bluuebunny/crossref_metadata_2025_split huggingface dataset:
Activation of Nucleic Acid-Sensing Toll-Like Receptors Induces Proliferation, Cytokine Production, Immunogenic Phenotype, and Plasma Cell Differentiation of CLL Cells and Immunoglobulin Production.
Authors: Paul Santanu , Chu Charles C. , McCarthy Brian A. , Boyle Erin , Steinberg Bettie M. , Kaufman Matthew , Kolitz Jonathan , Allen Steven L. , Rai Kanti R. , Chiorazzi Nicholas 
Published: October 2019

Abstract
Weak immune function of chronic lymphocytic leukemia (CLL) cells may contribute to disease progression and inhibit effective immunotherapy. Accordingly, agents that enhance the function of CLL cells may be useful in immunotherapeutic approaches to this disease. Since Toll-like receptors (TLRs) are major regulators of B-cell function in innate and adaptive immunity, we asked to what extent stimulating two nucleic acid-sensing TLRs affected the activation and differentiation of CLL B cells. Isolated CLL cells were treated with either the TLR7-activating ligand, gardiquimod, or the TLR9-activating ligand ODN 2006-G5. CLL cells were also treated with the contents of apoptotic leukemic B cells, produced by repeated freezing and thawing. Ability to induce proliferation, as defined by CFSE dilution studies, cytokine production, differentiation, and altered surface molecule expression was measured. Both TLR 7 and TLR 9 ligands individually induced high levels of proliferation and plasma cell differentiation of CLL B cells. A dramatic increase in percentage of dividing cells was seen with simultaneous TLR7 and TLR9 activation, while blocking the TLR 7/9 pathway by pre-treating CLL B cells with chloroquine or bafilomycin reduced the proliferative capacity by 90%. Furthermore, stimulating CLL B cells with TLR7 and 9 ligands significantly up-regulated activation (CD38, CD69 and CD83), adhesion (CD54), and other immunophenotypic (CD40, CD80 and CD86) markers. Enzyme immunoassays of culture supernatants after TLR 7 and TLR 9 stimulation indicated that the levels of soluble IgM increased considerably, although levels of cytoplasmic IgM were unchanged. Production of IL6 and IL10 was also induced. We propose that ligands for TLRs 9 and 7 stimulate CLL B cells to proliferate and differentiate into plasma cells in the absence of T-cell help. In addition these stimuli alter CLL cell surface phenotype to a more immunogenetic profile, providing a rationale for CpG oligonucleotides and gardiquimod as humoral vaccine adjuvants for both normal immune reactions and potentially for anti-leukemic therapy.
Secondary Prophylaxis in a Cohort of Adolescent and Adult Italian Hemophiliacs.
Authors: Tagliaferri Annarita , Rivolta Gianna Franca , Coppola Antonio , Santoro Cristina , Rossetti Gina , Dragani Alfredo , Feola Giulio , Zanon Ezio , Iannaccaro Pier Giorgio , Radossi Paolo , Franchini Massimo 
Published: October 2019

Abstract
Although primary prophylaxis is a well-established and widely recommended practice for the management of patients with severe hemophilia, there are limited data in the literature documenting the benefit of secondary prophylaxis initiated in adolescence or adulthood. In order to address this issue we have conducted a retrospective study among the 10 Italian Hemophilia Centers on adolescent and adult patients with severe hemophilia switched from on-demand treatment to a long term secondary prophylaxis regimen. On the whole, we collected information on 73 consecutive patients with severe hemophilia (66 hemophilia A and 7 hemophilia B). Their median age started prophylaxis was 12 (range 10–17) for 23 adolescents and 30 (17.4–72) for the 50 adults and continued for a median of years 5.8 (2–14.7) in adolescents and 4 years (1.5–12) in adults. The clinical impact of secondary prophylaxis with respect to the last year on-demand regimen before switching is showed in the following table for each group of patients.
