bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization Of Chromatin Dynamics In Mammalian Erythropoiesis
Authors: Zhao Baobing , Keerthivasan Ganesan , Yang Jing , Ji Peng
Published: October 2019

Mammalian erythropoiesis is a dynamic process in which erythroid progenitors proliferate and differentiate into mature enucleated red blood cells. In the late stage of erythropoiesis, erythroblasts undergo terminal cell-cycle exit, chromatin condensation and extrusion of the pycnotic nucleus via an asymmetric cell division. Recent genetic and biochemical studies illustrated that various signaling pathways, including histone deacetylations and other chromatin modifications, are involved in chromatin condensation and enucleation. However, it is unclear the global dynamic changes of the nucleosome and how different histones are regulated during chromatin condensation.

We proposed to directly characterize the expression levels and localization of different histones and their variants during erythropoiesis. We found that histone H2A and its variants are differentially expressed during erythropoieisis, while the levels of other histones including H2B, histone H3 and histone H4 remained unchanged. To determine their localization, we performed immunofluorescence experiments of these histones together with nuclear lamins. Strikingly, lamin B staining revealed a gradually enlarged nuclear opening through which most histones, except histone H2AZ, were released out of the nucleus. This nuclear opening was confirmed by electron microscopy. The same phenotype was observed in vivo in freshly purified late stage erythroblasts. Western blot analysis confirmed this release, however, many well known nuclear proteins remained in the nucleus in this process. Mechanistically, we demonstrated that caspase 3 was involved in the regulation of nuclear openings during erythropoieiss. Inhibition or knockdown of caspase 3 completely blocked lamin B opening and histone release, which led to inhibition of chromatin condensation and cell differentiation, and ultimate cell death.

In summary, our study demonstrated that the dynamic changes of various histones, with their selective and gradual release out of the nucleus, is required for chromatin condensation and differentiation during erythropoieisis. Studies of the detailed mechanisms are ongoing. Particularly, we are using micrococcal nuclease digestion of chromatin followed by next generation sequencing technology (MNase-seq) to determine genome-wide nucleosome location during the differention. Chromatin- immunoprecipitation (ChIP) will also be performed to determine H2A family protein locations during erythropoiesis.

Disclosures:

No relevant conflicts of interest to declare.
Deletion Of Samsn1 Underlies Genetic Susceptibility To Monoclonal Gammopathy Of Undetermined Significance (MGUS) In Mice
Authors: Amend Sarah , Chu Liang , Serie Daniel , Vachon Celine M , Lu Lan , Vij Ravi , Colditz Graham A , Weilbaecher Katherine N , Tomasson Michael H.
Published: October 2019

KaLwRij/C57Bl (KaLwRij, “Kal-ridge”) is a spontaneous mutant mouse strain predisposed to monoclonal gammopathy of uncertain significance (MGUS) and parallels many features of human pathology including progression to multiple myeloma (MM). In African Americans, an increased prevalence of MM is due to an increased risk of MGUS development (rather than increased rate of progression from MGUS) and is likely therefore mediated by germline MGUS risk alleles. In both humans and mice, the genetic basis for MGUS/MM risk is unknown. We sought to identify the genetic basis for MGUS/MM risk in KaLwRij mice to shed light on genes or pathways that might mediate risk in humans. We employed a novel integrative computational analysis, combining KaLwRij/B6 modified haplotype block analysis and low-power human MM/control single nucleotide polymorphism (SNP) data. We used high-density SNP arrays to establish genetic distances of KaLwRij from 11 another diverse inbred strains and confirmed close homology to parent C57Bl/6. Haplotype block analysis identified genetic loci that diverged from C57Bl/6 and included over 500 genes. We filtered the haplotype list using previously published RNA expression data from the bone marrow of C57Bl/6 and KaLwRij mice, to identify 71 differentially expressed genes. As a final filter, we used SNP data from a genome wide association study (GWAS) of human Caucasian MM case and normal volunteer samples (∼300 each) to identify SNPs differentially represented in MM patients. Combining these sets yielded four (4) candidate genes. Our top candidate, Samsn1 (Hacs1), is a negative regulator of B-cell activation and interestingly, was first identified as a gene differentially expressed in MM. Strikingly, we found that KaLwRij mice had a complete germline deletion of the Samsn1 gene. Samsn1 is expressed within the hematopoetic compartment, including B-cells, macrophages, and osteoclasts (OC), key cell types that participate in MM pathology. Consistent with the reported inhibitory function of SAMSN1, KaLwRij had enhanced naïve B-cell response and constitutive Lyn phosphorylation. Further, we found that KaLwRij macrophages and OC also have constitutive Lyn activation consistent activation of cytokine signaling pathways in stromal cells. Absence of Samsn1 may lead to unregulated B-cell and macrophage/OC activation, priming both a pro-tumor bone microenvironment and B-cells for development of MGUS and predisposition to MM. Our approach focused on germline loci rather than malignant cell genetic alterations, advancing our understanding of host predisposition. Identification of the gene or genes predisposing patients to MGUS/MM will provide novel targets for identifying at-risk individuals and will potentially allow the develop of preventative therapeutics for MM.

Disclosures:

No relevant conflicts of interest to declare.
Vitamin D Effect On Umbilical Cord Blood Characteristics: A Comparison Between African Americans and Caucasians
Authors: Eldjerou Lamis K , Rosenau Emma , Lu Xiaomin , Bennett Clayton , Sugrue Michele W. , Hoyne Jonathan , Lambert Amy , Hertel Lindsay , Sazama Kathleen , Wingard John R , Zubair Abba C
Published: October 2019

Background

Umbilical cord blood (UCB) is an important alternative source of stem cells for patients who lack matched adult donors, particularly in ethnic minorities. The infused total nucleated cell (TNC) and CD34+ cell dose are important prognostic factors on transplant outcomes. UCB units collected from African Americans (AA) have lower TNC and CD34+ cell counts and more likely to be disqualified for banking compared to other racial and ethnic groups. Furthermore, AA, including pregnant women, have increased prevalence of vitamin D deficiency. Vitamin D has an established role in hematopoiesis; therefore, we investigated the racial differences in UCB vitamin D content and its correlation with UCB cell composition and hematopoietic potential.

