bluuebunny/crossref_metadata_2025 huggingface dataset:
A Proteomic Approach to Discovery of Candidate Proteins Involved in the Transformation of Follicular Lymphoma.
Authors: Fan Guang , Zhong Yanping , Smith Cristina , Huang James , Braziel Rita
Published: April 2020

Background: Follicular lymphoma (FL) undergoes transformation to a high grade diffuse large B-cell lymphoma (tr-DLBCL) in about 50% of patients. During transformation, a more virulent subclone of tumor cells emerges, leading to a rapidly progressive clinical course and resistance to therapy. The identification of proteins involved in transformation is critical for understanding the mechanism of transformation and developing molecularly targeted therapy. In this study, we compared protein expression between grade 1- FL (G1-FL) and tr-DLBCL using 2D-gel electrophoresis and Western blot analysis.

Design: Frozen tissue and frozen cells were obtained from the Department of Pathology, Oregon Health and Science University tumor bank. The protein expression profiles of 3 G1-FL and 3 tr-DLBCL were compared using 2D-gel electrophoresis. Protein identification was done using a MALDI mass spectrometer. Frozen cells of an additional 11 non-paired GI-FL and 11 non-paired tr-DLBCL, and 2 pairs of G1-FL and tr-DLBCL specimens were used for Western blot confirmation of the initial 2D-gel findings.

Results: 2D-gel analysis and MALDI protein identification revealed 14 differentially expressed proteins between G1-FL and tr-DLBCL (figure 1), all of which are known to play important roles in cellular energy/metabolic pathways, signal transduction pathways, and protein and nuclear synthesis. The two most differentially expressed proteins on 2D-gel analysis were superoxide dismutase (MnSOD2) and growth factor receptor bound protein 2 (Grb2). Western blot analysis of MnSOD2 and Grb2 confirmed their relative over- or under-expression in frozen cells from multiple additional clinical lymphoma samples, including 2 paired- and 22 non-paired G1-FL and tr-DLBCL. Both 2D-gel analysis and Western Blot showed a significantly higher level of expression of MnSOD2 and a lower expression of Grb2 expression in tr-DLBCL (figure 2).

Summary: Using proteomic profiling, confirmed by Western blot analysis of clinical G1-FL and tr-DLBCL samples, we have confirmed 2 proteins (MnSOD2 and Grb2) that are expressed at significantly different levels in G1-FL and DLBCL. MnSOD2 is capable of protecting cells from reactive oxygen species and regulating signal transduction pathways to influence cell growth and apoptosis. Inhibition of MnSOD2 has been shown in studies of several cancer cell lines to render cancer cells more susceptible to apoptosis. Grb2 is a member of a critical signaling pathway leading to Ras activation in hematopoietic cells. Both proteins may play a critical role in FL transformation. These proteins have the potential to be therapeutic drug targets, diagnostic and/or prognostic markers, or biomarkers for monitoring therapeutic response.

Summary of Differentially Expressed Spots Summary of Differentially Expressed Spots Figure Figure
SELDI-TOF Identifies Specific Protein Patterns in Small Lymphocytic Lymphoma, Marginal Zone Lymphoma and Mantle Cell Lymphoma.
Authors: Rolland Delphine , Bouamrami Ali , Ballester Benoit , Grangeaud Samuel , Arlotto Marie , Paret Aurelie , Berger Francoise , Felman Pascale , Callet-Bauchu Evelyne , Coiffier Bertrand , Houlgatte Remi , Berger Francois , Thieblemont Catherine
Published: April 2020

Non-germinal centre small B-cell lymphomas represent a heterogeneous group of non-hodgkin lymphomas which most frequent histologic subtypes are small lymphocytic lymphoma (SLL), marginal zone lymphoma (MZL) and mantle cell lymphoma (MCL). These three lymphoma entities have very different clinical outcomes but may be difficult to distinguish either histologically or clinically. We previously identified transcriptomic signatures specific of these 3 lymphoma subtypes. We further analyzed these lymphomas using Surface-Enhanced Laser Desorption/Ionisation Time of Flight (SELDI-TOF). A total of 58 tumors, including 20 SLL (all lymph nodes), 20 MZL (1 lymph node and 19 spleens) and 18 MCL (19 lymph nodes and 1 spleen) were analyzed. In addition, we included 7 controls obtained from traumatic normal spleens. The spectra were generated on weak cation exchange (CM10), strong anion exchange (Q10) and reversed-phase (H50) ProteinChip arrays. Protein patterns of all samples were comparatively analysed using two distinct strategies. We first used a binary recursive partitioning method with the Biomarker Pattern software (Ciphergen®), and second a hierarchical clustering method to visualized patterns of protein peaks completed with a supervised method (discriminating score) to point out individual peaks distinguishing the three histological subtypes (SLL, MZL and MCL). Spectra analyses revealed a very homogeneous protein patterns among all lymphoma samples. However specific SLL, MZL and MCL signatures based on 34 protein peaks with differential expression could be identified and allowed to classify 95% of the samples in their respective entity. SLL signature included 9 peaks, MZL signature 16 peaks and MCL signature 9 peaks. The binary recursive partitioning analysis was concordant but identified only the five most discriminant peaks. Further identification of the discriminating peaks is currently realized using SELDI-assisted purification. We are focusing on peaks at 9942 Da for SLL and at 11324 Da for MCL. Functional genomic studies can distinguish non-germinal small B-cell lymphomas at the transcriptomic level (our previous study) and at the proteomic level. This will provide new markers for diagnosis and potentially new therapeutic targets.
MLL Translocation Partners AF4, AF5q31, and ENL Associate in a Higher Order Protein Complex with CDK9 and Cyclin T1.
Authors: Lin Min , Cleary Michael L.
Published: April 2020

