bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of Granulocyte Colony Stimulating Factor (G-CSF) On Monosomy 7 Aneuploidy and T Cell Subsets in Healthy Donors.
Authors: Olnes Matthew J. , Leitman Susan , Biancotto Angelique , McCoy J. Philip , Miranda Susan , Tucker Zachary , Pfannes Loretta , Furnari Megan , Young Neal S , Sloand Elaine M 
Published: October 2019

Abstract
Abstract 3147
Poster Board III-84
Recent reports of chromosomal and immunological abnormalities in healthy donors receiving granulocyte colony stimulating factor (G-CSF) have raised concerns among hematologists that this popular cytokine may promote genomic instability or alter immune surveillance (Pampilon D et al Transfusion 2008; 48(7):1495-501). We previously reported that G-CSF altered the Th1:Th2 ratio in healthy individuals following short-term administration (Blood 2000 Apr 1;95(7):2269-74), but reported no new karyotypic abnormities after in vitro culture of bone marrow mononuclear cells with pharmacological doses of G-CSF (Proc Natl Acad Sci 2006 Sep 26;103(39):14483-8). There is no systematic study of the long-term effects of administering granulocyte colony stimulating factor (G-CSF) to healthy individuals. We examined CD34 cells of 10 healthy stem cell donors after they had received 10 mcg/kg G-CSF for 4 days; fluorescent in situ hybridization (FISH) was the method employed to monitor chromosomal changes. We also studied 37 healthy granulocyte donors who received G-CSF (5ug/Kg x 1 day) and dexamethosone for up to 42 times (median= 15; range 6-42) using FISH to examine chromosomes 7 and 8 and flow cytometry to define their T cell subsets. FISH did not detect chromosomal abnormalities in the CD34 cells of 10 donors mobilized with G-CSF; neither could monosomy 7 cells be isolated after culturing cells in media with 400 ng/mL G-CSF (previously shown to support outgrowth of monosomy 7 cells) for two weeks. Furthermore, FISH did not detect aneuploidy in the 37 regular granulocyte donors. Evaluation of T cell subsets by flow cytometry demonstrated similar percentages of CD4+ T cells in 18 granulocyte donors as compared to 23 untreated controls (57.5% vs 56.5%). However, donors had increased numbers of CD4+TNFαa+ Th1 T cells and decreased CD4+IL-6+ Th2 T cells (4.2% vs 1.6%, P= 0.0003 and 11% vs. 35%, P=0.04 respectively), while the donor Th2 subset expressed significantly more IL-6 per cell (P<0.01). CD4+CD25+FoxP3+ regulatory T cells (Tregs) were significantly increased in G-CSF-treated donors (10.1% vs 6.0%, P<0.0001), while Th17 T cells were not significantly different (2.4% vs 0.7%, P=0.423). G-CSF does not produce chromosomal abnormalities of monosomy 7 or trisomy 8 in healthy SCT donors or in serially treated granulocyte donors. However, there are significant changes in T cell subsets that modulate the immune response.

Disclosures
No relevant conflicts of interest to declare.
Two Alleles with RHCE*nt818C>T Change Encode the Low Prevalence Rh Antigen STEM.
Authors: Halter-Hipsky Christine , Hue-Roye Kim , Coghlan Gail , Lomas-Francis Christine , Reid Marion E. 
Published: October 2019

Abstract
Abstract 3159
Poster Board III-96

Background
According to the original (and only) report, the low prevalence Rh antigen, STEM, is associated with an altered e phenotype. Approximately 65% of hrS– and 30% of hrB– RBCs from South African donors are STEM+. STEM has a variable expression, which is an inherited characteristic. Anti-STEM has induced mild HDFN (Marais, et al., Transf Med 1993;3:35-41). The purpose of this study was to determine the molecular basis associated with STEM expression.

Material and Methods
Blood samples and reagents were from our collections. Hemagglutination and DNA extraction were performed by standard methods. Molecular testing included direct sequencing and cloning of cDNA, AS-PCR, PCR-FRLP, and sequencing specific exons of gDNA.

