bluuebunny/crossref_metadata_2025_split huggingface dataset:
Vascular Abnormalities and Pulmonary Hypertension in the Berkeley Sickle Mouse.
Authors: Archer David R. , Elms Shawn , Boutwell Joshua , Perry Jennifer , Sutliff Roy 
Published: April 2020

Abstract
Clinically, pulmonary hypertension is a major risk factor for mortality in adults with sickle cell disease. Contributing factors probably include red cell hemolysis and vaso-occlusive injury with their associated oxidative and inflammatory stimuli. Previously, we have described RBC hemolysis and endothelial oxidative stress in the Berkeley sickle mouse model and extend those studies in this work to investigate cardiovascular and endothelial dysfunction. Eight to ten month old homozygous and hemizygous Berkeley sickle mice and C57BL/6 control mice were used for all aspects of these experiments. In vivo measurements of mean arterial pressure and right ventricular pressures were conducted in fully anesthetized mice using a pressure transducer inserted in the carotid and right ventricle respectively. Following in vivo readings hearts were excised for measurement of ventricular mass. The ascending aorta was removed and cut into 5 mm rings for in vitro studies of agonist- induced contractility and relaxation. The mean arterial pressure of the hemizygous sickle mice (70.6 ± 3.4) was significantly lower than the control mice (86.0 ± 3.1) and the mean arterial pressure of homozygous sickle mice (59.0 ± 2.2 mmHg) was significantly lower than the hemizygous and control mice (p≤0.05 and p≤0.001, respectively). The right ventricular pressure showed a trend that approached significance (p= 0.08) such that pressures in homozygous mice were ≥ than those in hemizygous which were ≥ than those in control mice. Increased basal cardiac output was suggested by significant left ventricular hypertrophy. In vitro examination of potassium chloride activation of voltage gated calcium channels showed no significant difference in sensitivity or maximal contraction. Similarly, there was no difference in sensitivity to the α1 agonist, phenylephrine. However, both hemi- and homozygous mice showed a significant reduction in maximal force of contraction (normalized to cross sectional area when compared to controls. Maximal acetylcholine induced relaxation of aortic rings was significantly reduced (p≤0.05) in homozygous sickle mice compared to controls. The same effect was not seen with sodium nitroprusside induced relaxation indicating that the acetylcholine effect was not due to effects on the smooth muscle but was endothelium-dependent. The Berkeley mouse model shows cardiac hypertrophy consistent with the increased cardiac output associated with chronic anemia and a reduced basal mean arterial blood pressure similar to that seen in humans. 8–10 month old mice have increased right ventricular pressure and RV mass indicative of pulmonary hypertension. Further endothelial dysfunction is characterized by a reduction in the maximal relaxation elicited by acetylcholine. Therefore, the Berkeley mouse is a good model for investigating sickle related endothelial dysfunction.
Lactate Dehydrogenase as a Biomarker of Hemolysis-Associated Nitric Oxide Resistance, Priapism, Leg Ulceration, Pulmonary Hypertension and Death in Patients with Sickle Cell Disease.
Authors: Kato Gregory J. , McGowan Vicki , Machado Roberto F. , Little Jane A. , Taylor James , Morris Claudia R. , Nichols James S. , Wang Xunde , Poljakovic Mirjana , Morris Sidney M. , Gladwin Mark T. 
Published: April 2020

