levels in BERK-SS mice. To determine if these changes in ALT levels were mirrored by changes in other biomarkers, we measured serum amyloid P (SAP) as a marker of acute inflammation (similar to CRP in humans) in the same plasma samples. We found up to 1.6-fold variation in SAP over baseline plasma levels over time. However, there was less overall variation in SAP levels in individual mice compared to the striking changes in ALT. In addition, we found that variations in SAP did not consistently precede, follow, or coincide with variations in ALT levels, suggesting that SAP is a marker of sickle cell-induced inflammation or vaso-occlusion independent of liver injury. We next sought to confirm our findings with daily serial ALT and SAP measurements from Townes sickle cell (TOWNES-SS) mice (n=8), a second commonly used mouse model of severe sickle cell disease. Our data showed that baseline ALT levels are higher and SAP levels are lower in TOWNES-SS as compared to BERK-SS mice, likely reflecting differences in strain background between the two models. However, we again found daily fluctuations in ALT and SAP levels in individual TOWNES-SS mice, similar to those seen in individual BERK-SS mice. We propose that these temporal variations indicate that both the Berkeley and Townes mouse models of sickle cell disease exhibit fluctuations in steady state health, similar to those seen in human individuals with sickle cell disease. In addition, the results suggest that studies in these mouse models should be designed and interpreted with these temporal fluctuations in mind.

Disclosures

No relevant conflicts of interest to declare.
Chd1 Regulates Primitive State of Bone Marrow Mesenchymal Stromal Cells without Affecting Hematopoietic Support; An Insight on the Dissociation Between Primitive State and Niche Function
Authors: Oh Il-Hoan , Choi Hyun-Kyung
Published: October 2019

Mesenchymal stromal cells (MSCs) are characterized by heterogeneity in the proliferation/self-renewal potentials and hematopoietic supporting activity among subpopulations. Numerous studies have suggested that a primitive state of MSC subpopulation are correlated to its niche function to support hematopoietic stem cells (HSCs), but the mechanisms regulating primitive state of MSCs remains poorly understood.

In the present study, we examined the role of a chromatin remodeling enzyme, chd1 in the maintenance of open chromatin and undifferentiated state of MSCs. We analyzed for expression in MSCs, the expression level of chd1 progressively decreased during in-vitro subculture (from 7 to 18 passages) in a manner proportional to the passage numbers. Moreover, chd1 expression was down regulated in the MSCs during their differentiation into adipogenic or osteogenic lineages, compared to proliferative state, indicating the correlations between MSC proliferation potentials and expression level of chd1. Next, we transduced human bone marrow-derived MSCs with shRNAs against chd1 and found that chd1 knock down MSCs (chd1-KD) exhibit significant loss of colony forming activity (CFU-F), decrease of cell proliferation and loss of multi-lineage differentiation towards osteogenic or adipogenic lineages. Moreover, chd1-KD MSCs exhibited lower level expression of pluripotency-related genes, oct-4, sox-2 and nanog, with concomitant increase of H3K9me3 on the promoters and decreased chromatin accessibility in the oct-4 promoter, suggesting that chd1 regulate open chromatin and multi-lineage potential of MSCs.

However, KD of chd1 in MSCs did not affect the HSC-supporting activity of MSCs; human cord blood-derived CD34+ cells co-cultured on chd1-KD MSCs exhibited rather higher maintenance of primitive phenotype (CD34+90+) and higher repopulating activity in NOD/SCID-ɤC KO mice compared to those co-cultured on control group MSCs. Together, these results show that, while primitive state of MSCs are regulated by chromatin remodeling complex,chd1, the hematopoietic niche activity of MSCs is not directly influenced by the primitive state of MSCs, raising a questions on the prevailing notion that undifferentiated MSCs can better support hematopoietic function.

Disclosures

No relevant conflicts of interest to declare.
Desoxyribonucleoprotein Thromboembolism Following Total Body Irradiation: Case Report
Authors: HRUBAN ZDENEK , PIERCE MILA I. , NAIR SUMATI , WARNER NANCY E.
Published: October 2019

A case of sudden death shortly after total body radiation is reported in a patient with acute stem cell leukemia. The sudden death was associated with capillary DNA thromboembolism.
OBSERVATIONS ON THE EFFECT OF IRRADIATION IN CHRONIC ACQUIRED HEMOLYTIC ANEMIA EXHIBITING HEMOLYTIC ACTIVITY FOR TRANSFUSED ERYTHROCYTES
Authors: EVANS ROBERT S. , DUANE ROSE T.
Published: October 2019

The history and course of a middle-aged woman with a chronic acquired hemolytic anemia have been presented. Splenectomy resulted in a complete remission of the disease for 18 months before a relapse occurred which led eventually to a fatal termination.

The original cause of the abnormal hemolytic process is not known, but the onset of the relapse was closely associated with the resumption of gold therapy.

The acceleration of the hemolytic process was associated with an increasing tendency toward spherocytosis and susceptibility of the erythrocytes to hemolysis in hypotonic solution.

