bluuebunny/crossref_metadata_2025_split huggingface dataset:
Activated Platelet-Derived Antimicrobial Peptides Exhibit Virucidal Properties against Vaccinia Virus in Plasma.
Authors: Ketha Krishna Mohan V. , Rao Shilpakala Sainath , Atreya Chintamani D 
Published: October 2019

Abstract
Abstract 2118
Poster Board II-95

Introduction:
Contamination of blood and blood components by bacteria, virus, fungi and parasites is a major safety risk in transfusion medicine. While there has been a tremendous success in inactivating virus contamination in blood products through UV-irradiation, new and novel proof-of-concepts for microbial reduction that enhance risk to benefit ratio of the treated products are still a public health need in transfusion medicine. In the present study, we tested four novel synthetic antimicrobial peptides originating from thrombin-induced human platelet-derived antimicrobial proteins named PD1-PD4 against an enveloped virus, Vaccinia Virus (VV) spiked in plasma as a model system. We have recently shown these peptides to be useful in reducing bacterial burden in plasma and platelets. These short synthetic peptides are human platelet-derived and known to cause no immune response in humans and non-hemolytic in nature as reported by others.

Methods:
The PD1-PD4 peptides were tested on plasma samples spiked with 10-fold dilutions of the wild-type lab strain of Vaccinia Virus (WR strain). Each spiked sample was pre-incubated individually with a peptide (PD1-PD4) for 1 hour at 37°C. Spiked sample without any peptide was included as control. A cell culture-based standard plaque reduction assay method was utilized to monitor the virucidal effectiveness of the peptides. Minimal inhibitory concentration of the peptides was also estimated by testing the peptides at doubling dilutions of 100 μg/ml, 50 μg/ml, 25 μg/ml and 12.5 μg/ml concentrations.

Results:
Our analysis revealed that peptides PD3 and PD4 were potent against vaccinia virus resulting in reduction of viral titers in the plasma. PD3 peptide demonstrated the highest virucidal activity by bringing about a 2-log reduction of VV titers. PD4 peptide treatment resulted in a 1-1.5 log reduction in viral titers. The minimal inhibitory concentration analysis revealed that at 50 μg/ml concentration both the PD3 and PD4 were able to bring about a log reduction in viral titers.

Conclusion:
The present study reports a novel antiviral agent for reducing vaccinia virus contamination in plasma. Safety profiles of these peptides as reported by others in conjunction with our current studies, provide a new proof-of-concept that could be useful as safer and simpler alternatives to the viral reduction agents in transfusion medicine.
The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

Disclosures:
No relevant conflicts of interest to declare.
Mechanisms of Human Neutrophil Elastase-Catalyzed Inactivation of the Factor VIII(a).
Authors: Nishiya Katsumi , Nogami Keiji , Matsumoto Tomoko , Ogiwara Kenichi , Takeyama Masahiro , Shima Midori 
Published: October 2019

Abstract
Abstract 2125
Poster Board II-103
Inflammation and coagulation are linked in a variety ways. Since both neutrophil elastase and cathepsin G bind to activated platelets, they can be localized on the platelet membrane providing negatively-charged phospholipid, that is essential for assembly of tenase complex. Although it has been reported that cathepsin G provides some procoagulant effect by activating factor (F)VIII, effect of elastase on FVIII is poorly understood. We now examine the effect of human neutrophil elastase (HNE) on FVIII(a). FVIII activity (used 100 nM) was rapidly decreased and was undetectable within 10 min after the addition of HNE (0.5 nM) in a one-stage clotting assay. This time-dependent inactivation was little or mildly affected (∼35% inhibition at 10 μg/ml) in the presence of phospholipid or von Willebrand factor, respectively. Rate constant of FVIII inactivation by HNE possessed ∼15-fold greater than that by APC/protein S (PS). SDS-PAGE analysis revealed that HNE proteolyzed the 90∼210-kDa heavy chain of FVIII to 47- and 37-kDa terminal products, via some proteolyses within the B domain. Western blot and N-terminal sequence analyses showed that these fragments derived from the heavy chain were identified as the A11-357 and A2375-708, respectively, by cleavages at Val357 and Val374 in A1 and Val708 in A2. The cleavages at Val374 and Val708 by HNE were faster than that at Val357. The 80-kDa light chain was proteolyzed to 75-kDa fragment by cleavage at Val1670, quite different with the pattern of FVIII cleavage by known serine proteases. Of note, HNE-catalyzed inactivation of FVIIIa was much slower (by ∼25-fold) than that of FVIII, supporting that HNE preferably attacked to FVIII. This discrepancy was attributed to much slower cleavage at Val708 in FVIIIa. Rate of inactivation of FVIIIa by HNE was ∼30-, ∼8-, and ∼3-fold lower than those by plasmin, APC/PS, and FXa, respectively. SDS-PAGE using FVIII(a) fragments revealed that each cleavage at Val357 and Val708 was regulated by the presence of the light chain and heavy chain (intact A1-A2), respectively. These results demonstrate that the importance of cleavage at Val708, constitutes a FIXa-interactive site in tenase complex, for the mechanism of HNE-catalyzed FVIII inactivation. Furthermore, this cleavage appears to be selectively modulated by the A1-A2 domain that might interact with HNE.

