bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet Release Occurs in the Bone Marrow Sinusoids and Spleen but Not in the Lungs Following Hematopoietic Stem Cell Transplant in the Mouse.
Authors: Kreinest Deanna , Sola Martha , Li Xiao-Miao , Sanders Ronald , Jorgensen Marda , Scott Edward , Slayton William B.
Published: October 2019

The steps that lead to platelet production are poorly understood. Current theories suggest that megakaryocytes mature under the influence of contact with sinusoidal endothelium, and release platelets either in the sinusoids or in the lungs. We hypothesized that platelet release would be accentuated following hematopoietic stem cell transplant, and that sites of platelet release would be apparent during the period of platelet recovery. We transplanted highly purified hematopoietic stem cells based on lack of expression of markers for mature lineages (Linneg) and expression of Sca-1, c-kit, and Thy-1.1 (KTSL cells), and subfractionated these cells based on low expression of Rhodamine 1-2-3, into lethally irradiated hosts expressing an allelic version of glucose phosphate isomerase to identify donor and host-derived platelets. We collected bones, lungs, livers and spleens on day 7, 14, 21, and 28 post-tranplant, and stained formalin/fixed tissue with anti-Von Willebrand Factor antibody to identify megakaryocytes (5–10 animals per cohort, 2 separate experiments). We scored megakaryocytes based on their location relative to endothelial cells, and whether they were releasing platelets based on extension of proplatelet processes into the vascular spaces. Almost every megakaryocyte was associated with the endothelium during the period of platelet recovery, and we did not identify megakaryocytes that were migrating to the endothelium. We saw numerous megakaryocyte releasing platelets in both the bone marrow and the spleen during the time of platelet recovery, which occurred on days 13–28 following transplant of purified stem cells. Some of these megakaryocytes had disrupted the endothelium and were incorporated into the sinusoidal wall. Others were completely within the sinusoidal spaces. Between 30 and 50% of megakaryocytes were releasing platelets in the spleen and bone marrow at any given time following transplant, and platelet release did not correlate with the platelet counts. These levels were similar to levels of platelet release seen in healthy control mice. In contrast, we saw no identifiable megakaryocytes in the liver and lung during the period of platelet recovery. Our results suggest that in the mouse, the bone marrow and spleen, and not the lung, are major sites of platelet release following stem cell transplant.
Profiling Retroviral Integration Sites in Donor T-Lymphocytes for Suicide Gene Therapy.
Authors: Giordano Frank A. , Laufs Stephanie , Nagy Katalin Z. , Fehse Boris , Hotz-Wagenblatt Agnes , Lauterborn Daniel , Fellenberg Kurt , Kuehlcke Klaus , Naundorf Sonja , Zander Axel R. , Zeller Jens W. , Fruehauf Stefan
Published: October 2019

Retroviral vectors encoding the herpes simplex thymidine kinase gene (HSV-Tk) have been employed to render T-lymphocytes (TLCs) sensitive to the prodrug ganciclovir. Such genetically modified T cells have been used for adoptive immunotherapy in the context of allogeneic hematopoietic stem cell transplantation (SCT). Infused T cells have been shown to be susceptible to elimination through exposure to ganciclovir in the event of graft-versus-host disease (GvHD). Recent reports on insertional “genotoxicity” in a mouse gene marking study and a clinical gene therapy trial for X-chromosomal severe combined immunodeficiency (X-SCID) reminded us the actual risk of insertional oncogene activation and subsequent leukemia development. Here we investigated retroviral integration sites in donor TLCs transduced with the MoLV-based TK/neoR vector Mo3TIN of four donors in a clinical HSV-Tk study. A total of 114 retroviral integration sites were detected using highly sensitive and specific ligation-mediated PCR (LM-PCR).

41.2% of all integrations appeared near the transcription start regions (+/−10kb) of genes. Further analysis showed that 57 (50%) of all integrations targeted RefSeq genes. 24 of those appeared in intron 1 (42% of all integrations into genes) while 18% (10/57) of all integrations into genes landed in exon sequences whereas 6 hit the first exon. 18 of the targeted genes (15.8% of all integrations) could be at last assigned to signal transduction pathways, whereas the transcription factor family was afflicted 13 times (11.4% of all integrations). The zinc ion binding group makes up 4 (resp. 6, minding that GTF2HII contains a C2H2 type and KIAA0427 a C-x8-C-x5-C-x3-H-type zinc finger) of them. Among the targeted genes we found integrations into the CD8, CD100, CD44, CX3CR1, HLA-DMP and IL10-receptor genes. Within at a range of 5kb up- and 5kb downstream of vector integrations 15 genes were located that were not hit. 5 are known as transcription factors, whereas two of those are involved in leukemia, namely the homo sapiens myeloid/lymphoid or mixed-lineage leukemia 5 gene (MLL5) and the homo sapiens ALL1 fused gene from 5q31 (AF5Q31).

Hit genes are currently examined more systematically in terms of function, e.g. involvement in signal transduction and transcription promoting processes. RISC (Retroviral Insertion estimate of Chromosomal Integration) scores and integration specific data will be submitted to a data warehouse, the collaborative RISC score database (CRSD). Such a systematic data collection similar to the IBMTR or EBMT databases will allow to recognize factors contributing to the safety, optimal transgene expression and persistence of transduced cells in the setting of allogenic matched donor transplantation.
Downregulating the Fibromodulin Gene by Short Interfering RNA Causes B-CLL Cell Death.
Authors: Choudhury Aniruddha , Derkow Katja , Mikaelsson Eva , Kokhaei Parviz , Österborg Anders , Rabbani Hodjattallah , Mellstedt Håkan
Published: October 2019

Development of targeted therapies against B-CLL is dependent on the identification of molecules that are essential for the proliferation and survival of the leukemic cells. One such molecule investigated by us as a putative leukemia-associated target is Fibromodulin (FIM). FIM is an extracellular matrix molecule belonging to the leucin-rich proteoglycan family. Our laboratory studies have verified that expression of FIM is upregulated in B-CLL cells. Moreover, this molecule is specifically overexpressed in B-CLL cells and not on normal peripheral blood mononuclear cells. Analysis of FIM expression on various other hematological tumor cells have revealed that with the exception of mantle cell lymphoma, FIM is not expressed in other hematological malignancies.

