bluuebunny/crossref_metadata_2025 huggingface dataset:
Recombinant Circularly Permuted TRAIL (CPT) for the Treatment of Relapsed or Refractory Multiple Myeloma: An Open-Label, Multicenter Phase II Clinical Trial
Authors: Chen Wenming , Qiu Lugui , Hou Jian , Zhao Yaozhong , Pan Ling , Yang Shifang , Leng Yun , Xi Hao , Zhang Xuejun , Cui Junsheng , Wei Na
Published: October 2019

Abstract 78

Background:

Circularly permuted TRAIL (CPT) is a recombinant mutant of human Apo2L/TRAIL developed by Beijing Sunbio Biotech Co., Ltd. as a targeted therapy for multiple myeloma and other hematologic malignancies. CPT is a dual pro-apoptotic receptor agonist that directly activates both pro-apoptotic receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). CPT selectively induces apoptosis in a variety of cancer cells, while sparing most normal cells in preclinical models.

Objective:

CPT has shown preliminary activities for patients with relapsed or refractory multiple myeloma (Rel/Ref MM) in phase I study. The aim of this phase II study is to investigate the effect and safety of CPT for Rel/Ref MM patients.

Methods:

In this multi-center, open-label, single arm phase II study, twenty-seven Rel/Ref MM patients were enrolled to receive CPT treatment alone at the dose of 2.5 mg/kg/day intravenously, once daily for 14 consecutive days of each 21-day cycle. Clinical responses to CPT after 2 cycle's treatment were assessed by an independent review committee according to the criteria of the European Group for Blood and Marrow Transplantation (EBMT).

Results:

Among the 27 patients, one patient (3.7%) achieved near complete response (nCR) and eight patients (29.63%) exhibited partial response (PR). The overall response rate (ORR) was 33.3%. The median PFS was not reached within 2 cycle's treatment. The drug related adverse events (≥10%) included fever, AST/ALT elevation, leucopenia, neutropenia, rash, thrombocytopenia, and LDH elevation. The severe adverse events were occurred in 3 patients (11%), one of them was CPT-related liver injury. Anti-drug antibodies were detected in 6 patients without reduced efficacy or increased toxicities.

Conclusions:

The CPT is a very effective and well-tolerated new agent for Rel/Ref MM, and it is worthy to be further studied.

Disclosures:

Yang: Beijing Sunbio Biotech Co. Ltd.: Employment. Cui:Beijing Sunbio Biotech Co. Ltd.: Employment.
CHD7 Negatively Regulates Hematopoietic Stem Cell Potency
Authors: Hsu Jingmei , Huang Hsuan-Ting , Lee Chung-Tsai , Yu Shuqian , Gerber Scott , Zon Leonard I. , Speck Nancy A.
Published: October 2019

Abstract 850

The molecular mechanisms governing HSC homeostasis and potency are incompletely understood. Runx1-CBFb is required for HSC formation in the embryo and for maintaining normal HSC and progenitor cell frequencies in the adult. To identify chromatin regulatory proteins that function with Runx1-CBFb during hematopoiesis, we performed co-immunoprecipitation and mass spectrometry using a FLAG-tagged CBFb protein, and as a control a FLAG-CBFb (G61A/N104A) mutant that cannot bind Runx1. Multiple unique peptides of the ATP-dependent chromatin remodeling enzyme Chromodomain Helicase DNA binding protein 7 (CHD7) were immunoprecipitated specifically with FLAG-CBFb. Autosomal dominant CHD7 mutations are associated with CHARGE syndrome (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and/or development, Genital and/or urinary abnormalities, and Ear abnormalities and deafness). Several subunits of the nucleosome remodeling and histone deacetylase (NuRD) complex were also identified, including p66a, p66b, MTA2, and histone binding protein RBBP4. Hence Runx1-CBFb may associate with distinct CHD7 and NuRD containing complexes to regulate HSC function.

The CHD7 interaction with Runx1 requires the Runx1 transactivation domain, which is critical for all stages of hematopoiesis. The interaction of CHD7 with Runx1 does not require heterodimer formation with CBFb. Runx1+/−; Chd7+/− double heterozygous mice are born in a sub-Mendelian ratio, indicating that Runx1 and Chd7 interact genetically. Knockdown of chd7 in zebrafish embryos increased the number of runx1 expressing cells specified in the AGM, and this effect is cell autonomous as determined by blastula transplantation. Downstream lineages such as myeloid and erythroid cells were also expanded. Consistent with these results, conditional pan-hematopoietic deletion of Chd7 in mice with Vav1-Cre did not cause mature lineage differentiation defects. Methylcellulose colony forming assays of CHD7 deficient cells resulted in increased granulocyte/monocyte progenitors. Although CHD7 deficient bone marrow had a normal frequency of CD48− CD150+ Lin−Sca-1+c-kit+(LSK) phenotypic long term repopulating HSCs (LT-HSCs), it has a three fold higher frequency of functional LT-HSCs as determined by whole bone marrow limiting dilution transplants. By serial transplantation, a higher percentage of tertiary recipient mice were engrafted with CHD7 deficient bone marrow cells compared to wild type. In summary, our study identifies CHD7 as a novel epigenetic regulator of HSC and progenitor function that functions with Runx1 and members of the NuRD complex to negatively regulate HSC numbers and potency.

