bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of Potent Anti-B-Cell Lymphoma CTL Responses by the Same Immunoglobulin Variable Region Subfamily Framework-Derived Peptides.
Authors: Guo Xiaoling , Zhu Ping
Published: October 2019

The immunoglobulin variable region (IgHV) which also be called the idiotype (Id) is the best-characterized tumor antigen expressed in B-cell lymphoma. It can function as a target for T-cell-mediated immune responses. However, the usefulness of this approach is limited because vaccines must be patient-specific. IgHV gene can be divided into seven subfamilies, which share the same gene sequence within their framework regions (FR). Here, we choice some peptides derived from the different gene subfamily FRs, and tried to identify if these peptides were capable of inducing cytotoxic T-lymphocyte (CTL) responses, and if the special CTL had possibility to kill B-cell lymphoma cells that belonging to the same subfamily. Meantime we explore if the limitations could be overcome by using the FR-derived peptides. In our study, we amplified seven respective IgHV gene subfamilies by PCR and directly sequenced for 108 cases of acute lymphoblastic leukemias. Complete VHDJH rearrangements were identified in 71(66%)out of 108 cases with 34 (47.89%) monoallelic rearrangements, 30 (42.25%) biallelic rearrangements and 7 (9.86%) oligoclonal rearrangements. SYFPEITHI and BIMAS software databases were used to predict T-cell epitopes in the seven different IgHV subfamily’s FR, and to find that these peptides have the same sequence as their germ line gene. Using an antigen-specific T-cell expansion system in vitro, we stimulated the PBMC from the health HLA-A0201 donor by the IgHV1 peptide(QLVQSGAEV) loading APC every week, and demonstrated that the predicted peptides could generate peptide-specific CTL successfully in healthy donors. The frequency of CD8+ and IgHV1 peptide(QLVQSGAEV)/tetramer+ double positive cells in the gated lymphocyte population was 0.38% before stimulation and increased to 49.38 % after forth stimulation . The cytokine release assay and the cytotoxicity assay indicate that these CTLs are capable of killing the lymphoma cell line belonging to the same family in a peptide-specific, and MHC-restricted way. Furthermore, the CTL stimulated by the IgHV1 peptide cannot kill the target cell pulsing a different subfamily peptide, indicating that the cytotoxicity is family-specific. Using the RT-PCR method, TCRBV expression of the CTLs were analyzed and the PCR products were sequenced directly. We found after four stimulations, the TCRBV expression were skewed in several families, and all the clones were found to have identical nucleotide sequences. In conclusion, our results demonstrate that the family-specific peptides derived from the IgHV FR can successfully lead to the generation of peptide-specific CTLs in vitro. Such defined antigenic epitopes may not only serve as candidates for novel lymphoma immunotherapy strategies, but also may help in discovering the interaction between the B-cell and T-cell clones.
The Expression of the CD21 Antigen in Non-Hodgkin’s Lymphoma Is Involved in LFA-1 Expression and Tumor Survival.
Authors: Otsuka Masaki , Yakushijin Yoshihiro , Hamada Makoto , Hato Takaaki , Yasukawa Masaki , Fujita Sigeru
Published: October 2019

CD21/complement receptor type 2 is a membrane protein expressed on B lymphocytes, follicular dendritic cells, early thymocytes, and a subset of mature T lymphocytes. This major B cell antigen appears later than CD19 and CD20 in cell differentiation and is lost from the cell surface during the early stages of B cell activation. CD21 is characterized not only as a complement ligand but also as an adhesion molecule of the cell surface that is dependent on CD23 expression, suggesting that it may be involved in lymphocyte trafficking and several immune responses. Recently two groups have reported that CD21 expression is significantly negatively associated with mortality in diffuse large B cell lymphomas. From these reports and clinical observations, we have transfected the CD21 gene into a CD21/CD23-negative lymphoma cell line (Namalwa). Interestingly, all established CD21+ transfectants (six clones from different bulks) showed homotypic aggregation during in vitro cell culturing, and none of the anti-CD21 antibodies prevented this cell aggregation. Moreover, these CD21+ transfectants were easily rejected in vivo when they were injected in nude mice compared to vector-only transfectants and parental cells as controls. These observations suggest that other adhesion molecules may be involved in CD21-induced cell aggregation and reduced tumorgenesis in vivo. After the screening of several integrins, CD21+ transfectants highly expressed LFA-1 (CD11a/CD18) on their cell surfaces compared to vector-only transfectants and parental cells. CD21+ transfectants displayed tight adhesion when in contact with recombinant human ICAM-1, and cell aggregation of CD21+ transfectants was blocked by an anti-LFA-1 monoclonal antibody. Northern and Western analysis and confocal laser scanning microscopic analysis suggested that the CD21 expression made the transfer of LFA-1 from the cytoplasm to the cell surface. We conclude that the expression of the CD21 molecule is strongly associated with the LFA-1 expression on the cell surface, independent of the CD23 molecule, and this phenomenon may be involved in tumor survival in non-Hodgkin’s lymphoma in vivo.
Early Allogeneic Hematopoietic Cell Transplantation during Induction Chemotherapy in High-Risk Acute Myeloid Leukemia.
Authors: Ehninger Gerhard , Platzbecker Uwe , Thiede Christian , Illmer Thomas , Schuler Ulrich S. , Schaich Markus A. , Bornhaeuser Martin
Published: October 2019

Objectives: In patients with acute myeloid leukemia and high-risk cytogenetic aberrations or minor response to the first cycle of induction chemotherapy (IC) the probability of achieving a sustained complete remission is low. Thus early treatment intensification may be warranted in order to achieve long-term disease control. We performed a prospective trial to evaluate whether reduced-intensity conditioning followed by allogeneic hematopoietic stem cell transplantation (HSCT) from related or unrelated donors can be performed during the aplastic phase of IC in patients with poor-risk AML.