The Mixed Lineage Leukemia (MLL) gene is frequently involved in chromosomal translocations that cause acute leukemia. More than 40 different genes have been identified as MLL translocation partners, with the expression of corresponding MLL fusion proteins. The MLL protein has histone methyltransferase activity and is required for embryonic development and hematopoiesis. Several proteins have been demonstrated to associate with MLL in a macromolecular complex, which is believed to have chromatin remodeling function. However, the C-terminal SET domain of MLL, which carries the histone methyltransferase activity, is lost in all MLL fusion proteins, thus making the biochemical functions of the fusion proteins unclear. Moreover, the promiscuity of MLL translocation partners, most of them with no known functions, further complicates an understanding of MLL leukemogenic mechanisms. In this study, we purified a protein complex containing AF4, the most common MLL translocation partner, using a combination of conventional column chromatography and immunoaffinity techniques. The AF4 protein complex contains AF5q31 and ENL, two other MLL translocation partners, as well as CDK9 and Cyclin T1, a heterodimer that regulates transcriptional elongation. Gel filtration confirmed that these five proteins co-fractionate with an estimated overall size of 0.8 MDa. All protein-protein interactions were further confirmed by immunoprecipitation-western blotting from K562 cell nuclear extract. To investigate whether these protein-protein interactions are retained in corresponding MLL fusion proteins, immunoprecipitation-western blotting assays were carried out in human leukemia cell lines harboring MLL chromosomal translocations. We found that MLL-AF4, MLL-AF5q31, MLL-ENL and MLL-AF9 each associate with wild type AF4 complex components, including CDK9 and Cyclin T1. In contrast, MLL-AF6 does not associate with any of the AF4 complex components. We propose that the four nuclear MLL translocation partner proteins (AF4, AF5q31, ENL/AF9), whose translocations are found in over 75% of MLL leukemias, associate in a higher order protein complex with CDK9 and Cyclin T1 and thus function in part to regulate transcriptional elongation. The association of CDK9 and Cyclin T1 with the four MLL fusion proteins suggests a common leukemogenic mechanism that may involve transcriptional elongation, which we are currently investigating. Conversely, MLL-cytosolic fusions, e.g. MLL-AF6, appear to function independently of association with the AF4 protein complex, possibly through a homo-dimerization pathway.
Cdx4 Upregulates Hox Gene Expression and Generates Acute Myeloid Leukemia Alone and in Cooperation with Meis1a in a Murine Model.
Authors: Bansal Dimple , Scholl Claudia , Frohling Stefan , Mcdowell Elizabeth , Lee Benjamin H. , Dohner Konstanze , Ernst Patricia , Davidson Alan J. , Daley George Q. , Zon Leonard I. , Gilliland D. Gary , Huntly Brian J.
Published: April 2020

HOX genes have emerged as critical effectors of leukemogenesis, but the mechanisms that regulate their expression in leukemia are not well understood. Recent data suggest that the caudal homeobox transcription factors CDX1, 2 and 4, developmental regulators of HOX gene expression, may contribute to HOX gene dysregulation in leukemia. We report here that CDX4 is normally expressed in early hematopoietic progenitors, and is aberrantly expressed in~25% of AML patient samples. Cdx4 regulates Hox gene expression in the adult murine hematopoietic system, and upregulates Hox genes that are implicated in leukemogenesis. Furthermore, bone marrow progenitors that are retrovirally engineered to express Cdx4 serially replate in methylcellulose cultures, grow in liquid culture and generate a partially penetrant, long-latency acute myeloid leukemia (AML) in bone marrow transplant recipients. Co-expression of the Hox co-factor Meis1a accelerates the Cdx4 AML phenotype and renders it fully penetrant. Structure -function analysis demonstrates that leukemic transformation requires intact Cdx4 transactivation and DNA-binding domains, but not the Pbx co-factor interaction motif. Taken together, these data indicate that Cdx4 regulates Hox gene expression in adult hematopoiesis and may serve as an upstream regulator of Hox gene expression in the induction of acute leukemia. Inasmuch as many human leukemias show dysregulated expression of a spectrum of HOX family members, these collective findings also suggest a central role for CDX4 expression in the genesis of acute leukemia.
Modification of rVWF with Polysialic Acid: Biochemical and Functional Characterization in Mice with VWD.
Authors: Turecek Peter L. , Siekmann Juergen , Weber Alfred , Gritsch Herbert , Váradi Katalin , Matthiessen H. Peter , Muchitsch Eva-Maria , Baumgartner Bernhard , Schwarz Hans Peter
Published: April 2020

Post-translational glycosylation determines the pharmacodynamic and pharmacokinetic properties of therapeutic proteins. While desialylation of FVIII/VWF (asialo-FVIII/VWF) has a significantly reduced half-life compared to normal FVIII/VWF complex (Sodetz et al 1977), we tried to introduce additional sialic acids on rVWF to obtain the opposite: prolonged survival of rVWF in the circulation. Additional sialic acid residues were introduced to rVWF by chemically attaching polysialic acid (PSA, colominic acid) to lysines on recombinant VWF. rVWF was purified from the cell culture supernatant of CHO cells co-expressing rFVIII and rVWF. Coupling of PSA was performed by terminal oxidation of the vicinal diols in PSA to aldehydes and binding to the primary amino groups in the VWF molecule by Schiff-base formation. Schiff-bases were stabilized by reduction with NaCNBH3 under formation of a secondary amine.

PSA-rVWF conjugates retained their multimeric structure but had slightly increased molecular sizes of the VWF sub-units due to bound PSA. The attachment of PSA to rVWF resulted in a decrease of RCo activity and a decrease in FVIII binding capacity to approximately 60%.

