bluuebunny/crossref_metadata_2025 huggingface dataset:
Fetal-to-Adult Hemoglobin Switching Is Associated with up-Regulation of Specific MicroRNA Species in Circulating Human Erythroid Cells.
Authors: Noh Seung-Jae , Miller Samuel , Lee Y. Terry , Marincola Francesco M. , Stroncek David , Reed Christopher , Wang Ena , Miller Jeffery L
Published: April 2020

MicroRNAs are ~22nt-long small noncoding RNAs that negatively regulate expression of target proteins through mRNA degradation or translational repression. They are functionally important in diverse biological processes including development, oncogenesis, and hematopoiesis. To study a potential role of microRNA for regulating cellular changes during erythroid ontogeny, we examined microRNA abundance patterns in circulating erythroid cells during the fetal and adult stages of human development. Expression profiling of microRNA was performed using total RNA from 4 adult peripheral blood samples compared to 4 cord blood samples after depletion of plasma, platelets, and nucleated cells. Labeled RNA was hybridized to custom spotted array containing 474 human microRNAs species (miRBase release 9.1). Total RNA from Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines was used as a hybridization reference for all samples to generate microRNA abundance profile for each sample. Among the differentially expressed probe-pairs, 40 microRNAs were significantly up-regulated in adult erythroid cells and only 4 microRNAs were down-regulated (p < 0.01 and fold change > 2). Two microRNAs, miR-144 and miR-451, previously reported as being GATA-1 regulated in erythroid cells, were detected above the dynamic range of the array technology in all eight samples. Quantitative PCR was performed to further quantitate differences in the microRNA abundance profiles of the 10 most differentially expressed microRNAs as well as miR-144 and miR-451. Per published recommendations, miR-103 was used for signal normalization. Among the studied microRNAs, 8 of 12 demonstrated a more than 4 fold increase in abundance in adult erythroid cells compared to cord blood by qPCR. In particular, let-7d and let-7e demonstrated more than 10-fold increased expression in adult cells. Of note, let-7 microRNAs regulate metamorphic processes in model organisms and inhibit ras-associated growth of cancer cells. According to miRBase predictions, over 1200 genes may be targeted by the 8 microRNAs (p < 0.001). Examination of cord and adult reticulocyte mRNA expression profiles (Goh et al. Phy. Gen. 2007) revealed 24 developmentally downregulated genes as predicted targets. Among globin genes, only gamma globin is a predicted target. These findings demonstrate dramatic up-regulation of specific erythroid microRNAs during the fetal-to-adult transition of ontogeny, and support the notion that microRNAs function as developmental regulators of erythropoiesis and globin gene switching.
Characterization of Histone Deacetyalses Involved in γ-Globin Gene Regulation.
Authors: Muralidhar Shalini A , Nimer Sadeieh , Pace Betty
Published: April 2020

Fetal hemoglobin (HbF; α2γ2) ameliorates vaso-occlusive symptoms in individuals with sickle cell disease (SCD) because of its ability to inhibit hemoglobin S polymerization. One mechanism for γ-globin reactivation likely involves chromatin modification and the release of repressor complexes in which histone deacetylases (HDACs) may be present. The objective of this study was to identify HDACs involved in γ-globin gene regulation. Experiments were performed in K562 cells to determine the ability of NaB (2mM), TSA (0.5μm) and the non-HDAC-inhibitor γ-globin activator hemin (50μM), to alter transcription levels of the HDAC genes during concomitant HbF induction. Gene expression was measured by reverse transcription (RT) of mRNA followed by quantitative PCR (qPCR) analysis using gene-specific primers. Treatment of K562 cells with TSA and NaB reduced transcription levels of both HDAC9 and HDRP (histone deacetylase related protein, a splice variant of HDAC9) from 20–80% as did the control agent hemin (p<0.05). By contrast, expression of HDAC7 and HDAC10 was enhanced in the presence of both HDAC inhibitors. The altered HDAC gene expression levels provided indirect evidence for a possible role in mechanisms of γ-globin response to drug inducers. Subsequent experiments were performed to delineate whether HDAC9 and HDRP are directly involved in γ-globin regulation. We performed siRNA knockdown of HDAC9 and HDRP in K562 cells to determine the effect on expression of endogenous γ-globin. siRNA oligonucleotides were transfected using Oligofectamine (Invitrogen) for 48 hrs and expression of targeted genes were quantified by RT-qPCR. siHDAC9 and siHDRP (Dharmacon) treatment resulted in dose-dependent γ-globin silencing and transactivation respectively at 80–320nM. Experiments were then performed with 160nM of siHDAC9 or siHDRP in the K562 cell lines which were stably transfected with a luciferase reporter (pGL4.17-Luc2-neo, Promega) under the control of Gγ-globin promoter (−1500 to +36) and the pGL4.17-Luc2-neo empty vector. We likewise observed a 30% decrease in luciferase activity with siHDAC9 and a 40% increase with siHDRP suggesting that HDAC9 and its variant are directly involved in regulation of γ-promoter activity. In summary, the effects on endogenous γ globin levels and Gγ globin promoter activity through HDAC9/HDRPspecific knockdown by siRNA experiments suggest that HDAC9 molecules play a role in regulating γ-globin gene expression. We conclude that HDAC9 and HDRP have opposite regulatory effect on γ-globin gene expression and may act by a feedback mechanism.
Evidence of Endothelial Dysfunction in Cocaine Users.
Authors: Pereira Jai me , Sáez Claudia G. , Pallavicini Julio , Olivares Paulina , Panes Olga , Moreno Natalia , Belmont Sabine , Massardo Teresa , Mezzano Diego
Published: April 2020

Background: Cocaine abuse is associated with an increased risk of cardiac and cerebrovascular events, such as myocardial infarction, sudden cardiac death, and ischemic stroke. The underlying mechanisms leading to these complications are not fully understood although intravascular thrombus formation and accelerated atherosclerosis are prominent findings. In this sense, in vitro studies have demonstrated that cocaine may induce damage and/or activation of endothelial cells. The structural and functional integrity of the endothelium is essential for the maintenance of vascular homeostasis and its damage plays a critical role in the pathogenesis of vascular disease. Endothelial dysfunction may be assessed by testing the impaired vasodilator response to a stimulus or by measuring the release of plasma markers of endothelial damage. Increased number of circulating endothelial cells (CECs) has been reported in several pathologic conditions associated with severe vascular damage and its enumeration in peripheral blood is currently considered a reliable method to assess endothelial damage. We hypothesized that chronic exposure to cocaine induces endothelial damage which could be expressed by increased CEC counts in peripheral blood.

