bluuebunny/crossref_metadata_2025 huggingface dataset:
A Role for Survivin in Enucleation of Erythroid Progenitors.
Authors: Keerthivasan Ganesan , Kang Jeong Ah. , Seferos Dwight S. , Mirkin Chad A. , Wickrema Amittha , Crispino John D.
Published: April 2020

Enucleation is the final step in the development of reticulocytes and this process is unique to mammalian red blood cells among vertebrates. Even though enucleation of erythroblasts was structurally studied by electron microscopy half a century ago, little is known about the underlying mechanism. Initially it was thought that enucleation involved degradation of DNA and other apoptotic processes. More recently it has been shown that enucleation of erythroid progenitors does not require major apoptotic machinery including caspase-3 activation (Krauss et al, 2005; Yoshida et al, 2005). Alternatively, it has been suggested that enucleation in erythroblasts is a result of asymmetric cytokinesis. In support of this later model, survivin, a regulator of chromosomal segregation and cytokinesis, which also has an anti apoptotic role, is highly expressed in orthochromatic erythroblasts, cells at the stage of differentiation just prior to enucleation (Gurbuxani et al, 2005). Moreover conditional heterozygous deletion of survivin in hematopoietic cells caused a decrease in enucleated cells in the spleen (Leung et al, 2007). This has led us to hypothesize that survivin has a role in enucleation of erythroblasts. To test this prediction, we first over expressed survivin in MEL (murine erythroleukemia) cells and then subjected them to DMSO induced differentiation. Normally MEL cells decrease their survivin expression level upon differentiation and at the same time undergo limited enucleation (<4%) even after 5 days of differentiation. In contrast, survivin overexpressing clones consistently enucleated 3–5 fold more than control cells, suggesting that levels of survivin are likely to be important for enucleation. We are currently harnessing the power of survivin antisense nanoparticle agents to assess the specific requirement of survivin in enucleation of primary human orthochromatic erythroblasts. Separately, we evaluated the localization of survivin and its known partners in enucleating primary human erythroid cells by confocal microscopy. Survivin is predominantly localized to the cytoplasmic compartment and to our surprise did not colocalize with Aurora B kinase and INCENP, two of its partners in the chromosomal passenger complex, which is essential for mitosis. On the contrary, we noticed that survivin partially colocalized with actin at the junction between the extruding nucleus and cytoplasm, as well as in the periphery of cytoplasm in these enucleating cells. Moreover enucleation assays performed in the presence of various inhibitors of cell cycle and cytokinesis did not prevent enucleation in erythroblasts. Taken together, these findings demonstrate that enucleation is not a form of cytokinesis and further suggest that survivin participates in enucleation by a novel mechanism.
A Novel Mutation of the α Spectrin Gene in a Family of Northern European Descent Is Associated with Three Different Phenotypes.
Authors: Agarwal# Neeraj , Swierczek# Sabina , Naidoo Kubendran , Prchal Josef T. , Coetzer Theresa L.
Published: April 2020

Mutations of α spectrin (Sp) involving the Sp heterodimer self-association site (the α I domain of Sp) represent the most common group of membrane skeletal defects in hereditary elliptocytosis (HE) and a closely related disorder, hereditary pyropoikilocytosis (HPP). HPP is characterized by extreme anisocytic microcytosis, a severe spectrin dimer self-association defect and spectrin deficiency. The conventional explanation for the different phenotypes is that HPP subjects are compound heterozygotes for an α sp defect that interferes with sp tetramer assembly and a second defect which results in decreased synthesis of functional α sp. In contrast, HE subjects have normal spectrin content and a less severe sp self-association defect. The clinical expression of HE/HPP is influenced by the inheritance of modifying factors such as the α Sp hypomorphic mutation, α LELY. α LELY is a low expression allele of the α Sp gene characterized by a C>G mutation in codon 1857 of exon 40 and a C>T-12 mutation in intron 45 that is responsible for partial skipping of exon 46, which is essential for the functional assembly of α/b sp dimers. Here we describe a family of northern European descent in which members had different erythrocyte morphology ranging from atypical HPP to HE to normal and a novel αSp mutation. Quantitation of RBC membrane proteins of the propositus with atypical non-microcytic HPP revealed 48% spectrin dimers (control 10%) due to a marked increase in the 74kD αI Sp peptide. There was only a slight decrease in the spectrin/band 3 ratio, which correlated with the normocytic morphology. There was also an abnormal α Sp peptide at 41kD suggesting presence of α LELY. Sequencing of his α Sp gene revealed heterozygosity for a novel mutation in exon 2, codon 34: CGG->CCG (Arg>Pro) and heterozygosity for α LELY. These mutations were also present in his brother and daughter who have HE, while another son, who had Arg34Pro, but not α LELY has repeatedly confirmed normal morphology (data in pedigree, figure 1). Ongoing clinical, α Sp peptide, DNA and quantitative Real Time PCR mRNA studies of extended family reveal a complex interaction of codon 34: G>C mutation with α LELY and different levels of transcripts of α Sp alleles. #equal contribution.

