bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced Efficacy of Recombinant Factor VIII in Non-Covalent Complex with PEGylated Liposome in Hemophilia A Mice.
Authors: Pan Junliang , Liu Tongyao , Kim Ji-Yun , Patel Chandra , Newgren Jim , Sommer Jurg , Pierce Glenn , Jiang Haiyan
Published: October 2019

BAY 79-4980, a non-covalent complex of rFVIII (Kogenate® FS) reconstituted in PEGylated Liposome (PEG-Lip) diluent, was reported to double the bleed-free days in severe hemophilia A patients from 7 to 13 days (35 IU/kg) and 6 to 11 days (25 IU/kg), as compared to respectively corresponding doses of rFVIII1. However, in a more recent phase I trial, the FVIII pharmacokinetics (PK) in plasma was virtually identical in patients who received either rFVIII or BAY 79-4980. To understand the mechanism of action of BAY 79-4980, we compared the pharmacological efficacy and PK of BAY 79-4980 to rFVIII in Hemophilia A (HemA) mice. A tail vein transection model was established, in which the survival of animals following the injury correlated to the rFVIII dose administered at 24 hrs prior to the injury, and a 2.5 to 3-fold difference in rFVIII dose led to significantly different survival rates (p<0.05). In this bleeding model, BAY 79-4980 (13 IU/kg) demonstrated significantly enhanced (p=0.018) survival compared to rFVIII at 24 hrs post dosing. The formation of a non-covalent complex of rFVIII with PEG-Liposome prior to the administration is required for the improved efficacy of BAY 79-4980, as sequential infusion of rFVIII and PEG-Liposome was no longer different from the identical dose of rFVIII alone. The plasma PK of FVIII in HemA mice treated with either BAY 79-4980 or rFVIII displayed two-compartmental decay with comparable incremental recovery, clearance and volume of distribution. However, BAY 79-4980 showed a modest (13%) but significant (p=0.024) increase in terminal half-life, and a noticeable (42%) but not statistically significant (p>0.05) increase in AUC, compared to rFVIII. Similar results were also observed in a whole blood PK study in HemA mice that received 125I-labeled rFVIII reconstituted in PEG-liposome versus buffer. While the difference in plasma FVIII levels resulting from different terminal half-lives of BAY 79-4980 and rFVIII may account for the improved efficacy of BAY 79-4980 in HemA mice, other mechanisms, such as the association of PEG-liposome with platelets and monocytes, thereby altering their pro-hemostatic functions, are under investigation.
The Use of Ideal Amphipathic Helical Peptides To Reduce Hemorrhage In Vivo.
Authors: Charbonneau Sophie , Ganopolsky Jorge G. , Pang Henry T. , Shek Pang N. , Blostein Mark D.
Published: October 2019

We have previously demonstrated that a 22 amino acid ideal amphipathic peptide (IAP) of K7L15 composition dramatically accelerates both factor IXa and factor Xa activity. In the present work, we investigate the activity of IAP attached to a surface in view of designing a procoagulant surface to reduce hemorrhage. Our results show that IAP maintains its catalytic enhancing properties for factor IXa and factor Xa when attached to a surface. This enhancement is dependent on the presence of the gamma-carboxyglutamic acid domain of factor X, consistent with the hypothesis that IAP behaves as a phospholipid membrane, providing a surface for the assembly of procoagulant enzymes and substrates. To further confirm this hypothesis, we demonstrate direct binding between surface-bound IAP and the Gla domain of factor X using an ELISA-based binding assay. Based on the aforementioned evidence that immobilized IAP enhances procoagulant activity, we conducted in vivo experiments using an ear-bleeding model in rabbits. We incorporated IAP into DuraSeal, a commercially available sealing agent, and found that the addition of IAP decreases the bleeding time in rabbits by 25% (p=0.0065). In conclusion, the above data provide a rationale for designing procoagulant surfaces in vivo. Further evaluation in larger animal models is warranted.
Effects of Direct Factor Xa Inhibitor, YM150, on Clot Formation and Clot Lysis In Vitro Compared with Other Anticoagulants.
Authors: Kaku Seiji , Suzuki Ken-ichi , Funatsu Toshiyuki , Saitoh Minori , Koshio Hiroyuki , Ishihara Tsukasa , Hirayama Fukushi , Kawasaki Tomihisa
Published: October 2019