Methods: Twenty cocaine-dependent subjects (aged 19–52 years, mean age 30 years) and 25 healthy, matched controls (aged 20–49 years, mean age 31 years) were studied; all patients fulfilled DSM-IV criteria for cocaine dependence with drug exposure within the 72 hours prior to blood sampling. CECs were isolated from peripheral blood by use of immunomagnetic beads coated with anti-CD146, stained for CD45 and Ulex Europaeus lectin 1 and counted under fluorescence microscopy. MCP-1, sICAM-1, von Willebrand factor and high-sensitivity C-reactive protein (hsCRP) were measured by enzyme-linked immunosorbent assay.

Results: Compared to controls, CECs were significantly elevated among cocaine users (632 ± 281 vs 67 ± 54 cells/mL, p<0.0001). Plasma levels of sICAM-1 (360±92 ng/mL) and MCP-1 (166±71 pg/mL) were increased in cocaine-dependent individuals compared to the controls (261±34 and 67±29, respectively; p< 0.01). The hsCRP levels were significantly increased (6.8 mg/L); however plasma von Willebrand factor concentration was not different between patients and controls (86.4±22 vs 70.5±16%, respectively; NS). Levels of CECs correlated positively with sICAM-1 (r: 0.7; p: 0.003) and hsCRP (r: 0.56; p: 0.0037).

Conclusions: Highly increased number of CECs and significant increments in soluble plasma markers of endothelial perturbation were found in cocaine dependent individuals. This is the first demonstration of endothelial dysfunction among these individuals and our findings support the notion that cocaine-induced endothelial damage may play a pathogenic role in the ischemic vascular disease observed in chronic cocaine users.
CXCR7 Is Expressed in B Acute Lymphoblastic Leukemia (ALL) Cells and Mediates Their Transendothelial Migration.
Authors: Jalili Ali , Shirvaikar Neeta , Mirza Imran , Ilnitsky Sara , Penfold Mark , Schall Thomas J , Janowska-Wieczorek Anna
Published: April 2020

The function of chemokine stromal cell-derived factor (SDF)-1 and its receptor CXCR4 in the pathobiology of hematological malignancies is known, but the role of another, newly identified receptor for SDF-1, RDC1/CXCR7, has not yet been defined. Thus we investigated whether CXCR7 plays a role in hematological malignancy. Using RT-PCR and flow cytometry analysis, we screened the expression of CXCR7 in 14 hematological cell lines and found that it is strongly expressed in all 5 B cell lines tested (Raji, NC- 37, NALM6, Ramos and REH) and weakly in two T cell lines( Jurkat and CEM) and 2 of 7 myeloid cell lines tested. Moreover, CXCR7 was also strongly expressed in all 9 patients diagnosed with B precursor ALL, very weakly in normal lymphocytes, and not at all in T precursor ALL samples, suggesting that CXCR7 is selectively expressed in B-cell ALL. Next we examined whether CXCR7 plays a role in the chemotaxis of these cells and found that its antagonist CCX733 (ChemoCentryx, Inc, Mountain View, CA) did not inhibit SDF-1-induced chemotaxis, indicating that CXCR7 does not play a significant role in the chemotactic activity of B-cell ALL. However, we found that CCX733 inhibited the transendothelial migration (TEM) of B cell lines and primary B precursor ALL cells in a dose-dependent manner, but the inactivated CXCR7 antagonist CCX226 did not. Moreover, transfection of the NALM6 with CXCR7 resulted in downregulation of CXCR4 and the TEM of such transfected cells was more inhibited by CCX733 than of wild-type cells. When we compared the roles of CXCR4 and CXCR7 in the TEM of these cells, we found that the CXCR7 antagonist totally inhibited the TEM of B cells at the low dose of 1mM, but the CXCR4 antagonist AMD3100 did not. On the other hand, the SDF- 1-induced TEM of T cell lines and primary T lymphoblasts was not significantly inhibited by CCX733, but was by AMD3100. CCX733 significantly prevented binding of NC-37 cell line to HUVEC (as shown by adhesion assay) which indicates that CXCR7 is likely required for the interaction of leukemic cells with endothelial cells and their extravasation. Moreover, because CXCR7 is known to be a receptor for the interferon-inducible T cell chemoattractant (I-TAC), we investigated whether the I-TAC/CXCR7 axis crosstalks with the SDF-1/CXCR7 axis in the trafficking of lymphoblastic cells. We found that

I-TAC itself did not induce the TEM of NC-37 cells; pretreatment of NC-37 cells with I-TAC, but not with other chemokines (MCP-1 and MIG), inhibited TEM towards SDF-1; in contrast, pretreatment of MOLT-4 (T cells) with I-TAC did not block the TEM of MOLT-4 cells.

