bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Influence of Polymorphisms of Interleukin-17F Genes on the Susceptibility and Clinical Significanse of Chronic Idiopathic Thrombocytopenic Purpura.
Authors: Saitoh Takayuki , Kasamatsu Tetsuhiro , Yokohama Akihiko , Handa Hiroshi , Tsukamoto Norifumi , Karasawa Masamitsu , Ogawara Hatsue , Nojima Yoshihisa , Murakami Hirokazu 
Published: October 2019

Abstract
Introduction: Recent several cytokine studies have shown Th1 polarization of the immune response in idiopathic thrombocytopenic purpura (ITP) patients. Interleukin-17 F(IL-17F) is a relatively new cytokine that regulates the adaptive and innate immune systems. In vivo studies in murine disease indicate that the Th17 lineage plays a pathogenic role in autoimmune disease. IL-17 polymorphism has been implicated in autoimmunity, including ulcerative colitis and asthma. Polymorphisms were studied, including the coding-region sequence variant single nucleotide polymorphism rs763780 (7488T/C), which causes a His-to-Arg substitution at amino acid 161 (H161R). We examined the single nucleotide polymorphisms (SNPs) in the promoter regions of the IL-17 genes in patients with ITP, and analyzed the relationship between IL-17 SNPs and clinical features.
Patients and methods: Seventy-eight patients (male/female; 19/59, median age; 59.4) diagnosed as chronic ITP and 202 healthy controls were included. ITP with severe thrombocytopenia was defined as thrombocytopenia (platelet count < 10X109/L) at initial diagnosis of ITP. ALL patients gave written informed consent about the study. The platelet count was ranged from 1X109/L to 100X109/L at an initial diagnosis. Genomic DNA was isolated from peripheral blood using the DNA Kit (QIAGEN, Hilden, Germany). Genotyping in IL-17F was determined by PCR based technique. Genotype and allele frequencies were compared between the study groups using χ2-test. The characteristics and laboratory features of the ITP patients with each IL-10 promoter polymorphism were compared using χ2- tests and student t-tests. Probability values <0.05 were considered statistically significant.
Results: The frequencies of the genotypes were as follows: TT (72%), TC (12%), and CC (16%). No significant differences in the genotype frequencies demonstrated between chronic ITP patients and control group. However, patients with TT/TC genotypes showed severe thrombocytopenic state at initial diagnosis compared to those with CC genotypes (42.2% vs. 23.1%, p<0.05). No significant difference was observed in treatment response according to IL-17 polymorphism.
Conclusion: No significant differences in the genotype frequencies demonstrated between chronic ITP patients and control. However, homozygosity of the H161R variant was inversely associated with severity of chronic ITP. Thus, these findings suggest that IL-17 polymorphism reflect the severity of chronic ITP.
Best Approach for Harvesting Bone Marrow to Maximize TNC and CD34+ Cell Counts
Authors: Pati Nalini K , Garvin Frances , Antonenas Vicki , Kerridge Ian , Bradstock Kenneth F , Gottlieb David J 
Published: October 2019

Abstract
Background: Bone marrow (BM) has been utilized as a source of stem cells for transplantation for many years. Although the use of BM has decreased with the advent of mobilized stem cells, utilization is increasing once again due to the lower rate of chronic GVHD associated with BM as a stem cell source. There is no generally accepted technique for harvesting BM. Protocols vary both in relation to the volume of each aspirate, the number of aspirates performed at each puncture site and the total volume of harvests.
Method: BM was collected from the posterior iliac crests (PIC) in 2 separate bags: 10ml aspirates from the left and 20 ml aspirates from the right. Samples taken at the start and after 100, 150, 200, 250, & 500 ml were analyzed for TNC, CD34+ and CD3+ cell counts.
Results: The following table shows cell number (mean ± SEM ×106) for the parameters indicated.