Methods: Seventeen patients (n=17) aged between 17 and 63 years (median 45) with acute myeloid leukemia and high-risk cytogenetic aberrations (n=14, complex, inv3 or t(3;3), t(3;5), −7 or del 7q, +8) or more than 10 % marrow blasts on day 15 after the first cycle of IC (n=3) were included so far. During aplasia a median of thirteen days (range 7–35) after the first (n=8) or second (n=9) cycle of IC patients received 5 x 30 mg/m2 fludarabine i.v. combined with either 8 mg/kg busulfan p.o. (n=4) or 150 mg/m2 melphalan iv. (n=13) followed by allogeneic G-CSF mobilized peripheral blood stem cells (PBSC, n=16) or bone marrow (n=1) from related (n=7) or unrelated (n=10) donors. Nine out of seventeen patients were not in complete remission before conditioning therapy was started. Patients with unrelated grafts received antithymocyte globulin (4 x 10 mg/kg ATG Fresenius). GvHD prophylaxis was performed with cyclosporine A (CSP).

Results: All patients engrafted (ANC > 0.5 Gpt/l on day 11, range 8–19, platelets > 50 Gpt/l day 15, range 11–32) and went into remission. Acute GvHD grade II-IV occurred in 8 patients and extensive chronic GvHD was documented in 5 patients with a follow-up of > 100 days. Two patients died while being in remission from infectious complications associated with acute (n=1) or chronic (n=1) GvHD and two patients died during relapse eight and twelve months after PBSC. With a median follow-up of 15 months (range 1–65) thirteen out of seventeen patients (76 %) are alive and in remission.

Conclusion: Early allogeneic HSCT as part of primary induction therapy seems to be an effective strategy in AML patients with either poor risk karyotype or minor response to the first induction cycle.
Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) for Patients Aged 65 Years or Older with AML and MDS.
Authors: de Lima Marcos , Shahjahan Munir , Alamo Jorge , Williams Patricia , von Wolff Brigitte , Couriel Daniel , Khouri Issa , Rondon Gabriela , Wheeler Brandy , Estey Elihu , Ueno Naoto , Kebriaei Partow , Champlin Richard , Giralt Sergio
Published: October 2019

Allogeneic HSCT is a potentially curative treatment for AML/MDS, but aging is generally associated with poorer outcomes. The incidence of AML/MDS, however, increases after the 7th decade of life, and there is limited data with transplantation in this age group. Here we review our experience treating such patients.

Methods: Retrospective analysis of outcomes of patients aged 65 or older treated from 1996 to 2004 with allogeneic HSCT (n=40; median age 67 years, range 65–75 years). Diagnosis was MDS in 5 cases and AML in 35 patients. Cytogenetics were high-risk in 50% and intermediate risk in 50%; 80% of the patients had active disease at HSCT (n=32). All preparative regimens contained fludarabine 100–150 mg/m2, combined with cytarabine 4 gm/m2, and idarubicin 36 mg/m2 (n=12); or with busulfan (n=8); with melphalan 140 or 180 mg/m2 (n=12); and with melphalan 140 mg/m2 and Mylotarg 2 or 4 mg/m2 (n=8). ATG was added in unrelated donor (MUD) HSCT. All but 2 patients received tacrolimus and methotrexate for graft-versus host disease (GVHD) prophylaxis. Stem cell source was bone marrow in 11 cases and peripheral blood in the others. Donors were related in 27 cases and unrelated in 13 cases (33%).

Results: 35 patients engrafted (88%); complete remission (CR) rate was 72%, 6 patients died early and 3 did not respond. Eleven patients are alive at a median of 12.5 mo (range, 2.6–59 mo), 10 of them in CR. One-year overall survival was 30% for the whole group, 26% for recipients of MUD and 32% for recipients of related donor HSCT (MUD x sibling, P=NS). One-year event-free survival was 28%. Median survival and disease-free survival was 4.5 and 2.5 mo, respectively; 42% of the patients in CR post HSCT have relapsed (n=13). Acute and chronic GVHD rates were 45% and 48%, respectively. Non-relapse mortality (NRM) was 40%.

Figure shows survival of patients with and without circulating blasts at the time of transplant. Figure shows survival of patients with and without circulating blasts at the time of transplant.

Conclusions: Here we expanded our previous observations indicating that allogeneic HSCT is a treatment option for selected patients in this age range. In this cohort with advanced stage disease (80% with active disease at transplant), NRM was high, but survival after sibling and unrelated donor transplants was similar.
Varicella-Zoster-Virus-Specific CD4+ Cytotoxic T Cells as Tumor-Specific Effector Cells in Cancer Immunotherapy.
Authors: Landmeier Silke , Pscherer Sibylle , Eing Bodo , Rooney Cliona M. , Juergens Heribert , Rossig Claudia
Published: October 2019

