bluuebunny/crossref_metadata_2025 huggingface dataset:
An RNA Interference Model of RPS19 Deficiency in Diamond Blackfan Anemia Recapitulates Defective Erythropoiesis and Rescue by Dexamethasone: Identification of Dexamethasone Responsive Genes by Microarray.
Authors: Ebert Benjamin L. , Lee Michele , Pretz Jennifer , Golub Todd R. , Sieff Colin
Published: October 2019

Diamond Blackfan Anemia (DBA), a congenital erythroblastopenia, is a model disease for the study of erythroid differentiation, but is poorly understood. RPS19 is the only gene yet to have been associated with DBA, but the relevance of this ubiquitously expressed ribosomal gene to erythroid differentiation is unclear. Glucocorticoids are the primary pharmacological therapy for patients with DBA, but the molecular basis for the activity of glucocorticoids in erythroid differentiation has not been identified. Through retroviral expression of short hairpin RNAs (shRNAs), we demonstrate that targeted degradation of the RPS19 gene in cultured human CD34+ cells blocks the proliferation and differentiation of erythroid progenitor cells. Decreased RPS19 expression limited production of erythroid colony formation on methylcellulose, decreased cell surface expression of glycophorin-A, and decreased cellular proliferation. Treatment of RPS19 deficient cells with dexamethasone restored erythroid differentiation to normal levels. We investigated the molecular basis of pharmacologic therapies for DBA using oligonucleotide microarrays to survey gene expression in CD34+ cells treated with combinations of erythropoietin, dexamethasone, IL-3, and SCF. None of these agents had a direct effect on the expression of RPS19 or on the coordinate expression of other ribosomal genes. Instead, dexamethasone activated a genetic program that includes a set of key hematopoietic regulatory genes, including Flt-3 and PLZF, that increase proliferation of hematopoietic progenitor cells. Genes specific to erythroid progenitor cells were up-regulated by dexamethasone, while genes specific to non-erythroid lineages were powerfully down-regulated. Deficiency of RPS19 therefore blocks proliferation of immature erythroid progenitor cells, and dexamethasone activates proliferation of the same cell population through mechanisms independent of RPS19. Identification of the key regulatory genes affected by dexamethasone may aid in the development of novel therapeutics that de-couple the beneficial hematopoietic effects of dexamethasone from detrimental non-hematopoietic side effects.
Monoubiquitinated FANCD2 Is Both Necessary and Sufficient for Mitomycin C Resistance in the Absence of a Functional Fanconi Anemia Core Complex.
Authors: Shimamura Akiko , Leary Rebecca , Stotsky Maria , Moreau Lisa A. , D’Andrea Alan D.
Published: October 2019

Monoubiquitination of the Fanconi anemia (FA) protein, FANCD2, is necessary for resistance to MMC/DEB-induced chromosomal breakage, which is the diagnostic feature of Fanconi anemia. The upstream Fanconi proteins FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, and FANCL are necessary for FANCD2 monoubiquitination and Fanconi pathway function. We posed the question whether FANCD2 monoubiquitination is sufficient to restore the Fanconi pathway function in the absence of the upstream FA proteins. We constructed a chimeric monoubiquitinated FANCD2 protein by fusing the FANCD2 cDNA sequence contiguously (in-frame) with a ubiquitin coding sequence. This FANCD2-ubiquition fusion protein (D2-ubi) was stably expressed in FANCD2−/−, FANCA−/−, FANCC−/−, and FANCG−/− human patient-derived cell lines. D2-ubi expression corrected the MMC hypersensitivity of these FA cell lines in the absence of a functional upstream FA core complex. By western blot analysis, the D2-ubi protein migrated at a slower rate than the endogenous monoubiquitinated FANCD2 protein, consistent with its higher molecular mass. An even slower migrating D2-ubi form (D2-ubi-L) was detectable in cells harboring an intact Fanconi core protein complex (i.e., FA-D2 cells) but was absent in cells lacking functional Fanconi proteins FANCA, FANCC, or FANCG. These results indicate the D2-ubi can be further monoubiquitinated on lysine 561, but only in the presence of an intact FA complex. Furthermore, D2-ubi-L was upregulated in response to DNA damage in transfected FA-D2 and wild type cells. Like endogenous FANCD2, D2-ubi formed nuclear foci in the presence of the upstream Fanconi core complex. In the absence of the FA core complex, D2-ubi was localized diffusely throughout the nucleus, but unlike endogenous FANCD2, D2-ubi was retained within the nucleus following detergent permeabilization of FA-A, FA-C, and FA-G cells. These results further support the epistatic relationship of the FA genes in a linear pathway. In summary, these data indicate that a constitutively monoubiquitinated FANCD2 polypeptide is able to bypass defects in the upstream FA core complex to restore Fanconi pathway function. These studies identify FANCD2 ubiquitination as a potential therapeutic target for patients lacking the upstream FA core complex.
The E1841K Mutation in MYH-9 of a Patient with May-Hegglin Anomaly Inhibits the Disassembly of Non-Muscle Myosin IIA (MYH-9) Responsible for the Phenotype of the Disease.
Authors: Miyazaki Koji , Komatsu Satoshi , Watanabe Mariko , Nakadate Naoya , Ikebe Mitsuo , Higashihara Masaaki
Published: October 2019