The objective of this study was to evaluate the effects of direct factor Xa inhibitor, YM150 and its major in vivo metabolite, YM-222714, on clot formation and clot lysis compared with other anticoagulants, such as a direct thrombin inhibitor (melagatran), a pentasaccharide (fondaparinux), low molecular weight heparin (enoxaparin) and unfractionated heparin. To assess clot lysis, the tissue plasminogen activator (tPA)-induced clot lysis assay was used with human plasma triggered by low and high levels of tissue factor (TF). Under low TF conditions, clot formation was completely prevented by melagatran at 1 μmol/L, by fondaparinux at all concentrations examined (0.1 to 1 μg/mL), by enoxaparin at 0.3 and 1 IU/mL and by heparin at 0.1 and 0.3 U/mL. Even under high TF conditions, 0.3 U/mL heparin prevented any clot formation. Although melagatran, fondaparinux, enoxaparin, and heparin potently prevented plasma clot formation under low TF conditions, under high TF conditions they were less effective at prolonging the clotting time. Under both low and high TF conditions, YM150 and YM-222714 prolonged the clotting time in a concentration dependent manner at concentrations between 0.3 and 3 μmol/L. YM150 and YM-222714 significantly accelerated clot lysis under both low and high TF conditions, but their effects were most evident under high TF conditions. Lower concentrations of melagatran (0.1 and 0.3 μmol/L) enhanced clot lysis under low TF conditions, but under high TF conditions, enhancement of clot lysis required higher melagatran concentrations (0.3 μmol/L or more). Under high TF conditions, fondaparinux enhanced clot lysis only at the highest concentration tested (1 μg/mL). Enoxaparin and heparin enhanced clot lysis under low TF conditions at the lowest test concentrations (0.1 IU/mL and 0.03 U/mL, respectively). Both also enhanced clot lysis under high TF conditions, but their effect reached statistical significance only at higher concentrations (1 IU/mL and 0.1 U/mL, respectively). These results suggested that direct factor Xa inhibitors, YM150 and YM-222714, exert stable anticoagulant effects independently of TF concentration. Both inhibitors enhanced tPA-induced fibrinolysis in human plasma clotted via the extrinsic coagulation pathway. Useful characteristics of YM150 and YM-222714, such as a linear dose response and reliable anticoagulation independent of TF concentration, may lead to the creation of an anticoagulant that is easier to use in the clinical setting than existing products. Potentially beneficial antithrombotic effects, which can be promoted by accelerating endogenous fibrinolytic pathways, may further aid in the prevention or treatment of thrombosis.
Comprehensive Molecular Characterization of Malignant and Microenvironmental Cells in Waldenstrom’s Macroglobulinemia by Gene Expression Profiling.
Authors: Hatjiharissi Evdoxia , Mitsiades Constantine S. , Bryan Ciccarelli T. , Lian Xu , Yang Cao , Adamia Sophia , Leleu Xavier , Roccaro Aldo , Ioakimidis Leukothea , Hunter Zachary R. , Patterson Christopher J. , Ghobrial Irene , Treon Steven P.
Published: October 2019

Waldenstrom’s Macroglobulinemia (WM) is an incurable B-cell malignancy characterized by bone marrow (BM) infiltration with a spectrum of clonally related cells, including small lymphocytes and lymphoplasmacytic cells (CD19+) as well as mature plasma cells (CD138+). The molecular pathogenesis of the disease remains to be defined. We therefore analyzed the gene expression profiles of CD19+ and CD138+ BM mononuclear cells from 30 untreated patients with WM and compared their gene expression profile to their normal counterparts from 10 healthy donors using Affymetrix microarrays (U133 plus 2.0). Since the microenvironment plays an important role in the pathogenesis of WM, we also profiled and compared gene expression profiling for CD19 and CD138 depleted BM mononuclear cells from the same patients and healthy donors. Gene expression analysis was performed using dChip software. Unsupervised hierarchical cluster analysis demonstrated distinct gene expression patterns between WM cells versus their normal counterparts. In supervised hierarchical cluster analysis selecting for genes with > 2 fold change in expression and a False discovery Rate (FDR) < 2%, a set of 1171, 582 and 360 genes were found to be differentially expressed between WM patient and healthy donor CD19+, CD138+, as well as CD19/CD138 depleted (microenvironmental) cells, respectively. Among the most significantly over-expressed genes in the CD19+ compartment in WM patients were: BCL2, TNFRSF13B, TNFRSF17, IGLL1, CCR2, CLLU1, whilst the AP1 family genes JUND and FOSB were among the most significantly down-regulated genes in both CD19+ and CD138+ cells in WM patients. Other interesting transcripts which were over-expressed in CD138+ cells from WM patients included those from genes involved in transcription regulation (ZKSCAN1, ZMYM1, ZNF189, ZNF19, and ZNF559) and interferon response (IFI16 and IFIH1). Of considerable interest was our observation that microenvironmental cells in WM patients demonstrated an overactive transcriptional profile composed of genes which are associated with immune and inflammatory responses including the Toll like receptors (TLR 1,5,7,8), interferon and cytokines (IFI16, IFNAR1, IL-10R, IL-8R), genes encoding extracellular matrix components (Fibronectin and Hepatocyte Growth Factor) as well as genes involved in apoptotic signaling (TNFSF10, TRAF4). These studies provide the first comprehensive molecular characterization of WM, dissecting the molecular features of the two immunophenotypically distinct populations of malignant cells, and providing for the first time evidence for a distinct molecular profile in BM microenviromental cells.
p53 Signaling in Response to Increased DNA Damage Sensitizes AML1-ETO Cells to Stress-Induced Death.
Authors: Krejci Ondrej , Wunderlich Mark , Geiger Hartmut , Chou Fu-Sheng , Schleimer David , Jansen Michael , Andreassen Paul R. , Mulloy James C.
Published: October 2019

Chromosomal translocation 8;21 is present in 10–15% of patients with acute myeloid leukemia and results in the expression of the AML1-ETO (AE) fusion protein. AE expression by itself is not sufficient for malignant transformation of bone marrow progenitor/stem cells, but the nature of the cooperating genetic alterations are unknown. It is unclear whether AE facilitates acquisition of these synergizing events. We have previously shown that retroviral transduction of primary human CD34+ cells with AE causes a dramatic expansion of CD34+ progenitor cells that retain the ability to differentiate into myeloid, lymphoid, and erythroid lineages and engraft NOD/SCID mice. Consistent with mouse studies, these transgenic human cells fail to cause disease in immunodeficient mice or become transformed in vitro. These cells serve as a highly relevant pre-leukemia model for t(8;21) leukemogenesis. Here we demonstrate that AE downregulates genes involved in multiple DNA repair pathways, an effect that is evident as early as two days after retroviral transduction of human CD34+ cells. The human AE cells show delayed repair of induced DNA damage as shown by kinetics of a comet assay. During in vitro culture, AE cells show spontaneous accumulation of DNA damage, as demonstrated by γH2AX staining. Using a transgenic mutation frequency assay based on the lacZ reporter gene, we show that the in vivo mutation frequency of murine BM cells was increased approximately two fold by AE expression. Possibly as a result of this chronic DNA damage, AE cells have increased p53 protein expression and an upregulation of multiple p53 response genes. This results in increased basal apoptosis and enhanced sensitivity to DNA damaging agents compared to control cells. Intriguingly, microarray data indicate that t(8;21) patient samples also exhibit increased expression of p53 response genes when compared to other AML patient samples. Inhibition of the p53 pathway by shRNA interferes with the G1 cell cycle checkpoint and increases the resistance of AE cells to DNA damage. We thus speculate that AML1-ETO may indeed facilitate accumulation of additional genetic alterations by suppressing endogenous DNA repair, but that this activity also renders the cells more susceptible to stress. It is possible that the superior outcome of t(8;21) patients is partly due to an activated p53 pathway, and that loss of the p53 response pathway in disease progression will consequently negatively impact overall outcome. We are currently investigating whether a “p53 activation” profile can be used as a prognostic tool in AML.
Influence of Protein Kinase C and Calcium in the Course of Thrombin Receptors Activation.
Authors: Xie Ying , Han Yue , Wu De Pei , Sun Aining , Zhang Wei
Published: October 2019

