bluuebunny/crossref_metadata_2025 huggingface dataset:
Thromboembolic Events among Adult Patients with Idiopathic Thrombocytopenic Purpura in the United Kingdom General Practice Research Database.
Authors: Sarpatwari Ameet , Bennett Dimitri , Logie John W , Shukla Amit , Beach Kathleen J , Newland Adrian , Sanderson Simon , Provan Drew
Published: October 2019

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease characterised by elevated peripheral platelet destruction and inadequate platelet production. Little published epidemiological data exists concerning the risk of thromboembolic events (TE) among adult patients with ITP.

The objective of our investigation was to estimate the risk of TE among adult patients with and without ITP in the United Kingdom (UK) General Practice Research Database (GPRD), which contains patient data from 6.4 million patients from more than 480 representative medical practices across the UK.

Using read [2871C] or Oxford Medical Information System (OXMIS) [D3130000, D313012, 42P2.11, D313.12] codes to define cases, incidence rates (IRs) per 10,000 patient-years of observation (PYO) with 95% confidence intervals (CIs) were estimated. Hazard ratios (HRs) of venous, arterial, and combined (venous and arterial) TE were modelled using Cox proportional hazards regression adjusting for history of prior TE, hypertension, splenectomy status, oral corticosteroids and intravenous immunoglobulin exposure.

840 adults (≥ 18 years) with codes for ITP first referenced between January 1, 1992 and September 30, 2005 and having at least one year pre-diagnosis and three months of post-diagnosis medical history were matched with 3,360 non-ITP patients by age, gender, primary care practice and pre-study follow-up time. Over a median 52 months of follow-up (range: 3–182 months), the cumulative incidence of TE among thromboembolic-free patients at baseline was higher within the ITP cohort [47 (6.7%) v. 172 (5.7%)]. As shown in the table below, the IRs of venous, arterial, and combined TE were additionally elevated among patients with ITP.

Outcome Incidence Rate/10,000 PYO 95% CI Adult Patients with ITP Adult Patients without ITP Venous TE 70.83 (46.67–103.05) 47.53 (37.39–59.58) Arterial TE 82.02 (55.73–116.42) 71.50 (58.87–86.04) Combined TE 134.74 (99–179.18) 116.32 (99.59–135.06)

Adjusted HRs of 1.58 (95% CI, 0.98–2.53), 1.02 (95% CI, 0.65–1.59), and 1.17 (95% CI, 0.82–1.66) were found for venous, arterial, and combined TE respectively. Further event categorisation revealed elevated HRs for each venous thromboembolic subtype [deep vein thrombosis 1.49 (95% CI, 0.62–3.61), pulmonary embolism 3.52 (95% CI, 1.23–10.04), and other TE 1.17 (95% CI, 0.64– 2.16)].

This study provides evidence of an increased risk in venous TE in adult patients with ITP relative to the general adult non-ITP population.
The Feature of TCR Vγ And TCR Vδ Repertoire Distribution and Clonality in Patients with Immune Thrombocytopeinc Purpura.
Authors: Zhang Xueli , Chen Shaohua , Yang Lijian , Zhu Kanger , Li Yangqiu
Published: October 2019