RNAi technology has recently emerged as a powerful method to specifically silence the expression of a gene. We have generated three siRNA against various segments of FIM and tested the effects of these siRNA on B-CLL cells. Transfection of B-CLL cells with these siRNA significantly diminished, or completely abrogated the expression of FIM mRNA as detected by RT-PCR. The figure below shows silencing of the FIM gene following siRNA transfection, as assayed by RT-PCR. “Untrans” and “ctrl” represents untransfected and control siRNA-transfected B-CLL cells respectively. As noted, transfection with each of the three siRNA against FIM completely abrogated the expression of FIM siRNA. 24–48 hours after siRNA transfection, a substantial fraction (30–70%) of the B-CLL cells, compared to cells transfected with control non-silencing siRNA, went into apoptosis as assayed by Annexin-V-propidium iodide staining. Time kinetic studies revealed that siRNA mediated silencing and apoptosis occurred between 18 and 48 hours. No such effect was noted when peripheral blood mononuclear cells of healthy donors or FIM-positive fibroblast cell lines were transfected with siRNA. In reconstitution experiments, siRNA treated B-CLL cells were co-cultured with various numbers of FIM-positive fibroblasts. The presence of these fibroblasts greatly diminished the apoptosis of B-CLL cells induced after siRNA treatment. Our results indicate that overexpression of FIM in B-CLL cells are critical for their survival and FIM may serve as a leukemia-specific target for B-CLL therapy.

Figure Figure
Efficacy wnd Bleeding with Ximelagatran and Warfarin after TKR: Pooled Subgroup Analysis of 3 Phase III Trials.
Authors: Berkowitz Scott D. , McElhattan Jennifer , Francis Charles W. , Colwell Clifford W.
Published: October 2019

Introduction: In patients undergoing total knee replacement (TKR), the post-operative period is a time of intense thrombotic stimulus often resulting in venous thromboembolism (VTE), precisely when surgical hemorrhage must be minimized: anticoagulant management requires a delicate balance. Ximelagatran (Exanta®, AstraZeneca), the first oral alternative to warfarin, has a rapid onset of action and requires no coagulation monitoring or dose adjustment. Three randomized, double-blind trials of ximelagatran vs. warfarin as VTE prophylaxis after TKR were completed: Study 236 (n=680), EXULT A (n=2301) and EXULT B (n=2303). Ximelagatran 36 mg BID provided efficacy superior to well-controlled warfarin (target INR 2.5) administered for 7–12 days, while 24 mg BID yielded numerically better but not statistically superior efficacy; neither dose increased bleeding.

Objective: We performed pooled subgroup analyses to assess pre-specified subgroup factors with regard to thromboembolic and adjudicated major/minor bleeding events.

Methods: Two comparison pools were created: 36-mg pool (ximelagatran 36 mg and corresponding warfarin groups from EXULT A and B) and 24-mg pool (ximelagatran 24 mg and corresponding warfarin groups from Study 236 and EXULT A). Prognostic factors were examined using multiple logistic regression analysis. Treatment by factor interactions are reported.

Results: In the 36-mg pool, no significant treatment interactions were observed for efficacy (i.e., the influence of each subgroup factor on ximelagatran and warfarin patients was similar). However, in the 24-mg pool, increases in age and CrCL indicated a disproportionate increase in the composite efficacy endpoint of total VTE and/or all-cause mortality for the 24-mg group compared to the warfarin group. Subgroup analyses of major/minor bleeding events showed no significant treatment interactions in the 24-mg pool. In the 36-mg pool, increases in age resulted in increased bleeding in the warfarin group compared to the 36-mg group. Men on ximelagatran 36 mg exhibited a greater risk of bleeding than did men on warfarin. No factors were identified to explain the observed increase in risk.

Conclusions: Subgroup analysis results for the ximelagatran 36-mg and 24-mg pools were generally consistent with those in the overall population, with the incidence of the composite efficacy endpoint consistently lower in the ximelagatran groups than in the warfarin group for most pre-specified subgroups. Patients expected to have higher ximelagatran exposure due to renal impairment and increased age showed no significant difference in bleeding risk compared with warfarin. This analysis supports a 36-mg dose for all patients undergoing TKR.
Monocyte-Derived Dendritic Cells from HLA-Matched Allogeneic Donors Showed Superiority To Induce Leukemic Cell-Specific T Cells in Comparison to Leukemic Cell-Derived Dendritic Cells or Monocyte-Derived Dendritic Cells from AML Patients.
Authors: Lee Je-Jung , Choi Bo-Hwa , Park Myong-Suk , Yang Deok-Hwan , Kim Yeo-Kyeoung , Kim Hyeoung-Joon
Published: October 2019

