bluuebunny/crossref_metadata_2025 huggingface dataset:
Detailed Mapping of the Binding Site within Platelet Glycoprotein Ibα for Leukocyte Integrin Mac-1 (αMβ2).
Authors: Han Qiang , Peng Yuandong , Chen Zhiping , Ustinov Valentin A. , Ugarova Tatiana P. , Plow Edward F. , Simon Daniel I. , López José A.
Published: October 2019

We have shown that the platelet counter-receptor for integrin Mac-1 (αMβ2, CD11b/CD18) is glycoprotein (GP) Ibα, the largest component of the GP Ib-IX-V complex, better known for its role in mediating platelet attachment to sites of vessel wall injury. The GP Ibα-Mac-1 interaction mediates the firm adhesion of leukocytes on platelet thrombi, enabling their subsequent transmigration into the vessel wall. Our previous studies showed that the I-domain of Mac-1 binds the C-terminal flanking sequences (Phe201–Gly268) of GP Ibα, as demonstrated by the fact that binding is almost completely inhibited by the I domain ligands fibrinogen and heparin, the I-domain monoclonal antibody LPM19c, and the GP Ibα-specific monoclonal antibody AP1. The latter antibody is of interest because it also inhibits the binding of von Willebrand factor and thrombin. We have mapped the AP1 epitope to a 10-amino acid sequence spanning Arg218 to Tyr228. In the current investigation, we constructed a series of cell lines expressing mutants of human GP Ibα, either by replacement of the human sequence with the corresponding dog sequence (dog GP Ibα does not bind human Mac-1) or by targeted mutagenesis, and tested their ability to bind the recombinant Mac-1 I domain. The GP Ibα region Phe201-Asn223 was crucial for Mac-1 binding, with residues Arg218, Asp222 and Asn223 playing vital roles. In addition, a peptide containing the AP1 epitope (Leu214-Val229) bound Mac-1 I-domain specifically and saturably. Peptide binding was blocked by LPM19c and soluble GP Ibα, and by the M2 peptide, which corresponds to the GP Ibα-binding site in the Mac-1 I domain (Phe201-Lys217). Peptide binding was also blocked by an antibody against the M2 sequence. The AP1 peptide inhibited the attachment of GP Ib-IX-V complex-expressing CHO cells to immobilized Mac-1 I domain, and the adhesion of THP-1 cells—a monocytoid line expressing Mac-1—to immobilized GP Ibα. This peptide did not inhibit attachment of the cells VWF surfaces, or thrombin-induced platelet aggregation. In summary, we have defined the GP Ibα sequence Arg214 to Val229 as a critical binding site for Mac-1. Because a peptide corresponding to this region inhibits GP Ibα binding to Mac-1 but blocks neither platelet adhesion to immobilized VWF nor thrombin-induced platelet aggregation, it has potential to guide the development of agents that will specifically inhibit leukocyte-platelet complexes that promote vascular inflammation.
Interaction of a SNARE-Associated Protein, Snapin with the Cytoplasmic Domain of the Platelet Membrane Glycoprotein V.
Authors: Nozoe Akiko , Fujimoto Tetsuro-Takahiro , Fujimura Kingo
Published: October 2019

Glycoprotein V (GPV) is a subunit of the platelet GPIb-IX-V receptor for von Willebrand factor (vWF) and thrombin. GPV is cleaved from the platelet surface during activation by thrombin, but its role in platelet activation is still unknown. It is reported that the GPV knockout mouse did not suffer from a bleeding syndrome, and the GPV-deficient platelets showed normal GPIb expression and vWF-mediated adhesion. One report showed an increased sensitivity of these platelets to activation by low concentrations of thrombin, suggesting that GPV may act as a negative modulator of the thrombin response. Another showed that GPV-deficient platelets exhibited defective adhesion to a collagen-coated surface, suggesting GPV binds to collagen and participates in platelet adhesion and aggregation responses to this agonist. To define the signaling pathway through GPV, we searched for proteins that bind to the GPV. We preformed a yeast two-hybrid screening in a human placenta library, using the sequence for GPV cytoplasmic domain as a bait. As a result, we isolated 2 independent positive clones, and the both clones encoded a partial and a full length sequence of the same protein, named snapin (SNAPAP), which was previously reported as one of the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor)-associated proteins. By RT-PCR analysis, snapin mRNA was detected in megakaryocyte cell lines and peripheral blood platelets. When the snapin was expressed as a GFP-fusin protein in K562 cells, which expressed intrinsic GPIb-IX-V complex, the protein localized at perinuclear region and cytosol. By immunoprecipitation-western blotting study, GPV was co-immunoprecipitated with GFP-snapin from the lysates of these cells by anti-GFP antibody, whereas GPV was not detected in the precipitates by a control antibody. Previous reports showed that snapin is a binding protein of SNAP-25 (synaptosome-associated protein-25) and SNAP-23, and influences the release response of neurons and chromaffin cells by enhancing the synaptotagmin binding to the SNARE complex. Therefore, the novel interaction between GPV and snapin, identified in this study, might be involved in the intracellular membrane-fusion events and the granule release reaction during the platelet activation.
The Interaction of Platelet Integrin αIIbβ3 and ICln Is Activation Dependent.
Authors: Larkin Deirdre , Moran Niamh
Published: October 2019

