bluuebunny/crossref_metadata_2025_split huggingface dataset:
High Doses of CD34+ Cells Chemomobilized with G-CSF PBPC Collection Had Greater CD34(+) Cells Collected Than G-CSF Alone and Trended toward Reduced Progression Rate after Autologous PBPC Transplantation for Breast Cancer in a Phase III Trial.
Authors: Gajewski James L. , Saliba Rima , Rondon Gabriela , Ueno Naoto T. , Donato Michele L. , Anderlini Paolo , Korbling Martin , McMannis John D. , Giralt Sergio A. , Khouri Issa F. , Mehra Rakesh , Champlin Richard E. 
Published: April 2020

Abstract
We evaluated intensive chemotherapy prior to PBSC mobilization vs. G-CSF alone for PBPC mobilization in a randomized controlled trial of 143 patients who received high dose chemotherapy with cyclophosphamide 6 g/m2, thiotepa 750 mg/m2 and BCNU 450 mg/m2 over 3 days with PBSC support for treatment of breast cancer. 74 patients were randomized to chemomobilization with cisplatin 135 mg/m2, etoposide 1200 mg/m2, and cyclophosphamide 4.5 g/m2 over 3 days plus G-CSF (6mcg/kg q12h)(CVP-G), while 69 patients received G-CSF alone with the collection goal &gt;4 x 106 CD34(+) cells/kg. 77 patients were stage II–III, 66 were stage IV. The CVP-G group had improved mobilization with a collection of 18 (range 3–141) x 106 CD34(+) cells/kg compared with 4(2–13) x 106/kg for the G-CSF group (p&lt;0.001). Granulocyte and platelet recovery post-transplant was 9/9 days for the CVP-G mobilized group, 10/11 days for the G-CSF group (p&lt;0.001). Actuarial risk of progression at 6 years in patients mobilized with CVP-G was 59±7% vs. 66±6% for G-CSF alone (p=0.2). Rate of progression post-transplant was evaluated according to quantities of collected CD34(+) cell dose [for CVP-G, first quartile ≤ 9.0, second ≤17, third ≤40, and fourth &gt;40]. Only one patient in the G-CSF alone group collected &gt;9 x 106 CD34(+) cells. In the CVP-G group progression trended toward being reduced in patients with CD34(+) cell doses collected of &gt;9 x 106/kg (57±9% vs. 65±11%, p=0.06) This effect was consistent for all stages. Overall survival was improved in the CVP-G patients collecting ≥9 x 106 CD34(+)/kg 62±7% vs. 27±11% those collecting less. For patients receiving CD34(+) doses ≤9 x 106/kg, the progression rate was 65±11% in the CVP-G group and 68±6% in the G-CSF group (p=NS). Higher doses of CD34(+) cells collected by chemomobilization than G-CSF alone and those patients with the higher doses of CD34(+) collected trended toward lower disease progression after high dose chemotherapy.
Localization of Binding Sites for Low-Density Lipoprotein Receptor in Coagulation Factor VIII.
Authors: Makogonenko Evgeny M. , Sarafanov Andrey G. , Ananyeva Natalya M. , Strickland Dudley K. , Saenko Evgueni L. 
