bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of a PML-RARα-IL-2 Recombinant Plasmid DNA for APL.
Authors: Zhou Yubing , Hu Gang , Cen Dongzhi , Yang Lijian , Chen Shaohua , Li Yangqiu 
Published: October 2019

Abstract
Specific active immunotherapy with DNA vaccines is one of the best approaches to eradicate minimal residual disease in cancer patients, when appropriate tumor-specific antigens are identified for this disease. However, little data are regarding the DNA vaccines in acute leukemia, recent reports has shown that a human PML-RARα breakpoint-drived DNA vaccine can prevent APL in mice model, but the host immune response were weakly, due to the weak immunogenicity. In order to improve the effect of DNA vaccine for acute promyelocytic leukemia (APL) therapy, we develop a vector coexpressing PML-RARα gene fused to human Interleukin- 2 (hIL-2) gene. PML-RARα fusion gene segment and the full-lenght hIL-2 gene were amplified from NB4 cells or Jurkat cells. Both PCR products were cloned into PIRES plasmid respectively to construct a recombinant plasmid PML-RARα-IRES-hIL-2. The recombinant plasmids were then transfected into K562 or A549 cells respectively. The expression of the PML-RARα/hIL-2 mRNA and protein in transfected cells were identified by RT-PCR, dot blotting, ELISA and Western-Blot respectively. By in vivo assays, BALB/c mice were vaccinated at 6–8 week of age with a total of 200 μg DNA in normal saline, injected into two sites in the quadriceps muscles on day 0, 7 and 21. The plasmid containing the same sequence of PML-RARα gene and blank plasmid served as controls. Two weeks after the final DNA boost, both PML-RARα/hIL-2 mRNA and protein, serum INF-γ and anti-NB4 cells specific cytotoxicity of splenocytes following 7 days of stimulation in vitro with freeze thawing NB4 cells and recombinant human IL-2 were assessed by ELISA and LDH assays. The results showed that the sequence of the fragments inserted in multi-clone site (MCS) A and MCS B of PIRES plasmid were absolutely correct by double restriction enzyme cutting analysis (Sal I/Not I) and sequence analysis, the PML-RARα/hIL-2 mRNA and protein could be identified in transfected K562 or A549 cells and in mice quadriceps muscles. The level of serum INF-γ and cytotoxicity of splenocytes against NB4 cells from immunized mice was significant increased than that from control groups. Our results indicated that the recombinant plasmid expressing PML-RARα and hIL-2 was successfully constructed, which can induce more effective immune response and anti-APL cells effect in animal models. The data suggest that effective vaccination approaches should be possible against APL. It could be farther used in the research as PML-RARα DNA vaccine for APL.
Potent Anti-Leukemic Activity of a Cationic Lipid-DNA Complex.
Authors: Herse Zachary , Chang Stella , Liggitt Denny , Fairman Jeffery , Claxton David F. 
Published: October 2019

Abstract
Cationic Lipid and DNA Complex (CLDC) is a chemically defined preparation of lipids and non-coding bacterial plasmid DNA able to activate antigen presenting and immune effector cells and thereby enhance cellular immune responses. We tested this preparation subcutaneously (SC) in two models of transplantable murine leukemia: 32D -(bcr-abl)p210 (leukemogenic in C3H/HEJ animals) and WEHI-3B cells (leukemogenic in BALB-C mice.) In multiple experiments using the 32Dp210 model a single SC dose of CLDC (200 ul) prevented or significantly delayed death from leukemia when delivered between -1 and 16 days from leukemic challenge. Specifically, control animals died at 28 days (SD=2.73) of leukemia whereas 20/24 similarly leukemia challenged but CLDC treated animals were alive >71 days (p<0.0001 − 4/20 animals died of leukemia). Animals dying of leukemia had swollen spleens and leukemic infiltration of spleen and liver on histopathological evaluation. Similarly Balb-C animals challenged with WEHI-3B cells died at a median of 30 days unless treated with CLDC (1 dose day 6 − 4/5 alive >day 40 p=0.0017). A green fluorescence protein (GFP) expressing subclone of 32Dp210 allowed quantitation of leukemia in peripheral blood (PB). PB leukocytes were 20% GFP positive on day 19. After CLDC on day 19, day 21 PB showed only 2% GFP. Animals receiving 32D-p210-GFP and treated with CLDC and CD8 antibody on day 19 showed higher GFP in followup and died earlier (median day 35) than animals treated with CLDC alone (>42 days) but later than animals receiving no treatment after leukemic challenge (median 23 days, p=0.0002.) See Figure below. We conclude that CLDC shows potent anti-leukemia activity in two murine models of AML. Antibody experiments suggest that CD8 positive effector cells contribute to this effect. Additional experiments are maturing and will be reported. This preparation has promise for activity in human acute leukemias.
Figure Figure
Y-Chromosome Genes Strongly Expressed in Acute Myeloid Leukaemia.
Authors: Gallo Antonio , Bund Dagmar , Mindnich Rebecca , Buhmann Raymund , Schmetzer Helga , Adamski Jerzy , Kolb Hans-Jochem 
Published: October 2019