Aspirate Volume (mls) Parameter Start (n=4) 100mls (2) 150mls (2) 200mls (2) 250mls (4) 500mls (4) 10 TNC 555 ± 28 286 ± 38 257 ± 40 226 ± 8 199 ± 11 158.5±18.5 CD34 5.8 ± 0.05 1.8 ± 0.1 1.7 ± 0.2 1.3 ± 0.4 1.1 ± 0.2 0.8 ± 0.1 CD3 65.8 ± 12.0 35.8 ± 6.9 32.8 ± 8.0 29.9 ± 1.5 28.4 ± 5.1 29.8 ± 6.2 20 TNC 914 ± 52 627 ± 137 458 ± 44 429 ± 113 391 ± 81 264 ± 24 CD34 9.1 ± 0.5 3.8 ± 0.2 2.4 ±0.2 2.7 ± 0.1 2.3 ± 0.7 1.0 ± 0.2 CD3 106.9 ±22.1 64.6 ± 5.3 55.0±14.1 56.5±14.7 53.7±14.6 41.2 ± 9.6
There is a rapid fall in the yield of CD34+ cells obtained with increasing harvest volume (19 and 25% of the initial number after 250 ml for 10 and 20 ml aspirates respectively; 14 and 11% respectively after 500 ml). In contrast the CD3+ cell numbers fall more slowly (43 and 50% after 250 ml, 45 and 38% after 500 ml). By the time 500 ml has been aspirated, there is no difference in the total number of CD34+ cells obtained from a 10 ml versus a 20 ml aspirate of bone marrow.
Conclusion: CD34+ cell yields fall rapidly when BM is harvested along the PIC. Using additional areas such as the anterior iliac crests may be preferable to a large volume PIC harvest for optimizing CD34+ stem cell collection. After 500 ml of BM has been harvested, 20 ml BM aspirates do not increase CD34+ cell numbers and 10 ml aspirates should be taken to minimize unnecessary blood loss and reduce T cell contamination.
Short-Term Ex Vivo Expansion of CD133+ Cells by Co-Culture with Mesenchymal Stem Cells: Role of SDF-1/CXCL12
Authors: Vaiselbuh Sarah , Lipton Jeffrey Michael , Liu Johnson M. 
Published: October 2019

Abstract
CD133 (prominin-1) is the first in a class of novel pentaspan membrane proteins identified in humans and mice, and studies have since confirmed the utility of CD133 as a marker of stem cells with hematopoietic and non-hematopoietic lineage potential. A number of human transplantation studies have documented hematopoietic reconstitution from CD133+ stem cells from mismatched donors, with a suggested advantage over standard grafts in avoidance of graft versus host disease. We have developed a novel hematopoietic culture system (Long-Term Stem Cell Culture or LTSCC) to investigate the potential of human mesenchymal stem cells (MSC) to form stroma that can support short- and long-term hematopoiesis derived from cord blood (CB)-derived CD133+ cells. In addition, we analyzed the effect of stromal derived factor-1 (SDF-1/CXCL12) on survival and short-and long-term colony-forming capacity of CD133+ hematopoiesis. LTSCC induced stroma-like changes in the MSC feeder layer, with adipocyte formation, thought to be needed for formation of stem cell niches, and supported long-term (>9 weeks) survival of CB-CD133+ cells. Cobblestone areas of active CD133-derived hematopoiesis were seen in LTSCC for up to 9 weeks of culture. SDF-1/CXCL12 acted as a survival factor for CB-CD133+ cells and induced a significant ex vivo cell expansion at weeks 3 and 4 of LTSCC (maximal 500-fold increase), while maintaining the capacity for CFU-Mix and BFU-E colony formation up to 7 weeks. Long-term hematopoiesis was assessed by enumeration of long-term culture initiating cells (LTC-IC). When SDF-1/CXCL12 was added to LTSCC, we found a significant increase in LTC-IC: 0.3% (+SDF-1/CXCL12) vs. 0.05% (-SDF-1/CXCL12). Finally, homing capacity, as defined by SDF-1/CXCL12-induced adhesion and migration of CB-CD133+ cells, was maintained and even increased during the first 3 weeks of LTSCC. In summary, MSC can be maintained in LTSCC medium, and this simplified feeder layer is able to provide niches for cobblestone area forming cells derived from CB-CD133+ cells. SDF-1/CXCL12 is critical to support the survival and expansion of CD133+ cells, either directly or indirectly by paracrinesignaled retention of CD133+ cells in contact with specialized MSC niches. We suggest that expansion of CD133+ cells from cord blood may be useful in clinical transplantation limited by insufficient numbers of stem cells.