Administration of PSA-rVWF at a dose of 100 units rVWF antigen per kg to VWF-deficient mice resulted in a 2-fold increase in the survival of rVWF as compared to non-modified rVWF. A secondary rise in endogenous murine FVIII after substitution of VWD mice with human VWF occurred elevating FVIII levels from 0.1 (baseline) to 0.4 IU FVIII/mL at 3 hours after application of both non-modified and polysialylated rVWF. Maximum levels of ~ 0.4 IU FVIII/mL were maintained for more than 20 hours in the group treated with PSA-rVWF despite the reduced FVIII binding capacity observed in vitro, while FVIII levels in mice treated with non-modified rVWF steadily declined and returned to their baseline level after 21 hours. The FVIII AUC was 5.3 and 3.3 IU/mL/hr for mice treated with PSA-rVWF and non-modified rVWF, respectively.

We conclude that polysialylation of rVWF represents a method to prolong the survival of rVWF in the circulation.
A Limited Number of Anti-Idiotypic Antibodies Is Required To Maintain FVIII Function in Presence of Polyclonal Anti-FVIII Antibodies of Haemophilia A Patients with Inhibitors.
Authors: Gilles Jean Guy G. , Grailly Sabrina E. , Jacquemin Marc G. , Saint-Remy Jean Marie M.
Published: April 2020

Replacement therapy using either plasma-derived or recombinant FVIII elicits FVIII-specific Abs in about 25% of the patients. The majority of such antibodies are directed towards specific FVIII regions on which major epitopes have been identified. Most of these epitopes are located in the carboxy-terminal end of the C2 domain of the FVIII light chain and in the amino-terminal end of the A2 domain of the heavy chain, whereas, antibodies directed towards the C1 domain have been described in cases of mild/moderate haemophilia A. From a functional point of view, epitopes have been mapped to the phospholipid and von Willebrand binding sites in the C1 and C2 domains and to the FIX binding site in A2.

We have derived human mabs (mabRhd5, mabBo2C11 and mabBoIIB2) that react with high affinity to the FVIII C1, C2 and A2 domains, respectively, and are representative of most of the specific inhibitors observed in haemophilia A patients.

Recently, we have generated mouse anti-Id mab (mab14C12) against mabBo2C11 and provided the first demonstration that an anti-Id mab directed towards a C2-specific inhibitor restored FVIII activity both in vitro and in vivo in the presence of the inhibitor. Futher more, we demonstrated that FVIII inhibition obtained by a mixture of two anti-FVIII mabs (mabBo2C11 (anti-C2) and mabBoIIB2 (anti-A2)) was neutralized up to 100% when a mixture of the corresponding anti-Ids (mab14C12 and mab30D1) was added to the assay establishing the proof of concept that combination of anti-Id mabs will be able to neutralize the anti-FVIII immune response of haemophilia A patients. Based on this observation, We have also generated an anti-idiotypic antibody (mab18B6) towards an anti-C1 domain inhibitor (mabRHD5). In ELISA, mab18B6 specifically prevents, in a dose-dependent manner, mabRHD5 binding to C1 and fully neutralized mabRHD5 inhibitory properties in a coagulation assay. With a mixture of anti-Ids (mab14C12, mab30D1 and mab18B6) we were able to completely neutralized the cumulative anti-FVIII activity obtained in functional assay by a mixture of the corresponding monoclonal anti-FVIII antibodies. We next evaluated whether a combination of anti-Ids (anti-anti-A2,-C1,-C2) had the ability to neutralize the inhibitory properties of polyclonal antibodies obtained from haemophilia A patients. To this end, eight patients with severe haemophilia A and inhibitor were tested. An average of 70% neutralization of the inhibition was obtained in 6 out of 8 cases. These data allow to conclude that potent polyclonal high-affinity FVIII inhibitors can be neutralized with corresponding anti-idiotypic antibodies and that only a limited number of anti-idiotypic antibodies is required for inhibitor neutralization in a majority of patients.
Factor XI Deficiency Confers a Survival Advantage in a Murine Sepsis Model.
Authors: Gailani David , Tucker Erik I. , Cheng Qiufang , Hanson Stephen R. , Gruber Andras
Published: April 2020

Bacterial sepsis is frequently accompanied by intravascular activation of coagulation and fibrinolysis, resulting in a coagulopathy with thrombotic, hemorrhagic, and inflammatory components (disseminated intravascular coagulation - DIC) that contributes significantly to morbidity and mortality. Recently, using vascular occlusion models, the plasma coagulation protease factor XI (FXI) has been shown to contribute to pathologic thrombus formation in primates, mice, and rabbits. These results are supported by epidemiologic studies in human populations that show an association between elevated plasma FXI levels and an increased risk for venous and arterial thrombosis. FXI has also been shown to contribute to the lethal consumptive coagulopathy in protein C deficient mice. To investigate the contribution of FXI to consumptive coagulopathy and mortality in sepsis, we compared wild type (WT) and FXI deficient (FXI−/−) mice in models of polymicrobial sepsis and endotoxemia. Polymicrobial sepsis was induced in WT and FXI−/− C57Bl/6 mice (n = 24 for each genotype) using an established model of cecal ligation and puncture (CLP). Overall survival was 21% for WT and 42% for FXI−/− mice (p < 0.01), with WT deaths occurring between days 2 and 4, and FXI−/− deaths between days 3 and 6. Average survival for mice succumbing to sepsis was 55 ± 10 hr for WT, and 88 ± 22 hr in FXI−/− mice (p < 0.01). 24 hrs after CLP, platelet count in WT and FXI−/− animals decreased 27 ± 13% and 13 ± 12% (p = 0.05), and leukocyte counts decreased by 64 ± 13% and 40 ± 23% (p < 0.01), respectively. Fibrinogen levels 24 hours post-CLP decreased slightly in WT mice (37 ± 34% from baseline), while increasing 212 % ± 74% in FXI−/− mice (p < 0.01). Mice were also challenged with intraperitoneal Escherichia coli endotoxin. Mortality 24 hrs after endotoxin administration was identical for WT and FXI−/− mice (n =15 for each dose) at doses of 50 mg/kg (mortality 80%) and 25 mg/kg (mortality 70%). Twelve hours after administering 25 mg/kg endotoxin, the mean platelet counts in WT animals decreased by an average of 60% (from 440 ± 159/mm3 at baseline to 175 ± 77/mm3, n = 10), and by 36% in FXI−/− animals (from 474 ± 218/mm3 to 303 ± 100/mm3). In summary, prognosis was better for FXI−/− mice in CLP-induced sepsis compared to WT mice, and the CLP and endotoxin data indicate that consumptive coagulopathy is less severe in the absence of FXI. The results suggest that therapeutic inhibition of FXIa activity could be beneficial in treating sepsis-related DIC. Humans with severe congenital FXI deficiency typically have, at most, a mild trauma-induced bleeding disorder. It is possible, therefore, that inhibition of FXIa in DIC may be associated with a lower risk of exacerbating bleeding when compared to anticoagulant therapies such as heparin or activated protein C.
Establishment of ES Cells Secreting Human Factor VIII for Hemophilia A-Targeted Cell Therapy.
Authors: Kasuda Shogo , Sakurai Yoshihiko , Kubo Atsushi , Shima Midori , Keller Gordon , Yoshioka Akira
Published: April 2020