The major platelet integrin αIIbβ3 plays a critical role in thrombus formation and has been a major target for anti-thrombotic therapy. The precise molecular mechanism of activation of the platelet integrin αIIbβ3 is not understood although a critical role for the conserved αIIb-integrin cytoplasmic motif, KxGFFKR, has been identified. We have recently shown, using a synthetic KVGFFKR peptide probe, evidence for a direct and specific interaction between the platelet integrin αIIbβ3 and a protein involved in regulatory volume decrease, ICln. ICln is a volume regulating chloride channel protein that is capable of forming a pore structure within the membrane. Previously we demonstrated by surface plasmon resonance that purified αIIbβ3 binds to His-tagged recombinant ICln (His-ICln). We now show that purified αIIbβ3 binds to His-ICln in protein pulldown assays using a western blot detection system. In addition, His-ICln selectively pulls down endogenous αIIbβ3 from platelet lysates, but in an activation dependent manner. These comparisons were carried out in lysates from resting platelets and platelets activated with U46619 (10μM), TRAP (5μM) and thrombin (0.2U/ml) for 3 minutes at 37°C, unstirred. Reduced integrin precipitation was observed in lysates from platelets treated with acyclovir (100μM), a known pharmacological inhibitor of ICln, consistent with an activation dependent interaction.

As the channel function of ICln is nucleotide sensitive we set out to further identify if ICln regulates the activation of αIIbβ3 or if the integrin contributes to the activation of ICln. Low dose nucleotides were found to effect platelet function. Gel-filtered platelets treated with ATP (2nM) and apyrase (1U/ml) showed an increase in PAC-1 binding that could be inhibited with acyclovir (1mM). U46619 stimulation caused an increase in PAC-1 binding and enhanced platelet aggregation in the presence of ATP/apyrase that could also be inhibited by acyclovir in a dose-dependent manner (10μM–1mM). Thus, activation of ICln by low dose nucleotides can induce αIIbβ3 activation without further stimulus. However maximum αIIbβ3 activation is observed upon stimulation of both αIIbβ3 and ICln. The presence of an ICln inhibitor, acyclovir, interferes with platelet aggregation and integrin activation with and without specific ICln stimulation, suggesting a co-dependence between αIIbβ3 and ICln to obtain maximum platelet activation. Taken together with results of the protein- protein interaction studies this data suggests an activation-dependent, functional interaction between the volume regulating ICln protein and the platelet integrin αIIbβ3.
Dose-Adjusted EPOCH-Rituximab Is Highly Effective in the GCB and ABC Subtypes of Untreated Diffuse Large B-Cell Lymphoma.
Authors: Wilson Wyndham H. , Dunleavy Kieron , Pittaluga Stefania , Grant Nicole , Steinberg Seth , Raffeld Mark , Chabner Bruce , Grossbard Michael , Jaffe Elaine , Staudt Louis , Janik John
Published: October 2019

Diffuse large B-cell lymphoma (DLBCL) is subdivided by microarray into germinal center B-cell (GCB) and activated B-cell (ABC) subtypes. These subtypes have distinct pathways of lymphomagenesis and outcome with 5-year survival of 59% and 31%, respectively, using CHOP. A molecular predictor showed high tumor proliferation and the ABC subtype are associated with a poor outcome. We showed DA-EPOCH overcomes the adverse effects of high tumor proliferation (BLOOD 99:2685, 2002) and rituximab overcomes the adverse effects of BCL-2, which is associated with the ABC subtype (Proc Am Soc Heme 102:abstr 356, 2003). To assess DA-EPOCH-R with G-CSF in GCB and ABC subtypes, we employed a validated immunohistochemistry (IHC) algorithm (BLOOD: 103:275, 2004). GCB was defined as CD10+ or BCL-6+ and MUM1-. ABC was defined as CD10- and BCL-6- or CD10-, BCL-6+ and MUM1+. Eligibility includes de novo DLBCL, no prior chemotherapy, any PS, HIV- and stage I thymic DLBCL > 5 cm and all stage II-IV. Characteristics of 77 enrolled pts include median (range) age 48 (12-85) and PS 1 (0-3); stage III/IV 48 (62%); and LDH > nl 42 (55%); extranodal sites >1 27 (35%); and HI/H IPI 26 (34%). Including all 77 pts, response is CR/CRu 72 (94%). At the median (range) follow-up of 28 (4-60) mos, PFS is 82% and OS is 83% (Fig 1). DFS is 87% at 28 mos with no relapses beyond 18 mos. PFS at 28 mos according to IPI risk is 95% for 0-2 factors and 54% for 3-5 factors. Using IHC, 44 pts were subdivided in GCB (35) and ABC (9) subtypes. The proportion of the ABC subtype vs. GCS is low and likely due to incomplete IHC for the ABC subtype. Specifically, 16 cases that were CD10- did not have adequate BCL-6 and/or MUM1 IHC for categorization. PFS at 28 mos for the GCB and ABC subgroups were 84% and 62%, respectively (Fig 2). DA-EPOCH-R employs a pharmacodynamic design where doses of doxorubicin, etoposide and cyclophosphamide are adjusted (i.e. normalized) to achieve neutrophil nadirs < 500/mm3. PK analysis shows that clearance of doxorubicin and etoposide varies by up to 1 log and that dose-adjustment compensates for these differences. This approach increases dose intensity in pts with high drug clearance while limiting toxicity. Based on 451 cycles, the target nadir ANC < 500/mm3 was achieved on 277 (61%) cycles but with 67 (15%) episodes of fever/neutropenia. Non-hematological toxicity was low and there were 2 treatment related deaths. These results suggest DA-EPOCH-R is highly effective in all subtypes of de novo DLBCL. A phase III study comparing R-CHOP and DA-EPOCH-R with microarray analysis of tumor biopsies is under development.

