bluuebunny/crossref_metadata_2025 huggingface dataset:
Murine Modeling of Human Severe Congenital Neutropenia and Non-Immune Chronic Idopathic Neutropenia.
Authors: Zarebski Adrian P. , Pawlak Wojciech , Baktula Avinash M. , Basu Sudeep , Trent John , Grimes H. Leighton
Published: April 2020

Severe Congenital Neutropenia (SCN) is a rare hematological disease manifested by a complete block in the maturation of neutrophils. The lack of neutrophils leads to recurrent bacterial and fungal infections. Most SCN patients have mutations in Ela2, the gene encoding neutrophil elastase; however, introduction of SCN-patient mutations into murine Ela2 or disruption of murine Ela2 does not recapitulate neutropenia in mice. Thus, there are no mouse models of SCN. Gfi1 is a transcriptional repressor leukemia oncoprotein involved in HSC maintenance as well as proper lymphoid and myeloid development. Mice lacking Gfi1 have profound neutropenia. We recently described four patients in which Ela2 was intact but mutant for Gfi1. The heterozygous single aminoacid substitutions were found in zinc finger 5 (N382S) and zinc finger 6 (K403R) correlating to either SCN or Non-immune Chronic Idiopathic Neutropenia of Adults (NICINA) respectively. Given that Gfi1 null mice lack neutrophils, and that people with Gfi1 mutations display SCN or NICINA, we asked whether the human mutations when introduced into murine Gfi1 are sufficient to model neutropenia in mice. To this end, we constructed murine Gfi1 cDNAs containing the SCN and NICINA patient mutations, retrovirally overexpressed them in murine Lin- bone marrow cells, then analyzed them by in vitro differentiation assays. Strikingly, cells overexpressing Gfi1-N382S produced no granulocytic colonies (similar to the profound phenotype of SCN). Whereas, the expression of Gfi1-K403R mutation resulted in a moderate reduction in granulocytic colonies (corresponding to the mild phenotype of NICINA). We have constructed a virtual model of Gfi1 zinc finger/DNA interaction to explore the molecular defect engendered by the N382S mutation. At the meeting, we will discuss the molecular basis of the profound phenotype engendered by Gfi1-N382S expression. These data demonstrate for the first time that human neutropenia can be modeled in murine cultures through the introduction of mutations in Gfi1 found in SCN and NICINA patients.
Neutrophil Elastase, but Not Myeloperoxidase, Depends on Serglycin Proteoglycan for Granule Localization in Neutrophils.
Authors: Niemann Carsten U. , Åbrink Magnus , Pejler Gunnar , Christensen Erik I. , Knight Stefan D. , Borregaard Niels
Published: April 2020

Granule proteins play a major role for the bactericidal activity of neutrophils. The mechanisms for sorting of proteins to granules in neutrophils remain unknown. Serglycin proteoglycan with glycosaminoglycan side chains is the major intracellular proteoglycan of hematopoietic cells. Neutrophil elastase, a resident of primary (azurophilic) granules, binds glycosaminoglycans. Serglycin has been proposed to play a major role in sorting and packaging of granule proteins in neutrophils and other cells.

