bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sequence Analysis in the BLYS and APRIL Receptor TACI Reveals Novel Variants with a Potential Pathogenetic Role in Waldenstrom’s Macroglobulinemia (WM).
Authors: Hunter Zachary R. , Leleu Xavier , Santos Daniel D. , Hamilton Susannah , Verselis Sigitas , Fox Edward A. , Grewal Iqbal , Treon Steven 
Published: April 2020

Abstract
B-lymphocyte stimulator protein (BLYS) and a proliferation inducing ligand (APRIL) are members of the tumor necrosis family of ligands which play an important role in normal and malignant human B-cell homeostasis through their receptors BCMA, TACI (for BLYS and APRIL) and BAFF-R (for BLYS only). We previously demonstrated via multicolor flow cytometric and RT-PCR analysis the expression of BLYS and APRIL, along with BCMA, TACI and BAFF-R on lymphoplasmacytic cells (LPC) as well as mast cells, which provide LPC growth support in patients with Waldenstrom’s Macroglobulinemia (WM) (Blood 104:917a). Based on these studies, we performed sequence analysis for BLYS, APRIL, BCMA, TACI and BAFF-R from DNA obtained from CD19+ selected bone marrow LPC from 15 patients with the consensus panel diagnosis of WM, who also demonstrated active disease. These studies demonstrated multiple variants in exons 3 and 5 of TACI in 5/15 patients, and exon 1 of BAFF-R in 2/15 patients. The finding of genetic variants in the TACI gene in WM patients is of particular interest given its recently described role as a potential tumor suppressor gene, and potential involvement in the generation of autoimmunity (Immunity2003; 8:279–88), which is a common complication in patients with WM. Moreover, variants in TACI have been reported in patients with common variable immunodeficiency (CVID) (Nat Genet2005;37:820–8), a finding significant to patients with WM who chronically demonstrate IgA and IgG hypogammaglobulinemia, even following successful therapeutic intervention as we recently reported (Blood 104:306b). The results of these studies suggest that variants in TACI, and possibly BAFF-R may play a significant role in the pathogenesis of WM.
PEG Modified rVWF Prolongs the Survival of Native rFVIII in Hemophilia A Knock-Out Mice.
Authors: Turecek Peter L. , Scheiflinger Friedrich , Gritsch Herbert , Baumgartner Bernhard , Váradi Katalin , Matthiessen H. Peter , Siekmann Juergen , Muchitsch Eva-Maria , Schwarz Hans Peter 
Published: April 2020

Abstract
Mature CHO cell derived and highly purified rVWF was chemically derivatized by binding of 20 kDa polyethylene glycol (PEG) succinimidyl glutarate, to the primary amino groups in rVWF with covalent bond formation, primarily with lysine residues. PEG conjugates of rVWF were further purified and pharmaceutically formulated for application into hemophilia A knock-out mice. Groups of 6 mice each were treated with respective test and control articles to measure pharmacokinetics. Mice were treated with chemically modified rVWF in combination with recombinant human FVIII (rFVIII, Advate). For control purposes, mice were treated with unmodified rVWF plus rFVIII. Citrated plasma was prepared from their blood and measured for FVIII activity with a chromogenic FVIII assay.
Mice received 300 IU/kg rFVIII together with 3.6 mg/kg PEG-rVWF or 1.6 mg/kg unmodified rVWF. FVIII levels were measured at time points 5 minutes and 3, 9, 24 and 32 hours after injection. FVIII levels found 5 minutes after injection were set as 100%. At all time points FVIII levels were substantially higher in the groups co-administered with PEG-rVWF. Mice co-administered with PEG-rVWF showed a FVIII level of 3% at 24 hours which was maintained at 2% after 32 hours, while in the controls treated with rFVIII and unmodified rVWF, FVIII levels were close to the limit of detection at 24 hours (~ 0,6%) and had completely disappeared at 32 hours. Assuming a one compartmental model, a mean half life of 4.4 hrs of rFVIII in the presence of PEG-rVWF and 1.2 hrs in the presence of unmodified rVWF was calculated (Scientist program, MicroMath, Saint Louis, MO, USA). The FVIII AUC was increased by a factor of 2.5 for the group treated with PEG-rVWF (MS EXCEL, trapezoidal model). Our study demonstrates that chemical modification by PEGylation of rVWF can be used to improve the survival of co-administered native rFVIII in the circulation of hemophilic mice.
Recombinant Human IL-11 (rhIL-11, Neumega®) Increases VWF Activity in Type 1 Von Willebrand Disease.
Authors: Jankowitz Rachel B. , Chapman Holly , Merricks Elizabeth , Kloos Mark , Dillow Aaron , Nichols Timothy C. , Ragni Margaret V. 
Published: April 2020