Hemophilia A is an X-linked bleeding disorder resulting from abnormality in the coagulation factor VIII (FVIII) gene. Although bleeding episodes can be prevented or treated by intravenous injection of FVIII concentrates, there are some drawbacks such as necessity of frequent intravenous transfusion and high cost of FVIII concentrates. Lots of basic studies for gene therapy have been reported but clinical trials for hemophilia have been interrupted by significant safety concerns. Cell-based therapy may overcome the disadvantages, without impairing the advantages of gene therapy. Therefore, we have tried to use embryonic stem (ES) cells for production of FVIII protein. For the stable expression of FVIII, we established tet-hFVIII ES cells, which induce human (h) FVIII mRNA (F8) by doxycycline (Dox) exposure. Tet-hFVIII ES cells were cultured in the serum containing and/or serum free media and then FVIII activity (FVIII:C) and FVIII antigen (FVIII:Ag) were measured in the supernatant by APTT-based FVIII:C assay and ELISA, respectively. In the previous study, we demonstrated that ES cells could be differentiated with serum into cells with mesoderm potentials such as hematopoietic and vascular endothelial cells. Although mRNA of hFVIII was detected by Dox induction, neither FVIII:C nor FVIII:Ag was detected in the conditioned media in undifferentiated ES cells and differentiated cells with mesoderm potentials. Next we cultured tet-hFVIII ES cells using endoderm and hepatocyte differentiation condition in which differentiated ES cells express Foxa2 and albumin genes (Kubo et al. Development 2003), since FVIII is known to be produced in the liver. Both FVIII:C (3.7 mIU/ml/mg protein) and FVIII:Ag (0.3 ng/ml/mg protein) were detected in the conditioned media for hepatocyte differentiation. These data demonstrated that ES cells could produce and secrete the functional hFVIII protein in our system, when ES cells cultured in the hepatocyte condition. The level of FVIII:C and FVIII:Ag were increased twice by addition of von Willebrand factor (2.5 μg/ml) into the culture media. Furthermore, Dox enhanced the level of FVIII:C and FVIII:Ag dose-dependently. In conclusion, we confirmed secretion of hFVIII in endodermal differentiation culture system. This system may be applicable for cell-based therapy targeting to hemophilia A.
Novel Large Deletion Is the Most Frequent Cause of Type 3 von Willebrand Disease in Hungary.
Authors: Mohl Adrien , Masszi Tamás , Nagy Eszter , Obser Tobias , Oyen Florian , Sallai Krisztina , Bodo Imre , Schneppenheim Reinhard
Published: April 2020

Background: Type 3 is the most severe form of von Willebrand disease (VWD) caused by the virtual absence of von Willebrand factor (VWF) in affected patients. The prevalence of type 3 VWD in Hungary is 2.6 per million. Capitalizing on a nationwide National Bleeding Disorder Registry, we designed a study to characterize the genetic background of the entire Hungarian type 3 VWD population. The current report focuses on the molecular characterization of a novel large deletion.

Methods: 24 patients from 23 unrelated families were studied by direct sequencing of the 52 exons of the VWF gene. The breakpoints of a large deletion were characterized by standard gene mapping. Breakpoint-specific PCR was used to confirm the presence of the deletion, and to screen for identical deletions in other populations from Germany, Russia, and Poland.

Results: A large partial deletion (delExon1-3) of the 5′-region of the VWF gene was detected in 10 alleles (19 percent of all type 3 mutations). Five patients from 4 unrelated families were homozygous, and 2 patients were heterozygous for the deletion. Consanguinity was known in one of the families. In comparison, 2435 delC in exon 18, a common cause of type 3 VWD in some European populations, was found on 6 alleles (12 percent; one patient homozygous). The large deletion resulted in the loss of a 35 kb fragment, incorporating exons 1, 2 and 3. The 5′ breakpoint is located in the 5′ untranslated region, while the 3′ breakpoint is in intron 3 of VWF. No other known gene is lost with the deletion. Clinically, all homozygous patients had serious bleeding episodes from infancy requiring frequent VWF substitutions. However, bleeding became much milder in all patients with no significant spontaneous bleeding after the age of 3-5 years. No inhibitor to VWF was detected. delExon1-3 was not detected in any of the other screened populations.