Figure Figure
Dematin and Adducin Provide a Critical Link between the Spectrin Cytoskeleton and the Erythrocyte Membrane Via Glucose Transporter-1.
Authors: Khan Anwar A. , Hanada Toshihiko , Gaetani Massimiliano , Li Donghai , Reed Brent C. , Speicher David W. , Chishti Athar H.
Published: April 2020

There is considerable interest in the elucidation of the mechanism that governs the linkage of elongated spectrin molecules to the erythrocyte plasma membrane. The mechanism by which the “head” region of the spectrin dimer, which participates in tetramer formation, binds to the membrane via ankyrin and band 3 has been reasonably well characterized. However, the mechanism by which the tail end of the spectrin dimer is anchored to the plasma membrane is not completely understood. Dematin and adducin are actin binding proteins located at the spectrin-actin junctions or “junctional complex” in the erythrocyte membrane. Individual suppression of their function in mice by the gene deletion exerts a modest effect on erythrocyte shape and membrane stability. In contrast, the combined deletion of dematin and adducin genes results in severe defects of erythrocyte shape, membrane instability, and hemolysis. Based on these findings, we proposed a model whereby dematin and adducin could function as a molecular bridge linking the junctional complex to the plasma membrane. Using a combination of cell surface labeling, immunoprecipitation, and vesicle proteomics, we have identified glucose transporter-1 as the receptor for dematin and adducin in the human erythrocyte membrane. This finding is the first description of a transmembrane protein that binds to dematin and adducin, thus providing a rationale for the attachment of the cytoskeletal junctional complex to the lipid bilayer via glucose transporter-1. Since homologues of dematin, adducin, and glucose transporter-1 exist in many non-erythroid cells, we propose that a conserved mechanism may exist that couples sugar and other related transporters to the actin cytoskeleton.
Comparable Outcome after Allogeneic Hematopoietic Cell Transplantation from Matched Unrelated and Sibling Donors in Elderly Patients with Acute Myeloid Leukemia - Results of a Retrospective Analysis in 368 Patients.
Authors: Schetelig Johannes , Bornhäuser Martin , Schmid Christoph , Hertenstein Bernd , Schwerdtfeger Rainer , Martin Hans , Stelljes Matthias , Hegenbart Ute , Schäfer-Eckart Kerstin , Füssel Monika , Wiedemann Beate , Thiede Christian , Baurmann Herrad , Ganser Arnold , Kolb Hans-Jochem , Ehninger Gerhard
Published: April 2020

Purpose: In patients with acute myeloid leukemia (AML) differential indications for matched sibling and unrelated hematopoietic stem cell transplantation (HCT) are considered and arbitrary age limits for HCT exist. We sought to determine whether donor type is a prognostic factor in elderly patients in the era of high-resolution DNA-based HLA-typing.

Patients and Methods: We performed a retrospective cohort analysis in patients with AML older than 50 years who had received an allogeneic HCT between 1995 and 2005. If available, DNA from donors and recipients of unrelated HCT was used for molecular retyping in order to get information on HLA-A, -B, -C, -DRB1 and DQB1 at the allele-level. Donor-recipient pairs with fully matched donors or one mismatch out of ten alleles were considered well-matched.

Results: We identified 368 patients with cytogenetic intermediate or high risk AML who fulfilled the entry criteria. The median age of this cohort of patients was 57 years (range 50 to 73 years). 46% of patients had matched sibling donors, 3% related non-sibling donors, 41% well-matched unrelated donors and 10% poorly matched unrelated donors. In the respective period the percentage of patients with unrelated donors increased from 0% in 1995 to 64% in 2004. High risk features were a history of prior myelodyplasia in 34% of patients, poor risk cytogenetic abnormalities in 33% of patients and a disease status beyond CR1 in 62% of patients. 72% of patients received reduced-intensity conditioning regimens. Peripheral blood stem cells were used as graft in 84% of patients. In multivariate analysis disease status at HCT (p<0.001) and cytogenetic risk (p<0.001) proved to be highly significant predictors, both, for EFS and OS. Whereas, the relative risk of a patient with a well-matched unrelated donor compared to a sibling donor was 0.9 (95% CI, 0.6 to 1.2) for EFS and 1.0 (95% CI, 0.7 to 1.4) for OS. In subgroup analyses EFS was better in AML patients with cytogenetic high risk disease beyond first remission (CR1) (p=0.0147) who had well-matched unrelated donors compared to those with sibling donors and not inferior in any of the other subgroups.

Conclusions: Allogeneic HCT from matched unrelated donors (>=9/10) should be considered equivalent to sibling HCT in terms of survival for patients above the age of 50 years with intermediate or high risk AML. In advanced stages of AML with high risk cytogenetics patients with matched unrelated donors may even have better EFS compared to those with sibling donors.
Nutritional Deficiencies in Chronically Transfused Hemoglobinopathy Patients.
Authors: Claster Susan , Wood John , Carson Susan , Hofstra Thomas , Nord Anne , Khanna Rachna , Coates Thomas
Published: April 2020

Vitamin and mineral deficiencies have been well described in non-transfused patients with sickle cell disease (SCD). Levels of biomarkers of oxidant damage are increased in both SCD and thalassemia major (TM), and levels of antioxidants have been shown to be lower in both diseases. Hypermetabolism, increased hemolysis and denaturation of unpaired globin chains contribute to oxidative stress in SCD and TM. Chronic transfusions, initially thought to be protective against oxidation by marrow suppression, may have the opposite effect due to the well-known propensity of iron to promote free radical generation. Methods: To test this hypothesis, we measured levels of vitamins A, B1, B6, C, D, E as well as selenium, zinc, copper and carnitine and compared them to liver iron concentrations in chronically transfused SCD and TM patients. All patients were required to fast overnight and to hold their iron chelator for 24 hours prior to blood draws. Samples were processed by routine clinical processing (Quest Nichols Institute, San Juan Capistrano, CA). Results were expressed as the percentage of patients outside the reference range for the referral laboratory. Twenty four patients with TM and 43 patients with SCD were studied.