Abstract
Von Willebrand disease (VWD) is the most common congenital bleeding disorder, affecting 1% of the population. Current treatment is suboptimal. Tachyphylaxis limits the use of DDAVP; potential transmissible agents limit the use of Von Willebrand Factor (VWF) concentrate; and both require venous access. Recombinant human IL-11 (rhIL-11, Neumega), a gp-130 signaling cytokine with hematopoietic and anti-inflammatory activity, has recently been shown to increase VWF activity with good tolerance in mice, dogs, and healthy volunteers. We, therefore, initiated a prospective Phase II open label, escalating dose trial of rhIL-11 in subjects with type 1 VWD. VWD was defined by the presence of a bleeding history with at least two of four abnormal tests: F.VIII:C, VWF:RCo, VWF:Ag, PFA-100, and normal distribution of multimers. A total of six subjects completed the study, three each at 25 mcg/kg and 50 mcg/kg dose levels, and all were given rhIL-1 subcutaneously daily for 7 days. On day 4 of rhIL-11, VWF:RCo increased 32% over baseline to 107±9% (mean ± SE) at the 25 mcg/kg dose, and 70% over baseline to 179±55%, at the 50 mcg/kg dose; VWF:Ag levels increased similarly, to 102±38% and 240±120%, respectively; and FVIII:C and VIII:Ag each increased similarly, by up to 143% and 200%. Intravenous DDAVP at a dose of 0.3 mcg/kg given 30 minutes after rhIL-11 on day 7, induced a further increase in VWF:RCo, to 114±48% and 359±152%, and in VWF:Ag, to 113±33% and 277±21%, each respectively at the two rhIL-11 doses; and very high molecular weight VWF multimers were detected by gel elecrophoresis on plasma samples. These data suggest that rhIL-11 acts on other than the DDAVP releasable pool of VWF. The drug was well tolerated. Minor adverse events included hypertension, fluid retention, and hypokalemia, each at less than grade 1 toxicity. There was a mild dilutional decrease in hemoglobin related to fluid retention. There was no significant increase in platelet count. In conclusion, rhIL-11 increases VWF activity to levels in the range associated with clinical hemostasis, suggesting the potential use of rhIL-11 in treatment of clinical bleeding in type 1 VWD.
Figure Figure
Polymorphisms in Coagulant and Inflammatory Genes Modify the Phenotype of Severe Hemophilia A and B.
Authors: Jayandharan G. , Devadharshini Mercy , Viswabandya Auro , Nair Sukesh C. , Shaji R.V. , Mathews Vikram , Chandy Mammen , Srivastava Alok 
Published: April 2020

Abstract
Among patients with severe hemophilia (&lt;1% factor level), 10–15% are known to have a clinically mild phenotype. The basis for this phenomenon is unclear. We hypothesized that functionally significant polymorphisms in the coagulant, inflammatory and immunoregulatory genes may affect the phenotype of severe hemophilia. A total 114 patients with hemophilia A (n=95) and hemophilia B (n=19) were studied. All these patients were on minimal on-demand treatment. Patients were evaluated for the frequency and site of hemorrhage. Their clinical and radiological joint scores were documented. They were categorized as ‘mild’ (&lt;1 affected joint and &lt; 5 bleeds in the preceding year, n=15) or ‘severe’ (&gt;1 affected joint and &gt;5bleeds, n=99). Functional polymorphisms in the coagulant system (human platelet alloantigen; tissue factor; fibrinogen; factors II; V; VII; XIIIA; thrombin activable fibrinolysis inhibitor (TAFI); endothelial protein C receptor; endothelial nitric oxide synthase 3; tissue plasminogen activator; plasminogen activator inhibitor; tissue factor pathway inhibitor; protein C and S; thrombomodulin), known procoagulant factors (methylene tetrahydrofolate reductase gene), inflammatory cytokine genes (tumor necrosis factor alpha; transforming growth factor beta; interleukin (IL) 10; IL 6; IL 1beta; IL 1 beta receptor antagonist; tumor necrosis factor beta), immunoregulatory cytokine genes (interferon gamma; HLA B27; FC gamma receptor), MDM2, angiotensin converting enzyme and HFE genes were genotyped. The mean age in the two groups was 18.5 & 14.85, p=0.124. The clinical features showing significant difference are shown in the table. Of the polymorphisms studied, the FVII RQ/QQ (lower levels) (RR-3.99, p=0.022, 95% CI 1.2–13.4), TNF alpha-308AA/AG (pro-inflammatory) (RR-3.4, p=0.037, 95% CI, 1.07–10.7), TGF beta Codon 10 CC/CT (pro-inflammatory) (RR-2.8, p=0.07, 95% CI, 0.91–8.3), have been associated with a severe phenotype while MDM2GG (anti-inflammatory, RR-0.3, p=0.038, 95% CI, 0.1–0.93) was associated with a milder phenotype. We hypothesize that the bleeding frequency in severe hemophilia may be increased due to relatively lower FVII levels and a combination of cytokine driven pro-inflammatory state involving TNF alpha, TGF beta and MDM2 would cause destruction of the cartilage resulting in elaboration of metalloproteinases from chondrocytes leading to the development of arthropathy.
Parameter Severe, n=99 Median (Range) Mild, n=15 Median (Range) p Value Number of bleeds /yr 15(3–74) 2(0–5) 0.000 Number of joints /yr 3 (1–6) 1 (0–1) 0.000 Age at first clinical symptom (months) 21(1–300) 60(6–90) 0.056 WFH clinical score 10 (0–27) 4 (0–21) 0.000 Pettersson score 13 (0–57) 6 (0–20) 0.001
Fertility in Male Hodgkin Lymphoma Patients - A Report of German Hodgkin Study Group.
Authors: Sieniawski Michal , Reineke Thorsten , Josting Andreas , Diehl Volker , Engert Andreas 
Published: April 2020