Disclosures:
No relevant conflicts of interest to declare.
Tissue Factor Is Frequently Expressed in Multiple Myeloma Cells.
Authors: Gupta Sameer , Hayes Tanisha R , Liu Yuchuan , Hurford Matthew T , Solomides Charalambos , Bromberg Michael 
Published: October 2019

Abstract
Abstract 2132
Poster Board II-109
Tissue factor (TF) is a 47 kDa transmembrane glycoprotein that initiates blood coagulation when complexed with factor VIIa. TF is constitutively expressed in a variety of tumor cells and has been shown to have a role in cellular signaling, angiogenesis and solid tumor progression. However, the role of TF in the hematologic malignancies is poorly defined. Multiple myeloma (MM) is associated with an increased risk of venous thromboembolic disease. However, whether increased TF expression contributes to the hypercoagulable state associated with MM remains controversial. In this study, we determined the expression of TF on archived bone marrow biopsies and plasmacytomas, and human MM cell lines. Immunohistochemical staining of TF was carried out on paraffin-embedded specimens from eighteen advanced stage MM patients. Staining for TF expression was scored as 0 (null), 1+ (weak), 2+ (moderate) and 3+ (strong). TF expression for the MM cell lines (U266B1, MM1.RL and MM1.S) was carried out by semi-quantitative real time RT-PCR. TF mRNA levels were normalized to 18S ribosomal mRNA levels. The TF: 18S ratios were then compared to that of a low TF expressing human breast cancer cell line cell line, MCF-7. Paraprotein distribution in the MM patient specimens was: IgG kappa (7), IgG lambda (5), IgA kappa (3), lambda light chain (2) kappa light chain (1). Overall, TF expression was observed in 10/18 (56%) of the patient specimens. Six specimens stained 1+, and two each stained 2+ and 3+. Staining was mainly cytoplasmic and did not correlate with the type of secreted paraprotein. TF expression (relative to MCF-7) was 17.3, 1.97 and 0.77 for the U266B1, MM1.RL and MM1.S cell lines, respectively. Results from these studies suggest that TF is frequently expressed in MM cells and might contribute to the hypercoagulability associated with this disease. In addition, TF may play a role in MM cell progression.

Disclosures:
No relevant conflicts of interest to declare.
Plerixafor and G-CSF Versus Cyclophosphamide and G-CSF for Stem Cell Mobilization in Patients with Multiple Myeloma.
Authors: Nazha Aziz , Cook Rachel , Vogl Dan T. , Mangan Patricia A. , Hummel Kimberly , Cunningham Kathleen , Luger Selina , Porter David , Schuster Stephen J. , O'Doherty Una , Sell Mary , Siegel Don L. , Stadtmauer Edward A. 
Published: October 2019

Abstract
Abstract 2146
Poster Board II-123

Introduction:
High dose melphalan and autologus stem cell transplant remains an effective treatment for patients with either early or refractory multiple myeloma (MM). Collection of sufficient numbers of stem cells for more than one transplant is optimal. G-CSF with chemotherapy, particularly cyclophosphamide (CY/G-CSF), has been a widely used and effective regimen for stem cell collection in MM. Plerixafor, a CXCR4 antagonist, when combined with G-CSF has been shown in a large randomized clinical trial to be superior to G-CSF alone. A comparison of plerixifor/G-CSF to CY/G-CSF is presented here.