Disclosures:

Zon: Fate Therapeutics: Founder Other.
Loss of Krüppel-Like Factor 4 (KLF4) Impairs the Self-Renewal of Leukemic Stem Cells
Authors: Park Chun Shik , Shen Ye , Yamada Takeshi , Suppipat Koramit , Lacorazza H. Daniel
Published: October 2019

Abstract 911

The identification of pathways that differentially regulate the self-renewal in normal hematopoietic stem cells (HSC) and leukemic stem cells (LSC) is critical for effective treatment of relapsed leukemia without affecting normal hematopoiesis. Krüppel-like factor 4 (KLF4) is a bi-functional transcription factor that can either activate or repress transcription and thus function as an oncogene or a tumor suppressor depending on the cellular context. We have previously shown that loss of KLF4 leads to increased self-renewal of HSC in serial bone marrow transplantation. In this work, we investigated whether KLF4 also regulates self-renewal in LSCs using a mouse model of somatic deletion of KLF4 in hematopoietic cells (Klf4fl/fl Vav-iCre+) and the BCR-ABL-induced chronic myeloid leukemia (CML) model. Mice transplanted with wild type bone marrow (BM) cells transduced with retrovirus carrying BCR-ABL (p210) (n=18) showed an expansion of myeloid cells (GFP+ Gr1+) with a median survival of 19 days. In contrast, mice transplanted with KLF4-deficient BM cells transduced with BCR-ABL retrovirus (n=12) exhibited prolonged survival with a median survival of 27 days (p<0.0011). Progression of leukemia was monitored in peripheral blood, BM and the spleen by flow cytometry. Mice transplanted with KLF4-deficient cells showed expansion of myeloid leukemic cells (GFP+ Gr1+) in the first two weeks after BM transplant, which was followed by a progressive loss of myeloid cells and an expansion of B cells (GFP+ B220+). In control group, 90% of leukemic mice succumbed from CML whereas more than 50% of KLF4-deficient leukemic mice developed mixed CML and B-ALL leukemia. These results suggest that loss of KLF4 impairs the maintenance of BCR-ABL-induced CML, while allowing expansion of BCR-ABL positive B cells (GFP+ B220+). Since KLF4 deletion prevented continuous expansion of myeloid leukemic cells (GFP+ Gr1+), we postulated that KLF4 may be critical to LSC maintenance. Thus, we analyzed the frequency of LSCs (GFP+ Lin–c-Kit+ Sca-1+and GFP+ Lin– c-Kit+ Sca-1+Flt3–) in BM and the spleen. We found that LSCs were significantly reduced in recipients of BCR-ABL-transduced KLF4-deficient BM cells 18 days after transplantation (n=6, p <0.001). These studies demonstrate that KLF4 has different roles in the maintainenance of LSCs and normal HSCs and that inactivation of KLF4 provides a therapeutic strategy for eradicating LSCs without damaging normal HSC.

Disclosures:

No relevant conflicts of interest to declare.
ULK1-Hsp90-Cdc37 and AMPK: Novel Insight Into the Regulation of Mitophagy
Authors: Joo Joung Hyuck , Kundu Mondira
Published: October 2019

Abstract 987

Autophagy plays an important role in maintaining mitochondrial integrity, directing lysosome-mediated destruction of cellular cargo, including damaged or dysfunctional mitochondria. Flux through the autophagy pathway is rapidly induced to promote survival in response to metabolic or proteotoxic stress resulting from exposure to noxious environmental cues, such as starvation, hypoxia or heat stress and likely contributes to the increase in mitochondrial turnover observed under each of these conditions. Dysregulation of this process has been linked to the pathogenesis of diseases, including anemia, diabetes, neurodegeneration and cancer.

Atg1 is a serine-threonine kinase that directs the autophagy machinery to appropriate cargo in responses to changes in the availability of carbon and nitrogen in yeast. Ulk1, one of the mammalian homologues of Atg1, is required for starvation-induced autophagy and clearance of mitochondria in terminally differentiating erythroid cells. The function of Ulk1 is also regulated by AMP dependent Kinase (AMPK)-mediated phosphorylation, however, the precise molecular consequence of this post-translational modification has not been explored. Our preliminary findings indicate that AMPK phosphorylates Ulk1 during red blood cell maturation and in response to mitochondrial uncoupling, and that this phosphorylation is critical for mitochondrial clearance. Therefore, we sought to use these systems to explore the mechanism by which AMPK phosphorylation regulates Ulk1 function.

We previously demonstrated that the stability and kinase activity of Ulk1 depends on its physical interaction with Hsp90 and the kinase-specific co-chaperone, Cdc37. Hsp90 is an abundant chaperone that directs the maturation and activation of a restricted group of metastable proteins, typically kinases and signaling molecules, and orchestrates a broad response to cellular stress. Here, we demonstrate that AMPK phosphorylation of Ulk1 does not affect Ulk1 kinase activity, but instead promotes its release from Hsp90 and its localization to damaged mitochondria. Preliminary studies indicate that the serine-proline rich domain of Ulk1, which contains at least 4 residues that are phosphorylated by AMPK, is an intrinsically disordered domain. We hypothesize that phosphorylation of Ulk1 by AMPK stabilizes a predicted alpha-helical structure within this domain and contributes to release of Hsp90. These findings are important because they provide significant insight into the regulation and function of Ulk1, a protein involved in mitochondrial turnover during red blood cell maturation and in proliferating cells.