Adoptive transfer of gene-engineered T cells expressing tumor antigen-specific chimeric receptors (chRec) is a promising tool in cancer immunotherapy. A major limitation is the failure of chRec to induce proliferative T cell responses, resulting in a rapid loss of function. To provide a strategy for reexpansion of tumor-reactive T cells in vivo, we generated dual-specific T cells that respond to varicella zoster virus while also possessing chRec-mediated tumor reactivity. We expanded VZV-specific cytotoxic T cell lines (VZV-CTL) from four seropositive donors by culturing peripheral blood-derived T cells with lysates extracted from VZV-infected fibroblasts. Repeated stimulation with VZV lysates resulted in efficient and continued expansion for 10–12 weeks. >1x109 T cells were routinely obtained from a starting number of 1x106 peripheral blood T cells. The T cells displayed a mainly CD3+CD4+ (90±5%) phenotype. ELISPOT assays showed specific, MHC class II-restricted IFN-γ release in response to CD40-activated B cells expressing the viral glycoproteins gE and IE62. VZV-CTL belong to a non-regulatory effector T cell subset, shown by their failure to exert antiproliferative effects against cocultured autologous T cells and lack of Foxp3 expression. Retroviral transduction with chRec recognizing the tumor ganglioside antigen GD2 (14.G2a-ζ) and the B cell lineage antigen CD19 (CD19-ζ) resulted in receptor surface expression on 29–74% and 39–45% of cells, respectively. Gene-modified VZV-CTL efficiently recognized antigen-expressing tumor targets in an MHC-independent manner, as demonstrated by antigen-specific secretion of IFN-γ in response to coincubation with GD2-expressing tumor targets. Furthermore, chRec-transduced VZV-CTL performed potent and antigen-specific tumor cytolysis. Antibody blocking experiments revealed that tumor cells were lysed in a granulysin-dependent manner. ChRec-transduced CD3+CD4+ cytolytic VZV-CTL may provide a source of highly potent tumor-reactive cells for adoptive immunotherapy cancer. Endogenous viral reactivations or administration of booster doses of varicella vaccine may lead to survival of these tumor-reactive T cells for prolonged periods of time in vivo.
Augmented Hyper-CVAD in Acute Lymphoblastic Leukemia (ALL): The MDACC Experience with Intensified L-Asparaginase and Vincristine in Adult ALL Salvage.
Authors: Faderl Stefan , Thomas Deborah A. , Garcia-Manero Guillermo , Ravandi Farhad , Beran Miloslav , Rytting Michael , Koller Charles A. , Verstovsek Srdan , Borthakur Gautam , Andreeff Michael , Kornblau Steve , Vu Khanh , Jarmon Rita , Kantarjian Hagop M.
Published: October 2019

Prognosis has improved in newly diagnosed adult ALL using dose-intense multiagent regimens. Outcome of 185 patients (pts) treated with Hyper-CVAD (fractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone) showed 91% CR rate with 5-yr. survival of 39% and low induction mortality (Kantarjian et al. JCO 18:547, 2000). Outcome remains poor in ALL salvage. Following leads in pediatric ALL, we hypothesized that “augmentation” of hyper-CVAD (AHCVAD) with vincristine, dexamethasone, and L-asparaginase during induction and consolidation may improve prognosis in adult ALL salvage. To evaluate efficacy and safety of AHCVAD we conducted a phase II study, in which pts received up to 8 courses of HCVAD alternating with methotrexate/high-dose cytarabine. Each course was augmented by L-asparaginase 20,000 units i.v. and vincristine 2 mg i.v. at days 1, 8, and 15 each, and dexamethasone 80 mg i.v./p.o. on days 1–4 and 15–18. From June 2003 to June 2005, 29 pts with relapsed/refractory ALL were treated. Median age was 32 yrs (range 14–70). Twenty-two pts (76%) had pre-B ALL (1 isolated CNS disease), 6 (21%) T cell ALL (2 with isolated extramedullary disease), and 1 (3%) pt had mature B ALL. Karyotype was diploid in 11 (44%) and abnormal in 14 (56%) pts (1 Ph+ ALL). No information is available in 4 pts. Median number of prior regimens was 1 (1–4). Median remission duration to initial induction regimen was 9 mos (0–68). Three pts were primary refractory. Of 29 pts, 22 are evaluable for response (2 too early, 5 off study for toxicity or pt. choice). Eight pts (37%) achieved CR, 1 (5%) PR, and 2 (10%) hematologic improvement (HI) for an OR of 52%. Six pts were able to proceed with stem cell transplant (5 CR, 1 HI). Of 29 pts evaluable for toxicity, 5 (17%) died on study from infectious complications. Uncomplicated neutropenic fever was common. Three pts (10%) were intolerant to L-asparaginase and had to be taken off study early. In summary, AHCVAD has activity in ALL salvage. However, asparaginase-related toxicities and neutropenic complications remain frequent and may limit the usefulness of this approach to selected patient populations.
Response Rates in Patients with Acute Myeloid Leukemia (AML), Treated with Azacitidine, Using WHO and International Working Group (IWG) Criteria for Myelodysplastic Syndrome (MDS).
Authors: Silverman Lewis R. , McKenzie David R. , Peterson Bercedis L. , Stone Richard M. , Powell Bayard L. , Mayo Christy , Backstrom Jay T. , Larson Richard A. ,   The Cancer Leukemia Group B (CALGB)
Published: October 2019

The CALGB conducted a series of clinical trials with azacitidine (Vidaza®) administered subcutaneously or intravenously in patients with MDS using the FAB classification (JCO2002;20:2429). Since completion of these CALGB studies (8421, 8921, 9221), a new classification system was developed by the WHO that distinguishes MDS from AML (blasts > 20%). Although studies with azacitidine in patients with AML had previously shown activity, the 75 mg/m2/day dose in the CALGB studies was lower than previously studied. Using the WHO system, the diagnosis for CALGB study patients was redefined and patients with AML were analyzed separately. Most of the 105 patients were previously considered refractory anemia with excess blasts in transformation (RAEB-T). Also, new treatment response criteria for MDS were published by the IWG (Blood2000; 96:3671). Using IWG response criteria, azacitidine patients with WHO AML in studies 8421, 8921, or 9221 had an overall response rate (CR+PR+HI) of 48% (12/25), 32% (9/28), and 37% (10/27), respectively.