It has been demonstrated that MYH-9 (non-muscle myosin IIA) is responsible for the hereditary macrothrombocytopenia, such as May-Hegglin Anomaly (MHA), Fechtner syndrome (FS) and Sebastian syndrome (SS). We identified the E1841K mutation of MYH-9 gene of a patient with May-Hegglin Anomaly. Immunofluorescent staining of her peripheral blood smear samples revealed that non-muscle myosin IIA and actin filaments were co-localized at the Döhle-like inclusion bodies of neutrophils. To investigate the mechanism by which the E1841K mutation causes the phenotype of MHA, we first expressed GFP-tagged wild type and E1841K mutant MYH-9 cDNA in NIH3T3 cells. The mutant was able to form filaments and some inclusion bodies such as Döhle-like bodies, although it seemed to make no influences on the shape of the fibroblasts. Next we expressed the GFP-tagged wild and E1841K mutant MYH-9 in HEL cells and induced differentiation with TPA. HEL cells expressing the E1841K mutant failed in full differentiation. They could not produce the proplatelet-like projections as the cells overexpressing wild type of MYH-9 did. These results demonstrate that overexpression of this mutant can induce some phenotype similar to MHA. To clarify what molecular characteristics of the mutant myosin can cause the MHA phenotype, we expressed the recombinant wild and mutant proteins of non-muscle myosin IIA using Baculo-virus system. The mutant myosin failed to dissolve even in high ionic strength, suggesting this mutant can assemble in cytoplasm even under the condition, in which wild type myosin changes the conformation and exchanges dynamically assembly to disassembly. Next we induced myosin filaments in HEL cells by over-expressing the mutant of myosin light chain (DD mutant), which mimics the double-phosphorylated form. The DD mutant of myosin light chain also inhibited the proplatelet-like projections. Moreover silencing of MYH-9 gene with siRNA could have no inhibitory effects on the differentiation of HEL cells, producing rather more proplatelet-like projections than the control. These results indicated that filament formation of myosin is not critical for proplatelet-like projections, but disassembly of myosin is rather essential. Collectively, the E1841K mutant inhibits the disassembly of myosin to prevent the proplatelet-like formation of HEL cells, and the assembled myosin forms the Döhle-like inclusion bodies probably with actin filaments.
A Novel and Disease Specific Gene in Chronic Lymphocytic Leukemia.
Authors: Buhl Anne Mette , Jurlander Jesper , Pedersen Lone B. , Josefsson Par , Geisler Christian H. , Leffers Henrik
Published: October 2019

One of the best predictors of poor outcome in CLL is the absence of somatic hypermutations in the Ig-genes of the leukemic cells. This prognostic dichotomy, between cases of mutated and unmutated CLL, enabled us to screen for a gene associated with poor outcome CLL using differential display RT-PCR. We identified a novel transcript from chromosome 12q22, which by RT-PCR and Northern blotting seemed to be selectively over-expressed in CLL patients with poor prognosis. No matching genes or ESTs were annotated in the region, but screening of a cDNA library from unmutated CLL patients resulted in cloning of 7 cDNAs, which probably derived from alternative splicing of a common transcript. The majority of the transcripts had no significant reading frames, but one splice variant may encode a protein of 121 amino acids. Despite very low primary sequence similarity, structure modelling revealed that the peptide potentially can fold into a structure remarkably similar to human IL-4. By RT-PCR the various transcripts were undetectable in a panel of normal tissues and cell lines. Using quantitative RT-PCR (QRT-PCR), we could however detect minute amounts of the mRNAs in normal B-cells; the level was similar or slightly higher in good prognosis patients and much higher in poor prognosis patients. The expression level of the two major mRNAs was 24–50 fold higher in patients with unmutated Ig-genes compared to patients with somatic hypermutation (p<0.0001), and 20–30 fold higher in ZAP-70 positive patients compared to ZAP-70 negative patients (p<0.0001). The median time to treatment for patients with overexpression of the mRNAs (n=26) was 11 months compared to 104 months for patients with normal expression (n=31) (p=0.0005). The median overall survival for patients with overexpression was 97 months while patients with low expression had not reached a median overall survival at their last follow up (p=0.03). This prognostic power was comparable to that provided by mutational status, and stronger than that provided by ZAP-70 or CD38 expression. Finally, in 56 patients the gene dosage as defined in QRT-PCR was inversely proportional with time to treatment (p= 0.006). This novel gene therefore has several features that make it a candidate to be the first disease specific gene identified in CLL.
VEGFR-1 (Flt-1) Tyrosine Kinase Signaling Enhances Hematopoiesis, Proliferation/Differentiation and Immunity of Monocyte/Macrophage from Bone Marrow Hematopoietic Stem Cells, and Promotes Rheumatoid Arthritis.
Authors: Murakami Masato , Iwai Shinobu , Hirasuka Sachie , Iwakura Yoichiro , Maru Yoshiro , Shibuya Masabumi
Published: October 2019