Conclusion: We report a large novel deletion including exons 1, 2 and 3 of VWF commonly causing type 3 VWD in the Hungarian population. This mutation, which is most probably due to a founder effect, seems to be unique to Hungarian patients with a high allele frequency. Together, delExon1-3 and 2435delC make up 31 % of genetic defects in Hungarian patients with VWD type 3. This offers a rational approach to molecular testing of respective families in Hungary.
Galectin-3 Binding Protein Contaminates the Purification of Recombinant Factor IX.
Authors: Blostein Mark D. , Cuerquis Jessica , Galipeau Jacques
Published: April 2020

The hemophilas are the most common severe hereditary bleeding disorders. The standard treatment of these disorders is to infuse plasma concentrates of the missing coagulation factor. However, the high cost of replacement therapy and the serious morbidity from the transmission of infectious agents have mandated a need for alternative therapeutic strategies. One of these strategies is gene therapy and we embarked on a novel gene therapeutic strategy involving the use of factor IX-transduced bone marrow stroma to treat hemopihlia B, a deficiency of the gamma-carboxylated protein, factor IX. The rate limiting step for the production of factor IX is gamma-carboxylation, a post translational modification carried out only in mammalian cells. In order to test the carboxylation efficiency of our gene therapeutic platform, we first transfected Human Embryonic Kidney (HEK) 293 cells with a retroviral construct containing factor IX. Factor IX was purified by immunoaffinity chromatography and then subject to hydroxyapatite chromatography to isolate biologically active properly carboxylated factor IX from inactive uncarboxylated factor IX. Unexpectedly, during hydroxyapatite chromatography, we discovered that purified factor IX was contaminated by a heretofore unknown protein. Further analysis by mass spectrometry sequencing revealed this protein to be galectin-3 binding protein (G3BP).G3BP contaminates media of HEK293 cells regardless of whether factor IX is transfected into these cells. Furthermore, the contamination of purified media is not specific for factor IX as G3BP is isolated regardless of the antibody used for purification. In summary, the above results provide important information regarding the production of recombinant factor IX and uncover a potentially new contaminant in recombinant products produced in mammalian cells.
The Genetics of Hemophilia: Analysis of Patients at the Hospital for Sick Children, Toronto, Canada.
Authors: Knox David , Samuel Christopher , Pazmino-Canizares Janneth , Barnes Chris , Floros Georgina , Stain Ann Marie , Carcao Manuel
Published: April 2020

There is great interest in identifying genetic mutations responsible for hemophilia and in determining if/how mutations correlate with disease phenotype. A hemophilia genetic database was created at SickKids in 2004. At the time <40% of hemophiliacs followed by the clinic had been genotyped. Currently mutations have been identified on 194/236 patients (82%) followed in the clinic. From this we are performing genotype/phenotype correlations. Preliminary analysis has revealed the following novel findings:

Most mothers of hemophiliacs are carriers; even when there is a no family history of hemophilia. Of the 199 mothers of the 236 children only 6 have been shown not to be carriers; 205 have been shown to be carriers; 15 have not been tested and 10 are unavailable for testing. We believe that most de novo FIX and FVIII mutations occur in females. Mutations responsible for hemophilia B show poor concordance with disease severity i.e. for any mutation the disease severity is not always the same. One example found in 10 hemophiliacs (5 families) who despite having the same missense mutation in exon H have shown FIX levels anywhere between 0 and 11%. Given that disease severity is assigned according to factor levels these patients have been labeled as either severe (n = 3), moderate (n = 5) or mild (n = 2). Clinically these patients all appear to behave as moderate. This points to the fallibility of using FIX levels (which varies according to patient age and health state) in labeling patients. Other aspects of the hemophilia genetic data base are being analyzed. We believe that a detailed study of the genetics of hemophilia will point to novel findings that will eventually translate into patient care.
Defibrotide Does Not Cross-React with HIT Antibodies. Implications in the Management of HIT.
Authors: Fareed Jawed , Hoppensteadt Debra A. , Iacobelli Massimo , Walenga Jeanine M.
Published: April 2020

Defibrotide is a mammalian DNA derived antithrombotic and anti-ischemic agent that does not produce systemic anticoagulation. Heparin-induced thrombocytopenia (HIT) is an immune disease related to heparin exposure, in which patients are at risk of developing life- and limb-threatening thrombosis. Studies were performed to determine whether defibrotide cross-reacts with HIT antibodies. Sera from 141 clinically confirmed HIT patients were tested for platelet activation in the presence of defibrotide (1 – 100 μg/ml) and unfractionated heparin (1 – 100 μg/ml). 103 sera (73%) produced platelet aggregation and serotonin release activities with heparin. Only 4 samples (2%) showed a weak reactivity with defibrotide which was eliminated by heparinase treatment indicating heparin contamination in the patient sample. Further studies revealed that defibrotide does not complex with platelet factor 4 (PF4), and that prolonged incubation of defibrotide with platelet rich plasma does not mobilize PF4. Studies of patients treated with extended dosing of intravenous or oral defibrotide (n=270) demonstrated that HIT antibodies are not generated with defibrotide treatment. Studies carried out on the effect of purified IgG from HIT patients revealed that defibrotide blunts platelet activation and microparticle formation as measured by flow cytometry. These studies suggest that defibrotide may be a useful antithrombotic agent for the management of patients with HIT. Moreover, unlike heparin, defibrotide does not promote platelet activation, rather it is capable of suppressing the hypercoagulable state associated with HIT. Defibrotide is orally bioavailable and can be used for extended anticoagulant management of HIT patients.
Pancreatitis Preceding Acute Episodes of Thrombotic Thrombocytopenic Purpura: Report of Five Patients with a Systematic Review of Published Reports.
Authors: Swisher Karen K. , Doan John T. , Vesely Sara K. , Kwaan Hau C. , Kim Benjamin , Lammle Bernhard , Hovinga Johanna Kremer , George James N.
Published: April 2020