Figure Figure

We have investigated the expression and localization of granule proteins in neutrophils from a serglycin knock out mouse. We found neutrophil elastase absent from neutrophils of serglycin knock out mice as shown by activity assay (fig. 1), Western blotting (fig. 2), and immunocytochemistry. Northern blotting (fig. 1) demonstrated the presence of neutrophil elastase mRNA both in serglycin knock out mice and in wild type mice. No other granule proteins tested (myeloperoxidase, cathepsin G, proteinase 3, bacterial permeability increasing protein (primary granules), lactoferrin, 24p3 (secondary (specific) granules), MMP-9/gelatinase B (tertiary (gelatinase) granules)) showed differences in expression or localization in neutrophils from wild type mice compared to serglycin knock out mice. Three-dimensional models of neutrophil elastase revealed a cluster of positively charged amino acids around the active site of the enzyme. This cluster may be engaged in binding the negatively charged glycosaminoglycan side chains of serglycin and thus forms the basis for serglycin mediated sorting of elastase to primary (azurophilic) granules. No counts for both bone marrow cells and peripheral blood are similar for the wild type and serglycin −/− genotypes; indicating that the lack of sorting of elastase to granules does not induce neutropenia itself. These findings disclose an important role for serglycin proteoglycan in targeting neutrophil elastase to neutrophil primary granules. This is in accordance with the recently published significance for serglycin in granule formation of mast cells. Our results also indicate that different mechanisms are responsible for sorting of different granule proteins to the same neutrophil granule subset, as none of the other tested granule proteins showed differences in localization between wild type and serglycin knock out neutrophils.
Cytokine Gene Expression Profile Distinguishes CD4+/CD57+ T-Cells of Nodular Lymphocyte Predominance Type of Hodgkin Lymphoma from Their Tonsillar Counterparts.
Authors: van den Berg Anke , Atayar Cigdem , Visser Lydia , Poppema Sibrand
Published: April 2020

Little is known about the cytokine profile of nodular lymphocyte predominance Hodgkin lymphoma (NLPHL) and the significance of the characteristic rosetting CD4+/CD57+ T-cells. We analyzed the T-lymphocyte populations isolated from lymph node suspensions of 5 patients with NLPHL, 2 with follicular hyperplasia and progressive transformation of germinal centres (PTGC), 3 with classical Hodgkin lymphoma (CHL) and 5 with hyperplasia of the tonsil. We sorted the T-cells based on expression of CD3, CD4 and CD57 by flow cytometry and evaluated the cytokine mRNA profiles of the T-cells with quantitative RT-PCR. NLPHL cases were as rich in T-cells as the CHL cases, but all NLPHL cases with a much higher frequency of CD4+/CD57+ T-cells. In contrast to the CD4+/CD57+ T-cells from tonsils, IL2 and IL4 mRNA was consistently absent from the CD4+/CD57+ T-cells of NLPHL. Even after stimulation, no IL4 transcripts could be detected in the CD4+/CD57+ T-cells of NLPHL. On the other hand, IFNγ transcripts were elevated in NLPHL and PTGC T-cell subsets as compared to tonsillar T-cell subsets. IL13 mRNA was exclusively produced by the T-cells of CHL cases, indicating that IL13 may be a key cytokine in CHL. In conclusion, elevated levels of CD4+/CD57+ T-cells are characteristic for NLPHL and these T-cells display a distinct cytokine mRNA profile.
6q Deletion in Waldenström Macroglobulinemia Is the Most Common Cytogenetic Abnormality and Is Associated with Aggressive Disease with a Trend towards Worse Survival.
Authors: Schop Roelandt F.J. , Ocio Enrique M. , Gonzalez Belen , Gutierrez Norma C. , Moro Maria Jesus , Aguilera Carmen , Hernández Jose , García-Sanz Ramon , Xu Ruifang , Hernandez-Rivas Jesus M. , Greipp Philip R. , Dispenzieri Angela , Jalal Syed M. , Lacy Martha Q. , Gonzalez-Paz Natalia , Gertz Morie A. , San Miguel Jesús F. , Fonseca Rafael
Published: April 2020

