bluuebunny/crossref_metadata_2025_split huggingface dataset:
Exosomes Derived from AML/MDS Cells Is Involved in Stromal Dysfunction and Bone Marrow Failure
Authors: Horiguchi Hiroto , Kobune Masayoshi , Kikuchi Shohei , Jomen Wataru , Murase Kazuyuki , Ibata Soushi , Iyama Satoshi , Sato Tsutomu , Kamihara Yusuke , Miyanishi Koji , Sato Yasushi , Hayashi Tsuyoshi , Takimoto Rishu , Kato Junji 
Published: October 2019

Abstract
The failure of normal hematopoiesis in myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) could be induced by a variety of mechanism such as the alteration of property of hematopoietic stem cells and stem cell niche. However, it has not yet been clarified precise mechanism how MDS stem/progenitor cells could replace normal hematopoietic stem/progenitor cells especially regarding involvement of mesenchymal stromal cells (MSCs). To gain insight into the mechanism of stromal dysfunction, comparative analyses of transcriptomes were conducted between normal and MDS/AML-derived MSCs. Further, we attempted to identify certain effectors originated from MDS/AML cells could alter the function of bone marrow (BM) MSCs.
The MSCs derived from healthy volunteer (HV)-derived (normal) and MDS/AML-derived stromal cells were established and analyzed mRNA expression by quantitive PCR (qPCR) array. Additionally, the supporting activity of MSCs for BM CD34+ progenitor/stem cells was examined using serum free coculture system. The interaction between MDS/AML cells and MSCs were evaluated by using Boyden Chamber and the changes of mRNA expression were analyzed.
The results of qPCR array revealed that the expression of hematopoietic factors was drastically altered in MDS/AML-derived MSCs as compared with normal MSCs. Among these factors, the expression of SCF and JAG1 mRNA were significantly and consistently reduced in all MDS/AML patients examined. Functional assay of these MSCs demonstrated that the number of colony-forming units (CFU) mixed cells (MIXs) and cobblestone area-forming cells (CAFCs) derived from CD34+ cells was significantly reduced after coculture with MDS/AML-derived MSCs as compared with normal MSCs. Even non-contact culture using Boyden Chamber between leukemic cells and MSCs induced the reduction of SCF and JAG1 mRNA, indicating that certain inducers could be soluble factors. Interestingly, this effect of transcriptomes alteration was negated by nSMase2 inhibitor (GW4869). Exosome transfer assay using Boyden Chamber revealed that GFP and PKH26 in leukemic cells transmit onto MSCs in non-contact coculture system and this transfer of exosome was significantly inhibited by GW4869 or nSMase siRNA. The multiple type of microRNA in exosome derived from MDS/AML cells was transferred into MSCs, suggesting that exosome could contribute to the alteration of mRNA expression in stromal cells.
Collectively, these results indicated that exosome derived from MDS/AML cells could be involved in the reduction of SCF/JAG mRNA and the stromal supporting activity of normal hematopoietic stem/progenitor cells.

Disclosures
No relevant conflicts of interest to declare.
Gene Editing of CCR5 in Hematopoietic Stem Cells in a Nonhuman Primate Model of HIV/AIDS
Authors: Peterson Christopher W. , Wang Jianbin , Polacino Patricia , Holmes Michael C. , Hu Shiu-Lok , Gregory Philip D , Kiem Hans-Peter 
Published: October 2019

