bluuebunny/crossref_metadata_2025_split huggingface dataset:
Predicting Response and Survival for Severe Aplastic Anemia Patients Treated with Immunosuppression.
Authors: Scheinberg Phillip , Wu Colin O. , Nunez Olga , Young Neal S. 
Published: April 2020

Abstract
Prognosis in acquired aplastic anemia is predicted by blood counts. The popular “Camitta criteria” were developed about 40 years ago, prior to the introduction of immunosuppressive therapies (IST) and the wide application of hematopoietic stem cell transplantation (HSCT), both of which have markedly improved survival in bone marrow failure. The hematologic response rate to anti-thymocyte-globulin (ATG) has been reported by many centers to be 60–70%; however, a practical and reliable predictor of response to IST is not available. Therefore, the decision to pursue HSCT or IST in SAA often relies on the patient’s age, availability of an HLA-matched sibling donor and in the presence of comorbidities. We conducted a retrospective analysis of 316 patients who received initial IST with a horse ATG-based regimen at the NIH Clinical Center from 1989 to 2005 for criteria that were predictive of hematologic response at 6 months and survival long-term. In multivariate analysis, younger age and higher baseline absolute reticulocyte count (ARC) and absolute lymphocyte count (ALC) were predictive of response at 6 months. Patients with a baseline ARC ≥ 25,000/uL (96 patients) had a much greater probability of response at 6 months following IST compared to the 91 patients with an ARC &lt; 25,000/uL and an ALC &lt; 1,000/uL (80% vs. 41%, respectively; p &lt; 0.001). Those with an ARC &lt; 25,000/uL and an ALC ≥ 1,000/uL (129 patients) formed an intermediate risk group with a probability of response to IST of 62%. This higher likelihood of response translated to better survival in patients in the high ARC and ALC group (92% at 5 years) compared to those with a low ARC and ALC (53%). The probability of response in patients younger than 18 years of age was 74% regardless of the pre-treatment blood counts. When the predictive criteria of the ARC and ALC was used in pediatrics patients only, the ALC was not predictive, however a high baseline ARC remained a significant predictor of response in the pediatric cohort with a response rate of 90% (in those with an ARC ≥ 25,000/uL) vs. 65% (in those with an ARC &lt; 25,000/uL; p=0.02). In the post-ATG era, baseline ARC and ALC together serve as simple predictor of response following IST, which should guide in stratifying risk among patients with SAA. These criteria should be useful for comparison between studies and in clinical decision making, particularly regarding timing of transplantation, as the indication for matched sibling HSCT (now offered to older patients) and alternative donor HSCT (in patients who lack an HLA-matched sibling) broadens.
Long Term Outcome of Pediatric Patients with Severe Aplastic Anemia Treated with Anti-Thymocyte Globulin and Cyclosporine.
Authors: Scheinberg Phillip , Wu Colin O. , Nunez Olga , Young Neal 
Published: April 2020

Abstract
Acquired severe aplastic anemia (SAA) is a fatal bone marrow failure disorder which commonly affects the young. An increased incidence of SAA is observed in children and young adults between the ages of 15 and 25. In those who have a histocompatible sibling donor, hematopoietic stem cell transplantation (HSCT) is often performed; for the remainder of patients immunosuppressive therapy (IST) with an anti-thymocyte globulin (ATG) based regimen is the treatment of choice. The overall response rate (children + adults) to ATG is 60–70%, with prolonged survival achieved in 70–80% of cases. Most of the reports on the efficacy of IST with ATG in children are of small cohorts. Here we report the outcome of children under the age of 18 treated with horse ATG + cyclosporine (CsA) at our institution from 1989 to 2006. Overall 77 received horse ATG as the initial IST. The overall response rate at 6 months was 74% (57/77); the cumulative incidence of relapse at 10 years was 33% and the median time to relapse was 558 days. The cumulative incidence of evolution following IST was 8.5%; all 3 cases occurred among partial responders. Cytogenetic abnormalities that were detected after IST were: monosomy 7 in two patients and deletion 13q in one patient. Overall there were 13 deaths (17%): four occurred within the 3 months following IST in patients who had a pre-treatment ANC of less than 100/uL and nine deaths occurred further than 6 months of IST. The median time to death was 570 days. The overall 10-year survival for the entire cohort was 80%; long term survival in patients who responded to IST was 89%. We conducted a separate analysis in the age group of 18–21. In total, 35 patients in this age group were treated with h-ATG-based IST from 1989 to 2006. The overall hematologic response rate was 60% (21/35) of which 7 relapsed (33%). The overall response rate among children (&lt; 18) was about 75% which is higher than what is observed in older patients at our institution (response rate of about 60%). The response rate in young adults approximates the overall adult response rate. The long term survival in pediatrics patients who respond to IST is very favorable at about 90%. IST remains a very good alternative in pediatric patients who lack an HLA-matched sibling donor and should be offered as initial therapy prior to an alternative HSCT.
TCR Vbeta Repertoire Analyses in Childhood Aquired Servere Aplastic Anemia: A New Predictor for IST Response?.
Authors: Hubner Bernd , Fuhrer Monika , Gombert Michael , Schuster Friedhelm R. , Harbott Jochen , Borkhardt Arndt 
Published: April 2020