In conclusion, we demonstrated that CXCR7 is expressed on B lymphoblasts, and the SDF-1/CXCR7 axis plays a crucial role in the trafficking of these cells, indicating that CXCR7 could be a therapeutic target in patients diagnosed with B precursor ALL.
Outcome after Frontline Therapy with the Modified Hyper-CVAD Regimen with or without Rituximab for De Novo Acute Lymphoblastic Leukemia (ALL) or Lymphoblastic Lymphoma (LL).
Authors: Thomas Deborah A. , Kantarjian Hagop , Faderl Stefan , Wierda William G. , Ferrajoli Alessandra , Burger Jan A. , Cortes Jorge , Koller Charles A. , Borthakur Gautam , Estrov Zeev , Konopleva Marina , Ravandi Farhad , Manero Guillermo Garcia- , O’Brien Susan
Published: April 2020

The hyper-CVAD regimen is an effective frontline program for de novo adult ALL and LL [Kantarjian, JCO18:547, 2000; Kantarjian, Cancer101:2788, 2004, Thomas, Blood104:1624, 2004]. Intensive chemotherapy with hyper-CVAD (fractionated cyclophosphamide, vincristine [VCR], doxorubicin, dexamethasone) alternates with high dose methotrexate and cytarabine every 21 days for 8 courses, followed by maintenance with POMP (6-mercaptopurine, methotrexate, VCR, prednisone). Historical CR rate was 92% with 3-year disease-free survival (DFS) rate of 38%. The regimen was modified in 1999 to address the following:

higher induction mortality in patients (pts) aged 60 years or older (17% vs 3%); possible benefit of early anthracycline intensification; worse survival with CD20 expression [Thomas D, Blood, e-pub]; and late relapses after completion of POMP therapy.

Hyper-CVAD Modified Hyper-CVAD Laminar air flow rooms No For age ≥60 yrs Dose-intensive anthracycline No C2 lipo DNR & ara-C Rituximab No For CD20 ≥20% Intrathecal CNS prophylaxis 4, 8 (16 BL) 6, 8 (16 BL) POMP maintenance 24 mos 30 months Intensifications Months 7, 11 Months 6, 7 & 18, 19

Newly diagnosed or primary refractory (1 course only) pts with ALL (n=184) or LL (n=27) were treated on two sequential studies. Burkitt-type leukemia/lymphoma (BLL) and Philadelphia positive ALL were treated on alternative protocols. From May 2000 to December 2001, 69 pts were treated with the modified regimen as detailed above (9 courses of intensive chemotherapy). Course 2 was then eliminated from the regimen (8 courses of intensive chemotherapy), with an additional 145 pts treated to date. Median age was 40 yrs (range 15–83). CD20 expression was noted in 40%. Overall CR rate of the group (n=211) was 90%; 4 pts achieved PR, five failed to respond, and 3 died during the induction phase. After a median follow-up of 42 months (range, 2–80), outcome appeared favorable for the younger group (30 yrs or less) with 3-year remission duration (CRD) and survival (OS) rates of 70% and 80%. In the CD20 positive precursor B-cell ALL subset (n=88), rituximab improved outcome compared to historical experience, with 3-yr CRD rates (68% vs 28%, p<.001) and OS rates (65% vs 35%, p=.01) approaching those of the CD20 negative counterparts. In contrast to the Burkitt experience, rituximab was not beneficial for the elderly subgroup (OS rates 48% vs 35%, p NS). Anthracycline intensification appeared to worsen outcome. Rituximab appears to benefit the younger pts with CD20 positive precursor B-cell ALL. Incorporation of rituximab and other monoclonal antibodies (e.g., ofatumumab, epratuzumab) into frontline chemotherapy regimens should be investigated systematically.
CD4+ Regulatory T Cells Specific for the WT1 Antigen Are Present in Acute Myeloid Leukemia Patients: Implication for Immunotherapy.
Authors: Dermime Said , Lehe Cynthia , Ghebeh Hazem , Al-Sulaiman Abdullah , Qudaihi Ghofran Al , Al-Hussein Khaled , Almohareb Fahad , Chaudhri Naeem A. , Alsharif Fahad Z. , Al-Zahrani Hazza , Mohamed Said Y , Tbakhi Abdelghani , Aljurf Mahmoud
Published: April 2020

Compelling evidences indicate a key role for regulatory T cells (Tregs) on the host response to cancer and recent studies indicated that the generation of effective WT1-specific cytotoxic T cells can be largely affected by the presence of Tregs. This is the first study to describe human Tregs generated specifically against the WT1 antigen which is overexpressed in several human leukemias and provide the mechanism by which these anti-WT1 Tregs inhibit the immune response in leukemia patients. We have generated T cell lines and clones that specifically recognized a WT1-84 peptide in an HLA DRB1*0402/TCR-Vb8-restricted manner. Importantly, they recognized HLADRB1* 04-matched fresh leukemic cells expressing the WT1 antigen. These clones exerted a Th2 cytokine profile, had a CD4+CD25+Foxp3+GITR+CD127− Tregs phenotype, and significantly inhibited the proliferative activity of allogeneic T cells independently of cell-contact. Priming of allo-reactive T cells in the presence of Tregs strongly inhibited the expansion of NK; NK-T and CD8+ T cells, had an inhibitory effect on NK/NK-T cytotoxic activity but not on CD8+ T cells. Furthermore, priming of T cells with the WT1- 126 HLA-A0201-restricted peptide in the presence of Tregs strongly inhibited the induction of anti-WT1-126 CD8+ CTL responses as evidenced by both very low cytotoxic activity and IFN-g production. Moreover, these Tregs clones specifically produced Granzyme-B and selectively induced apoptosis in WT1-84 pulsed-autologous APCs but not in apoptoticresistant DR4-matched leukemic cells. Importantly, we have also detected anti-WT1-84 IL-5+/Granzyme-B+/Foxp3+ CD4+ Tregs in 5 out of 8 HLA-DR4+ AML patients. These findings suggest a critical role for anti-WT1 Tregs in the inhibition of T cell responses against leukemia. This study may have important implications for the clinical manipulation of Tregs such as immuno-targeting of TCR-Vb-8 with mAbs to eliminate anti-WT1 Tregs in leukemia patients in order to enhance GVL before vaccination with the WT1 antigen. On the other hand, leukemia patients with GVHD should be clinically-tried for vaccination with the current WT1-84 peptide or adoptively-treated with ex-vivo anti-WT1 Treg cells to specifically enhance their frequency, which is known to be very low in these patients.
Relapsed and Refractory Acute Myeloid Leukemia (AML) Treated with AS1411 and Cytarabine: A Randomized Phase II Trial.
Authors: Stuart Robert Kenneth , Acton Gary ,   AS1411-201 Investigator Group
Published: April 2020

Background: AS1411 is an aptamer that binds to nucleolin, which is upregulated and overexpressed in the cytoplasm and on the cell surface of cancer cells. Combination of AS1411 with cytarabine produces synergistic effects on AML cell lines and in vivo models. A phase I trial of AS1411 monotherapy in 30 patients with various advanced cancers revealed no dose-limiting toxicities and showed evidence of activity, including 2 responses among 12 patients with metastatic renal cancer. This phase II study evaluates the addition of AS1411 to high-dose cytarabine in relapsed and refractory AML.