VEGF and its receptor family including VEGFR-1(Flt-1) are well known to be a crucial regulatory system for normal development and pathological angiogenesis. Rheumatoid arthritis(RA) is a chronic systemic disease characterized by an inflammatory erosive synovicitis, which show marked neovascularization, inflammatory cell infiltration and synovial hyperplasia, then produce a pannus of inflammatory vascular tissue and lead to irreversible cartilage and bone destruction. We have already shown VEGFR-1 is expressed not only in vascular endothelial cells but also in inflammatory cells, especially in monocyte/macrophage. A recent report suggests the involvement of VEGFR-1 in RA by using collagen induced RA mouse model. To examine whether the signaling from VEGFR-1 is important for the pathological process of RA, we used VEGFR-1 tyrosine kinase(−/−) mice which cannot generate the signaling from this receptor, and an arthritis mouse model system carrying Human T-cell leukemia virus(HTLV-1) pX transgene. VEGFR-1 TK(−/−) mice with pX gene clearly showed a reduction in the incidence and the degree of clinical symptom of arthritis. Furthermore, the heterozygote VEGFR-1 TK(+/−) with pX transgene showed a partial decrease in the degree of clinical as well as pathological scores. To explain the reason of reduction of clinical symptoms, we investigated involvement of VEGFR-1 TK signal in lineage of bone marrow hematopoietic stem cell(HSC) to monocyte/macrophage proliferation and differentiation and their immunity. VEGFR-1 TK activities are not associated in number of HSC in bone marrow. However, VEGFR-1 TK(−/−) HSC toward multi-lineage proliferation is suppressed in colony-formation. In addition, failures of monocyte/macrophage faculties are observed in immunological reaction, phagocytosis, cytokine secretion(IL-6, VEGF) and migration. Furthermore, expressions of hematopoiesis and inflammation related genes in VEGFR-1 TK(−/−) macrophage are downregulated by microarray analysis. Next we treated with small molecule inhibitors of VEGF receptor(VEGFR-1 and VEGFR-2) of tyrosine kinase, KRN951, in RA model(pX transgenic model and type II collagen Ab cocktail model) for treatment. Treatment with KRN951 strongly attenuated the disease symptom through inhibiting recruitment of BM hematopoietic cells into peripheral inflammatory cells. These observations indicate that VEGFR-1 signals play an important role in both RA mouse model. The tyrosine kinase activity and the signaling of VEGFR-1 enhances hematopoiesis, proliferation/differentiation and immunity of monocyte/macrophage from bone marrow HSC, and promotes rheumatoid arthritis, which may be a new possibilities for the treatment of RA in humans.

Figure Figure
Myeloma Cell Lines Are Not Sensitive to Modulation of Cyclin D1 Level.
Authors: Nickerson Helen D. , Chesi Marta , Bergsagel Peter Leif
Published: October 2019

Dysregulation of D type cyclins is an almost universal event in multiple myeloma. Overexpression of cyclin D1 may occur by chromosomal translocation of t(11:14)(q13:32), and also in association with hyperdiploidy. To investigate the role of cyclin D1 in the growth of human myeloma cell lines, we used both shRNA plasmid constructs, and an siRNA Smartpool against cyclin D1 and compared to control cells transfected with anti-GFP or GAPDH constructs. siRNA was electroporated into multiple myeloma cell lines overexpressing cyclin D1 (KMS12PE, U266, H929) which gave a 50–60% transfection efficiency, assessed using a GFP plasmid.

Successful reduction in levels of cyclin D1 was confirmed by western blotting and normalization to a beta actin standard. Cyclin D1 protein in sh- or siRNA transfected cells was reduced on average to 32% of that in cells transfected with an anti-GAPDH or anti-GFP siRNA. In individual experiments reduction of cyclin D1 protein to as little as 10% of control was observed. However, we found no sign of increased apoptosis by Annexin V/Propidium iodide staining. Furthermore, cell cycle analysis by ethidium bromide staining and flow cytometry revealed no significant change in the cell cycle of sh- or siRNA transfected cells when compared to control cells. qPCR analysis revealed no acute compensatory increase in the RNA levels of other D-type cyclins (D2, D3) following reduction of cyclin D1 levels.

These results may be explained by the residual presence of sufficient cyclin D1 protein for cell cycle progression. An alternative explanation is that changes that frequently occur in myeloma to other cell cycle regulators, for example p16(INK4a) and Rb are able to circumvent cell cycle effects of reducing cyclin D1protein. These results suggest that the human myeloma cell lines tested are not acutely sensitive to cyclin D1 level.
The Transcriptional Program of Polycythemia Vera Revelas Altered Expression of Multiple Putative Tumor Suppressor Genes.
Authors: Berkofsk-Fessler Windy D. , Licht Jonathan D. , McConnell Melanie-Jane , Neuberg Donna S. , Bowler Timothy S. , Fruchtman Steven M.
Published: October 2019

Polycythemia vera (PV) is a myeloproliferative disease characterized by accumulation of erythrocytes and cells of the myeloid and megakaryocyte lineages. Although genes like PRV-1 and PTP-MEG2 have been implicated in the pathology of PV, there is no consensus on their importance in the disease process. Progenitor cells from PV patients can grow in the absence of erythropoietin, and are hypersensitive to a variety of other growth factors. This suggests that polycythemic hematopoietic progenitor cells possess a significantly different genetic program. We tested this idea by molecular profiling hematopoietic progenitor cells (CD34+) from PV specimens and normal donors. We purified CD34+ cells from the marrow of 10 PV patients and harvested total RNA. Biotinylated cRNA was made through two rounds of linear amplification, and hybridized to Affymetrix HGU133A genechips. CD34+ cells from marrow mononuclear cells of 5 normal controls were processed similarly. The resulting datasets were normalized to the median across chips and across genes. Unsupervised hierarchical clustering showed that PV samples had a distinct gene expression profile from the controls. We then performed supervised clustering using a non-parametric t-test (Wilcoxon rank sum test) using the Benjamini and Hochberg multiple testing correction held to a p-value of 0.01 to determine genes that were significantly different between disease and normal samples. Using these stringent criteria, there were 331 genes that reached significance. Strikingly most of these were decreased in expression compared with control CD34+ cells and only 34 genes were upregulated in PV. A 35 gene predictor set was discovered through the use of a k-nearest neighbor metric. This set was 100% accurate for the prediction of PV in a leave one out cross-validation approach. Among these genes are EVI1, a known oncogene and one of only two genes upregulated in PV on this list, and the putative tumor suppressor genes TUSC4 (NPR2), NDRG1 and KLF4. Also among the predictor genes is BAALC, a gene expressed in normal CD34+ cells and known to be a prognostic indicator gene for acute myeloid leukemia.
Early Tumor Detection Using Platelet Uptake of Angiogenesis Regulators.
Authors: Klement Giannoula , Kikuchi Lena , Kieran Mark , Almog Nava , Yip Tai-Tung , Folkman Judah
Published: October 2019

We report a new function for platelets: selective sequestration of tumor-derived angiogenesis regulatory proteins above the concentration of these molecules in plasma.