Published: April 2020

Abstract
Clearance of coagulation factor VIII (fVIII) is mediated by a hepatic receptor low-density lipoprotein receptor-related protein (LRP), a member of low-density lipoprotein receptor (LDLR) family. It has been recently discovered that LDLR acts in concert with LRP in regulating fVIII level. FVIII has the domain structure A1-A2-B-A3-C1-C2, and to identify the portions providing the interaction with LDLR, in surface plasmon resonance-based assay we studied the binding of fVIII and its fragments to immobilized recombinant ligand-binding domain of LDLR (residues 1-292). The affinity values were determined from the families of binding signals obtained for five concentrations (10–150 nM) of each analyte. The binding signals for full-length fVIII, and its portions A3-C1–C2 (or light chain, LCh) and A1/A3-C1–C2 heterodimer (derived from activated fVIII) were best fitted to a two-site model, with equilibrium dissociation constants KD(1) ~1, 4, 14 nM and KD(2) ~14, 45 and 37 nM, respectively. Noteworthy, we did not observe any significant binding for the isolated C2 domain (at 300 nM). This suggests that the LDLR-binding site within LCh is likely located within the A3 domain, similar to that found previously for LCh-LRP interaction. The binding signals for A1-A2-B (heavy chain, HCh) were best fitted to a one-site model, with KD ~60 nM. We registered a dose-dependent, high-affinity binding of the isolated A2 domain to LDLR, with KD ~14 nM whereas the A1 domain showed no appreciable binding. This suggests that within HCh, A2 domain bears the LDLR-binding site. Von Willebrand factor did not significantly block the binding of fVIII to LDLR as compared to a 3-fold inhibition of fVIII binding to LRP. This indicates that within fVIII/vWf complex, the A2 binding site for LDLR is more available than that for LRP. Anti-A2 monoclonal antibody 413 (epitope 484–509) inhibited the A2 binding to LDLR in a dose-dependent manner, similarly to that demonstrated for fVIII-LRP interaction. A number of A2 point mutants with substitutions of the residues critical for A2 binding to LRP, megalin and VLDL were found to have significantly reduced affinity also for LDLR. The obtained data indicate that fVIII interacts with LDLR preferentially via the binding sites located within the A2 domain of HCh and within the A3 domain of LCh, and that the A2 site is likely to be universal for the interactions with four tested members of LDL receptor family.
Expression of Protein Z (PZ) and Protein Z-Dependent Protease Inhibitor (ZPI) In Situ in Human Malignant Tissues.
Authors: Sierko Ewa , Wojtukiewicz Marek , Tokajuk Piotr , Zimnoch Lech , Ramlau Rodryg , Kisiel Walter , Broze George J. 
Published: April 2020

Abstract
The activity of the hemostatic system components facilitates tumor growth and dissemination. There exists a mechanism of direct inhibition of factor Xa that involves protein Z (PZ)/protein Z-dependent protease inhibitor (ZPI) system. ZPI also attenuates the activity of factor XIa. Data on the presence of PZ and ZPI in malignant tissue in situ are lacking.
The aim of this study was to assess the localization of PZ and ZPI in situ in colon, breast, gastric, laryngeal, pancreatic, renal, endometrial cancer, non-small cell lung cancer (NSCLC), malignant melanoma and glial neoplasms. Studies were performed on tumor tissues obtained at surgical treatment of previously untreated patients. Immunohistochemical procedures according to the ABC method and Envision Double Staining System (Dako) employed a polyclonal antibody against PZ and a monoclonal antibody against ZPI. Protein Z was localized in cancer cell bodies in all types of malignant tumors examined. Staining intensity was more pronounced in less differentiated cancer cells of anaplastic gliomas. In contrast, more differentiated cancer cells of gastric cancer revealed stronger staining than less malignant ones. Cancer cell bodies of NSCLC and renal cancer were characterized by weak intensity of staining for PZ. In pancreatic and renal cancer, as well as in malignant melanoma, the intensity of staining for PZ in cancer cells was irregular: both high and low PZ expression was observed independently of the degree of malignancy. Protein Z was also revealed in association with macrophages in colon cancer, NSCLC and malignant melanoma. Expression of ZPI was observed in cancer cell bodies in all examined specimens. The staining intensity for ZPI was irregular: both strong and weak expression of ZPI was observed. Various percentages of ZPI-positive cancer cells were observed in different specimens of the tissues examined. In colon cancer, ZPI expression was detected in tumor infiltrating macrophages. Double staining studies revealed co-expression of both proteins in cancer cell bodies in all examined tumor types, although there were areas of tumor foci where only one protein (either ZP or ZPI) was localized. Our results suggest that PZ and ZPI, apart from being inhibitors of blood coagulation, may play an additional regulatory role in tumor progression.
Intracellular Trafficking of Factor V Subsequent to Its Endocytosis by Megakaryocytes.
Authors: Seifert Lorna W. , Wahle-Ritchie Alexa , Bouchard Beth A. , Tracy Paula B. 