Regulatory T Cells (Tregs) Can Be Isolated from G-CSF Mobilized PBSC after Monocyte Depletion and Inhibit Anti-Stem Cell T Cell Alloreactivity
Authors: Mahmud Dolores , Rondelli Damiano 
Published: October 2019

Abstract
Small numbers of human CD4+CD25+FoxP3+ Tregs can be isolated from normal peripheral blood, thus their potential clinical application is limited. In this study we tested whether Granulocyte Colony-Stimulating Factor (G-CSF)-mobilized peripheral blood stem cells (PBSC) from healthy donors can represent a useful source of CD4+CD25+ cells with regulatory activity. We utilized antibodies conjugated to microbeads (Miltenyi Biotec Inc, Auburn, CA) to immunomagnetically separate the cells on a MidiMACS device (Miltenyi) and checked the purity after isolation by flow cytometry. Starting from on average 4.0±1.8 × 108 unseparated PBSC (n=3) we positively selected 2.4±0.8 × 106 CD34+ cells (>90% purity). We then utilized the CD34− cell fraction to isolate Tregs. Due to the large content of CD4dim monocytes in the initial cell product, we initially depleted the PBSC of CD14+ cells and then utilized a two-step process that includes a CD4+ cell negative selection (using a cocktail of biotin-conjugated antibodies against CD8, CD14, CD19, CD16, CD36, CD56, CD123, TCR g/δ and Glycophorin-A) followed by a positive selection of CD25+ cells (Treg isolation kit, Miltenyi). This process allowed us to obtain 1.2±1 × 106 CD4+CD25+ cells with a purity of >70%. Intracellular expression of FoxP3 was also detected in purified CD4+CD25+ cells by flow cytometry. Primary mixed leukocyte cultures (MLC) were performed with irradiated CD34+ cells isolated from PBSC and HLA mismatched blood CD3+ responders for 6 days and T cell response was measured by a 3H-thymidine uptake assay. Tregs isolated from PBSC were added to the MLC at 1:2 Treg:responder ratio to test their regulatory function. Control experiments were performed using CD4+CD25− cells. Addition of Tregs isolated from PBSC resulted in 76±17% inhibition of anti-CD34 T cell alloreactivity (cpm: 19000±530 vs 4590±1880) (n=3), while control CD4+CD25 neg cells did not show suppressive activity. These findings show that after isolation of CD34+ cells, adequate numbers of Tregs can be obtained from the CD34− cell fraction of PBSC by using a three-step process. In addition, since Tregs isolated from PBSC suppressed in-vitro T cell alloreactivity against CD34+ cells, these findings will prompt the design of pre-clinical studies to test the combination of PBSC-derived CD34+ cells and Tregs in HLA mismatched transplantation.
Insulin-Like Growth Factor 1 Protects against Lethal Irradiation
Authors: Chen Benny J. , Deoliveira Divino , Chao Nelson 
Published: October 2019

Abstract
Ionizing irradiation can cause bone marrow failure leading to death. Effective therapeutic agents capable of promoting or accelerating the recovery of the hematopoietic and/or immune compartment following radiation injury are limited. We and others have previously demonstrated that recombinant human growth hormone promotes hematopoietic and immune recovery following stem cell transplantation and irradiation. Published data suggest that growth hormone elicits its pro-hematopoietic effects via action of insulin-like growth factor 1 (IGF1). Since IGF1 has recently been approved by the Federal Drug Administration to treat other conditions, IGF1 could be brought to the clinic rapidly upon demonstration of its activity. In this study, we sought to determine whether IGF1 has radioprotective activity. The studies were performed using BALB/c mice. Recombinant human insulin-like growth factor 1 (rhIGF1) was administered at a dose of 100 mcg/dose, i.v., once a day, starting within one hour after irradiation. BALB/c mice were irradiated with 7.5 Gy and treated with saline or rhIGF1 for 5 days. In the saline control group, all mice (10 out of 10 mice) died within 25 days following irradiation. By contrast, four out of 10 mice (40%) in the rhIGF1-treated group survived more than 100 days after irradiation (Figure, P<0.01). These data indicate that rhIGF1 can protect against lethal irradiation. Because one of the primary causes of death following irradiation is infections, we next sought to determine the effects of rhIGF1 on immune recovery. Compared with the saline control group, treatment with rhIGF1 for 5 days on irradiated (7.5 Gy) BALB/c mice significantly accelerated the recovery of CD4 and CD8 T cell subsets, B cells, and NK cells following irradiation. Our data demonstrate that rhIGF1 could potentially serve as a medical countermeasure in radiation emergencies.
Figure Figure
Emergence of Dominant T-Cell Populations in Patients with CLL after Allogeneic Transplantation: Mediators for Gvl Effects?