Methods: This open-label randomized phase II trial compares AS1411 plus cytarabine with cytarabine alone in patients with primary refractory or relapsed de novo or secondary AML who have received up to 3 previous induction cycles. Patients in cohort I were randomized in a 1:2 ratio to receive cytarabine (3,000 mg/m2/day) for 4 days or AS1411 (10 mg/kg/day) on days 1–7 plus cytarabine on days 4–7. Following safety assessment, a second cohort was to be randomized 1:2 to receive cytarabine 3,000 mg/m2/day) for 4 days or AS1411 at a higher dose (40 mg/kg/day) on days 1–7 plus cytarabine on days 4–7. Key study objectives are comparison of response rates and safety and tolerability between treatment groups. Patients not achieving a CR after one cycle of single agent cytarabine are eligible for a subsequent cycle of AS1411 plus cytarabine.

Results: Thirty-three patients had been randomized by July 2008, all to the first cohort. Safety findings were available for 27 patients (9 in the cytarabine arm and 18 in the combination arm). Numbers of grade 3 and 4 adverse events per patient were 19 among 9 patients on cytarabine alone and 35 among 18 patients on AS1411 plus cytarabine. The main grade 3/4 toxicities reported were blood and lymphatic system disorders, particularly febrile neutropenia, neutropenia and thrombocytopenia; and infections and infestations. Induction deaths were 1/9 and 0/18, respectively. Response data were available for 20 patients. In the cytarabine-alone group, 0/7 patients achieved a CR, CRp or cytogenetic response. In the cytarabine plus AS1411 group, 3/13 (23%) patients showed responses to treatment, with one CR, one CRp and one cytogenetic response. Three patients from the cytarabinealone group who failed to achieve remission with cytarabine alone crossed over to receive AS1411 plus cytarabine, one of whom experienced a 90% reduction in leukemic blast count.

Conclusion: The combination of AS1411 at 10 mg/kg/day for 7 days with high-dose cytarabine 3,000 mg/m2/day over 4 days appeared to be well tolerated and showed signs of activity in patients with relapsed/refractory AML. Further patients are now being randomized to cytarabine or cytarabine plus the higher dose of 40 mg/kg/day AS1411.
Lenalidomide Enhances Leukaemia Specific T Cell Responses to Dendritic Cell Based Immunotherapy for Myeloid Leukaemias.
Authors: Duncan Caroline J , Johnson Peter R. E. , Roddie Huw
Published: April 2020

Dendritic cell (DC) vaccines in leukaemia show promise as a novel treatment modality aimed at reducing relapse rates by augmenting immune responses when patients are in a state of minimal residual disease. DC vaccination studies to date have not been as successful as hoped and this may be because of insufficient cytotoxic T cell (CTL) responses. Immunomodulatory agents such as 4-IBB ligand, and cytokines such as IL2, IL6 and GM-CSF have been reported to improve vaccine therapy however these agents have to be tolerated clinically and this limits the use of some preparations. Our study aims to generate a more efficient DC vaccine process by using Lenalidomide, a thalidomide analogue and immunomodulatory agent which is a powerful potentiator of CTLs and natural killer cells. It has the advantage of clinical tolerability and is already in use for treating haematological malignancies.

In this in vitro study DCs were generated from monocytes of eight patients in remission following chemotherapy for acute myeloid leukaemia (AML) or chronic myeloid leukaemia (CML) with standard cytokine protocols. The immature DCs were loaded with autologous leukaemia cells from the patients’ presentation samples. The presentation leukaemia cells were treated with UVB irradiation to induce apoptosis. DCs were matured with TNF alpha for two days then co-cultured with autologous T cells for one week with or without the addition of Lenalidomide at ten micromolar. A control group of unstimulated T cells were kept in culture for the same time period. The T cells from the three groups were harvested and their cytoxicity assessed in an Interferon Gamma (IFNγ) ELISPOT assay where the stimulators used were the unmodified blasts. Results in all eight patients show the leukaemia specific CTL responses (as measured by IFNγ release) were markedly improved in the DC/irradiated blasts groups compared with the unstimulated T cells. In six out of eight patients there was a further, significant improvement in the groups where Lenalidomide was added (see Figure 1).

Figure Figure

These results show promise for the use Lenalidomide as an agent to optimise CTL responses to DC based Immunotherapy for myeloid leukaemia and should be investigated further in a clinical trial.
Rituximab for Graft-Versus-Host-Disease-Prophylaxis after Allogeneic Stem Cell Transplantation Given as Treatment of High Risk Relapse of Aggressive Lymphoma: Results of a Randomized Phase II Study.
Authors: Glass Bertram , Hasenkamp Justin , Görlitz Anke , Dreger Peter , Schubert Jörg , Wulf Gerald , Nickelsen Maike , Truemper Lorenz H. , Schmitz Norbert
Published: April 2020

Background: High dose therapy (HDT) followed by autologous stem cell support has poor outcome in patients with primary progressive lymphoma or relapse after primary HDT. Allogeneic SCT may help these patients by exerting an GVL effect. Its use however is hampered by high incidence of severe GVHD and non-relapse mortality in this population. Rituximab has been claimed to reduce the incidene of GVHD without negative impact on the relapse rate.