Iodinated VEGF in a Matrigel pellet (from 100 to 600 ng/100 microl), implanted subcutaneously in mice, accumulates almost exclusively in platelets in a dose-dependent manner over a period as long as 2–3 weeks, without raising plasma levels of VEGF. Similarly, platelet VEGF increases in the presence of a single microscopic VEGF-secreting human tumor of up to only 1 mm3 in SCID mice without any increase of VEGF in plasma. In addition to VEGF, other factors such as bFGF, PDGF, BDNF, endostatin and other regulators of angiogenesis are taken up by platelets in a selective and quantifiable manner which is dependent on tumor generation of these molecules. Our data show that these proteins are not simply associated with the platelet surface, but are internalized. Furthermore, they are protected from degradation within the platelet, and are not released by classical degranulating agents, such as thrombin, ADP or epinephrine. Incubation of human platelets with endostatin at above physiological levels results in decrease of the majority of platelet-associated VEGF and bFGF in a concentration-dependent manner.

Using SELDI-ToF mass spectroscopy of platelet extracts, we have found that this novel property of platelets enables the detection of microscopic tumors that undetectable by any presently available diagnostic method. The platelet angiogenic profile is more inclusive than a single biomarker because it can detect a wide range of tumor types and tumor sizes. Relative changes in the platelet angiogenic profile permit the tracking of a tumor throughout its development, beginning from an early in situ cancer.

Conclusions: (i) While the half-life of mouse platelets is approximately 3 days, the platelet angiogenic profile persists for as long as the tumor (or Matrigel pellet) is present. This indicates that platelets may continuously scavenge proteins which regulate angiogenesis. (ii) The fact that the presence of a human tumor can now be detected at microscopic size, suggests that it may not be necessary to know the type and location of a tumor before initiating treatment, especially since it is feasible to use anti-cancer therapies of little or no toxicity.

Figure Figure
Antiphospholipid Antibodies, Platelet and Endothelial Activation in Acute Thrombotic Thrombocytopenic Purpura (TTP).
Authors: Bidot Carlos J. , Jy Wenche , Jimenez Joaquin J. , Horstman Lawrence , Ahn Eugene , Lander Gabriela , Yaniz Miriam , Fontana Vincenzo , Ahn Yeon S.
Published: October 2019

BACKGROUND. Although the etiology of TTP remains controversial, some notable associated conditions have been identified. These include genetic predisposition, infections, drugs, pregnancy, systemic lupus erythematosus (SLE) and deficiency of ADAMTS13. These associations underscore the suspected immunological nature of this disorder. Some authors have reported the presence of antiphospholipid antibodies (APLA) with onset of fulminate severe TTP. Also, platelet and endothelial activation has been described in TTP but their possible relation with APLA is unknown.

METHODS. We measured APLA in 19 patients with acute TTP and 17 in remission. Fifteen were female and four were male, mean age of 37 ± 15. All met the diagnostic criteria of TTP and were treated with standard therapy including exchange plasmapheresis. The severity of TTP was classified based on duration of hospitalization and numbers of plasmapheresis needed. Recurrence of TTP was assessed by history and review of medical records. CBC, platelet counts, blood chemistry and LDH were monitored. We investigated APLA antibodies IgG and IgM against 6 antigens (β2GP1, FVII/VIIa, CL, PC, PS, PE) by ELISA. To investigate platelet activation, we measured by flow cytometry: CD62P on platelets, and platelet microparticles (PMP) by CD41 (PMP41). We also measured endothelial microparticles (EMP) using CD62E.

RESULTS.

Lab study : In acute TTP, 42% (8/19) were APLA positive for at least one IgG antibody. The most frequent was aPS (33%) followed by aCL(28%), aPC(28%), aβ2GP1 (28%), FVII/VIIa (21%) and PE (21%). IgM antibodies were not detected. APLA was two-fold more prevalent in acute TTP (42%) than in remission (21%). In acute TTP multiple antibodies (two or more) were seen in 75% (6/8) while in remission only one (12.5%) had multiple antibodies. Platelet activation measured by CD62P was higher in APLA+ than in APLA− (102 ± 25 vs 33 ± 8 fluorescence units, p<0.02, X ± SEM). PMP41 was higher in APLA+ than in APLA− but this difference was not significant. We also found that EMP62E was significantly higher in APLA+ than in APLA− patients (p<0.01).

Clinical observations : When comparing the two groups (APLA+ vs APLA−), APLA+ group had higher incidence of recurrence of TTP than APLA− (75% vs 36%), p<0.05. Platelet counts, LDH, severity and incidence of CNS syndrome were not significantly different between APLA+ and APLA− groups.

CONCLUSIONS: APLA are frequently seen in acute TTP (42%) and they tend to decline in remission. Their presence is associated with increased activation of platelets and endothelial cells, and also with clinical recurrence of TTP. APLA may play a role in excerbation and recurrence of TTP.
Proteomics Used To Identify the Target of a Platelet Aggregating Antibody in a Patient with HUS.
Authors: Rock Gail , Kolajova Maria
Published: October 2019

Hemolytic Uremic Syndrome (HUS) has considerable overlap with Thrombotic Thrombocytopenia Purpura (TTP). Recently, evidence is emerging to suggest that TTP, like HIT, is an immune mediated disorder. We have investigated the plasmas of patients with Hemolytic Uremic Syndrome to determine the presence of antibodies and the specificity of the related antigens.

Methods: A 20 year old female presented with repeated episodes of HUS. She is dialysis dependent. Plasma was taken for VWF, multimers, platelet aggregation, metalloprotease and inhibitor, western blot and 2D PAGE analysis.