Abstract
Pulmonary hypertension is prevalent in adult patients with sickle cell disease, and is strongly associated with markers of hemolysis, in particular serum lactate dehydrogenase (LDH), and early mortality. Intravascular hemolysis leads to a state of resistance to nitric oxide (NO), mediated by NO scavenging by plasma oxyhemoglobin and by arginine degradation by plasma arginase. We hypothesized that serum LDH may represent a convenient biomarker of intravascular hemolysis and NO bioavailability, characterizing a clinical subphenotype of hemolysis-associated vasculopathy. In a cohort of 213 patients with sickle cell disease, we found statistically significant associations of LDH with low levels of hemoglobin and haptoglobin and high levels of reticulocytes, bilirubin, plasma hemoglobin, aspartate aminotransferase, arginase and soluble adhesion molecules. LDH isoenzyme fractionation confirmed predominance of LD1 and LD2, the principal isoforms within erythrocytes. In a subgroup, LDH levels closely correlated with plasma cell free hemoglobin, accelerated nitric oxide consumption by plasma and impaired vasodilatory responses to an NO donor. Remarkably, this simple biomarker was statistically associated with a clinical subphenotype of pulmonary hypertension, leg ulceration, priapism, and risk of death in patients with sickle cell disease. We propose that LDH elevation identifies patients with a syndrome of hemolysis-associated NO-resistance, endothelial dysfunction and end-organ vasculopathy.
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Delayed Wound Healing in Hemophilia B Mice.
Authors: Hoffman Maureane , Lenkowski Angie , Brock Jacqueline , Hedner Ulla , Monroe Dougald M. 
Published: April 2020

Abstract
These studies use our previously reported wound healing model to establish the mechanisms behind the impaired wound healing we observed in hemophilia B animals. We hypothisized that thrombin mediated signals generated in response to the initial wound would be absent or reduced in hemophilia B mice, leading to an impaired course of wound healing and poor angiogenesis. All work was done in accord with the US Amended Animal Welfare Act and was monitored by the Institutional Animal Care and Use Committee of UNC. Three mm circular dermal wounds were made in normal and hemophilia B mice. All wounds were treated with an antibiotic gel at the time the wound was made. The mice were monitored for bleeding both at the time the wound was made and for delayed bleeding. Hemophilia B mice with excessive bleeding were treated. The wounds were monitored for regrowth of the epithelial layer. The size of the area not covered by epithelial tissue was measured and expressed as square millimeters (wound size). We have previously reported that there is a significant difference in the time to wound healing between untreated hemophilic (10 days) and normal animals (7 days). At the end of the time course, wound areas were excised and the skin was formalin fixed, paraffin embedded, and sectioned. Essentially all hemophilia but no normal animals showed unresolved subcutaneous hematoma. During the wound healing process, macrophage infiltration into the granulation tissue was significantly delayed. Unexpectedly, hemophilia B mice relative to wild type mice showed twice as many blood vessels in the healing wound. This difference persisted out to two weeks after the wound was made and well after reepithelialization. Pretreatment (30 minutes prior to wounding) of hemophilia B mice with a single bolus dose of human factor IX sufficient to give 1 U/mL corrected the wound healing time. Pretreated hemophilia B mice did not have obvious subcutaneous hematomoas. The observation that a single bolus dose of factor IX prior to wounding a hemophilia B animal normalized the wound healing time provides a starting point for determining how different doses or different administration times effect wound healing. It also provides a starting point from which to compare the effects of bypassing therapy on wound healing.
Epitope Mapping of Inhibitors in Acquired Hemophilia by Phage Display.
Authors: Königs Christoph , Kessel Christoph , Scholz Sabine , Stumpf Susanne , Krause Manuela , Klingebiel Thomas , Kreuz Wolfhart 
Published: April 2020

Abstract
In acquired hemophilia polyclonal autoantibodies (inhibitors) to coagulation factor VIII are generated. Inhibitors functionally interfere with interaction of molecules involved in the coagulation cascade for example the interaction of FVIII and the von Willebrand factor. Inhibitors in acquired hemophilia have been reported to bind either to the A2 or to C2 domain of FVIII.
Random peptide phage display libraries were screened with patient plasma to identify small peptides mimicking inhibitor epitopes. Peptide sequences were used to map the inhibitor epitope to the surface of FVIII. The phage libraries included libraries with linear or cyclic 7mer or 12mer inserts. Biopanning was performed including positive selections and also negative selections to deplete irrelevant binders. Phages bound to inhibitors were eluted by pH shift or by competition with FVIII. Single phage clones were tested for their specificity for inhibitor positive plasma by ELISA. For specific binders, the sequence of the inserts was identified by DNA sequencing. For one patient 33 phage clones were analysed from three libraries and 28 peptide sequences mimicking the inhibitor epitopemimotopes - were determined. The peptide sequences map to two conformational epitopes on the A2 and A1 domain of FVIII. Clusters of six and four amino acids are the proposed binding regions on the A2 and A1 domain respectively. The matching conformational motifs contain PPLRQ and PPLS. Synthetic peptides corresponding to the sequence displayed on phage were generated and binding of inhibitors to peptides was confirmed by different ELISA formats.
Currently, functional studies with synthetic peptides are being performed to further analyse the epitopes. Additionally, more patient samples are being screened to analyse further epitopes and understand immuno dominant regions of FVIII in acquired haemophilia and compare them to the immune response in hemophilia A. The mimotopes isolated could be a basis for the development of ligand effector conjugates to target inhibitor specific B cells.
Ligand Mediated Targeting of FVIII Inhibitor Specific Primary B Cells Via Surface Immunoglobulin.
Authors: Kessel Christoph , Kreuz Wolfhart , Brassat Katharina , Klingebiel Thomas , Königs Christoph 
Published: April 2020