Fluorescence in situ hybridization (FISH) is an effective alternative for the analysis of cytogenetic abnormalities of low proliferative entities such as Waldenström macroglobulinemia (WM) but, to date, no studies have been carried out to explore the potential impact of molecular cytogenetics on disease evolution. Deletion of the long arm of chromosome 6 (6q-) is the most frequent cytogenetic abnormality in WM. In this study we have analyzed the incidence of this aberration (using both conventional cytogenetics and FISH analysis) in 102 WM patients, and we have correlated it with disease characteristics. Deletion of 6q21 was detected in 6% of patients by conventional cytogenetics, while FISH studies increased this incidence up to 39%. Moreover, in those patients analyzed by cIgM-FISH, the incidence of 6q- was higher (54%). Interestingly, 6q- patients displayed features associated with adverse prognosis (higher levels of ESR, CRP and beta-2 microglobulin), and a greater tendency to display anemia and hypoalbuminemia. Other differentiating variables without reaching statistical significance were a tendency of displaying a higher bone marrow lymphoplasmacytoid infiltration and the amount of monoclonal component in 6q- patients (table). In addition, patients with 6q- displayed shorter survival from time of diagnose (log-rank; p=0.09). In summary, our results show that 6q deletion in WM patients is currently the most frequently found cytogenetic abnormality. Patients with this deletion also shows a trend towards an aggressive disease and poorer outcome in WM. Further studies should focus on possible tumor suppressor genes in the minimal area of deletion at 6q and to determine their role in disease progression.

table del6q present del6q absent p-value n 40 (39%) 62 (61%) β2M ≥4mg/dL 48% 16% 0.005 CRP ≥3.5 mg/dL 47% 11% 0.006 Albumin <4mg/dL 86% 55% 0.006 ESR ≥100 mm/h 73% 44% 0.02 Hb <11 g/dL 63% 40% 0.02 serum IgM >20 g/L 86% 73% 0.10 BM infiltration >40% 44% 31% 0.20
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

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

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

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

Among patients with severe hemophilia (<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’ (<1 affected joint and < 5 bleeds in the preceding year, n=15) or ‘severe’ (>1 affected joint and >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

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<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<0.001). Azoospermia was more frequent after BEACOPP then after COPP/ABVD - 85% (34/40) vs. 63% (37/59) (p<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>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<0.001). LH and testosterone were normal in most of patients; these results were similar to pre-treatment levels (p>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<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

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 > 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

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 <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

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 >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

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 > 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.
A Prospective Comparision of Intranasal (I.N.) and Subcutaneous (S.C.) Desmopressin (DDAVP) Therapy in Type 2M Von Willebrand Disease (VWD).
Authors: Herbert Scott , Parameswaran Rekha , Shapiro Amy D. , Di Paola Jorge , Winay Jon , Roberson Chris , Greist Anne
Published: April 2020

DDAVP is effective therapy for VWD patients with documented adequate biologic response. Few data evaluating DDAVP exist in the less common type 2M VWD Seventy one members of an Amish kindred diagnosed with type 2 M VWD with abnormal VWF:Rco <35% and C-to-T transition at nucleotide 4120 in exon 28 of the VWF gene have been identified; 11 previously participated in an evaluation of weight-based trial dose of I.N. DDAVP(Stimate® Nasal Spray: 1.5 mg/ml concentration, 2 puffs weight >50 kg); 9/11 were further evaluated for response to S.C. DDAVP (0.3 microgram/kg). These 9 patients included 5 males, 4 females; age range: 21 to 58 years; weight range: 59–101.8 kg. Response utilizing ristocetin cofactor activity (VWF:Rco), factor VIII activity (VIII:C), and VWF antigen (VWF:Ag). Was assessed at 90 minutes post Stimate® administration and at 60 and 120 minutes post S.C. administration; values were also obtained at 4 and 6 hours post S.C. DDAVP to assess pharmacokinetics. Response to DDAVP was defined as VWF:Rco >40%. Five people (55%) responded to Stimate®; three (33%) responded to S.C. DDAVP. There were no major adverse effects. Two males who had responded to I.N. route did not respond to S.C. route; one female who responded to S.C. DDAVP did not respond to Stimate®. Responses fell below 40% at 4 and 6 hours post-dose. Responses to DDAVP are thus observed in a subset of individuals with type 2M VWD. Although all individuals tested exhibit the 4120C>T mutation, the response to DDAVP as measured by VWF: Rco levels varied from 1.7 to 4.9 fold. Therefore response to DDAVP in these patients appears not to be solely based upon the mutation causing the VWD phenotype but may be modified by other environmental and genetic factors. DDAVP responsiveness is an interesting phenotypic trait to be studied. Response may vary based on route of administration; this report highlights the necessity of assessing response to a trial dose by the intended route of administration prior to clinical use. Both I.N. and S.C. DDAVP may be used for minor bleeding episodes in those patients with type 2M VWD with adequate response to a trial dose. Both may be self administered as home based first line hemostatic therapy and are well tolerated.