Results: At the time of the acute episodes, the VWF was increased three fold; the VWF multimer patterns on two occasions showed the presence of unusually large VWF forms. The metalloprotease was normal at all times and no inhibitor was present. Her platelets showed a normal response to aggregation, however, the plasma caused spontaneous aggregation of normal washed human platelets. This reactivity was removed after absorption with Staph A. On western blot analysis of platelet and microvascular endothelial cell lysate vs. patient plasma, multiple reactions were detected with a strong and persistent reactivity against antigens of 200,00 kDa and 55 kDa. Two dimensional PAGE of the whole platelet proteome identified strong immunoreactivity with two target spots in the 55 kDa area. Mass spectroscopy, using the nano-LC-MS/MS analysis, with the MASCOT search program confirmed the target antigen as beta fibrin with a molecular weight of 50.731 and an isoelectric point of 7.95 with a MASCOT score of 859 and 750 respectively.

Conclusions: Anti-platelet antibodies which cross-react with platelet and microvascular endothelial cells are present in the plasma of patients with recurrent episodes of HUS. These antibodies cause aggregation of normal human platelets and are removed following specific absorption of IgG from the plasma. 2D proteomic analysis indicates that the antibody in our patient was directed against a specific antigen, B fibrin. The data suggests that HUS, like TTP and HIT has an immunological basis.
Distinction of Inherited from Acquired Thrombotic Thrombocytopenic Purpura Applying the Cone and Plate(Let) Analyzer.
Authors: Shenkman Boris , Budde Ulrich , Angerhaus Dorothea , Schneppenheim Reinhard , Lubetsky Aharon , Savion Naphtali , Seligsohn Uri , Varon David
Published: October 2019

Measurement of von Willebrand factor-cleaving protease (VWF-CP) activity is useful for diagnosis of thrombotic thrombocytopenic purpura (TTP), but the current methods are cumbersome. We recently introduced a simple method of TTP detection using the Cone and Plate(let) Analyzer (CPA), that was based on the observation that mixing of acute TTP plasma (50 μL) with normal whole blood (150 μL) induces a significant increase in platelet deposition (surface coverage, %SC) on polystyrene surface under flow condition (1800 s−1) (Brit J Haematol, 120: 597, 2003). The test was specific for TTP, since plasma from patients with HUS, APLS, ITP or HIT did not affect normal platelet deposition. In this study, we further explored the potential use of the CPA method in differential diagnosis of inherited and acquired TTP by increasing the shear rate and by inducing the VWF-CP activity with BaCl2. TTP or normal plasma was mixed with normal blood (type O+) and the surface covered by platelets was tested under shear rates ranging between 1800 s−1 to 2500 s−1. Maximal difference in SC between TTP and normal plasma was observed at a shear rate of 2050 s−1. Under these conditions, plasmas of 5 patients with inherited TTP (ITTP), and 11 patients with acquired TTP (ATTP) yielded comparable increase in SC of normal platelets in whole blood, i.e. 77±19% and 78±17%, respectively. However, experiments in which BaCl2 was added to allow activation of VWF-CP resulted in a substantial distinction between inherited and acquired TTP plasma. While addition of BaCl2 to normal or inherited TTP plasma, both mixed with normal blood, yielded similar reductions of SC by 41±12% and 51±19%, only 11±4% reduction of SC was observed when acquired TTP plasma was used. These results suggest that when normal or inherited TTP plasma is mixed with normal blood, activation of VWF-CP cleaves large VWF multimers giving rise to substantial reduction in SC, whereas in the case of acquired TTP plasma the inhibitor of VWF-CP abolishes BaCl2-induced activation of VWF-CP and the ensuing reduction in SC. We conclude that introduction of BaCl2 in the CPA at high shear conditions may be useful for differentiation between inherited and acquired TTP.
Preliminary Findings from the Prospective Multicenter Surveillance, Epidemiology and Risk Factors for Thrombotic Thrombocytopenic Purpura (SERF-TTP) Study.
Authors: Zakarija Anaadriana , Bandarenko Nicholas , Pandey Dilip K. , Kwaan Hau , McCaffrey Kathryn , Finley Denise , Carson Ken , Yarnold Paul , Bennett Charles L.
Published: October 2019

Background: Due to the rarity of TTP (approximately 4 cases per million), epidemiologic data and correlative laboratory samples from a prospectively identified incident cohort of TTP patients are needed.

Methods: SERF TTP is a NHLBI-funded 15-site study targeted to identify 300 incident TTP patients and 600 age-/gender-matched community controls.

Results: From nine IRB-approved collaborating sites, 22 incident TTP cases and 17 controls have been interviewed. Data and plasma (acute and convalescent samples) are available for 19 TTP patients (Table 1). Their mean age is 42.5 years (range, 20 – 80 years), and 90% are female. Using the Rose-Eldor TTP Scoring system based on platelets, creatinine, hemoglobin, and neurologic function, 37% would be classified as severely ill (score > 4 of a maximum 8 points). Mean time from symptom onset to diagnosis was 9.2 days, median 5.5 days (range, 0 to 33 days). Severity of TTP was poorer for the 5 patients whose treatment did not begin for at least 16 days (16–33 days) versus the other 14 patients (mean Rose-Eldor score of 4.6 versus 4). Medications which had been prescribed to the patients prior to TTP onset included herbal supplements (n= 5) and hormone replacement therapy or oral contraceptives (n= 6). All patients received at least daily therapeutic plasma exchange (TPE). A platelet count >150,000 was reached at a mean of 9.4 days following TPE initiation. At 30-days follow-up, all patients were alive, although 3 had an exacerbation requiring daily TPE reinstitution. Fourteen patients (78%) experienced an adverse event, including allergic reactions to plasma (n=9), citrate-related toxicity (n=9) and venous access complications (n=2) including one with major hemorrhage requiring transfer to the intensive care unit.