Abstract
In hematopoietic stem cell transplantation (HSCT) the gender of the donors has a substantial impact on the relapse rate, graft-versus-host reaction and overall therapeutic outcome. In several studies including our own HLA-haploidentical setting female to male transplantation seemed to be superior to other gender combinations, suggesting the dominance of Y-encoded minor histocompatibility antigens for graft-versus-leukemia reactions. In order to explore the therapeutic possibilities we compared the Y-chromosomal gene expression between isolated AML blasts (FAB M4/M5) and isolated CD14+ cells from healthy male donors. RNA was isolated and subsequently analyzed by low density arrays (LDAs), a real time-PCR based method. This way we could identify four Y-encoded genes found to be strongly up-regulated in the AML samples. Bcorl2, a pseudogene; pcdh11y, known to be involved in acquisition of apoptotic resistance in prostate cancer; tgif2ly and vcy, both putative transcription factors. The genes were cloned in plasmids and transferred in 293 cells. The expression of these proteins was analyzed by western blot. Biostatistical soft ware was used to predict the proteasomal cleavage site and binding capacity to HLA-A 0201 of these peptides. Five peptides were found to bind strongly to HLA-A 0201; they are now studied for recognition by female T cells. In summary, the genes identified might be involved in the immune recognition of AML blasts and other tumor cells. These peptides may help to identify and select T memory cells specific for AML blasts.
Ganoderma lucidum Polysaccharides Can Induce Human Monocytic Leukemia Cells into Dendritic Cells with Immunotolerogenic Function.
Authors: Chan Wing Keung , Cheung Christopher , Law Helen Ka Wai , Lau Yu Lung , Chan Godfrey Chifung 
Published: October 2019

Abstract
Previous studies demonstrated Ganoderma lucidum polysaccharides (GL-PS), a form of bioactive β-glucan can stimulate the maturation of monocyte-derived DCs (Mo-DCs). However, the question of how leukemic cells especially in monocytic lineage respond to GL-PS stimuli remains unclear. We hypothesized that GL-PS may also have an effect on leukemic monocytic cells In this study, we used human dendritic cell (DCs) in vitro culture model with leukemic monocytic cell-lines THP-1 and U937 as cell sources. We treated the cells with purified GL-PS (100 μg/mL) or GL-PS with GM-CSF/IL-4 (cocktail). GL-PS alone induced proliferative response on both cell-lines with more markedly on U937. But it induced THP-1 cells to differentiate into cells with typical DC morphology. Additive maturation effect can be shown when THP-1 cells were stimulated with the cocktails shown by significant increase in HLA-DR, CD40, CD80 and CD86 which express in normal peripheral blood Mo-DCs. The GL-PS THP-1 DCs had similar antigen-uptake ability as the Mo-DCs positive control. However, we found that GL-PS THP-1 DCs could not induce allogeneic T cell proliferation in the one-way mixed lymphocyte reaction as Mo-DCs or THP-1, suggesting GL-PS THP-1 DCs were immunotolerogenic in nature. Our data indicated that the GL could induce monocytic leukemic cell proliferations as well as differentiate particular leukemic cells to DCs with immunosuppressive action. The possible clinical impact has to be carefully investigated.
Leukoreduction System Chambers as a Source of Viable Human Peripheral Blood Mononuclear Cells.
Authors: Kim Sinyoung , Kim Han-Soo , Lee Yangsoon , Song Jaewoo , Kim Hyun Ok , Min Yoo-Hong 
Published: October 2019