Patients and Methods : We initiated a randomized phase II study using intermediate conditioning (Fludarabine 125 mg/m2, Busulfan 12 mg/kg and cyclophosphamide 120 mg/kg) followed by GVHD prophylaxis with short term mycophenolat mofetil plus tacrolimus. Anti-thymozyte globulin (ATG) was given due to center decision. Patients were randomized to receive two times four doses of rituximab (375 mg/m2) post transplant starting day +21 and day +175 or no additional GVHD prophylaxis. From June 2004 to July 2007 sixty five patients with aggressive NHL were enrolled and 59 were eligible for analysis. Thirty one pts had diffuse large B cell NHL, 3 patients follicular lymphoma grade 3, 7 pts blastic mantle cell lymphoma, 2 pts aggressive marginal zone lymphoma, 2 patients lymphoblastic B cell lymphoma, and 12 T cell lymphoma. 42 (71%) pts received at least one cycle of HDT and autologous SCT prior to alloSCT; 78% had early relapse ( < 12 months) or primary progressive disease, 59% chemo-refractory disease and 48% had progressive disease with high or high intermediate age adjusted IPI immediately prior to conditioning.

Results: Allo-PBPC were obtained from HLA-identical siblings in 17 pts, from matched unrelated donors in 32 pts and from one locus mismatched unrelated donors in 10 pts. 33 patients did receive ATG. Median observation time of surviving patients is 1,8 years. 30 pts died, in 19 patients death was attributed to treatment related causes. After one year, estimated overall survival is 49%, progression free survival is 39%, relapse rate is 28 % and incidence of GVHD > grade 1 is 73%. The last documented lymphoma progress occurred at day 288 after alloSCT. There are no significant differences in OS, PFS and GvHD in relationship to the application of Rituximab.

Conclusion: Intermediate intensity conditioning followed by allogeneic SCT is a valuable treatment option in patients with high-risk relapse of aggressive NHL. The incidence of GVHD and non-relapse mortality is high. On the background of this very high basic incidence of GVHD, we did not find a significant impact of post transplant rituximab on GVHD, relapse rates or survival, respectively. Therefore, ATG will be added as an obligatory part of the conditioning regimen in the next study phase in additional 30 patients
The Unmet Need for Extended Thromboprophylaxis in Acutely Ill Medical Patients: The Findings of IMPROVE, ENDORSE and EXCLAIM.
Authors: Anderson Frederick A , Hull Russell D , Schellong Sebastian M , Deslandes Bruno , Tapson Victor F , Samama Meyer Michel , Turpie Alexander GG , Yusen Roger D , Decousus Herve , Cohen Alexander T , Monreal Manuel
Published: April 2020

Introduction: Findings from the recent EXCLAIM study demonstrate that selected acutely ill medical patients with reduced mobility benefit from extended-duration enoxaparin venous thromboembolism (VTE) prophylaxis (Blood2007;110:1862). However, it is unknown whether the EXCLAIM population is representative of acutely ill medical patients admitted to hospitals worldwide. In this subanalysis, we assessed whether the EXCLAIM population was representative of the medical patients enrolled in the multinational IMPROVE (Chest2007;132:936–45) and ENDORSE (Lancet2008;371:387–94) registries known to be at risk for VTE.

Methods: Using patient data from IMPROVE and ENDORSE, we applied the EXCLAIM eligibility criteria to a representative, multinational population of acutely ill medical patients at risk for VTE. The IMPROVE and ENDORSE studies assessed VTE-risk and prophylaxis use of enrolled patients according to the 2004 American College of Chest Physicians (ACCP) guidelines (Chest2004;126:338S–400S). ACCP criteria for VTE risk were met in 46% (6,907/15,156) of IMPROVE and 43% (15,487/36,351) of ENDORSE study patients (Chest2007;132:936–45; Lancet2008;371:387–94). The EXCLAIM eligibility criteria required patients to be confined to total bed rest (level 1 immobility), or bed rest with bathroom privileges (level 2 immobility) and have at least 1 of 3 predefined risk factors for VTE (i.e., age > 75 years; history of VTE; active or prior cancer).

Results: EXCLAIM eligibility criteria were met in 30% (2,072/6,907) IMPROVE and 36% (5,535/15,487) ENDORSE medical patients considered at risk for VTE by ACCP. During hospitalization, ACCP-recommended prophylaxis was provided to 69% (1,426/2,072) and 46% (2,548/5,535) of EXCLAIM-eligible patients enrolled in IMPROVE and ENDORSE, respectively. Following discharge from hospital (median length of stay 5 days for US, 8 days for non-US), only 9% (153/1,719) of EXCLAIM-eligible patients in the IMPROVE study received any type of ACCP-recommended VTE prophylaxis.

Conclusions: Evidence from the EXCLAIM study demonstrated that the benefit-to-risk ratio in selected hospitalized medical patients favors the use of prolonged VTE prophylaxis. This population corresponds to one in three of the representative hospitalized patients with acute medical illness enrolled in IMPROVE and ENDORSE. Data on prophylaxis use from the IMPROVE registry suggest that only 9% of such patients are currently receiving optimal VTE prophylaxis following hospital discharge.
Elevated Procoagulant Activity Due to Phosphatidylserine Exposure on Red Blood Cells during Storage.
Authors: Zhou Jin , Lu Chengfang , Yu Hongjuan , Hou Jinxiao , Liu Shuchuan , Ma Ming , Gilbert Gary , Shi Jialan
Published: April 2020