Leukemic-dendritic cells (leukemic-DCs) have certain limitations, which include difficult generation in 30–40% of patients, and low levels of expression of several key molecules. To overcome these limitations, autologous mononocyte-derived DCs (autologous-DCs) from the patients can use as an alternative approach, but the DCs may have some defective functions. In this study, we investigated the feasibility of immunotherapy for AML using leukemic-cell specific cytotoxic T lymphocytes that were stimulated by allogeneic monocyte-derived DCs (allogeneic-DCs) pulsed with leukemic cell lysates in vitro. To generate autologous- or allogenic-DCs, CD14+cells isolated from mononuclear cells (MNCs) of the patients or of HLA-matched donors were cultured in the medium with GM-CSF and IL-4 for 6 days. Their maturation was induced by adding TNF-α, IL-1β, IL-6, and PGE2 for 2 days, and then, leukemic cell lysates were pulsed. To generate leukemic-DCs, MNCs obtained from AML patients were cultured in the presence of GM-CSF, IL-4, and TNF-α for 8–12 days. The expressions of several key molecules on allogeneic-DCs were significantly higher than that on leukemic-DCs or autologous-DCs. Allogeneic-DCs exhibited a higher capacity to stimulate allogeneic CD3+ T cells compared to leukemic-DCs or autologous-DCs in allogeneic mixed lymphocyte reaction assay. Autologous CD3+ T cells stimulated by HLA-matched allogeneic-DCs pulsing with leukemic cell lysates showed more potent cytotoxic activities against autologous leukemic cells than those stimulated by leukemic-DCs or autologous-DCs. In addition, autologous-DC showed an intermediate level between leukemic-DCs and allogeneic-DCs in phenotypic expressions, allogeneic T-cell stimulatory capacities, or cytotoxic activities. In conclusion, these results suggest that monocyte derived-DCs from HLA-matched allogeneic donors can alternatively use to generate leukemic cell-specific cytotoxic T cells and to overcome the limitation of leukemic-DCs or autologous-DCs.
AML Induction Therapy with Outpatient Azacitidine.
Authors: Shadduck Richard K. , Rossetti James M. , Faroun Yacoub , Kaplan Robert B. , Lister John
Published: October 2019

15 patients with newly diagnosed acute myelogenous leukemia were treated with outpatient 5-aza-2′-deoxycytidine (azacitidine, 75 mg/m2 subcutaneously for seven days every four weeks) as primary induction therapy. The average age was 68 years (range = 44–80). Bone marrow blast counts ranged between 20% and 38%. The overall response rate (complete remission, CR + parital remission, PR) was 53% (8/15). CR was achieved in 4 patients (27%) in an average of 3 cycles (range = 2–5). Duration of response averaged 8 cycles (range = 6–13) from the time CR was documented. Two of these patients went on to allogeneic hematopoietic stem cell transplantation (one in CR, one in PR). PR was achieved in 4 patients (27%) in an average of 3 cycles (range = 2–3). Duration of response averaged 7 cycles (range = 1–15) from the time PR was documented. Two patients continue therapy, maintaining PR, at 9 and 15 cycles. One patient met criteria for stable disease with a survival of 17 months. One patient with biphenotypic leukemia survived 12 months with a significant reduction in transfusion requirements. One patient demonstrated a major platelet response, but discontinued therapy after 3 cycles due to anorexia. Four patients had disease progression after 1, 1, 2, and 3 cycles. The average survival for patients who achieved at least stable disease and did not go on to hematopoietic stem cell transplantation was 14+ months (range = 8–24). None of the responders had an ECOG performance status worse than 1 while being treated. The most common toxicity was febrile neutropenia (4 patients, 2 deaths). Azacitidine administered in the outpatient setting can induce remission in AML. This therapy appears to be well tolerated. Further study is warranted in poor risk patients to document activity and toxicity.
Organic Arsenic Lipid Derivatives Are More Potent and Less Toxic Than Inorganic Arsenic Trioxide in Preclinical Testing.
Authors: Cheng Xiaodong , Golemovic Mirna , Giles Francis , Zingaro Ralph , Gao Ming-Zhang , Freireich Emil J. , Andreeff Michael , Kantarjian Hagop M. , Verstovsek Srdan
Published: October 2019

Arsenic trioxide (ATO) is an inorganic arsenic derivative that is effective in patients with relapsed acute promyelocytic leukemia. It is being investigated as therapy for other cancers but the risk/benefit ratio for its use still has to be determined due to its significant side effects. In contrast, organic arsenic derivatives are known to be much less toxic. We have synthesized a series of eight organic arsenic lipid derivatives and have tested them in NIH 60 cell line screen. Based on its activity, we selected GMZ27 [3-S-di(n-propyl)arsino-3-thio-1,2-propanediol] for further study in our laboratory and have confirmed its potent activity against human acute myeloid leukemia cell lines HL60 and NB4, which was significantly higher than that of arsenic trioxide. The IC50 (concentration that kills 50% of the cells) in MTS assay is 0.5 and 0.9 μm for GMZ27. Assessment of the mechanisms of action of GMZ27 in cell lines has shown that the GMZ27 was more potent inducer of superoxide than ATO. GMZ27 caused dissipation of mitochondrial transmembrane potential, cleavage of caspase 9, caspase 3 activation, PARP cleavage, and compromise in cell membrane integrity. However, its treatment also resulted in caspase 8 cleavage, suggesting that it affects both intrinsic and extrinsic apoptotic pathway. Its activity was related to the level of glutathione in the leukemic cells as pretreatment of the cells with BSO, which depletes intracellular glutathione, results in cells sensitivity to GMZ27. On the other hand, pretreatment with DDT, which increases intracellular glutathione, results in their resistance to GMZ27. GMZ27 had no effect on cells maturation and differentiation, and cell cycle. GMZ27 when tested against healthy donor mononuclear cells in a colony forming assay showed significantly less toxicity than arsenic trioxide. In vivo toxicity testing in Swiss Webster mice showed LD50 (dose that kills 50% of mice) to be 100 mg/kg for GMZ27, comparing to 10 mg/kg for ATO. In conclusion, organic arsenic lipid derivatives, and in particular GMZ27, may have more potent antileukemic activity and significantly less toxicity in vivo and in vitro than ATO and therefore further development of these medications is warranted.
Monitoring Immune Responses Following Vaccination with Autologous Dendritic like Leukaemia Cells.
Authors: Klammer Matthias , Waterfall Martin , Samuel Kay , Thomas Charlotte , Johnson Peter R.E. , Turner Marc L. , Roddie Patrick H.
Published: October 2019