Disclosures:

No relevant conflicts of interest to declare.
Stress-Mediated Activation of Dormant Hematopoietic Stem Cells In Vivo
Authors: Trumpp Andreas , Essers Marieke
Published: October 2019

Abstract SCI-41

Maintenance of the blood system is dependent on dormant hematopoietic stem cells (HSCs), which are characterized by pluripotency and lifelong self-renewal capacity. In order to both maintain a supply of mature blood cells and not exhaust HSCs throughout the lifespan of the organism, most adult HSCs remain deeply quiescent during homeostasis, and only a limited number are cycling at any given time. The balance between self-renewal and differentiation of HSCs is controlled by external factors such as chemokines and cytokines, as well as by interactions of HSCs with their niche environment. The transcriptome of dormant CD34-CD150+CD48-LSK- HSCs significantly differs from that of active HSCs with the same phenotype, while the latter are highly similar to MPP1 progenitors which express CD34. One of the genes differentially expressed is the cylindromatosis (CYLD) gene, which encodes a negative regulator of the NF-κB signaling pathway. HSCs failing to express functional CYLD show various defects associated with a disturbed balance between dormant and active HSCs, suggesting a role for NF-κB signaling in establishing dormancy in HSCs. In addition, our studies have recently shown that the cytokine interferon-α (IFNα) very efficiently activates dormant HSCs in vivo. Within hours after treatment of mice with IFNα, HSCs exit G0 and enter an active cell cycle. In general, IFNα is produced in response to viral infections by cells of the immune system, and plays an important role in the antiviral host defense. We now questioned whether endogenous IFNα is also produced in response to other types of bone marrow stress and whether this affects the proliferation rate of HSCs. To monitor IFNα production in the bone marrow in vivo, we have generated MxCre ROSA-R26-EYFP mice and found that treatment with both the chemotherapeutic agent 5-FU as well as the endotoxin LPS leads to the production of IFNα in the vicinity of HSCs and progenitors. In addition, LPS treatment in vivo induced a strong increase in HSC cycling. Surprisingly, since mice lacking the IFNα receptor (Ifnar−/−) still respond to LPS, this effect is independent of IFNAR signaling. Strikingly, LPS-induced HSC activation correlated with increased expression of Sca-1, similar to what occurs upon IFNα treatment. Moreover, as for IFNα, the upregulation of SCA-1 is required for LPS-induced proliferation, since Sca-1−/− mice fail to respond to LPS stimulation. In summary, these data suggest that not only virus-inducible IFNα, but also infections by gram-negative-bacteria-produced LPS induce cycling of progenitors and otherwise dormant HSCs.

Disclosures:

No relevant conflicts of interest to declare.
Prostaglandin E Synthase Is Upregulated By Gas6 during Cancer-Induced Venous Thromboembolism
Authors: Aghourian Meghedi N , Lemarie Catherine A , Bertin Francois-Rene , Blostein Mark D
Published: October 2019

Venous thromboembolism (VTE) is the most common morbid complication related to cancer and its treatments. Although malignancies are characterized by a hypercoagulable state leading to VTE, the pathophysiology of this state has not been well studied. Growth arrest specific 6 (Gas6) is a protein that has pro-coagulant properties. Gas6 deficient mice develop smaller venous thrombi as compared to wild type mice, and express less tissue factor in the endothelium when challenged with thrombotic stimuli. We hypothesize that Gas6 may be involved in cancer-induced venous thrombosis. In order to test this hypothesis, venous thrombi were induced in wild type (WT) and Gas6 null (-/-) mice injected with M27 murine lung cancer cell lines. Thrombus size was measured using ultrasonography, thrombus weight and histology. We observed that WT mice with cancer developed larger thrombi than their healthy counterparts (p<0.05). However, these larger thrombi induced by cancer were not seen in Gas6-/- mice, suggesting that Gas6 has a pathophysiologic role in promoting malignancy associated VTE. Whole genome microarray analysis was then used to identify differential gene expression in WT and Gas6-/- endothelial cells co-cultured with M27 murine lung carcinoma cells. Microarray analysis revealed that prostaglandin E synthase (PTGES) was increased in WT endothelial cells but not in Gas6-/- cells co-cultured with M27. These results were confirmed using real-time PCR and immunofluorescence staining (p<0.05). In WT endothelial cells, PTGES expression was regulated through ERK1/2 phosphorylation. We also show that co-culture of WT endothelial cells with M27 augments the secretion of PGE2, the enzymatic product of PTGES. PGE2 activates platelets in vitro after binding to its receptor, EP3. In vivo, EP3 receptor antagonism reversed the effect of cancer-induced thrombosis in WT mice. These results show that Gas6, through upregulation of PGE2, contributes to cancer-induced VTE.

Disclosures

No relevant conflicts of interest to declare.
Ex Vivo Expansion of Cord Blood Hematopoietic Stem and Progenitor Cells in a Wharton’s Jelly and Bone Marrow Stromal Cell Based Co-Culture System
Authors: Li Dandan , Aljitawi Omar S. , Hopkins Richard A.
Published: October 2019

Umbilical cord blood (UCB) is of great value by providing transplantable hematopoietic stem and progenitor cells (HSPCs). Compared with HSPCs from adult bone marrow and periferial blood, UCB cells are more primitive with higher proliferation ability, and UCB HSPC transplantation requires less HLA matching. The major problems in UCB transplantation is the limited number of transplantable cells in each unit which are often insufficient for transplantation in adults

In order to elucidate the effects of the main components of bone marrow on cord blood CD34+ expansion, freshly enriched cord blood CD34+ cells were cultured in contact with bone marrow stromal cell (BM-MSC) monolayer, BM-MSCs pre-seeded in decellularized Wharton's jelly matrix (DWJM), and in DWJM alone, as well as separated by a transwell preventing the physical contact between CD34+ cells and BM-MSCs in a medium supplemented with a cytokine cocktail including Flt-3 igand, stem cell factor, and thrombopoietin. Expansion patterns were analyzed by CD34+ and CD45+ expansion, as well as colony-forming unit (CFU) assay. We found that the number of CFU increases significantly in cord blood CD34+ cells co-cultured with BM-MSCs in DWJM compared to that of control. Particularly, the increase in CFU-GEMM, BFU-E and CFU-GM number is more significant compared to other colonies. Importantly, DWJM alone is not able to increase CFU numbers compared to that of the control indicating there is a synergistic effect between DWJM and BM-MSCs on cell stemness. Surprisingly, BM-MSCs in DWJM also increses CD34+ cell expansion by 2 to 3 fold after one week culture, presumably from enhanced ablilty of self-renewal from CD34+ cells. Therefore, our data suggest DWJM synergizes with BM-MSCs to increase CD34 cell stemness, which can potentially be used in the clinic therapy.