Abstract
Acquired Severe Aplastic Anemia (aSAA) is a rare disease characterized by pancytopenia and bone marrow hypocellularity. There is good clinical and laboratory evidence that a T-cell mediated autoimmune mechanism directed against stem- and progenitor cells located in bone marrow plays a major role in the pathogenesis of aSAA. Immunosuppressive therapy (IST) and bone marrow transplantation (BMT) are the two treatment options. One of the key issues selecting the appropriate treatment is the long latency period of 4–6 months before a response to IST. In a large multicenter prospective study in children we could show that the severity of the disease (i.e. degree of granulocytopenia &lt; 200/μl) is predictive for response to IST. The spectratyping (immunoscope)-technique reflects the T-cell diversity by determining the T-cell receptor (TCR) CDR3 length polymorphisms. Since the grade of polymorphism is proportional to the number of peaks detected, it is possible to determine a complexity score which reflects the diversity of the T-cell population. Herein, we prospectively analyzed the clonality of Vbeta TCR-repertoires in bone marrow of children with aSAA (n=6). After treatment with IST we correlated the initial diversity of the T-cell repertoire with their response 4–6 months after the start of IST. Moreover, we addressed the question weather the bone marrow T-cell repertoire is skewed by the expansion of specific cells, which indicates an antigen driven immune response located in BM. For that we compared the complexities of bone marrow (BM) and respective peripheral blood (PB) derived lymphocytes. The Vbeta profiles in bone marrow compared to PB were skewed in aSAA patients (n=4) most apparent in the CD8 population (figure). The complexities in the healthy control group (n=3) were similar in both compartments. Furthermore, in patients with a haematological response (n=3) a decrease in complexity of T-lymphocytes was present in BM compared to IST non-responders (n=3). The decreased complexity reflects a high number of monoclonal expansions in BM as determined by sequencing analysis of 37 (CD4) and 55 (CD8) CDR3 regions. However, we could not detect preferential usage of specific Vbeta families, J-beta segments or uncommon features in CDR3 size compared to V(D)J-rearranged sequences in the NCBI Prot database. Our findings presented here provide evidence for BM specific proliferation of lymphocytes possibly due to an antigen driven immune response. Moreover, the TCR complexity at diagnosis is associated with a later IST response and could thus be used as an additional predictor for the appropriate treatment.
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Oxidative Metabolism in Red Cells Deficient in Antioxidant Enzymes.
Authors: Johnson Robert M. , Ho Ye-Shih , Yu Dae-Yeul , Kuypers Frans A. , Ravindranath Yaddanapudi , Goyette Gerard W. 
Published: April 2020