It is demonstrated that transfused normal erythrocytes shared in the hemolytic process and were eliminated from the circulation at an abnormal rate although no atypical isohemolysin was demonstrated by in vitro tests.

An attempt was made to alter the rate of hemolysis in several ways. The injection of thorium dioxide was followed by signs of transitory slowing of red cell destruction. Injections of Congo red were apparently without effect. Irradiation of the mediastinal and periaortic nodes was begun to ascertain if the production of a hemolytic antibody in lymphatic tissue could be altered. Irradiation was followed by a fall in leukocyte and lymphocyte counts in the peripheral blood. Transfusions were then followed by partial remissions in the hemolytic process for varying periods of time in two instances. Irradiation of a second patient with a chronic hemolytic anemia of similar character produced a fall in leukocyte count and lymphocyte count, but in the absence of transfusions no slowing of the hemolytic process occurred.

No definite conclusions can be drawn from these fragmentary observations on the effect of irradiation, but further investigation as to the character of the hemolytic substance and possible methods of modifying its production are indicated when the opportunity is afforded.
TREATMENT OF PERNICIOUS ANEMIA BY A SPECIAL DIET
Authors: MINOT GEORGE R. , MURPHY WILLIAM P.
Published: October 2019

The dietetic treatment of pernicious anemia is of more importance than hitherto generally recognized.

Forty-five patients with pernicious anemia observed essentially in sequence are continuing to take a special diet that they have now been living on for from about six weeks to two years but which was temporarily omitted by three. This diet is composed especially of foods rich in complete proteins and iron—particularly liver —and containing an abundance of fruits and fresh vegetables and relatively low in fat.

Following the diet, all the patients showed a prompt, rapid and distinct remission of their anemia, coincident with at least rather marked symptomatic improvement, except for pronounced disorders due to spinal cord degeneration. Improvement was often striking, so that where the red blood cell count averaged for all before starting the diet 1,470,000 per cubic millimeter, one month afterward it averaged 3,400,000; and for the twenty-seven cases observed from four to six months after the diet was begun, the average count was 4,500,000 per cubic millimeter.

Patients having had two or more relapses showed on the average slightly lower red blood corpuscle counts about one and two months after commencing the diet than did those who had started it in their first or second relapse.

Change in the frequency of bowel movements, temporary increase of reticulocytes in the peripheral blood, and decrease of the icterus index of the blood serum were among the earliest signs that heralded the patient’s better health.

All the patients have remained to date in a good state of health except three, who discontinued the diet; two rapidly improved on resuming it and the other has just commenced it again. As the diet was advised for most of the patients less than eight months ago, enough time has not yet elapsed to determine whether or not the remissions will last any longer than in other cases.
THE DISTRIBUTION CURVE OF ERYTHROCYTE FRAGILITY
Authors: BOLTON J. H.
Published: October 2019

1. A simple method of representing erythrocyte fragility as a distribution curve of actual cellular fragility is described.

2. The importance of deciding the form of a distribution before using summarizing statistics is emphasized.

3. The danger of concluding that abnormal fragility is present in the presence of increased marrow activity is pointed out.

4. Possible applications in diagnosis are suggested.
HYPOPLASTIC ANEMIA DUE TO ATABRINE
Authors: FISHMAN ALFRED P. , KINSMAN J. MURRAY
Published: October 2019

Seven patients with severe hypoplastic anemia were studied at an army Tropical Disease Center. Four of the 7 patients had concomitant dermatitis. The relationship of the prolonged administration of atabrine to the anemia and dermatitis is presented. A hematologic remission could not be induced by specific therapeutic measures. Four of the 7 cases recovered spontaneously.
Comparative transcriptomics of Gymnosporangium spp. teliospores reveals a conserved genetic program at this specific stage of the rust fungal life cycle
Authors: Tao Si-Qi , Cao Bin , Morin Emmanuelle , Liang Ying-Mei , Duplessis Sébastien
Published: October 2019

Background

Gymnosporangium spp. are fungal plant pathogens causing rust disease and most of them are known to infect two different host plants (heteroecious) with four spore stages (demicyclic). In the present study, we sequenced the transcriptome of G. japonicum teliospores on its host plant Juniperus chinensis and we performed comparison to the transcriptomes of G. yamadae and G. asiaticum at the same life stage, that happens in the same host but on different organs.

Results

Functional annotation for the three Gymnosporangium species showed the expression of a conserved genetic program with the top abundant cellular categories corresponding to energy, translation and signal transduction processes, indicating that this life stage is particularly active. Moreover, the survey of predicted secretomes in the three Gymnosporangium transcriptomes revealed shared and specific genes encoding carbohydrate active enzymes and secreted proteins of unknown function that could represent candidate pathogenesis effectors. A transcript encoding a hemicellulase of the glycoside hydrolase 26 family, previously identified in other rust fungi, was particularly highly expressed suggesting a general role in rust fungi. The comparison between the transcriptomes of the three Gymnosporangium spp. and selected Pucciniales species in different taxonomical families allowed to identify lineage-specific protein families that may relate to the biology of teliospores in rust fungi. Among clustered gene families, 205, 200 and 152 proteins were specifically identified in G. japonicum, G. yamadae and G. asiaticum, respectively, including candidate effectors expressed in teliospores.