Object In order to compare the functions of protein kinase C (PKC) and calcium (Ca2+) in platelet aggregation and platelet membrane surface glycoproteins GPIb expression after thrombin receptors activation, then to investigate the role of Gq signal transmission pathway in the course of thrombin receptors activation.

Methods Peptide SFLLRN (PAR1-AP) and AYPGKF (PAR4-AP) were used for stimulating platelet at different time point (0, 1, 2, 5, 10, 30min), then the alterations of platelet aggregation and GPIb were analyzed in the involvement of Ro-31-2220 (inhibitor of PKC) and BAPTA/AM (calcium chelator).

Results Either PAR1 or PAR4 peptide can induce absolute platelet aggregation, together with a reversible internalization of GPIb. Platelet aggregation was inhibited by Ro-31-2220 or BAPTA/AM while the shape change curve still occurred upon PARs activation. In addition, Ro-31-2220 decreases GPIb centralisation upon PAR1 stimulation (P <0.05 at 1, 2 min), though it blocks the pool of GPIb inside platelet in PAR4 activation (P <0.05 at 10, 30 min). Meanwhile, GPIb internalization was blocked by BAPTA for both peptides (P <0.05 at 1∼10 min).

Conclusion All the results confirm a critical role of Gq pathway in thrombin signal transmission through the involvement of protein kinase C and calcium. Calcium is closely correlated with the thrombin receptors activation, seemed to be similar for two PARs signal pathways. Protein kinase C urges GPIb centralisation in PAR1 pathway and accelerates GPIbα return in PAR4 pathway.
Prevention of Complete TTP Relapses by Immediate Initiation of Rituximab Treatment.
Authors: von Auer Charis , Hess Georg , Scharrer Inge
Published: October 2019

Plasma exchange (PE) is the treatment of choice in thrombotic thrombocytopenic purpura (TTP) patients. The use of PE has decreased the mortality rate of TTP from 90 to 10–30%. However PE is costly, technically demanding and often associated with serious complications such as infection and/or transfusions reactions. Some patients are also unresponsive to PE. In recent studies patients with refractory TTP have achieved sustained remission after Rituximab treatment. Patients with chronic TTP are very often afraid of PE. We cared for 4 anxious patients, who refused PE treatment on the one side and demanded treatment in their early phase of relapse on the other side. Therefore we treated 4 patients with a beginning relapse of TTP with Rituximab (375 mg/m2/week) before onset of symptoms. The 4 patients had already suffered from 10, 3, 4 and 5 relapses. Due to close control of laboratory data in an 8 or 12 weekly interval it was possible to detect deterioration of laboratory data (decrease of platelets <50.000, increase of LDH>500 U/l, decrease of ADAMTS-13 activity to <6.5%, increase of inhibitor to ADAMTS-13 >4BU and detection of schistocytes in the blood smear). Already after the first or second Rituximab dose a significant effect on the laboratory data was seen (increase of platelets >100.000, decrease of LDH to normal values, increase and normalization of ADAMTS-13 activity, decrease of inhibitor titer and disappearance of schistocytes). In the mean observation time (3–12 months) no deterioration of laboratory data and no TTP symptoms were observed. No side effects of Rituximab occurred. Based on our experience we recommend close control of laboratory parameters and an immediate treatment before onset of symptoms in patients with impending relapses. Up to now the reasons for these rapid effects of Rituximab are not fully understood. Other mechanisms of Rituximab treatment in TTP compared to lymphoma therapy might be possible.
ADAMTS13 Antigen Levels and the Risk of Relapse during Long-Term Clinical Follow Up of Patients with Idiopathic Thrombotic Thrombocytopenic Purpura.
Authors: Cataland Spero R. , Jin Ming , Lin Shili , Kennedy Melanie , Kraut Eric H. , George James N. , Wu Haifeng M.
Published: October 2019

Background: Although nearly all patients with idiopathic TTP will achieve remission, a majority of patients will suffer a recurrence at some point in the future. Our group has had an ongoing interest in using laboratory biomarkers to predict the risk of recurrence of idiopathic TTP after patients achieve remission and complete their initial therapy.