Abstract
Background: Hematopoietic stem cell (HSC) transplantation remains the only clinically observed path to functional cure of HIV infection. To better understand the mechanism of HSC-driven HIV control, and apply this therapy to a greater number of patients, we have developed a model of combination antiretroviral therapy (cART)-suppressed HIV infection in the pigtailed macaque, applicable to both gene therapy- and allogeneic transplant-based cure strategies. Following transplantation of HIV-resistant, autologous cells into conditioned animals, we are evaluating the extent to which protected cell progeny impede infection by SIV/HIV (SHIV) chimeric virus in vivo.
Methods: Animals are challenged with SHIV virus containing an HIV envelope, after which a 3-drug cART regimen is initiated. Autologous HSCs are engineered to resist infection through targeted disruption of the CCR5 genetic locus using Zinc Finger Nucleases (ZFNs). Engraftment, persistence, and SHIV response of these autologous stem cells, and stem cell-derived lymphoid and myeloid cells, are measured in vivo.
Results: SHIV infection in the pigtailed macaque model results in sustained viremia with consequent reduction in CD4+ T cells. Moreover, administration of three-drug cART leads to rapid and durable suppression of plasma viremia to <30 copies/mL plasma - suggesting that this model recapitulates key features of HIV infection and treatment in humans. CCR5 targeting experiments yield up to 60% gene disruption in CD34+ cells ex vivo, translating to approximately 5% disruption in vivo following transplant. Importantly, up to 10% of transplanted cells carry two disrupted alleles of CCR5; these cells should preferentially reconstitute CD4+ T-cell pools and other susceptible subsets following SHIV challenge. Consistent with this prediction, our preliminary data suggest that CCR5-deleted cells undergo positive selection following SHIV challenge in vivo.
Conclusions: Our pigtailed macaque model of HIV infection and cART represents a promising platform for modeling functional cure strategies. Here we show that CCR5 deletion does not impair HSC engraftment or differentiation, and that CCR5-deleted cells can undergo SHIV-dependent positive selection even when present at low levels. Our model enables the evaluation of novel therapeutic approaches in the clinically relevant context of cART controlled SHIV infection - a setting of particular importance to approaches aimed at addressing the viral reservoir.

Disclosures
Wang: Sangamo Biosciences: Employment. Holmes:Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment.
Glycopeptide-Specific Chimeric Antigen Receptor Targeting of T Cell Leukemia
Authors: Posey Avery D. , Schwab Robert D. , Boestaneau Alina , Johnson Laura A. , June Carl H. 
Published: October 2019

Abstract
Background
Adoptive transfer of T cells redirected with chimeric antigen receptors (CARs) has shown tremendous efficacy in targeting CD19-expressing leukemic cells; however, these CAR T cells also deplete normal CD19-expressing B cells. Although B cell lymphopenia can be overcome by gamma-globulin supplementation, other forms of on-target, off-site toxicity emphasizes the need for cancer cell-specific epitopes in CAR T cell therapy. To date, T cell leukemia has not been targeted due to possible fratricide because of shared antigens expressed on leukemic T cells and CAR T cells. Cellular transformation often produces altered glycosylation patterns, making cancer-specific glycoantigens attractive targets for CAR development. Here we demonstrate the successful eradication of the Jurkat T cell leukemia with Tn-specific CAR T cells.
Methods
The Jurkat E6.1 T cell leukemia line contains a frameshift mutation in the T synthase chaperone protein Cosmc that prevents core 1 structure formation on O-linked protein glycosylation, resulting in the presentation of Tn antigen on many glycoproteins. Here we describe the development of a CAR using a scFv sequence derived from the 5E5 mAb, which was developed against the Tn antigen of Mucin-1 (Muc1). A CAR was constructed by linking the 5E5 scFv to CD8alpha hinge and transmembrane region to the signaling endodomain comprised of 4-1BB and CD3zeta. The resultant Tn specific CAR was expressed in a lentiviral vector.
Results
In preliminary experiments Tn-CAR T cells had cytotoxicity to a variety of primary ovarian, breast and pancreatic tumor cells. Specific killing of Jurkat T leukemic cells was demonstrated by lysis of wild type Jurkat while the CAR T cells failed to kill Jurakat after overexpression of Cosmc. In specific, expression of full-length Cosmc expression in the Jurkat E6.1 cell line abolished staining by the 5E5 mAb, restoredT synthase activity, and protected the cells from lysis by 5E5-CAR T cells. Human T cells expressing the 5E5-CAR demonstrate rapid clearance of established Jurkat tumors in NSG mouse xenograft models, as assessed by serial bioluminescence imaging and prolonged survival compared to mice expressing a control CD19-specific CAR T cells (P<0.005).
Conclusions
These results demonstrate that the 5E5-CAR is specific for the Tn antigen and provides a potent cancer-specific immunotherapy for the targeting of non-dispensable tissues such as T cell leukemias.