Abstract
Many blood banks are using apheresis machines to collect blood components such as platelets (PLTs), RBCs, or plasma. Especially, leukoreduced plateletpheresis using apheresis instrument (Trima Accel, Gambro BCT, Lakewood, CO) provided subsidiary cell products retained in leukoreduction system (LRS) chamber that was originally discarded. The LRS chamber is a conical-shaped chamber that uses saturated, fluidized, particle bed filtration technology to remove WBCs from PLTs. In the current study, a total of 24 LRS chambers from different donors were investigated to determine it would be a valuable source of viable human peripheral blood mononuclear cells (MNCs). The proportions of CD3+, CD19+, CD16+/CD56+, CD14+, CD45+ cells, and absolute CD34+ cell count within the LRS chambers were determined by flow cytometry. Dendritic cells (DCs) were generated from the immunomagnetically purified CD14+ cells from LRS chamber and characterized by phenotypic surface marker and stimulatory capacity in an allogeneic mixed lymphocyte reaction. In the LRS chamber, the total number of WBC count was 1.1 × 109 ± 0.3 × 109 and the mean percentage of MNCs was 80.6 ± 13.1%. The mean proportion of T cells, B cells, NK cells, CD14+ monocytes among CD45+ cells was 54.3 ± 11.5%, 6.4 ± 3.1%, 14.6 ± 3.9%, 12.9 ± 7.5%, respectively. Total absolute CD34+ cell count in LRS chamber was 0.95 × 106 ± 0.65 × 106. Also, we could demonstrate CD14+ cells isolated from LRS chamber was capable of differentiating into functionally mature DCs in vitro. LRS chambers are a valuable and convenient source of viable human peripheral blood mononuclear cell population and could replace standard buffy coat preparations for research applications.
0.1 Grams of Sub-Cutaneous Adipose Tissue (SCAT) as Source of Adult Multipotent Mesenchymal Stromal Cells (MSC) for Cell-Based Therapies.
Authors: Dominici Massimo , Sternieri Rita , De Fazio Domenico , Cafarelli Luigi , Guaraldi Giovanni , Spano Carlotta , Rasini Valeria , Paolucci Paolo , Cilli Michele , Piccardi Federica , Astori Giuseppe , Hofmann Ted J. , Horwitz Edwin M. , Conte Pierfranco 
Published: October 2019

Abstract
The purification of multipotent mesenchymal stromal cell (MSC) from sub-cutaneous adipose tissue (SCAT) represents a promising approach for several clinical applications. The isolation procedures have been applied in humans only to fairly large amount (5–300 g) of harvested SCAT (Zuk PA et al., 2001; Van Harmelen V et al., 2004; Meyerrose TE et al., 2007). Thus, we tested whether it might be possible to isolate MSC from far smaller tissue volumes. SCAT specimens from healthy donors which underwent to plastic surgery operations (n=5; mean weight ± SD=116.3±91.9 mg) were processed with a protocol modified from Zuk et al. 2001. The released cells were filtered, centrifuged and seeded with expansion medium. Solely small, undifferentiated and adherent proliferating fibroblasts were soon evident in the cultures and cells reached the first confluence within 12.6±5.1 days. After 40 days of expansion, the SCAT-MSC displayed a robust proliferative potential, with a cell count of 1.32±0.53x108. FACS analyses showed that SCAT-MSC were positive (>95%) for CD90, CD105, CD73 and negative (<1%) for CD45, CD14, HLA-DR, CD31 and 3G5. CD34 antigen was expressed (<50%) in 2 samples. In vitro differentiation assays successfully drove the cells towards osteogenic, adipogenic and chondrogenic phenotypes. Long-term cryopreservation (2 years) did not affect both phenotype and functional properties. Additionally, SCAT-MSC were gene-modified (57±12%) by retroviruses encoding for GFP and subcutaneously injected into NOD/SCID mice without evidence of uncontrolled proliferation. In 20% of these mice, the transplantation of GFP+ cells was followed by the finding of human derived SCAT three months after the trasplants. We here report a novel and safe approach to isolate multipotent cells from small SCAT specimens to be used as platform for cell based therapies with minimal donors’ discomfort.
CD133 Immunomagnetic Separation: How Effective Is the Method for CD133+ Cell Isolation from Umbilical Cord Blood?.
Authors: Pelagiadis Iordanis , Dimitriou Helen , Kalmanti Maria 
Published: October 2019