Disclosures

No relevant conflicts of interest to declare.
A Genetic Variant in the CD53 Gene Is Associated with Clinical Outcomes after Unrelated Bone Marrow Transplantation
Authors: Espinoza Luis Jorge , Matsuo Keitaro , Toda Saki , Morishima Yasuo , Onizuka Makoto , Kashiwase Koichi , Fukuda Takahiro , Kodera Yoshihisa , Nakao Shinji , Takami Akiyoshi
Published: October 2019

Background: CD53, a member of the tetraspanin family of proteins, broadly expressed in hematopoietic cells, is implicated in leukocyte activation and cell survival responses to stress conditions. In a recent genome linkage scan, the single nucleotide variation, rs6679497 (C>G), in the intron of the CD53 gene was identified as an important regulator of TNF release from innate immune cells in response to inflammatory stimuli. (Bos SD: Eur J Hum Genet. 2010). Tumor necrosis factor alpha (TNF-ƒ¿) which is released pretransplant due to irradiation and cytotoxic preconditioning regimens, has been implicated in transplant complications.

Methods: To assess the impact of rs6679497 variation of the CD53 gene, on transplant outcomes, we evaluated 322 of patients with hematologic malignancies transplanted through the Japan Marrow Donor Program. The functional relevance of the rs6679497 variation was investigated in a whole blood culture system using samples from healthy individuals. To investigate the effect of rs6679497 on mRNA transcription activity we performed allele-specific quantitative RT-PCR with a TaqMan probe of rs6679497 using cDNA from in vitro activated leukocytes from healthy donors with heterozygous genotypes.

Results: There was a significantly lower overall survival in the recipients with the GG genotype in the univariate analysis (29% vs. 53% at 5-y; P=0.01) and multivariate analysis (relative risk=1.97; 95% confidence interval, 1.22-3.18; P=0.01, Figure 1). The donors s6679497 genotypes did not significant influence the transplant outcomes. In the allele-specific quantitative PCR, the mean ratio (G allele transcripts vs. C allele transcripts) was 1.5 which is higher than that of DNA amplicons, suggesting that the G allele has higher transcriptional activity than the C allele. Using the TF-SEARCH online tool, which predict putative transcription factor binding sites, the genomic region containing the G allele of s6679497 was found to create a novel consensus sequence corresponding to the putative binding element of GATA-1. In vitro stimulated leucocytes from healthy individuals possessing the G allele secreted higher levels of TNF-ƒ¿ than those without the G allele. Consistent with these results, flow cytometry studies revealed that monocytes from donors possessing the G allele express higher levels of CD53 on their surface (p=0.02) which may account for their higher TNF-ƒ¿ secretion. These findings substantiate the functional relevance of the rs6679497 variation, and indicate that the increased TNF-ƒ¿ secretion by individuals with the G allele likely account for their worse OS.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Meta-Analysis Evaluating Efficacy of High-Dose Therapy and Autologous Hematopoietic Cell Transplantation As Front-Line Consolidation in Chronic Lymphocytic Leukemia: Results Do Not Justify This Treatment Approach
Authors: Reljic Tea , Kumar Ambuj , Djulbegovic Benjamin , Kharfan-Dabaja Mohamed A
Published: October 2019

Background: High-dose therapy (HDT) followed by autologous hematopoietic cell transplantation (auto-HCT) has been offered to patients with chronic lymphocytic leukemia (CLL), both as front-line consolidation and in the relapsed setting. Uncertainty remains in regards to the role of HDT in the front-line consolidation setting in CLL. Accordingly, we performed a systematic review and meta-analysis aiming at evaluating the totality of evidence pertaining to the efficacy (or lack thereof) of HDT and auto-HCT as front-line consolidation in subjects with CLL.

Materials and methods: A total of 475 references were identified through a systematic search of PUBMED/MEDLINE and Cochrane through June 26, 2014. Only four randomized controlled trials which included a total of 600 subjects were eligible for inclusion in this meta-analysis. Data was meta-analyzed for benefits: progression-free survival (PFS) (data from 2 studies (total n=178)), event-free survival (EFS) (data from 2 studies (total n=422)), PFS or EFS (data from 4 studies (total n=600)), and overall survival (OS) (data from 4 studies (total n=600)); and harms: treatment related mortality (TRM) (data from 2 studies (total n=276)), development of any secondary malignancy ((data from 4 studies (total n=581)), or development of secondary MDS/AML in particular (data from 4 studies (total n=581)).

Results: These results are reported in accordance with Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines. Four studies enrolled a total of 301 subjects in the HDT/auto-HCT arm and 299 subjects in the control arm. Offering front-line HDT/auto-HCT did not improve PFS [Hazard ratio (HR)=0.70 (95%CI= 0.32, 1.52), p=0.37] or OS [HR=0.91 (95%CI= 0.62, 1.33), p=0.64]. An advantage favoring HDT/auto-HCT was observed in terms of EFS (HR=0.46 (95%CI= 0.26, 0.83), p=0.01] or when analysis included PFS or EFS [HR=0.54 (95%CI= 0.35, 0.82), p=0.004]. Moreover, HDT/auto-HCT did not result in higher TRM (Risk ratio (RR)=1.32 (95%CI= 0.43, 4.06), p=0.63]. When analyzing development of any secondary malignancy, no difference was observed whether subjects were offered HDT/auto-HCT or not [RR=1.06 (95%CI=0.55, 2.05), p=0.86]. A two-fold higher incidence of secondary MDS/AML, albeit not statistically significant, was observed with the HDT/auto-HCT approach [RR=1.95 (95%CI=0.60, 6.34), p=0.27].