Authors: Ritgen Matthias , Brueggemann Monika , Boettcher Sebastian , Raff Thorsten , Pott Christiane , Hallek Michael , Kneba Michael , Dreger Peter 
Published: October 2019

Abstract
Allogeneic stem cell transplantation (SCT) is the only known curative treatment for high-risk CLL. We have recently shown that minimal residual disease (MRD) monitoring can identify patients with graft versus leukemia (GvL)-induced disease response to either reduction of immunosuppression (IS) or to administration of donor lymphocyte infusions (DLI), suggesting that those patients are potentially cured by an ongoing immunologic antileukemic effect induced by donor immune cells (Leukemia 22:1377). It is uncertain, however, which cell population maintains this process; although T as well as NK-cell mediated effects are discussed. The present study addressed the question whether disease response upon immunomodulation after SCT is associated with the occurrence of dominant T cell clones.
Methods: 32 patients allografted for high-risk CLL who had MRD follow-up by clone-specific PCR or MRD-flow available were included in this investigation. We used the BIOMED T-cell receptor multiplex PCRs (TCR-PCR) to search for T cell clones which might be involved in the documented GVL effects. TCR rearrangements were sequenced and analyzed using the IMGT database.
Results: 16 of 32 patients showed MRD response after IS reduction or DLI. GVL-induced MRD clearance was associated with onset of chronic GVHD in almost all instances. Twenty-four different dominant TCR rearrangements could be identified in 15/32 patients by BIMOD TCR-PCR. Most of the T cell populations show rearranged gamma/delta TCRs suggesting that regulatory gamma/delta T cells might be involved in this process. TCR sequences employed were TRGV9 (13), TRGV2 (2) and TRGV1, TRGV4, TRGV8, TRGV10, TRGV11, TRBV5, TRBV6, TRBV12, TRBV15. In 4 patients with a potential productive TCR rearrangement (TRGV4+TRDV1, TRBV6, TRGV2, TRGV11+TRGV9) we were able to design a TCR-specific real-time PCR for quantitative follow-up of this clonal T cell population. This data was compared to flow cytometric monitoring of T-cell subpopulations and MRD kinetics post SCT. In those 4 patients we could demonstrate an inverse correlation of the kinetics of MRD and the kinetics of clonal T cell expansions. T cell clones emerging during this phase remained on a stable level throughout the whole follow-up in patients showing durable MRD negativity.
Conclusion: In CLL, MRD clearance after SCT is correlated to the emergence of dominant T cell clones, suggesting that GVL activity is based on allo- or CLL-specific T cell expansion. Further studies are needed to clarify the role of these T cell clones for GVL and GVHD development.
SOCS3 Regulates Th1 Dependent Acute Graft-Versus Host Disease
Authors: MacDonald Kelli P A , Kuns Rachel D , Don Alistair LJ , Olver Stuart D , Raffelt Neil C , Markey Kate A , Alexander Warren , Roberts Andrew W. , Hill Geoff R 
Published: October 2019

Abstract
G-CSF mobilized peripheral blood has emerged as the dominant source of stem cells for allogeneic stem cell transplantation. G-CSF modulates T cell function and Suppressor of Cytokine Signaling-3 (SOCS3) is the major regulator of signaling by this cytokine, although the downstream effects in vivo remain unclear. We have therefore examined the effect of SOCS3 in the well established B6 → B6D2F1 murine model of acute GVHD directed to major histocompatibility antigens. Using SOCS3−/Δvav mice in which SOCS3 deficiency is restricted to the haematopoietic compartment we transplanted splenocytes from G-CSF mobilized wild-type (WT) or SOCS3−/Δvav donors and demonstrated that the absence of SOCS3 within the graft accelerated GVHD (median survival 23 vs. 39 days, P=0.04). By using SOCS3−/ΔLysM and SOCS3−/Δlck donors in which SOCS3 deficiency was restricted to the myeloid or T cell lineage respectively we confirmed SOCS3 deficiency enhanced GVHD via effects only within the donor T cell (median survival 19 vs. 35 and 39 days in SOCS3−/Δlck vs. WT and SOCS3−/ΔLysM, P<0.0001). This protective effect of SOCS3 was also seen in the absence of G-CSF mobilization since bone marrow and purified donor T cells from SOCS3−/Δvav donor mice significantly accelerated GVHD mortality (median survival 29 vs. 44 days, P=0.04). In mixing experiments this acceleration of GVHD was again seen only when the donor T cell was SOCS-3 deficient.