Abstract
Purpose: We performed semen and hormone analysis before and after treatment to investigate the influence of disease and therapy on the fertility status of male patients with Hodgkin lymphoma (HL).
Patients & Methods: Patients (pts) with first diagnosis of HL without previous chemotherapy or radiotherapy who were enrolled into trials of German Hodgkin Study Group (GHSG) between 1988 and 2003 were analyzed. 202 pts were evaluated prior to treatment and 112 pts were evaluated after treatment.
Results: The median age in the pre-treatment evaluation group were 26 years and 27 years in the post-treatment evaluation group. In pre-treatment evaluation group 51% of pts were advance stage of disease, 31% in intermediate stage and 11% in early stage. In the post-treatment evaluation group 45% of pts were in intermediate stage of disease, 44% in advanced stage and 11% in early stage.
Before treatment 20% (40/202) of pts had normozoospermia and 80% (162/202) had dyspermia. After treatment, 64% (72/112) of pts had azoospermia, 30% (33/112) other dyspermia and 6% (7/112), the differences are significant (p&lt;0.001). Azoospermia was observed in 67% (62/93) of pts treated with combined modality, in 90% (9/10) of those treated with chemotherapy alone and in 11% (1/9) of those treated with radiotherapy alone (p&lt;0.001). Azoospermia was more frequent after BEACOPP then after COPP/ABVD - 85% (34/40) vs. 63% (37/59) (p&lt;0.001). There was no difference between patients treated with 8 cycles of BEACOPP escalated (n=15) and those treated with 8 cycles BEACOPP baseline (n=21); with 93% and vs. 86% (p&gt;0.05). The median time of onset of spermatogenesis was 27 months. During the first year after the treatment the onset of spermatogenesis was found in 18% of pts, during the second year in 23%, during the third year in 35% and in 35% after the third year. In univariate risk factors analysis, we found exttranodal involvement, risk groups, treatment with chemotherapy and BEACOPP being significantly predictive for asevere damage of fertility, none of these factors was significant in multivariate analysis.
In the contrast to the pre-treatment analysis, most of the pts (79%) showed abnormal FSH-levels (p&lt;0.001). LH and testosterone were normal in most of patients; these results were similar to pre-treatment levels (p&gt;0.005). There was the relationship between the post-therapeutic FSH level and sperm count; in the group with normal FSH levels, 23% of pts showed azoospermia and in the group with abnormal FSH level 78% of pts were azoospermic (p&lt;0.001). The correlation between sperm count and LH and testosterone level was not so pronounced.
Conclusion: The majority of patients with HL were azoospermic after treatment, but recovery of spermatogenesis was observed depending on the treatment received. The FSH level appears to be helpful in diagnosis of the fertility status.
Biochemical and Functional Characterization of a Serum-Free rVWF Drug Candidate.
Authors: Turecek Peter L. , Matthiessen H. Peter , Mitterer Artur , Gritsch Herbert , Váradi Katalin , Plaimauer Barbara , Purtscher Martin , Woehrer Wilfried , Muchitsch Eva-Maria , Schwarz Hans Peter 
Published: April 2020