RESPONSE TO I.N. DDAVP(2005) AGE/SEX WEIGHT(KG) PRE VWF:RCo(%) POST VWF:RCo(%) POST/PRE VWF:RCo RATIO 58/M 94.8 11 42 3.8 26/M 69.7 10 48 4.8 48/M 83.2 14 68 4.9 29/M 79.4 10 28 2.8 49/F 58.8 12 20 1.7 43/F 79 10 35 3.5 21/F 59.2 10 40 4.0 22/F 51.8 10 38 3.8 27/M 76.2 11 52 4.7 RESPONSE TO S.C. DDAVP (2006) AGE/SEX WEIGHT (KG) PRE VWF:RCo(%) PEAK POST VWF:RCo(%) POST/PRE VWF:RCo RATIO 58/M 101.8 13 42 3.2 26/M 72.7 10 33 3.5 48/M 88.1 14 61 4.4 29/M 82.7 10 31 3.1 49/F 70.5 11 21 1.9 43/F 79.5 10 46 4.6 21/F 59 10 49 4.9 22/F 62.7 10 39 3.9 27/M 79 10 37 3.7
Recombinant Factor VIIa Combined with Plasma Factor XIII Stabilize Clot Formation and Increase Clot Stability in Whole Blood from Patients with Severe Hemophilia A.
Authors: Sorensen Benny , Rojkjaer Rasmus , Ingerslev Jorgen
Published: April 2020

Patients with severe hemophlia A and inhibitors suffer from significantly compromised clot formation as well as reduced clot stability. Recombinant factor VIIa (rFVIIa - NovoSeven®, Novo Nordisk, Bagsvaerd, Denmark) has proven safe and efficacious for securing haemostasis in hemophilia patients with inhibitors. Recently, it was proposed that the reduced thrombin generation in severe haemophilia hinders sufficient activation of factor XIII and thereby result in insufficient covalent lateralization of fibrin (Blood2005; 106: 11, Abstract 321). The present study aimed at exploring the effect of rFVIIa and rFVIIa + plasma-derived FXIII (Haematological Technologies Inc) on whole blood clot (WB) formation and WB clot stability in severe hemophilia A. In total, 14 patients with a verified FVIII:C < 0.01 IU/ml were enrolled. Ex vivo studies were performed with rFVIIa (2 μg/ml), rFVIIa+FXIII (2+10 μg/ml), and a buffer control. Dynamic WB coagulation profiles describing initiation (clotting time=CT[sec]), propagation (maximum velocity=MaxVel [mm*100/sec]) and clot strength (maximum clot firmness=MCF[mm*100]) were recorded using thrombelastography and activation with a minute amount of tissue factor (TF, Innovin, final dilution 1:50000). WB clot stability was evaluated using a reaction mixture containing TF and tPA (1nM), followed by evaluation of the MCF and the total area under the elasticity curve after 120 min analysis time (AUEC[mm*100*sec]). Data are presented as mean and Wilcoxon statistical results. In the absence of tPA, Both rFVIIa+FXIII and rFVIIa significantly shortened the CT (Buffer=1424, rFVIIa+FXIII=739 (p=0.010), rFVIIa=881, (p=0.0005)) and accelerated WB MaxVel (Buffer=3.8, rFVIIa+FXIII=10.5 (p=0.0001), rFVIIa=9.2, (p=0.0002)). The standard deviation (SD) of the CT was significant lower in WB spiked with rFVIIa+FXIII than rFVIIa (Buffer SD=697, rFVIIa+FXIII SD=289 vs rFVIIa SD=655, p=0.007). In the absence of tPA, rFVIIa+FXIII increased the MCF significantly more than rFVIIa (Buffer=4441, rFVIIa+FXIII=6414 vs rFVIIa=5943, p=0.04) and the SD of the MCF was significant lower in WB spiked with rFVIIa+FXIII than rFVIIa (Buffer SD=2174, rFVIIa+FXIII SD=331 vs rFVIIa SD=948, p=0.0006). In the presence of tPA, rFVIIa+FXIII induced higher clot strength and stability than rFVIIa alone (MCF: Buffer=1313, rFVIIa+FXIII=3295 vs rFVIIa=3023, p=0.10 (N.S.); AUEC: Buffer=3.8*106, rFVIIa+FXIII=12.8*106 vs rFVIIa=10.2*106, p=0.0269). In conclusion, both rFVIIa (2μg/mL) and FXIII (10 μg/ml) added to rFVIIa (2 μg/ml), significantly increased WB clot formation and stability in this ex vivo evaluation of the clotting potential of WB from patients with severe hemophilia A.
Immunoregulatory Mechanisms of PF4/Heparin Antibody Production In Vivo.
Authors: Arepally Gowthami , Qi Rui , Suvarna Shayela
Published: April 2020