Conclusions

This comprehensive comparative transcriptomics study of three Gymnosporangium spp. identified gene functions and metabolic pathways particularly expressed in teliospores, a stage of the life cycle that is mostly overlooked in rust fungi. Secreted protein encoding transcripts expressed in teliospores may reveal new candidate effectors related to pathogenesis. Although this spore stage is not involved in host plant infection but in the production of basidiospores infecting plants in the Amygdaloideae, we speculate that candidate effectors may be expressed as early as the teliospore stage for preparing further infection by basidiospores.
Attitudes of Chinese health sciences postgraduate students’ to the use of information and communication technology in global health research
Authors: Huang Kaiyong , Abdullah Abu S. , Ma Zhenyu , Urmi Dilshat S. , He Huimin , Quintiliani Lisa , Friedman Robert H. , Yang Jun , Yang Li
Published: October 2019

Background

Information and communications technology (ICT) has been suggested as an important tool for improving global health education and building research capacity in developing countries. However, the existing curricula do not have adequate emphasis on global health research and training. This study was carried out to examine health sciences postgraduates’ attitudes and practices regarding curriculum for ICT use in global health research and training in China.

Methods

A cross-sectional study was conducted among health sciences postgraduates from six universities in southern China, during December 2016 to March 2017. A self-administered online questionnaire was used to collect data through an online survey platform. Data were analyzed using SPSS for Windows 13.0.

Results

A total of 1065 participants successfully completed the questionnaires. More than 90% of the students have not had any training about ICT, three quarters have not taken an online course, and 31% of the students do not use ICT in their current research. More than 65% thought that, in an ICT research training curriculum, it was important to learn: ICT utilization related knowledge, ICT research methods/resources, knowledge of databases, ways of data use and acquisition, and informatics search methods (ICT users compared to non-users were more likely to agree to these learning components (all p <  0.05)). Many of the respondents used or planned to use mobile phones (80%), Internet (59%), use computer and WeChat (> 40%), and QQ (a popular chat tool in China) (30%) as ICT tools in research activities. ICT users compared to non-users were more likely to consider using ICT and/or biomedical informatics methods in decision-support or support for information seeking, healthcare delivering, academic research, data gathering, and facilitating collaboration (all p <  0.05).

Conclusions

The findings of this study showed that ICT utilization was very important to health sciences postgraduates for their research activities in China, but they lacked ICT-related training. The results suggested the need for specialized curriculum related to ICT use in global health research for health sciences postgraduates in China.
How to evaluate the clinical outcome of joint-preserving treatment for osteonecrosis of the femoral head: development of a core outcome set
Authors: Xue Zhipeng , Sun Jigao , Li Taixian , Huang Zeqing , Chen Weiheng
Published: October 2019

Background

This study aimed to develop a core outcome set (COS) for clinical trials of joint-preserving treatment for osteonecrosis of the femoral head (ONFH), that is, to define a minimal set of outcomes that should be reported in such trials.

Methods

A mixed research method was adopted in this study. First, clinical trials of hip preservation therapy were systematically researched and analyzed. Second, a three-round Delphi survey involving both doctors and patients was carried out to obtain the core outcome indicators. Round 1 was a modified Delphi questionnaire for doctors and patients to determine which outcomes are important to these stakeholders, round 2 determined what clinical evaluation core outcomes should be included for the joint-preserving treatment of ONFH, and round 3 determined how core outcomes should be measured. Finally, a consensus meeting was held to discuss and vote on the established COS.

Results

The results of the systematic review showed that 42 outcome indicators were classified according to common signs and symptoms, quality of life, long-term outcomes, radiological evaluation, blood biochemistry, and indexes of safety. The three rounds of Delphi surveys completed the selection of indicators for the COS and the determination of the corresponding measurements.

A total of 73 orthopedic doctors and 103 patients participated in round 1, and the top 10 indicators selected were basically the same. In round 2, 32 experts identified the following indicators: pain, range of motion (ROM) of hip flexion, walking distance, and stable rating of X-ray images. In round 3, 35 experts defined the measurement of each indicator. Finally, the consensus meeting identified the four indicators aforementioned that constituted the COS.

The scores for pain, ROM of hip flexion, and walking distance are from 0 to 10; 0 represents the best scores, while 10 represents the most serious impairment. The stable rating of X-ray images is determined by the morphology of the femoral head and the change in the density of the necrotic area.

Conclusions

We established a COS for hip-preserving treatment of ONFH that includes four indicators: pain, ROM of hip flexion, walking distance, and stable rating of X-ray images.