Abnormal Values In Transfused Patients SC (%abn) TM (%abn) A 73.7 52.4 B1 38.5 37.5 B6 34.2 34.8 Folate 28.6 37.5 C 56.7 66.7 D25 74.4 50.0 D1, 25 48.6 39.1 Se 67.5 75.0 Zn 24.3 8.3 Carnitine (Free) 22.2 41.7

Results: The results in the table demonstrate that over half the patients were deficient in A, C, D and selenium. A third of the patients had low levels of B vitamins and folate (despite replacement doses in the latter), as well as carnitine. B12 levels(not shown) were normal. E levels (not shown) were low in only a few patients. Deficiency profiles were remarkably similar between the two diseases: only copper was significantly higher in the SCD patients (34.2% elevated vs 0% in TM). B6 and folate were weakly correlated with hepatic iron concentration (r2 = −0.12, −0.18). B6, folate, D25-OH, and D1,25 OH levels declined with age. These results demonstrate that chronically transfused patients have significant deficiencies in nutrients which are involved in buffering oxidant stress, regardless of their diagnosis. Severity of iron overload was not a strong predictor of these abnormalities; although the precise explanation is not known, chronic anemia may be the unifying factor. The magnitude of B1, B6, C and D deficiencies in some patients was well into the range known to cause serious complications. These combined abnormalities may contribute to the morbidity of chronically transfused patients with SCD and TM.
Rituximab Therapy for Autoimmune Hemolytic Anemia and Immune Thrombocytopenia: A Belgian Registry.
Authors: Dierickx Daan , Mineur Philippe , Triffet Agnes , Kentos Alain , Keppens Carine , Pierre Pascal , Ferster Alina , Boulet Dominique , Vermylen Christiane , Deprijk Bernard , Beghin Yves , Dresse Marie-France , Kargar Khalil , Bries Greet , Verhoef Gregor , Delannoy Andre
Published: April 2020

Background. Immune thrombocytopenic purpura (ITP) and autoimmune hemolytic anemia (AIHA) may respond to the chimeric anti-CD20 monoclonal antibody rituximab, even when refractory to conventional therapy.

Aims. To collect data on Belgian patients given rituximab in the setting of ITP or AIHA in order to assess the response rate and the factors predictive for response in a multicenter study.

Method. Belgian hematology centers were invited to fill a questionnaire specifying the major characteristics and quality of response of ITP and AIHA patients given rituximab. For ITP, complete response (CR) was defined as a platelet count >150,000/μL, and a partial response (PR) as a platelet count 50–100,000/μL. For AIHA, Response (R) was defined as a 2g increase of the hemoglobin concentration and achievement of transfusion independence.

Results. All the patients were given rituximab after relapse or after failing at least one previous line of treatment. Except in 10 cases, rituximab was given at the dose of 375mg/m2 weekly for 4 weeks. In 31 assessable episodes of ITP a CR was achieved in 18 patients (58%) and a PR in 2 patients (6%). In 53 episodes of AIHA, R was achieved 42 times (79%). In both ITP and AIHA patients we could find no significant correlation between response and sex, age, prior splenectomy, platelet count or hemoglobin concentration when rituximab was started, number of previous treatments, response to previous treatments, duration of disease before rituximab was given, presence of an underlying malignant or autoimmune condition. In patients with AIHA, response rates were similar in cold agglutinin disease (8/10) and in warm antibody mediated hemolysis (29/38). Progression-free survival in responding patients is presented graphically. At one year, 83% of ITP patients and 61% of AIHA patients were alive without progression.

Conclusion. In this still open registry, we could confirm that rituximab induces responses in a majority of previously treated patients with ITP or AIHA. Responses could not be predicted from pre-treatment patient characteristics. In most patients response duration exceeded one year.

Progression-free survival of responding patients Progression-free survival of responding patients
Coexistence of Congenital Red Cell Pyruvate Kinase Deficiency and Hereditary Stomatocytosis.
Authors: Marcello Anna P. , Vercellati Cristina , Fermo Elisa , Bianchi Paola , Barcellini Wilma , Zanella Alberto
Published: April 2020

We describe a case of chronic hemolytic anemia due to the co-presence of pyruvate kinase (PK) deficiency and Hereditary Stomatocytosis (HSto). The propositus was a 30 years old adopted male with no known family history; he had severe neonatal jaundice requiring exchange transfusion, followed by a life-long history of moderate to severe chronic hemolytic anemia (Hb 7–10 g/dL), with jaundice and splenomegaly. At the age of 6 months hemoglobin screening was made and a beta trait was found. At the age of 20 splenectomy and cholecystectomy were performed. Surgery resulted in an increase of 1.5 g/dL in haemoglobin, and in a conspicuous rise of reticulocytes (from 125×109/L to 562×109/L). Two thrombotic events occurred thereafter, the former 6 days after surgery, and the latter two years later, during a toxoplasmosis infection. At the time of the study Hb was 10.8 g/dL, MCV 82.2 fL, reticulocytes 562×109/L, unconjugated bilirubin 2.19 mg/dL, LDH 335 U/L, haptoglobin <20 mg/dL, serum ferritin 342 ng/mL and transferrin saturation 71%. The peripheral blood smear examination showed the presence of echinocytes (13%), stomatocytes (11%), acantocytes (10%), schistocytes (7%), elliptocytes (6%), spherocytes (4%), target cells (4%) and a few erythroblasts. Erythrocyte osmotic fragility was decreased; screening test for unstable hemoglobins and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of red cell membrane gave normal results. The study of the most important red cell enzymes revealed reduced PK activity (6.0 UI/gHb, normal range 11.1–15.59 UI/gHb) and thermal stability (43%, normal range 57–100%). Direct sequencing of PK-LR gene showed a compound heterozygosity for mutation 1456T (Arg486Trp) and the new variant −73g>c. Mutation −73g>c occurs in the most proximal of the four GATA motifs in the R-type promoter region and possibly result in a decrease of mRNA synthesis, as already reported for the variant −72a>g (Manco et al, 2000). Molecular analysis of HFE gene showed heterozygosity for H63D mutation. The history of post splenectomy thrombosis and the presence of stomatocytes in peripheral blood smear prompted us to investigate for the coexistence of hereditary stomatocytosis. The determination of plasma potassium and sodium concentration revealed an increase in intracellular sodium (16.3 mmol/LRBC, reference range 5.0–12.0) and a decrease in intracellular potassium (74.73 mmol/LRBC, reference range 90–103), suggestive for a diagnosis of dehydrated HSto, or hereditary xerocytosis. This defect likely accounts for the thrombophilic state in this case, since HSto is known to be associated with hypercoagulability, particulary after splenectomy.
Upregulation of Microparticles and CD40 Ligand in Antiphospholipid Syndrome: Potential Implication in Inflammatory Responses and Thrombosis.
Authors: Fabbrini Nancy , Hoppensteadt Debra , Messmore Harry , Bick Rodger , Adiguzel Cafer , Fareed Jawed
Published: April 2020