Abstract
Since 1–2% of the red cell’s oxyhemoglobin undergoes spontaneous heterolytic dissociation into metHb and superoxide every day, and superoxide is readily converted to H2O2 by superoxide dismutase, the red cell is unavoidably exposed to high levels of reactive oxygen species (ROS). We have used a series of gene-disrupted mice to examine the oxidative defenses of the red cell. Work with glutathione peroxidase deficient [GSHPx(−/−)] red cells has been reported earlier. We here add results with red cells from mice that were catalase deficient (−/−), catalase heterozygotes (+/−), double knockouts [GSHPx(−/−) and catalase (−/−)], or peroxiredoxin II deficient. Catalase(−/−) cells were readily oxidized by exogenous H2O2, as monitored by methemoglobin formation, but no methemoglobin was formed when these cells were exposed to organic peroxides. Catalase heterozygotes were not distinguishable from wild type cells in these assays. This is consistent with the clinical finding that partial catalase deficiency is a benign condition. Red cells lacking both GSHPx and catalase exhibited more metHb formation in response to an H2O2 generating system than did either single knockout, indicating that both enzymes contribute to the defense against exogenous H2O2. Adding catalase deficiency to GSHPx deficiency did not increase Hb oxidation by organic peroxides. With peroxiredoxin-deficient cells, PrxII(−/−), preliminary results found an increased flux of the endogenously generated H2O2 through catalase, which could be detected using 3-amino-1,2,4-triazole (3-AT) inhibition. Earlier work (Lee et al, Blood101:5033, 2003) has demonstrated increased ROS with cell and membrane damage in PrxII(−/−) red cells, but exposure to a flux of H2O2 generated by glucose oxidase produced no increase in metHb formation over that seen in wild type cells. Thus, Hb may be less sensitive to oxidation than is the cell membrane. Prx(−/−) red cells were not more sensitive than wild type red cells to Hb oxidation by organic peroxides. These findings show that all three oxidant defense enzymes play multiple roles in the erythrocyte. All participate in detoxifying endogenously generated H2O2. Calculations with a model of erythrocyte oxidative reactions (Johnson et al, Free Rad. Biol. Med.39:1407, 2005) are consistent with the experimental findings, predicting H2O2 disposal by all three enzymes. Thus, it is not accurate to identify any one of these enzymes as the sole oxidant defense mechanism in the erythrocyte, as all participate in H2O2 removal. GSHPx in addition has a unique role in detoxifying organic peroxides. The data indicate that peroxiredoxin plays a minor role in the red cell’s defense against Hb oxidation. However, PrxII(−/−) mice are anemic, in contrast with the hematologically normal GSHPx(−/−) and catalase(−/−) mice. This suggests that the primary role of PrxII in the red cell may lie in protecting the cell membrane against oxidative damage.
Microvascular Endothelial Cells Express Phosphatidylserine Receptor: A Functionally Active Receptor for Phosphatidylserine-Positive Erythrocytes.
Authors: Setty Yamaja B.N. , Betal Suhita Gayen 
Published: April 2020

Abstract
Phosphatidylserine (PS)-positive erythrocytes adhere to both endothelial cells and the sub-endothelial matrix components. While thrombospondin mediates these interactions, it is not known whether PS-associated erythrocyte-endothelial adhesion occurs in the absence of plasma ligands. Using ionophore-treated PS-expressing control erythrocytes, we demonstrate that PS-positive erythrocytes adhered directly to human lung micro-endothelial cells in the absence of plasma ligands, that this adhesion was further enhanced following endothelial activation with IL-1α, TNF-α, LPS, and hypoxia (2.5- to 8-fold increase), and that this adhesive interaction was selective to erythrocyte-PS. We next explored whether micro-endothelial cells express an adhesion receptor that recognizes cell surface expressed PS (PSR) similar to that expressed on activated macrophages. Using RT-PCR and Western blotting, we demonstrate constitutive expression of both PSR mRNA and protein which were up-regulated in a time-dependent manner following endothelial activation (with maximal increases of 3-fold in mRNA and 2-fold in protein noted at 4 and 6 hour, respectively). While minimal PSR expression was noted on un-stimulated cell surface (8% positivity), endothelial activation up-regulated surface expression of this receptor (35–40% positivity in IL-1α and TNF-α activated cultures). In antibody blocking studies, using both artificially generated PS-positive erythrocytes and also using PS-positive erythrocytes from patient with sickle cell disease, we demonstrate that PSR was functionally active supporting PS-mediated erythrocyte adhesion to activated endothelial cells. Our results demonstrate the existence of a novel functional adhesion receptor for PS on the micro-endothelium which is up-regulated by such pathologically relevant agonists as hypoxia and cytokines.
Cell Membrane Sulfatide Promotes Sickle Cell Adhesion to Endothelium.
Authors: Guchhait Prasenjit , Thiagarajan Perumal , Lopez Jose A. 
Published: April 2020