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Normal Prion Protein Colocalises with the Tetraspanin CD63 in Cultured Human Erythroblasts.
Authors: Griffiths Rebecca E. , Anstee David J.
Published: October 2019

Detailed information regarding the intracellular distribution and trafficking of prion protein (PrP) in human haemopoietic cells is lacking. In order to address this deficiency we have investigated the expression of PrP in cultured erythroid cells. CD34+ cells derived from peripheral blood were cultured in the presence of IL3, SCF and EPO and aliquots removed from the cultures at different time points for examination by confocal microscopy with monoclonal antibodies. PrP was demonstrated in the plasma membrane and, by colocalisation with organelle-specific markers, in Golgi, ER and lysosomes at all time points. PrP mRNA expression was profiled throughout the culture by QRT-PCR. PrP mRNA expression is high in the immature cells and decreases throughout the culture. PrP mRNA expression is different from erythroid specific protein expression profiles. Monoclonal antibodies to PrP and another GPI-linked protein DAF (CD55) showed distinct, non-overlapping, staining in the plasma membrane suggesting they are located in different microdomains. CD63 is a widely expressed tetraspanin thought to function in trafficking membrane proteins between the plasma membrane and intracellular organelles. All CD63 staining observed in cultured erythroid cells was colocalised with PrP. A similar concordance was not observed with CD63 and DAF. These results disclose a previously unrecognised property of PrP and suggest a general mechanism whereby PrP is recycled within cells. This mechanism may be relevant to the pathogenesis of transmissible spongiform encephalopathies.
E47 Binds to PU.1 Inhibiting Its Ability To Bind DNA and Activate Gene Expression.
Authors: Dahl Richard , Iyer Sangeeta R. , Simon M. Celeste
Published: October 2019

The transciption factor PU.1 is required for the development of macrophages, granulocytes, and B lymphocytes. Additionally its protein concentration in multipotential hematopoietic progenitor cells regulates cell fate decisions with high levels of PU.1 directing myeloid cell fate acquisition and low levels directing B cell fate acquisition. Potentially high levels of PU.1 are required for myeloid development in order to overcome repressive effects of B cell lineage specific factors. The essential B cell factor BSAP (Pax-5) was shown to associate with PU.1 and repress its transactivation activity. Here we investigate whether two other essential B cell factors, E2A and EBF could affect PU.1 activity. We observed that the E2A component, E47, but not EBF could associate with PU.1. Additionally E47 could repress PU.1 dependent transactivation of the M-CSFR promoter and that this repression is mediated by inhibiting PU.1 DNA binding. Exogenous E47 expression in an IL-3 dependent multipotential cell line could block PU.1 induced differentiation and expression of endogenous M-CSF receptor protein. Our data suggests that high PU.1 concentration is required for myeloid development in order to overcome repressive actions of other lineage specific transcriptional regulators such as E47.
Severely Impaired Erythropoiesis in Mice Lacking Mediator Subunit Med1/TRAP220.
Authors: Borggrefe Tilman , Waskow Claudia , Roeder Robert G. , Stumpf Melanie
Published: October 2019

GATA transcription factors are essential for diverse developmental processes including erythropoiesis. Med1/TRAP220 was shown to interact with GATA factors in vitro (Crawford et al. (2002): J Biol Chem. 277(5):3585-92.). Med1/TRAP220 is part of the multiprotein Mediator complex that forms a bridge between gene-specific regulatory proteins and the RNA polymerase II (Borggrefe (2004): Research Adv. in Biol. Chem. (2: 9-20), Bourbon et al. (2004) Mol. Cell 14(5):553-7). Mediator subunit Med1/TRAP220 deficient mice die during mid-embryonic development (day 11.5dpc) due to defects in placental, cardiac and hepatic development (Ito et al. (2000): Mol Cell. 5 (4):683–93., Landles et al. (2004): Mol Endocrinol. 17(12):2418–35). In order to analyze hematopoiesis in TRAP220-deficient embryos, AGMs (aorta-gonad-mesonephros) and Yolk-sacs were isolated from day 10.5 dpc embryos and analyzed by FACS and methylcellulose assays. TRAP220-deficient embryos contain c-Kit positive cells and BFU-E (Burst forming units erythrocytes) activity. However, CFU-E (Colony-forming units erythrocytes) activity was 10-fold reduced. This correlated with severe decrease of the CD71/Ter119 double-positive cells, the CFU-E containing fraction, in FACS-analysis.

Our data shows that Mediator subunit Med1/TRAP220 is required for erythropoiesis just before the CFU-E stage. Because GATA factors affect early erythropoiesis and because MED1/TRAP220 binds to GATA factors in vitro, our data suggest that the Mediator of transcriptional regulation forms the bridge between GATA factors and the RNA polymerase II machinery.
Lyn Kinase Supports Primary Erythroid Progenitor Cell Development.
Authors: Karur Vinit , Vincent Bethany , Lowell Clifford , Wojchowski Don M.
Published: October 2019