Antiphospholipid syndrome (APS) represents a complex pathphysiologic state where vascular endothelium, platelet activation, coagulation and inflammatory processes are contributory to the overall outcome. Cellular apoptosis and immune mediated fragmentation results in the generation of microparticles (MP). Platelet associated CD40 Ligand contributes to the inflammatory complications, which may be further augmented by MP. To test the hypothesis that both the MP and CD40 ligand are increased in patients with APS, 90 samples received at Hines VA/Loyola Medical Center laboratories for antiphospholipid screening were profiled for MP utilizing a functional method and CD40 ligand employing an ELISA method (R&D, Minneapolis, MN). ProteinChip Array analysis was also carried out for biomarker profiling to identify a previously described inflammatory biomarker at 11.9 kDa utilizing surface enhanced laser desorption/ionization (SELDI). Fifty normal healthy male and female plasma samples were used as controls for comparative purposes. Of the 90 samples screened, 30 were found to be positive for the antiphospholipid antibody (APA) titer using an ELISA method (American Diagnostica, Stamford, CT). All of the 30 positive samples have elevated DRVVT; however, wide variations in the APTT were noted. The remainder of the 60 samples, which were negative for APA, also showed variability in the DRVVT and APTT values. In contrast, the DRVVT and APTT values in the normal were within the expected range. None of the normal samples were positive for the APA antibody. Most strikingly, the CD40 ligand in the samples positive for APA titer were markedly higher (480 ± 230 pg/ml) than the 60 which were negative (180 ± 80 pg/ml). The normal values were much lower (90 ± 30 pg/ml). The MP titer in the APA positive samples was also much higher (34 ± 14 nM) in contrast to the APA negative samples (18 ± 12 nM). The normal levels were even lower (9 ± 4 nM). In the ProteinChip Array analysis, the APA positive samples showed a higher prevalence of the unique biomarker at 11.9 kDa. This unique biomarker was absent in the normal human plasma. These findings clearly suggest that APA represents a complex syndrome where hemostatic activation involving the formation of the MP, upregulation of inflammatory process as evident by the generation of CD40 ligand and protease activation resulting in the formation of unique biomarkers contribute to its overall pathophysiologic outcome. Sequential profiling of these parameters may be helpful in the risk stratification of these patients.
Two-Hybrid Screening Identifies an Interaction between the CUB Domains of Human ADAMTS13 and Factor XII.
Authors: Yu Zheyong , Sadler J. Evan
Published: April 2020

The metalloprotease ADAMTS13 regulates the size of released von Willebrand factor (VWF) multimers by cleaving a peptide bond in the VWF A2 domain. Unlike the other ADAMTS family members, ADAMTS13 contains two C-terminal CUB domains, and mutations in this region have been found in patients with congenital thrombotic thrombocytopenic purpura (TTP). In vitro studies indicate that CUB domains are dispensable for ADAMTS13 catalytic activity under static conditions with denatured VWF substrate, but they may be crucial for recognition of native VWF under flow. CUB domains in many proteins mediate protein-protein interactions, and to identify potential binding partners we conducted yeast two-hybrid screening using MATCHMAKER GAL4 two-hybrid system 3 (Clontech Laboratories Inc., CA, USA). A cDNA fragment encoding both CUB domains was inserted into the pGBKT7 vector. The resulting GAL4BD-CUB fusion protein was used as bait to screen a human liver cDNA library. Both bait and library vectors were simultaneously introduced into Saccharomyces cerevisiae AH109 and double transformants were selected on minimal synthetic dropout media lacking Trp, Leu and His. A total of approximately 3 × 106 clones were screened. Putative positive colonies were re-streaked and tested for β-galactosidase activity. DNA fragments from positive colonies were sequenced and 26 proteins were identified, 10 of which were plasma proteins including factor XII (FXII), β2-glycoprotein I, and fibrinogen β chain. Interactions were further characterized with a binding assay employing purified ADAMTS13 immobilized on microtiter plates. The FXII-ADAMTS13 interaction was saturable and reversible, with Kd = 17 nM, which is significantly lower than the plasma concentration of FXII (∼ 375 nM). Deletion of CUB domains in ADAMTS13(ΔCUB) reduces its affinity toward FXII by ∼10-fold (Kd = 164 nM). Activated FXII (FXIIa) bound saturably and reversibly to ADAMTS13 with Kd = 19 nM. Binding of FXIIa to ADAMTS13(ΔCUB) was undetectable, suggesting that the FXIIa-ADAMTS13 interaction is more specific than the FXII-ADAMTS13 interaction. This interaction of FXIIa and ADAMTS13 was totally blocked by a heat and acid stable inhibitor in plasma, whereas the interaction of FXII and ADAMTS13 was decreased only 50% by plasma. These data suggest that FXII can bind either to ADAMTS13 CUB domains or with lower affinity to a distinct site that does not involve the CUB domains; FXIIa, a two-chain enzyme formed by a single cleavage of FXII, only retains the structure involved in high affinity binding to ADAMTS13 CUB domains. Fibrinogen and β2-glycoprotein 1 also bound to ADAMTS13 in vitro, but with much lower affinity. However, the plasma concentrations of both proteins (about 9 μM and 4 μM, respectively) are at least 10 times higher than their apparent Kd values. Whether these CUB domain-interacting proteins are important for the regulation of ADAMTS13 activity in vivo remains to be established.
A Small Fragment of ADAMTS13 Enhances Cleavage of von Willebrand Factor by the Intact Enzyme.
Authors: Majerus Elaine M.
Published: April 2020