SOCS3−/Δlck donor T cells underwent enhanced alloantigen dependent proliferation after transplantation as determined by CFSE dilution and demonstrated increased IL-10, IL-17 and IFNγ generation but reduced IL-4 secretion. In contrast no effects were seen in IL-6 and TNF secretion, or CD8 cytotoxicity against host-type targets. Consistent with the increased mortality, SOCS3−/Δlck donor T cells induced significantly more severe gastrointestinal GVHD as determined by semi-quantitative histopathology (GI tract scores 18 ± 1.7 vs. 10.2 ± 1.1, P=0.008). Blockade of the IL-10 receptor or IL-17 neutralization from the day of transplant or after the initiation of GVHD failed to attenuate the enhanced GVHD mortality induced by SOCS3−/Δlck donor T cells. The suppression of acute GVHD by SOCS3 is thus consistent with its ability to suppress IFNγ generation by the donor T cell, a cytokine which we have shown to be a key and direct mediator of GVHD within the GI tract (Burman A et al, Blood2007: 110:1064).
These studies highlight the previously unrecognized role of the SOCS3 molecule in regulating alloreactive T cell responses and suggest that the delivery of small molecule SOCS3 agonists may prove to be useful for the inhibition of Th1 dependent acute GVHD.
Differentially Cleaved Forms of Osteopontin by Thrombin and Carboxypeptidase B (TAFIa) Regulates Neutrophil Viability and Synoviocyte Binding in Rheumatoid Arthritis
Authors: Sharif Shadi , Du Xiaoyan , Myles Timothy , Song Jason Jungsik , Price Elizabeth A , Lee David , Goodman Stuart , Morser John , Robinson William , Leung Lawrence L. 
Published: October 2019

Abstract
Osteopontin (OPN) is a pro-inflammatory cytokine that plays a significant role in rheumatoid arthritis (RA). We have recently shown that OPN is a bona fide substrate for thrombin. OPN can be cleaved by thrombin, leading to OPN-Arg (OPN-R) and exposing the cryptic C-terminal a4b1 and a9b1 integrin-binding motif (SVVYGLR). Procarboxypeptidase B (proCPB or thrombin-activatable fibrinolysis inhibitor TAFI), upon activation to CPB (TAFIa) by the thrombin/thrombomodulin complex on endothelial cell surface, removes the C-terminal arginine from OPN-R, generating OPN-Leu (OPN-L) and abrogating its enhanced cell-binding. We have developed specific ELISAs for OPN-R and OPN-L. While intact OPN (OPN-FL) was significantly elevated in RA synovial fluid (SF) (n=26, median level 352.5 ng/mL), as compared to its normal plasma level, it was not significantly higher in osteoarthritis (OA, n=13, 157.9 ng/mL, .142) or psoriatic arthritis (PsA, n=10, 143.4 ng/mL, .074) SF samples. On the other hand, highly significant elevation of OPN-R and OPN-L levels was detected in the RA SF, as compared to OA and PsA (median values of OPN-R 138.6 ng/mL, 10.6 ng/mL and 2.2 ng/mL, p <0.003 and OPN-L 205.3 ng/mL, 25.9 ng/mL and undetectable, p <0.006 in RA, OA and PsA respectively). Significant disease heterogeneity was noted. The median value for the ratio of OPN-R and OPN-L to total OPN was approximately 0.5, indicating extensive enzymatic cleavage activity in a significant subset of patients. In RA SF, elevated OPN-R and OPN-L levels were significantly correlated with multiple inflammatory cytokines, including IL-6, IL-12p40, and FGF-2 while OPN-FL levels only correlated with IL-6. Fibroblast-like synoviocytes, in addition to producing OPN locally, released proCPB into SF and expressed functional cell surface thrombomodulin that supports the activation of proCPB to CPB by thrombin. Immunohistochemical studies demonstrated robust expression of OPN-FL, but minimal OPN-R, in RA synovium, suggesting that cleaved OPN is released into the SF. OPN had an anti-apoptotic effect on neutrophils, which was retained by but diminished in OPN-R and OPN-L. Cultured synoviocytes expressed a4b1 integrins and adhesion of synoviocytes to immobilized OPN-R, but not OPN-L, was significantly enhanced compared to OPN-FL. Synoviocytes bound to immobilized SVVYGLR, but not SVVYGL, dose-dependently, consistent with binding via the a4b1 receptor on synoviocytes. Our results suggest that the sequential thrombin activation of OPN (OPN-R) and its subsequent inactivation by CPB (OPN-L) may represent an important local homeostatic mechanism that regulates neutrophil viability and synoviocyte adhesion and play an important role in the pathogenesis of RA in a subset of patients.