Serious complications and mortality after cardiac surgery are increased when transfused red cells are stored for more than 2 weeks (Koch, N Engl J Med. 2008). However, the mechanism remains unclear and the relationship of procoagulant activity to PS exposure on banked RBCs has not been clarified, although prior studies suggest that banked cells went to apoptosis. Procoagulant activity of RBCs was measured in a modified prothrombin time in which RBCs replaced thromboplastin. The presence of PS in neutrophils and mononuclear cells from healthy donors and banked RBCs was investigated by flow-cytomety and confocal microscopy. The assembly of extrinsic tenase, intrinsic tenase, and prothrombinase complexes on these cells was studied using enzymatic assays employing plasma or purified proteins. Lactadherin, a glycoprotein, was utilized as a probe to track PS exposure and as an agent to block exposed PS. Coagulant activity increased approx 5-fold after cells were banked for 14 days and progressively increases. The percent of phosphatidylserine (PS)-positive cells increased from 7.4% on day 14 to 30% on day 42. RBCs with exposed PS were nearly absent in samples at 7 days but readily evident at 14 days on confocal microscopy. Banked RBCs exhibited patched, a rim, and diffuse-pattern that stained with lactadherin but neutrophils and MNC did not, indicating more PS exposure on banked RBCs from 7 days than the other two cell types. These data reveal a positive correlation between PCA and PS exposure on stored RBCs. Inhibition of PCA by lactadherin further confirms that PS supports the PCA in stored RBCs. Thus, we propose the possible role of PCA due to increased PS exposure on aged RBCs as a potential pathogenetic factor leading to organ injury, aggravated infections and finally higher mortality.
Alemtuzumab (Campath 1-H) in Patients with HTLV-1 Associated Adult T-Cell Leukemia/Lymphoma.
Authors: Sharma Kamal , Janik John E. , O’Mahony Deirdre , Stewart Donn , Pittaluga Stefania , Stetler-Stevenson Maryalice , Jaffe Elaine S. , Raffeld Mark , Fleisher Thomas A. , Lee Cathryn C. , Urquhart Nicole , Steinberg Seth , Waldmann Thomas A. , Morris John C.
Published: April 2020

This study investigated the antitumor activity of alemtuzumab (Campath 1-H) with regard to response rate, time to progression, overall survival and adverse events in patients with human T-cell lymphotrophic virus-1 (HTLV-1)-associated adult T-cell leukemia/ lymphoma (ATL). In a single institution non-randomized open-label phase II trial, 24 patients with chronic (n=2), acute (n=13) and lymphomatous (n=9) ATL were treated with intravenous alemtuzumab 30 mg three times weekly for a maximum of 12 weeks. Patients received antimicrobial prophylaxis from the initiation of treatment continuing for a minimum of 2 months after treatment, or until the peripheral blood CD4+ T-cells were ≥ 200 cells/μL. Tumor responses were assessed at the end of 4, 8, and 12 weeks. All patients were evaluable for toxicity and one patient was not evaluable for response due to early withdrawal. The overall objective response rate was 10 out of 23 patients (95% CI: 23 to 66%). Based on the 10 patients who responded, median time to response was 1.1 months (range, 1.0 to 3.0 months). The median response duration was 3.9 months. Median progression free survival of patients with chronic subtype was 2.0 months, 1.0 month for lymphomatous subtype and 2.4 months for acute subtype. Median time to treatment failure was 1.7 months. At data cut-off, (April 24, 2008), 6 (25%) patients were alive. Overall median survival was 5.9 months. Median overall survival by subtypes was 3.9 months for chronic, 4.8 months for lymphomatous, and 7.6 months for acute. Median survival for all responders from on study date was 8.4 months. Clinical benefit was observed both in responders and in patients with stable disease. The most common adverse events were related to infusion and were generally grade 1 or 2, occurring mainly in the first week of alemtuzumab administration. All patients developed CMV antigenemia; however, only 3 (12.5%) were symptomatic. Grade 3 or 4 infections were reported in 6 (25%) patients. Median time to recovery of ALC to >200 cells/μL was 1.9 months.
Comparative Outcomes Following CP-R, CVP-R and CHOP-R in Patients with Waldenstrom’s Macroglobulinemia.
Authors: Ioakimidis Leukothea , Patterson Christopher , Soumerai Jacob , Manning Robert , Sheehy Patricia , Treon Steven
Published: April 2020

Since the adoption of rituximab as a mainstay of therapy in non-Hodgkin’s lymphoma, there has been growing debate on the importance of adriamycin and vincristine as treatment components used in the therapy of indolent non-Hodgkin’s lymphoma, including Waldenstrom’s macroglobulinemia (WM). We therefore examined the outcome of symptomatic WM patients who required therapy based on consensus guidelines and received treatment at our Institution with CP-R (n=20), CVP-R (n=17), or CHOP-R (n=23). Baseline characteristics for all 3 cohorts were as follows:

Median Age Median Prior Therapies Bone Marrow Involvement sIgM (mg/dL) Hct PLT B2M CP-R 65 (range 42–74) 0 (range 0–2) 45% (range 5–95%) 2620 (range 551–6750) 33.4 270 2.3 CVP-R 60 (range 32–81) 1 (range 0–2) 50% (range 20–90%) 2220 (range 185–8430) 30.0 169 3.3 CHOP-R 54 (range 42–72) 0 (range 0–2) 50% (range 5–90%) 5150 (range 241–12400) 31.0 239 3.6

Responses to therapy, including median decrease in serum IgM and best response for IgM, Hct, and PLT counts were as follows:

ORR(CR+PR+MR) ≥PR CR/nCR % decrease sIgM Post-sIgM Post-Hct Post-PLT p=N.S. for all treatment cohorts. CP-R 90% 80% 0% −54% 1150 38.0 300 CVP-R 88% 71% 12% −67% 790 36.1 219 CHOP-R 83% 70% 17% −63% 794 38.3 230

Adverse events attributed to therapy, including rituximab related IgM flare were as follows:

Neutropenic fever Hospitalizations Treatment related neuropathy IgM flare IgM flare requiring plasmapheresis P=N.S. except as follows: (a) p=0.02; (b) p=0.00006; (c) p=.0.004 versus CPR. CP-R 0% 0% 0% 25% 10% CVP-R 18% 12% 59%b 29% 11% CHOP-R 26%a 17% 35%c 23% 17%