Abstract
Umbilical cord blood (UCB) is a rich source of stem cells. Characterisation of stem cell subpopulations residing in UCB requires specific stem cell markers and reliable and reproducible protocols. We assessed the feasibility of CD133 positive cells’ isolation using immunomagnetic cell sorting protocols. The separation was based on the manufacturer’s instructions (Myltenyi Biotec). We compared these results with the corresponding ones of CD34 isolation. Two additional protocols were also applied to increase the efficiency of the CD133+ yield. The first one included an extra-labeling of the selected CD133 subpopulation from the first column with CD133 microbeads before their application to the second column. The second protocol was an indirect labelling procedure, using CD133-PE monoclonal antibody and either anti-PE microbeads or goat anti-mouse IgG microbeads as well as a combination of them. The CD133 isolation was performed in 18 UCB samples, while the CD34 in 17 samples. The volume of the samples used for CD133+ isolation was 38.5 ± 2.1ml and the respective one for the CD34+ protocol was 40.4 ± 2.8ml. The mononuclear cell fraction had 1.29 ± 0.16 x108 cells and 0.53 ± 0.06% of them expressed CD133. The corresponding values for samples used for CD34 isolation were 2.8 ± 0.3 x108 mononuclear cells and 1.64 ± 0.15% CD34. Following the manufacturer’s instructions for CD133 isolation, the number of cells obtained was 21.33 ± 3.3x104 (median 18 x104) and CD133 expression ranged from 10–85% (median=60%). Concerning the CD34 isolation, the number of cells obtained was 54.3 ± 2.1x104 and 93 ± 1.25% expressed CD34. Applying the extra-labeling method, the number of cells obtained was 8±4 x104 and these cells were CD133+ at a percentage of 32.2±4.1%. Applying the indirect immunomagnetic separation the results were as follow: using anti-PE microbeads only, 3 ± 3 x104 cells were retrieved and the percentage of CD133+ was 13.8±9.7. With the use of goat anti-mouse IgG microbeads the cell number obtained was 13±1 x104 of which CD133+ were just 2.0± 2%. In the combination method, the cells initially isolated with anti-PE microbeads and subsequently with goat anti-mouse IgG microbeads were 20 x 104, of which only 18.5% were CD133+. Finally, labeling the cells initially with anti-PE microbeads and consecutively with goat anti-mouse IgG microbeads followed by column isolation yielded 16x104 cells and 19.1% CD133 positive. In conclusion CD34 isolation by the immunomagnetic method results in highly pure CD34+ population confirming the reliability and reproducibility of the method. On the other hand, with the currently available CD133 isolation kit, efficient and reproducible yield of pure CD133 positive population is not feasible.
Stem Cell Recovery Following Implementation of an Automated Cord Blood Processing System in a High Volume Laboratory.
Authors: Harris David T. , Paulson Ingrid E. , Brown Heather , Rosenthal Joseph 
Published: October 2019