Abstract
Sickle cell disease (SCD) affects millions of people worldwide, and is associated with significant morbidity and mortality. Although the clinical manifestations of the disease are very complex, much of the cause can be ascribed to occlusion of small vessels by the sickle red blood cells (RBCs). More than 30% of all deaths in SCD are due to the vasoocclusion, which results in ischemia, multiorgan failure and strokes. The proximate cause for vasoocclusion appears to be an increased adhesiveness of sickle cells to the vessel wall, and we postulate that the exposure of sulfatide on sickle cells accounts for their adhesive phenotype. Sulfatide binds with high affinity to many of the adhesion proteins known to be involved in cell adhesion to subendothelium and endothelium, including von Willebrand factor (VWF), thrombospondin (TSP), laminin and P-selectin. We therefore compared the expression and distribution of sulfatide in sickle cells to that in normal RBCs. When examined by flow cytometry using a previously described Alexa fluor-conjugated single-chain variable fragment (scFv) antibody, PA38, we found that sickle cells displayed more sulfatide on surface than normal RBCs (mean fluorescence 1.6±0.5 Vs. 0.9±0.3, p&lt;0.05, n=6). When we examined sulfatide distribution by confocal microscopy using the labeled PA38, we found it to label more intensely in sickle cells than the normal RBCs and to be distributed heterogeneously, with areas of intense staining. The heterogeneous distribution suggested that the sulfatide might exist within membrane-microdomains/lipid rafts. We tested this possibility by sucrose density centrifugation of detergent lysates (1% Triton X-100) of erythrocyte ghosts from sickle and normal cells and found that sulfatide was distributed in raft fractions, as defined by being in the fractions containing the raft marker flotillin-1. Consistent with an important role for sulfatide in sickle vaso-occlusion, we found that both normal and sickle RBCs attached under flow to the surface of histamine-activated human umbilical vein endothelial cells (HUVEC). The sickle RBCs adhered more avidly, as they were able to rest higher shear stresses (1.86 and 2.5 dyne/cm2) than the normal RBCs before detaching Greater than 50% of the initial adhesion was inhibited by treatment with the anti-sulfatide scFv, PA38. We obtained similar results in terms of the greater shear resistance of sickle cells and the ability of PA38 to inhibit adhesion when we compared the adhesion of sickle and normal RBCs to surfaces coated with the adhesive ligands such as VWF (the ultra-large form) and laminin. Thus, our study elucidates an important role of red cell membrane sulfatide in sickle cell adhesion to the endothelium and to adhesive ligands, and suggests that this mechanism is important pathophysiologically in the development of sickle vaso-occlusion. Sulfatide distribution into lipid rafts may allow the formation of adhesive patches that facilitate adhesion.
The Ligand Occupancy of Endothelial Protein C Receptor Switches the PAR-1 Dependent Signaling Specificity of Thrombin from a Disruptive to a Protective Response in Endothelial Cells.
Authors: Rezaie Alireza R. , Bae Jong-Sup , Yang Likui , Manithody Chandrashekhara 
Published: April 2020

Abstract
It has been hypothesized that activated protein C (APC) exerts its cytoprotective and antiinflammatory activities through the endothelial protein C receptor (EPCR)-dependent cleavage of protease activated receptor 1 (PAR-1) on vascular endothelial cells. Noting that
the activation of protein C on endothelial cells requires thrombin, relative to APC, thrombin cleaves PAR-1 with ∼3–4-orders of magnitude higher catalytic efficiency, and PAR-1 is a target for the proinflammatory activity of thrombin, it is not understood how APC can elicit a protective signaling response through the cleavage of PAR-1 when thrombin is present.
In this study, we demonstrate that EPCR is associated with caveolin-1 in lipid rafts of endothelial cells and that its occupancy by the Gla-domain of protein C/APC leads to its dissociation from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway through coupling of PAR-1 to the pertussis toxin sensitive Gi-protein. Thus, when EPCR is bound by protein C/APC, the PAR-1 cleavage-dependent protective signaling responses in endothelial cells can be mediated by either thrombin or APC. These results provide a new paradigm for understanding how PAR-1 and EPCR participate in protective signaling events in endothelial cells.
Identification of a New Genetic Determinant Controlling Human Hematopoietic Stem Cell Engraftment.
Authors: Takenaka Katsuto , Prasolava Tatiana K. , Wang Jean C.Y. , Mortin-Toth Steven M. , Khalouei Sam , Gan Olga I. , Dick John E. , Danska Jayne S. 
Published: April 2020