Conclusion: The clinical characteristics of the SERF-TTP cohort are similar to those reported from large single-site studies, although the survival rate (100% versus 71% and 83%) is higher. Only a minority of incident TTP cases in the modern era present with renal insufficiency or neurologic findings - highlighting the importance of developing reliable diagnostic laboratory testing.

Clinical Characteristics at Presentation

Study Platelets <100,000 Hgb <10 or Hct <.3 Cr > 2mg/dl Fever Neurologic symptoms Mortality (1) Zheng XL, Kaufman RM et al. Blood.2004; 103: 4043; (2)Vesely SK, George JN et al. Blood.2003; 102: 60. HSCT: Hematopoetic stem cell transplantation, FK-506: Tacrolimus. SERF-TTP n=19 95% 79% 32% 11% 47% 0% Washington University (1) (excluding HSCT/FK506 cases) n= 28 96% 82% 29% 32% 50% 29% Oklahoma TTP-HUS Registry (2) (ADAMTS13 < 5%) n=18 100% 100% 22% NA (Not Available) 56% 17%
Clinical Observation on the Efficacy of All-Trans Retinoic Acid (ATRA) Combined with Arsenic Trioxide (As2O3) in Newly Diagnosed Acute Promyelocytic Leukemia (APL).
Authors: Liu Y.F. , Shen Z.X. , Hu J. , Zhu Y.M. , Li J.M. , You J.H. , Shen Y. , Shi Z.Z. , Chen S.J. , Chen Z. , Wang Z.Y.
Published: October 2019

PURPOSE: To determine the efficacy of front-line use of all-trans retinoic acid (ATRA) combined with arsenic trioxide (As2O3) in patients with newly diagnosed acute promyelocytic leukemia (APL).

PATIENTS AND METHODS: Since 2001, 61 patients have received ATRA (25mg/m2) and As2O3 (0.16mg/kg) daily till CR and all patients received 3 consolidation chemotherapy and then received 5 cycles of sequential treatment of ATRA, As2O3 and 6-MP/MTX. The efficacy of treatment protocol in induction remission, molecular response and relapse-free survival were compared with our historical control.

RESULT: 58 (95.1%) patients achieved CR and all remain relapse free with the current protocol. Though ATRA/As2O3 duet did not improve the hematological/molecular remission rate and reduced the early mortality after induction, it did induce an early hematological response (26.1±4.1 days). During limited follow-up (20 to 39 months), both RFS and OS are significantly increased in patients who received the ATRA/As2O3/chemotherapy triad as post-remission therapy compared to the historical control. PML-RARa mRNA was retrospectively assessed by quantitative real-time reverse transcription-polymerase chain reaction (RQ-RT-PCR) before treatment, after CR, after consolidation and during follow-up period in 36 patients with ATRA and As2O3 combination treatment. PML-RARa normalized dose was found to be more significantly decreased after remission induction (median fold reduction: 335.8), and after consolidation (median fold reduction: 358362.2), as compared with ATRA or As2O3 mono-therapy. The addition of As2O3 into post-remission therapy can further increase the post-remission molecular response through either qualitative or quantitative RT-PCR measurement.

CONCLUSION: Our current follow-up data suggested a potential benefit of front-line combination of ATRA and As2O3, which might translate into better chance of curing the disease.
High Dose Thiotepa, Busulfan, Cyclophosphamide (TBC) and Autologous Hematopoietic Stem Cell Transplantation (ASCT) without Whole Brain Radiotherapy (WBRT) for Primary Central Nervous System Lymphoma (PCNSL).
Authors: Stewart Douglas A. , Forsyth Peter , Chaudhry Ahsan , Jeje Oluyemi , Morris Donald , Savoie Lynn , Brown Chris , Russell James R.
Published: October 2019

Introduction: Treatment of PCNSL with high dose methotrexate (HD-MTX)-based chemotherapy and WBRT is associated with severe neurotoxicity, but high relapse rates are associated with the use of either modality alone.

Patients and Methods: In an attempt to improve upon these dismal results, we treated eleven PCNSL pts aged 40–68 years (median=55) with TBC/ASCT without WBRT. These pts all received prior induction therapy with HD-MTX 3.5 or 5g/m2 q14d x 4-5 cycles +/− procarbazine 100mg/m2 po d1-7 q28d x 2 cycles concurrently with HD-MTX, and then underwent stem cell mobilization with Ara-C 3g/m2 IV d1 and 2, G-CSF d7-14, and apheresis d15 or 16. The TBC regimen consisted of thiotepa 300mg/m2 d-8 and -7, busulfan 3.2mg/kg IV daily d-6 to -4, and cyclophosphamide 2g/m² d-3 and -2. Poor prognostic features included relapsed/progressive disease (n=2), immune-deficiency (SLE, Imuran/Prednisone = 1), ECOG 2-4 (n=9), age >60 yrs (n=4), deep brain involvement (n=8), elevated LDH (n=2), elevated CSF protein (n=2).