The results of this study demonstrate comparable response characteristics among WM patients treated with CP-R, CVP-R, or CHOP-R though a trend for attainment of more CR/nCR was observed among those patients receiving CVP-R and CHOP-R. Importantly, significantly more toxicity was observed, particularly neutropenic fever and treatment related neuropathy among patients treated with CVP-R and CHOP-R versus CP-R. The results of this study suggest that in WM, the use of CPR may provide analogous treatment responses to more intense cyclophosphamide based regimens, while minimizing treatment related complications.
Lysine-Binding Site-Dependent Interaction with Plasmin and Factor Va Responsible for Plasmin-Catalyzed Factor Va Inactivation.
Authors: Nishiya Katsumi , Nogami Keiji , Okada Kiyotaka , Matsuo Osamu , Ogiwara Kenichi , Shima Midori
Published: April 2020

Plasmin (Plm), an active form of plasminogen (Plg), functions as a key enzyme in the fibrinolytic system. Furthermore, this enzyme directly inactivates various coagulation factors such as factor V (FV) and factor VIII (FVIII) by limited proteolysis, suggesting another role of Plm in the regulation of the coagulation system. We recently reported that Plm/Plg interacts with FVIII and its active form (FVIIIa), both dependently and independently of lysine-binding site (LBS) (Blood2007; 110, 522a). In this study, we attempted to localize a factor Va (FVa)-interactive region on Plm (and Plg) using Plm/Plg kringle fragments. Surface plasmon resonance-based assays showed that FVa directly bound to active-site modified Plm (anhydro-Plm) with an ~2-fold higher affinity, compared to Plg (Kd; 97 and 198 nM, respectively). In particular, FVa bound to the immobilized-Plg fragment consisting of kringle 1-2-3 domains (K1-3) (Kd: 706 nM), whilst FVa failed to bind both the kringle 4 domain (K4) and Plg fragment consisting of kringle 5 and catalytic domains (K5-CD). A similar experiment using immobilized FVa also revealed that the K1-3 solely bound to FVa. These results were quite different from those obtained by FVIII and Plm/Plg binding experiment that the K5-CD bound to FVIII(a) more preferably. Competitive binding assay using 6-aminohexanoic acid (6-AHA), a competitor of LBS of Plm/Plg, showed that 6-AHA markedly inhibited (by >90%) the K1-3 binding to FVa (IC50; ~25 μM), suggesting that interaction of FVa with Plm is mostly dependent upon LBS. According to the one stage-clotting assay, 6-AHA inhibited (>90%) Plm-catalyzed inactivation of FVa in a dose-dependent manner (IC50; ~10 μM). Furthermore, Plm-catalyzed inactivation of FVa was blocked by an anti-K1-3 monoclonal antibody (mAb), not by either anti-K4 or anti-K5-CD mAb, although Plm-catalyzed inactivation of FVIII was blocked by anti-K5-CD mAb. In order to confirm that the inhibitory effect of 6-AHA on the Plm-catalyzed inactivation, we performed SDS-PAGE experiment. Plm cleaves FVa at Lys309 and Arg348 in the heavy chain, and at Arg1752 in the light chain. SDS-PAGE analysis revealed that 6-AHA blocked Plm-catalyzed cleavages of the light chain more prominently than that of the heavy chain (IC50; ~10 and ~>100 μM, respectively). These findings suggest that the K1-3 of Plm (and Plg) interacts with the FVa through the LBS-dependent mechanisms, and these interactions likely contribute to the FVa-catalyzed inactivation by proteolytic cleavages at Arg1752 in the light chain. Present study indicated that plasmin-catalyzed protelytic inactivation of coagulation factor is complicatedly regulated by the LBS dependency in the protein and protein interaction.
Non-Classical Anti-Factor VIII C2 Domain Antibodies Are Pathogenic in a Murine in Vivo Bleeding Model.
Authors: Meeks Shannon , Healey John F. , Parker Ernest T. , Barrow Rachel T. , Lollar John (Pete)
Published: April 2020

Approximately 30% of patients with severe hemophilia A will develop inhibitory antibodies to factor VIII (fVIII inhibitors). In addition, autoimmune antibodies to fVIII can develop in non-hemophiliacs, producing acquired hemophilia A, which frequently produces life- or limb-threatening bleeding. Patients with congenital hemophilia who develop inhibitors usually have a polyclonal antibody response directed against the A2 and C2 domains of fVIII. Patients with acquired hemophilia typically have a more limited B-cell epitope response with antibodies directed against the A2 or C2 domain not both. Classical anti-C2 antibodies inhibit the binding of fVIII to phospholipid membranes and to von Willebrand factor. We recently have identified anti-C2 antibodies that inhibit the activation of fVIII, but do not inhibit the binding of fVIII to phospholipid membranes or to von Willebrand factor. These non-classical inhibitors are found in the plasmas of most inhibitor patients (Meeks, S.L. et al. Blood112, 1151-1153, 2008). The pathogenicity of classical and non-classical murine anti-human fVIII monoclonal antibodies (MAbs) was tested in a murine in vivo bleeding model. MAbs were injected into the tail veins of –hemophilia A mice to a peak plasma concentration of 60 nM followed by an injection human B domain-deleted fVIII to a concentration of 2 nM. At 2 hours the mice were anesthetized and a 4 mm tail snip was made. The amount of blood lost into a collection tube of normal saline over 40 minutes was measured. 4A4 is a type I anti-A2 inhibitor with an inhibitory titer of 40,000 Bethesda units (BU)/mg IgG. I54 and 1B5 are classical type I anti-C2 inhibitors with inhibitory titers of 1300 and 930 BU/mg IgG, respectively. 2-77 is a non-classical type II anti-C2 inhibitor that produces a residual fVIII level of 40% at saturating concentrations and whose titer is 21,000 BU/mg IgG. 2-117 is a non-classical anti-C2 MAb with inhibitory activity less than 0.4 BU/mg IgG. All of these MAbs except 2-117 significantly increased blood loss over control mice injected with fVIII alone (p= 0.01-0.02, Mann-Whitney Test) (Fig .1). The amount of blood loss was similar at these saturating concentrations of antibody despite inhibitory titers ranging from 930-40,000 BU/mg IgG. Increasing the dose of fVIII to 4 nM could overcome the bleeding diathesis produced by the non-classical MAb 2-77, but not the type I antibodies, 4A4 and I54. Similar results were seen in the in vitro Bethesda assay where 4A4 completely inhibited both 1 U/ml and 3 U/ml fVIII at saturating concentrations, while 2-77 had 40% residual activity with either 1 or 3 U/ml fVIII (0.4 U and 1.2 U respectively) (Fig. 2). These results suggest that high-dose fVIII rather than bypassing agents may be warranted in patients with an inhibitor response dominated by non-classical anti-C2 antibodies.