Results
Three STEM+ samples (including the original index case) had RHCE*ceBI [ce 48C (16C), 712G (238V), 818T (273V), 1132G (378V)] (Noizat-Pirenne, et al., Blood 2002;100:4223-31) and 6 had a new allele, which we name RHCE*ceSM (ce 48C, 712G, 818T). In contrast, 8 STEM– samples (which included hrS– and hrB– samples) did not have the RHCE*818C>T change. RBCs with the ceBI phenotype expressed STEM more strongly than those with the ceSM phenotype.

Conclusions
The previously reported allele RHCE*ceBI and a new allele, RHCE*ceSM, encode the STEM antigen. This study also revealed other new findings: (i) ceSM encodes a weaker expression of STEM than does ceBI, which explains the previously reported variable expression, (ii) provides an explanation for why not all hrS– and hrB– RBCs express STEM. RBCs with ceAR, ceMO, and ceEK, phenotypes are hrS– STEM–, and RBCs with ceS phenotypes type hrB–, STEM–, (iii) ceBI and ceSM do not express hrS but do express hrB. It is likely that anti-hrS made by hrS– STEM– people (ceAR, ceMO, ceEK) will be incompatible with hrS– STEM+ RBCs, and vice versa. Our findings provide a means to positively identify the STEM+ phenotypes, which, by hemagglutination, is virtually impossible due to the dearth of anti-STEM. Further, it provides a tool to provide suitable antigen-negative RBC products to a patient who has made an ‘e-like’ antibody.

Disclosures
No relevant conflicts of interest to declare.
Comparative Decay Rates of Human, Rhesus Macaque, Cynomolgus, and Porcine Activated Factor VIII.
Authors: Healey John F , Parker Ernest , Lollar John (Pete) S. 
Published: October 2019

Abstract
Abstract 3164
Poster Board III-104
The proteolytic conversation by thrombin of factor VIII (fVIII) to fVIIIa produces a A1/A2/A3-C1-C2 heterotrimer that spontaneously dissociates into inactive A1/A3-C1-C2 and A2 species. Human mutations that increase the rate of A2 subunit dissociation produce hemophilia A, indicating that A2 subunit dissociation is physiologically relevant and is an important regulatory feature of the blood coagulation mechanism. The A2 subunit dissociation rate from human fVIIIa is significantly faster than the corresponding dissociation rates from porcine or murine fVIIIa. The fast decay rate of human fVIIIa raises the question whether the f8 gene is under positive selection for this trait. To determine whether fast A2 dissociation occurs elsewhere in the primate lineage, we cloned cDNAs encoding B-domain deleted (BDD) fVIII from rhesus macaque and cynomolgus monkey liver. The deduced BDD amino acid sequences of rhesus and cynomolgus fVIII were 97.9 % and 98% identical to human fVIII, respectively, and were 99.9% identical to each other. The expression of rhesus and cynomolgus fVIII from baby hamster kidney-derived cells was similar to human fVIII and ten-fold lower than porcine fVIII. BDD human, rhesus, cynomolgus, and porcine fVIII molecules were purified to homogeneity by tandem ion-exchange chromatography. Concentrations of the purified constructs were calculated using a molar extinction coefficient at 280 nm based on their predicted tyrosine, tryptophan and cysteine compositions. Human, rhesus, and cynomolgus fVIII displayed similar specific coagulant activities by one-stage coagulation assay (6800, 4500, and 5200 units per mg, respectively). The kinetics of decay of human, rhesus, cynomolgus and porcine fVIIIa were measured following rapid activation of 1 nM fVIII by thrombin using a chromogenic substrate assay of purified intrinsic fXase complex under conditions in which fVIIIa was limiting. Decay curves were fit using nonlinear least-squares regression to a first-order model (Fig. 1). Decay rate constants for rhesus and cynomolgus fVIIIa were similar (0.31 and 0.27 min-1, respectively) and were slightly, but significantly lower than human fVIIIa (0.40 min-1). In contrast, the decay rate constant for porcine fVIIIa, 0.17 min-1, was 2.3-fold lower than human fVIIIa, consistent with previous observations. These results suggest that fast A2 subunit dissociation rates evolved before evolution of the primate lineage.