Methods

119 UCB units that did not meet the TNC count banking criteria were analyzed. 51 UCB units were collected from AA mothers and 68 from Caucasian mothers. We analyzed UCB variables including volume, hematocrit (HCT), TNCs, mononucleated cells (MNC), CD34+ cells, plasma 25-Hydroxy vitamin D [25(OH)D] and in vitro progenitor cell hematopoietic potential measured by Colony-Forming Cell (CFC) assay.

Results

The median values of 25(OH)D in UCB units were significantly lower in AA compare to Caucasians (p= <.0001). Severe vitamin D deficiency < 10 ng/mL was exclusively detected in UCB units from AA (19/51; 37%) and plasma 25(OH)D concentration < 20 ng/mL was more prevalent in AA (43/51 units; 84%), compare to Caucasians (15/68 units; 22%). UCB units from AA had lower absolute numbers of TNC (p= .0016), MNC (p= .0261), and CD34+ cells (p= .0257) compare to Caucasians. No difference in median CFC/10,000 MNC was detected between AA and Caucasians (p= 0.661) (Table 1).

Univariate analysis of 25(OH)D effect on UCB variables revealed a correlation between 25(OH)D level and TNC (r= 0.193, 95% CI 0.013-0.36; p= .0353) and UCB HCT (r= 0.196, 95% CI 0.016-0.363; p= .0327). A significantly higher MNC count was noted when 25(OH)D concentration was ≥ 20ng/mL (4.5x 108, range (1.7-9.5) vs. 3.9x 108, range (0.8-9); p= .0329). This correlation between 25(OH)D level and TNC and MNC was not detected after adjustment for race (Figure 1). However, 25(OH)D concentration ≥ 20 ng/mL significantly correlated with CBU HCT in AA race (median 31.9% (24-42) vs. 27.8%, range (21.4 - 40); p= .0124).

Conclusion

25(OH)D level, TNC and HCT are all significantly lower in UCB units from AA compare to Caucasians. Independent of race, 25(OH)D directly correlated with TNC and MNC. These data suggest vitamin D and other yet to be determined factors play an important role in the mechanism of low UCB cell and progenitor counts.

Disclosures:

No relevant conflicts of interest to declare.
Carfilzomib, Rituximab and Dexamethasone (CaRD) Is Highly Active and Offers a Neuropathy Sparing Approach For Proteasome-Inhibitor Based Therapy In Waldenstrom’s Macroglobulinemia
Authors: Peter Treon Steven , Tripsas Christina K , Meid Kirsten , Cropper Steven J , Mostyn Patrick , Warren Diane , Paba-Prada Claudia E. , Patterson Christopher , Yang Guang , Hunter Zachary , Kunsman Janet , Kanan Sandra , Ghobrial Irene M.
Published: October 2019

Bortezomib is active in Waldenstrom’s macroglobulinemia (WM) but associated with considerable peripheral neuropathy (PN). The proteasome inhibitor carfilzomib (CFZ) is approved in the USA for relapsed/refractory myeloma. Herein, we examined the efficacy and safety of carfilzomib, rituximab, and dexamethasone (CaRD) in 31 proteasome inhibitor and rituximab naive WM patients with symptomatic disease. Median baseline characteristics: age 61, prior therapies 0 (range 0-1), hematocrit 32.3%, hemoglobin 10.7 g/dL, serum IgM 3375 mg/dL, serum M-protein 2.185 g/dL, B2M 3.6 mg/L, and bone marrow disease involvement 60%. Therapy consisted of IV CFZ 20 mg/m2 (cycle 1) then 36 mg/m2 (cycles 2 and beyond) with IV dexamethasone (dex) 20 mg given on days 1,2,8,9 and rituximab 375 mg/m2 on days 2,9 of each 21-day cycle. Treatment consisted of six induction cycles, then maintenance beginning 8 weeks after induction (given every 8 weeks for eight cycles; consisted of CFZ 36 mg/m2, and IV dex 20 mg on days 1,2 and rituximab 375 mg/m2 on day 2). Patients with IgM level >4000 mg/dL underwent plasmapheresis and/or had rituximab held until IgM <4000 mg/dL to prevent symptomatic IgM flare. Patients received oral acyclovir (400 mg twice daily) and famotidine (20 mg twice daily) as concomitant medications. For all 31 patients, median serum IgM levels and M-protein declined to 749 mg/dL and 0.7 g/dL, respectively (p<0.00001). Median hematocrit and hemoglobin rose to 40.9% and 13.7 g/dL, respectively (p<0.00001). A total of 30 patients concluded induction therapy with bone marrow tumor involvement reduced to a median of 7.5% (p=0.0003). The best overall response rate using criteria adapted from the 3rd International Workshop on WM was 81% (1 CR, 8 VGPR, 12 PR, 4 Minor Responses). With a median follow-up of 8 cycles, 22 patients remain on study, including 20 currently on maintenance therapy. Median time to response (for minor responders or better) was 2.1 months. Grade >2 treatment related toxicities included asymptomatic elevation in lipase (12.9%), dex-related hyperglycemia (6.45%), reversible neutropenia (9.67%), and cardiomyopathy (3.22%). There were no grade 2 or greater PN events. Treatment discontinuation occurred for non-response (n=8), cardiomyopathy in a patient with multiple cardiac risk factors (n=1), and progressive disease (n=1). CaRD is highly active and offers a neuropathy sparing approach for proteasome-inhibitor based therapy in WM.