Published: April 2020

Abstract
Recent studies by our laboratory have identified physical and functional differences between plasma- and platelet-derived factor V. Additional studies indicate that the platelet-derived molecule originates from megakaryocyte endocytosis of the plasma-derived cofactor via a receptor-mediated, clathrin-dependent mechanism, and is subsequently packaged and stored in platelet α-granules. We hypothesize that plasma-derived factor V is modified intracellularly following its endocytosis by megakarycytes to generate the unique platelet-derived cofactor molecule. Thus, the time-dependent, intracellular trafficking of fluorescently-labeled factor V by the megakaryocyte-like cell line, CMK, was determined by confocal microscopy using various organelle-specific, fluorescent markers. Previously, we had demonstrated that subsequent to its endocytosis factor V partially co-localizes with two other proteins known to be endocytosed by megakaryocytes, fibrinogen, an α -granule protein, and transferrin, an iron transport protein. In the current study, we demonstrated that subsequent to their endocytosis, factor V and transferrin partially co-localized to early endosomes as determined using an antibody directed against Rab5. Complete co-localization of anti-Rab5 with an antibody against early endosomal antigen-1 (EEA-1) confirmed the specificity of the anti-Rab5 antibody for early endosomes. Endocytosed factor V was also shown to partially co-localize with von Willebrand factor, an α -granule protein that is synthesized by megakaryocytes. Its synthesis by megakaryocytes was confirmed by partial co-localization of this antibody with anti-Golgi antibodies against GM130, a structural element of the Golgi apparatus, and p230 trans, a protein involved in vesicular transport from the trans-Golgi network. Factor V also partially co-localized with these Golgi markers, consistent with the hypothesis that factor V is modified intracellularly subsequent to its endocytosis. Co-localization studies were also performed using LysoSensor Blue, which partitions into acidic organelles with a pH ~5.1 exhibiting an increase in fluorescence intensity upon acidification. Neither factor V nor transferrin co-localized with LysoSensor Blue confirming that they are not trafficked to lysosomes subsequent to their endocytosis. In conclusion, these combined observations suggest that subsequent to its endocytosis by megakaryocytes factor V is trafficked through early endosomes and ultimately stored in the α -granule with vWF and fibrinogen. Further, these data suggest that prior to its packaging into α -granules factor V may undergo retrograde transport through and O-linked glycosylation in the trans-Golgi network, which is consistent with our previous observations that purified, platelet-derived factor V contains an N-acetyl glucosamine or galactosamine at Thr402 that is not observed in its plasma counterpart.
Different Prevalence of Drug Metabolism Gene Polymorphisms (GSTP and GSTT) in FA Patients Compared to a Normal Population.
Authors: Santos Moema Nene , Rocha Vanderson , Zanette Dalila , Porcher Raphael , Ionescu Irina , Tavela Marli , Soulier Jean , Leblanc Thierry , Fisher Gustavo , Baruchel André , Zago Marco Antonio , Gluckman Eliane 
Published: April 2020

Abstract
Fanconi Anemia patients have an increased toxicity of drugs used in conditioning regimen (CT) for hematopoietic stem cell transplantation (HSCT), due to defective DNA repair processes. This observation has led to diminish the doses of drugs (such as cyclophosphamide and busulphan) and irradiation in the preparative regimen. We and others have reported that genetic polymorphisms, which interfere with metabolisms of drugs used in CT, are associated with toxicities after HSCT. In order to test the hypothesis that probably Fanconi patients have a difference in prevalence of pharmacogene polymorphisms, we have compared the samples of 49 Fanconi patients (median age of 6,2 years) with 107 samples of normal population of the same ethnic group. Polymerase chain reaction (PCR) and restriction-digestion protocols were used to identify most of the polymorphisms. We have selected pharmacogenes knonwn to interfere with the metabolisms of drugs used in the CT or GVHD prophylaxis. The following gene polymorphisms were analyzed: P450 cytochrome family [CYP2B6*2(C64T), *4(A785G), *5(C1459T), *6(G516T)]; glutathion-S-Transferases [GSTM1 (null), GSTT1(null), GSTP1(G313A)], Thiopurine-S-methyl transferase [TPMT *3B (G460A) and *3C (G238C)], Multiresistance-drug [MDR1 (C3435T)], Methylenetetrahydrofolate reductase [MTHFR (C677T, A1298C)], Vitamin D receptor [VDR (ApaI, TaqI and BsmI)]. All gene polymorphisms studied were found in the same frequency in FA as in normal controls, except polymorphisms of GSTP and GSTT. In fact, FA patients had a higher frequency of mutant alleles (AG or GG) of GSTP (86%; n=42) compared to 51% (n=55) in normal controls (p&lt;0.001). GSTP polymorphisms are associated with reduced ability to detoxify chemotherapeutic agents thereby increasing the effective dose of the drug within the cell. It has also been associated with increased risk of breast, prostate, bladder and testicular cancer. We have also observed a decreased frequency of GSTT wild type (null; 8%, n=4) in FA patients compared to normal controls (25%, n=27) (p=0.01). In conclusion, this study shows that prevalence of GSTP and GSTT gene polymorphisms in FA is different from the normal population.