It has been demonstrated that in a proportion of patients with Acute Myeloid Leukaemia (AML) their leukaemic blasts can be induced to differentiate into Dendritic Like Leukaemic Cells (DLLC) in vitro under the influence of GMCSF, IL-4 and TNF alpha. DLLC hold promise as cellular vaccines aimed at eradicating Minimal Residual Disease following successful induction of remission by chemotherapy. In A Phase I/II Multi-Centre Study we assessed leukaemic blasts of 21 patients with AML for their cytokine driven potential to differentiate into DLLC in vitro. 7 out of 21 (33%) patients showed DLLC conversion of their AML blasts based on morphological appearances and immunophenotype. 5 patients have completed chemotherapy and were then eligible to receive 4 escalating doses of subcutaneous DLLC vaccine. Immune responses to the administration of the vaccine are monitored by a combination of methods. The emergence of leukaemia specific T-cells following vaccination is demonstrated using Elispot assaying of Interferon gamma release in an in vitro re-stimulation assay. In WT1 expressing patients HLA-Tetramers allow to determine the frequency of WT1 specific T-cells. Regulatory T-cells (CD4/CD25 positive) are monitored by flow-cytometry. Monitoring of Minimal Residual Disease (MRD) is undertaken by means of real-time quantitative RT-PCR for leukaemia specific fusion transcripts or WT1 gene expression.

4 patients have so far completed the DLLC vaccination course. Vaccination was well tolerated with minimal side effects. A more than two-fold increase of leukaemia specific cytotoxicity was demonstrated following DLLC vaccination in one patient, while reduction of MRD was seen in several patients during the vaccination. The increase in CD4 /CD25 positive regulatory T-cells observed in several patients post vaccination may serve to dampen the induced anti-leukaemic immunity.
A Phase II Trial of Busulfan, Melphalan, and Fludarabine followed by Allogeneic T-Cell Depleted Hematopoietic Stem Cell Transplants from HLA-Identical, or HLA-Non Identical Related or Unrelated Donors for the Treatment of Advanced Hematopoietic Malignancies.
Authors: Boulad Farid , Jakubowski Ann , Castro-Malaspina Hugo , Hsu Katharine C. , Kernan Nancy A. , Papadopoulos Esperanza B. , Perales Miguel A. , Prockop Susan E. , Small Trudy N. , van den Brink Marcel R.M. , Young James W. , Ro Michelle , Collins Nancy , O’Reilly Richard J.
Published: October 2019

Twenty five patients have been enrolled on this trial to date. There were 14 males and 11 females aged 0.6–54 years. Patients’ diagnoses and stage included: NHL in CR2 or refractory (n=2), AML (n=7), including 5 pts with secondary AML, ALL > CR3 (n=4), CML in CP2 (N=1) and high risk MDS (n=11) including 5 pts with secondary MDS. Eight pts had a matched related donor, 14 pts an unrelated donor and 3 pts a mismatched related donor. Cytoreduction consisted of busulfan (Bu) (0.8–1 mg/Kg/dose x 10 doses), melphalan (Mel) (70 mg/Kg/day x 2) and fludarabine (Flu) (25 mg/m2/day x 5). Graft rejection prophylaxis included rabbit ATG (Thymoglobulin) (2.5 mg/Kg/day x 2). Four pts tolerated only one of two doses of the ATG, 2 pts received equine ATG and one pt Alemtuzumab. Twenty one pts received G-CSF mobilized peripheral blood stem cell transplants that were T-cell depleted by CD34 selection and E-rosetting while the other four pts received Soybean agglutinin E-rosette depleted marrow grafts. Cell doses were 1.3–20.5 x 106 CD34 cells/Kg. and 0 -100 x 103 CD3 cells/Kg. Engraftment occurred in 24 pts. One pt suffered a graft failure; This pt had initial low busulfan levels, and received bone marrow derived stem cells with a low cell dose from a 5/6 HLA-matched unrelated donor. Acute graft-versus-host disease occurred in four pts: grade 1 (n=2) and grade 2 (n=2) and no pts developed any grade 3-4 severe GvHD. Two patients were diagnosed with chronic GvHD: localized (n=1) and extensive (n=1). Two patients developed sepsis early post BMT, with secondary multi organ failure and early mortality, while for the rest of the patients, regimen-related toxicity was acceptable. Relapse occurred in 9 pts. Mortality included 7 pts from relapse, two pts from sepsis and multi-organ failure, 3 pts from infections, and one pt from unknown causes. The overall survival (OS) and disease-free survival (DFS) at 2 yrs for the entire patient cohort were respectively 44% and 42 %; The DFS was 50% for patients with secondary MDS or AML. In summary, the cytoreduction with Bu Mel and Flu allowed consistent engraftment of T-cell depleted grafts and was associated with acceptable outcome for patients with secondary MDS or AML.
Reduced Intensity Conditioning Therapy Using Campath -1H Is Successful for Stem Cell Transplantation in Non-Malignant Disorders.
Authors: Rao Aarati , Hayashi Robert , Grossman William , Wilson David , Yu Lolie , DiPersio John , Barnes Yvonne , Shenoy Shalini
Published: October 2019