Conclusion: Offering HDT/auto-HCT as front-line consolidation in patients with CLL does not improve OS. At the present time, this approach should be offered only in the context of a clinical trial.

Disclosures

No relevant conflicts of interest to declare.
A Study of Phenotypic Heterogeneity in Hb E-Beta Thalassemia and Its Correlation with Hb E Level
Authors: Bhattyacharyya Maitryee , Bagchi Basab , Kumar Meet
Published: October 2019

INTRODUCTION: Hb E-beta thalssemia has variable clinical phenotype, varying from NTDT to severe transfusion dependent thalassemia. Factors influencing the severity include HbF level, type of beta mutation, co-inheritance of alpha mutation (including triplication and quadruplication), and various polymorphisms. We evaluate the possible correlation between HbE levels and clinical severity in patients in Hb E-beta thalassemia.

AIMS AND OBJECTIVES: To study the phenotypic heterogeneity of Hb E-beta thalassemia patients with respect to Hb E levels.

MATERIALS AND METHODS: Patients >18years were enrolled and evaluated for clinical phenotype as per Mahidol score (age of onset, age of first transfusion, requirement for transfusion, spleen size, growth retardation and steady-state hemoglobin), and classified into mild, moderate and severe. Further biochemical data was evaluated including serum ferritin. The clinical phenotype severity was correlated with HbE and HbF levels. Patients on disease modifying drugs (eg hydroxyurea) were excluded.

RESULTS: A total of 120 patients of Hb E-beta thalassemia were enrolled with M:E = 1.1:1. 65.8% patients were 18-30 years age range, while 5.8% were above 50years of age. As per Mohidol score, 39.2% (47) patients were mild, 45.8% (55) were moderate and 15% (18) were severe phenotype. Observations amongst patients of different clinical severity are as shown in Table 1. Abstract 1365. Table 1.CharacterMildModerateSevereRangeP valueNumber475518Baseline Hb (g/dl)7.9+-0.96.4+-1.16.2+-0.73.8-10.30.000Age at presentation (years)18.3+-8.7613.1+-8.03.16+-2.081-440.000Age at 1st transfusion (years)22.1+-11.415.1+-8.763.9+-1.91-600.000Freq of transfusion (annual)2.5+-4.512.2+-7.619.3+-11.40-480.000Spleen size (cms)5.6+-2.47.8+-2.910.4+-3.40-150.000Serum ferritin (ng/ml)792.5+-534.61206.5+-523.91570.5+-581.264.2-2924.20.000

Hb E levels of the patients ranged from 28% to 83.5%, and HbF levels ranged from 3.1% to 49.4%. On statistical analysis, difference in HbE levels was significant between severe vs mild and intermediate groups (p=0.0005 and 0.012 respectively), but not amongst mild vs intermediate groups (p=0.21). Also, difference in HbF levels was significant between mild vs moderate and severe groups (p=0.007 and 0.004 respectively), but not between moderate vs severe groups (p=0.26). Regression analysis showed HbF and HbE to be independent parameters of severity and both have negative correlation with disease severity.

CONCLUSION: HbE is an independent parameter of severity and correlated negatively with disease severity in HbE-beta thalassemia.

Disclosures

No relevant conflicts of interest to declare.
Functional Analysis of Phage Display Selected Single-Chain Variable Antibody Fragments (scFvs) Specific for Anti-FVIII Antibodies
Authors: Brettschneider Kerstin , Naumann Anja , Neimanis Sonja , Kahle Joerg , Heller Christine , Klingebiel Thomas , Schwabe Dirk , Koenigs Christoph
Published: October 2019

The development of inhibitory antibodies against coagulation factor VIII (FVIII) is currently the most serious complication for hemophilia A patients that undergo FVIII replacement therapy. In addition, non-hemophilia A patients can spontaneously develop inhibitory auto-antibodies to FVIII, which results in acquired haemophilia A. The control of the allo- or autoimmune response to FVIII apparently includes the elicitation of anti-idiotypic antibodies. In this study the capacity of anti-idiotypic single-chain variable antibody fragments (scFvs) for neutralization of inhibitory anti-FVIII antibodies (FVIII inhibitors) was evaluated in vitro and in vivo.

Anti-idiotypic scFvs were selected from phage-displayed libraries against murine monoclonal FVIII-specific inhibitors. As the majority of inhibitory antibodies is directed against the A2 or C2 domain of FVIII, strongly inhibitory A2- and C2-specific antibodies served as targets. Selected scFvs were expressed as scFv-Fc fusion proteins.

Analysis of the scFv-Fcs by ELISA confirmed the specific binding to the cognate targets and binding studies via surface plasmon resonance revealed high affinities within the nanomolar range. Further characterization showed that binding of inhibitors to immobilized FVIII was blocked by specific scFv-Fcs in vitro. The ability of scFv-Fcs to neutralize their corresponding inhibitors was analyzed in a functional clotting assay. By adding scFv-Fcs to plasma spiked with inhibitors, FVIII activity was restored to 80% in a concentration dependent manner.

FVIII knockout mice served as model organism for testing the capacity of scFv-Fcs to restore coagulation in vivo. Subsequent injection of FVIII following the injection of the inhibitors resulted in a largely reduced FVIII activity. However, FVIII activity was recovered in a concentration dependent manner by adding cognate anti-idiotypes. The scFv-Fcs were either preincubated with the corresponding inhibitor or added to the FVIII mixture without preincubation. The latter represents an adaption to a therapeutic setting.