Disclosures:

Off Label Use: Carfilzomib is a novel proteasome inhibitor, which offers a neuropathic sparing approach on waldenstrom macroglobulinemia disease when used in a combination therapy with rituximab and dexamethasome.
Whole Genome and Exome Sequencing Defines The Genetic Landscape Of Hepatosplenic T-Cell Lymphoma
Authors: Scotland Paula , Gaulard Philippe , Love Cassandra L , Fataccioli Virginie , Travert Marion , De Leval Laurence , Weisenburger Dennis D. , Czader Magdalena , Parihar Mayur , Nair Reena , Sengar Manju , Beaven Anne W , Crow Jennifer H. , Miles Rodney R. , Gordon Leo I. , Chadburn Amy , Evens Andrew M , Gill Javed , Fedoriw Yuri D. , Richards Kristy L. , Srivastava Gopesh , Choi William W. L. , Flowers Christopher R. , Bernal-Mizrachi Leon , Mann Karen P. , Naresh Kikkeri , Hsi Eric D. , Horna Pedro , Tao Jianguo , Sun Zhen , Long Kevin , Zhang Jenny , Dave Sandeep
Published: October 2019

Background

Hepatosplenic T-cell lymphoma (HSTL) is a rare form of lymphoma, comprising less than 1% of the cases. However, HSTL extracts a highly disproportionate toll on patients with a median age of diagnosis of 35 years and an expected median survival of less than two years. The vast majority of HSTL patients eventually succumb to their disease. The genetic basis of the disease is largely unknown. Although abnormalities of chromosome 7, including isochromosome 7q occur commonly in the disease, the role of specific genes and genetic mutations to the disease remains essentially unknown.

Methods

In this study, we sought to define the genetic features of HSTL through the whole genome sequencing and exome sequencing of 32 HSTL tumors and germline DNA (where available) from the same patients. Exome enrichment of DNA was carried out using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes as well as ∼700 human microRNAs. Both whole genome and exome sequencing were performed using the Illumina platform.

Results

We identified 28 candidate cancer genes that were recurrently mutated in HSTL. Commonly implicated biological processes comprising these genes included signal transduction (e.g. PIK3CD, KRAS) and chromatin modification (e.g. TET1, SETD2 and MLL3), accounting for 16% and 23% of the total genetic events, respectively. Nearly all of these genes have been implicated in HSTL for the first time and provide new insights into the pathogenesis of the disease and potential targets for therapy. Whole genome sequencing confirmed isochromosome 7q as the most common recurrent chromosomal abnormality in HSTL and additional structural genetic alterations in chromosome 7.

Conclusion

Our study provides the most comprehensive genetic portrait of HSTL to date, and is a significant step in defining the genetic causes of this disease.

Disclosures:

No relevant conflicts of interest to declare.
Next-Generation Sequencing Suggests Complex, Heterogeneous Pathogenesis In Peripheral T-Cell Lymphoma Unspecified
Authors: Schatz Jonathan H. , Horwitz Steven M. , Lunning Matthew A. , Dolgalev Igor , Huberman Kety , Heguy Adriana , Viale Agnes , Socci Nicholas D. , Pirun Mono , Teruya-Feldstein Julie , Wendel Hans-Guido
Published: October 2019

Peripheral T-cell lymphoma (PTCL) makes up about 12 percent of non-Hodgkin lymphoma, comprising 18 diseases that are poorly understood and carry a generally worse prognosis than B-lymphomas. PTCL not otherwise specified (PTCL-NOS), a diagnosis of exclusion, is most common, making up 25-30 percent. Gene-expression studies suggest a heterogeneous origin of this diagnosis, with overlap to other PTCL types, but the genetic factors underlying its pathogenesis are undefined. Current therapy for PTCL-NOS is empiric and ultimately ineffective for most patients. Identification of specific therapeutic targets is therefore a high priority. We have sought better understanding of pathogenesis through next-generation sequencing of PTCL-NOS tumor DNA. Whole-exome sequencing revealed candidate genes but low availability of fresh-frozen samples limited our ability to draw conclusions by this method alone. We therefore sequenced the coding regions of 237 candidate genes in a collection formalin-fixed paraffin-embedded samples. We used Nimblegen Sequence Capture for PCR amplification of exons and Illumina hiSeq for raw sequence generation. Results were aligned to hg19 and compared to dbSNP and the 1,000 genomes data to exclude germline variants. Analysis, including comparison to the COSMIC database of cancer-specific mutations, revealed high-confidence mutations affecting more than 60 known cancer-related genes in 25 PTCL-NOS cases. Recurrent mutations pointed to frequent activation of three key signaling pathways: NF-kB (TNFAIP3), WNT/B-Catenin (APC, CHD8, CELSR2), and NOTCH (NOTCH1, FBXW7). Recurrent deregulation of epigenetic processes was indicated by mutations in genes affecting histone acetylation (EP300, CREBBP), histone methylation (MLL2, KDM6A), and DNA methylation (TET2, DNMT3A). In addition, components of core tumor suppressor pathways showed evidence of frequent inactivation (TP53, ATM, RB1, CUL9, PRKDC). In all, 22 of 25 cases had mutations in at least one of these 17 recurrently mutated genes. Multiple additional candidate disease mechanisms also were suggested by lower-confidence mutations but require confirmation studies, which are under way. In sum, analysis of the coding region of PTCL-NOS tumor DNA suggests a complex and heterogeneous pathogenesis, in line with gene-expression profiling. This work provides an opportunity to better sub-classify entities within the diagnosis of PTCL-NOS and identify specific therapeutic targets and their associated biomarkers.