Heparin-Induced Thrombocytopenia (HIT) is a prothrombotic disorder caused by antibodies directed to complexes of platelet factor 4 (PF4) and heparin. Little is known about the immune basis of HIT, such as why serologic responses develop commonly in certain clinical settings or why immune responses may be transient in humans. To gain insight into the immune mechanisms governing PF4/heparin antibody production in vivo, we initiated studies in a recently developed murine immunization model of HIT, in which mice are injected with PF4/heparin antigen by retro-orbital injection daily for five days without adjuvant. PF4/heparin antibody development in mice is monitored weekly by a murine PF4/heparin (mP+H) ELISA. Immunizations of identical doses of PF4/heparin antigen in BALB/c and C57BL/6 (C57) mice resulted in marked strain-dependent differences in immune susceptibility, temporal kinetics and secondary recall. BALB/c mice showed more variable sensitization to mP+H injections (See Figure below) and manifested lower peak antibody levels (mean peak A450nm± SD: 0.153±0.1) than C57 mice (0.91±0.73). Time course of antibody formation was also strain-dependent. Peak antibody levels occurred ~ 45 days after start of immunization in BALB/c mice as compared to 15 days in C57 mice. Antigen rechallenge with one dose of antigen 65–85 days after primary sensitization revealed presence of variable immune recall in BALB/c mice (increased antibody levels in 3/7 mice) as compared to uniform recall in C57 mice (5/5 mice). We then initiated studies to investigate the immunoregulatory mechanisms underlying strain-dependent responses to mP+H antigen. Because isotype switching and expression of IgG subclasses is influenced by T helper (Th) cell subsets, we examined IgG subclasses in BALB/c and C57 mice as a surrogate marker of Th cytokine expression. C57 mice showed IgG2b>IgG1 anti-mP+H indicative of Th1 cytokine response, while BALB/c mice showed an IgG1>IgG2a response, a Th2 predominant response. Taken together, these findings of strain-dependent differences to mP+H indicate that variable expression of human HIT may be similarly governed by differences in peripheral immunoregulatory mechanisms.

Figure Figure
Improved Clinical Outcome and ADAMTS13 Activity after Concurrent Cyclosporine and Plasma Exchange in TTP.
Authors: Cataland Spero R. , Kraut Eric H. , Benson Don , Ferketich Amy K. , Horne April , Jin Ming , George James N. , Wu Haifeng M.
Published: April 2020

We previously reported that concurrent cyclosporine and plasma exchange(CSA/PE) is superior to corticosteroids and PE for acute TTP in terms of a decreased exacerbation rate (0% v. 47%) after stopping PE.(ASH 2005,Abstract# 1235) We now present similar data and serial analysis of ADAMTS13 activity and inhibitor concentration from two patient cohorts treated with CSA/PE.