Thrombotic thrombocytopenic purpura (TTP) is a syndrome with multiple recognized etiologies, associated conditions, and risk factors but the events that may precipitate acute episodes are unclear. Patients may have undetectable ADAMTS13 activity without evidence for TTP until a precipitating condition, such as pregnancy, infection, or surgery, occurs. These clinical observations are similar to experimental observations on transgenic mice with absent ADAMTS13 in which a TTP-like condition only occurred when an additional stimulus, Shiga toxin or collagen/thrombin, was administered. Also patients may have acute episodes of typical TTP without apparent ADAMTS13 deficiency. We report 5 patients (Table) who had the onset of an acute episode of TTP 1–13 days following the onset of pancreatitis. Three patients are from The Oklahoma TTP-HUS Registry, a series of 360 consecutive patients with TTP, 1/1/1989 – 6/30/2006. Two patients are from Northwestern University. In each patient there was an apparent etiology for the pancreatitis: bile duct obstruction in patients 1, 4 and 5 and alcoholism in patients 2 and 3. There was no evidence for TTP when the pancreatitis was diagnosed. In patients 1 and 2, the pancreatitis was clearly resolving when the TTP occurred. ADAMTS13 was measured in patients 2–5 and was severely deficient with a high-titer inhibitor in patients 3 and 5. Patient 5 had a recurrent episode of pancreatitis that was also followed by an acute episode of TTP.

No. Laboratory data on days of diagnosis of pancreatitis and of TTP Hgb (g/dL) Plt (103/μL) Cr (mg/dL) LDH (U/L) Amylase (U/L) Days1 (Pan-TTP) (Pan-TTP) (Pan-TTP) (Pan-TTP) (Pan-TTP) 1 Days between onset of acute pancreatitis and diagnosis of TTP 1 6 12.8–8.0 346–40 0.9–2.1 272–1526 1077–154 2 5 14.3–9.0 291–24 0.7–7.7 345–1668 1704–140 3 3 15.4–10.1 301–53 1.2–2.0 --1093 140— 4 1 12.5–9.6 359–74 1.5–4.1 --1195 --- 5a 13 10.3–8.0 588–34 0.8–2.1 --997 627–433 5b 7 13.8–10.0 340–45 1.3–1.1 --541 1584–40

A systematic review of published case reports identified 16 additional patients who had TTP following pancreatitis. Seven patients were alcoholic; 5 patients had gallbladder disease. Coagulation tests were reported in 12 patients and all were normal. Recurrent TTP following a recurrence of pancreatitis was reported in 3 of the 16 patients. Our 5 patients and the 16 previously reported patients all recovered from TTP.

CONCLUSION: Pancreatitis, a disorder that results in an intense systemic inflammatory response, may provoke acute episodes of TTP. Inflammatory cytokines may precipitate acute episodes of TTP by stimulating endothelial cell release of ultralarge von Willebrand factor multimers and inhibiting the cleavage of these multimers by ADAMTS13.
Disseminated Malignancy Misdiagnosed as Thrombotic Thrombocytopenic Purpura: A Report of 10 Patients and a Systematic Review of Published Cases.
Authors: Francis Kristin K. , Kalyanam Nalini , Terrell Deirdra R. , Vesely Sara K. , George James N.
Published: April 2020

Patients with disseminated malignancy who present with microangiopathic hemolytic anemia and thrombocytopenia may be misdiagnosed as thrombotic thrombocytopenic purpura (TTP), resulting in inappropriate plasma exchange treatment, a procedure with major risk, and delay of appropriate chemotherapy. We report the 17 year experience of The Oklahoma TTP-HUS Registry, 1989–2005, and a systematic review of previously published case reports. Ten of 335 consecutive patients in the Oklahoma Registry who were initially diagnosed with their first episode TTP and treated with plasma exchange were subsequently discovered to have disseminated malignancy; only one had a history of cancer. These 10 patients were compared to the 133 concurrent patients with idiopathic TTP.

Disseminated malignancy (n=10) Idiopathic TTP (n=133) P Data are median values. P-value for neurologic symptoms compares distribution of abnormalities. Number of patients who had ADAMTS13 measured is in parentheses. 8 idiopathic TTP patients never had plasma exchange (PE). Age (years) 56 47 0.106 Sex (% female) 30% 73% 0.008 Duration of sx (days) 21 8 0.005 Presenting sx Weakness 70% 53% 0.347 cough, dyspnea 70% 26% 0.007 Fever 50% 31% 0.292 abdominal pain 40% 35% 0.745 Nausea/vomiting 20% 53% 0.052 Neurologic abnormalities Severe 20% 47% 0.128 Minor 60% 29% None 20% 24% Laboratory data hematocrit (%) 21 22 0.868 platelet count (103/ml) 21,000 13,000 0.328 creatinine (mg/dL) 2.4 2.0 0.761 LDH (U/L) 2894 1260 0.045 ADAMTS13 Median activity (%) 50(8) 23 (81) 0.167 <5% activity (% of pts) 0 30% 0.102 Response to PE 10% 82% (125) <0.001 Death (within 30 days) 90% 20% <0.001

Patients with disseminated malignancy had a longer duration of symptoms, were more often men, had more frequent presence of respiratory symptoms, higher LDH levels, more often failed to respond to plasma exchange treatment, and had a higher mortality. Neurologic abnormalities, hematocrit, platelet count, and serum creatinine were not different between the two groups. Diagnosis of malignancy was made by bone marrow biopsy in 6 patients but not until autopsy in 2 patients. A systematic review identified 19 additional patients, reported from 1965 to 2005, in whom TTP or HUS was initially suspected and systemic malignancy subsequently discovered. Only 5 patients had a history of cancer. Malignancy was not diagnosed until autopsy in 6 patients. Fourteen different malignant disorders were diagnosed in these 29 patients.