Disclosures
No relevant conflicts of interest to declare.
Kallikrein Inhibitors as Antiinflammatory Therapeutics.
Authors: Bryant John , Kolti Dhaval , Holsworth Daniel , Akbari Parmiss , Shariat-Madar Zia 
Published: October 2019

Abstract
Abstract 3179
Poster Board III-118
Plasma kallikrein is a novel target for antiinflammatory inhibitor design since it plays a role in the initiation of the intrinsic clotting, fibrinolysis, bradykinin-forming and neutrophil activation. The goal of this study was to synthesize a potent and selective inhibitor of kallikrein for the prevention and/or treatment of hyperfibrinolytic states, systemic inflammation, sepsis as well as cardiopulmonary bypass. Using a focus library of compounds, UK-097820, CP-013118 and PF-04886847 were found not to be cytotoxic and had the ability to abolish the hydrolysis of S2302 by kallikrein both in fluid phase and on the surface-mediated activation. In a competition study, PF-04886847 (3 mM) reduced the formation of a complex between C1-inhibitor and kallikrein by 40% following the activation of prekallikrein (kallikrein zymogen) on bound high molecular weight kininogen (HK) to human pulmonary artery endothelial cell (HPAEC). It was also mechanistically important to distinguish between direct kallikrein inhibition caused by the binding of kallikrein inhibitor to kallikrein versus the inhibitor's indirect effect on HK, prekallikrein or perturbation of the membrane function. The binding of PK to HK or HK to HPAEC surface was unaffected by the kallikrein inhibitors. To determine the selectivities of the kallikrein inhibitors, the effects of UK-097820, CP-013118 and PF-04886847-00 on factor XI and factor XII were examined. PF-04886847 was found to be a potent and selective inhibitor of kallikrein at a concentration ranging from 1 to 3 mM. BK stimulates cultured endothelial cells to release various mediators. The effects of the kallikrein inhibitors on the generation of bradykinin (BK), nitric oxide (NO) and prostacyclin and intracellular calcium signaling were determined. The generation of BK, NO formation and 6-keto-PGF1a release were blocked by PF-04886847, indicating that PF-04886847 might have potential applications for controlling unwanted inflammation provoked by BK-induced endothelium activation. In situ study, PF-04886847 blocked PK activation on intact aorta precontracted by phenylephrine. Taken together, we propose that PF-04886847 treatment might be beneficial in tissue injury and have potential clinical application.

Disclosures
No relevant conflicts of interest to declare.
Multiple Mapping of Peptide Sequences Recognized by Various Monoclonal Anti-ADAMTS13 Antibodies with Functional or Nonfunctional Effects On the Catalytic Activity.
Authors: Nakagawa Terumichi , Moriki Takanori , Yamaguchi Yusuke , Igari Atsuko , Soejima Kenji , Matsumoto Masanori , Fujimura Yoshihiro , Murata Mitsuru 
Published: October 2019