Best Response using IWG Response Criteria for WHO AML Patients in Studies 8421, 8921, and 9221

Median duration of any response (CR, PR or HI) in the 33 azacitidine-treated responders was 279 days (range: 61 to 724 days). The median duration of CR in the 8 azacitidine-treated responders was not achieved; however, the 25th percentile was 115 days (range: 92 to 274+ days). In Study 9221, the median duration of transfusion independence (defined as ≥56 days) in patients independent at baseline was significantly longer in the azacitidine group compared with supportive care for red blood cells (azacitidine [n=8]: 411 days vs. supportive care [n=9]: 133 days, p=0.02) and platelets (azacitidine [n=13]: 363 days vs. supportive care [n=18]: 125 days, p=0.004). In the azacitidine group, 22% (6/27) of patients had a hemoglobin improvement to >11 g/dL that was maintained for ≥56 days compared with 8% (2/25) in the supportive care group (p=0.2). The proportions of patients with ANC >1500/m3 and platelets >100,000/mm3 lasting for ≥56 days were similar between the treatment arms. Azacitidine patients with WHO AML had a longer median survival (19.3 months) compared with the supportive care group (12.9 months) (p=0.2). Further studies investigating azacitidine in patients with AML with dysplasia are warranted.
Fludarabine Based Regimen (FLAI) Is an Effective Treatment for Induction of Multidrug Resistant Pgp-Positive Acute Myeloid Leukemia Patients.
Authors: Russo Domenico , Damiani Daniela , Malagola Michele , De Vivo Antonio , Fiacchini Mauro , Martinelli Giovanni , Piccaluga Pier Paolo , Candoni Anna , Michelutti Angela , Fanin Renato , Castelli Maurizio , Testoni Nicoletta , Ottaviani Emanuela , Rondoni Michela , Pricolo Giancarla , Mazza Patrizio , Zuffa Eliana , Zaccaria Alfonso , Raspadori Donatella , Bocchia Monica , Lauria Francesco , Bonini Alessandro , Avanzini Paolo , Gugliotta Luigi , Pierri Ivana , Gobbi Marco , Visani Giuseppe , Baccarani Michele
Published: October 2019

Induction treatment of acute myeloid leukemia (AML) is conventionally based on regimens containing cytarabine (Ara-C), one anthracycline and, sometimes, a third drug, such as etoposide. Primary P-glycoprotein (Pgp) overexpression is the most important mechanism of multidrug resistance (MDR) in AML cells and it is almost always associated with less response to treatment. To deal with this problem, in 1997, we started a treatment program with a regimen including Fludarabine (FLUDA) for the induction of newly diagnosed AML patients. FLUDA showed to be toxic against the MDR cells, in vitro, and able to enhance Ara-C cytotoxicity by increasing cell concentration of Ara-C 5′ triphosphate and inhibiting DNA repair. Between 1997 and 2004, 110 newly diagnosed AML patients aged less than 60 years were induced with FLAI (Fludarabine 25 mg/sqm/day days 1–5, Ara-C 2 gr/sqm/day days 1–5, Idarubicine 10 mg/sqm/day days 1, 3, 5) in the context of three consecutive prospective multicentric trials. At diagnosis, all the patients were assessed for the Pgp expression by an indirect immunofluorescence method with the anti-p170 monoclonal antibody MRK-16. The results were expressed as the mean fluorescence index (MFI) and patients with a MFI > 6 were setted as MDR+ve. We correlated the Pgp-expression, with the response to the induction.

Interestingly, the Pgp+ve (MFI > 6) patients treated with FLAI entered CR as well as the Pgp-ve (MFI < 6). Twenty-four out of 39 Pgp+ve patients (61%) and 54 out of 71 Pgp-ve patients (76%) achieved a CR after a single induction course of FLAI (p= 0.1). This observation strongly supported the original hypothesis that fludarabine could play a favourable role against MDR+ve cells. In order to validate this finding we compared the results obtained in an hystorical group of 136 newly diagnosed AML patients younger than 60 years treated with a non-fludarabine containing regimen AI (Ara-C 200 mg/sqm/day c.i. days 1 - 7, Idarubicine 10 mg/sqm/day days 1, 3, 5). In the non-fludarabine group, 22 out of 69 Pgp+ve patients (32%) and 42 out of 67 Pgp-ve patients (63%) achieved a CR after one course of the induction therapy. This difference was stastically significant (p= 0.0006). Based on these observations, we decided to conduct a match pair study to confirm the superiority of FLAI for overcoming the primary multidrug resistance Pgp-mediated in AML.
Comparative Pharmacodynamics of TGN 255, a Novel Direct Thrombin Inhibitor and Unfractionated Heparin in Healthy Volunteers.
Authors: Combe Sophie , Allen Graham , Kennedy Antony
Published: October 2019

Background: TGN255 is a synthetic direct thrombin inhibitor undergoing development for the prevention of arterial and venous thrombosis in high risk patients.

Methods: An open-label, heparin-controlled, cross-over study was performed in 12 healthy male volunteers to evaluate the pharmacokinetics, pharmacodynamics and safety of TGN255 and heparin given as an infusion over a 3 hour period. Doses of TGN255 were given as a bolus of 25 mg over 60 sec followed by a 3 h infusion of 40, 80, 160 mg/h. Heparin was administered as a 5000 IU bolus followed by 15 IU/kg/h infusion for 3 h. Plasma concentrations of TGN255 were evaluated using a validated LC-MS/MS method. The dynamic activity was assessed through measurement of the Activated Clotting Time (ACT), thrombin time (TT) and activated Partial Thromboplastin Time (aPTT).