Disclosures:

Horwitz: Seattle Genetics, Inc.: Consultancy, Research Funding; Millennium: Consultancy, Research Funding.
A Review Of Computed Tomography Pulmonary Angiograms In Three Teaching Hospitals: The Rise Of Sub-Segmental Pulmonary Emboli and Their Management
Authors: Goy Jennifer , Lee Justin Y. , Levine Oren , Chaudhry Salman , Crowther Mark A.
Published: October 2019

Background

The availability of Computed Tomography Pulmonary Angiography (CTPA) has led to an increase in the number of investigations for Pulmonary Embolism (PE). With more widespread use of these high resolution scans, the frequency of identification of isolated Small Sub-segmental Emboli (SSPE) is also expected to increase. Current clinical practice guidelines do not make any treatment distinctions for SSPE, though the benefits of anticoagulation for SSPE have not been established.

Aims

To review the frequency of Pulmonary Embolism and Sub-segmental Pulmonary Embolism identified through CTPA as well as their management

Methods

Retrospective review of 2213 patient charts who underwent CTPA in three Hamilton teaching hospitals from 2009-2011. In depth chart review of patients with SSPE was undertaken to determine the frequency with which patients who received anticoagulation therapy for SSPE. The frequency of bleeding complications and recurrent thrombosis were also investigated in this detailed SSPE chart review.

Results

Our patient population (mean age 65) consisted of 1099 medical inpatients (50%), 702 surgical inpatients (32%) and 412 (18%) emergency department patients.

PE was identified in 26 % of scans (n=576). Of these, SSPEs were the only identified thrombus in 82 patients (4% of total scans and 14% of identified PEs). In 55 of these 82 SSPEs, in addition to the SSPE, an alternative diagnosis that might explain the PE symptoms was found.

Fifty-two percent (n=43) of the patients with an SSPE received therapeutic anticoagulation. In these life threatening bleeding occurred in 2 patients. There was no documented recurrent thrombosis or thrombosis-related deaths in three month follow-up among the 39 patients who did not receive anti-coagulation for SSPE.

Of the 1,608 CTPAs that did not identify PE, an alternative diagnosis to account for the patient’s symptoms was identified on CT in 1078 (67%) and no alternative cause was found in 531 (33%).

Summary/Conclusions

Our study demonstrated a much lower frequency of pulmonary embolism in comparison to approximate 50 % pre-test probability of a positive scan seen in studies which validated CTPA for the diagnosis of PE. Isolated SSPEs accounted for 14% of all PEs found in our study population – and were present in 4% of all patients undergoing CTPA. A substantial proportion of patients were anti-coagulated SSPE (52%) and two developed life-threatening bleeding complications. No recurrent VTE was documented in patients who were not anticoagulated for PE, though follow-up was limited to hospital records. Randomized controlled trial data is needed to further investigate the risks and benefits of anticoagulation in patients with SSPE.

Disclosures:

No relevant conflicts of interest to declare.
Upregulation Of Hepcidin Expression By Lactoferrin Administration To Pre-Weanling Mice
Authors: Elif Memisoglu , Cooper Jeffrey A. , Fleming Robert E.
Published: October 2019

Background

Hepcidin is a peptide hormone produced in hepatocytes which regulates dietary iron absorption and systemic iron distribution. The hepatic expression of the hepcidin gene is regulated by signals which reflect body iron status and erythropoietic activity. The regulation of hepcidin by iron status includes a signal from the circulating transferrin via hepatocellular transferrin receptor (TfR2). Lactoferrin is a member of the transferrin family of iron-binding glycoproteins found in high concentrations in milk. Lactoferrin has a well described role in host defense; however, its in iron metabolism is less clear. Like transferrin, lactoferrin will deliver iron to hepatocytes but unlike transferrin, lactoferrin cannot deliver iron to erythroid cells. Lactoferrin does not interact with TfR1 or TfR2. We took advantage of these properties of lactoferrin to determine if delivering iron to the liver (but not erythron) in a form which is independent on transferrin and its receptors could regulate hepcidin.

Methods

To take advantage of the naturally-occuring low iron and low hepcidin states at this developmental stage, experiments were performed on pre-weanling mice. Mice at 12-14d (pre-weanling) were administered human lactoferrin equivalent to 2 mg/kg iron (n=10) or carrier (n=10) intraperitoneally (IP) and sacrificed 6 h later. Other mice were administered a comparable IP dose of transferrin (n=5) or carrier (n=4). Additional groups of mice were delivered Lf (n=8) or carrier (n=7) enterally (by orogastric tube), and the sacrificed 6h or 24h later. Liver hepcidin (Hamp1) mRNA was measured by real-time RT-PCR, normalized to beta actin expression, and analyzed by commercial software (Rest 2007, Qiagen). Liver iron distribution was determined histochemically in tissue sections by modified Perls' staining.

Results

Parenteral administration of lactoferrin in pre-weanling mice was associated with a 32-fold increase in liver Hamp1 expression after 6 hours (P<0.001). This compares with an only 4.5 fold increase in Hamp1 expression 6h after a comparable dose of transferrin. Enteral lactoferrin likewise resulted in a ∼30-fold increase in Hamp1 expression (P<0.005) after 24 hours. Iron delivered to the liver from lactoferrin was distributed in sinusoidal lining cells as well as hepatocytes.

Conclusions

Exogenously administered iron-rplete lactoferrin increases liver Hamp1 expression in pre-weanling mice. These observations demonstrate that iron delivery to the liver in a form not dependent upon transferrin or its receptors upregulates hepcidin expression. These findings moreover raise the possibility that lactoferrin-mediated changes in hepcidin expression may contribute to reported benefits of supplemental lactoferrin on host defense.