Several lines of investigation have implicated Lyn as an important positive effector of red cell development: Deregulated Src kinases promote erythroleukemia, and Lyn is the predominant Src kinase of erythroid cells; as critical erythropoietic factors, Kit and the Epo receptor each stimulate Lyn kinase; and in an insightful set of investigations in J2E cells, Lyn has been shown to be required for Epo-dependent late erythroid development. Based on these considerations, adult bone marrow-derived primary erythroid progenitor cells from Lyn −/− mice presently were assessed for their ex vivo growth, survival and differentiation potentials. Lyn −/− erythroid progenitors expanded efficiently in serum-free media, and showed essentially wild-type Epo dose-dependent proliferative responsiveness. When transferred to BSA/insulin/transferrin differentiation medium, however, Lyn −/− erythroid progenitor cells clearly faltered in their development to Ter119-high, CD71-low erythroblasts. For these Lyn −/− cells, annexin-V binding studies revealed that this defect was associated, in part, with a stage-specific loss in survival potential. Interestingly, however, this defect was not Epo-dose dependent. In addition, MACS-isolated Kit-positive early erythroid progenitor cells prepared from Lyn −/− mice (unlike preparations from wild-type mice) failed to support synergistic effects of SCF-plus-Epo in 3HdT incorporation assays. In response to phenylhydrazine, Lyn −/− mice exhibited expanded erythroid progenitor cell pools (including BFUe and CFUe), and this hyper-expansion may occur in response to the compromised survival of late Lyn −/− erythroblasts. Analyses of pp60-Src expression revealed elevated levels of activated PY416-Src specifically in Lyn −/− EPC, a finding that is consistent with the activation of apparent compensatory mechanisms. In contrast, no significant changes in the levels of GATA1 or other assessed erythroid defining factors were detected. In response to phenylhydrazine, Lyn −/− mice showed ≥2-fold enhanced splenomegaly, as well as enhanced frequencies of BFUe, CFUe and Ter119(+) cells. Overall, these studies in primary erythroid progenitor cells from Lyn −/− mice reveal a previously undiscovered positive role for Lyn as a late-stage specific positive effector of erythroid cell survival, and regulator of Epo receptor and Kit co-signaling.
Extracellular Nucleoside Diphosphate Kinase NDPK/Nm23 Protein Isoforms A and B Support Erythropoiesis and Have a Burst-Promoting Activity.
Authors: Berneman Zwi N. , Willems Roel , Nijs Griet , Lenjou Marc , Van Bockstaele Dirk R.
Published: October 2019

We previously demonstrated the in vitro hematopoietic effects of different nucleoside diphosphate kinase NDPK/Nm23 proteins, i.e. increase of burst-forming units erythroid (BFU-E) and decrease of colony-forming units macrophage (CFU-M) (Willems R et al. Experimental Hematology2002;30: 640–648). The effect was especially pronounced in the marrows that had a particular low BFU-E count. Since these experiments were performed under optimal erythroid growth factor stimulation including adequately high concentrations of erythropoietin (Epo) and stem cell factor (SCF 100 ng/mL), subtle effects of NDPK/Nm23 proteins on erythropoiesis could have been missed. In the present series of experiments on 7 normal bone marrow mononuclear cell suspensions, we deliberately created suboptimal in vitro conditions for early (BFU-E) erythropoiesis: i.e. serum free conditions with a limited number of cytokines (only Epo and SCF) and a low concentration of burst-promoting activity (SCF at 5 ng/ml). The results show that addition of NDPK A/Nm23-H1 and NDPK B/Nm23- H2 (but not of NDPK C) at high (40 μg/mL), but not low, extracellular concentration is sustaining and improving in vitro erythropoiesis in a statistically significant manner (P<0.05). Red cell colonies increased by a factor of 1.9 and 1.4 following addition of NDPK A/Nm23-H1 and NDPK B/Nm23- H2 respectively. Especially with NDPK A/Nm23-H1, this approaches levels attained under optimal conditions for erythropoiesis, i.e. at SCF concentration of 100 ng/mL, where a 2.1-fold increase was seen, as compared to cultures with low concentrations of SCF (5 ng/mL) without addition of extracellular NDPK/Nm23. In cultures without Epo no erythroid colonies were seen, even following addition of NDPK A/Nm23-H1 or NDPK B/Nm23-H2. This study strongly suggests that NDPK/Nm23 has a burst-promoting activity. The fairly high concentrations of NDPK/Nm23 constitutively present in plasma supports the physiological role of this protein in stimulating early erythropoiesis. Of the (known) cytokines or proteins with burst-promoting activity (eg. SCF, interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), NDPK/Nm23 (this study)), only SCF and NDPK/Nm23 are produced in a constitutive fashion, strongly supporting their role in the steady-state support of early erythropoiesis.
Serum Erythropoietin and Aging: A Longitudinal Analysis.
Authors: Ershler William B. , Shan Sheng , McKelvey Julie , Artz Andrew S. , Denduluri Neelima , Tecson Josephine , Taub Dennis D. , Brant Larry , Ferrucci Luigi , Longo Dan L.
Published: October 2019

There are conflicting data regarding expected changes in serum erythropoietin (EPO) with advancing age, primarily because prior reports have been based upon cross-sectional analyses of diverse populations. To examine changes in serum EPO with age, we reviewed the clinical history, hematological parameters and serum EPO levels of 143 participants in the Baltimore Longitudinal Study on Aging (BLSA) who were studied at 1 – 2 year intervals for eight to thirty years. None had anemia at the time of the first visit (by WHO criteria: <12g/dL for women; or <13 g/dL for men) but it developed in 10 (7%) over the period of evaluation. An additional 20 individuals (14%) had a fall in hemoglobin of 1.5 g/dL or more, sustained over at least two visits, and for the purpose of this analysis were also considered anemic. EPO levels rose significantly with age for the group as a whole, and the slope of the rise was found to be greater for those who did not have associated diabetes or hypertension. Subjects who developed anemia but did not have hypertension or diabetes had the greatest slope in EPO rise over time. In contrast, those who had, or developed hypertension or diabetes during the period of follow-up within the BLSA, had significantly higher initial EPO levels and a reduced EPO slope over time. The increase in serum EPO with aging may be compensating for increased RBC turnover or increased EPO resistance of red cell precursors. We suspect that with very advanced age, or in those with compromised renal function (e.g., diabetes or hypertension), the compensatory mechanism becomes inadequate.