Materials and Methods:
We performed a single institution retrospective analysis of 365 patients with MM who underwent stem cell mobilization and harvest at the University of Pennsylvania Abramson Cancer Center from January 2002 to December 2007. All patients were harvested early in the course of their disease. 76 patients were excluded from this analysis (23 had incomplete data on induction regimen, 19 had incomplete data on stem cell collection, 16 had incomplete data on mobilization regimen, 10 underwent allogeneic transplants, 2 had bone marrow rather than peripheral blood harvests, 2 had stem cells collected at an outside institution, 2 had chemotherapy mobilization other than CY and 2 had medical complications prior to harvest and after mobilization). Therefore, 289 patients were included in the analysis; 16 received plerixafor/G-CSF, 198 received CY/G-CSF, and 75 received G-CSF alone.

Results:
The median number of collected stem cells was 7.95 × 106 CD34+/kg in plerixafor/G-CSF group, 7.7 × 106 CD34+/kg in Cy/G-CSF group and 4.5 × 106 CD34+/kg in G-CSF alone group. The median number of apheresis days was 2 days, 2 days and 4 days respectively. The percentage of the patients who collected ≥ 6 × 106 CD34+/kg in < 3 apheresis was 63% (10/16), 62% (123/198) and 19% (14/75) respectively. The percentage of the patients who collected ≥ 6 × 106CD34+/kg <5 apheresis was 81% (13/16), 69% (136/198) and 23% (17/75) respectively. The mean CD34+/kg collected erither after CY/G-CSF or plerixafor/G-CSF was higher than G-CSF alone (p<0.0001 for each analysis).

Conclusion:
This analysis suggests that plerixafor/G-CSF and CY/G-CSF mobilization result in similar and adequate stem cell harvest numbers for autologous stem cell transplantation for MM. Both approaches are superior to G-CSF alone. The choice of plerixafor/G-CSF vs CY/G-CSF for stem cell mobilization will therefore depend on further analysis of the relative costs, toxicities and long term outcome of these regimens.

Disclosures:
Stadtmauer: genzyme: Consultancy.
Wnt-Pathway Directed Compound Targets Blast Crisis and Chronic Phase CML Leukemia Stem Progenitors.
Authors: Peterson Luke F , Turbiak Anjanette J , Giannola Diane M , Donato Nicholas , Showalter Hollis H.D. , Fearon Eric R , Talpaz Moshe 
Published: October 2019

Abstract
Abstract 2168
Poster Board II-145
The deregulated tyrosine kinase associated with t(9;22) fusion protein BCR-ABL is efficiently targeted by tyrosine kinase inhibitors (Imatinib, Nilotinib and Dasatinib). It is generally believed that most patients have residual disease. The inability to eliminate the refractory leukemia stem cell in chronic myeloid leukemia (CML) is not well understood. The refractory stem cell is present despite effective inhibition of the BCR-ABL kinase in these cells, and dissociation between kinase inhibition and cell death implies lack of dependency on BCR-ABL aberrant kinase activity, unlike the one seen in more mature CML cells. Thus the oncogene addictive dependency of these leukemia stem cells may be overcome by intrinsic and/or extrinsic factors/pathways. One such pathway involved in hematopoeitic stem cell maintenance is the Wnt/β-catenin signaling pathway. BCR-ABL signaling is known to directly activate the Wnt/β-catenin pathway. The loss of function of a negative regulator of the Wnt/β-catenin pathway, known as GSK3β is associated with CML progression from chronic phase to the blast phase. Lastly, the loss of β-catenin in murine models impairs the self renewal capacity of both the normal and the BCR-ABL+ leukemia stem cell. Thus this pathway is important during various stages of the disease.
We explored the effect of Wnt inhibition using a novel Wnt pathway inhibitor (AG-214, University of Michigan). AG-214 is structurally related to a previously reported Wnt-inhibitor. AG-214 is able to antagonize β-catenin/TCF in luciferase reporter assays, and expression of Wnt targets in colon cancer cell lines. We utilized a serum free culture system with 5 added cytokines and treated both CML blast crisis CD34+ as well as chronic phase CD34+ cells with AG-214. Our in vitro experiments show that primary blast crisis CD34+ cells are induced to undergo apoptosis at an IC-50 of approximately 2 μM. Furthermore, combination of 1.25 μM of AG-214 with 2 μM Imatinib achieved greater than 50% apoptosis. Analysis of chronic phase CML CD34+ cells showed that targeting of Wnt/β-catenin pathway requires higher concentration of the Wnt-inhibitor (>2.5 μM), and that the addition of Imatinib can cooperate to enhance the apoptotic response. Furthermore, chronic phase progenitors (Lin-CD38+/CD34+) are more sensitive to lower concentrations of AG-214 whereas 5 μM is required to induce significant apoptosis of ∼70% in the primitive leukemia stem cell (Lin-/CD38-/CD34+) population, and addition of 2 μM Imatinib increased their apoptotic response to ∼84%. Normal CD34+ cells do not undergo significant apoptosis with 5 μM of AG-214 (∼10%) even when combined with 2 μM Imatinib (∼20%). Targeting of the Wnt/β-catenin pathway enhances apoptosis in both blast crisis and chronic phase CML progenitors and leukemia stem cells.