Abstract
Abstract 3182
Poster Board III-119
Fourteen mouse anti-ADAMTS13 monoclonal antibodies (MoAb#1∼#13, A10) were individually analyzed for their precise epitope peptide sequences in each domain using lambda phage surface display system. A phage library expressing random peptide fragments of ADAMTS13 on its surface was constructed, thereby selecting phage clones bound to each MoAbs immobilized on microtiter plates. Binding epitope sequences for eleven MoAbs were defined, although MoAb#3, #4 and #5 were not clarified. Among 11 epitope-determined MoAbs, epitopes were relatively short (6 to 23 amino acids) in MoAb#1, #2, #8, #11, #12 and #13, recognizing metallopretease, disintegrin-like, TSP1-4, TSP1-8, CUB1 and C-terminus domains, respectively. On the other hand, epitopes were relatively long (49 to 72 amino acids) in A10, MoAb#6, #7, #9 and #10, recognizing disintegrin-like, TSP1-2, TSP1-3, TSP1-5 and TSP1-7 domains, respectively. MoAb#1, #2 and A10 demonstrated inhibitory effects on the cleavage activity of ADAMTS13 evaluated by FRETS-VWF73 assay. MoAb#1 recognized Gln159 to Asp166 in the metalloprotease domain, and MoAb#2 and A10 recognized Asn308 to Glu327, Tyr305 to Glu376 in the disintegrin-like domain, respectively. From findings using C-terminal truncated mutants of ADAMTS13, MoAb#3 and #5 were supposed to recognize TSP1-1 and spacer domain, respectively, although only C-terminal tail peptide sequences were selected from both of the screening, suggesting the possibility of intramolecular association between the C-terminal region and TSP1-1/spacer domains. MoAb#4 was supposed to recognize disintegrin-like domain, although we could not obtain any significant ADAMTS13 peptide sequence from the screening. We speculate that these 3 epitope-undetermined MoAbs may recognize complex conformational structure of ADAMTS13. Alternatively, intact peptide structure of ADAMTS13 might not be expressed properly on the phage surface. In conclusion, we defined precise epitope sequences of 11 monoclonal anti-ADAMTS13 antibodies. Three of them, recognizing metalloprotease or disintegrin-like domains inhibited the cleavage activity of ADAMTS13. Analysis of the epitope sequences may elucidate the correlation between the molecular conformation and the catalytic activity.

Disclosures
No relevant conflicts of interest to declare.
HYC750: A Novel Stem Cell Mobilizer.
Authors: Schönekess Brett , Campion Rosalia De Necochea , Dardov Victoria , Sonjara Srdjan , Josephs Steven F. , Ramos Famela , Ichim Thomas , Minev Boris , Carrier Ewa 
Published: October 2019

Abstract
Abstract 3220
Poster Board III-157
Hyaluronic acid (HA) is a repeating disaccharide polymer having various biological activities depending on size: high molecular weight HA performs structural functions, whereas low molecular weight HA induces systemic cytokine-like effects. HYC750 is a Good Manufacturing Practices (GMP)-generated HA with a maximum molecular weight of 750 kDA that is currently in development as a hematopoietic stem cell mobilizer. Given the potential for proinflammatory activities of various very low MW HA fragments we sought to determine whether HYC750 may possess properties detrimental to clinical use. Dose-dependent administration of HYC750 to bone marrow stromal cells, the monocytic cell line RAW, and differentiated THP-1 cells did not result in alteration of proliferation or viability. Treatment of said cells with HYC750 did not elicit production of TNF-alpha as assessed by the L929 assay. In contrast, LPS treatment induced a dose dependent cytokine response. These data, combined with dose escalation studies in BALB/c mice without induction of inflammatory reactions or adverse effects suggested HYC-750 does not cause biological changes that are conventionally seen with low molecular weight HA administration. In order to quantify stem cell mobilization activity, 240 ug/mouse of HYC750 was administered for 5 consecutive days intraperitoneally and subsequently marrow, spleen and blood were sacrificed on day 6. A profound increase in cellularity of circulating cells was observed with the HYC750 treatment by forward and side scatter FACS profiles. A >2 fold increase in lineage negative, stem cell antigen (SCA-1) positive cells, c-kit positive cells was observed in the periphery in the treated animals. In contrast to G-CSF mobilization, HYC750 treatment was not associated with decreased marrow cellularity or local macrophage activation. These data suggest HYC750 is a novel mobilizing agent that does not elicit inflammatory responses. Recently, HYC750 received approval to be used in a Phase I, open-label, single-ascending-dose, safety study in healthy male volunteers, by the European Competent Authority in Seville, Spain.