Chronic idiopathic/immune thrombocytopenic purpura (ITP) is an autoimmune disorder in which anti-platelet antibodies induce platelets destruction due to an imbalanced immune response. Recently, data indicated the γδ+T cells may play an important role in mediated autoimmune disease. Our previous study has showed the restricted expression of TCR Vβ subfamilies and the alteration of peripheral TCR Vβ repertoire pattern in the majority of ITP patients. In the present study, we farther analyze the feature of TCR Vγ and TCR Vδ repertoire distribution and clonality in patients with ITP. The CDR3 size of three TCR Vγ and eight TCR Vδ subfamily genes were analyzed in peripheral blood mononuclear cells (PBMCs) from 8 cases with ITP and 10 healthy individuals, using RT-PCR and genescan technique. To determine the expression level of TCR Vγ subfamily genes, quantitative analysis of TCR Vγ I–III subfamilies was performed by real-time PCR. TCR Vγ I to III subfamilies could be detected in the most samples from ITP as well as in healthy control. However, oligoclonal expansion of TCR Vγ I was identified in 3 cases with ITP, which display polyclonality in all of samples from healthy control. The expression level of all TCR Vγ I–III subfamilies in PBMCs from ITP was significant lower than that from healthy control (P=0.003,P=0.000,P=0.005 respectively). the pattern of TCR Vγ I–III repertoire in ITP was TCR VγI> TCR VγIII> TCR VγII, in contrast, TCR VγII> TCR VγI> TCR VγII was found in healthy control. TCR Vδ1 and TCR Vδ2 could be detected in most samples from ITP as well as in healthy control, whereas TCR Vδ3 could be detected only in 2 out of 8 cases with ITP, which could be found in 90% of healthy controls (p=0.02), TCR Vδ8 could not be identified in all of samples from ITP, which could be found in 30% of healthy control. Oligoclonal expanded TCR Vδ1and TCR Vδ2 T cells could be identified in the half of samples from ITP, similar results were found in healthy control. In conclusions, the alteration of peripheral TCR Vγ and TCR Vδ repertoire pattern might be important in the pathogenesis of immune-mediated platelet destruction in some cases with ITP. Our report is the first description of feature of TCR Vγ and TCR Vδ repertoire pattern in ITP patients.
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

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

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

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

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

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

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

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

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.
Bone Marrow Endothelial Cell Support of Primitive Hematopoietic Cells
Authors: Zhou Xiaoying , Rosa Dilani , Cunningham Cynthia , Adams Gregor B.
Published: October 2019

Hematopoietic stem cells (HSCs) in the bone marrow (BM) reside in specialized microenvironments known as the stem cell niche. Many reports have found the HSCs to be resident next to the endosteal surface of bone where cells of the osteoblastic lineage are a key component of the so-called endosteal niche. However, HSCs have also been found to reside adjacent to sinusoidal blood vessels. These observations have led to the proposal that HSCs in the adult BM may also reside in a vascular niche. However, the functional role of the vascular niche in hematopoiesis remains to be determined. We wished to evaluate the role that BM endothelial cells (BMECs) play in HSC physiology. To examine this we cultured BMEC-enriched cells in vitro, identified by expression of CD31, Tie-2, VE-cadherin and LDL uptake. We compared these cells to spleen derived ECs and BM stromal cells (BMSCs) in their ability to support primitive hematopoietic cells for extended periods in in vitro culture. We found that BMECs were superior in their ability to support the cobblestone area forming cell activity of VEGF-R1+ HSCs than spleen ECs or BMSCs. We also found that the number of cobblestone area cells was markedly reduced when VEGF-R1− HSCs were cultured on any of the supportive cell layers, however this may be due to an intrinsic difference between these cells as a much higher proportion of VEGF-R1+ HSCs were found to be in the G0 phase of the cell cycle than VEGF-R1− cells. To evaluate the supportive role of BMECs, spleen ECs or BMSCs on hematopoietic progenitor cells (HPC) we cultured purified primitive cells on these supportive layers and the total number of colony-forming unit-culture (CFU-C) cells were examined after 4-days or 7-days co-culture with the feeder cells. The results showed that BMECs or spleen ECs can promote the generation of CFU-C from VEGFR1+ HSCs or VEGFR1− HSCs, yet the tot al number of CFU-C produced from VEGFR1+ HSCs was greater than that from VEGFR1- HSCs. However, both of these cell types were able to support the generation of CFU-Cs to a greater degree than BMSCs. To examine the mechanism of enhanced support of VEGF-R1+ HSCs by the BMECs, we performed real-time PCR analysis for the expression of the VEGF-R1 ligands. Both BMECs and spleen ECs were found to express VEGF-A and –B to similar levels, however the expression of placental growth factor was higher in the BMECs. Whether the increased expression of this factor plays a functional role in the support of the HSCs in currently being evaluated. Our findings suggest that the ECs from BM or spleen can promote the proliferation of hematopoietic progenitor cells, while BMECs can maintain the long-term culture of VEGFR1+ HSCs in vitro. The functional relevance of this in vivo is currently being investigated.
Ajuba Functions as An HDAC-Dependent Co-Repressor for Autoregulation of the Growth Factor Independent-1 Transcription Factor
Authors: Rojas Meghan E. B. , Velu Chinavenmeni S. , Montoya-Durango Diego E. , Kazanjian Avedis , Jay Chris M. , Longmore Gregory D. , Grimes H. Leighton
Published: October 2019