In conclusion, phage display selected anti-idiotypic scFvs are able to bind and effectively neutralize their target inhibitors in vitro and in vivo. Based on these promising results the potential of anti-idiotypic scFvs for the development of specific cell based immunotherapies for hemophilia A patients with inhibitors is currently under investigation.

Disclosures

No relevant conflicts of interest to declare.
CHRONIC NEUTROPENIA
Authors: HATTERSLEY PAUL G.
Published: October 2019

A case of severe chronic neutropenia without splenomegaly is reported. The disorder was characterized by chronic fatigue and many pyogenic infections, and by a persistently low neutrophile count. The marrow was cellular, with a defect in maturation in both granulocytic and megakaryocytic series. There was no improvement on the usual types of medical therapy, and splenectomy, while it was followed by transient thrombocytosis, likewise failed to induce a remission.

It is suggested that in the consideration of therapy for severe neutropenia, a lack of splenomegaly should be considered a contraindication to splenectomy.
CHRONIC LEG ULCER IN DISEASES OF THE BLOOD
Authors: GENDEL BENJAMIN R.
Published: October 2019

The literature pertaining to the association of chronic leg ulcer with diseases of the blood other than sickle cell anemia has been reviewed.

Two patients with this association have been presented. One patient had a hemolytic anemia and the other a pancytopenia (secondary hypersplenism) in association with congestive splenomegaly due to cirrhosis of the liver.

It is suggested that the association of leg ulcer with these various diseases of the blood is related in an unknown manner to either splenomegaly or hyperfunction of the spleen (hypersplenism).
THE OCCURRENCE AND SIGNIFICANCE OF "MOTILE" ERYTHROCYTES IN HUMAN BLOOD AND MARROW IN ANEMIC STATES
Authors: RALPH PAUL H.
Published: October 2019

1. With the aid of supravital studies and dark-field illumination, "motile" erythrocytes (capable of self initiated amoeboid-like change in form and, in some cases, movement from place to place) may be found in the peripheral blood of cases of anemia, particularly those of the macrocytic variety.

2. These motile erythrocytes are usually hypochromic, contain mitochondria and neutral red-stainable granules, all of which are attributes of normal but immature red cells.

3. Motile erythrocytes similar to those found in the blood of human patients with anemias are found in the blood of normal rabbits and mice.

4. In the marrow of patients with macrocytic anemias, megaloblasts and megalocytes may be found which give rise to motile erythrocytes by budding.

5. Poikilocytosis, motility, paleness, content of mitochondria and neutral red stainable granules, and origin by budding from a large parent cell do not differentiate the erythrocytes of pernicious anemia from those of other anemic states.
STUDIES ON HYPOPROTEINEMIA. II. FAMILIAL IDIOPATHIC DYSPROTEINEMIA
Authors: HOMBURGER F. , PETERMANN M. L.
Published: October 2019

1. A new syndrome, idiopathic familial dysproteinemia, is described in 4 adult members of one generation, in 2 of their paternal uncles and in 4 members of the second generation. The syndrome is characterized by hypoproteinemia and/or abnormalities in the electrophoretic patterns of the blood plasma (dysproteinemia). These are accompanied in the adult by peripheral vascular changes (ulcers of the legs in the men, low oscillometric indices in the women) and edema. There are also malformations of the thoracic cage and of the occipital hair distribution in some of the cases.

2. The idiopathic nature of the disease was ascertained in some of the patients by study of the nutritional history, of the renal, hepatic and adrenal functions, and of the response to a high-protein diet under controlled conditions.

3. In one case detailed studies of the mechanisms of plasma protein regulation resulted in findings that indicate a disturbance in the production of certain protein components. The disappearance rate of injected albumin and the rate of replacement of acutely withdrawn plasma protein were normal.

4. The clinical and physio-pathologic significance of this syndrome and the possible role of genetic factors are discussed.
Congenital neutropenia: neutrophil proliferation with abnormal maturation
Authors: Parmley RT , Ogawa M , Darby CP Jr , Spicer SS
Published: October 2019

A child with congenital neutropenia was studied using bone marrow culture and ultrastructural and cytochemical techniques. The patient's marrow cells formed a large number of granulocytic colonies of normal size in culture, and her peripheral blood leukocytes produced adequate colony-stimulating factor. No serum inhibitors were identified. The patient's promyelocytes from direct marrow and culture appeared normal in ultrastructure, and primary granules, contained peroxidase and acid phosphatase activity. Myelocytes and rare segmented neutrophils from direct marrow specimens demonstrated atypical notched nuclei, myelin figures in Golgi lamellae and primary (azurophilic) granules, and no identifiable secondary (specific) granules. These data indicate an intrinsic neutrophil defect which allows normal proliferation of precursor cells, but results in abnormal granulogenesis and apparent inability to form secondary granules.
Bovine factor v: a calcium-containing metalloprotein
Authors: Greenquist AC , Colman RW
Published: October 2019