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Gene Therapy of the Human Erythrocyte Pyruvate Kinase Deficiency.
Authors: Segovia Jose C. , Meza Fellow Nestor W. , Fellow Oscar Quintana-Bustamante , Puyet Antonio , Rio Paula , Bueren Juan A. , Bautista Jose M.
Published: October 2019

Human Erythrocyte Pyruvate Kinase (hRPK) deficiency, an autosomal recessive disorder produced by mutations in the PKLR gene, is known to be the most common cause of chronic non-spherocytic haemolityc anaemia (CNSHA). Patient survival and clinical severity have been associated with the type of mutation found and to a compensatory expression of the broadly tissue distributed M2PK isozyme in their erythrocytes. Mutations in the RPK gene with severe clinical symptoms have been described in our laboratory. The transduction of the wild-type RPK to hematopoietic stem cells from deficient patients may become an alternative therapy in those patients where the mutation cause clinical symptoms. In an attempt to address this hypothesis we constructed retroviral vectors based on the pSF11 backbone and expressing the hRPK and the green fluorescent protein cDNAs in a unique messenger RNA separated by an eukaryotic IRES (SF11RPKXEG). In a first step, murine and human cell lines were transduced with infective supernatants expressing this construct. FACS and western-blot analysis demonstred stable RPK expression in all transduced cell lines. In murine erithroleukemia cell line (MEL) the stability of the expression was screened for more than 20 weeks by EGFP flow cytometry evaluation. The percentage of transduced cells and the intensity of the expression, evaluated by quantification of the mean of fluorescence (MFI) remain unaltered over time. Moreover, the hexamethylene bisacetamide-induced differentiation of MEL cells neither produce any change in RPK or eGFP expression. Secondly, Lin−Sca-1+ murine hematopoietic stem cells were infected in vitro with SF11RPKXEG supernatants and transplanted into myeloablated recipients. Three months post-transplant, very high expression of both RPK and EGFP proteins was detected in mononuclear cells, platelets and, most importantly, mature erythrocytes, the cell target for the treatment of this disease. These findings show that retroviral vectors could be a successful system for RPK delivery and expression in erythroid cells and provide evidences of the gene therapy potential in the phenotypic correction of erythropoietic defects related to human RPK deficiency.
Impact of Prior Pregnancy on Outcomes of Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Loren Alison Wakoff , Bunin Greta R. , Boudreau Christian , Champlin Richard E. , Cnaan Avital , Horowitz Mary , Loberiza Fausto , Porter David L.
Published: October 2019

Allogeneic hematopoietic stem cell transplantation (HSCT) can cure adults with hematologic malignancies, but results in significant morbidity and mortality. Graft-vs.-host disease (GVHD) is a major complication; attempts to reduce the risk of GVHD include selecting donors based on several characteristics, including parity, a criterion which has been controversial. This retrospective cohort study using data from the Center for International Blood and Marrow Transplant Research (CIBMTR) is the first multi-center analysis of the effects of donor and recipient parity on outcomes of HSCT in the modern transplant era.

Methods: We studied patients at least 18 years old who received a non-T-cell-depleted, myeloablative HLA-identical sibling HSCT between 1995 and 1999, for acute lymphoblastic or myelogenous leukemia or chronic myelogenous leukemia. The study endpoints included acute and chronic GVHD, overall survival, and relapse. There were 2,626 patients who met inclusion criteria and had complete information on both donor and recipient pregnancy status. Donors and recipients were categorized as: males, nulliparous females, or parous (one or more pregnancies) females.

Analysis: We compared all possible combinations of donor and recipient pregnancy status (9 groups in total); the reference group was male donor/male recipient pairs. Multivariate Cox proportional hazards regression was used to adjust for other prognostic factors. Because multiple groups were compared, significant p-values were considered to be less than or equal to 0.006.

Results: After controlling for important patient-, disease-, and transplant-related covariates, the risk of chronic GVHD was significantly increased in parous female donor/male recipient pairs (HR 1.56, 95% CI 1.23 – 1.94, p < 0.0001). Neither donor nor recipient parity had an impact on overall survival, acute GVHD, or relapse risk.

Conclusions: This multi-center retrospective registry study showed that parous female donors resulted in a significantly increased risk of chronic GVHD in male recipients, but without concomitant reduction in relapse. Thus, H-Y antigens may be important targets of GVHD, but not of a graft-versus-leukemia effect. As when selecting unrelated donors, avoidance of parous female donors, particularly for male patients, in HLA-identical sibling transplants is recommended when possible.
Analysis of T-Cell Repertoire before and 21 Days after Autologous Peripheral Blood Stem Cell Transplantation for Systemic Sclerosis.
Authors: Donnenberg Albert D. , Donnenberg Vera S. , Medsger Thomas A. , BuchBarker Diane M. , Betters Dawn M. , Yeager Andrew M.
Published: October 2019