Abstract
Recombinant VWF was co-expressed with recombinant FVIII in CHO cells. In order to obtain fully processed, mature rVWF, rVWF was exposed to recombinant CHO-cell-derived furin for VWF pro-peptide removal. Fermentation for both rVWF and furin and downstream processing were performed under serum-free conditions.
Mature rVWF was highly purified by a series of conventional chromatography steps to a purity of &gt; 95%. rVWF was formulated in a protein-free buffer.
Several large scale batches of rVWF were produced and characterized in comparison to Humate-P, Wilfactin and a highly purified plasma-derived (pd)VWF, an experimental pd VWF product obtained from cryoprecipitate. Residual pro-VWF levels were approximately 2% of total VWF antigen compared with 0.12, 0.05 and 0.13 for the rVWF product, experimental pd VWF, Humate-P and Wilfactin, respectively. VWF pro-peptide levels were approximately 0.5 pmol per unit VWF antigen, compared with 0.6 and 0.9 for Humate-P and Wilfactin, respectively; in the experimental pd VWF pro-peptide was below the limit of detection. Residual FVIII content was approximately 0.02 units (chromogenic assay) per unit VWF antigen, compared with 0.4 units in Humate-P, and 0.04 units in Wilfactin, and ≤ 0.01 in the experimental pd VWF product.
Ristocetin cofactor activity was around 0.7 VWF:RCo units per unit antigen for all rVWF and pd VWF products tested. Collagen-binding activity was also similar between rVWF and pd VWF controls (around 0.8 – 0.9 units per unit). FVIII-binding capacity of rVWF was compared by setting the mean pd VWF as 100% and was found to also be ± 100%. The VWF affinity for FVIII was similar in rVWF and pd VWF (Biacore). rVWF showed high molecular weight multimers in low resolution agarose gels, with a pre-dominance in the high molecular weight range. In contrast to pd VWF, rVWF showed homogenous multimer bands without triplet structure formation due to the non-exposure to ADAMTS13 during the manufacturing process.
The pharmacokinetic properties of rVWF were compared with pd VWF in VWF knock-out mice. The half-life or rVWF in VWD mice was 4.3 and 3.5 hours for 2 batches investigated, which seems longer than the half-lives obtained for pd VWF, 2.9 hours for Humate-P and 2.2 hours for a highly purified pd VWF control. Both VWF preparations stabilized endogenous murine FVIII which rose over time to the levels observed in normal C57BI/6J mice.
Overall, these results showed that the properties of mature, furin-processed rVWF in vitro and in vivo are equivalent to those of plasma-derived VWF.
Coated-Platelet Levels May Explain Some Variability in Clinical Phenotype of Severe Hemophilia.
Authors: Saxena Kapil , Pethe Kalpana , Dale George L. 
Published: April 2020

Abstract
Objective: To determine if variability in clinical phenotype of severe hemophilia patients is influenced by the percentage of coated-platelets.
Background: Co-activation of platelets with thrombin and collagen results in a unique subset of platelets, with high levels of alpha granule proteins on their surface, such as Factor V, fibrinogen, von Willebrand factor, and thrombospondin. This subset of activated platelets is referred to as coated-platelets (J. Thromb. Haemostasis3:2185, 2005). The high concentration of adhesive and prohemostatic proteins observed on coated-platelets provides a unique procoagulant locus that may influence the number of bleeding episodes in patients with severe hemophilia.
Methods: After informed consent, 3–5 ml of blood was drawn from patients with severe hemophilia (Factor VIII &lt;1%) and healthy controls. The number of bleeding episodes reported in the last 6 months was taken as an indicator of clinical phenotype.
Results: In 6 patients with more than 3 bleeds reported in the last 6 months (14 ± 4.9 bleeds; mean ± 1SD), the average coated-platelets percentage was 22.3 ± 11.2%. In 17 patients with 3 or less bleeds (1.6 ± 1.3) in the last 6 months, the average coated-platelets percentage was 37.6 ± 12.0%, a difference that was statistically significant (p=0.012). In healthy controls (n=12), the mean coated-platelets were 30.2 ± 9.5%.
Conclusion: A higher percentage of coated-platelets may provide a better procoagulant locus for residual Factor VIII, thereby reducing the number of clinical bleeding episodes and partially explaining some variability observed in clinical phenotype of severe hemophilia.
Biochemical and Functional Characterization of PEGylated rVWF.
Authors: Turecek Peter L. , Scheiflinger Friedrich , Siekmann Juergen , Váradi Katalin , Matthiessen H. Peter , Weber Alfred , Gritsch Herbert , Muchitsch Eva-Maria , Baumgartner Bernhard , Schwarz Hans Peter 
Published: April 2020