Method: Twenty patients (Table 1) with idiopathic TTP were analyzed beginning from the time of their initial presentation to our institution. 8 patients had a history of previous episodes of TTP. Patients were followed prospectively and monitored for relapse of TTP. Nine patients have maintained a continuous remission (Non-relapse) and 11 patients (Relapse) had a relapse of TTP. 139 serial samples from both groups, a median of 6.5/patient, were analyzed and compared in terms of ADAMTS13 activity, ADAMTS13 antibody concentration, and ADAMTS13 antigen. Median follow-up for the "Non-relapse" group was 39 months (range, 12 to 50). The median time to relapse was 11 months (range, 1.5 to 34) in the "Relapse" cohort. Data for the relapse group was censored at the time of relapse accounting for the shorter median follow-up of that cohort. The serial biomarkers from the "Relapse" group presented below reflect only samples obtained during continuous remission.

Results: Serial measurements of laboratory biomarkers are presented for both the "Relapse" and "Non-relapse" patients in Figure 1 below. During the first year of follow-up, the "Non-Relapse" patients had significantly higher ADAMTS13 Ag levels (p = 0.0065) compared to the patients who relapsed. There was no significant difference in the ADAMTS13 activity and antibody concentration between the two groups during the first year of follow up. In the 11 patients who relapsed, the mean ADAMTS13 activity was 1.2% (range, 0.5 – 3.2%), the mean antibody concentration was 958 ug/ml (range, 147 – 4,988), and the mean ADAMTS13 antigen was 221 ug/ml (range, 73 – 468) at the time of disease recurrence.

Conclusion: These data suggest that ADAMTS13 antigen determinations in the first year of follow-up may predict which patients will have a sustained remission of their disease. ADAMTS13 antigen measurement, compared to ADAMTS13 activity or antibody concentrations, may provide additional information to more accurately predict a patient’s future clinical course after achieving remission.

Demographic and Laboratory Data at Presentation Mean Laboratory Data at Presentation (range) N Age Sex Previous TTP ADAMTS13 % ADAMTS13 Ab(ug/ml) ADAMTS13 Ag. (ng/ml) Relapse 11 37 (19–56) 4M/7F 4/11 2.5% (0.6 – 7.9) 1,221 (190 – 3,397) 360 (99 – 1,195) Non-Relapse 9 47 (21–59) 2M/7F 4/9 4.2% (<0.5 – 9.1) 891 (59 – 4,040) 266 (84 – 668) Figure Figure
The Incidence of Idiopathic Thrombocytopenic Purpura (ITP) among Adults in the United Kingdom’s General Practice Research Database (GPRD), 1992–2005.
Authors: Feudjo-Tepie Maurille A. , Hall Susan A. , Logie John W. , Bennett Dimitri
Published: October 2019

ITP is a disease caused by inadequate platelet production as well as increased platelet destruction. Population-based literature on the epidemiology of ITP is currently sparse, but incidence has been estimated to be between 1.6 and 3.0 per 100,000 person years of observation (PYO) (Neylon AJ et al., Br J Haematol. 2003; Satia J et al., Eur Hematol Assoc [poster presentation] 2006). The objective of this study was to estimate the incidence rate of ITP in adults in the United Kingdom using the GPRD, which contains patient data from over 370 representative medical practices. Using diagnostic codes for ITP (D313000, D313012, 42P2.11, or D313.12 or the Oxford Medical Information System code 2871C) to define cases, incidence rates per 100,000 PYO with 95% confidence intervals (CI) were estimated. Incidence by age, gender, and time period (1992–1998, 1999–2005) was evaluated. Eight hundred forty adult cases of ITP (aged 18 years or more) during 21.7 million PYO from 1992 to 2005 were identified. The overall incidence rate (per 100,000 PYO) during this time was 3.9 (95% CI: 3.6, 4.1) with higher rates among women (4.6 [95% CI: 4.2, 4.9]) compared with men (3.2 [95% CI: 2.8, 3.5]). Incidence rates (per 100,000 PYO) increased with age (from 2.5 [95% CI: 2.2, 2.7] to 4.1 [95% CI: 3.6, 4.6] to 8.2 [95% CI: 7.3, 9.2] in the age groups 18–49, 50–69 and 70+ respectively), and in later study years (from 2.9 [95% CI: 2.5, 3.2] in the period 1992–1998 to 4.5 [95% CI: 4.1, 4.8] in the period 1999–2005). In summary, ITP was diagnosed in approximately 4 adults per 100,000 PYO in the General Practice Research Database. Incidence rates of ITP in this study are slightly higher than in previous estimates, possibly due to a code-based disease definition without validation by clinical and lab data. More population-based studies of the natural history of adult ITP are needed to better elucidate the burden of this disease.
Natural History of 33 Patients with Upshaw-Schulman Syndrome Has Revealed That All the Gravida Develop Thrombocytopenia, Often Followed by Thrombotic Microangiopathy with Stillbirth.
Authors: Fujimura Yoshihiro , Matsumoto Masanori , Kokame Koichi , Yagi Hideo , Isonishi Ayami , Matsuyama Tomomi , Kato Seiji , Ishizashi Hiromichi , Shida Yasuaki , Kenji Nishio , Akiyama Nobu , Tomiyama Junji , Natori Kazuhiro , Kuraishi Yasunobu , Imamura Yutaka , Inoue Nobumasa , Higasa Satoshi , Seike Masako , Kozuka Teruhiko , Hara Masamichi , Sugimoto Mitsuhiko , Wada Hideo , Murata Mitsuru , Miyata Toshiyuki , Ikeda Yasuo
Published: October 2019