CONCLUSIONS: Disseminated malignancy may be occult and may mimic TTP. A search for systemic malignancy, including a bone marrow biopsy, is appropriate when patients diagnosed with TTP have atypical clinical features or fail to respond to plasma exchange.
Cross Reaction of Antibody Against Helicobacter Pylori Urease B with Platelet Glycoprotein IIIa and Its Significance in the Pathogenesis of ITP.
Authors: Wang Zhaoyue , Bai Yanyan , Bai Xia , Zhang Wei , Ruan Changgeng
Published: April 2020

The data about association of Idiopathic Thrombocytopenic Purpura (ITP) with Helicobacter pylori (H pylori) infection are always contradictory, and the factors that incite ITP remain uncertain. Several hypotheses have been suggested about the striking discrepancies between the reported results, one of which is variable effects of genetically diverse H pylori strains in remote geographic regions. The sequence of gene for H pylori urease B is relatively constant, and its protein production is believed to be a crucial factor resulting in some diseases. We therefore conducted a study to assess the role of H pylori in the pathogenesis of ITP, and to investigate its immune mechanisms. Firstly, bacteria H pylori were isolated from gastric mucosal tissue of a patient with peptic ulcer and cultured. H pylori urease B gene was amplified, then ligated into expression plasmid. The latter was transfected into E coli. The recombinant urease B was used to develop monoclonal antibody (MoAb), 1F11, against urease B of H pylori. In Western blot analysis, both 1F11 and SZ21 (MoAb against GP IIIa) bound to the band of protein 95 KDa of normal platelet lysate, but not to that of platelet lysate prepared from a patient with Glanzmann thrombobasthenia (GT), in whom platelet membrane GP IIb/IIIa were absent. Flow cytometry analysis also showed that normal platelets were reacted with both 1F11 and SZ21, whereas platelets of the GT patient showed a low reactivity with 1F11 or SZ21. In immunoradiometric assay, in which four MoAbs against different platelet membrane GPs (GP Ib, GP IIb, GP IIIa and GP IV) were each coated on microtiter plates, and platelet lysate and then 125I-labeled 1F11 were separately added, the radioactivity value (count per minute) in plates coated with SZ21 was the highest among all coated plates. All the three different experimental results showed that there exists a cross reaction between MoAb 1F11 against Helicobacter pylori urease B and platelet glycoprotein IIIa. In addition, MoAb 1F11 could inhibit ADP induced platelet aggregation in a dose-dependent manner. However, 1F11 had no effect on plasma P-selectin and TXB2 levels induced by ADP, because platelet P-selectin expression and TXB2 synthesis could occur before aggregation, the “final step” of platelet aggregation, just like what reported in Glanzmann thrombocytopenia. Finally, we performed a prospective clinical study on H pylori infection by means of immuno-radiometric assay of urease B in 164 ITP patients, 88 (54%) were found H pylori-positive. platelet counts in 15 of 38 H pylori-positive patients were significantly increased after eradication treatment, and were completely recovered in 7 patients. In conclusion, our results first indicate that antibidy against H pylori urease B could cross-react with platelet GP IIIa, and inhibit platelet aggregation. H pylori eradication may improve the platelet counts in some chronic ITP patients.
Profile of Cell-Derived Microparticles (C-MP) in ITP: Red Cell Microparticles (RMP) Correlate with Severity of ITP.
Authors: Fontana Vincenzo , Dudkiewicz Pamela , Ahn Eugene , Jy Wenche , Horstman Lawrence L. , Yaniz Myriam , Ahn Yeon-Soong
Published: April 2020

BACKGROUND: The roles of cell-derived microparticles (C-MP) released from platelets (PMP), endothelial cells (EMP), leukocytes (LMP) and red cells (RMP) in hemostasis, thrombosis and inflammation have been appreciated in recent years. Although PMP were shown to be hemostatically active, the roles of other C-MP, especially RMP, have not been studied in ITP. We investigated C-MP in patients with ITP.

MATERIAL AND METHODS: One-hundred-six patients with ITP were studied. They consisted of 73 pts with active ITP (ITP-A, 30M/43F, mean age 53.8 yr) and 33 pts in remission (ITP-R, 4M/29F, mean age 53.4 yr). ITP-A was defined by plt count <140,000 for >3 months; ITP-R was defined by plt count >140,000 for >3 months. Mean plt counts was 78,000 for ITP-A and 224,000 for ITP-R. The two groups were similar in ages. CBC with plt count, aPTT and activities of FVIII, FIX, FXI, were measured. Using flow cytometry, PMP were identified by CD41+, EMP by CD31+/CD41−, LMP by CD45+, and RMP by glycophorin+. Effects of intravenous immunoglobulins (IVIG) on C-MP were also evaluated.

RESULTS: There was a strong inverse correlation between plt counts and RMP (p=0.002). RMP were higher when plt counts were lower. A significant correlation was found between platelet counts and PMP (p<0.0001) and EMP (p<0.0001), but not LMP (p>0.05). The mean value of RMP was higher in ITP-A than ITP-R (p= 0.009). Conversely, PMP and EMP were higher in ITP-R (P<0.0001). No difference was found in LMP. FVIII and IX were higher in ITP-A than ITP-R (p=0.006, p=0.001, respectively).The aPTT was shorter and FXI higher in ITP-A but they did not reach statistical significance. Only RMP (not PMP, EMP or LMP) correlated with elevated factors VIII, IX and XI (P=0.005, 0.001 and 0.005 respectively) and inversely correlated with aPTT (p=0.009). Infusion of IVIG reduced RMP (RMP pre/post=1519/1018), not the other C-MP, in 9 pts

DISCUSSION/CONCLUSIONS: (i) RMP inversely correlated with plt counts and appear to be a sensitive marker of severity of ITP. However mechanisms of RMP generation, their reduction following IVIG and their roles remain to be elucidated. (ii) Among C-MP, only RMP, not other C-MP, were associated with shortening aPTT and elevated FVIII, IX, XI. (iii) RMP are positive for annexin V, providing anionic phospholipids for clotting factors and generating thrombin (data not shown). These findings suggest that RMP associated with these factors are involved in hemostasis to prevent bleeding in severe ITP.