Disclosures
June: Novartis: IP licensed by University of Pennsylvania to Novartis. Author entitled to royalties from the University. Patents & Royalties, Research Funding.
Pulmonary Embolism Begets Pulmonary Embolism?: A Meta-Analysis Depicting the Type of Recurrence Following an Index Pulmonary Embolism
Authors: Coleman Craig I , Kohn Christine G , Patel Dhaval 
Published: October 2019

Abstract
INTRODUCTION: Conflicting evidence suggests a person who suffers an index pulmonary embolism (PE) is more likely to develop a recurrent PE than a deep vein thrombosis (DVT). We performed a meta-analysis of randomized controlled trials (RCTs) to determine the proportion of recurrent events that will be PE in such people.
METHODS: A literature search of MEDLINE, EMBASE and CENTRAL (through 7/2014) was performed to identify RCTs of acute treatment and prevention of recurrent VTE in persons suffering an index PE, enrolling ≥50 subjects, requiring anticoagulation for ≥3 months and reporting the proportion of recurrent events that were DVT or PE (±DVT). RCTs not in English, enrolling only cancer, surgical or thrombophilia patients, primary prevention and extended treatment trials were excluded. We extracted recurrent VTE event data; and used a random-effects approach to pool the proportion [along with 95% confidence intervals (CIs)] of recurrent events that were PE and fatal PE, as well as, the proportion of recurrent PEs that were fatal.
RESULTS: Nine RCTs (n=13,606 subjects; 413 recurrent events) were included. In persons presenting with an index PE, 67%; 95%CI, 62-71% of their recurrent events were PE and 27%; 95%CI, 22-33%) were fatal PE. Of all recurrent PEs observed, 41%; 95%CI, 33-48% resulted in death. No statistical heterogeneity was observed in any analysis (I2<32% for all).
CONCLUSION: Our meta-analysis suggests a majority of recurrent events in those experiencing an index PE will be PEs. Moreover, recurrent PEs are associated with a high mortality rate.

Disclosures
No relevant conflicts of interest to declare.
Increased Oxygen Supply in Metabolic Syndrome Patients Treated with Gingko Biloba
Authors: Siegel Guenter , Mockenhaupt Felicitas , Ermilov Eugeny 
Published: October 2019

Abstract
Background: In Swiss mountaineers, it was observed that they did not suffer from O2 deficiency even in high altitude with diminished O2 partial pressures, if they had taken gingko. In a pilot study with 11 metabolic syndrome patients, who received as therapy 2 × 120 mg/d Gingko biloba special extract EGb 761 (Rökan novoÒ: Spitzner Arzneimittel, Ettlingen; TeboninÒ: Schwabe Pharmaceutical, Karlsruhe, Germany) over 2 months, it should be proven whether a change of the blood parameters MCH, MCHC, MCV and Hct could eventually supply a mechanistic explanation.
Methods: Photometric methods, commercial ELISAs and EIAs, and clinical routine chemistry were applied.
Results: MCH was increased from 27.7 ± 0.9 to 28.6 ± 1.0 pg (p < 0.0003), a change of +3.2% (p < 0.0044), MCHC from 317.7 ± 2.5 to 333.0 ± 3.1 g/L (p < 0.0), a change of +4.8% (p < 0.001), while Hct was decreased from 0.463 ± 0.011 to 0.407 ± 0.009 vol/vol (p < 0.0), a change of -12.0% (p < 0.001). Additionally, alterations in several biomarkers were measured: SOD +17.7% (p < 0.009), oxLDL/LDL -21.0% (p < 0.002), MPO -29.6% (p < 0.013), hs-CRP -39.3% (p < 0.004), HOMA-IR -14.0% (p < 0.024), CREA -11.3% (p < 0.001), ALT -16.0% (p < 0.0208). The table demonstrates the results of a linear correlation analysis between these biomarker changes and the changes in Hct and MCHC showing calculated Pearson’s correlation coefficients r and error probabilities p.
Discussion: Even though the additional load of the single erythrocyte with an increased hemoglobin concentration seems insignificant, it is, however, decisive for the O2-saturation of the blood. Decrease of the hematocrit from 46.3% to 40.7% signalizes a normalization of the initially elevated value in these metabolic syndrome patients. Possibly, this lowering by 12.0% would not have been so distinct in a normal person. Reduction of oxidative stress, inflammatory parameters as well as decrease in insulin resistance offer an explanation for the increase in MCH and MCHC.
Conclusion: With the intake of ginkgo, a better O2-supply for organs and tissues is guaranteed not only through an increased blood perfusion but also through an improved load of the single erythrocyte with hemoglobin (MCHC) by 4.8% as well as through reduction of the hematocrit (Hct) by 12.0%.
Figure 1 Figure 1.