Upshaw-Schulman syndrome (USS) is a congenital deficiency of the activity of von Willebrand factor (VWF)-cleaving protease or ADAMTS13 due to its gene mutations. USS is a complex thrombo-hemorrhagic disease, but its hallmark is severe neonatal jaundice soon after birth that often requires for exchange blood transfusion, and subsequently during childhood they have repeated episodes of chronic thrombocytopenia and hemolytic anemia that are reversed by infusions of fresh frozen plasma. However, after a discovery of ADAMTS13, the presence of two phenotype expression on USS-patients was described; one is the early-onset type as abovementioned, and the other is the late-onset type which is asymptomatic during childhood and the first bout develops after adolescent or during adulthood, in association with infections or pregnancy. During the past 8 years, we diagnosed 33 patients with USS. Through analyzing the natural history and the phenotype-genotype expression in these patients, and more specifically to 9 USS-patients who had their first bout at pregnancy, we found that two clinical phenotypes of USS are mostly attributable to misdiagnosis or overlook of thrombocytopenia during childhood, and excluded a possibility of the concern on a trace amount of ADAMTS13 activity which has not been evaluated by the previous methods. Further, in vitro studies we have clearly shown that platelet aggregation or thrombi formation under high shear stress is tremendously up-regulated in the absence of ADAMTS13, that occurs proportionally to the amount of high VWF multimers, that in part is assumed to be an in vitro reflection of the circulation in USS-gravida.

Figure Figure
Incidence of HPA-9b in Testing for Neonatal Alloimmune Thrombocytopenia.
Authors: Smith James W. , Chamberlain Kristen , Moffatt Diana , Santos Aurelio , Neutel Denise , Moore Jane , Warkentin Theodore E. , Arnold Donald M. , Kelton John G.
Published: October 2019

Background: Neonatal alloimmune thrombocytopenia (NAT) is caused by maternal alloantibodies that cross the placenta and cause increased clearance of fetal platelets. The alloantibodies are directed to incompatible human platelet antigens (HPA) inherited from the father and expressed on fetal platelet glycoproteins. Most cases of NAT are caused by HPA-1a, -5a, -5b, -15a, or -15b. Some are associated with immunization to a low frequency antigen found only in the paternal lineage. We investigated the incidence of the low frequency antigen HPA-9b in a population with suspected NAT, and compared the frequency to that found in a normal control population. HPA-9b is caused by a Val837Met polymorphism on alpha-IIb and is expressed only in a rare subset of individuals with a corresponding serine at position 843 that defines the HPA-3b antigen.

Methods: We used PCR-SSP for HPA-9b genotyping. Antibody studies were performed by incubating maternal serum with intact platelets using flow cytometry, monoclonal antigen capture enzyme immunoassay, and radioimmunoprecipitation.

Results: Of 294 normal control blood donors, 4 typed positive for HPA-9b, yielding a gene frequency of 0.0068. However, if only HPA-3b positive donor controls are considered (n=177) the gene frequency for HPA-9b is 0.011. Archived DNA from HPA-3b positive (HPA-3a3b or -3b3b) fathers of children with NAT were tested for HPA-9b. Of 94 fathers, 3 (3.2%) typed HPA-9a9b, yielding a gene frequency for HPA-9b of 0.016, not significantly different from the frequency for HPA-9b found in the HPA-3b-positive healthy controls (p>0.1). Similarily, when DNA from HPA-3b positive mothers was tested in tandem, 3 of 60 were positive for HPA-9b, yielding a gene frequency of 0.026. Of the 3 cases of suspected NAT (platelet count range 15,000 to 63,000) in which the mother and father were incompatible for HPA-9b, 2 had no other platelet antigen incompatibility, and one was incompatible for HPA-15b. None of the 3 maternal sera was positive for anti-platelet reactivity using radioimmunoprecipitation and paternal platelets as a target, and no anti-platelet antibody was detected either by flow cytometry or by GPIIb/IIIa antigen capture immunoassay with platelets from a HPA-9a9b donor.

Interpretation: These results support the findings that HPA-9b is distributed in the general population at a frequency higher than initially suspected. However, we also found that the frequency of HPA-9b in fathers of children with suspected NAT was similar to that in the normal control population when only HPA-3b-positive (HPA-3a3b or -3b3b) targets were analyzed. Therefore, in the absence of a positive anti-platelet antibody, the significance of a maternal/paternal incompatibility for HPA-9b alone is not predictive of an increased risk of NAT.
Mutation Screening of GPIIb and GPIIIa Exons by Conformation Sensitive Gel Electrophoresis.
Authors: Kannan Meganathan , Ahmad Firdos , Yadav Birendra K. , Kumar Rajive , Saxena Renu
Published: October 2019

Glanzmann’s Thrombasthenia (GT) is an inherited platelet function disorder characterized by a severe reduction in, or absence of, platelet aggregation in response to multiple physiologic agonists due to abnormalities of platelet glycoprotein GPIIb and / or GPIIIa. The clinical complications in GT include life long bleeding with easy bruising, epistaxis, menorrhagia and gastrointestinal bleeding. GT is caused by mutations in the genes encoding GPIIb or GPIIIa that result in qualitative or quantitative abnormalities of the platelet membrane proteins. For the detection of mutations affecting GPIIb and GPIIIa genes, screening method like single stranded conformation polymorphism (SSCP) is being used in most of the developing countries like India, but is a less sensitive technique. The current study was done to screen for mutations in GT patients using conformation sensitive gel electrophoresis (CSGE), an enhanced method of mutation screening. The work was carried out on 45 GT patients that included 25 males and 20 females with an age ranging from 5 months to 46 years. GT was diagnosed in these patients on the basis of clinical and hematological parameters. Platelet receptor GPIIb/IIIa protein was analyzed first by flow cytometry followed by western blot. Flow cytometry allowed the characterization of GT in to types I, II and III and western blot allowed measuring the presence of aIIb and b3 protein in the platelet lysates. Platelet GPIIb/IIIa analysis by flow cytometry revealed 62.2%, 15.5% and 22.2% of GT to have types I, II and III respectively. Western blot showed the absent or reduced amount of aIIb or b3 protein. Nucleotide changes in either of GPIIb or GPIIIa gene was screened first by CSGE followed by sequencing of defective exons. A total of 25 exons of GPIIb and 12 exons of GPIIIa showed band sift by CSGE. 5 out of 30 exons (Exons 2, 3, 7, 27 and 29) of aIIb gene and 3 out of 15 exons (Exons 4, 8 and 11) of GPIIIa gene showed no band shift by CSGE. Sequencing revealed mutations in 36 of 45 (80%) unrelated GT patients. Of these, 22 patients (48.8%) showed defect in aIIb gene and 14 patients (31.1%) showed defect in b3 gene. In 9 out of 45 (20%) GT patients, no gene alterations were identified and these patients though their mutation status were not identified, their hematological tests including platelet aggregation and flow cytometry revealed that they were definite GT. GPIIb gene was the most common defective gene identified in GT patients in India. CSGE can be used not only to screen for mutations in GPIIb/IIIa exons, but also in carrier detection. Once the defective exon and the mutation causing GT in the patient is identified, the same defect can be looked for in the family members using CSGE. Thus CSGE is an effective technique to screen for mutation in patients as well as to predict carrier status in family members.
Pregnancy Associated Thrombotic Thrombocytopenic Purpura: A Case Report.
Authors: Ye Xu , Feng Ying , Pang Ying , Wang Zhaoyue
Published: October 2019