Patients: Ten patients failing initial therapy with corticosteroids and PE (exacerbations or refractory) were treated with CSA/PE. This study led to a second cohort study of upfront therapy with CSA/PE in 11 patients with acute TTP.

Methods: CSA (2–3 mg/kg) orally was administered concurrent with daily PE. After remission (normal platelet count and LDH), 2 additional exchanges were performed on an every other day schedule. CSA was administered for 6 months and discontinued.

Results: In the exacerbation/refractory patients, 9/10 achieved remission after a median of 11 exchanges. One patient suffered an exacerbation and required one additional exchange to achieve a sustained (>30 d.) remission. Four patients relapsed 2,3,7, and 10 months after completing 6 months of CSA. In the CSA/PE upfront group, 10/11 achieved remission. One patient died of infectious and disease-related complications. Remission occurred after a median of 5 exchanges. No patients(0/10) suffered an exacerbation. Three patients recurred, 2 while taking CSA and one 2 weeks after stopping CSA. Median ADAMTS13 activity and the change in inhibitor concentration over time for all CSA/PE patients is shown in Figure 1.

Conclusions: CSA/PE improves clinical outcome and prevents exacerbations after tapering PE. CSA/PE also increased in vitro ADAMTS13 activity and suppressed the inhibitor, with both occurring well after the last PE. These findings coupled with the decline in median ADAMT13 activity after discontinuing CSA(Fig. 2) suggest the improvement in ADAMTS13 activity was a result of CSA therapy.

Table 1. Presenting ADAMTS13 and Inhibitor Data N Med. ADAMTS13% Prior to Initial Therapy Med. ADAMTS13% Prior to CSA and PE Med. Inhibitor Conc. Prior to Initial Therapy(ug/mL) Med. Inhibitor Conc. Prior to CSA and PE(ug/mL) Remission Rate Exacerbation (30 d. Recurrence) Rate CSA and PE Exacerbation and Refractory 10 2 (0–8) 4 (0–20) 587 (275–1,111) 3,173 (372–9,845) 9/10 1/10 CSA and PE Upfront Therapy 11 N/A 2 (0–60) N/A 571 (147–4,988) 10/11 0/10 Table 2. Effect CSA and PE on ADAMTS13 Activity and Inhibitor Concentration. Time (weeks) 0 4 10 24 CSA and PE Exac. and Refractory N 9 9 7 8 Median ADAMTS13 % 4 (0–20) 24 (4–39) 42 (5–72) 49 (7–116) Median Inhibitor Conc. (ug/mL) 3,173 (372–9,845) 294 (165–6,548) 191 (41–4,228) 204 (55–756) CSA and PE Upfront Therapy N 10 8 5 4 Median ADAMTS13% 2 (0–60) 20 (5–104) 32 (4–37) 49 (7–116) Median Inhibitor Conc. (ug/mL) 571 (147–4,988) 270 (82–2,637) 302 (110–378) 204 (55–756) Figure Figure
Dapsone as Second-Line Drug in Non-Splenectomized Patients with Chronic Idiopathic Thrombocytopenic Purpura (ITP): A Highly Cost Effective Option in a Developing Country.
Authors: Sharma Ajay , Nair Velu , Das Satya R. , Mishra D.K. , Kotwal J. , Agarwal Mohan B.
Published: April 2020

The management of chronic refractory ITP continues to be a frustrating experience for the patients as well as treating hematologists. Many patients relapse after steroid therapy as well as splenectomy. Even though latter modality leaves them susceptible to life-threatening infections, without making them symptom free. Immunoglobulins and anti-D vaccine do show a good but brief response. Retuximab is a newer drug to be used in ITP, but it is out of reach for most of the patients in a developing country like India. Of late Dapsone has been seen to be an effective drug in such situations and probably acts through reticulo-endothelial blockade.