Disclosures
Siegel: Schwabe Pharmaceutics, Karlsruhe, Germany: Research Funding.
Autoimmune Hemolytic Anemia Induced By Kangaroo Essence Pills (Report of Two Cases)
Authors: Meng Yuesheng , An Zengmei 
Published: October 2019

Abstract
The autoimmune hemolytic anemia (AIHA) is characterized by the autoimmune destruction and shortened life span of red blood cells. The kangaroo essence pills (KEP)are marketed worldwide as an aphrodisiac agent. They may contain muscles and genital tissues of Australian kangaroo as the key active components. We report here two cases of autoimmune hemolytic anemia induced by the KEP. Patient One: A 68 years old female presented with malaise, dizziness, jaundice and deep-colored urine for three weeks. Before this, she had taken three brand names of KEP for several weeks. An anemia was diagnosed in a local hospital but the underlying cause was obscure. Treatment with iron, folic acid and mecobalamin was ineffective. Laboratory analysis revealed anemia with a hemoglobin level of 34.4g/L, reticulocyte count of 3.0%, elevated white blood cell count of 22.7×109/L with neutrophiles of 81.4%, and normal platelet count (98.4×109/L). The urine was positive for occult blood, proteinuria (+++), red blood cells (11-15/HP) and bilirubin (++). Total serum bilirubin was 93.3 umol/L and direct bilirubin 69.7umol/L. Both of direct and indirect antiglobulin tests (Coombs' tests) were positive. Cold agglutinins of IgM were negative. Ham’s test was negative. Activity of Glucose-6-phosphate dehydrogenase was normal. Bone marrow morphology showed erythroid hypercellularity, with a predominance of polychromatic normoblasts and orthochromatic normoblasts and abnormality of double-phases. A diagnosis of warm antibody autoimmune hemolytic anemia was made. Intravenous methylprednisolone with a daily dose of 80 mg was administered immediately in addition to transfusion of saline-washed red blood cells. Due to the severity of anemia, three regimens of daily plasmapheresis were given. Clinical conditions were improved rapidly. Two weeks later, the hemoglobin level was returned to normal range and she was discharged. Follow-up for one year proved no evidence of relapse. The patient’s history had no record of allergy and other health problems. The KEP was the only possible cause to induce hemolysis. Besides kangaroo tissues, some KEP may have other addictives such as ginseng, Chinese wolfberry, deers’ sinew, snowdeer’s penis, yak penis, wild donkey penis, cordyceps sinensis, and so on. It is yet known which ingredient, or even some impurities, were the prime culprit in this case. Patient Two: A 25 year old young woman presented with pale and dizziness for a week. CBC test showed decreased hemoglobin level 76.5g/L, reticulocyte count of 4.5%, increased direct bilirubin 48.2 umol/L, and a positive Coombs' test. Two weeks ago she had tried a brand name of KEP as analeptic agent. She was diagnosed with autoimmune hemolytic anemia and immediately cured with oral methylprednisolone. To our knowledge, the AIHA induced directly by “kangaroo essence pills” has yet been reported. It is suggested that caution should be made to take aphrodisiacs of unknown ingredients.

Disclosures
No relevant conflicts of interest to declare.
Hepcidin Levels in Patients with Lung Cancer
Authors: Mandadi Mounika , Rios Jorge Arturo , Schade Andrew , Kloecker Goetz H 
Published: October 2019

Abstract
Background:
Hepcidin is a 25 amino acid peptide produced by the liver and plays a key role in iron homeostasis. It regulates the serum iron concentration by binding and inhibiting ferroportin, a transmembrane protein causing iron efflux. Anemia is a common complication in patients with cancer and is caused by the interaction of the tumor cells and the immune system resulting in the expression of various inflammatory cytokines which cause changes in iron metabolism resulting in anemia. Elevated levels of hepcidin have been noted in patients with various cancers. A better understanding of anemia in cancer patients may lead to a more effective and safe treatment of cancer induced anemia, possibly by targeting hepcidin.
Methods:
Blood samples of patients with lung cancer before treatment, with any histology or stage were obtained from the Brown Cancer Center’s Clinical Data and Specimen Repository. Ferritin and hemoglobin level were performed on each sample at the University of Louisville laboratory. The deidentified samples of plasma were then tested by a chemiluminescence kit for determination of the hepcidin level.
Results:
Plasma of 30 patients with lung cancer was available for testing.
There was no correlation seen between the hepcidin level and age, race, sex, lung cancer histology and stage. Hepcidin has a Pearson correlation of 0.66 with ferritin levels. The range of ferritin was 8.7 to 765 ng/ml and hepcidin 349 to 411321 pg/ml.
Conclusions:
Hepcidin correlates with ferritin in lung cancer patients. Targeting hepcidin in lung cancer patients may be a treatment option for cancer induced anemia.