Adolescents (n=23) Adults (n=50) On-Demand Prophylaxis On-Demand Prophylaxis Values are reported as mean (standard deviation) Bleedings 32.4(24.8) 2.8 (3.2) 42.7 (24.5) 5.5 (5.2) Joint bleedings 25.6 (22.1) 1.9 (2.3) 36.4 (24.5) 4.2 (4.1) Orthopaedic score 8.4 (7.5) 3.7 (2.4) 21.6 (12.2) 17.7 (12.2) Pettersson score 5.1 (3.4) 5.0 (3.3) 14.7 (13.8) 15.2 (14.3) Work/school days lost 35.1 (34.5) 1.0 (1.5) 33.8 (20.9) 2 (0–17) Concentrate consumption U/Kg/y 2449.8 (1761.9) 3508.3(803.1) 3222.6 (2241.8) 4058.5 (952.3)
The decreased joint bleeding, better orthopaedic score, less work/school days lost, delayed progression of arthropathy and improved well-being observed with secondary prophylaxis show the benefit of this therapeutic approach and appear to counterbalance its higher clotting factor consumption. In conclusion large, prospective, long-term randomized studies are needed to definitevely assess the clinical impact of this strategy in adolescent and adult hemophiliacs and to identify those groups of patients who would benefit from this therapeutic approach.
Flt3-Ligand Plays a Critical Role in Restoring Function of NOD Facilitating Cells.
Authors: Huang Yiming , Fugier-Vivier Isabelle J. , Miller Thomas , Elliott Mary J. , Tanner Michael K. , Xu Hong , Atay Safinur , Hussain Lala-Rukh , Bozulic Larry D. , Chilton Paula M. , Ildstad Suzanne T. 
Published: October 2019

Abstract
CD8+/TCR− facilitating cells (FC) enhance engraftment of purified hematopoietic stem cells (HSC) in syngeneic and allogeneic recipients. FC also induce the production of regulatory T cells (Treg) in vivo and in vitro. The B220+/CD11c+/CD11b− precursor plasmacytoid dendritic cell (p-preDC) subpopulation in FC (p-preDC FC) is critical to FC function. However, p-preDC FC are significantly less efficient in function compared to FC total. In this study, we evaluated the phenotype and function of FC from diabetes-prone nonobese diabetes (NOD) mice. We found that NOD FC contain subpopulations similar to those previously described in B6 FC, including p-preDC, CD19+, NK1.1+DX5+ and myeloid cells. P-preDC represent the major FC subpopulation in NOD mice. The CD19+, DX5+ and B220−/CD11c+/CD11b+ subpopulations were significantly decreased in NOD FC compared to those from B6 or NOR mice (Figure 1a; * = P< 0.05; ** = P< 0.007). To test the function of NOD FC, 500 HSC (c-Kit+/Sca-1+/Lin−) were sorted and transplanted with or without 30,000 FC into conditioned 950 cGy recipients. MHC-matched diabetes-resistant NOR mice were served as a control strain for NOD mice. 5 (31%) of 16 recipients of NOR HSC were engrafted and survived up to 130 days. 7 (70%) of 10 recipients of NOR HSC plus FC have long-term engraftment and survival over 130 days, indicating that the NOR FC significantly enhanced engraftment of NOR HSC compared to HSC alone. In striking contrast, NOD FC were functionally impaired and did not enhance HSC engraftment in NOD recipients as evidenced by similar engraftment of HSC with FC (31%, n = 13) compared to the HSC alone (24%, n = 17; P = 0.579). Notably, when NOD mice were treated with Flt3 ligand (FL; 10 μg/ subcutaneous/daily, 10 days), FC were expanded in peripheral blood (PB). The DX5+ and B220−/CD11c+/CD11b+ subpopulations were significantly increased (Figure 1b). The FL-PB FC significantly facilitate engraftment of allogeneic HSC in vivo in the NOD → B10 model (Figure 1c; P = 0.02). These data demonstrate that NOD FC exhibit significantly impaired function and FL plays an important role in regulation and development of FC function. We propose that the defective function of NOD FC is mechanistically due either to the abnormal activation status of the p-preDC FC population and/or the absence of collaborative subpopulations in FC such as NK FC. This hypothesis offers an attractive explanation for the mechanism of FC to enhance HSC engraftment and to induce tolerance. Studies are underway to evaluate the mechanism by which FC contribute to diabetes-pathogenesis and/or prevention.