Results: The plasma concentration of TGN255 rapidly increased following the bolus injection, and the pharmacokinetic profile of TGN255 was dose proportional. The effective half-life of TGN255 was 1.7 h. This short half-life was consistent with the rapid decline in the observed TT, aPTT and ACT ratios after administration was completed. The elevations in TT ratios were considerably higher than for the aPTT ratios across TGN255 groups (6.9, 11.7, 19.7 times baseline for TT and 1.7, 2, 2.4 times baseline for aPTT). The relationship between TT ratio and TGN255 plasma concentration was very well defined with a correlation coefficient of 0.94. For heparin, the TT ratios reflected a saturated system (measurements >300 s) and the aPTT ratios showed a marked increase compared to TGN255 groups (mean of 9.3 times baseline) indicating less selective behavior than TGN255 for this clotting parameter. The ratios for ACT showed a measurable elevation above baseline: mean of 1.9, 2.2, 2.7 for TGN255 groups versus 1.7 for heparin. No significant adverse events have been observed of either a general or cardiovascular nature (including hemorrhage, ECG changes, or liver function tests).

Conclusions: This study will support the entry of TGN255 into the clinical arena of cardiovascular procedures, and support its use in patients undergoing coronary artery bypass grafting.
Comparison of Enoxaparin to the Thrombin Variant Protein C Activator, W215A/E217A, in a Primate Model of Thrombosis.
Authors: Marzec Ulla M. , Bush Leslie , Gruber Andras , Hanson Stephen R. , Di Cera Enrico
Published: October 2019

Antithrombotic therapy by thrombomodulin-dependent activation of endogenous protein C might be safer than currently used therapies, since endogenous activated protein C (APC) is generated and probably concentrated on the endothelial surface and not in blood that escapes the blood vessels during injury. We compared the antithrombotic and antihemostatic activities of a protein C selective recombinant double mutant human thrombin (W215A/E217A; WE) that activates endogenous protein C on the endothelial surface, in vivo, with a low molecular weight heparin, enoxaparin in awake non anticoagulated baboons. Thrombosis was initiated by interposing a two part device consisting of a 2cm, 4mm ID, polyethylene terephthalate vascular graft (DVG) followed by a 2cm silicone tubing chamber, 9mm ID, into a chronic femoral arteriovenous shunt in baboons. Upon initiation of blood flow at 100mL/min, wall shear rate of 265sec−1 in the 4mm ID segment (arterial type flow) and 29sec−1 in the expansion chamber (venous type flow), platelet thrombus growth was monitored by gamma camera imaging of autologous 111In-labeled platelets for 1 hour. Fibrin accumulation was quantified by homologous 125I-labeled fibrinogen. Bleeding time and aPTT were monitored. WE was administered as a loading IV bolus (1/3 dose) 10 minutes before thrombus initiation followed by maintenance infusion (2/3 dose) for the duration of the study at 3 dose levels: 1.8 μg/kg (dose-1), 3.75 μg/kg (dose-2) and 7.5 μg/kg (dose-3). Results were compared to controls given normal saline and to 3 doses of IV enoxaparin (1/2 dose loading + 1/2 dose maintenance): 0.3 mg/kg (dose-1), 0.6 mg/kg (dose-2) and 1.2mg/kg (dose-3) (3–5 animals in each study group). WE inhibition of platelet deposition in the chamber was dose dependent and reduced by 57%, 76% and 95% for dose 1, 2 and 3 respectively, compared to controls (p < 0.05 each). Enoxaparin likewise reduced thrombosis in the chamber by 49%, 83% and 91% (p< 0.05 each) respectively at the 3 doses tested. Fibrin accumulation paralleled the platelet deposition data in the chamber. Thrombus growth on DVG was only reduced at dose level 3 for both WE and enoxaparin by 18% and 47%, respectively as determined by platelet deposition. Fibrin accumulation was reduced by 35% by WE and 51% for enoxaparin at dose 3 on the arterial thrombosis segment. Bleeding times did not increase significantly with any of the treatments except for dose-3 of enoxaparin which prolonged the bleeding time from 3.5±1.3 minutes to 5.3 ± 1.4 minutes. Prolongation of APTT was 1.14, 1.33, and 2.04 fold for WE doses 1, 2 and 3, respectively and enoxaparin prolonged the APTT by 1.25, 1.6 and 1.9 fold at dose 1, 2 and 3, respectively. We conclude that low dose WE infusion is safe and about two orders of magnitude more antithrombotic than enoxaparin infusion in the baboon model of thrombosis.
Extra-Cellular DNA in Cord Blood (CB) Plasma: Use for Sample Identification [short Tandem Repeat (STR) Analysis] and for Infectious Disease Screening in CB Banks.
Authors: Albano Maria S. , Andrus Linda , Lee Dong H. , Stevens Cladd E. , Rubinstein Pablo
Published: October 2019

Introduction: A commitment of CB banks is to test CB units and maternal blood for multiple genetic and infectious disease markers. As a consequence, several aliquots of blood, mononuclear cells and plasma are taken during processing for immediate and delayed testing. Whereas control of identity between aliquots containing cells can be performed by DNA assays, identity controls for plasma aliquots rely only on bar-coded label systems. We hypothesize that there is soluble extra-cellular DNA (EC-DNA) and that it can be used for identity control of plasma aliquots. The aims of our study were;

to determine the concentration of EC-DNA in CB-plasma, to evaluate whether genetic identity testing by STR analysis is feasible and to investigate whether EC-DNA allows infectious disease screening by nucleic acid testing (NAT).

Methods: CB (n=20) was collected, processed (Rubinstein et al, PNAS 1995) and EC-DNA from 200 μl of 0.45μm-filtered plasma was extracted both with the Qiagen and the High Pure Viral Nucleic Acid method (Roche). Presence of EC-DNA was evaluated by detection of HLA-DR and ALU gene sequences and by STR analysis (AmpFlSTR-Profiler Plus-Applied Biosystems). Purified HBV was added to CB and peripheral blood (PB) plasma, EC-DNA recovered as above and HBV-DNA assessed by real time PCR.