Growth factor independent-1 (Gfi1) is mutated in patients with Severe Congenital Neutropenia. Gfi1 is a zinc finger protein with a SNAG-transcriptional repressor domain. The SCN-associated Gfi1 mutant proteins function as dominant negatives to sequester limiting SNAG-dependent corepressor proteins from the remaining wild type Gfi1 protein, and deregulate a subset of Gfi1 target genes such as CSF1. The identity of the critical Gfi1 SNAG-domain-associated co-repressor proteins is unknown. Ajuba is a LIM-domain protein that shuttles between the cytoplasm and the nucleus. Ajuba functions as a co-repressor for synthetic Gfi1 SNAG-repressor-domain containing constructs, but a role for Ajuba co-repression of the cognate DNA bound Gfi1 protein has not been defined. Coimmunoprecipitation of synthetic and endogenous proteins, and co-elution with gel filtration identified an endogenous Ajuba-Gfi1-HDAC multiprotein complex. Active histone deacetylase activity co-immunoprecipitates with Ajuba or Gfi1, and both proteins depend upon histone deacetylases for full transcriptional repression activity. Ajuba LIM domains directly bind to Gfi1, but the association is not SNAG domain dependent. ChIP analysis and reciprocal knock-down experiments suggest that Ajuba selectively functions as a co-repressor for Gfi1 autoregulation. The data suggest that Ajuba is utilized as a corepressor selectively on a subset of Gfi1 target genes, but is not the critical SNAG-dependent cofactor sequestered by SCN-associated Gfi1 mutant proteins.
Treatment of High Grade B-Cell Lymphoma with Chemotherapy Plus Rituximab: A Survival Benefit Can Be Demonstrated in the Routine Data of a Regional Cancer Registry
Authors: Krause Stefan W , Andreesen Reinhard , Hofstädter Ferdinand , Gerken Michael
Published: October 2019

Introduction: Combination of standard chemotherapy with Rituximab led to improved disease control in pts. with B-cell lymphoma and a clearcut survival benefit in diffuse large B cell lymphoma (DLBCL) in a multitude of clinical trials. We wanted to know, if a similar benefit could be demonstrated in the routine follow-up data of the regional population-based cancer registry of the Regensburg cancer center (“Tumorzentrum Regensburg”, TUZ). The TUZ spreads evidence-based treatment recommendations, but beyond this, treatments are performed according to the discretion of individual physicians and institutions.

Methods: We searched the registry for B-NHL diagnosed between 1998 and 2005 entered as DLBCL, mantle cell lymphoma (MCL), follicular lymphoma (FL), undefined low grade lymphoma and corresponding terms from the ICD-O3. Furthermore, death certificates were systematically matched to the patient data and additionally searched for the terms C82, 83, 85, 88 of the ICD-10. Using these data, we compared overall survival of patients receiving any first line chemotherapy plus rituximab and patients receiving chemotherapy alone.

Results: Comparing registry data to death certificates an 85% coverage of lymphoma patients within the registry can be estimated. In the high grade lymphoma group, 450 patients have been entered into the registry. Of these, 49 were insufficiently documented, 29 patients had a delayed start of their treatment and 41 received no chemotherapy at all. 122 pts. received rituximab-containing chemotherapy resulting in a 2 year survival of 87%, whereas 209 pts. received chemotherapy alone with a significantly inferior 2 year survival of 70%, p=0,004 by log rank test (fig). 307 pts with low grade lymphoma were found. Of these, 31 were insufficiently documented. 84 pts. did not receive any therapy at all during the observation period and showed the best survival as expected (watch and wait). 69 and 123 pts. received first line chemotherapy with or without Rituximab, respectively. Regarding all low grade NHL together, no significant difference was found, however, FL pts. survived longer, when Rituximab was given (2 year survival 91% vs. 72%, p=0,046).

Conclusions: A survival benefit comparable to the results within clinical trials can be observed in registry data in an unsupervised community setting for patients with DLBCL treated first line with a Rituximab containing chemotherapy. A strong trend for improved survival was also observed for FL. These data confirm the standard use of Rituximab as first line therapy in DLBCL and FL and they support the collection of an extended set of treatment data containing exact descriptions of systemic therapy in cancer registries in order to answer similar questions in the future.

