bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.