Stem cell transplantation (SCT), indicated for many non-malignant disorders, is limited by donor availability, graft rejection (GR), toxicities of conditioning, morbidity and mortality (TRM), and graft versus host disease (GVHD). To overcome these barriers, we tested a novel conditioning for SCT. It was designed to support engraftment by deleting host immune reactive lymphocytes and macrophages. Campath -1H (anti-CD52 mab) was given on days -21, -20, and -19 (total dose 48 mg), fludarabine (day -8 to -4) (total 150 mg/m2) and melphalan on day -3 (140 [n=15] or 70 [n=1] mg/m2). Stem cell sources were related/unrelated bone marrow (BM) (8), peripheral blood (PB) (5) and umbilical cord blood (UCB) (3). GVHD prophylaxis was cyclosporine (tapered after 3 months), methylprednisone (tapered after day +28) and methotrexate on days +1, +3, and +6. Methotrxate was not used in UCBT. End points of the study included engraftment and TRM. Sixteen patients (1.5–40 yrs) diagnosed with aplastic anemia (5), Hurler’s (2), sickle cell anemia, XLAAD, histiocytosis (3), thalassemia, adrenoleukodystrophy, Evan’s syndrome and dyserythropoietic anemia were transplanted. Median follow-up was 219 days (66–845). The regimen was well tolerated. All patients that survived >1 month engrafted. Neutrophils (ANC >500/dL) engrafted at a median of 12.5 (10–36) days and platelets (>50,000/dL) at a median of 21 (12 –63) days. Skin GVHD developed in 3 patients and resolved early. Four are tapering immune suppression; 8 are successfully off immune suppression. All survivors have either stable disease or are cured. One recipient had a normal pregnancy and delivered twins. Two patients died prior to engraftment from previously acquired Pseudomonas infection. A third patient died of CMV disease (day +112) and another recipient died of intracranial hemorrhage/refractory thrombocytopenia (day +43) after engraftment. Other complications were bacterial and viral infections occurring within the first 100 days post-transplant. All but 1 CMV+ recipient reactivated CMV as assessed by PCR. Profound lymphopenia was present in all recipients on day +30. NK cells recovered by day +100, CD8+ cells by day +180, CD4+ and B cells between days 180–270 after transplant. Immunoglobulin (Ig M and A) levels dropped post transplant and normalized by 9 months. Ig A recovered later than IgM. In summary, successful engraftment despite varied stem cell sources, was achieved without significant toxicity or GVHD. Lymphopenia resulted in a significant infection risk within the first 100 days post transplant, requiring close surveillance and early intervention. This transplant regimen is well tolerated and may preserve fertility, making it a promising alternative to conventional SCT.
Argatroban, Not Lepirudin or Bivalirudin, Treatment Results in the Generation of Nitric Oxide during Parenteral Administration.
Authors: Walenga Jeanine M. , Fareed Daniel , Schultz Chris , Neville Brian , Hoppensteadt Debra , Iqbal Omer , Fareed Jawed
Published: October 2019

While all direct thrombin inhibitors (DTIs) produce their anticoagulant effect by inhibiting thrombin, there are differences in their mode of action, not only in protease generation/inhibition, but also in modulating cellular mechanisms such as the release of endothelial mediators including nitric oxide (NO) and prostacyclin. Preliminary studies have revealed that argatroban treatment, unlike bivalirudin and lepirudin, results in vasomodulation and other pharmacological effects unrelated to thrombin inhibition [Pathophysiol Haemost Thromb2002;Suppl 3:56–65]. To validate this observation primates (Macaca mulatta; n=6/group) were treated with 100 to 500 μg/kg iv BID of these three DTIs. Blood samples were drawn at 5, 15, 30, 60, 120, 240 and 360 minutes. Total NO levels were measured by two different methods. The Griess reaction was performed by a chromogenic substrate method (R&D Systems, Minneapolis, MN) based on the two-step diazotization reaction where NO2 is acidified to produce a nitrosating agent, which is complexed with sulfanilic acid to produce a diazonium ion, which is then coupled with chromogenic reagents. A chemiluminescent assay was also used. Plasma NO reacts with oxygen to form nitrite. Using the Ionics Instrument 208I (Sievers Instrument, Boulder, CO), the nitrite is converted into NO by chemical reactions, and the resulting NO level is determined using a highly sensitive gas phase. DTIs did not have any non-specific interfering affect on the assays. Primates treated with argatroban showed a dose- and time-dependent increase in plasma NO levels. Baseline levels ranged from 4–8 μM (mean ± SD = 6.1 ± 1.8 μM), whereas 5 min after the administration of argatroban NO levels were 6–11 μM (7.2 ± 1.8 μM) for 100 μg/kg, 8–14 μM (11.8 ± 2.6 μM) for 250 μg/kg, and 11–19 μM (15.7 ± 3.1 μM) for 500 μg/kg argatroban. This increase in NO was observed for 30 min. The area under the curve (0–6 hrs) revealed a dose-dependent increase in a non-linear fashion. No difference between the two methods was noted. For lepirudin and bivalirudin, the NO levels did not differ from baseline during the study period, and no differences between these treatment groups were observed. Flow cytometric studies showed comparable inhibition (29–37%) of tissue factor mediated platelet activation 5 min after dosing for all DTIs. However, the % P-selectin expression was lower in argatroban treated primates than with lepirudin and bivalirudin treatment (17.1 ± 5.2 vs 29.3 ± 7.1; p <0.026). The 5 and 60 min argatroban samples assayed for 6-keto-PGF1 alpha showed 20–30% higher levels (not significant) compared to lepirudin and bivalirudin. These results support the hypothesis that argatroban is distinct from lepirudin and bivalirudin in that it produces an increase in NO and prostacyclin which may partly contribute to its therapeutic efficacy by stabilizing platelets.
Antithrombotic Properties of DU-176b, a Novel, Potent and Orally Active Direct Factor Xa Inhibitor in Rat Models of Arterial and Venous Thrombosis: Comparison with Fondaparinux, an Antithrombin Dependent Factor Xa Inhibitor.
Authors: Fukuda Toshio , Matsumoto Chikako , Honda Yuko , Sugiyama Nobutoshi , Morishima Yoshiyuki , Shibano Toshiro
Published: October 2019