The goal of autologous peripheral blood stem cell transplantation (PBSCT) in autoimmune diseases is to ablate the clones of autoimmune lymphocytes. We evaluated the T-cell repertoire in 5 patients (pts) with systemic sclerosis (SSc) who received T-depleted (mean T-cell reduction, 5.2 log) autologous PBSCT after a conditioning regimen of cyclophosphamide (200–400 mg/m2/day x 5), fludarabine (25 mg/m2/day x 5), and rabbit anti-thymocyte globulin (2.5 mg/kg/day x 3). One pt has had a recurrence of SSc at 12 months after PBSCT; the other pts are progression-free at 4.9+, 5.1+, 6.5+ and 11.5+ months, respectively, after PBSCT. We used 5-color flow cytometry to assess T-cell receptor V-beta repertoire in CD4+ and CD8+ peripheral T-cells susbsetted into central memory (CM; CD45RA−/CD27+), naïve (N, CD45RA+/CD27+), effector/memory (EM; CD45RA−/CD27−) and effector (E; CD45RA−/CD27−) populations. Pts were studied before and at 21 days after PBSCT. The pt with recurrence of SSc had 2 large expansions in CD4+ T-cells (Vbeta 1 and 14) before PBSCT, which were conserved and amplified at 21 days post PBSCT. These expansions were limited to the EM and E populations before PBSCT but were evident in all except the E subset at 21 days after PBSCT. Of the remaining 4 pts, none had evidence of CD4 expansions, and 3 had multiple CD8 expansions before PBSCT. In all cases, the expansions were limited to the EM and E populations. None of these T-cell restrictions were conserved in the 21-day post-PBSCT sample. These data suggest that EM and E T-cell expansions present before PBSCT in pts with SSc can be ablated by an immunosuppressive conditioning regimen. Although our observations require confirmation in a larger series of pts, the conservation of restrictions at 21 days after PBSCT may be a poor prognostic sign for efficacy of autologous PBSCT in SSc.
Immunotherapy with Donor Leukocyte Infusions (DLIS) in Relapsed Hodgkin’s Disease (HD) Following Allogeneic Stem Cell Transplantation (ALLO-SCT): CD3+ Cell Dose, GVHD and Disease Response.
Authors: Anderlini Paolo , Acholonu Sandra A. , Okoroji Grace-Julia , Saliba Rima , Giralt Sergio A. , Ueno Naoto , Donato Michele L. , Andersson Borje S. , Champlin Richard E.
Published: October 2019

Background: There is paucity of data on DLIs for the management of recurrent HD following allo-SCT, and on whether CD3+ cell dose is relevant to response (and the development of GVHD) as is the case in chronic myeloid leukemia.

Patients: Nine patients with advanced HD who had undergone allo-SCT received DLIs as immunotherapy for treatment of progressive disease (PD). A total of fifteen DLIs were performed, with four patients receiving more than one DLI. Their median age was 30 years (18–42), and 7/9 had failed a prior autologous SCT. In four patients prior chemotherapy was administered.. Seven had a matched sibling donor and two a matched unrelated donor, and in all but one case the conditioning regimen included fludarabine plus cyclophosphamide or melphalan (plus/minus antithymocyte globulin). The pre-DLI chimerism status was 2/9 mixed and 7/9 full donor.

Results: The CD3+ cell dose administered was 77.5 x 10E6/kg (5–285). GVHD developed following the DLI in all but one patient and after 9/15 DLIs (60%). The response rate (complete/partial response) was 4/9 (44%). Three of these four responders developed GVHD and 3/4 had received chemotherapy. After the DLI mixed chimerism (n=2) was converted to complete (or near-complete) donor chimerism. The median response duration was 7 months (range 4-9+). No correlation (or threshold) was observed between CD3+ cell dose infused and disease response. The correlation between CD3+ cell dose and GVHD could not be evaluated statistically as all but one patient developed GVHD. At the latest follow-up three patients are alive (one in ongoing complete remission) and six have expired (PD n=3, non-relapse mortality n=3).

Conclusions: These data suggest that DLIs for immunotherapy of recurrent HD following allo-SCT have significant activity, although they frequently lead to GVHD (and, if not already present, full donor chimerism). The small sample size does not allow any firm conclusion as to whether chemotherapy administration increases the chance of response. The CD3 cell dose infused does not seem to correlate with disease response.
The Relationship of Pulmonary Hypertension and Survival in Sickle Cell Disease.
Authors: Ataga Kenneth I. , Jones Susan , Olajide Oludamilola , Strayhorn Dell , Lail Alice , Orringer Eugene P.
Published: October 2019

Introduction: Pulmonary hypertension (PHT) is a common complication of sickle cell disease (SCD). Most SCD patients who have PHT appear to have only mild elevations in their pulmonary artery systolic pressure (PASP). Although SCD patients with PHT have been reported to have an increased mortality, the effect of mild PHT on survival in patients with SCD remains uncertain.

Methods: The study subjects represent a cohort of patients followed at the Sickle Cell Clinic at UNC, Chapel Hill. Doppler echocardiography was used to estimate the PASP. The PASP was determined by applying the modified Bernoulli equation (PASP = 4V2 + right atrial pressure), if an adequate tricuspid regurgitant velocity was observed. A subject was considered to have PHT if his/her PASP exceeded the upper limits of normal in the age- and body mass index-adjusted reference ranges. Subjects with PHT were sub-classified into mild (above normal values up to 44 mm Hg), moderate (45 – 74 mm Hg) or severe pulmonary hypertension (≥ 75 mm Hg). Patients’ data were censored at the time of their death or loss to follow-up.

Results: The 60 subjects enrolled in this study have been followed for a period of 9 to 47 months (mean [± SD] of 29.8 ± 10.7 months). Pulmonary hypertension was observed in 18 subjects on initial evaluation, while the remaining 34 subjects had no evidence of PHT. The 18 subjects with PHT have been followed for 24.9 ± 10.6 months, while the 34 subjects without PHT have been followed for 31.8 ± 10.1 months. Six patients have died to date. All of these 6 patients had evidence of PHT, with estimated PASP values of 44, 44, 48, 54, 74 and 84 mm Hg (mean = 58 mm Hg) respectively. The presence of PHT was strongly associated with an increased risk of death (p = 0.0001). No other variables were associated with the risk of death in these patients. Although more patients with moderate and severe PHT died when compared to patients with mild PHT, the difference was not statistically significant.

Conclusion: Pulmonary hypertension is strongly associated with an increased risk of death in patients with SCD. While our study did not find a significant difference in the risk of death when patients with mild PHT were compared to patients with more severe disease, the number of patients followed was relatively small and the duration of follow-up was short. More patients and a longer period of follow-up would be required to ascertain whether the effect of mild pulmonary hypertension on mortality is different from that of more severe PHT in patients with SCD.