Table 1. ITP-A ITP-R p-value No. Patients 73 33 Plt count 78,000 224,000 <0.0001 C-MP: PMP 8074 16403 <0.0001 EMP 292 667 <0.0001 LMP 1718 1705 n. s. RMP 2292 1505 <0.01 Coag: aPTT 24.9 25.7 n. s. FVIII 1.95 1.50 0.006 FIX 1.53 1.28 0.001 FXI 1.29 1.12 n.s.
Two Mouse Genetic Models of Thrombocytopenia.
Authors: Chan E. Ricky , Lavender Heather , Trevarthen Karrie , Haviernik Peter , Bunting Kevin D. , Adams Mark D.
Published: April 2020

Thrombocytopenia is a disorder in which there is a low level of circulating platelets. There are two main underlying causes; a hematopoietic defect in which there is a decreased production of platelet cells or an autoimmune disorder in which platelets are actively destroyed. Two mutant mouse strains have been obtained from the mutagenesis program at The Heart, Lung, Blood and Sleep Disorders Center at The Jackson Laboratory that each exhibit a dramatic decrease in the level of platelets in comparison to the C57BL/6J wild-type strain making them potentially valuable models for thrombocytopenia. The mutations were crossed to BALB/cByJ and to 129Sv/ImJ and an F2 intercross strategy was used to map the mutation in each strain. Candidate gene sequencing of strain HLB219 revealed a point mutation in the Mpl gene on chromosome 4, which encodes the thrombopoietin (TPO) receptor. This mutation causes an amino acid change from a conserved cysteine to an arginine at codon 40. This ligand-receptor complex plays a pivotal role in megakaryocyte proliferation and maturation and platelet production. The mutation in HLB219 is consistent with other recessive loss-of-function mutations in Mpl that cause hematopoietic defects and thrombocytopenia. HLB219 homozygotes have an elevated circulating TPO level and decreased numbers of hematopoietic progenitors in a colony formation unit (CFU) assay. A competitive repopulation assay showed that HLB219 bone marrow is unable to contribute to hematopoiesis in a lethally-irradiated mouse recipient. Interestingly, HLB219/C57BL/6J heterozygotes show a unique overdominance effect in which platelet levels are elevated compared to wild-type C57BL/6J mice despite having normal TPO levels and reduced megakaryocyte levels in the CFU assay. The mutation in the second mouse model of thrombocytopenia, strain HLB381, has been mapped using a genome-wide linkage scan of 50 F2 intercross progeny. The candidate interval has been narrowed by screening >240 F2 progeny with additional microsatellite and SNP markers. The candidate interval spans 4.2 megabases at the centromeric end of chromosome 8. In contrast to results obtained with HLB219, a competitive repopulation assay demonstrated that HLB381 has normal hematopoietic capability. However, chimeric mice from these assays have low platelet levels, indicating that an autoimmune defect may be responsible for the phenotype. Furthermore, HLB381 mice have normal levels of circulating TPO. Current studies are focused on determining whether HLB381 may serve as a heritable model for idiopathic thrombocytopenic purpura (ITP), an autoimmune disorder.
Unusual Bone Marrow Findings in a Patient with Congenital Amegakaryocytic Thrombocytopenia.
Authors: Rose Melissa J. , Nicol Kathleen K. , Gross Thomas G. , Kerlin Bryce A.
Published: April 2020

BACKGROUND: Congenital Amegakaryocytic Thrombocytopenia (CAMT) is a rare bone marrow failure syndrome that presents with isolated thrombocytopenia within the first year of life. Classic diagnostic bone marrow findings reveal absent or significantly decreased megakaryocytes with otherwise normal marrow cellularity. We present a case of a newborn with CAMT whose initial bone marrow aspirate showed an appropriate number of megakaryocytes.

CASE: A caucasian male presented on day-of-life (DOL) 1 with petechiae and a platelet count of 13,000/mcL. Physical exam, WBC count and hemoglobin were otherwise normal. Alloimmune thrombocytopenia evaluation and CMV serology were negative. Platelet transfusions were required almost daily to maintain platelet counts >100,000/mcL in the neonatal period. Tibial bone marrow aspirate performed on DOL 21 revealed normocellular marrow with a normal number of immature megakaryocytes.

Figure Figure

Cytogenetics were normal and DNA breakage studies were negative. Beyond 1-month-of-age, platelet counts were maintained >25-30,000/mcL with 1–2 transfusions per week. Repeat bone marrow aspirate and biopsy on DOL 102, from the iliac crest, again was normocellular, but was now significant for marked megakaryocytic hypoplasia.

Number of Megakaryocytes Found on Serial Bone Marrow Aspirates Age Patient Age-Matched Control 4 slides per aspiration site were examined at the feathered edge at 40× magnification. Data is expressed in average number of megakaryocytes/10 hpf 21 days 3 1.6 102 days 0 2.1

Mutation analysis of the gene responsible for the thrombopoietin receptor MPL was notable for a homozygous point mutation in exon 3, resulting in a substitution of proline for arginine at amino acid position 102. This missense mutation has been previously associated with CAMT.

CONCLUSION: This is the third reported case of normal megakaryocyte numbers found on the initial bone marrow aspirate, in a patient who was later diagnosed with CAMT. CAMT should not be excluded from the differential diagnosis of persistent thrombocytopenia within the first year of life, solely based on normal numbers of megakaryocytes on the initial bone marrow. The presence of immature megakaryocytes may represent early morphological findings in CAMT.