Abstract
Covalent modification of therapeutic proteins by polyethylene glycol derivatives is an established method for improving pharmacokinetic properties of therapeutic proteins.
Highly purified rVWF expressed in CHO cells, was chemically modified via PEGylation of lysine residues at mild alkaline pH with PEG succinimidyl succinate (linear 5 kDa PEG). Increasing the amount of PEG used in the coupling procedure, the molecular size of VWF increased, as demonstrated in SDS-PAGE and by agarose gel electrophoreses indicating an increase in size of the bands resembling the VWF multimers.
PEGylation of rVWF reduced platelet-aggregating and collagen-binding functions by 60% (VWF:RCo activity) and 40% (VWF:CB activity). While FVIII-binding capacity, measured by a FVIII binding ELISA, was reduced and reduction correlated with the amount of PEG bound to VWF, there was almost no effect on FVIII binding affinity which remained in the same order of magnitude as measured with non-PEGylated VWF. Similar results were obtained when rVWF was PEGylated via carbohydrate moieties after oxidation and subsequent derivatization with monomethoxy-PEG hydrazide.
PEGylated rVWF was applied to VWF-deficient mice at a dose of 40 VWF:Ag U/kg and plasma levels were monitored for up to 24 hours. As a control, non-modified rVWF was applied to the animals. PEGylated VWF had substantially prolonged survival in the circulation compared with non-modified rVWF with an increase of the AUC by a factor of &gt;10. VWD mice substituted with human VWF show a secondary rise in FVIII bringing them into the FVIII levels measured in C57Bl/6 control mice. This secondary rise was sustained after treatment with PEGylated rVWF where FVIII levels above the starting level were measurable even 48 hours after injection while in the control group base line FVIII levels were reached already after 24 hours.
rVWF is the largest protein ever PEGylated and PEGylation results in prolonged survival in the circulation while maintaining FVIII stabilizing functions of the VWF molecule in vivo.
Development of a Screening Tool in Women Presenting with Unexplained Menorrhagia.
Authors: Philipp Claire S. , Faiz Ambarina , Dowling Nicole F. , Beckman Michele , Owens Sally , Ayers Charletta , Bachmann Gloria 
Published: April 2020

Abstract
Menorrhagia is a common clinical problem among reproductive age women and 5% of women in the US seek medical attention annually for menorrhagia. Recent studies suggest that underlying bleeding disorders, particularly Von Willebrand disease and platelet dysfunction, are prevalent in women presenting with menorrhagia. However referral for comprehensive hemostatic evaluation of the substantial numbers of women with otherwise unexplained menorrhagia is problematic given the complexity, availability, and cost of hemostatic testing and the lack of effective point of care screening tests for this population. In the present study, we evaluated 146 women (ages 13–55 yrs) with unexplained menorrhagia in order to develop a simple screening tool for stratifying women for referral for comprehensive hemostatic evaluation and testing. Women with a physician diagnosis of menorrhagia were studied on days 3–9 of their menstrual cycle. ASA, NSAIDS, herbals and other potentially platelet function impairing medications were discontinued at least 14 days prior to study and estrogen based treatment was discontinued for at least 1 menstrual cycle. Women underwent comprehensive hemostatic testing including VWF, platelet aggregation and ATP release, and factor assays. A 12 page questionnaire was developed based on significant bleeding symptoms in a CDC study of women with known VWD (Drews CD, Dilley AB, Lally C, Beckman MG, Evatt B. J Am Med Womens Assoc2002;57:217–218) and was administered to study subjects. A combination of 8 questions in 4 categories, including history of duration/extent of menorrhagia, anemia treatment, excessive bleeding with hemostatic challenges, and family diagnosis, resulted in a sensitivity of 81% (95% CI 74–89) and positive predictive value 71% (95% CI 63–71) for hemostatic abnormalities. Adding a pictorial blood assessment chart score &gt; 100 increased the sensitivity of the screening tool to 93% (95% CI 89–98). The data suggest that a simple 8 question screening tool with implementation of a pictorial blood assessment chart may be useful for physicians evaluating women presenting with menorrhagia to help determine which women to refer for comprehensive hemostastic testing and evaluation.