Disclosures:

No relevant conflicts of interest to declare.
The HU Inducible SAR1 Gene Acts Through GαI /JNK/JUN Pathway To Activate γ-Globin Expression
Authors: Zhu Jianqiong , Chin Kyung , Aerbajinai Wulin , Kumkhaek Chutima , Li Hongzhen , Rodgers Griffin P.
Published: October 2019

Hydroxyurea (HU) has been effectively used in the treatment of β-hemoglobinopathies by augmenting γ-globin chain to compensate for the impaired or defective β-globin chain, but the molecular mechanism underlying HU-mediated γ-globin regulation remains unclear. We previously reported that over-expression of the HU-induced SAR1 gene increased γ-globin expression in primary erythroid cells, and SAR1 is required for HU's effects in this process. Here we present further mechanistic details underlying SAR1’s effects. First, we examined the role of COPII pathway in HbF expression by genetically silencing the key downstream COPII components Sec12/Sec23 or by chemical inhibition of vesicle protein transport from ER to Golgi with BFA. We found that blocking this pathway through siRNA Sec12/23 or BFA only reduced HU-induced γ-globin expression by 32% and 28%, respectively, without a change in basal HbF level (as determined by QT-PCR or flow cytometry). These data suggested that other signaling pathways might account for HU- and SAR1-mediated HbF induction. Additional studies also indicated that stable tranfected SAR1 was able to activate Gαi /JNK/JUN signaling in erythroid cell. Inhibition of JNK/JUN activation by the JNK inhibitor SP600125 was able to reduce by 8.79% and 38.63% HbF positive cells in vector- and SAR1-transfected-cells, respectively. Silencing of Gαi2 or Gαi3, but not Gαi1 or Gαq expression in SAR1-stable transfected cells could significantly eliminate SAR1-stimulated JUN protein phosphorylation, and resulted in the 54% and 46% reduction of γ-globin expression, respectively. Reciprocal co-immunoprecipitation assays revealed a direct association between Sar1 and Gα (Gαi and Gαq) protein in both erythroid and non-erythroid cells. These data taken together with our previous findings indicate that HU induces SAR1 which in turn activates γ-globin expression predominantly through the Gαi/JNK/JUN pathway. These findings illuminate an alternative approach to targeted therapy for the beta-globin disorders.

Disclosures:

No relevant conflicts of interest to declare.
Treatment of Relapsed ACUTE Leukemia after ALLO-HSCT By Salvage Chemotherapy with Low-Dose Cytarabine and Aclarubicin Combined with G-CSF Priming: More Effective and LESS Toxic?
Authors: Zhou Hua , Ge Ling , Zhu Xiaming , Qiu Huiying , Jin Zhengming , Miao Miao , Fu Chengcheng , Ma Xiao , Chen Feng , Xue Shengli , Ruan Changgeng , Depei Wu , Tang Xiaowen
Published: October 2019

Background: Recurrence is a major cause of treatment failure after allogeneic hematopoietic stem cell transplantation (allo-HSCT) for acute leukemia, and subsequential treatment options are very limited.

Aims: We evaluate the efficacy and toxicity of cytarabine and aclarubicin combined with granulocyte colony-stimulating factor priming (CAG regimen), consisting of concurrent use of granulocyte colony-stimulating factor (G-CSF) with low-dose cytarabine and aclarubicin, as a salvage therapy for acute leukemia patients who relapsed after allo-HSCT.

Methods: Fifty-nine patients (32 male and 27 female) with acute leukemia, with a median age of 27 years, relapsed post allo-HSCT and received salvage chemotherapy. Twenty-seven patients received CAG regimen while 32 patients received non-CAG regimen such as intensive chemotherapy.

Results: 1.The overall response rate (ORR) of CAG was significantly superior than that of non-CAG groups (55.6% vs 28.1%, P= 0.033). Especially for AML patients (64.3% vs 20%, P= 0.025). However, ORR of ALL in CAG group was similar with that in non-CAG group (50% and 35.2% respectively; P = 0.471).

2. Median overall survival (OS) from the starting of CAG chemotherapy and the estimated 2-year OS of CAG group were 9 (1-27) months and 16.1%. Meanwhile, median OS and the estimated 2-year survival of non-CAG group were 4 (1-49) months and 8.8% (Figure1). For the patients who received donor lymphocyte infusion (DLI) as a subsequential therapy, two-year OS of CAG and non-CAG group were 17.2% and 12.5% respectively (P=0.577).

3. The median duration of neutropenia and thrombocytopenia of CAG group were significantly shorter than that of non CAG group, 6 (1-12) vs 11 (5-28) days (P=0.000) and 8 (1-14) vs 14 (7-35) days (P=0.000). Treatment related mortality (TRM) was found in 2 cases in CAG group compared with 10 cases in non-CAG group.

4. For CAG group, univariate analysis results showed that lower leukocyte level and lower medullar blast percentage at relapse post-transplant were significantly associated with a higher overall response rate (P=0.005 and P=0.000).Furthermore, multivariate analysis results showed that the response to chemotherapy was the only factor that correlated with better survival (HR 0.426, P=0.016)

Conclusion: CAG regimen as a salvage chemotherapy for relapsed acute leukemia post allo-HSCT could effectively reduce tumor burden with mild toxicity, especially for hypoplastic acute leukemia patients. For certain type of relapsed acute leukemia patients post allo-HSCT, CAG regimen may be an optimal choice as the bridge therapy followed by DLI or second allo-HSCT.