Results: All 20 samples were positive for HLA-DR by PCR and for ALU sequence by real time PCR, confirming the presence of EC-DNA in CB-plasma in concentrations of 0.022 +/− 0.012 ng/μl at the time of CB collection, rising to 0.124 +/− 0.025 ng/μl 33 hrs later; p<0.016. Concentrations observed in CB-plasma were higher than those of adult donor plasma (PB-plasma 0.005+/−0.0065 ng/μl). STR analysis was validated on cellular DNA and, when applied for EC-DNA, showed good signal strengths and low backgrounds, allowing accurate automatic allele identification with no manual corrections (genemapper software, Applied Biosystems). An increase in STR-PCR product concentration was observed when we analyzed EC-DNA samples from CB after 33hs (7017.6 +/− 2014 RFU/μl at time of CB collection and 30290.9 +/− 3164 RFU/μl after 33 hs; RFU = relative fluorescence units). HBV was recovered from all spiked units, in correspondence with the viral concentrations added. Recovery of HBV was 96 +/− 21% with high and low viral concentrations. Three mothers with HBAg/HBcore positive had also HBV-DNA in PB-plasma. Six with anti-HBcore only and 70 with anti-HBs were negative. HBV-DNA was also negative in CB-plasma for all seventy nine respective newborns.

Conclusions: there is infant EC-DNA in CB-plasma and viral nucleic acids can be obtained using the same extraction method. There is an increase of EC-DNA in CB over time that may reflect cell death. STR assay of EC-DNA can be a useful molecular tool for the identification of plasma aliquots used for infectious disease testing in CB banks.
RNA-Binding Protein VICKZ Is Expressed in a Germinal Center Associated Pattern among Lymphoma Subtypes.
Authors: Natkunam Yasodha , Vainer Gilad , Zhao Shuchun , Amir Gail , Pikarsky Eli , Hammer Anne S. , Hamilton-Dutoit Stephen , Levy Ronald , Yisraeli Joel K. , Lossos Izidore S.
Published: October 2019

Recent effort in the molecular characterization of diffuse large B-cell lymphoma (DLBCL) has led to the recognition that patients with DLBCL of germinal center origin exhibit a better overall survival. Thus, identification and characterization of markers of germinal center derivation are of importance in dissecting prognostic subclasses of DLBCL. The VICKZ (Vg1 RBP/Vera, IMP1, 2, 3, CRD-BP, KOC, ZBP-1) family members are RNA-binding proteins that recognize specific RNA targets and have been implicated in diverse cellular functions including cell polarity, migration, proliferation and tumorigenesis/metastasis. We generated an antibody that recognizes all three isoforms of VICKZ protein and characterized its expression in normal lymphoid tissue and in lymphoma subtypes. In normal tonsils, VICKZ protein showed a germinal center-specific pattern of expression with staining localized to the cytoplasm. Among 868 non-Hodgkin and Hodgkin lymphomas tested by immunohistochemistry on tissue microarrays, staining for VICKZ protein was present in 76% (126/165) of follicular lymphoma, 78% (155/200) of DLBCL, 90% (9/10) of mediastinal large B-cell lymphoma, and 100% (2/2) of Burkitt lymphoma. A subset of mantle cell lymphoma (11%, 2/19), extranodal (8%, 2/25), and nodal (20%, 1/5) marginal zone lymphoma and lymphoblastic lymphoma (25%, 4/13), showed VICKZ staining. The majority of lymphocyte predominant Hodgkin (92%, 12/13) and classical Hodgkin (94%, 101/108) lymphoma were found to be positive. Among T cell lymphoma, anaplastic large cell lymphoma were positive (75%, 6/9). Additional work is in progress to correlate VICKZ protein expression with other germinal center markers such as HGAL, BCL6 and CD10 as well as with prognostic subclasses of DLBCL. The differential expression pattern of VICKZ protein in lymphoma subtypes suggests a potential utility for VICKZ in the identification of subgroups of DLBCL associated with different prognoses.
Clinical Features and Outcome of 147 Patients with Diffuse Large B-Cell Lymphoma and Hepatitis C Virus Infection.
Authors: Visco Carlo , Arcaini Luca , Merli Michele , Andreoli Annalisa , Burcheri Sara , Ambrosetti Achille , Brusamolino Ercole , Lazzarino Mario , Pizzolo Giovanni , Rodeghiero Francesco
Published: October 2019

Hepatitis C virus (HCV) has been implicated in the pathogenesis of a subset of low-grade non-Hodgkin lymphomas. Furthermore, diffuse large B-cell lymphoma (DLBCL) has been correlated to HCV infection in several series from our geographical area (north-east of Italy), but little is known about the characteristics of such high-grade tumors. We analyzed presentation features of 147 previously untreated HCV-positive patients with DLBCL who presented to the three participating centers between 1993 and 2004. All patients were provided with complete clinical information, were HIV negative, and had been tested at tumor onset for HCV antibodies by ELISA and RIBA.

Median age at presentation was 64 years old (range 29–88), 47% were males, ECOG performance status was >1 in 20%, Ann Arbor stage was I in 20%, II in 27%, III in 26%, IV in 27%, and B-symptoms were present in 37% of patients. The International Prognostic Index (IPI) value at diagnosis was low in 18%, int/low in 23%, int/high in 32%, and high in 27% of patients. Surprisingly, DLBCL transformed from a low-grade histology represented only 7% of the whole population, while primary mediastinal DLBCL were extremely rare (1/147, <1%). Patients frequently presented as primary extranodal DLBCL (65/147, 44%). Most involved extranodal sites were skin, liver, stomach, and spleen, with the latter being the most represented syte (33% of patients). Remarkably, spleen was the only extranodal involved organ in 20% of patients. Treatment was delivered with cure-intent, and consisted of CHOP-like regimens +/− Rituximab for the large majority of patients, except for 16 (11%) patients with cirrhosis or severe hepatic dysfunction, who received mono-chemotherapy or radiotherapy. Only three (2%) HCV-positive patients had to discontinue chemotherapy due to liver function impairment. The addition of Rituximab to chemotherapy did not seem to affect patients’ tolerance to treatment. With a median follow-up of 48 months for survivors, 5-year overall survival (OS) was 75%, while 5-year failure-free survival (FFS) was 51%. In particular, the 65 patients with primary extranodal DLBCL shared a better 5-year OS (83% vs 71%, p=0.01) and FFS (75% vs 39%, p=0.009) than their nodal counterpart. Nodal origin of the tumor resulted the strongest independent adverse factor both in terms of OS and FFS in multivariate analysis.