Results: Stem Cell Collection and Engraftment: A median of 24 (5–45) x 106 CD34+ cells/kg were collected with a median 14.1L (8.5-30L) apheresis. Engraftment to ANC>0.5 and platelet>20 both occurred at a median of day +9 (7–12). TBC Toxicity: Two early treatment-related deaths occurred in pts 65 and 66 years of age, who both originally presented with ECOG of 4. In addition, a 68 yr old man developed Bearman grade 3 regimen-related toxicity (RRT) requiring ICU admission for encephalopathy and respiratory failure. He survived and returned home to function independently. The remaining pts experienced significant Bearman grade 1–2 RRT including generalized skin rash, peripheral edema, mucositis, delirium and asthenia. All surviving pts experienced improvement in neurological function and ECOG. Six pts returned to work including a 62 yr old pianist. Only one pt developed late neurotoxicity with symptoms of mild dementia. Survival: One patient relapsed 30 months post-TBC and died 1 month post-cranial radiotherapy. Eight pts (73%) are currently alive and relapse-free at 9, 15, 17, 18, 25, 43, 52, and 55 months post-TBC/ASCT. Two pts received TBC/ASCT as the only treatment after rapid disease progression following initial chemotherapy. One of these pts relapsed 7 mo after prior HD-MTX/Ara-C and BEAM/ASCT, and the other progressed within 1 mo of completing induction HD-MTX/Ara-C. Both these 2 pts remain relapse-free 43 and 52 months post-TBC/ASCT.

Conclusion: TBC/ASCT is capable of inducing prolonged remissions in pts with poor prognosis PCNSL, but is poorly tolerated in pts over 65 years. High dose chemotherapy for PCNSL should include drugs that penetrate the CNS well such as busulfan and thiotepa rather than standard lymphoma regimens such as BEAM.
Total Therapy 2 (TT 2) for Newly Diagnosed Patients with Multiple Myeloma (MM): Examination of Dose Effect of Thalidomide (T) among Those Randomized to T.
Authors: Thertulien Raymond , Barlogie Bart , Zangari Maurizio , Fassas Athanasios , Stover Julie , Milner Teresa , Rasmussen Erik , Anaissie Elias , Lee Choon-Kee , Hollmig Klaus , Talamo Giampaolo , Tricot Guido
Published: October 2019

TT 2 has completed accrual of 668 patients as of February 2004, of whom 323 were randomized to T. All patients received intensive reduction with VAD, DCEP, CAD, DCEP, melphalan-based tandem autotransplants; consolidation chemotherapy with DPACE; and interferon maintenance. T randomization data still blinded. We report, however, on the effect of total cumulative T dosing through induction (n=185; median T dose 26g, range 0 to 100 g), tandem transplants (n=141; median T dose 33 g, range 0 to 160 g) and consolidation therapy (n=69, median T dose 74 g, range 5 to 250 g). No significant differences in T dose distribution according to quartiles at the 3 observation points existed when examined according to age, gender and prognostically potentially relevant features such as cytogenetic abnormalities, CRP, B2M, LDH and albumin. EFS and OS were then compared, according to T dosing quartiles, from first transplant, consolidation and maintenance initiation. T dose effects were not observed for the first two observation times. When considered from initiation of maintenance, there was one event among 31 patients in the 2 upper quartiles of the cumulative T dose compared to 7 of 35 for the 2 lower quartiles (p=.02).

We conclude that, among patients randomized to the T arm of TT 2, cumulative dosing does not reveal an effect until after consolidation therapy, probably attesting to the marked efficacy of the cytotoxic components of TT 2.

Figure Figure
Decreased Transplant Related Mortality and Better Survival in HLA Matched (12/12 A, B, C, DRB1, DQB1, DPB1) Unrelated Bone Marrow Transplants.
Authors: Filion Alain , Loiseau Pascale , Rocha Vanderson , Tamouza Ryad , Chevret Sylvie , Busson Marc , Esperou Helene , Traineau Richard , Socie Gerard , Gluckman Eliane , Charron Dominique
Published: October 2019

In order to study the association of HLA high resolution typing with outcomes after unrelated bone marrow transplants (UBMT), we have retrospectively analyzed 121 consecutive recipients of unmanipulated UBMT performed in Hôpital Saint-Louis from 1994 to 2002 with a minimum follow-up of 6 months. The median age was 24 years (22 children, 99 adults). Ninety-six patients had malignant disorders (69% leukemias) and 25 had non malignant disorders (bone marrow failure syndromes). High resolution typing was performed in donors and recipients pairs for HLA-A,-B,-C,-DRB1,-DQB1 and-DPB1. According to this typing definition, patients were classified for transplants as 10 out of 10 (<8/10=18 patients, 9/10=32 patients and 10/10=71 patients) and 12 out of 12 (including DPB1 disparities, <9/12=25, 10/12=34, 11/12=48 and 12/12=14 patients). KIR ligand incompatibilities were observed in 19 cases. All transplants were performed using bone marrow cells; TBI and ATG were administered in 80% of cases. Graft versus host disease (GvHD) prophylaxis consisted in CsA+MTX in 90%. Univariable and multivariable analyses, using death as competing event, were performed to study risk factors for outcomes, including number of HLA compatibilities (10 out of 10 and 12 out of 12), KIR incompatibilities and disparities in each locus.

Results: In univariate analysis, we found that patients receiving a graft with less than 10 out of 10 HLA compatibilities (p=0.005) and any disparity in HLA-Class I (p=0.0009) were associated with delayed platelet recovery. Disparities in HLA-B were associated with high incidence of acute GvHD (51% versus 80%, p=0.03) and more importantly, a decreased 180-day transplanted related mortality (TRM) was associated with HLA 12/12 compatibilities instead of HLA 10 out of 10. In fact, TRM was 69% in patients receiving HLA graft <9/12, 41% in 10/12, 38% in 11/12 and 29% in 12/12. Two years survival was also associated with the number of HLA disparities with better survival in those patients receiving a UBMT 12 out of 12 HLA compatible donor. KIR ligand incompatibilities were not associated with any outcomes studied.