Disclosures
Schönekess: Orcrist Bio: Employment, Equity Ownership.
Age Related Expression of hMLH1 but Not hMSH2 in Human Hematopoietic Stem Cells Predicts Genomic Instability.
Authors: Kenyon Jonathan , Saurastri Anshul , Gerson Stanton L. 
Published: October 2019

Abstract
Abstract 3235
Poster Board III-172
DNA damage repair pathways have been shown to be important for hematopoietic function and longevity. A number of studies focusing on the loss of DNA damage repair genes have reported age related loss of hematopoietic stem cells, reduced hematopoietic function, and increased rates of hematologic cancer. A poorly studied pathway with respect to its effect on hematopoietic function is the mismatch repair (MMR) pathway. Microsatellite instability (MSI), one marker of MMR failure, has been observed in acute lymphoblastic leukemia, myelodysplastic syndrome, and lymphocytes obtained from the peripheral blood of the elderly. Further, studies investigating MMR pathway disruption in mice show an increased incidence of age related hematologic disorders and failure to repopulate in competitive transplant experiments. These reports led to the formation of the hypothesis that age related loss of MMR in a subset hematopoietic stem/progenitor cells (HSC) leads to a progenitor population with a mutator phenotype potentially leading to cell death or leukemogenesis.
To test this hypothesis we clonally expanded HSC from cord blood, bone marrow, and bone core samples from various aged individuals most of whom had normal CBCs. These colony forming units (CFU) were then tested for MSI at five microsatellite loci previously used in the diagnosis of the MMR defective disease HNPCC (BAT 25, BAT 26, D2S123, D5S346, and D17S250). These results showed a statistically significant increase in high grade instability (measured as MSI observed at >20% of loci tested) in CFU obtained from individuals over the age of 50 compared to those in the age range between 0 and 50 years (p=0.0034). Next we sought to determine if loss of gene expression in MMR genes could account for the observed MSI. Gene expression of two genes known to be transcriptionally downregulated in MMR failure, hMLH1 and hMSH2, were assessed by qRT-PCR in CFU obtained from pure CD34+ immunomagnetic separations of HSC from various aged donors. Further, individual CD34+ cells from these same donors were fixed to slides and immunostained for hMLH1 and hMSH2. We observed a significant loss of hMLH1 gene expression in a subset of HSC obtained from older donors above age 42, but no pattern of loss of hMSH2 gene expression. The age related loss of MMR in HSC suggests that MMR pathway proficiency is important for hematopoiesis. These results suggest that loss of MMR might precede myelodysplastic syndromes and leukemia in older individuals.

Disclosures
No relevant conflicts of interest to declare.
Association of Increased Thymidine Uptake Relative to Tumor Cell Proliferation in Low-Grade Follicular Lymphoma and DNA Repair.
Authors: Juweid Malik E. , Syrbu Sergei I. , Bleile Michelle , Buck Andreas , Wondergem Marielle , Vose Julie M. 
Published: October 2019