Although coagulation factor Xa requires Ca2+ for binding to phospholipid, factor V, the other protein component in the prothrombinase enzyme complex, binds tightly to phospholipid in the absence of Ca2+. To explore the possibility that calcium might be present in the fact V molecule, the effect of several chelators, including oxalate, citrate, pyrophosphate, and EDTA, on factor V activity has been studied. A time- and concentration-dependent inhibition of factor V which reflects the respective association constants for calcium of each chelator is observed. The inhibition can be prevented by the prior addition of calcium and manganese but not magnesium. Reversal of the activity loss can be accomplished at high protein concentrations by the addition of calcium, the removal of the chelator by gel filtration, or an increase in temperature. Factor V contains 1 g atom of calcium per 300,000 daltons which is not removed by incubation with EDTA under nondenaturing conditions. Thus, the inhibition by EDTA is due to binding to calcium associated with factor V. In 8 M urea, EDTA can remove over 80% of the calcium, demonstrating the importance of the native structure in maintaining the calcium binding site. Prior binding of phospholipids to factor V prevents inhibition by EDTA. The results suggest that phospholipids complex at the calcium site on the factor V metallopretein.
Management of insomnia in sleep disordered breathing
Authors: Janssen Hennie C.J.P. , Venekamp Lisette N. , Peeters Geert A.M. , Pijpers Angelique , Pevernagie Dirk A.A.
Published: October 2019

Both obstructive sleep apnoea (OSA) and chronic insomnia disorder are highly prevalent in the general population. Whilst both disorders may occur together by mere coincidence, it appears that they share clinical features and that they may aggravate each other as a result of reciprocally adverse pathogenetic mechanisms. Comorbidity between chronic insomnia disorder and OSA is a clinically relevant condition that may confront practitioners with serious diagnostic and therapeutic challenges. Current data, while still scarce, advocate an integrated and multidisciplinary approach that seems superior over the isolated treatment of each sleep disorder alone.
Low-protein diets supplemented with casein hydrolysate favor the microbiota and enhance the mucosal humoral immunity in the colon of pigs
Authors: Wang Huisong , Shen Junhua , Pi Yu , Gao Kan , Zhu Weiyun
Published: October 2019

Background

High-protein diets can increase the colonic health risks. A moderate reduction of dietary crude-protein (CP) level can improve the colonic bacterial community and mucosal immunity of pigs. However, greatly reducing the dietary CP level, even supplemented with all amino acids (AAs), detrimentally affects the colonic health, which may be due to the lack of protein-derived peptides. Therefore, this study evaluated the effects of supplementation of casein hydrolysate (peptide source) in low-protein (LP) diets, in comparison with AAs supplementation, on the colonic microbiota, microbial metabolites and mucosal immunity in pigs, aiming to determine whether a supplementation of casein hydrolysate can improve colonic health under very LP level. Twenty-one pigs (initial BW 19.90 ± 1.00 kg, 63 ± 1 days of age) were assigned to three groups and fed with control diet (16% CP), LP diets (13% CP) supplemented with free AAs (LPA) or casein hydrolysate (LPC) for 4 weeks.

Results

Compared with control diet, LPA and LPC diet decreased the relative abundance of Streptococcus and Escherichia coli, and LPC diet further decreased the relative abundance of Proteobacteria. LPC diet also increased the relative abundance of Lactobacillus reuteri. Both LP diets decreased concentrations of ammonia and cadaverine, and LPC diet also reduced concentrations of putrescine, phenol and indole. Moreover, LPC diet increased total short-chain fatty acid concentration. In comparison with control diet, both LP diets decreased protein expressions of Toll-like receptor-4, nuclear factor-κB, interleukin-1β and tumor necrosis factor-α, and LPC diet further decreased protein expressions of nucleotide-binding oligomerization domain protein-1 and interferon-γ. LPC diet also increased protein expressions of G-protein coupled receptor-43, interleukin-4, transforming growth factor-β, immunoglobulin A and mucin-4, which are indicators for mucosal defense activity.

Conclusions

The results showed that supplementing casein hydrolysate showed beneficial effects on the colonic microbiota and mucosal immunity and barrier function in comparison with supplementing free AAs in LP diets. These findings may provide new framework for future nutritional interventions for colon health in pigs.
First report of the entomopathogenic nematode, Steinernema carpocapsae, from Moghan region of Iran and its efficacy against the turnip moth, Agrotis segetum Denis and Schiffermuller (Lepidoptera: Noctuidae), larvae
Authors: Ebrahimi Laleh , TanhaMaafi Zahra , Sharifi Parviz
Published: October 2019

During a survey of entomopathogenic nematodes (EPNs) in Moghan region of Iran in 2015, a steinernematid species was isolated, using the Galleria-baiting method. Based on its morphological and phylogenetic analysis of molecular data, the isolate was identified as Steinernema carpocapsae. The ITS rDNA sequence was deposited in NCBI (National Center for Biotechnology Information) with accession number of MF187616.1. Nucleotide row data was edited, using MEGA 6.0 software, and homologous sequences were involved in analysis, using Blast software. Sequences were aligned using Clustal W. Bootstrap analysis. The phylogenetic tree was constructed by the maximum likelihood method, using MEGA 6.0 software, and Steinernema feltiae was used as out-group. The turnip moth, Agrotis segetum Denis and Schiffermuller (Lepidoptera: Noctuidae), is one of the most important and destructive cutworm pests in Moghan. Therefore, the lethal effect of S. carpocapsae isolate Moghan (IRMoghan) was evaluated in a soil assay against the last instar larvae of A. segetum under laboratory conditions. The bioassay results showed high susceptibility of the larvae to S. carpocapsae. The LC10, LC50, and LC90 values were 9.9, 54.13, and 246.2 IJs (infective juveniles) per larva of the pest, respectively (χ2 = 7.36; df = 3, P value = 0.061). Reproduction of the EPNs within the dissected cadavers was observed. The bioassay results indicated that the new isolate is a promising biocontrol agent against A. segetum larvae with success recycling through them.
Intraparenchymal supratentorial arachnoid cyst: a case report
Authors: Dlaka Domagoj , Raguž Marina , Muller Danko , Romić Dominik , Almahariq Fadi , Dlaka Jelena , Kaštelančić Anđelo , Chudy Darko
Published: October 2019

Background

Intracranial arachnoid cysts are collections of cerebrospinal fluid within the arachnoid membrane and subarachnoid space of the cisterns and major cerebral fissures that account for about 1% of all intracranial lesions. Expansion of the cyst and compression on surrounding structures may became symptomatic, which reflects its size and anatomic distribution.