Figure Figure
Density-Dependent Regulation of Hematopoietic Stem Cell Fate by the Notch Ligand, Delta1.
Authors: Dallas Mari H. , Delaney Colleen , Varnum-Finney Barbara , Bernstein Irwin D.
Published: October 2019

Notch signaling regulates multiple cell fate decisions by hematopoietic precursors. Previously, we found that endogenous Notch signaling in cultures of murine hematopoietic precursors (Lin-Sca-1+ c-Kit+) leads to a multi-log increase in the number of Sca-1+ c-Kit+ cells, inhibition of myeloid differentiation, and promotion of T/NK differentiation. To activate Notch signaling in those studies, a single dose (10μg/ml) of engineered Notch ligand consisting of the extracellular domain of Delta1 fused to the Fc domain of human IgG1 (Delta1ext-IgG) was immobilized to the plastic tissue culture surface. To investigate quantitative effects of Notch signaling, bone marrow Lin-Sca-1+ c-Kit+ (LSK) cells were cultured with plates coated with increasing concentrations of Delta1ext-IgG in media supplemented with 20% FBS, SCF (100 ng/mL), Flt3L (100 ng/mL), IL6 (100ng/mL) and IL11 (10ng/mL). LSK cells cultured for 14 days with control human IgG1 underwent terminal myeloid differentiation (determined by expression of GR1 and F4/80) with no further increase in cell number, whereas at all densities of Delta1ext-IgG there was approximately a 3 log greater number of cells than in control cultures. Furthermore, the portion of cells that maintained Sca-1 and c-Kit expression increased at greater densities of Delta1ext-IgG (10%, 32%, 77%, 71%, 71% and 71% for plates coated with ligand at 0.6, 1.25, 2.5, 5, 10 and 20 μg/ml, respectively, and 5% for human IgG1 control at 10μg/ml), whereas the portion of cells undergoing myeloid differentiation decreased at greater ligand densities (48%, 33%, 5%, 3%, 3% and 3% respectively, and 40% for control). In contrast, a substantial increase in the portion of cells expressing B220+ was observed at relatively low densities of Delta1ext-IgG (30% at 0.6 μg/ml and 19% at 1.25 μg/ml) compared to control (4%), but was no longer evident with further increases in ligand density (1.8%, 2%, 1.2%, m1.6% at 2.5, 5, 10 and 20 μg/ml respectively). Furthermore, promotion of early T cell differentiation was observed in ligand containing cultures with the generation of increased number of cells co-expressing Thy1.2 and CD25 (14%, 24%, 22% and 24% at 2.5, 5, 10 and 20 μg/ml respectively). Further evidence for T cell commitment was established by quantitative RT-PCR in which increased expression of CD3ε and pre-Tα was observed by 28 days of culture. Thus these studies demonstrate that culture with different densities of the Notch ligand, Delta1ext-IgG results in differential cell fate outcome with inhibition of myeloid differentiation and promotion of early T cell induction that is maximal at high ligand densities and of B220+ cells at relatively lower densities.
The Heparin Binding Properties of Heparin Cofactor II Suggest an Antithrombin -Like Activation Mechanism.
Authors: Okeeffe Denis J. , Baglin Trevor P. , Gallagher John T. , Olson Steve T. , Huntington James A.
Published: October 2019

Heparin cofactor II(HCII) is a serpin which specifically inhibits thrombin. Like antithrombin(AT) it requires glycosaminoglycan(GAG) binding before full activation. However unlike antithrombin no specific activators have been identified. What currently prevents selective therapeutic activation of HCII is the lack of knowledge of the determinants of GAG binding specificity. We have investigated the heparin binding properties of HCII using size fractionated heparins. The affinity of heparins for HCII, ranging in size from 8 to 26 saccharide units, were determined by following change in fluorescence of the extrinsic probe TNS. The effect of heparin concentration(from 1 to 100nM) on the rate of thrombin inhibition was determined for the same heparins under psuedo first-order conditions. Based on these studies we can conclude that binding is non specific with a minimal heparin length of 13 monosaccaride units required. The observed dependence of catalytic activity on heparin length is caused entirely by the size dependence of heparin affinity for HCII. By studying affinity at different ionic strengths it is clear that binding is critically dependent on ionic strength with a very weak non-ionic contribution to binding. Rapid binding experiments were conducted under psuedo first-order conditions in heparin using a stopped-flow fluorometer. These studies of heparin binding indicate an induced- fit mechanism which involves a conformational change in HCII. The low affinity of HCII can now be understood as a failure of interactions to reduce the rate of the reverse conformational, in a manner analogous to the interaction of AT with low affinity heparin. HCII is activated by a fully allosteric mechanism which produces a 2000 fold increase in activation on heparin binding. We conclude that the heparin binding mechanism of HCII is closely analogous to that of AT, and that the design or discovery of high affinity, HCII-specific GAGs is thus feasible.
Molecular Models of the Procoagulant Intrinsic Tenase Complex.
Authors: Autin L. , Lee W. H. , Miteva M. A. , Mertens K. , Radtke K. P. , Villoutreix B. O.
Published: October 2019