The peculiar clinical behavior shared by HCV-positive DLBCL may disclose relevant biological features of these tumors, and may be relevant for future studies aiming to clarify the link between HCV infection and aggressive lymphoproliferative disorders.
Complete Loss or Severely Compromised Expression of the Pro-Apoptotic Protein Bax Leads to Resistance to Curcumin-Induced Apoptosis in Burkitt’s Lymphoma Cells.
Authors: Hussain Azhar R. , Al-Rasheed Maha , Manogaran Pulicat S. , Al-Kuraya Khawla S. , Uddin Shahab
Published: October 2019

This report compares in detail, the role of curcumin treatment in a panel of Burkitt’s lymphoma (BL) cell lines that are either expressing full length Bax with a group of cell lines that are either completely deficient or have decreased expression of Bax. Multiple apoptotic stimuli induce conformational changes in Bax, a pro-apoptotic protein from the Bcl-2 family and its deficiency is a frequent cause of chemo-resistance in a variety of malignancies including Burkitt’s lymphoma. We extent our previous studies on Burkitt’s lymphoma to determine the role of curcumin treatment on BLs. Curcumin (diferuloymethane) is a naturally occurring yellow pigment isolated from the rhizomes of the plant curcuma longa. The medicinal value of curcumin has been well recognized with its anti-oxidant, anti-inflammatory and anti-tumor activities. Curcumin is also known to induce apoptosis in a variety of cancer cells including multiple myeloma and primary effusion lymphomas. To understand the role of Bax in curcumin-induced apoptosis, we used two groups of Burkitt’s lymphoma cell lines, one that expressed the Bax protein (AS283A, KK124 and Pa682PB) and the other group either did not or had decreased expression of Bax (BML895 CA46, and LW878). Cell viability decreased in a dose-dependent manner in AS283A, PA682PB and KK124 with curcumin treatment ranging between 0–40mM whereas only minimal changes in viability was observed in BML895, CA46 and LW878 after treatment. Curcumin induced a dose-dependent apoptosis in the Bax expressing group of cell lines while the cell lines that were either completely deficient or had decreased expression of Bax did not respond to curcumin treatment and remained refractory. In AS283A, KK124, and PA682PB, curcumin induced apoptosis through truncation of BID, loss of mitochondrial potential as determined by JC1 staining with subsequent activation of caspase3 followed by cleavage of PARP. However, in the curcumin resistant cell lines, there was no change in the mitochondrial potential after curcumin treatment and therefore apoptosis did not occur. In addition, zVAD-fmk, a universal inhibitor of caspases prevented caspase3 cleavage as well as cell death in the sensitive cell lines after curcumin treatment suggesting that curcumin-induced apoptosis is caspase dependent. Our findings suggest that Bax integrity is necessary for curcumin to induce apoptosis in Burkitt’s lymphoma cells. These results provide the molecular basis and preliminary data for new treatment strategies that may incorporate curcumin in regimens for Burkitt’s lymphoma treatment.
Factor VII Deficiency Remains Post-Puberty Due to Point Mutation in the HNF4 Binding Site in the Coagulant Factor VII Gene.
Authors: Zheng XingWu , Tidd Theresa M. , DiMichele Donna , Pollak Eleanor S.
Published: October 2019

A novel T to C point mutation at −60 in the gene for coagulant Factor VII results in life-long severe coagulant Factor VII deficiency in post-pubertal twin males. The clinical course of these patients provides an informative in vivo example of the regulation of expression of vitamin K-dependent clotting protein Factor VII. An analogous point mutation in the HNF4 binding site in the Factor IX gene results in the clinical phenotype Hemophilia B Leyden, a sex-linked antigen-negative Factor IX deficiency that resolves post-puberty. The affected Factor VII deficient patients have prolonged prothrombin times (46 and 52 secs), normal aPTTs and decreased FVII levels of FVII:Coagulant activity: < 1% and FVII:Antigen: < 3%. The −60 mutation, ACTTTG → ACTCTG occurs 9 base pairs before the start site of transcription and 59 bps before the before the start site of translation. The twins are compound heterozygotes and also possess a mutation in exon 8 at amino acid 348, a mutation that has previously been reported to cause FVII deficiency. Both affected individuals have recurrent target joint hemorrhage (shoulder, elbow, ankle) requiring replacement therapy 6–12 times/year.

Results: Gel mobility shift assays using a radio-labeled probe spanning from −76 to −46 in the FVII promoter region demonstrate the loss of binding of transcription factor HNF-4. Transient transfection assays in HepG2 cells using 186 bps of the mutant and the wildtype promoters (−185 to +1) revealed a loss of expression with the mutant allele. Co-transfection with an HNF4 expression plasmid resulted in an increase in expression of the wildetype construct in HeLa cells, a non-hepatic cell line. However, co-transfection of the HNF4 expression plasmid failed to increase expression with the construct containing the mutant allele sequence.