Disclosures

No relevant conflicts of interest to declare.
Third Party Invariant Natural Killer T Cells Protect from Graft-Versus-Host Disease Lethality through Expansion of Donor CD4+FoxP3+ Regulatory T Cells
Authors: Schneidawind Dominik , Baker Jeanette , Buechele Corina , Meyer Everett H. , Negrin Robert S.
Published: October 2019

Graft-versus-host disease (GVHD) is driven by extensive activation and proliferation of alloreactive donor T cells causing significant morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Invariant natural killer T (iNKT) cells are potent regulators of immune responses in both humans (TCRα Vα24-Jα18) and mice (TCRα Vα14-Jα18). As the iNKT cell receptor and the glycolipid-presenting molecule CD1d interaction is highly conserved, we explored the role of adoptively transferred third party CD4+ iNKT cells in a murine model of allogeneic HCT. BALB/c (H-2Kd) recipient mice were irradiated with 8 Gy and transplanted with T cell-depleted bone marrow together with 1x106 CD4+/CD8+ T cells (Tcon) from C57BL/6 (H-2Kb) donor mice. Adoptive transfer of purified (>95%) 5x104 CD4+ iNKT cells from FVB/N (H-2Kq) third party mice resulted in a significant survival benefit (p<0.001) while retaining Tcon mediated graft-versus-tumor (GVT) effects against A20 lymphoma cells (p=0.002). Consistently, weight and GVHD scores improved in mice that received a single injection of third party CD4+ iNKT cells as compared to animals that received Tcon alone. Notably, CD4+ iNKT cells from third party mice were as protective as CD4+ iNKT cells from donor mice (p=0.50). Signal intensity deriving from expanding luciferase expressing alloreactive Tcon was significantly lower in animals treated with third party CD4+ iNKT cells (p=0.003). Interestingly, inhibition of Tcon proliferation was similar to animals that received CD4+ iNKT cells from donor mice (p=0.90). In addition, adoptive transfer of third party CD4+ iNKT cells promoted a Th2-biased cytokine response of alloreactive donor T cells. Although we found that third party CD4+ iNKT cells were rejected by day +10 after allogeneic HCT, adoptive transfer of these cells resulted in a robust expansion of luciferase expressing donor CD4+FoxP3+ regulatory T cells (Treg) as measured by bioluminescence imaging (p=0.006). Using FoxP3DTR C57BL/6 donor mice, depletion of Treg from the graft abrogated both donor Treg expansion and protection from GVHD lethality through third party CD4+ iNKT cells. We conclude that low numbers of highly purified and adoptively transferred third party CD4+ iNKT cells protect from lethal GVHD through activation and expansion of donor Treg with retained GVT effects. Despite the fact that iNKT cells are a rare cell population, the in vivo activity of small numbers of cells and feasibility of in vitro expansion provide the basis for clinical translation.

Disclosures

No relevant conflicts of interest to declare.
Safety of Peripheral Hematopoietic Stem Cell Mobilization with Lenograstim in Allogeneic Healthy Donors ≥ 50 Years Old: A Single-Centre Experience
Authors: Martino Massimo , Dattola Antonella , Fedele Roberta , Pontari Antonella , Moscato Tiziana , Cuzzola Maria , Martinello Tiziana
Published: October 2019

Background and Objectives

The use of allogenic hematopoietic stem cell transplantation (HSCT) is increasing in older patients because of reduced ablative regimens and improved supportive care. An old patient usually has a old sibling healthy donors (HDs). The safety of G-CSF for mobilization in elderly HDs has not been provided. In our institution a long-term active follow-up study of G-CSF-mobilized healthy volunteer donors has been implemented.

Patients and Methods

After a median follow up of 94 months (7.8 years) we reviewed and analyzed safety data of our HDs database according to HDs age: HDs-1 (162), patients younger than 50 years old, HDs-2 (62), patients 50-59 years old and HDs-3 (23), patients 60 or more years old. Long-term follow-up included monitoring of neoplastic, cardiac or autoimmune diseases.

Results

Two hundred and forty-seven successive donors were evaluated and their characteristics are well balanced among age-groups and no statistical differences have been detected: most of them were male (55.9% ), sibling (97.2%) and HLA matching (93.1%). Mobilization failure rate has been 4.3% (7/162), 1.6% (1/62) and 13% (3/23) in HDs-1, HDs-2 and HDs-3, respectively (p=0.075).

Short- and long-term safety was not different among age-groups. Bone pain was reported as the most frequent short-term adverse event (76.5%). Other commonly observed short-term symptoms included headache (31.6%), fatigue (28.7%), insomnia (21.5%), nausea (13.0%) and fever (6.1%). For long-term safety surveillance no haematological malignancies were observed. The observed rate of solid tumors, cardiovascular and autoimmune events, was the expected incidence for these diseases in the western countries.

Conclusion

In our study, mobilization with lenograstim in elderly HDs seems to be as safe as for young HDs safe. This data contribute to the growing body of evidence of the long-term safety of G-CSF for allogenic donor stem cell mobilization also for elderly HDs.

Disclosures

No relevant conflicts of interest to declare.
Hypoferremia of Fasting in Mice Is Associated with Increased Hepcidin and Decreased Erythroferrone Expression
Authors: Li Yihang , Booth Gregory R , Feng Qi , Fleming Robert E
Published: October 2019

Background: Protein-calorie restriction in humans is associated with changes in iron metabolism that differ from dietary iron-deficiency. Dietary iron absorption and tissue iron distribution and are regulated by the hepatocellular peptide hormone hepcidin, which mediates a decrease in circulating iron concentrations and increase in cellular iron stores. Hepcidin is upregulated in response to increased serum and tissue iron, upregulated in response to ER-stress via the transcription factor CREB-H, and down-regulated in response to erythroid iron demand via the circulating signaling molecule erythroferrone/myonectin (ERFE). An overnight fast was reported to decrease expression of skeletal muscle ERFE in mice.