Introduction: In clinical setting, cases with thrombotic thrombocytopenic purpura (TTP) are easily mis-diagnosed and the clinical outcome may be influenced by the mis-diagnosis.

Case report: The patient is a 28-year-old woman in her first pregnancy. She had transient thrombocytopenia two years ago and was diagnosed idiopathic thrombocytopenic purpura (ITP) based on her bone marrow results but recovered quickly without immunosuppressive therapy. Her blood routine test was normal before the pregnancy. But she was found to have asymptomatic thrombocytopenia during her first pregnancy check-up in the first trimester of her pregnancy. A diagnosis of ITP was made after bone marrow examination. Then, she was put on a course of oral prednisolone and her platelet count transiently increased to normal but decreased to 18×109/L after she cut down on the dose. After that, she only showed partial response to increased dose of oral prednisolone and her hemoglobin (Hb) level began to fall slowly. She was admitted during her second trimester of gestation complaining of headache and low fever with signs of agitation. Blood routine, blood smear analysis, urine routine, liver function and blood bilirubin analysis, renal function tests were performed regularly. Bone marrow smear examination, tests for detecting various causes of hemolysis and tests detecting autoimmune antibodies were also performed. Her lab tests were indicative of severe hemolytic anemia and thrombocytopenia. Her renal function was continuously normal. As other causes of hemolysis and autoimmune diseases were excluded, a diagnosis of Evan’s syndrome was made although the Coombs’ test was negative. She showed partial response to high-dosage of prednisolone and IgG with increased but not normal platelet count and her fever disappeared. After transfusion of packed RBC and platelets, her symptoms and signs of the central nervous system disappeared and the fetus was removed through cesarean section. 22 days later, she underwent an emergent exploratory laparotomy confirming acute necrotic cholecystitis under supportive transfusion, and splenectomy was also performed. But the hemolytic anemia and thrombocytopenia still went on although she was still administered prednisolone and high-dosage of IgG. As a diagnosis of TTP was suspected, she received plasma exchange. The next day, her Hb level increase to 97g/L and her platelet count increased to 500×109/L without transfusion. More than one month later, her plasma sample was sent to another center to detect ADAMTS13 level and was found to be deficient in ADAMTS13 (7%). But antibody to ADAMTS13 was not detected.

Conclusion: The cause of anemia and thrombocytopenia in this case was pregnancy associated TTP. Further experiment for detection of mutation in her ADAMTS13 gene is being done. Mis-diagnosis in this case may be related to lack of recognition of variation in the course, precipitating factors of the disease and unavailability of ADAMTS13 level test.
Prediction of Heparin Induced Thrombocytopenia: Validation of an Experience-Based Computer Model.
Authors: Whitlatch Nicole L. , Kong David F. , Ortel Thomas L.
Published: October 2019

Background: Diagnosis of heparin-induced thrombocytopenia (HIT) requires certain clinical features along with the detection of platelet activating antibodies induced by heparin interaction with platelet factor 4 (PF4). Although the PF4 enzyme-linked immunoassay (ELISA) is a highly sensitive laboratory test, it is not specific. Adding excess heparin to the sample can confirm a positive PF4 ELISA result when it decreases antibody binding by 50% or more, potentially improving the specificity of the assay. We sought to determine the clinical value of the PF4 ELISA and confirmatory test by developing a predictive algorithm for HIT.

Methods: We retrospectively identified 116 patients with positive anti-PF4/heparin antibodies at Duke University Medical Center in 2005. We collected data on all patients to allow for clinical classification as HIT positive or HIT negative. HIT positive patients had at least a 30% decline in platelet count after 4–14 days of heparin exposure, or within 48 hours if recent (within last 100 days) heparin exposure. Anti-PF4/heparin antibody titers were determined by ELISA using a confirmatory step with excess heparin. A multivariate logistic regression model was fitted to the 2005 data, to estimate the relationship between patient characteristics (including age, gender, race, and clinical service), laboratory findings (including peak PF4 titer, confirmatory positive status), and clinical HIT status (HIT positive versus HIT negative). The model was then validated on an independent sample of 97 patients with positive anti-PF4/heparin antibodies retrospectively identified at Duke University Medical Center from January to July of 2006.