Disclosures:
No relevant conflicts of interest to declare.
A Common but Overlooked Mechanism of BCR-ABL1 Kinase Inhibitor Resistance in Chronic Myeloid Leukemia.
Authors: Bruey Jean-Marie , Kantarjian Hagop M. , Ma Wanlong , Yeh Chen-Hsiung , Lee Tai-Sung , O'Brien Susan , Giles Francis , Estrov Zeev , Cortes-Franco Jorge , Albitar Maher 
Published: October 2019

Abstract
Abstract 2179
Poster Board II-156
Point mutations in the BCR-ABL1 protein are the most commonly reported mechanism of resistance to imatinib mesylate (Gleevec or STI-571) treatment in patients with chronic myelogenous leukemia (CML). We have reported that some patients with imatinib-resistant CML express an alternatively spliced BCR-ABL1 mRNA with a 35-bp insertion (BCR-ABL135INS), but the prevalence and effect of this mutation have not been well delineated. We sought to improve understanding of this mutation in order to provide insight into mechanisms of resistance to tyrosine kinase inhibitors. Using a sensitive PCR method, we determined the prevalence of the alternatively spliced BCR-ABL135INS mRNA in a group of 168 patients with chronic-phase CML resistant to imatinib. Expression of truncated protein was confirmed by Western blot in patient samples. The effects of various kinase inhibitors on human K562 CML cells transfected with BCR-ABL135INS cDNA were tested by measuring cell survival, caspase-3 activation, and the BIM activation pathway. In our analysis of BCR-ABL1 genotypes in 168 imatinib-resistant chronic-phase CML patients, (56%) of patients expressed various levels of BCR-ABL135INS whereas only (25%) had detectable point mutations in BCR-ABL1. Expression of this truncated BCR-ABL1 fusion protein was also confirmed by immunoprecipitation at levels proportional to those predicted by mRNA quantification. In vitro, BCR-ABL135INS expression conferred resistance to the tyrosine kinase inhibitors imatinib, dasatinib, and nilotinib in a concentration-dependent fashion, but had no effect on the protein-synthesis inhibitor homoharringtonine (HHT or omacetaxine) or the aurora kinase inhibitor MK-0457 (VX-680). The combination of imatinib with nilotinib or HHT was synergistic in overcoming BCR-ABL135INS-induced resistance. Moreover, the resistance to imatinib induced by BCR-ABL135INS was associated with loss of BIM expression. This loss of BIM was overcome by pretreatment with HHT. In conclusion, these findings emphasize the importance of the overlooked alternatively spliced BCR-ABL135INS found in imatinib-resistant CML patients. Optimal patient management in the future will likely require periodic testing for the expression of BCR-ABL-135INS mRNA during therapy. This may help in predicting various levels of resistance or the potential for eliminating the disease by adjusting the treatment strategy.

Disclosures:
No relevant conflicts of interest to declare.
Alefacept Treatment for Graft Vs. Host Disease May Not Suppress the Graft Vs. Leukemia Effect.
Authors: Dray Liliane , Matusevich Aliza , Resnick Igor , Aker Memet , Stepansky Polina , Or Reuven , Shapira Michael Y. 
Published: October 2019

Abstract
Abstract 2223
Poster Board II-200

Introduction:
In general it is expected that strong immune suppression that alleviates GVHD, will increase the risk of relapse. Alefacept (Amevive®) is an immunosuppressive dimeric fusion protein that is used for psoriasis control. We showed its remarkable effect in acute steroid resistant/dependent and chronic extensive GVHD.

Methods:
To date, 42 patients with a median age of 31.5 years (range 3-66) were treated by us with alefacept due to acute (n=28) or chronic (n=14) steroid resistant/dependent GVHD (27 males, 15 females). Twenty were transplanted from HLA matched family members, 14 from HLA matched unrelated donors, 8 from mismatched donors and 2 from unrelated cord blood units. Pretreatment acute GVHD grade ranged 2-4 (median 3) and involved the skin (30), gut (19) and liver (7). All the patients with chronic GVHD had extensive involvement prior to therapy.