Conclusion: The lack of phenotypic change of the FVII:C in 19 yo twin boys provides dynamic support of the necessity of an overlapping androgen binding site in the homologous Factor IX gene as responsible for the phenotypic resolution of Factor IX deficiency (Hemophilia B Leyden) post-puberty. It is of interest that an increase in FVII:C did not occur with advancing age in FVII deficiency due to this HNF4 binding site mutation.
Human Factor VIIai(K62E) Is a More Effective Inhibitor of Coagulation Than Wild-Type FVIIai.
Authors: Clarke Bryan J. , Sridhara Sampath , Blajchman Morris A.
Published: October 2019

Background: In previous work we provided evidence for a linked conformational change of the recombinant factor VII(rFVII) first epidermal growth factor-like(EGF1) domain associated with chloromethylketone(ck) covalent modification of the wild-type(WT) rFVIIa active site (J Biol Chem275:34894,2000). All naturally-occurring mutations of the human FVII gene hitherto described encode plasma FVII with both reduced activity and affinity for their cell-surface receptor tissue factor (TF). We have recently characterized a mutant rFVII protein, rFVIIa(K62E), which has both increased enzymatic activity and a 5-fold greater affinity for TF than wild-type rFVIIa (J Thromb Haemost3:1250, 2005). In the present report we provide data about the TF-binding characteristics and conformational state of rFVIIa(K62E) before and after inactivation with a variety of chloromethylketones.

Methods: Purification of both the rFVIIa(K62E) mutant protein and rFVIIa(WT) and their ck-inactivated derivatives was achieved by anion-exchange chromatography. FVII antigen concentration was determined by ELISA and biological activity by modified prothrombin time (PT) assay. Affinity of inactivated rFVIIa(rFVIIai) for both TF and a FVII EGF1 domain conformation-specific monoclonal antibody(Moab 231–7) was determined by competitive ELISA (IC50).

Results: The affinity of rFVIIai(K62E)DEGR and rFVIIai(K62E)FFR for TF were increased 24.4-fold and 7.7-fold respectively versus rFVIIa(WT)[shown below]. Notably, the enhanced affinity of both rFVIIai(K62E) and unmodified rFVIIa(K62E) for TF was associated with substantially increased affinity for Moab 231–7 as compared to rFVIIa(WT). The clotting time of diluted human plasma was significantly more prolonged by rFVIIai(K62E)DEGR and rFVIIai(K62E)FFR than rFVIIai(WT).

Comparison of rFVIIai(K62E) and rFVIIai(WT) rFVIIa Preparation Affinity for TF(IC50) Relative Increase in TF Affinity Relative Increase in Affinity for Moab 231–7 Prolongation of Plasma Clotting time(sec) Reduction of Coagulant Activity(%) Affinity of rFVII for TF (IC50 expressed as molar excess of rFVII) was determined by competitive ELISA. Plasma clotting times were determined at 2.5-fold molar excess of each rFVIIai inhibitor. Control plasma clotting time was 81 sec. rFVIIai(K62E)DEGR 0.13 24.4 3.1 68 90 rFVIIai(K62E)FFR 0.41 7.7 4.9 57 86 rFVIIa(K62E) 0.46 6.9 4.1 ND ND rFVIIai(WT)DEGR 0.98 3.2 3.5 1 4 rFVIIai(WT)FFR 0.62 5.1 1.2 27 65 rFVIIa(WT) 3.17 1 1 ND ND

Conclusions: We provide evidence that both active-site modification and the substitution K→E in human rFVIIa(K62E) causes conformational change(s) within the FVII EGF1 domain associated with a substantially increased affinity of rFVIIa(K62E) and rFVIIai(K62E) for TF. Both rFVIIai(K62E)DEGR and rFVIIai(K62E)FFR show considerable promise as more effective inhibitors of coagulation than rFVIIai(WT).
Factor IX Substrate Binding Exosite on the Light Chain of Factor Xia.
Authors: Sinha Dipali , Marcinkiewicz Mariola , Navaneetham Duraiswamy , Walsh Peter N.
Published: October 2019

Binding of factor IX (FIX) to an exosite on the heavy chain of factor XIa (FXIa) is essential for the optimal activation of FIX (Biochemistry26:3768–3775, 1987). We now provide evidence that a second substrate binding exosite resides on the light chain (LC) of FXIa. To understand the mechanism of activation of FIX by LC we have investigated 1) the kinetics of S-2366 hydrolysis by FXIa and its isolated LC in the presence and absence of a reversible inhibitor of serine proteases (p-aminobenzamidine, PAB); 2) the kinetics of activation of the macromolecular substrate FIX by FXIa and its isolated light chain in the presence and absence of either PAB or the alternative substrate S-2366; and 3) the effect of active site inhibited FXIa LC (LCi, i.e., FXIa LC treated with phenylmethylsulfonyl fluoride) on the activation of FIX by FXIa. Kinetic parameters for the hydrolysis of S-2366 by FXIa and its isolated LC are comparable. However kcat for the activation of the macromolecular substrate FIX by LC compared to full-length FXIa is reduced by ~300-fold whereas the Km was only slightly higher (2- to 3-fold). PAB inhibited the hydrolysis of S-2366 both by FXIa and LC in a classical competitive fashion. If an exosite on the heavy chain of FXIa is solely responsible for formation of the Michaelis complex then PAB should have inhibited the activation of FIX by LC in a competitive fashion. However, PAB and S-2366 were found to be noncompetitive inhibitors of FIX activation by FXIa or LC demonstrating the presence of an exosite for FIX binding on the LC remote from its active site. The presence of the second exosite on the light chain of FXIa was further confirmed by the fact that the V vs. S as well as the V vs. I plots of the activation of FIX by FXIa in the presence of LCi were sigmoidal suggesting formation of a nonproductive SI complex. We conclude that for FIX-activation, two macromolecular substrate-binding exosites, one on the heavy chain and one on the light chain of FXIa are required to mediate the formation of the Michaelis complex.