Human CIK Maintain Their In Vitro and In Vivo Anti-Tumor Ability after Cryopreservation.
Authors: Baker Jeanette , Sheehan Kevin , Monterola Gina , Staines Nancy , Negrin Robert S. 
Published: April 2020

Abstract
Adoptive cellular therapy holds promise for improving the outcome of hematopoietic cell transplantation (HCT). At present, donor lymphocyte infusion post-HCT is efficacious for only a limited number of diseases, yet can induce significant graft versus host disease (GVHD). To improve the outcome of this approach, it would be beneficial to identify populations of T cells that retain graft versus tumor (GVT) effects with reduced propensity for GVHD. We have previously described studies of murine expanded Cytokine Induced Killer (CIK) cells which are ex vivo activated and expanded T cells that express both T and NK markers. CIK cells mediate cytotoxicity both in vivo and in vitro in a non- MHC restricted NKG2D dependent manner. Human CIK cells were expanded from PBMC from 9 healthy donors, cultured with IFNg, CD3 and IL-2 and maintained in AastromRepliCell® biochambers for 21–28 days. We aimed to determine whether cryopreservation of the CIK affects viability, cytotoxicity and phenotype. Cells were cryopreserved immediately after harvest at 10x106/ml and stored in liquid nitrogen vapor phase. CIK viability was not compromised with cryopreservation and cells thawed at 1, 2, 4, 8, 10 and 28 weeks after freezing were 96% viable (range 95%–99%). Immediately upon thawing, CIK cells showed diminished cytotoxicity against the B cell lymphoma cell lines DB and SUDHL4 with 6–10% killing at the 40:1 E:T ratio. However, thawed CIK cells regained their pre-freeze cytotoxic activity against these targets within 5 hours of being placed in reactivation medium containing IL-2 at 300 IU/ml. Reactivation of the CIK cells was extended up to 48 hours but showed no further increase in cytotoxicity beyond that attained at 5 hours; nor did increasing the IL-2 concentration to 1500 IU/ml in the reactivation medium improve CIK cell activity over the same time course. Cell viability declined during reactivation, decreasing from an average 96% upon thawing to 60% over 48 hours. Thawed CIK cells placed in reactivation medium maintained their cytotoxic activity up to 14 days in vitro. The cytotoxicity of reactivated CIK cells was assessed in vivo using SCID mice inoculated IP with 1x106 human ovarian cancer UCI-101 cells expressing the firefly luciferase gene. The mice were treated weekly with 2x107 cryopreserved and thawed human CIK cells that were re-cultured for 5 hours before injection. Following each administration of CIK cells, there was a reduction of tumor signal. Weekly treatments resulted in a better survival outcome for the mice receiving CIK cells as compared to PBS control mice. This study demonstrates that human CIK cells may be reactivated after cryopreservation and regain their cytotoxic potential. These finding have important implications for the application of these cells as adoptive cellular therapy.
Interferon-gamma Expressing EBV LMP2A-Specific T Cells for T Cell Therapy Against EBV-Positive Hodgkin’s Lymphoma.
Authors: Sun Qi , Niyazova-Brewer Nargisa , Bao Lei , Chen Lipai , Lucas Kenneth G. 