ADAMTS13 regulates hemostasis by cleaving von Willebrand Factor (VWF) to smaller less thrombogenic fragments. Absence of ADAMTS13 activity leads to thrombotic thrombocytopenic purpura, an often fatal disease that is characterized by microangiopathic hemolytic anemia, thrombocytopenia and microvascular thrombosis especially in renal and central nervous system vasculature. ADAMTS13 like other ADAMTS family members has a characteristic domain structure consisting of a metalloprotease domain, a disintegrin domain, a thrombospondin type 1 repeat (TSR), a cysteine-rich domain, a spacer domain, 7 additional TSRs and 2 carboxyl terminal CUB domains. Regions in the spacer and C-terminal thrombospondin type 1 repeats (TSR) and CUB domains have been shown previously to be important for the interaction of ADAMTS13 and VWF. To further characterize the interaction of ADAMTS13 and VWF, we generated a construct consisting of the spacer domain and the second TSR of ADAMTS13. This construct is called ST2 for Spacer and Tsr2. Although many constructs of ADAMTS13 fail to be secreted because of poor folding, ST2 is folded since it is secreted from human embryonic kidney 293 cells. Previous work has shown that the spacer domain alone can bind VWF under static conditions, therefore ST2 was included in VWF cleaving assays. Unexpectedly, ST2 was found to enhance the cleavage of VWF by ADAMTS13 by approximately 10-fold in assays using either guanidine or urea-denatured VWF. To understand how ST2 enhanced the cleavage of VWF by ADAMTS13, interactions of ST2 with ADAMTS13 and VWF were studied. ST2 was found to specifically co-immunoprecipitate with VWF but not with ADAMTS13. A Kd calculated from these co-immunoprecipitation experiments was 8.7 nM and the stoichiometry of binding was approximately 1 mole/mole. One explanation for the enhanced cleavage of VWF by ADAMTS13 in the presence of ST2 is that ST2 may induce a conformational change in VWF that makes the cleavage site more accessible. To test this hypothesis, VWF in the presence and absence of ST2 was subjected to trypsin digestion at different concentrations of trypsin. Differential cleavage of VWF was seen in the presence and absence of ST2 at all concentrations of trypsin tested. This is consistent with induction of a conformational change in VWF by ST2. These studies provide additional information on the structure-function relationship of these two key proteins in the control of hemostasis.
Thrombin-Cleaved Osteopontin Is Regulated by Thrombin-Activatable Carboxypeptidase B in Rheumatoid Arthritis.
Authors: Myles Timothy , Sharif Shadi A. , Du Xiao-Yan , Song Jason J. , Elizabeth Price , Robinson William H. , Leung Lawrence L.
Published: April 2020

The pro-inflammatory cytokine osteopontin (OPN) is present in rheumatoid arthritis (RA) synovial fluids (SF). OPN can be cleaved by thrombin exposing the cryptic C-terminal α4β1/α9β1 integrin-binding motif (SVVYGLR). We have previously shown that thrombin-activated proCPB (CPB) can remove the C-terminal arginine from the SVVYGLR motif abrogating its α4β1 binding. Using peptides based on the C-terminus of thrombin-cleaved OPN (OPN-R) and thrombin/CPB-cleaved OPN (OPN-L), rabbit anti-OPN-R and anti-OPN-L antibodies were generated. Specificity of these antibodies was demonstrated by western blot analysis. Specific ELISAs for OPN-R and OPN-L showed significant elevation of OPN-R and OPN-L levels in SF from patients with RA (n=26) as compared to osteoarthritis (OA, n=18) and psoriatic arthritis (PA, n=10). (OPN-R median values: 138.6 ng/mL (RA), 10.6 ng/mL (OA), and 2.2 ng/mL (PA) p <0.001; OPN-L median values 205.3 ng/mL (RA), 25.9 ng/mL (OA), and undetectable in PA p <0.003). Multiplex cytokine analysis showed that elevated OPN-R and OPN-L levels in RA were significantly correlated with multiple inflammatory cytokines including IL-6, IL-12p40 and FGF-2, while OPN-FL levels only exhibited a statistical correlation with IL-6. Synovial lining cells expressed functional TM that supported the activation of proCPB to CPB by thrombin. ProCPB activation by thrombin, which may occur either systemically or locally within the inflamed joint space, inactivates thrombin-cleaved OPN and C5a and represents a homeostatic response in modulating the inflammatory process in RA.
The Severity of Bleeding Tendency in 5 Patients with Factor V Deficiency Due to Distinct Kinds of Factor V Mutations.
Authors: Shinozawa Keiko , Amano Kagehiro , Suzuki Takashi , Inaba Hiroshi , Fukutake Katsuyuki
Published: April 2020

Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. It is poor correlation between FV levels in plasma and the severity of bleeding tendency. In the present study, we identified 5 mutations in the FV gene (F5) in 5 unrelated Japanese patients with reduced plasma FV activities associated with inherited FV deficiency. Their bleeding tendencies varied in severity from asymptomatic to severe. We hypothesized that the severity of bleeding symptoms with severe FV deficiency is correlated with FV levels in platelets, and performed recombinant mutant proteins expression experiments and analyzed of platelet FV. The data concerning 5 patient’s FV levels in plasma and the bleeding symptoms are given in Table. The F5 mutations in 5 Japanese patients were identified by direct sequencing. One of the 5 patients was a compound heterozygote for FV mutations and carried a 5-base pair (bp) deletion in exon 22 (del.ACCCT) and V1813M. The other 4 patients were homozygous for a D68H, N468S, V1813M, and R2174L mutations, respectively. Four mutations except V1813M are newly identified mutations. These mutations were introduced independently by site-directed mutagenesis into a pMT2/FV mammalian expression plasmid containing the full-length FV cDNA, and the wild-type and mutant FV proteins were expressed in HEK293 cells. In the conditioned media, FV specific activities of the FV-D68H, FV-N468S, FV-V1813M, FV-R2174L, and 5bp del. mutants were an approximately 22%, 81%, 28%, 40%, and 19% of wild-type, respectively. On the other hand, analysis of platelet from patient by using RT-PCR showed that platelet F5 mRNA of FV-R2174L and FV-N468S was equal amount to that of normal subjects, although the amount of FV-V1813M platelet F5 mRNA was reduced. Platelet FV protein from patients was analyzed by western blotting and ELISA. Although the amount of platelet FV-R2174L protein was equal to that of normal platelets, platelet FV-V1813M protein was considerably reduced. In addition, the amount of FV-N468S protein in platelet was observed between that of normal subjects and FV-V1813M. These results indicate that the fact that sufficient amounts of FV are stored in platelet is required for local hemostasis. The results of FV-R2174L suggest that both functionality and amount of FV-R2174L in platelet is enough to cope with local bleeding, resulting very mild bleeding tendency. On the basis of present findings, we conclude that the severity of bleeding due to severe FV deficiency is correlated with not only plasma FV level, but also platelet FV.

Five patient’s FV levels in plasma and the bleeding symptoms Patient No. Mutation FV activity (%) FV antigen (%) Bleeding symptoms 1 D68H 4 4 asymptomatic 2 N468S 3 3 asymptomatic 3 V1813M & 5-bp deletion <1 9 severe 4 V1813M <1 4 moderate 5 R2174L 1 5 very mild
Effective Factor VIII Endocytosis Is under Conformational Control of von Willebrand Factor.
Authors: Meijer Alexander B. , Roosendaal Sigrid D. , de Laat Bas , van den Biggelaar Maartje , Limburg Vincent , van der Zwaan Carmen , Rodenburg Kees W. , Mertens Koen
Published: April 2020

Von Willebrand Factor (VWF) protects Factor VIII (FVIII) from rapid clearance of the cofactor from the circulations. This notion may agree with the observation that VWF blocks FVIII binding to LDL receptor (LDLR) and LDL receptor-related protein (LRP) in vitro. In spite of the presence of VWF, however, we have previously demonstrated that LRP and LDLR are involved in the catabolism of FVIII in vivo. This suggests that dissociation of the FVIII-VWF complex in plasma or at the cell surface drives the LRP/LDLR dependent clearance of FVIII. We therefore first assessed the effect of VWF on the binding of FVIII to the cell surface employing confocal microscopy. To this end, a functional FVIII derivative containing the yellow fluorescent protein (FVIIIYFP) was incubated with LRP-expressing or LDLR-expressing CHO cells at 4 °C in the presence and absence of VWF. The result showed that there was a distinct yellow fluorescent staining of the cell surface irrespective of the presence of VWF. When the same experiment was performed at 37 °C, however, yellow fluorescent focal spots rapidly appeared inside the cells but only in the absence of VWF. Co-localization studies demonstrated that these spots originated from FVIIIYFP present in early endosomes. These findings suggest that VWF blocks the transfer of FVIII to its endocytic receptors but not the binding of FVIII to the cell surface. Intriguingly, in the presence of ristocetin, FVIIIYFP was localized inside the cells not only in the absence of VWF but also in its presence. In agreement with this finding, flow cytometric analysis confirmed that VWF was no longer able to prevent endocytosis of FVIIIYFP in the presence of ristocetin. These results suggest that switching VWF in the VWF-FVIIIYFP complex into its active conformation triggers the endocytic uptake of FVIII. The restored endocytosis of FVIII was not the consequence of a reduced affinity of “active-VWF” for FVIII. The latter was concluded from solid phase binding studies that showed that FVIII-VWF complex formation is indistinguishable in the presence and absence of ristocetin. We propose that switching VWF in the VWF-FVIII complex into its active conformation initiates at the cell surface a sequence of molecular events, which ultimately lead to the endocytic uptake of FVIII by cells expressing LRP or LDLR.
Ikaros Drives Human Haemoglobin Switching by Facilitating Active Chromatin Hub Formation.
Authors: Perkins Andrew C. , Tallack Michael , Zhan Ye , Papathanasiou Peter , Goodnow Crhistopher , Gaensler Karin , Crossley Paul M. , Dekker Job , Keys Janelle R.
Published: April 2020