Factor Xa (FXa) is an attractive target for the treatment of thrombosis due to its crucial role in the blood coagulation cascade. Fondaparinux, a selective FXa inhibitor, has been approved for clinical use to prevent deep vein thrombosis after orthopedic surgery; however, it requires antithrombin (AT) to exert its antithrombotic effect. It is reported that AT dependent anticoagulants such as heparin are less effective to suppress platelet-rich arterial-type thrombus due to its inaccessibility to thrombus-bound FXa/thrombin. We have developed a potent direct (i.e. AT independent) FXa inhibitor, DU-176b. The objective of this study is to compare the antithrombotic properties of a direct selective FXa inhibitor, DU-176b, with an AT dependent selective FXa inhibitor, fondaparinux. We evaluated the antithrombotic effects of DU-176b and fondaparinux in rat models of arterial and venous thrombosis. The arterial and venous thrombosis was induced by topical application of ferric chloride to the carotid artery and by insertion of a platinum wire into the inferior vena cava, respectively. DU-176b (0.05 – 1.25 mg/kg/h) and fondaparinux (1 – 10 mg/kg/h for arterial thrombosis and 0.03 – 1 mg/kg/h for venous thrombosis) were intravenously administered as continuous infusions. DU-176b prevented both arterial and venous thrombosis in the same dose range. In contrast, the effective doses of fondaparinux markedly differed between these models. A higher dose of fondaparinux more than 100 times was required to inhibit arterial thrombosis compared with venous thrombosis. These results suggest that direct inhibition of FXa is a preferable strategy to AT dependent inhibition for the prevention of thrombus formation in the arteries.
In Vitro Characteristics, Anticoagulant Effects and In Vivo Antithrombotic Efficacy of a Novel, Potent and Orally Active Direct Factor Xa Inhibitor, DU-176b.
Authors: Morishima Yoshiyuki , Furugohri Taketoshi , Isobe Koji , Honda Yuko , Matsumoto Chikako , Shiozaki Yoko , Muramatsu Sumie , Sugiyama Nobutoshi , Shibano Toshiro
Published: October 2019

Factor Xa (FXa) is a key serine protease in the coagulation cascade and is a promising target enzyme for developing a new antithrombotic agent. Our first clinical candidate for a small molecular direct FXa inhibitor DX-9065a potently inhibits FXa (Ki = 41 nM) and exerts antithrombotic effects in animal models. However, due to its poor bioavailability (10% in monkeys) the compound is used only as an injectable formulation in clinical studies. Here we report in vitro characteristics of serine proteases inhibition, anticoagulant effects and in vivo antithrombotic efficacy of DU-176b, a novel, potent and orally active direct FXa inhibitor. DU-176b competitively inhibited human FXa with a Ki value of 0.561 nM, indicating 70-fold increase in FXa inhibitory activity compared with DX-9065a. DU-176b demonstrated 10,000-fold selectivity relative to inhibition of thrombin (Ki = 6.00 μM), and had no effects on the enzymatic activities of factor VIIa, t-PA, plasmin, trypsin and chymotrypsin. In human plasma, DU-176b prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT) in a concentration-dependent manner. Its concentrations for doubling these clotting times were 0.256 and 0.508 μM, respectively. After oral administration of DU-176b to rats, significant anti-Xa activity was observed in plasma over 4 h. The oral bioavailability of DU-176b (approximately 50%) was significantly higher than that of DX-9065a (10%) in monkeys. The antithrombotic efficacy of DU-176b was examined by oral administration to rats 30 minutes prior to thrombogenic stimuli. In a venous stasis thrombosis model, DU-176b (0.5 – 12.5 mg/kg, p.o.) dose-dependently inhibited thrombus formation, prolonged PT, and revealed plasma anti-Xa activity. DU-176b also exerted significant anticoagulant effect in a rat model of tissue factor-induced disseminated intravascular coagulation at doses of 0.1 – 2.5 mg/kg, p.o. These results demonstrate that DU-176b is a potent and selective factor Xa inhibitor that possesses antithrombotic effect after oral administration. DU-176b has the potential to be clinically useful for prophylaxis and treatment of several thrombotic diseases.
Supplemental Alpha-2-Antiplasmin Prevents Plasmin-Induced Hemorrhage.
Authors: Stewart Daphne , Marder Victor J. , Kong Mansze , Quon Doris , Cook Scott A. , Jesmok Gary , Radtke Peter , Baumbach George A.
Published: October 2019