Abstract
Hemophilia A is an X-linked bleeding disorder. Mutations in the Factor VIII (FVIII) gene result in reduced or non-functional expression of FVIII. In hemophilia A recombinant (rFVIII) or plasma derived (pdFVIII) preparations are substituted to restore coagulation activity. However, a major problem of current treatment in hemophilia A is the development of an antibody response (inhibitors) to FVIIII. Various Immune Tolerance Therapies (ITTs) are being applied to overcome the FVIII specific immune response. In this study, phage displayed random peptide libraries were screened with various patients’ plasma samples. We recently reported about the identification of a conformational epitope in the A2 subunit of FVIII. Phages as well as corresponding synthetic peptides specifically block inhibitor binding and crossreact with heterologous plasma. Peptide ligands specifically stained primary B cells (CD19+/CD27+ double positive cells) of the inhibitor patient, but not of a healthy control by FACS analysis. To increase the potential of small synthetic peptide ligands as tools for the development of novel inhibitor elimination strategies, the peptide was expressed in a multimeric form in an eukaryotic expression system. In multimeric form the peptide ligand is highly stable and reveals better inhibitor blocking capacities than the synthetic peptide we sowed before. The multimer now serves as basis for the construction of LECs (Ligand Effector Conjugates) for the specific targeting and destruction of inhibitor specific B cells.
Rituximab for the Treatment of High Titer Acquired Factor VIII Inhibitors Refractory to Conventional Chemotherapy.
Authors: Field Joshua J. , Fenske Timothy S. , Blinder Morey A. 
Published: April 2020

Abstract
Acquired factor VIII inhibitors are a rare cause of serious bleeding which are associated with a high mortality rate. Individuals with high titer antibodies (&gt;100 Bethesda units) often have difficulty achieving a complete sustained remission and although different treatment strategies have been advocated for these patients, no approach has been well defined. Rituximab is a monoclonal antibody targeting the CD20 antigen on B lymphocytes which has demonstrated efficacy in the treatment of a variety of immune-mediated hematologic diseases including patients with acquired factor VIII inhibitors. However, there is limited data in patients with high titer inhibitor antibodies. In this study, we prospectively studied 4 patients with acquired high titer factor VIII inhibitors who have had long-term follow up. All patients were resistant to initial therapy with cyclophosphamide, vincristine, and prednisone. The patients ranged from 40 to 71 years of age and three of the four were women. The male patient had an associated autoimmune disease but the other patients were thought to have an idiopathic inhibitor. The patient’s inhibitor titers ranged from 249 BU to 836 BU at the time of treatment with rituximab and all received 4 weekly infusions of rituximab at 375 mg/kg. No significant treatment-related complications were noted. Each patient initially responded to rituximab therapy with a decline in inhibitor titer. Subsequently, three of the four patients relapsed. The relapses occurred 4, 8 and 15 months following rituximab therapy and were associated with an elevation in inhibitor titers and clinically significant bleeding in all 3 patients. The individual who did not relapse achieved a partial response with a decline in her inhibitor titer from 836 BU to 5 BU over 13 months and then died of causes unrelated to her coagulopathy. After control of the clinical bleeding, the three remaining patients received an additional course of rituximab with responses observed in all patients. One patient has been in a sustained remission for 19 months following the second dose of rituximab. A second patient had a notable improvement in his inhibitor titers from 418 BU to 10 BU over 9 months after the second dose. The third patient continues to have high inhibitor titers with intermittent bleeding following the additional dose. We conclude that in patients with acquired high titer acquired factor VIII inhibitors, treatment with rituximab alone results in improvement of inhibitor titers but is not sufficient to achieve a sustained response. Rituximab in combination with other therapies may provide a better result in this high risk population.
Inhibitor Haemophilia A - Cases with Reduced Haemostatic Response to Wildtype rFVIIa Who Achieved a Normal Clotting Profile with a Novel Potent Analogue of rFVIIa.
Authors: Sørensen Benny , Persson Egon , Ingerslev Jørgen 
Published: April 2020