Abstract
Background: Many cord blood (CB) processing facilities are transitioning to automated systems to standardize methods and decrease human error. The AutoXpress Platform™ (AXP™) is an automated, functionally closed, sterile CB processing system. When placed within the AXP device and centrifuged, the whole blood product is separated into its composite cell populations and the TNC fraction of the CB unit is separated and automatically delivered into a blow-molded freezing bag at a uniform volume of 21 mL. Objective: This study evaluated the use of the AXP system in a directed donor (DD) family cord blood bank, targeted at producing consistently high TNC and MNC recovery rates regardless of broad variability in collection volume.
Methods: Cord blood units were collected between 12/5/2006 and 2/24/2007 from 1414 consenting mothers who elected to preserve and bank CB at Cord Blood Registry (CBR). Collection kits were provided at the time of enrollment, and after delivery, CB was collected from the umbilical cord and transported to CBR’s processing facility in Tucson, Arizona. During the study period, all units arriving at the CBR laboratory were allocated to either ficoll or AXP processing based on the volume, age, and the degree of clotting in each unit. Units processed using AXP had a volume of 40–130 mL, an age of less than 48 hours since collection, and a clotting score of 0 to 2+ (based on an internal scale). TNC and MNC counts were measured both pre- and post-processing, using the Sysmex analyzer.
Results: The mean age of cord blood units arriving at the laboratory was 23.59 hours, and the mean collection volume was 72.93 mL (±18.10mL). The mean TNC count post-processing was 9.94x108, the TNC percent recovery was 96.19%, and the mean MNC percent recovery was 98.65%.
Volume (mL) Mean Post-Processing TNC (x108) TNC Percent Recovery (%) Mean Post-Processing MNC (x108) MNC Percent Recovery (%) Mean 72.93 9.94 96.19 4.05 98.65 Standard Deviation 18.10 4.43 11.87 1.59 9.16
Conclusions: AXP automated cord blood processing provides consistently high TNC and MNC recovery rates, which has important implications for stem cell dose if the sample is used in transplant. Because limited cell dose is frequently cited as an obstacle to CB transplantation, processing results could impact the usability of each unit. Because DD family CB banks process all units, regardless of collection volume, percent recovery becomes particularly important in evaluating the differences between processing centers. The AXP system yields the highest published cell recovery rate to date and can be easily integrated into a CB processing center such that it decreases the labor and time required for processing while maintaining MNC recovery of greater than 98%.
Characterization of Ex Vivo Expanded Cord Blood Graft Treated with Chromatin Modifying Agents for Potential Clinical Use.
Authors: Yoshinaga Kazumi , Mahmud Dolores , Baluchamy Sudhakar , Parajuli Ritesh , Hiroto Araki , Lindgren Valerie , Rondelli Damiano , Mahmud Nadim 
Published: October 2019

Abstract
The lack of optimal number of repopulating hematopoietic stem cells (HSC) and graft failure following transplantation remain as obstacles to using cord blood (CB) as a true alternative graft for adult patients. Using chromatin modifying agents {5aza-2-deoxycytidine (5azaD) and trichostatin A (TSA)}, we have developed a culture regimen that permits expansion of primitive CB CD34+ CD90+ cells resulting in a 10 fold expansion of in vivo repopulating cells without jeopardizing their multilineage differentiation capacity. Our results also indicate that the chromatin modifying agents likely mediate this function by inducing expression of endogenous genes (HoxB4, Bmi-1, GATA-2, etc.) previously implicated in self-renewal of HSC (Araki H et al. Blood2007:109:3670–3678). In our current studies we have further characterized the allostimulatory function of these expanded CB to assess its potential as grafts to mount an immune response. The percentage of 5azaD/TSA treated expanded CB cells co-expressing HLA-DR and CD86, or CD11c was less than cells expanded in cytokines alone. The expression of HLA-DR/CD40 was not altered following 5azaD/TSA treatment. In a mixed lymphocyte reaction (MLR), the irradiated CB cells (stimulator) were co-cultured with allogeneic peripheral blood T cells (responder) at 1:2 ratios. The MLR results indicate that 5azaD/TSA treated expanded CB grafts possessed diminished allostimulatory response as revealed by their relatively lower stimulation index in comparison to cells expanded in cytokines alone. Interestingly, the stimulation index of 5azaD/TSA treated cells (day 9) was even less than unmanipulated primary CB CD34+ cells (day 0). The reduced allostimulatory capacity of 5azaD/TSA expanded CB cells may favor in the reduction of incidence of graft rejection / graft failure following infusion. Alterations in cell fate regulatory mechanisms raise concerns for tumorigenesis, so the safety of such grafts was further assessed by examining the telomere length and cytogenetic analysis. Both cytogenetic analysis and examination of cell-cycle showed normal karyotype and diploid nature of expanded grafts, respectively. The telomere length of ex vivo expanded grafts as a putative indicator for their proliferative potential did not reveal significant alteration in their length in comparison to unmanipulated CB grafts. Also, in vivo repopulation assays with xenogeneic NOD/SCID mouse model showed no apparent tumor development following transplantation of ex vivo expanded 5azaD/TSA treated CB cells. In summary, our results indicate that the chromatin modifying agents not only result in expansion of in vivo repopulating CB cells but are capable of suppressing immunostimulatory capacity of expanded grafts and these grafts retain their telomere length and normal ploidy and can be expanded from a single unit for potential clinical use.