We report the efficacy of Dapsone in 46 consecutively treated patients in our hospital, who had continued to be symptomatic despite adequate steroid therapy. They were chosen to be treated with Dapsone without being subjected to splenectomy. Their median age was 21 years(range : 8–46 years). The patients having associated connective tissue disorders were excluded. All the patients were screened for hemolytic tendency and splenectomy. Dapsone was started at an initial dose of 50 mg/day and escalated to100 mg/day, if the response was sub-optimal. Children received 25 mg/day initially and subsequent dose was adjusted to a minimal level required to achieve symptom free status. The aim of the therapy was to eliminate purpura rather than to achieve a particular level of platelet count. The duration of treatment ranged from six months to 48 months.36 patients (80%) showed an overall response with disappearance of purpura and a significant rise (> 40,000/cmm) in platelet count. Treatment was well tolerated and no adverse effects were encountered even though dose related hemolysis has been reported. The drug was continued in 36 responders for a median period of 32 months and they all continued to be asymptomatic throughout. The treatment was intentionally withdrawn in 12 responders, but thrombocytopenia recurred in all these patients. In our experience Dapsone provided an inexpensive and well tolerated alternative for patients with ITP who showed suboptimal response to steroid therapy. The splenectomy was successfully avoided in these patients. Its use is cost effective as well. Both these factors make this a great boon to the patients in a developing country. We recommend that all patients be started on Dapsone once they fail to show response to steroids. Splenectomy should be reserved for the patients who do not respond to Dapsone.
Single Dose of Anti-CD20 Monoclonal Antibody (Rituximab) Treatment in Adults with Refractory Immune Thrombocytopenic Purpura (ITP).
Authors: Tanaka Eri , Hayashi Shuji , Fujimaki Katsumichi , Fujita Hiroyuki
Published: April 2020

Refractory Immune Thrombocytopenic Purpura (ITP) is a difficult disease to treat effectively and the mortality approaches nearly 10% over 10 years. Moreover, the side effect profile of chronic steroid administration is undesirable due to the multi-systemic actions of these drugs. Recently, it has been reported in the literature that Rituximab is effective treatment for chronic ITP and it has been used at a dose of 375mg/m2 weekly for up to four weeks, as with lymphoma therapy. Rituximab is an expensive treatment, but according to previous data, patients treated with this drug have responded with increased and sustained platelet counts following only one infusion. Based on this, we treated five refractory ITP patients with single-dose Rituximab and all responded well.

Patient 1 is with a 15 year history of ITP and Patient 2 is with a 34 year long history of ITP. Following treatment with Rituximab, although there was an interval of up to 7 months, both eventually responded well. Patient 3 is a 93 year old male who presented to our hospital with an acute presentation of ITP which involved severe gastrointestinal bleeding and this proved to be refractory to both steroids and intravenous immunoglobulins, but responded very quickly to Rituximab within 24 days. Patient 4 is a 75 year old female with diabetes mellitus and three-vessel coronary artery disease who presented with a bleeding diathesis. She was treated with steroids, intravenous immunoglobulins, danazol and azathioprine but with no response. Single-dose Rituximab was effective within 20 days with an improvement in platelet counts. Patient 5 is a 51 year old male with a six year history of ITP who presented to our hospital with massive intraabdominal and gastrointestinal bleeding and after Rituximab therapy his platelet count responded appropriately after 45 days.

These patient responses were not associated with prior response to therapy, age, previous splenectomy, duration of ITP or platelet count. All patients tolerated treatment well except one patient who developed SLE-nephrotic syndrome 15 months later although it cannot be proven that such therapy induced this collagen-vascular diseases because Rituximab can be used to actually treat such conditions. Three patients remained in remission for more than one year after just one dose of the Rituximab therapy. Even from our small number of refractory ITP patients treated with Rituximab, it is our experience that single-dose treatment is also effective in some cases of refractory ITP and its effect may continue to provide long-term remission whereby it is now possible to decrease and even stop long-term steroid treatment in such patients.