Results: We found no significant relationship between age, race, or gender and clinical HIT status. In multivariate analysis, the peak PF4 titer and confirmatory positive status were independent predictors of HIT. Figure 1 depicts median, 25th, and 75th percentiles of the predicted probabilities for the HIT-positive and HIT-negative patients in the 2005 training and 2006 test populations. The predictive accuracy on the 2005 training set (c-index 0.783, Somer’s Dyx 0.566) was maintained when the algorithm was applied to the independent 2006 test population (c-index 0.799, Somer’s Dyx 0.597). Figure 2 depicts the ROC curves for both patient populations.

Conclusions: This is the first study to our knowledge that has demonstrated the clinical utility of the Heparin/PF4 ELISA confirmatory test for the diagnosis of HIT. Based upon the PF4 ELISA and confirmatory assays, a predictive computer algorithm can distinguish patients likely to have HIT from those who are not. Accurate predicted probabilities of HIT will enable clinicians to more rapidly initiate appropriate therapy.

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Primary Study on the Reconstitution of CD4+CD25high Regulatory T Cells Early after Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Sun Daoping , Wu De Pei , Li Caixia , Liu Yuejun , Yang Mingzhen
Published: October 2019

Background: CD4+CD25+ regulatory T cell has been found to play an important role in suppressing allogenetic immune response and controlling GVHD recently. Our study was to explore the reconstitution of CD4+CD25high regulatory T cells early after allogeneic hematopoietic stem cell transplantation(allo-HSCT), and to evaluate its role in the development of acute Graft-versus- host disease(aGVHD)

Methods :27 patients undergoing allo-HSCT were enrolled. Detect the CD4+CD25high T cells subpopulation ratios in CD4+ T lymphocytes(CD4+CD25high/CD4+)with Flow cytometry, and the expression of FOXP3 mRNA with Real-time PCR assays in the donor grafts and periperal blood frequently after transplantation(at 2 weeks,1 month,2 month,3 month post-transpant,at the onset of aGVHD,and when aGVHD was controlled.).

Results:

In without aGVHD group, the CD4+CD25high/CD4+ ratio increased significantly at 2 weeks post-transplant, and decreased later(P<0.05); the expression of FOXP3 increased significantly early after transplantation, but remained below the healthy-control level within 3 months after transplantation(P<0.05). In aGVHD-group, the CD4+CD25high/CD4+ ratio also increased significantly at 2 weeks post-transplant,but exhibited no decrease tendency later(P<0.05);moreover the expression of FOXP3 remained low(P<0.05),and was lower than the without-aGVHD-group at 1∼3month post-transplant(P<0.05). At the onset of aGVHD, the CD4+CD25+ /CD4+ and the CD4+CD25high/CD4+ ratios were both higher than the healthy control and without-aGVHD group(P<0.05),but the expression of FOXP3 were lower than that of healthy control and without-aGVHD group(P<0.05);when aGVHD was controlled, the expression of FOXP3 raised compared with that at the onset of aGVHD(P=0.025), also the CD4+CD25high/CD4+ ratio exhibited an increase tendency(P=0.050).

Conlusion:

The CD4+CD25high regulatory T cells subpopulation undergo a significant expansion early after transplantation, but its complete reconstitution is a long-term process. The lower number of CD4+CD25high regulatory T cells and/or their lower expression of FOXP3 gene may be related with the development of aGVHD.
Successful Translation of a GMP-Based, Clinical Scale Selective Allodepletion Approach for Matched Donor-Recipient Pairs from Bench-to-Bedside.
Authors: Mielke Stephan , Nunes Raquel , Rezvani Katayoun , Fellowes Vicki S. , Venne Annie , Solomon Scott R. , Fan Yong , Scotto Christian , Read Elizabeth J. , Barrett A. John
Published: October 2019

Selective allodepletion (SD) is a strategy to eliminate host-reactive donor T-cells from hematopoietic stem cell allografts to prevent graft versus host disease (GvHD) while conserving useful donor immune functions. To overcome fluctuations in activation-based surface marker expression and achieve a more consistent and effective allodepletion, we investigated a photodepletion (PD) process targeting activation-based changes in p-glycoprotein that result in an altered efflux of the rhodamide-based photosensitizer TH9402. Expanded lymphocytes, generated using anti-CD3 and IL-2, were co-cultured with responder cells from HLA-matched or mismatched donors. Optimal results were achieved when co-cultured cells were incubated with 7.5 μM TH9402, followed by dye-extrusion and exposure to 5 Joule/cm2 light energy at 5x106 cells/ml.

Figure Figure

In six mismatched stimulator-responder pairs, the median reduction of allo-reactivity was 474-fold (range, 43–864) when compared to the unmanipulated responder. Third-party responses were maintained with a median 1.4-fold (range, 0.9–3.3) reduction. In three matched pairs alloreactive helper T-lymphocyte precursors (HTLps) were reduced below 1:100,000 while third party responses remained above 1:10,000. CMV-specific T cells and responses to SEB were preserved. The intracellular TH9402 amount after extrusion correlated significantly with the depletion efficacy obtained against the original stimulator (r2=0.76; p=0.0235) but not against the 3rd party (r2=0.05; p=0.68). This establishes a clinical scale process capable of highly efficient, reproducible, selective removal of alloreactive lymphocytes from lymphocyte transplant products performed under current good manufacturing practice (cGMP) being reliably capable of generating at least 107/kg SD T cells for transfusion using a single PD session. In establishing this SD approach, we set out to design a technique that could ultimately be adopted in many cell transplant processing centers. Recent review of our Investigational New Drug (IND) application by the Food and Drug Administration (FDA) and review of our clinical protocol by our Institutional Review Board have been successful, and a clinical trial is in progress where recipients with hematological malignancies are given a myeloablative preparative regimen followed by a transplant of selected CD34+ cells together with 5 x 106 SD T cells/kg from HLA-matched sibling donors.
Enhanced T Cell Function in Patients with Hematological Malignanicies Undergoing Mobilization for Autologous Stem Cell Transplantation.
Authors: Colovai Adriana I. , Schwartz Joseph , Slotky Ronit , Liu Zhouru , Fan Jianshe , Kabbaj Nida , Savage David G. , Chari Ajai , Spitalnik Steven L. , Suciu-Foca Nicole
Published: October 2019