In conclusion, decreased TRM and better survival were associated with a compatible unrelated donor using HLA high resolution typing for A, B, C, DRB1, DQB1 and DPB1.
Outside-In Signaling upon Assembly on Endothelial Cells of the Proteins of the Plasma Kallikrein/Kinin System.
Authors: Mahdi Fakhri , Shariat-Madar Zia , Schmaier Alvin
Published: October 2019

Investigations from our laboratory have shown that there is a multiprotein receptor system on endothelial cells (HUVEC) consisting of gC1qR, uPAR, and cytokeratin 1 for high molecular weight kininogen (HK) and factor XII (XII) (Blood97:2342; 99:3585) that serves as a presentation receptor for prekallikrein (PK) activation. When PK binds to HK on HUVEC, it is activated to plasma kallikrein by the serine protease prolylcarboxypeptidase (JBC277:17962; Blood103:4554). HK is also known to have anti-proliferative and anti-angiogenic activity. We asked if there is outside-in signaling through this HUVEC receptor complex. Initial investigations determined if single chain urokinase (ScuPA) or XII stimulates Erk 1 and 2 (MAPK42 and 44) (MAPK) in HUVEC. Independently ScuPA (5–200 nM) or XII (15–200 nM) in the presence of 0.05 mM zinc ion stimulates HUVEC MAPK expression and it is blocked by 0.1 mM PD98059, 30 nM wortmann, or 0.05 mM LY294002. Two chain uPA or APMSF-treated ScuPA upregulates MAPK to the same extent as ScuPA. Similarly, XIIa or APMSF-treated XII upregulates MAPK like XII. Since HK, ScuPA, XII and vitronectin (VN) all bind to the same region on uPAR (JBC279:16621), studies focused on the role of uPAR in these activities. Mab3B10 to the HK, ScuPA, XII, and VN binding site on uPAR’s Domain 2 (D2) blocks ScuPA or XII upregulation of MAPK. Peptides LRG20, YLP20, PGS20, or FHN20 from uPAR’s D2 (L166-N200) block ScuPA or XII upregulation of MAPK. Similarly, HKa (1 micromolar) and peptides from the HK Domain 5 (G469-H498) HUVEC binding region (GGH18, HKH20) (JBC270:19256) block ScuPA- or XII-induced MAPK expression. Treatment of HUVEC with 5-20 mM methyl-beta-cyclodextran (MBCD) or fillipin (1 microgram/ml), agents known to disrupt lipid rafts, do not interfere with ScuPA- or XII-induced MAPK upregulation. These combined data indicate that zymogen ScuPA or XII, two proteins with growth factor regions, directly signal through uPAR to upregulate MAPK to possibly induce cellular proliferative activities. HK or its activated fragments bound to uPAR inhibits these potential growth promoting activities. These studies imply that HKa’s anti-proliferative and anti-angiogenic activities are mediated through its interaction with uPAR. These investigations also indicate that, in addition to a presentation complex for PK activation, the HUVEC multiprotein receptor complex for HK, XII, and ScuPA has an auto-regulating outside-in signaling system in endothelials cells.
Oral Mucositis (OM) and Outcomes of Autologous (AU) Hematopoietic Stem Cell Transplantation (HSCT) Following High-Dose Melphalan (MP) Conditioning for Multiple Myeloma (MM).
Authors: Oster Gerry , Vera-Llonch Montserrat , Ford Colleen , Lu John , Khazanov Irina , Sonis Stephen
Published: October 2019

Background. OM is a common toxicity of conditioning regimens for HSCT that has been associated with adverse clinical and economic outcomes.

Methods. A retrospective chart review study of ~400 consecutive HSCT recipients was undertaken at a single academic center. OM severity was assessed across eight oropharyngeal sites using a validated scale which was scored as follows: no erythema/ulceration = 0; erythema only = I; ulceration one site = II; two sites = III; three sites= IV; and four or more sites = V. OM assessments began on the day of conditioning and continued bi-weekly until hospital discharge. Outcomes of interest included number of days of fever, total parenteral nutrition (TPN), and parenteral narcotic therapy, incidence of grade III or IV (Common Toxicity Criteria) infection, and inpatient days and charges. We report data here for the 115 MM patients in the study cohort who underwent AU HSCT after high-dose MP conditioning.

Results. Mean age was 54 yrs. Seventeen percent of patients received total body irradiation. Bone marrow was the source of most grafts. Forty-eight percent of patients experienced OM ≥ grade II. The relationship between worst recorded grade of OM and each of the outcomes of interest is reported below.

Conclusions. OM is associated with worse clinical and economic outcomes in MM patients undergoing AU HSCT following high-dose MP conditioning.

Outcomes of AU HSCT following high-dose MP conditioning for MM, by worst recorded grade of OM Worst Grade of OM (N=115) 0 I II III IV V *mean, SD; **test for linear trend; Cochran-Armitage for dichotomous measures, GLM for continuous measures Outcome n=37 n=23 n=24 n=15 n=8 n=8 -value p Fever days* 1.2 (2.8) 2.5 (3.1) 2.1 (2.3) 1.9 (1.4) 3.0 (2.1) 3.6 (6.2) 0.057 TPN days* 0.0 (0.0) 0.1 (0.4) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 2.3 (6.0) 0.003 Narcotic days* 1.5 (3.1) 4.7 (3.8) 4.6 (4.4) 4.4 (3.7) 7.9 (3.3) 9.9 (7.4) <0.001 Infection, % 51.4 73.9 62.5 80.0 100.0 75.0 0.014 Mortality, % 0.0 0.0 4.2 0.0 0.0 12.5 0.085 Inpatient days* 18.6 (3.4) 21.0 (3.5) 19.6 (3.6) 19.7 (2.3) 23.3 (2.4) 23.4 (5.6) <0.001 Inpatient charges* (x 1,000 USD) 119 (36) 130 (35) 118 (25) 121 (28) 141 (31) 184 (92) <0.001