Plasmin is a direct-acting thrombolytic agent with an impressive safety margin in experimental models, as it is tolerated without bleeding at a 6-fold greater dose than needed for thrombolysis. At higher plasmin dosages, above a reproducible dose threshold, alpha-2-antiplasmin (AP) is consumed, accompanied by complete depletion of fibrinogen and factor VIII. As a result of clotting factor exhaustion, the primary bleeding time is prolonged. We assessed whether supplemental AP could prevent bleeding induced by a purposefully toxic dosage of plasmin, and whether AP could arrest such bleeding once it occurs. Using the rabbit ear puncture model, plasmin (8 mg/kg) alone was compared with plasmin plus supplemental AP (5 mg/kg), injected either prior to or after plasmin infusion. We dosed plasmin 8-fold higher than that required for thrombolytic efficacy, based upon our previous observations of its capacity to prolong primary bleeding in greater than 80% of treated animals. Parameters for comparison between treatment groups were: primary bleeding time at timepoints prior to, during, and after experimental infusion, incidence of rebleeding at previously stable hemostatic sites, and quantification of AP, fibrinogen, and factor VIII. Pre-treatment AP doubled the plasma concentration, preserved circulating fibrinogen and factor VIII, and prevented bleeding. Post-plasmin AP corrected the plasmin-associated depletion of AP, but fibrinogen and factor VIII concentrations remained very low, and prolonged bleeding was uncorrected. To test whether AP could be supplemented with coagulation factors in order to arrest bleeding following high dose plasmin, human cryoprecipitate was co-administered. While the fibrinogen and factor VIII levels increased to 200% and 50% of baseline values, respectively, prolonged bleeding persisted. Unexpectedly, administration of human cryoprecipitate led to excess bleeding in association with acute thrombocytopenia and gross hemolysis, presumably mediated by xenographic antibody present in the product. Thus, we find that supplemental AP administered prior to a toxic dose of plasmin prevents coagulation factor consumption and preserves normal hemostasis. Once coagulation factor depletion occurs, AP alone is insufficient to correct the hemorrhagic state that ensues. Clinically, plasmin may be useful as a regional thombolytic agent, and AP supplementation may permit higher dosing as well as protect against accidental overdose. Further studies are needed to determine whether combined AP and coagulation factor repletion may correct the prolonged bleeding induced by massive plasmin infusion.
Respiratory Synctial Virus (RSV) Infection in Hematopoietic Stem Cell Transplant (HCT) Recipients: Risk Factors for Acquisition and Lower Respiratory Tract Disease, and Impact on Mortality.
Authors: Boeckh Michael J. , Gooley Ted , Englund Janet , Chien Jason W. , Crawford Stephen W. , Bowden Raleigh , Lawrence Corey
Published: October 2019

RSV lower respiratory tract disease (LRTD) is a serious complication after HCT. Risk factors for virus acquisition and progression from RSV upper respiratory tract infection (URI) to RSV LRTD are poorly defined. A targeted surveillance system consisting of routine virologic evaluation for RSV (DFA, shell vial assay, culture) in HCT recipients with URI symptoms was instituted in 10/89. We retrospectively analyzed risk factors of RSV acquisition and the development of RSV-LRTD among patients with RSV-URI between 10/89 and 7/02. From 10/94 to 7/97, patients with RSV-URI received preemptive aerosolized ribavirin (2g/day) for 5–7 d; some patients received full-dose preemptive ribavirin (6g/day). All results are from multivariable models. One hundred forty-seven of 4717 (3.1%) patients were diagnosed with RSV URI and/or LRTD during the first 100 days after HCT. Risk factors for RSV acquisition included bone marrow vs. peripheral blood stem cell (PBSC) (1.7, P=0.01), winter season (P<0.0001), the years 1993 and 1997 when 2 outbreaks occurred (HR 1.7, P=0.01), male sex (HR 1.4, P=0.06). Laminar airflow rooms (HR 0.5, P=0.004) and the period after 1997 (HR 0.65, P=0.09) were protective against acquisition (after 1997, we started to restrict patient contact with staff and caretakers who had an URI with uncontrolled secretions). Risk factors for RSV-LRTD among infected patients included age > 20 year (OR 3.2, P=0.02) and lymphocytopenia < 100/mm3 (OR 4.7, P=0.0005). To assess risk factors for progression from RSV-URI to LRTD, patients who presented with RSV-URI were examined. Lymphocytopenia < 100/mm3 was the only statistically significant factor (OR 14, P<0.001) associated with progression; use of preemptive ribavirin (low- or high-dose) was not statistically significantly associated with a lower progression rate. The development of RSV LRTD as a time-dependent variable was associated with increased mortality after HCT (HR 2.2, p < 0.001), after controlling for age, underlying disease status, donor match and type, conditioning regimen, stem cell source, and CMV serostatus. Thus, RSV acquisition is less common with PBSC transplantation and in a strict isolation setting. Restricting recipients’ contact to people with uncontrolled respiratory secretions in the outpatient setting may be beneficial; further studies are needed to confirm this. Lymphocytopenia is an important risk factor for progression from URI to LRTD and RSV-LRTD is independently associated with mortality after HCT.
AMP Deaminase Activation Contributes to Accelerated Adenine Nucleotide Pool Depletion during Periods of Energy Imbalance in Sickle Cell Erythrocytes.
Authors: Sabina Richard L. , Wandersee Nancy J. , Hillery Cheryl A.
Published: October 2019

Mature erythrocytes are unable to synthesize AMP from IMP due to the developmental loss of adenylosuccinate synthetase, which also effectively prevents the synthesis of adenine nucleotides from circulating hypoxanthine, the major salvageable purine compound. Consequently, AMP deaminase (AMPD) activity must be tightly regulated in order to avoid irreversible depletion of the erythrocyte adenine nucleotide pool during periods of energy imbalance. However, increased intracellular calcium promotes activation of erythrocyte AMPD (AMPD3) through a protein-protein interaction with calmodulin (Mahnke and Sabina, Biochemistry44:5551, 2005). Therefore, we hypothesized that Sickle Cell Disease (SCD) erythrocytes would more rapidly accumulate IMP and deplete the adenine nucleotide pool during periods of energy imbalance. In order to test this hypothesis, acid-soluble pools of these metabolites were quantified by anion-exchange HPLC in SCD and control erythrocytes during autoincubation and glucose starvation, both at 37C. Data in the table show that SCD erythrocytes accumulate more IMP during a 6-hour autoincubation compared to control cells, and this is accompanied by a decrease in AMP.