Results:
The median time from transplantation to alefacept was 42.5 days and 13 months in acute and chronic GVHD respectively (range 18-110d and 3-47.5m) and a median of 9 (range 1-25) injections that were given per patient. Thirty-four out of the 41 evaluable patients (83%) responded to the treatment (23/28 and 11/13 in the acute and chronic group respectively). Despite this high response rate, demonstrating the deep immunosuppressive and immunomodulative effect of alefacept, only 5/41 evaluable patients (figure 1) have relapsed (with a median follow-up of 30.8 months on the 17 survivors). Other than the 5 patients that relapsed (all treated with calcineurin inhibitor and steroids), full-donor peripheral blood chimerism (100% donor cells and no residual host-type DNA) was stable throughout the treatment period and later in all but one patient, that developed mixed chimerism under alefacept treatment. His chimerism returned to full-donor chimerism with taper-down of immune suppression. Currently, 17/42 (40.5%) patients are alive (figure 1), most with improved or stable GVHD. Twenty-six patients died due to either: GVHD progression (n=14), progression of the basic disease (n=4), infections (n=5) or other causes (n=3).

Conclusions:
Alefacept is effective and safe for the treatment of acute or chronic steroid resistant/dependent GVHD and may discriminate between GVHD and GVL.

Disclosures:
No relevant conflicts of interest to declare.
High Levels of B-Cell Activating Factor During the Peri-Transplant Period Are Associated with the Reduced Incidence of Acute Graft-Versus-Host Disease Following the Myeloablative Allogeneic Stem Cell Transplantation.
Authors: Cho Byung Sik , Min Chang Ki , Kim Hee Je , Lee Seok , Kim Yoo Jin , Jeong Dae-Chul , Cho Bin , Kim Hack Ki , Eom Ki-Seong , Cho Seok Goo , Kim Dong Wook , Lee Jong Wook , Min Woo Sung , Park Jong Won , Kim Chun Choo , Chung Nak Gyun 
Published: October 2019

Abstract
Abstract 2229
Poster Board II-206
To determine whether serum B-cell activating factor (BAFF) levels that were previously found to be elevated in patients with chronic graft-versus-host disease (GVHD), and C-reactive protein (CRP) levels known as a predictor for aGVHD as well as a marker for systemic inflammation during the peri-allogeneic stem cell transplantation (SCT) period, could be used to predict the occurrence of acute GVHD (aGVHD), 45 consecutive patients who had undergone myeloablative allogeneic SCT for hematologic malignancies were assessed. Serum BAFF and CRP levels were measured using ELISA (R&D Systems, Minneapolis, MN, USA) before conditioning and on day 0, day +7, and day +14 after transplant. Thirty-three of 45 patients (cumulative incidence, 73=) developed aGVHD between 16 days and 98 days after transplant. Analyses using repeated measures of ANOVA revealed that the serum BAFF levels were significantly lower in patients with aGVHD than in those without aGVHD (P=0.001), whereas no association was detected between CRP levels and aGVHD (P=0.508). Receiver operating characteristic curve (ROC) analysis showed that serum BAFF levels at every time point were available for the prediction of development of aGVHD (pre-conditioning; P=0.005, day 0; P=0.002, day +7; P=0.004, and day +14; P=0.005). Using ROC curve analysis, the identical cutoff value of 43 pg/ml at every time point that divides patients into two groups, high (> 43 pg/ml) or low (≤ 43 pg/ml) BAFF group, was determined, which could assure 75= sensitivity and 73-82= specificity for the prediction of aGVHD at every time point. The analyses of the cumulative incidence of aGVHD at each time point by BAFF groups (high vs. low) showed that serum BAFF level at every time point plays a significant predictive role for the occurrence of aGVHD (pre-conditioning; P=0.040, day 0; P=0.023, day +7; P=0.003, and day +14; P=0.026). This study is, to the best of our knowledge, the first to show that high serum BAFF levels during the peri-transplant period may play a protective role against aGVHD in humans. The results of this study also show that the BAFF levels during peri-transplant period may be considered to be a predictor for aGVHD. Further trials with a larger cohort will be necessary to determine a definite cutoff value of high BAFF level as well as to construct an index with previously known factors for the prediction of aGVHD.

Disclosures:
No relevant conflicts of interest to declare.