Abstract
Development of inhibitory antibodies to coagulation factor VIII represents a serious complication in patients with congenital or acquired haemophilia A. Recombinant factor VIIa (rFVIIa, NovoSeven®, Novo Nordisk, Bagsvaerd, Denmark) has proven efficacious in management of bleeding episodes in patients with inhibitors. Recently, a novel analogue of rFVIIa (NN1731, Novo Nordisk) has been developed. NN1731 has increased potency to generate factor Xa on the surface of activated platelets. We have developed a thrombelastographic (TEG) whole blood (WB) coagulation model, employing activation with minimal amounts of tissue factor. This global clotting assay reflects the clinical severity of haemophilia A and can be used to ex vivo assess the haemostatic capacity of rFVIIa and NN1731. Adopting the TEG model, we report on two cases where ex vivo addition of rFVIIa only induced minor improvements of the WB coagulation profile. However, ex vivo addition of NN1731 revealed a normalization of the WB coagulation signature. The first case was a 74 years old female with acquired haemophilia A (APTT=51 sec, FVIII:C = 0.08 IU/mL, Inhibitor = 3.6 BU/mL. The second case was a patient with congenital haemophilia A and inhibitors (APTT=101 sec, FVIII:C&lt;0.01 IU/mL, Inhibitor=9 BU/mL). In both cases, the WB thrombelastographic analysis demonstrated a severely compromised haemostatic capacity. Ex vivo dose titration studies with rFVIIa induced minor improvements only of the suppressed WB clot formation profile. However, ex vivo addition of NN1731 at 2 μg/mL normalized the coagulation signature. The observations that the analogue NN1731 normalized the haemostatic WB clotting profiles in cases where rFVIIa induced minor changes stimulates further pre-clinical investigations.
Central Venous Access Device-Related Infections in Hemophilia.
Authors: Journeycake Janna M. , Brambilla Don , Ragni Margaret V. 
Published: April 2020