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High-Dose Methotrexate and Cytarabine Chemotherapy May Be Effective and Safe in Solid Organ Transplant Recipients with Primary CNS Lymphomas (PCNSL)
Authors: Moise Laura , Matta Clemence , Pilorge Sylvain , Moldovan Marius , Ferme Christophe , Durrbach Antoine , Ribrag Vincent
Published: October 2019

Background: Post transplant lymphoproliferative disorder (PTLD), especially primary CNS lymphoma (PCNSL), is a serious complication of solid organ transplantation. Treatment has proved difficult and includes dose reduction of immune suppression (RIS), combination chemotherapy and radiotherapy. Recently the monoclonal antibody Rituximab, has been added to the therapeutic choice. Chemotherapy used in PCNSL is based on high-dose methotrexate (HDMTX), but need to be carefully monitored in adult patients (pts), especially with renal transplant. HDMTX could be associated with deleterious effects on renal graft function. We report here the results of HDMTX in pts with PCNSL after solid organ transplantation treated at the Institut de Cancérologie Gustave Roussy.

Methods: From February 2000, 18 pts with PTLD were referred to our Institute. 9 of them had PCNSL following renal (6), hepatic (1) or both renal and pancreatic (2) transplants. In all cases, RIS was not associated with PCNSL regression. Except in one case, all pts were treated with chemotherapy with HDMTX in 3-hour infusion. Folinic acid rescue was given IV every 6 hours (50 mg/m2) combined with alkaline hyper-hydration until MTX serum level was below 1 X 10–7 M. Immune suppressive therapy (IST) was reduced to solupred (10 mg/day) during chemotherapy and was reduced for 8 months after chemotherapy. 4 cycles of chemotherapy was planned and efficacy assessed every two cycles. If CR was reached with chemotherapy alone, no radiotherapy was delivered.

Results: From March 2007, 9 pts (4 males) developed PCNSL with a median of 6 years after transplant (1–26 years). Median age was 55 (range 33–70). PS was > 2 in 8/9 and 3 in one case. 8/9 pts had glomerular filtration rate > 60 ml/mn and received HDMTX without renal toxicity. One pt with GFR < 60 ml/mn was treated with 3 cycles of HD cytarabine + rituximab and adryamicin and is alive in complete remission (CR) 31 months after therapy. With a median follow up of 15 months, 4/9 pts are alive and in CR 45, 31, 29 and 7 months after therapy. 3 of them received radiotherapy. 5 pts died, 2 of lymphoma progression, 2 of chemotherapy toxicity (sepsis) and one of a gastric cancer. No graft rejection was observed.

Conclusions: our results suggest HDMTX can be given in pts with PCNSL and long term CR can be reached without graft failure. As chemotherapy regimens used for in those pts are in themselves immunosuppressive, IST may be reduced during chemotherapy without major risk.
Depletion of the C3 Component of Complement Enhances the Ability of Rituximab-Coated Target Cells to Activate Human NK Cells and Improves the Efficacy of Monoclonal Antibody Therapy in An in Vivo Model
Authors: Wang Siao- Yi , Cagley Jeffrey , Fritzinger David , Vogel Carl-Wilhelm , John William St. , Weiner George J
Published: October 2019