Objective: The aim of this study was to investigate the regulation of hepcidin in the hypoferremia of fasting.

Design/Methods: We analyzed parameters of iron homeostasis in five 5 week old male AKR mice fasted for 18 hours overnight (and allowed access to water) compared with mice with access to chow ad libitum. Serum and tissue iron concentrations were measured. Liver hepcidin mRNA was quantified by real-time RT-PCR and verified by Northern blot analysis. Marrow ERFE and liver CREB-H mRNA expression was quantified by RT-PCR and normalized to beta actin.

Results: As anticipated, fasted mice had significantly lower serum iron concentrations (203 vs 321 mcg/dL, P<0.001) and transferrin saturations (64.4 vs 79.6%, P<0.05). Duodenal iron concentrations were increased in fasted mice, 2.3-fold (P<0.01). Histochemical staining demonstrated iron retention in the absorptive enterocytes. Liver (916 vs 735 mcg/g, P=0.1) and splenic (480 vs 414 mcg/g, P=0.001) iron concentrations were modestly higher in the fasted mice. Despite hypoferremia, the fasted mice demonstrated increased liver hepcidin expression (1.9 fold, P < 0.05). Liver CREB-H mRNA expression was increased ~10 fold, P<0.01; however there was no change in liver XBP-1 processing (marker of ER stress). Marrow ERFE expression was downregulated by approximately one third (P<0.01).

Conclusions: The fasting state in mice is associated with increased hepcidin expression, iron retention in absorptive enterocytes, and decreased circulating iron. These observations suggest that the hypoferremia of fasting is not consequent to limited iron availability, but rather to hepcidin-mediated retention of iron in the duodenum and storage tissues. They moreover support a role for nutritional state, independent of iron status, as a regulator of hepcidin and mediated by changes in expression of ERFE and CREB-H.

Disclosures

No relevant conflicts of interest to declare.
Thrombospondin-1 Stimulates Calcium Influx and Echinocytosis in Sickle Cell-Derived Red Blood Cells
Authors: Kanias Tamir , Isenberg Jeffrey , Bullock Grant C , Schrott Valerie M , Stolz Donna B , Novelli Enrico M
Published: October 2019

Thrombospondin-1 (TSP1) is a secreted matricellular protein found preformed in platelet α-granules and upregulated in the plasma of patients with sickle cell disease (SCD). In preclinical models TSP1 enhances sickle red blood cell (RBC) binding to endothelial cells, while in murine sickle mice TSP1 promotes RBC echinocytosis and vaso-occlusion. Recently we reported elevated plasma TSP1 was associated with vaso-occlusive complications in SCD patients. Herein we tested the hypothesis that TSP1 compromises RBC membrane integrity via enhanced calcium influx leading to morphological changes, such as echinocytosis. RBC were collected via centrifugation of heparinized blood from human HbAA donors (n=3), SCD patients with homozygous HbSS SCD (n=4), and wild type C57BL/6J mice (n=2). Cells were then re-suspended in glucose and calcium-supplemented PBS, and treated with several concentrations of exogenous TSP1 (2.75 nM, 11 nM, 22 nM and 44 nM) for 2 hours at 37°C. RBC morphology was assessed by both light and electron microscopy. TSP1 modulation of RBC calcium influx was studied using the calcium fluorophore Fluo-3, AM. TSP1 treatment, in a dose-dependent manner, stimulated echinocytosis in human SCD and non-SCD RBC and in murine cells. In some instances electron microscopy showed echinocyte membrane projections with a string-of-beads appearance, suggestive of imminent microparticle shedding. TSP1 treatment, again in a dose-dependent manner, also significantly increased calcium influx in SCD (Figure, right panel) and non SCD RBC (Figure, left panel) and murine RBC (not shown). Interestingly, SCD RBC displayed more sensitivity to TSP1-mediated increases in cellular calcium as compared to normal cells. Our results show for the first time that TSP1, at doses found in patient plasma, stimulates significant echinocytosis and calcium influx in both normal and SCD RBC. These data identify a novel role for plasma TSP1 in promoting RBC pathology. Future studies are underway to elucidate this mechanism and its pathogenicity in humans with and without SCD.

Figure 1 Figure 1.

Disclosures

Isenberg: Vasculox: Membership on an entity's Board of Directors or advisory committees; Radiation Control Technologies: Membership on an entity's Board of Directors or advisory committees.
Leukemic Reticuloendotheliosis
Authors: BOURONCLE BERTHA A. , WISEMAN BRUCE K. , DOAN CHARLES A.
Published: October 2019

The clinical, hematologic, and pathologic findings in 26 cases of leukemic reticuloendotheliosis are presented.

A histopathologic correlation of the various forms of leukemia is suggested. Clinically the disease may follow an acute, subacute, or chronic course There is no idiopathic clinical sign or symptom, but the hematologic picture is pathognomonic and is characterized by the presence of reticulum cells in the peripheral blood and bone marrow. These cells are also present in the organs where the reticuloendothelial system predominates: spleen, liver, and lymph nodes.

The study of these cases supports the concept that leukemic reticuloendotheliosis is an independent hematologic and pathologic entity.
Paroxysmal Nocturnal Hemoglobinuria: The Role of Properdin in the Diagnostic Thrombin Test
Authors: CROSBY WILLIAM H. , BENJAMIN NAOMI R.
Published: October 2019

1. The "thrombin test" for PNH consists of adding thrombin to the standard acid-hemolysis test, thereby increasing hemolysis of PNH red cells. It has been suggested that the effect of the thrombin solution is not due to involvement of the coagulation system but to contamination of the thrombin by properdin, a globulin which is essential in the acid-hemolysis reaction of PNH.