Figure Figure
Feasibility of Rapidly Generating Sufficient Autologous Human Marrow Stromal Cells for Cellular Therapy.
Authors: Lane Thomas A. , Garls Davina , Mackintosh Ellen , Kohli Sanjivan , Cramer Steven C. 
Published: October 2019

Abstract
Marrow stromal cells (MSC) are increasingly employed for human cellular therapy protocols. In some cases, it may be advantageous to use autologous MSC, but the feasibility of quickly generating sufficient MSC for autologous therapy has not been established. We investigated the feasibility of rapidly generating large numbers of autologous, early passage, MSC as a prerequisite for the clinical investigation of MSC in human ischemic stroke. Bone marrow (25–35 mL) was collected from 8 healthy volunteers under an IRB-approved protocol; a mononuclear cell (mnc) fraction prepared by ficol density gradient, a portion of the mnc fraction was suspended in low glucose DMEM with 20% pre-screened FBS and plated in T175 culture flasks at 0.2–0.4 × 106 mnc/cm2; maintained at 37 C in a 5% CO2 incubator; non-adherent cells were removed at 48 h and a portion of the cells were passaged when 80% confluent into additional flasks (12–14 days) at 5700 cells/cm2 and MSC were harvested with trypsin at 21 days. The MSC were washed x3 and resuspended in sterile saline for enumeration and suitability testing. MSC were identified according to ISCT criteria (Dominici M, Cytotherapy2006;8(4):315), including plastic adherence, morphology, growth characteristics, and phenotype by flow cytometry (CD73, CD90, CD105 positive; CD14, CD34, CD45, HLA-DR negative) and differentiation into adipocytes. The expected total cell yield was calculated based upon actual cell yields minus the volume of cells required for testing based upon a 70 kg patient.
Results: In 8 experiments using different donors, a median of 40 (range 20–225) × 106 mnc were available for plating from the original marrow. The median mnc recovery after ficol density gradient was 37% (range 23–130), but the median CFU-F recovery after ficol from the original marrow was 96% (94–96). The calculated MSC yield after primary culture was a median of 25 (10–53) × 106 cells. The calculated yield of MSC after 1st passage at 21 days was a median of 2.1 (0.9–4.4) × 106 MSC/kg (70 kg patient weight), and the median cell viability was 98.5% (92–97.9). At the end of 1st passage all samples yielded negative gram stains and cultures for mycoplasma, bacteria, fungal growth and the median endotoxin level was 0.027 EU/mL (0.02 – 0.10). The viability of the harvested MSC was well maintained in saline at 1 × 106 MSC/mL for 24 h at 4 C (85% +/− 9%) but not after 48 h (37% +/− 9%) or if stored at room temperature. The effects of supplementation with 5% human serum albumin on MSC storage for 24 h at 4 C were variable, ranging from 27% to 95% viability, depending on the albumin manufacturer (3 mfrs; n=4 each).
Conclusion: These studies suggest that sufficient MSC that are suitable for autologous human cellular therapy can be rapidly and reliably generated under GTP conditions, that the resultant MSC may be preserved for up to 24 h if stored at 4 C in either saline or pre-screened human serum albumin.
Enrichment of CD34+ Hematopoietic Stem and Progenitor Cells from Human Bone Marrow Using a P-Selectin-Coated Microtube.
Authors: Narasipura Srinivas D. , Liesveld Jane L. , Wojciechowski Joel C. , Charles Nichola , Rosell Karen , King Michael R. 