Abstract
Central venous access devices (CVAD) are commonly used in young children with hemophilia who need frequent factor infusions, including prophylactic infusions given every other day to prevent joint disease and immune tolerance therapy given every day to neutralize inhibitors, both of which result in better health outcomes. CVAD use, however, may be complicated by infection or sepsis, catheter replacement, and rarely, subacute bacterial endocarditis (SBE) or death. Infection rates have varied widely among institutions, possibly in part because of variable diagnostic and reporting methods. In order to identify risk factors for CVAD-related infection in children with hemophilia, we distributed an electronic survey to 14 U.S. hemophilia treatment centers participating in the TMH network regarding children with hemophilia whose initial CVAD was placed between January 1, 2002 and December 31, 2004. Thirteen (94%) sites responded, providing data on 94 patients with CVAD. All but two (98%) were ports. CVADs were placed for prophylaxis in 66 (70%) patients and for immune tolerance therapy in 28 (30%) patients. Thirty-one (33%) patients developed CVAD-related infections, all in the first year following insertion. One subject developed SBE, and one died of CVAD-related sepsis. Infection was significantly more common in subjects receiving immune tolerance, 19 of 28 (68%), than on prophylaxis 12 of 66 (18%) (odds ratio = 9.5, p&lt;0.001). The time from CVAD insertion to initial post-operative CVAD access did not influence the risk for infection (p = 0.13), but the duration of factor replacement following CVAD insertion did. Factor replacement for fewer than 7 days following insertion was associated with an increased incidence of CVAD-related infection (odds ratio = 2.2, p= 0.04). We conclude that individuals using CVAD for immune tolerance and/or receiving fewer than 7 days of factor replacement following CVAD placement are at greater risk for first CVAD-associated infection. The reasons for these findings are not known. However, the more frequent access of CVAD in patients on immune tolerance (daily vs. every other day) increases exposure to skin organisms, which could account for the higher rate of infection in these patients. Those receiving fewer than 7 days of factor replacement after CVAD placement may have insufficient wound healing or suffer local hematoma formation, which is a potential culture medium for local skin organisms. The high rate of CVAD infection in hemophilic children, &gt;30% overall, constitutes a major public health problem for which prevention trials are in development.
High Efficacy of Combination Therapy with Pegylated Interferon and Ribavirin in Hemophiliacs with Chronic Hepatitis C.
Authors: Mancuso Maria E. , Santagostino Elena , Rumi Maria G. , Linari Silvia , Coppola Antonio , Rocino Angiola , Iorio Alfonso , Dragani Alfredo , Barillari Giovanni , Tagariello Giuseppe , Nicolin Roberto , Colombo Massimo , Mannucci Pier M. 
Published: April 2020

Abstract
Pegylated interferon (Peg-IFN) plus ribavirin is the standard treatment for patients with chronic hepatitis C. In a multicenter open trial, we assessed the efficacy and tolerability of this treatment in 78 HIV negative hemophiliacs (age: 20–64 years) with persistently high transaminase values. Sixty-four were naïve and 14 were relapsers to IFN monotherapy. Cirrhosis was clinically detected in 12 patients (15%). HCV genotype was 1 in 69%, 2a/c in 14%, 3a in 14% and 4 in 3%. Peg-IFN alpha-2b was given subcutaneously at doses of 1.5 mcg/Kg/week for 48 weeks in genotypes 1 and 4 and for 24 weeks in genotypes 2 and 3; oral ribavirin at 800–1200 mg/day based on body weight. Treatment was stopped in patients with polymerase chain reaction positive HCV-RNA at month 6.
Results: 11 patients (14%) withdrew for side effects (4) or non-compliance (7). Neutropenia (&lt;500 cells/mmc), decompensated diabetes, ALT flares, and vomiting not responding to antiemetic drugs were reasons for treatment discontinuation. The median fall in hemoglobin levels was 3.1 g/dL. Weight loss (38%), fatigue (33%) and cephalalgia (15%) were frequent side effects. Thirty-two patients (41%) required dose reduction of ribavirin (23, 29%) or Peg-IFN (20, 26%). At the end of the 6-month follow-up, sustained virological response (SVR) was achieved in 43 patients (55%): 40/64 naïve (63%) and 3/14 relapsers (21%, p = 0.007). Five patients (6%), all relapsers to IFN monotherapy, had a virological breakthrough during treatment and 4 (5%) relapsed during the post-treatment follow-up period. SVR was obtained in 86% genotypes 2/3 and 43% genotypes 1/4 (p = 0.001). Predictors of SVR were evaluated by univariate analysis in the 64 naïve patients. SVR was significantly associated with HCV infection type 2 and 3 (86% vs 50% in HCV type 1 and 4; p = 0.008), absence of cirrhosis (97.5% vs 75% in non-responders; p = 0.02) and higher pre-treatment serum ALT levels (111 vs 75 IU/L in non-responders; p = 0.02). SVR rates did not differ in relation to patient’s age, pretreatment HCV viremia, median disease duration and compliance to full-dose treatment.
Conclusions: combination therapy with Peg-IFN plus ribavirin is the recommended therapeutic option for hemophiliacs with hepatitis C chronic infection. Relapsers to IFN monotherapy may benefit of re-treatment with Peg-IFN plus ribavirin achieving at least 20% SVR. Genotype 2 and 3 infection is the most significant predictor of SVR.