Abstract
Transplantation of human hematopoietic stem cells (HSC) has been the most important clinical application of stem cell biology. A key discovery enabling successful HSC transplantation was the recognition that HSC engraftment is controlled by human leukocyte antigen (HLA) polymorphism. Although HLA disparity plays a key role in graft rejection, graft failure can occur even in patients receiving an HLA-identical transplant, suggesting that additional, as yet uncharacterized, factors modulate engraftment. We have identified a new genetic determinant that controls the outcome of human HSC transplantation. Xenotransplantation in the non-obese diabetic/severe combine immune-deficient (NOD.SCID) mouse is the best functional assay for human HSC, based on their ability to repopulate recipient animals. We show that NOD.SCID mice provide significantly better support for human hematopoietic grafts than mice of different strain backgrounds carrying equivalent immunodeficiency mutations. To identify the molecular basis for this strain-specificity, we used positional genetics combined with in vitro and in vivo assays of human hematopoiesis. We generated reciprocal congenic strains between NOD and the related non-obese diabetes resistant (NOR) strain which is 88% identical to NOD, and found that support of human hematopoiesis is conferred by variation in a single gene, signal regulatory protein α (Sirpα) on chromosome 2. Human HSC were unable to engraft NOD.SCID mice carrying the NOR allele of Sirpα. NOD SIRPα displays 24 amino acid variations compared to NOR and C57BL/6, concentrated in the immunoglobulin IgV-like domain of this Ig-superfamily member. SIRPα is expressed on myeloid cells and mediates signals that modulate diverse macrophage functions. Indeed, lentiviral gene transfer of the NOD Sirpα variant into NOR macrophages restored their ability to support human hematopoiesis in long-term chimeric stroma-based assays. CD47, the only known cellular ligand of SIRPα, is ubiquitously expressed and modulates multiple cellular actions on hematopoietic cells including platelet activation and adhesion, and leukocyte adhesion and cytokine production. NOD SIRPα demonstrates enhanced binding to human CD47 compared to the NOR protein, suggesting that differential interaction with CD47 underlies the strong effect of Sirpα variation on support of human hematopoiesis in vitro and in vivo. Our findings reveal a novel SIRPα-dependent, macrophage-mediated mechanism critical in HSC transplantation. Future analysis of Sirpα variants within human populations could identify alleles associated with hematopoietic support in BM failure syndromes or correlated with outcomes in clinical transplantation.
Glutamic Acid 192 of Activated Protein C Restricts the Specificity of PAR-1 Cleavage in Endothelial Cells.
Authors: Bae Jong-Sup , Rezaie Alireza R. 
Published: April 2020

Abstract
It is known that residue 192 (chymotrypsin numbering) plays a key role in determining the P3 binding specificity of coagulation proteases. This residue is a Glu in both thrombin and activated protein C. Previous studies have indicated that the substrates containing an acidic residue at the P3 position are not recognized efficiently by either thrombin or APC in the absence of a cofactor. Protease activated receptor 1 (PAR-1), a common substrate for both thrombin and APC contains an acidic Asp at the P3 position. In this study we examined the ability of Glu-192 to Gln (E192Q) substitution mutants of both thrombin and APC to recognize and cleave PAR-1 in human umbilical vein endothelial cells (HUVEC) transfected with a PAR-1 cleavage reporter plasmid in which the exodomain of the receptor has been fused to a cDNA fragment encoding the soluble alkaline phosphatase. Thrombin E192Q cleaved PAR-1 with a catalytic efficiency that was approximately 5-fold higher than that of wild-type thrombin. On the other hand, the activity of APC E192Q toward PAR-1 was improved approximately 100-fold. Thus, unlike an approximately 1000-fold lower activity for APC in cleaving PAR-1 relative to thrombin, the activity of APC E192Q toward PAR-1 was only 10-fold lower than that of thrombin. These results suggest that the inhibitory interaction of Glu-192 of APC with P3-Asp of PAR-1 is responsible for its poor activity toward PAR-1. It is known that the cleavage of PAR-1 by thrombin in endothelial cells elicits a proinflammatory response. However, the cleavage of the same receptor by APC in complex with endothelial protein C receptor (EPCR) invokes a protective antiinflammatory response. The mechanism of the paradoxical effect of PAR-1 signaling by the two proteases is not known. We used these mutants to investigate the possibility that the level of PAR-1 activation by either thrombin or APC dictates the type of the response in endothelial cells. Furthermore, we used these mutants in TNF-a-stimulated endothelial cell permeability and apoptosis assays to understand the mechanism by which EPCR enables APC to activate PAR-1 in endothelial cells. We discovered that the dose of receptor activation is not responsible for the paradoxical effect of PAR-1 signaling by APC and thrombin in endothelial cells. Furthermore, the interaction of APC with EPCR is not associated with an improvement in the catalytic efficiency of the protease toward PAR-1. The possible EPCR and PAR-1 dependent recognition and signaling mechanism of APC and thrombin is discussed.