Published: October 2019

Abstract
Enrichment and purification of hematopoietic stem and progenitor cells (HSPCs) is important in transplantation therapies for hematological disorders and for basic stem cell research. Primitive CD34+ HSPCs have demonstrated stronger rolling adhesion than mature CD34- mononuclear cells on selectins (Blood2000; 95:478–486). We have exploited this differential rolling behavior to capture and purify HSPCs from bone marrow, by perfusing mononuclear cells through selectin-coated microtubes. Bone marrow mononuclear cells were perfused through the cell capture microtubes coated with adhesion molecules. These utilized a parallel plate flow chamber (Glycotech), and the P-selectin was adsorbed with laboratory tubing of appropriate lengths attached to the inlet, outlet, and vacuum ports of the gasket chamber. After perfusion, the device lumen was washed and captured cells were visualized and estimated by video microscopy. “Rolling” cells were defined as cells translating at less than 50% of the calculated hydrodynamic free stress velocity. Velocities of single cells were determined using a MATLAB program designed to measure the change in position of the cell centroid in a given time period. Adherent cells were eluted by high shear, calcium free buffer and air embolism. Immunofluorescence staining followed by flow cytometry was used to analyze CD34+ HSPCs. CD34+ HSPC purity of cells captured in adhesion molecule-coated devices was significantly higher than the fraction of CD34+ cells found in bone marrow- mononuclear cells (2.5 ± 0.8%). P-selectin coated surfaces yielded 16–20% CD34+ cell purity, while antibody coated surfaces yielded 12–18%. Although the CD34+ cell purities were comparable between selectin and antibody surfaces, the total number of CD34+ HSPCs captured was significantly higher in P-selectin devices (∼5.7–7.1 × 104) when compared to the antibody device (∼1.74–2.61 × 104). Furthermore, analysis for cells positive for CD133, a surface marker for more primitive HSPCs, depicted approximately 10–14 fold enrichment in P-selectin samples over control bone marrow mononuclear cells. The captured cells were viable and exhibited in vitro colony forming capabilities. Thus, P-selectin can be used in a compact flow device to capture and enrich HSPCs. This study supports the hypothesis that flow-based adhesion molecule-mediated capture may be a viable physiologic approach to the capture and purification of HSPCs.
Reciprocal Activation of GATA-1 and PU.1 Marks Initial Specification of Hematopoietic Stem Cells into Myelo-Erythroid and Myelo-Lymphoid Lineages.
Authors: Arinobu Yojiro , Mizuno Shin-ichi , Chong Yong , Shigematsu Hirokazu , Iino Tadafumi , Iwasaki Hiromi , Graf Thomas , Mayfield Robin , Chan Susan , Kastner Philippe , Akashi Koichi 
Published: October 2019

Abstract
Understanding how multipotent cells commit to each of their terminal fate potentials is an important aspect of stem cell biology. In adult murine hematopoiesis, HSCs with long-term self-renewal potential reside within the Lin −Sca-1+c-Kit+ (LSK) fraction having CD34−, Thy1lo, and Flt3/Flk2−phenotypes. The LSK cells having CD34+, Thy1−, and/or Flt3+ phenotypes are capable of multi-lineage reconstitution only for a short-term, and therefore should contain multipotent progenitors (MPPs). In terms of developmental steps downstream of MPPs, there has been a controversy. The existence of prospectively isolatable progenitors capable of generating only myeloerythroid cells (common myeloid progenitor: CMP) or only lymphocytes (common lymphoid progenitors: CLP) outside the LSK fraction suggests that the first commitment step after the MPP stage is the strict bifurcation of lymphoid vs. myeloid pathway. Recently, however, several studies have suggested that the lineage commitment could occur within the LSK fraction, preceding the proposed bifurcation of myeloid and lymphoid pathways. For example, MPPs expressing Flt3 at a high level retained granulocytes/monocytes (GM) but not megakaryocyte/erythrocyte (MegE) potential together with lymphoid potential, suggesting the existence of common progenitor for GM and lymphoid lineages within the LSK fraction. Based on this data, the coupled loss of self-renewal activity and MegE potential in the early HSC commitment has been proposed. How can we reconcile the controversy in the early hematopoietic lineage map? By utilizing mice having GATA-1 or PU.1 transcriptional reporters, we here present a high-resolution map containing new lineage-restricted progenitor populations within the MPP population of CD34+ LSK phenotype. Initial upregulation of GATA-1 and PU.1 occurred independently at the MPP stage. The GATA-1+ MPP displayed potent myeloerythroid potential without giving rise to lymphocytes, whereas the PU.1+ MPP showed granulocyte/monocyte/lymphoid-restricted progenitor activity without megakaryocyte/erythroid differentiation. Furthermore, GATA-1+ and PU.1+ MPPs possessed huge expansion potential, and differentiated into the original CMPs and CLPs, respectively. Thus, the reciprocal activation of GATA-1 and PU.1 primarily organizes hematopoietic lineage fate decision to form the earliest hematopoietic branchpoint that comprises new, isolatable myeloerythroid and myelolymphoid progenitor populations.