Antibody-dependent cellular cytotoxicity (ADCC) is felt to play an important role in mediating the anti-tumor effects of Rituximab (R). We previously reported that C3b deposition on R-coated target cells interferes with the binding of the Fc portion of R to NK cell CD16. This interaction inhibits the activation of NK cells and NK cell-mediated ADCC of 51Cr-labeled R-coated target cells. The current studies were designed to determine whether C3 depletion enhances the ability of mAb-coated targets to activate NK cells in vitro and improves mAb therapy in vivo. Normal human serum inhibited the ability of R-coated lymphoma cells to activate NK cells as previously reported. NK activation was increased when serum was pre-incubated with cobra venom factor (CVF) to deplete C3. Similar results were found when non-malignant ascites or transudative pleural fluid, as a surrogate for extravascular fluid, was used as the source of complement. For in vivo analysis, we utilized a syngeneic, immunocompetent murine model in which ADCC has been previously demonstrated to be a key mechanism of action. CVF or a human C3 derivative with CVF-like functions (HC3-1496) was used to deplete C3 in vivo. In this model, C3H/HeN mice were inoculated with murine 38C13 lymphoma cells (day 0) and treated with 2 doses of CVF or HC3-1496 (day 3 and day 5). Four hours after the initial dose of CVF or HC3-1496, mice were treated with a single dose of an anti-lymphoma mAb directed against the 38C13 idiotype (MS11G6). Untreated mice all developed tumor and died with a median survival of 28 days. All mice treated with mAb alone eventually developed tumor and died with a median survival of 42 days. Survival following treatment with CVF plus mAb was superior to that of mAb alone (Fig 1, p=0.0312) with 50% of mice remaining tumor free. Survival following treatment with HC3-1496 plus mAb was also superior to that of mAb alone (Fig 2, p=0.0002) with 80% of mice remaining tumor free. In summary, depletion of the C3 component of complement enhanced the ability of R-coated target cells to activate human NK cells, and improved the efficacy of mAb therapy in an in vivo model of lymphoma. Furthermore, these studies suggest the inhibitory effects of complement on NK activation and ADCC may be seen in the extravascular compartment such as within involved lymph nodes. We conclude that depletion of complement through use of agents such as CVF or HC3-1496 could be considered as an approach to enhancing the efficacy of R therapy.

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Increased Reactive Oxygen Species Induced by NUP98-HOXD13 Fusion Gene in MDS Mouse Model
Authors: Chung Yang Jo , Aplan Peter D.
Published: October 2019

The myelodysplastic syndromes (MDSs) comprise a group of pre-malignant hematologic disorders characterized by ineffective hematopoiesis, dysplasia, and transformation to acute myeloid leukemia (AML). The causative agents for de novo or therapy-related MDS (t-MDS) include exposure to known genotoxins, such as benzene and other solvents, anti-cancer chemotherapy, and ionizing radiation. It is known that MDS is closely related to chromosomal aberrations including deletions, amplifications, inversions, and translocations, however, the molecular mechanism of disease progression from MDS to AML not been completely elucidated. Recently, several studies have reported that oxidative stress contributes to the disease progression mechanism in MDS. Reactive oxygen species (ROS) play a role in regulating several biologic phenomena involving activation of signaling pathways in response to cytokines, and the gene expression induced by this signaling. ROS is also known to induce oxidative DNA damage which generates DNA base modifications and DNA helix alterations. Therefore, increased intracellular levels of ROS can induce mutations which lead to transformation of MDS to AML. A mouse model for MDS, generated by expressing a NUP98-HOXD13 (NHD13) fusion gene was exploited to investigate ROS levels in this study. Lineage negative (Linneg) bone marrow mononuclear cell (BMNC) from NHD13 mice with MDS had a 7.8±5.6 fold increased level of ROS compared with wild-type (WT) Linneg BMNC, while ROS levels of unfractionated BMNC were similar in NHD13 and WT mice. The increase ROS level in NHD13 Linneg BMNC was associated with an increase in G2/M phase (2 fold). Interleukin 3 (IL-3) dependent NHD13 ‘knock-in’ cell lines also showed an increased level of ROS (3 to 5 fold), in comparison with other IL-3 dependent cell lines, BaF3 and 32D. To determine if the increase in ROS could be generated by an NHD13 fusion, BaF3 and 32D cell were transfected with an NHD13 expression vector. The BaF3 and 32D transfectant showed 2 fold (2.1±0.3 and 1.9±0.6 fold) increase ROS level compared to controls transfected with an empty vector. These findings suggest that expression of an NHD13 fusion gene can induce ROS in hematopoietic progenitor cells, which may contribute to the development of MDS and subsequent disease progression to AML through DNA damages.
Induction of Sustained Early G1 Arrest by Selective Inhibition of CDK4 and CDK6 Primes Myeloma Cells for Synergistic Killing by Proteasome Inhibitors Carfilzomib and PR-047
Authors: Huang Xiangao , Bailey Kathryn , Di Liberto Maurizio , Parlati Francesco , Niesvizky Ruben , Demo Susan , Chen-Kiang Selina
Published: October 2019