Various regimens used for hematopoietic stem cell mobilization have been optimized to mobilize a maximum number of CD34+ hematopoietic stem cells (HSC) into the peripheral blood. However, the effects of mobilization regimens on other bone marrow derived cell populations have not been carefully examined. Since T cells from HSC grafts play an important role in anti-tumor responses, we studied T cell function in 11 adult patients with hematological malignancies undergoing mobilization for autologous stem cell transplantation (SCT). Mobilization regimens consisted of G-CSF alone (3 patients) or in combination with: GM-CSF (1 patient), chemotherapy (2), GM-CSF and chemotherapy (3), or AMD3100 (2). T cell function was determined using ImmuKnow (Cylex) assay, an FDA approved test, which measures immune cell function in whole blood by quantifying the amount of ATP released by PHA-stimulated CD4+ T helper (Th) cells. Prior to mobilization treatment, average Th reactivity was 242±128 ng ATP/ml (normal range: 200–525 ng ATP/ml). With mobilization, Th reactivity increased gradually in all patients, reaching peak reactivity values (>525 ng ATP/ml) on day 4–6 of treatment. Efficient stem cell mobilization (≥10 CD34+ cells/ul of blood) was accomplished in 8 out of 11 patients. These 8 patients exhibited an average Th peak reactivity level of 701±126 ng ATP/ml. Stem cell mobilization failed in the remaining 3 patients (<10 CD34+ cells/ul). Two of these patients had received AMD3100 (a CXCR4 inhibitor) and G-CSF, and showed unusually high ATP levels (>1000 ng/ml). Chi-Square analysis indicated that there was an inverse correlation between CD34 stem cell counts and peak ATP levels (p=0.01). However, there was no direct relationship between the number of CD3+ or CD3+CD4+ T cells and Th reactivity. Immunophenotypic studies performed in 4 patients with CD34 counts ≥10 cells/ul and 2 patients with CD34 counts <10 cells/ul indicated that the agents used for stem cell mobilization triggered the expansion of CD4+CD25+ T cell population. However, the vast majority of these T cells did not co-express FoxP3, the characteristic marker of regulatory T cells, consistent with the enhanced T cell reactivity detected in these patients. It, therefore, results that mobilization agents have a “priming” effect on CD4+ T helper cells, inducing their robust activation. This effect may lead to improved anti-tumor surveillance and might contribute to the higher rate of complete remission and overall survival observed in patients with hematological malignancies, who have received autologous SCT in addition to chemotherapy. Thus, monitoring of T cell reactivity by Immuknow assay might help tailor therapy to maximize the patient’s potential to mount an immune response against the tumor and, at the same time, obtain efficient stem cell mobilization.
Defective Expression of Neutrophil Serine Proteases in Myeloid Progenitors of Congenital Neutropenia Patients Carrying Either ELA2 or HAX1 Mutations.
Authors: Skokowa Julia , Fobiwe John-Paul , Lan Dan , Germeshausen Manuela , Welte Karl
Published: October 2019

Severe congenital neutropenia (CN) is a heterogeneous disorder of myelopoiesis with two major types of inheritance:

autosomal dominant CN defined by mutations in ELA2 gene encoding neutrophil elastase (NE) (Horwitz M., et al., Nat Genet.1999;23:433) and autosomal recessive CN (including Kostmann syndrome) carrying HAX-1 mutations (Klein C., et al., Nat Genet.2007;39:86), both characterized by a maturation arrest of granulopoiesis at the level of promyelocytes.

In the present study we aimed to evaluate the expression profile of genes specifically expressed in the CD33+ bone marrow promyelocytes of both patient groups harbouring ELA2 or HAX1 mutations. In healthy individuals mRNA expression levels of neutrophil serine proteases (neutrophil elastase (ELA2), cathepsin G, cathepsin D, proteinase 3 and azurocidin) as well as of myeloperoxidase (MPO) and defensins reached highest levels in the azurophil granules at the promyelocytic stage of neutrophil differentiation (Borregaard N., et al., Curr Opin Hematol.2001;8:23). We found downregulation of mRNA expression levels of ELA2 (8.9 fold), cathepsin G (7.6 fold), cathepsin D (11.2 fold), proteinase 3 (9.2 fold) and defensin B1 (6.5 fold) in both groups of CN patients (with ELA2 or HAX1 mutations), in comparison to G-CSF-treated patients with idiopathic neutropenia (IN) and G-CSF-treated healthy controls. In contrast, there were no difference in mRNA expression levels of azurocidin and only slight decrease in the expression of MPO mRNA in CN patients. Additionally, we found significantly reduced protein levels of neutrophil elastase (NE) in plasma of CN patients irrespective of “ELA2 or HAX1” inheritance, in comparison to cyclic neutropenia (CyN) patients, IN patients and G-CSF-treated healthy controls. Taken together, both ELA2 and HAX1 mutations are associated with defective expression of neutrophil serine proteases such as NE, cathepsin G, cathepsin D, proteinase 3 as well as of defensin B1 in CD33+ myeloid progenitor cells of CN patients, suggesting a common pathway for both patient groups. Intriguingly, ELA2 expression is directly regulated by LEF-1, suggesting that abrogated LEF-1 expression in CN promyelocytes (Skokowa J., et al., Nat. Med.2006;12:1191) may be responsible for defective serine proteases expression in both groups, since all are regulated by a similar mechanism.