FIGURE 1. FIGURE 1. FIGURE 2. FIGURE 2.
A Unique Recombinant Plasmin Molecule Lacking Kringles 2-5 Possesses Equivalent Hemostatic Safety as Full-Length Human Plasmin.
Authors: Marder Victor J. , Novokhatny Valery , Manyak Steve , Gruber Theresa , Hunt Jennifer , Bromirski John , Goyal Abha , Scuderi Philip , Petteway Steve
Published: April 2020

Background: All thrombolytic agents in current clinical use are plasminogen activators (PA). While effective, PA have a downside because of unavoidable and potentially lifethreatening bleeding complications in vulnerable patients. We previously demonstrated a striking margin of hemostatic safety for the “direct-acting” thrombolytic enzyme, plasmin. Now, a novel recombinant derivative of plasmin has been synthesized which lacks kringles 2-5, consisting only of kringle 1 linked to the serine protease domain.

Methods: We evaluated escalating dosages of recombinant D (K2-K5) plasmin in a randomized and blinded manner in comparison with molar equivalent dosages of full-length plasmin in an animal model of fibrinolytic hemorrhage. Hemostatic safety was measured by primary bleeding time (PBT) and plasma concentrations of a-2 antiplasmin, factor VIII and fibrinogen.

Results: A dose-related nadir in a-2 antiplasmin, factor VIII and fibrinogen followed administration of 4, 6, 8 and 10 mg/kg of D (K2-K5) plasmin, with residual fibrinogen of 65%, 40%, 30% and 0% of initial pre-treatment concentrations. Hemostasis was undisturbed with 4 mg/kg of D (K2-K5) plasmin (mean PBT 2.2 +/- 0.7 min), minimally so at the 6 or 8 mg/kg dosages (5 +/- 2.9 and 4.4 +/- 2.2 min). Hemostasis was markedly abnormal at the purposefully toxic 10 mg/kg dose (12.8 +/- 18.8 min), in association with complete depletion of plasma fibrinogen. Comparison of agents at 10 mg/kg showed no difference, but at 4 mg/kg (2-4-fold more than needed for efficacy), D (K2-K5) plasmin showed slightly less prolongation of PBT (2.2 +/- 0.7 vs 3.3 +/- 2.1 min). Both agents were equally potent for in vitro whole blood clot lysis, indicating that the safety advantage of D (K2-K5) plasmin was not due to decreased fibrinolytic activity.

Conclusions: Recombinant D (K2-K5) plasmin has equivalent hemostatic safety as full-length plasmin in an animal model of fibrinolytic hemorrhage and warrants evaluation in clinical thrombotic disease.
Mutations of the SBDS Gene Result in Nuclear Mislocalization.
Authors: Yamaguchi Masafumi , Fujimura Kingo , Toga-Yamaguchi Hanae , Svetic Valentina , Okamura Naoki , Chopra Rajesh
Published: April 2020

Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11. We have previously reported that Shwachman-Bodian- Diamond syndrome (SBDS) gene is not required for neutrophil maturation. However, SBDS knockdown cells were sensitive to apoptotic stimuli, indicating that SBDS acts to maintain survival of granulocyte precursor cells. (Exp Hematol35; 579, 2007). A wide variety of mutations in SBDS gene has been identified, and almost of all patients show truncated immature proteins, p.K62X (c.183_184TA>CT) or p.C84fsX3 (c.258+2T>C). However, it is not yet clear how these truncated proteins affect cellular processes that result in the SDS phenotype. The SBDS protein is localized to the nucleoli but does not have the canonical nuclear localization signal. In order to clarify the molecular basis of pathogenicity of mutated SBDS proteins, we explored the subcellular distribution of normal and mutant SBDS proteins in Hela and 32Dcl3 cells. Using various N-terminal and C-terminal deletion constructs, we found N-terminal region, domain I (1-87 amino acid residue) in particular, was necessary to localize to the nucleus. The disease related mutations (C31W, K33E, N34I, L71P) and the mutations which are conserved among the species in the domain I (E44K, K62E, D70N, E82K) were generated. C31W and N34I mutants failed to localize SBDS to the nuclei. The SV40 derived nuclear localization signal was fused to these mutated SBDS protein, and these proteins were clearly localized to the nuclei. In addition to the mislocalization, the protein expression level of these mutants showed a dramatic decrease compared to the wild type. We also established SBDS wild type and domain I overexpressed 32Dcl3 cell. SBDS wild type overexpressed cells could differentiate to normal neutrophils in the presence of mG-CSF, however domain I overexpressed cells did not differentiate. Almost of all cells showed apoptosis in this domain I overexpressed cells in the presence of mG-CSF, and this was very similar like SBDS RNAi knockdown cells. The localization of endogenous SBDS protein was also analyzed in this domain I overexpressed cells. The domain I was concentrated to nuclei, however endogenous SBDS protein was diffused to cytosol.

Conclusions: The present findings enable us to document the nuclear localization signals in SBDS domain I, and that the shuttling protein would promote SBDS to nuclei. These results also showed that mislocalization and/or low expression level of mutated SBDS protein would cause SDS.