Targeting the cell cycle in combination with cytotoxic killing is a rational approach to cancer therapy. Dysregulation of the cyclin-dependent kinases CDK4 and CDK6 precedes uncontrolled proliferation of myeloma cells in vivo, in particular during relapse and drug resistance. This finding reinforces the critical importance of targeting CDK4/6 in myeloma, but success with broad-spectrum CDK inhibitors has been modest. Using the only known selective inhibitor of CDK4/6, PD 0332991, we have developed a novel approach to prime chemoresistant myeloma cells for synergistic killing by diverse cytotoxic agents. We show that selective inhibition of CDK4/6 by PD 0332991 leads to sustained cell cycle arrest in early G1 in the absence of apoptosis. However, it markedly augments cytotoxic killing by PR-171 (carfilzomib), a selective inhibitor of the chymotrypsin-like activity of the proteasome, or PR-047, an orally bioavailable analog of carfilzomib. Synergistic killing of myeloma cells arrested in early G1 by carfilzomib (or PR-047) is caspase-dependent, and requires only a brief (one hour) exposure to the proteasome inhibitor at concentrations as low as 60 nM. This effect is mediated by synergistic and rapid induction of mitochondrial membrane depolarization and activation of downstream caspase-9 within 6 hours of removal of carfilzomib or PR-047. As PD 0332991 acts as an ATP-competitive inhibitor of the CDK4/6 kinase domain, inhibition of CDK4/6 and the cell cycle by PD 0332991 is reversible. Importantly, targeting CDK4/6 with PD 0332991 in combination with either carfilzomib or PR-047 leads to complete eradication of myeloma cells ex vivo, in contrast to the combination of PD 0332991 with other proteasome inhibitors. Selective inhibition of CDK4/6 in combination with carfilzomib (or PR-047), therefore, not only halts cell proliferation but also potently induces synergistic killing that is likely to eliminate cell cycle reentry and generation of resistant cells. PD 0332991 is a small molecule with bio-availability and proven tumor suppressing activity in both human myeloma xenograft and immunocompetent mouse myeloma models. It is well tolerated in humans as shown by the ongoing Phase I/II clinical trials in myeloma and previous phase I trials in mantle cell lymphoma and solid tumors. Evidence from Phase I trials of carfilzomib indicates that it is also well tolerated, in fact, the peripheral neuropathy that is commonly observed with proteasome inhibitor bortezomib appears to be less severe and possibly less frequent. Mechanism-based targeting of CDK4/6 in combination with selective proteasome inhibitors, like carfizomib and PR-047, thus represents a new and promising therapeutic strategy for multiple myeloma and potentially other hematopoietic malignancies.
Structure of the Notch1 Negative Regulatory Region: Implications for Normal Activation and Pathogenic Signaling in T-ALL
Authors: Gordon Wendy Ryan , Roy Mondeepa , Vardar-Ulu Didem , Garfinkel Megan , Mansour Marc R , Aster Jon C , Blacklow Stephen C
Published: October 2019

Proteolytic resistance of Notch prior to ligand binding depends on the structural integrity of a negative regulatory region (NRR) of the receptor that immediately precedes the transmembrane segment. The NRR includes the three Lin12/Notch repeats and the juxtamembrane heterodimerization domain, the region of Notch1 most frequently mutated in T cell acute lymphocytic leukemia. Here, we report the X-ray structure of the Notch1 NRR determined to 2.0 Å resolution in its autoinhibited conformation. A key feature of the Notch1 structure that maintains its closed conformation is a conserved hydrophobic plug that sterically occludes the metalloprotease cleavage site. Crystal packing interactions involving a highly conserved, exposed face on the third Lin12/Notch repeat suggest that this site may normally be engaged in inter- or intra-molecular protein-protein interactions. The vast majority of known T-ALL-associated point mutations of human Notch1 map to residues in the hydrophobic interior of the NRR. A novel mutation (H1545P), which alters a residue at the crystal-packing interface, leads to ligand-independent increases in signaling in reporter gene assays despite only mild destabilization of the NRR, suggesting that it releases the autoinhibitory clamp on the heterodimerization domain imposed by the Lin12/Notch repeats. The Notch1 NRR structure should facilitate a search for antibodies or compounds that stabilize the autoinhibited conformation.