The human β globin locus consists of an upstream locus control region (LCR) and five functional genes arranged sequentially in the order of their expression during development: 5′-ε-Gγ-Aγ- δ- β-3′. Haemoglobin switching entails the successive recruitment of these genes into an active chromatin hub (ACH). Although much is known about the cis elements and transcription factors involved in globin gene regulation, less is known about ACH formation. Here we show that the transcription factor Ikaros plays an essential role in both the formation of the β-globin ACH, and in haemoglobin switching. In Plastic mice, where the DNA-binding region of Ikaros is disrupted by a point mutation (H191R), there is concomitant marked (10 fold) down-regulation of human β-globin, and up-regulation of γ-globin gene expression. We show Ikaros binds to a critical cis elements in the LCR near the HS3 core and upstream of the δ-globin gene in the β-globin locus by electormobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) and that this DNA binding activity is lost in Plast mice. This latter site is implicated in deletional hereditary persistence of fetal haemoglobin (HPFH). Furthermore, chromatin conformation capture (3C) data suggest Ikaros facilitates long range looping between the LCR and a region upstream of the δ-globin gene. This study provides new insights into the mechanism of adult stage-specific assembly of the β-globin ACH. In addition the findings could lead to the development of novel drugs to reactivate HbF in adults with β-thalassemia and sickle cell disease.
TARC and RANTES Enhance Immunological Antitumor Effect Induced by GM-CSF Transduced Leukemia Vaccine in Mouse Model.
Authors: Inoue Hiroyuki , Iga Mutsunori , Xin Meng , Kurita Ryo , Nakayama Masaharu , Suehiro Yoko , Nakazaki Yukoh , Takayama Koichi , Nakanishi Yoichi , Tani Kenzaburo
Published: April 2020

Granulocyte-macrophage colony stimulating factor (GM-CSF) has been postulated to be a critical mediator in the initial tumor immune response and obvious antitumor immunomodulationg activity of autologous GM-CSF gene-transduced tumor vaccine therapy for various tumors including leukemia has been reported. Previously we demonstrated GM-CSF gene transduction into murine monocytic leukemia cell line of WEHI3B eliminated the tumorigenesity in vivo and the tumor regression started reproducibly around 10 days after tumor injection followed by disappearance within a week. We analyzed the gene expression of the transiently established tumor masses by the serial analysis of gene expression (SAGE) method to identify molecules associated with the antitumor effect. We identified that chemokine genes of TARC and RANTES were preferentially expressed in the GM-CSF-transduced tumors and the contribution of the two genes to the regression of GM-CSF-transduced tumors was suggested. To confirm the finding, we observed in vivo tumor formation of WEHI-3B cells transduced with a retroviral vector expressing murine GM-CSF, TARC, RANTES, GM-CSF+TARC or GM-CSF+RANTES, respectively. One million cells of each transduced cell group and non-transduced cell group were inoculated subcutaneously into syngeneic BALB/c mice. Then tumorigenecity and survival rate were observed. Both of the combination of GM-CSF with TARC or RANTES gene transduction profoundly inhibited tumor formation. In addition, the 2 groups demonstrated significantly longer survival than GM-CSF transduced group. To examine their therapeutic potency, 3×10^6 of irradiated WEHI3B cells retrovirally transduced with murine GM-CSF cDNA in combination with either recombinant TARC or RANTES were subcutaneously inoculated every 4 days for 4 times into syngeneic BALB/c mice preliminarily inoculated subcutaneously with 1×10^5 of wild type WEHI3B cells. Vaccinations were well tolerated without any major adverse events. The mice treated with the GM-CSF transduced cells with chemokines demonstrated significantly longer survival than the mice treated with GM-CSF transduced cells. Splenocytes harvested from mice treated with the former vaccines produced higher levels of IL-4, IL-6, IFN-γ, and TNF-α, suggesting a reinforcement of innate immunity as well as adaptive immunity. Immunohistochemical findings of the tumor section after vaccine treatment revealed more significant contribution of CD4+, CD8+ T cells to tumor repression in the combined vaccine groups than GM-CSF vaccine group. In coclusion, TARC and RANTES enhance immunological antitumor effect induced by GM-CSF in mouse WEHI3B tumor models and would be useful as the second generation GM-CSF immune gene therapy targeting leukemia.
Aberrant Expression in Human Epithelial Cancers of the P2X5-Encoded Minor Histocompatibility Antigen LRH-1: Implications for Graft-Versus-Tumor Immunity Against Solid Tumors.
Authors: Dolstra Harry , Levenga Henriette , Overes Ingrid , Vos Johanna C.M. , van Horssen-Zoetbrood Agnes , van der Voort Robbert , de Mulder Pieter , de Witte T.M.
Published: April 2020