2. In the present experiments the thrombin test was positive using preparations of thrombin which were free of properdin activity.

3. Thromboplastic materials devoid of properdin activity may be substituted for thrombin in the thrombin test.

4. It is concluded that the properdin which contaminates the commercially available thrombin preparations has little to do with the results of the thrombin test for PNH.
Factors Influencing the Incidence of Leukemia: Special Consideration of the Role of Ionizing Radiation
Authors: SCHWARTZ E. E. , UPTON A. C.
Published: October 2019

The recorded incidence of leukemia and lymphomas has markedly increased during the past 30 years. During the same interval the amount of ionizing radiation in the environment has been steadily increased by man-made additions to the natural background.

The possibility that radiation may account in part for an increasing incidence of leukemia is suggested by the more frequent development of the disease in those exposed to large amounts of radiation.

Although the extent of increase in the risk of leukemia cannot be determined accurately from the recorded rates and the relation between leukemia incidence and radiation dose cannot be estimated precisely, it appears probable that radiation is only one of many environmental agents influencing the occurrence of the disease.

Other factors associated with the changing standard of living, such as the increasing use of marrow-depressing drugs and the growing contamination of the atmosphere with chemical pollutants, deserve further investigation as leukemogenic influences.
On the Interaction of Dead Leukocytic Nuclei, L.E. Factor and Living Leukocytes in the L. E. Cell Phenomenon
Authors: NATHAN DANIEL J. , SNAPPER I.
Published: October 2019

1. Isolated leukocyte nuclei quantitatively adsorb lupus factor from serum. In the presence of viable leukocytes such nuclei which have adsorbed the lupus factor become transformed into typical L.E. cells.

2. Leukocytes with an intact cytoplasm fail to adsorb L.E. factor.

3. These studies suggest that the living leukocytes, apart from simple phagocytosis, serve other functions in the L.E. phenomenon:

(a) the disruption of the integrity of the cytoplasmic envelope of dead leukocytes so as to allow for the adsorption of L.E. factor to the cell nucleus.

(b) the transformation of the nuclei, to which L.E. factor has been adsorbed, into swollen homogenous lupus bodies.
Absence of functional messenger RNA activity for beta globin chain synthesis in beta 0-thalassemia
Authors: Benz EJ Jr , Swerdlow PS , Forget BG
Published: October 2019

Functional human globin messenger RNA was isolated from reticulocytes of two patients with homozygous beta 0-thalassemia, three patients with sickle cell beta 0-thalassemia, andone patient doubly heterozygous for beta 0-thalassemia and hemoglobin Lepore. When incubated in the Krebs type II mouse ascites tumor-cell-free system, messenger RNA from these patients actively directed the synthesis of human beta s and/or alpha- and gamma-globin chains but failed to stimulate the synthesis of any beta A-chains, even though nonthalassemic human globin mRNA preparations consistently stimulated two to four times as much beta A- or beta S-globin chain synthesis as alpha-chain synthesis when incubated in the same system under the same conditions. These results strongly suggest that functional beta A-chain-specific globin mRNA is absent in beta 0-thalassemia.
The response of red cell hexose monophosphate shunt after sulfhydryl inhibition
Authors: Sagone AL Jr , Balcerzak SP , Metz EN
Published: October 2019

In this investigation, we studied the importance of cellular glutathione (GSH) in the hexose monophosphate shunt (HMPS) activity of unstimulated human erythrocytes and the mechanism by which pyruvate stimulates the HMPS. The rate of HMPS activity was measured by the production of radioactive CO2 from 14C-1-glucose or 14C-1-ribose using a vibrating reed electrometer and ionization chamber. HMPS activity was not significantly impaired by N-ethylmaleimide (NEM) in concentrations which bound all red cell GSH. Red cells incubated under carbon monoxide (CO), an experimental condition which eliminates peroxide production, still had HMPS activity which was 44% of the value under air. Pyruvate stimulation of the HMPS was unaffected by doses of NEM which bound all cellular GSH or by incubation under CO. These data indicated that pyruvate stimulation of the HMPS occurs by pathways which do not involve peroxide formation, GSH, or oxygen. This study indicates that sulfhydrylblockade of GSH does not necessarily inhibit HMPS activity and that HMPS activity in red cells may respond to reactions not linked directly to glutathione reduction.
Studies on the mechanism of ristocetin-induced platelet aggregation
Authors: Kattlove HE , Gomez MH
Published: October 2019

Adenine nucleotide metabolism and the release reaction were studied during ristocetin-induced platelet aggregation. Decreasing platelet ATP by incubation with metabolic poisons did not decrease ristocetin- induced aggregation. ADP and ATP were released from platelets during ristocetin-induced aggregation, and ATP was converted to hypoxanthine. However, these occurred after aggregation was almost complete. Aggregation was inhibited by p-choromercuribenzoic acid. By studying platelet suspensions, we were able to determine that this effect was on platelets and not on the plasma cofactor needed for aggregation. We postulate that ristocetin and its cofactor aggregate platelets by binding platelet membranes and that the platelet plays a passive role in this reaction.
Heparin-induced thrombocytopenia: confirmation of diagnosis with in vitro methods
Authors: Fratantoni JC , Pollet R , Gralnick HR
Published: October 2019

Profound thrombocytopenia developed in a patient during treatment with heparin for venous thrombosis. The platelet count increased toward normal when heparin administration was stopped, but fell abruptly when the drug was again given. Platelet aggregation occurred when heparin was added to the patient's platelet-rich plasma, or to normal platelets plus the patient's serum. This serum also effected release of 3H- serotonin from normal platelets. This pattern of aggregation was clearly different from that occasionally caused by heparin in a control population. The data is consistent with an effect of heparin on platelets, possibly mediated by on immune mechanism.