Myeloid Gene Activation and T Cell/Natural Killer Cell Gene Repression in Cells Expressing Two Distinct PU.1 Concentrations.
Authors: Kamath Meghana , Houston Isaac , Janovski Alexander , Zhu Xiang , Gowrisankar Sivakumar , Jegga Anil , DeKoter Rodney 
Published: October 2019

Abstract
The Ets transcription factor PU.1 (encoded by the gene Sfpi1) functions in a concentration-dependent manner as a hematopoietic cell fate determinant. PU.1 levels are uniform in early hematopoiesis, increase during myeloid differentiation, and decrease after erythrocyte and T cell/natural killer cell commitment. It is unknown how downstream target genes respond to changes in PU.1 concentration. To address this, we generated mice with two distinct hypomorphic alleles of Sfpi1 and analyzed interleukin-3 dependent cell lines from fetal liver cells homozygous for either allele. PU.1 was produced in these cells at ∼20% (Sfpi1BN/BN) or ∼2% (Sfpi1Blac/Blac) of wild type. These cells fail to terminally differentiate as a consequence of low PU.1 expression and can be maintained as cell lines. To determine what groups of genes are expressed in response to two distinct PU.1 concentrations, we performed whole-genome microarray analysis and compared gene expression in Sfpi1BN/BN and Sfpi1Blac/Blac cell lines to Sfpi1−/− cell lines. Groups of downstream target genes were activated or repressed in four modes in response to the two discrete concentrations of PU.1:
at higher but not lower PU.1 concentration, at lower but not higher PU.1 concentration, at both lower and higher concentration, and in a gradient fashion.
We decided to focus on genes regulated in a gradient manner, because dose-dependency suggests that these may be direct targets of PU.1. Genes activated in a gradient manner were mostly myeloid-specific and enriched for target genes of PU.1. Genes repressed in a gradient manner included erythroid-specific genes and, unexpectedly, T cell and natural killer cell-specific genes. T cell genes were also repressed by PU.1 in cultured progenitor-B cells. With this unique allelic system, we can study three discrete concentrations of PU.1 at 20%, 2%, and 0% to examine concentration-dependent effects of PU.1 on target genes and lineage decisions. Overall, our results suggest that PU.1 functions in a concentration-dependent manner to promote myeloid differentiation and repress T cell or natural killer cell development.
Inhibition of VPAC1 Signaling Raises Platelet Numbers after Myelosuppressive Therapy and in GATA1 Congenital Thrombocytopenia.