Case presentation

Here, we present a very rare case of patient with supratentorial intraparenchymal arachnoid cyst placed in the left frontal lobe without any communication with the subarachnoid space and ventricle and presented with clinical symptoms. The patient underwent fenestration of the lesion and was clinically improved.

Conclusions

Although the etiology and the enlargement mechanism of arachnoid cysts remain unclear, both conservative and surgical treatments are optional. According to size, anatomical location, neuroimaging, and clinical symptoms, an arachnoid cyst should be included in the differential diagnosis of primary intracerebral cysts.
Building an Infrastructure and Standard Methodology for Actively Engaging Patients in Advance Care Planning
Authors: Stepan Karen , Bashoura Lara , George Marina , Griffith Wendy , Meyer Margaret , Nortjé Nico , Price Kristen , Zhukovsky Donna S. , Rodriguez Maria Alma
Published: October 2019

PURPOSE:

With little to no infrastructure or standardized methodology in place to actively engage patients in advance care planning (ACP), The University of Texas MD Anderson Cancer Center set out to identify needed resources, develop an intervention to improve ACP, and evaluate the intervention’s effects.

METHODS:

With the support of executive leadership, a multidisciplinary workgroup enlisted the support of ACP champions, performed a root-cause analysis, developed a detailed ACP process flow by provider role, developed patient and family education resources, and developed faculty and staff training materials. The workgroup also implemented two Plan-Do-Study-Act intervention cycles, which identified difficulty using the ACP note function in our electronic health record (EHR) as a barrier to ACP adoption. By educating patients, families, and providers and improving the EHR’s functionality, the workgroup aimed to increase the percentage of ambulatory patients with a diagnosis of advanced or metastatic cancer who had a documented ACP conversation with a provider by their third office visit. Our goal was to improve this percentage from 20% at baseline to 50% after the intervention. Data were obtained from our institution’s EHRs.

RESULTS:

The percentage of patients who had documented ACP conversations increased from 20% at baseline to 34% at the end of fiscal year 2017 and 54% at the end of fiscal year 2018.

CONCLUSION:

Owing to the dedicated efforts of many individuals across the institution, the postintervention goal was surpassed. Additional efforts to facilitate ACP conversations are ongoing.
Spine Injuries in Soccer
Authors: Plais Nicolas , Salzmann Stephan N. , Shue Jennifer , Sanchez Carlos Diez , Urraza Fabian J. , Girardi Federico P.
Published: October 2019

Soccer is the most popular sport in the world, with more than 270 million participants. It is characterized by repetition of short and intense actions that require high-coordination capacity. It is a sport where interactions with other players put the athletes at risk for traumatic injuries. Lower-limb injuries are the most prevalent injuries in soccer. Spine injuries are less frequent; however, they can impose serious and debilitating sequelae on the athlete. These injuries can be associated with long recovery periods preventing return to play. Moreover, specific repetitive activities (heading, kicking, etc.) can lead to chronic injuries. The cervical spine is particularly at risk for degenerative changes. Considerations for when an athlete should undergo spinal surgery and the timing of return to play present a difficult challenge to spine specialists. The objective of this article is to review the epidemiology, diagnosis, treatment, and prevention of spinal injuries in soccer.
MRI evaluation of extramural vascular invasion by inexperienced radiologists
Authors: Wang Shuai , Li Xiao-Ting , Zhang Xiao-Yan , Sun Rui-Jia , Qu Yu-Hong , Zhu Hui-Ci , Guan Zhen , Sun Ying-Shi
Published: October 2019

Objective:

We proposed to determine whether the performance of inexperienced radiologists in determining extramural vascular invasion (EMVI) in rectal cancer on MRI can be promoted by means of targeted training.

Methods:

230 rectal cancer patients who underwent pre-operative chemoradiotherapy were included. Pre-therapy and post-therapy MR images and pathology EMVI evaluation were available for cases. 230 cases were randomly divided into 150 training cases and 80 testing cases, including 40 testing case A and 40 testing case B. Four radiologists were included for MRI EMVI evaluation, who were divided into targeted training group and non-targeted training group. The two groups evaluated testing case A at baseline, 3 month and 6 month, evaluated testing case B at 6 month. The main outcome was agreement with expert-reference for pre-therapy and post-therapy evaluation, the other outcome was accuracy with pathology for post-therapy evaluation.

Results:

After 6 months of training, targeted training group showed statistically higher agreement with expert-reference than non-targeted training group for both pre-therapy and post-therapy MRI EMVI evaluation of testing case A and testing case B, all p < 0.05. Targeted training group also showed significantly higher accuracy with pathology than non-targeted training group for post-therapy evaluation of testing case A and testing case B after 6 months of training, all p < 0.05.

Conclusion:

The diagnostic performance for MRI EMVI evaluation could be promoted by targeted training for inexperienced radiologist.

Advances in knowledge:

This study provided the first evidence that after 6 month targeted training, inexperienced radiologists demonstrated improved diagnostic performance, with a 20% increase in agreement with expert-reference for both pre-therapy and post-therapy MRI EMVI evaluation and also a 20% increase in or accuracy with pathology for post-therapy evaluation, while inexperienced radiologists could not gain obvious improvement in MRI EMVI evaluation through the same period of regular clinical practice. It indicated that targeted training may be necessary for helping inexperienced radiologist to acquire adequate experience for the MRI EMVI evaluation of rectal cancer, especially for radiologist who works in a medical unit where MRI EMVI diagnosis is uncommon.