Published: April 2020

Abstract
Hodgkin’s lymphoma (HL) accounts for approximately 67% of lymphomas. Patients with HL refractory to standard intervention have poor prognoses. LMP2A is one of the Epstein-Barr virus (EBV) oncoproteins persistently expressed in approximately 50% of HL cases, providing an ideal target for T cell therapy against EBV-positive HL. However, polyclonal EBV-specific T cells have achieved only limited responses in clinical trials, with eventual disease progression or relapse. While multiple possibilities could underlie the unsatisfactory efficacy, the most obvious shortcoming of the current generation of therapeutic T cells is the low frequency of HL-relevant effectors in the T cell preparations. We designed a strategy to enrich LMP2A-specific T cells from polyclonal EBV-specific T cell preparations. With an “Interferon (IFN) Capture” procedure, LMP2A-specific T cells were isolated from T cell cultures generated with stimulation by autologous EBV immortalized B lymphoblastoid cells based on their antigen-specific expression of IFN-gamma. The enriched LMP2A-specific T cells were subsequently expanded with the anti-CD3 antibody OKT3 to a scale applicable for clinical use. Our results showed that a ten fold enrichment can be achieved for T cell cultures containing as low as 0.5% LMP2A T cells, and 5 fold for those containing higher than 10%. Thus, 2 consecutive expansions increased the LMP2A-specific T cells to nearly 50% from 0.5% in the starting culture. Cytokine expression analysis showed that the enriched T cells retained their antigen specific production of IFN-gamma, and a subpopulation upto 10% of the IFN-gamma producing T cells were capable of expressing interleukin (IL)-2. In contrast, no cells expressing immuno-suppressive cytokines IL-4, IL10 and TGF-beta were detected. The enriched T cells were mostly CD45RO+/CD45RA-, indicating that they were proliferation competent. Chromium release assays confirmed that the enriched T cells maintained antigen-specific cytotoxicity. With a panel of four HLA A02 restricted tetramers, we could further demonstrate that the enriched T cells were polyclonal recognizing multiple LMP2A epitopes and retained their spectrum of specificity throughout the enrichment and expansion. This system provided the foundation for T cell therapy against EBV-positive HL with new generation therapeutic T cells.
Better Molecular Response (MR) to Imatinib (IM) in Early Chronic Phase (CP) Versus Late CP Chronic Myeloid Leukemia (CML) Patients (pts) in Complete Cytogenetic Response (CCR): A Comparison at 24 Months of 2 Clinical Trials of the GIMEMA Working Party on CML on Behalf of the GIMEMA Working Party on Chronic Myeloid Leukemia (GIMEMA-CML).
Authors: Poerio Angela , Amabile Marilina , Iacobucci Ilaria , Soverini Simona , Colarossi Sabrina , Ottaviani Emanuela , Terragna Carolina , Grafone Tiziana , Luatti Simona , Castagnetti Fausto , Pane Fabrizio , Saglio Giuseppe , Russo Domenico , Rosti Gianantonio , Baccarani Michele , Martinelli Giovanni 
Published: April 2020

Abstract
We sought to determine the differences in molecular response between early and late CP pts with CML who achieved a CCR after treatment with IM at the standard dose of 400mg/d. We studied 2 different cohorts of patients in CCR: 67/191 (35%) pts after α-Interferon (α-IFN) failure enrolled on the CML/002/STI571 protocol
53/76 (70%) pts treated front line with a combination of IM and pegilated IFN-α (PEG-IFN) enrolled on the CML/011/STI571 protocol
Cytogenetic response was monitored on bone marrow (BM) metaphases and molecular response was assessed by real time RT-PCR (TaqMan) BM and peripheral blood (PB) samples, collected at baseline, 3, 6, 9 and 12 months during the first year, and every 6 months thereafter. Molecular response was expressed as the ratio between BCR/ABL and β2-microglobulin (β2-M) x100. The lowest level of detectability of the method was 10−5. Negative results (i.e. undetectable transcript) were confirmed by nested PCR performed 4 times (sensitivity 10−6).
For the purpose of this analysis, a major molecular response (MMR) was defined as a BCR-ABL/β2M value &lt;0.0001%, which turned out to be roughly equivalent to a 3-log reduction and a complete molecular response (CMR) was defined as negative (undetectable) BCR/ABL levels confirmed by nested PCR.
We observed a progressive decrease of the amount of BCR/ABL transcript in pts who achieved a CCR. At 24 months the median reduction in BCR/ABL transcript level was:
a 3-log reduction in late CP pts
a 4-log reduction in early CP pts
In the latter group of pts MR was assessed also at 36 months. So we observed that 36 months after the first dose of IM and PEG-IFN pts who were still in CCR had the median value of BCR/ABL transcript of 0.00001% both in BM and PB. Therefore all these pts achieved a MMR. However only 8/53 (4%) pts were in CMR (undetectable BCR/ABL at least once as assessed by nested PCR).
We conclude that front-line treatment with IM results in a better quality MR (4-log reduction in BCR/ABL transcript levels in early CP pts, as against a 3-log reduction in late CP pts).
Figure Figure