Authors: Peeters Karen , Freson Kathleen , Wittevrongel Christine , Thys Chantal , Stassen Jean-Marie , Hoylaerts Marc F. , Van Geet Chris 
Published: October 2019

Abstract
Thrombocytopenia is a serious complication in patients undergoing myelosuppressive chemotherapy. Therefore, the development of new strategies to reduce thrombocytopenia or accelerate platelet recovery without adverse effect on the bone marrow (BM) is still warranted. Previously we found that the pituitary adenylate cyclase activating peptide (PACAP) is a physiological inhibitor of platelet activation (JCI, 2004). The receptor for PACAP on megakaryocytes (MKs) and platelets is the Gs coupled receptor VPAC1. We have presently investigated whether the neutralization of the regulatory role of VPAC1 can affect thrombopoiesis, independently of thrombopoietin (TPO). We found that inhibition of VPAC1 signaling stimulates in vitro megakaryopoiesis, since more MKs could be generated from Sca1+ cells, isolated from normal mouse BM by incubation with a neutralizing anti-VPAC1 antibody (Ab) (23A11). 23A11 was also capable of enhancing the later stages of MK proliferation of human cord blood derived CD34+ cells. These in vitro findings were validated in two in vivo animal models of myelosuppressive therapy. The subcutaneous injection of a neutralizing Ab directed against PACAP (PP1A4) or 23A11 (1 mg/kg on days 0,3 and 7) boosted platelet recovery in mice after chemotherapy-induced thrombocytopenia (20 mg/kg busulfan intraperitoneally on days 7 and 10) in a TPO-independent manner. Platelet recovery in mice, injected with PP1A4, was also significantly increased compared to non-treated mice exposed to 8 Gy total body irradiation. Plasma TPO concentrations did not differ between the two groups. We next questioned whether VPAC1 inhibition could rescue the GATA1-defective phenotype due to a developmental arrest in the megakaryoblast stage. FACS analysis of BM Sca1+ cells from GATA1 deficient mice cultured during 12 days in MK differentation medium in vitro showed an increased MK DNA ploidy when 23A11 was added to the culture. A similar observation was made with CD34+ cells from BM of a GATA1 deficient patient with severe thrombocytopenia. GATA1 deficient mice are thrombocytopenic, with a platelet count of 50 +/− 15 x 10(9) plt/L. This platelet number increases to 150 +/− 40 x 10(9) plt/L after subcutaneous injection with 23A11 whereas injection with PBS had no effect. Again, TPO plasma levels in these injected GATA1 deficient mice showed no association between anti-PACAP or anti-VPAC1 treatment. Thus, our data indicate that VPAC1 signaling tempers normal megakaryopoiesis and that elimination of this pathway facilitates platelet recovery after myelosuppressive therapy and in GATA1 deficiency.
The Indoleamine 2,3-Dioxygenase Pathway Is Essential for Human Plasmacytoid Dendritic Cell-Induced Adaptive T Regulatory Cell Generation.
Authors: Chen Wei , Liang Xueqing , Peterson Amanda J. , Munn David H. , Bruce Blazar R. 
Published: October 2019

Abstract
Plasmacytoid dendritic cells (PDCs) are a unique DC subset that plays a critical role in regulating innate and adaptive immune responses. Recently, we have shown that human PDCs activated by CpG oligodeoxynucleotide (CpG ODN) can drive naive, allogeneic CD4+CD25− T cells to differentiate into CD4+CD25+Foxp3+ regulatory T cells (Tregs). However, the intracellular mechanism(s) underlying PDC-induced Treg generation is unknown. Here we show human PDCs express high levels of indoleamine 2,3-dioxygenase (IDO), an intracellular enzyme that catabolizes tryptophan degradation. Triggering of Toll-like receptor 9 (TLR9) with CpG oligodeoxynucleotides activates PDCs to upregulate surface expression of B7 ligands and HLA-DR antigen, significantly increases the expression of IDO, and results in the generation of inducible Tregs from CD4+25− T cells with potent suppressor cell function. Blocking IDO activity with a pharmacologic inhibitor 1-methyl-D-tryptophan (1MT) significantly abrogates PDC-driven inducible Treg generation and suppressor cell function. Adding kynurenine (KYN), the immediate downstream metabolite of tryptophan, bypasses the 1MT effect, and restores PDC-driven Treg generation. Our results demonstrate that IDO pathway is essential for PDC-driven Treg generation from CD4+25− T cells, and implicates the generation of KYN-pathway metabolites as the critical mediator of this process.