Disclosures
No relevant conflicts of interest to declare.
A Case of Kaposi’s Sarcoma in a HIV Negative Patient with CLL Treated with Rituximab
Authors: Belur Anuradha Avinash , Arumugam Raajasekar Arun Kumar , Nannapaneni Srikant , Chelliah Thandavababu 
Published: October 2019

Abstract
Case Description: - A 76 year old lady was diagnosed with Chronic Lymphocytic Leukemia (CLL) with 11 q deletion after she presented with generalized lymphadenopathy and anemia. She was treated with rituximab 375mg/m2 day1 and bendamustine 60mg/m2 on day 1 and day 2 and completed six cycles of treatment. After the sixth cycle she developed multiple itchy, papular lesions with bleeding on both lower extremities. She was evaluated multiple times by vascular surgery and dermatology without a definitive diagnosis. She underwent a biopsy with staining for HHV-8, CD31 and CD34 which was positive confirming the diagnosis of Kaposi sarcoma. ELISA test for HIV was negative. She was started on treatment with Doxorubicin 20 mg/m2every 3 weeks and with 3 cycles there was significant regression of the lesions.
Discussion-: We describe a case of CLL which was initially started on treatment with rituximab and bendamustine. She tolerated the treatment well, but a few months later presented with skin lesions which on biopsy was diagnosed as Kaposi sarcoma. It is very uncommon for Kaposi sarcoma to develop in a HIV negative patient. This patient was immunocompromised with recent chemotherapy. Rituximab specifically depletes B cells and leads to impaired T cell mediated immunity. This case illustrates the importance of a high index of suspicion in patients treated with rituximab as it is used for a number of hematologic malignancies like leukemia, lymphoma as well as non-malignant conditions like autoimmune disorders. While infusion reactions and reactivation of hepatitis B are side effects physicians are aware of and cautious of while using rituximab, Kaposi’s Sarcoma remains a less known side effect. Awareness of this possibility is important in physicians prescribing rituximab.
Footnotes
* Asterisk with author names denotes non-ASH members.

Disclosures
No relevant conflicts of interest to declare.
Inhibition of Protein Kinase a Induces Platelet Apoptosis
Authors: Zhao Lili , Wang Zhicheng , Zhang Weilin , Chen Mengxing , Dai Kesheng 
Published: October 2019

Abstract
Background:
Platelet apoptosis elucidated by either physical or chemical compound or platelet storage occurs wildly, which might play important roles in controlling the numbers and functions of circulated platelet, or in the development of some platelet-related diseases. However, up to now, little is known about the regulatory mechanisms of platelet apoptosis. Protein kinase A (PKA) is highly expressed and plays central roles in signal transduction in human platelets. The certain level of PKA activity is indispensability for maintaining circulating platelets in resting state. In nucleated cells, PKA stimulated proliferation or promoted cell death via apoptosis in different cell types. However, it is still unclear whether PKA plays a role in platelets apoptosis. The aim of the current study is to investigate the role of PKA in platelets apoptosis.
Methods and Results:
Apoptotic events were assessed in platelets by flow cytometry, western blotting. PKA inhibitor H89 dose-dependently elucidated depolarization of mitochondrial inner membrane potential (ΔΨm), activation of caspase-3, -9, cleavage of gelsolin, phosphatidylserine (PS) exposure and membrane shrinkage in platelets. Incubation of platelets with PKA catalytic subunit-α (PKACα)-siRNA specific reduced the production PKACα and obviously elucidated platelet apoptosis. Platelet aggregation induced by ADP, botrocetin, or α-thrombin was significantly reduced in platelets treated with H89. However, H89 does not incur P-selectin surface expression and PAC-1 binding in platelets. After intraperitoneal injection of PKA inhibitor (Rp-cAMPS), the circulating platelets were reduced at 8 hour time point and recovered within 24 hours. H89 down-regulated the expression of Bad in platelets, and phosphorylation of Bad at Ser 112 was reduced in platelets. Cyclosporin A and ROS inhibitor (NAC) significantly reduced H89-induced platelet ΔΨm depolarization. However, z-DEVE-fmk, GM6001, MDL28170, forskolin, SB203580, BAPTA had no obvious effect on depolarization of ΔΨm.
Conclusions:
These data indicate that inhibition of PKA results in ROS-mediated platelet apoptosis. The findings suggest a novel mechanism for PKA in regulating platelet numbers and functions, which has important pathophysiological implications for thrombosis and hemostasis.

Disclosures
No relevant conflicts of interest to declare.