Effect of Autoincubation on Erythrocyte AMP and IMP Pools GROUP HOURS AMPa IMP IMP/AMP a, metabolite concentrations reported as μmol/g Hb; †, p<.05 when compared to 0 time in a paired student’s t-test; ‡, p<.05 when compared to control in an unpaired student’s t-test Control (N=4) 0 .29±.08 .15±.06 .58±.28 6 .29±.01 .33±.11† 1.15±.35† SCD (N=5) 0 .22±.04 .08±.03 .36±.10 6 .14±.03† .74±.24†‡ 5.15±1.25†‡

Data in the figure show an accelerated depletion of ATP and increased accumulation of IMP in glucose-starved SCD erythrocytes compared to control cells (*, p<.05 when compared to control in an unpaired student’s t-test).

Figure Figure

These combined results demonstrate AMPD3 enzyme activation and accelerated adenine nucleotide pool depletion during periods of energy imbalance in SCD erythrocytes. This metabolic imbalance in SCD erythrocytes could contribute to 1) an increased rate of hemolysis, 2) cation channel dysregulation, 3) modulation of adhesive membrane components, and 4) diminished protein phosphorylation. Furthermore, strategies directed against this metabolic imbalance, e.g., diffusible AMPD inhibitors and adenine supplementation, may have a therapeutic benefit in SCD.
Hsp90 Is Critical for the Regulation of Phenotype and Functionality of Human Dendritic Cells.
Authors: Bae Jooeun , Mitsiades Constantine , Yu-Tzu Tai , Martinson Jeff , Batchu Ramesh Babu , Bertheau Robert , Li Cheng , Prabhala Rao , Masood Shammas , Hideshima Teru , Catley Lawrence , Anderson Kenneth , Munshi Nikhil
Published: October 2019

Hsp90, a molecular chaperone, plays a critical role in protein folding and transport, and thereby it modulates cellular activity. Pre-clinical data shows over-expression of Hsp90 in multiple myeloma (MM) and efficacy of Hsp90 inhibitor in myeloma has been determined in vitro. Based on these results, phase I/II trial evaluating clinical efficacy of the Hsp90 inhibitor is underway in MM. Although Hsp90 inhibitor shows significant effects on tumor cells, there is limited information concerning its effects on the immune system. The objective of this study was to evaluate the effects of Geldanamycin on activity of antigen-presenting cells. Immature and mature monocyte derived dendritic cells (DC) from normal human donors were used as the source of antigen-presenting cells in this study. Geldanamycin treatment of DC for 24 hours had no effect on cell viability (>90%), however, it led to a significant down-regulation of surface antigens associated with activation (CD86, CD80), maturation (CD83) and antigen presentation (HLA-ABC, HLA-DPQR). This decline was associated with changes in gene expression levels of these antigens, however the protein expression analyzed by % positive cells was not down-regulated with the treatment. Exposure to Hsp90 inhibitor was associated with significant decreases in IL-12 secretion (untrt vs. trt = 135 vs. 21 pg/ml), antigen uptake (MFI untrt 798 vs. MFI trt 449, Dextran-FITC), and antigen processing. These changes were associated with decline in DC function, which were demonstrated by significant decrease in Hsp90-treated DC compared to untreated DC in presentation of Tetanus Toxoid to autologous T lymphocytes (untrt vs. trt = 73 % vs. 47 %, CFSE proliferation), allogeneic T lymphocytes stimulation (untrt vs. trt = 232795 cpm vs. 116876 cpm, 3H-thymidine incorporation), and induction of IFN-g secretion from allogenic T lymphocytes (untrt vs. trt = 500 vs. 30 pg/ml). Taken together, these results show significant decline in DC function following Hsp90 inhibitor treatment. Further studies are underway using MM patient samples pre- and post-Hsp90 inhibitor treatment to understand in vivo effects on the immune system. Our pre-clinical data suggests the need to consider proper sequence of various therapeutic modalities, including immunotherapy, to optimize and improve clinical outcome.
Targeting MLL-AF4 with Short Interfering RNAs (siRNAs) Induces Apoptosis Related Genes APAF-1 and SEPT4 in t(4;11)-Positive Human Leukemic Cells.
Authors: Greil Johann , Thomas Maria , Gessner Andreas , Heidenreich Olaf
Published: October 2019

The chromosomal translocation t(4;11) marks infant acute lymphoblastic leukemia associated with a particularly dismal prognosis. We have recently shown that transfection of leukemic cells with MLL-AF4 siRNAs reduces leukemia-associated morbidity and mortality in xenotransplanted SCID mice suggesting that MLL-AF4 depletion negatively affects leukemia-initiating cells. In cell culture, transient suppression of MLL-AF4 expression impairs the proliferation of both t(4;11) cell lines SEM and RS4;11 more than tenfold. Clonogenicity of SEM cells was reduced fivefold and of RS4;11 cells more than twofold. One major reason for the antiproliferative consequences is the induction of apoptosis associated with caspase-3 activation and diminished BCL-XL expression. Caspase-3 was induced 3-fold and BCL-xL showed a 60% reduction after siRNA treatment compared to Mock-transfected controls in SEM cells. Analysis of intrinsic signaling pathways of apoptosis showed that MLL-AF4 depletion induces the expression of the proapoptotic genes APAF-1 and SEPT4 (ARTS). In both cell lines APAF-1 was induced more than twofold and SEPT4 more than fourfold. Our findings demonstrate that MLL-AF4 negatively interferes with an intrinsic apoptotic response and is, thus, important for the survival of these highly aggressive and chemoresistant leukemic cells. SiRNA-mediated MLL-AF4 inhibition may sensitize these cells for apoptosis-inducing agents. Therefore, complementing existing treatment strategies with a targeted inhibition of MLL-AF4 expression may lead to an improved and more specific treatment of this therapy-resistant leukemia.