Formation of the intrinsic tenase-complex, is an essential event in the procoagulant reactions that lead to clot formation. The tenase-complex is formed when the activated serine protease, factor IXa (FIXa), and its cofactor factor VIIIa (FVIIIa) assemble on the phospholipid surface of activated platelets in the presence of calcium ions to proteolytically convert the zymogen factor X (FX) into its active form FXa. The physiological relevance of the tenase complex is evident in hemophilia A or B patients, who present with bleeding disorders due to quantitative and/or qualitative deficiencies of FVIII or FIX. In this work we present preliminary models of the FVIIIa-FIXa-FX complex that were generated based on the published X-ray structures of FIXa (PNAS1995; 92:9796–800.), factor Va (PNAS2004; 101:8918–23.), factor Xa (PNAS1995; 95:6630–5.) and the FVIII homology model (Blood2002; 99:1215–23.). In a first step we developed new, refined, theoretical models of FVIIIa and FX zymogen via homology modeling, inter-domain docking/simulation and new loop simulation algorithms. This was followed by Pseudo-Brownian protein-protein docking in internal coordinates between FVIIIa and FIXa and between FIXa and FX with internal coordinate mechanics (Proteins2003; 52:113–7.). Flexibility of the molecules was partially accounted for by the use of a soft interaction energy function precalculated on a grid. Several models of the tenase-complex were selected based on structural criteria and agreement with known experimental data such as site directed mutagenesis, naturally occurring FVIII and FIX mutations reported in hemophilia, and overall location of the membrane plane with regard to the enzyme active site and peptide studies. These 3D models of the tenase-complex will be useful tools to guide future site directed mutagenesis and drug design studies aimed at improving functionality and half-life of FVIII and FIX and will contribute a better understanding of the role of the tenase-complex in the blood coagulation cascade.
Factor VIII:E113A Represents a High Specific Activity Factor VIII Arising from a Single Point Mutation within a Ca2+ Binding Site.
Authors: Wakabayashi Hironao , Fay Philip J.
Published: October 2019

We recently identified an acidic-rich segment in the A1 domain of factor VIII (residues 110-126) that functions in the coordination of Ca2+, an ion necessary for cofactor activity (Wakabayashi et al., J. Biol. Chem.279:12677–12684, 2004). Using Ala-scanning mutagenesis, it was determined that replacement of residue E113 with Ala yielded a factor VIII point mutant that possessed an ~2-fold increased affinity for Ca2+ as compared with wild type, suggesting that this residue did not directly contribute to Ca2+ coordination but rather modulated the affinity of the ion at this site. Furthermore, the E113A factor VIII possessed twice the specific activity of wild type as determined by a one-stage clotting assay. This increased activity was not likely a result of increased affinity for Ca2+, since assays were performed at saturating Ca2+ levels. Saturation mutagenesis at position 113 revealed that substitution at this position with relatively small, nonpolar residues were well-tolerated, whereas replacement with a number of polar or charged residues was detrimental to activity. Ala-substitution yielded the greatest activity increase of ~2-fold and this level was observed over a wide range of factor VIII concentrations. Time course experiments of factor VIII activation following reaction with thrombin revealed similar rates of activation and inactivation of E113A as observed for the wild type. Interestingly, results from factor Xa generation assays using purified reactants showed the mutant possessed <10% greater specific activity than wild type and yielded similar values for Km for substrate factor X, kcat for factor Xa generation and Kd for factor IXa. Thus the single amino acid substitution minimally altered cofactor structure or inter-molecular interactions relating to its participation in factor Xase. These results indicate that mutations within this Ca2+ coordination site may selectively enhance cofactor specific activity as measured in a plasma-based assay compared to activity determined in a purified system. The enhanced activity observed for E113A factor VIII may derive from a subtle alteration in conformation affecting a yet to be identified functional parameter.
Irreversible Blockade of Factor VIII Antibodies.
Authors: Planque Stephanie , Escobar Miguel A. , Taguchi Hiroaki , Karle Sangeeta , Nishiyama Yasuhiro , Donnachie Elizabeth , Paul Sudhir
Published: October 2019

High titer antibodies (Abs) to Factor VIII (FVIII) can render FVIII replacement therapy ineffective in severe hemophilia A patients. An antigen capable of specific and covalent binding is predicted to reverse the effects of pathogenic Abs potently compared to conventional noncovalently binding antigens, as dissociation of the former type of antigen from the immune complexes is precluded. Recently, we described the apparently universal presence of enzyme-like nucleophiles in the antigen binding sites of Abs (Planque et al, J Biol Chem, 2003). Here, we report a covalently reactive antigen analog (CRA) of FVIII that inactivates anti-FVIII Abs specifically. Recombinant FVIII was derivitized at Lys side chains with phosphonate diester groups, positioning these electrophiles within the antigenic epitopes expressed by FVIII. Comparable binding of FVIII-CRA and underivitized FVIII by a monoclonal Ab and polyclonal Abs from 5 hemophilia patients was evident, suggesting maintenance of the FVIII epitope structure necessary for noncovalent Ab recognition. FVIII-CRA formed immune complexes with polyclonal anti-FVIII Abs from hemophilia patients that were not dissociated by sodium dodecyl sulfate (2%), a detergent that disrupts noncovalent interactions. Removal of free FVIII-CRA from the reaction mixtures by affinity chromatography on Protein G did not restore the FVIII-binding activity of the Abs, confirming irreversible occupancy of the Ab binding sites. No binding of the Abs by an irrelevant peptidyl CRAs was observed, suggesting the role of noncovalent antigen binding in imparting specificity to the covalent reaction. Ab inhibition of the functional role of FVIII in coagulation was studied by the chromogenic assay. Treatment of the Abs with the FVIII-CRA followed by removal of free FVIII-CRA relieved the inhibitory effect of the Abs on FVIII mediated activation of FIXa/FX. An irrelevant peptidyl CRA did not relieve the Ab inhibitory effect. Compared to FVIII, the FVIII-CRA activated FIXa/FX poorly, suggesting that the integrity of the FIXa binding site had been disrupted. These observations encourage the development of electrophilic CRA analogs of FVIII for treatment of patients with inhibitory Abs to FVIII.