Abstract
Abstract 3236
Poster Board III-173
We have observed that the uptake of the thymidine analog 18F-fluorothymidine (FLT) is highly disproportional to cellular proliferation in untreated low-grade follicular lymphoma (FL). Since the uptake of thymidine/thymidine analogs is also increased with enhanced DNA repair synthesis, we investigated whether the “excess” uptake of FLT in FL may be related to DNA repair. We stained tumor samples from 20 patients each with grade 1 FL and DLBCL for Ki-67, a marker of cell proliferation and DNA replication but not DNA repair and 2 DNA replication and repair biomarkers: proliferating cell nuclear antigen (PCNA) and replication protein A (RPA). Median %Ki-67-positive cells (Ki-67 index) was 10% (range; 5-20%) in FL compared to 80% (range 60-90%) in DLBCL (P<4×10-20). In contrast, median %positive cells for PCNA and RPA were 90% and 100% in FL and 100% and 100% in DLBCL. In both FL and DLBCL, PCNA showed a characteristic staining pattern with 3+ or 4+ staining of the proliferating Ki-67-positive cells vs. 1+ to 2+ staining of quiescent cells. Similar results were obtained when staining for thymidine kinase I (TK1). Interestingly, similar staining pattern to that seen in lymphoma samples was seen in germinal center but not mantle zone cells of normal tonsils and reactive lymph nodes indicating that the DNA repair seen in both FL and DLBCL is likely related to somatic hypermutations (SHM) generated by error-prone DNA repair known to occur in FL, most DLBCLs and normal germinal center cells. Comparison of FLT uptake in FL and DLBCL indicated that > 70% of FLT uptake in FLs with a Ki-67 index of ≤10% was due to DNA repair. In contrast, contribution of DNA repair to overall FLT uptake in DLBCL with a Ki-67 index of ≥ 80% was <10% presumably due to the 4-fold lower fraction of quiescent compared with proliferating cells with high replicative DNA synthesis. This is the first demonstration of a high contribution of SHM/DNA repair to overall uptake of thymidine/thymidine analogs in low-grade FL. Our data further suggest that FLT use for assessing response to cytostatic therapy in low-grade FL will be confounded by this high contribution and highlights the pitfalls associated with the use of PCNA or RPA to assess proliferative activity in FL and DLBCL.

Disclosures
No relevant conflicts of interest to declare.
Cell Lines and “Knock-in” Mice Bearing FLT3/ITD Mutations Exhibit Elevated Repair Errors Generated through Alternative DNA Double Strand Break Repair Pathways: Implications for Genomic Instability.
Authors: Fan Jinshui , Li Li , Small Donald , Rassool Feyruz V. 
Published: October 2019

Abstract
Abstract 3237
Poster Board III-174
Activating mutations of the FMS-like tyrosine kinase-3 (FLT3) receptor occur in approximately 30% of acute myeloid leukemia (AML) patients and, at least for internal tandem duplication (ITD) mutations, are associated with poor prognosis. We previously reported that FLT3/ITD mutations are associated with increased reactive oxygen species (ROS) production which leads to increased DNA double strand breaks (DSBs) and decreased repair fidelity, that may in part explain aggressive AML in FLT3/ITD patients. While the DNA-PK and Ku-dependent non homologous end-joining (NHEJ) pathway is one of the main routes for repairing DSBs in normal mammalian cells, recent reports have suggested that an alternative and less well defined NHEJ pathway (Alt NHEJ) involving DNA ligase IIIαa and PARP1, characterized by DNA sequence microhomology at the repair junctions, may play a role in the generation of deletions and translocations that can lead to cancer progression. Here, we report that in hematopoetic progenitor cells expressing FLT3/ITD (Ba/F3/ITD), the frequency of errors and the size of deletions during DSB repair is increased compared with the Ba/F3 control cells. Moreover, these data are confirmed in bone marrow mononuclear cells (BM MNCs) from FLT3/ITD “knock-in” mice, compared with WT control cells. Strikingly, we show that Ku proteins (Ku70 and Ku86), key components of the main NHEJ pathway, are down-regulated in FLT3/ITD-positive cell line and primary mouse BM MNCs. Concomitantly, DNA ligase IIIαa, a component of Alt NHEJ pathway is up-regulated in FLT3/ITD-expressing cells and plays a role in abnormal DSB repair. Importantly, after treatment with a FLT3 inhibitor, AML (MOLM-14, MV4-11) cells containing FLT3/ITD mutations demonstrate a reduction of misrepair and deletion size, and down-regulation of DNA ligase IIIαa, suggesting that FLT3 signaling regulates the pathways by which DSBs are repaired. Thus, therapy to inhibit FLT3/ITD may lead to repair that reduces repair errors and genomic instability.

Disclosures
No relevant conflicts of interest to declare.