Allogeneic stem cell transplantation (SCT) in combination with donor lymphocyte infusion (DLI) is an experimental treatment for patients with metastatic solid tumors. The therapeutic efficacy is attributed to the graft-versus-tumor (GVT) response during which donor-derived T cells eliminate malignant cells via recognition of minor histocompatibility antigens (MiHA). To reduce accompanying GVHD, it is crucial to identify MiHA which are selectively expressed on hematopoietic cells and solid tumor cells. Previously, we identified a hematopoietic cell-restricted MiHA, designated LRH-1, which is presented by HLA-B7 and encoded by the P2X5 purinergic receptor gene (J. Clin. Invest.2005:115:3506–3516). Here, we report that LRH-1, in addition to its hematopoietic cell-restricted expression, is aberrantly expressed on epithelial tumor cell lines. We observed that P2X5 mRNA is significantly expressed in 14 out of 42 (33%) solid tumor cell lines tested by real-time quantitative RT-PCR. We detected P2X5 transcripts in 3 out of 11 renal cell carcinoma cell lines, 2 out of 4 melanoma cell lines, 3 out of 7 colorectal carcinoma cell lines, 4 out of 10 brain tumor cell lines and 2 out of 10 breast cancer cell lines. To determine whether P2X5 mRNA expression in solid tumor cell lines results in susceptibility to lysis by LRH-1-specific CTL, we performed flow cytometry-based cytotoxicity assays using P2X5-expressing tumor cell lines. Based on LRH-1 genotyping analysis, we selected six solid tumor cell lines for the cytotoxicity studies. Remarkably, LRH-1-specific CTL efficiently lysed and inhibited the growth of DAOY brain tumor cells up to 3 days of co-culture. The renal cell carcinoma cell lines SKRC-33 and SKRC-18 and the melanoma cell line BLM were also susceptible to LRH-1 CTL-mediated lysis, although less effectively. However, pre-incubation of these tumor cell lines with IFNγ and TNFα significantly increased the susceptibility to LRH-1-specific CTL and resulted in complete target cell lysis. Furthermore, these cytokine-stimulated cell lines induced higher levels of CTL degranulation as determined by CD107a staining. No cytotoxicity was observed against LRH-1-negative FM3 melanoma and SKRC-24 renal cell carcinoma cell lines. These findings illustrate that the GVT reactivity observed in solid tumors after allogeneic SCT may be selectively enhanced by LRH-1-specific immunotherapy.
Role of Glucocorticoid-Induced TNF-Related Protein (GITR) Ligand in the Interaction of Acute Myeloid Leukemia with NK Cells.
Authors: Krusch Matthias , Baltz Katrin M. , Baessler Tina , Kanz Lothar , Salih Helmut R.
Published: April 2020

NK cells play an important role in the reciprocal interaction of tumor cells with the immune system and participate in the surveillance and eradication of hematological malignancies. The activity of NK cells is governed by a balance of activating and inhibitory surface receptors. Glucocorticoid-induced TNF-related protein (GITR) and its ligand (GITRL) are members of the TNF/TNF receptor (TNFR) superfamily, which mediates multiple cellular functions including proliferation, differentiation, and cell death. Recently we reported that NK cells express GITR while cancer cells express GITRL and GITR-GITRL interaction down regulates NK cell-mediated anti-tumor immunity (Baltz et al., FASEB J 2007). Here we demonstrate that GITRL is expressed on 6 of 7 investigated acute myeloid leukemia (AML) cell lines and on primary AML cells in 30 of 52 (59%) patients, while no GITRL expression was detected on CD34+ cells of healthy donors (n=5). GITRL expression was not restricted to a specific French-American-British (FAB) subtype, but was significantly (p<0.05, one-way ANOVA) associated with monocytic (FAB M4, M5) differentiation. In addition, no association with a particular cytogenetic abnormality or with expression of MHC class I was observed. Reverse signaling via GITRL led to phosphorylation of ERK and JNK resulting in significantly (p<0.05, Mann-Whitney U-test) enhanced production of IL-10 and TNF by patient AML cells (n=10). In line, specific inhibitors for JNK and ERK1/2 blocked the cytokine release by AML cells demonstrating that activation of MAP kinases is responsible for the production of the immunoregulatory cytokines following GITRL stimulation. Importantly, blocking GITR-GITRL interaction in cocultures of AML and NK cells significantly (both <0.05 Mann-Whitney U-test) increased cellular cytotoxicity about 70% and IFN-γ production about 60%, and this was due to restored NK cell NF-κB activity. Thus, GITRL substantially influences immunoediting by AML cells and enables the escape of AML cells from NK cell-mediated immune surveillance. The correlation found between GITRL expression and NK cell susceptibility may provide useful information for NK cell-based immunotherapy.
Interplay of CXCR4 and FLT3-ITD in the Regulation of Stem Cell Migration and Proliferation.
Authors: Jacobi Angela , Ryser Martin F. , Koch Sina , Lehmann Romy , Bornhaeuser Martin , Brenner Sebastian
Published: April 2020

Activating mutations of the FLT3-receptor tyrosine kinase such as the short internal tandem duplication (ITD) represent the single most common genetic aberration in patients with acute myeloid leukemia (AML). FLT3-ITD mutations are present in about 25% of AML patients and are associated with high blast counts and a higher rate of relapse after conventional chemotherapy. CXCR4 and its ligand SDF-1 are of central importance for stem cell homing to the bone marrow. In FLT3-ITD positive AML, CXCR4 expression on stem cells is negatively correlated to overall survival. Using retroviral vector transduction we achieved overexpression of the human FLT-ITD transgene, the FLT3-wt or the GFP control gene in greater than 70% of cord blood hematopoietic progenitors (HP). In contrast to previously published data in murine cell lines, we found that FLT3-ITD positive human cells display strongly reduced migration towards the CXCR4 ligand SDF-1. Analysis of SOCS3 (suppressor of cytokine signaling 3) expression revealed strongly increased mRNA expression in FLT3-ITD overexpressing CD34+HP and FLT3-ITD positive AML blasts. Since SOCS3 is a known antagonist of CXCR4 signaling, increased SOCS3 levels might cause the observed inhibition of SDF-1 dependent migration. For individually tested CD34+HP from 6 cord blood donors, co-cultivation of FLT3-ITD overexpressing HP on the irradiated mouse stromal cell line M210-B4 or on primary mesenchymal stromal cells resulted in an increased formation of early cobble stones and a dramatic proliferation advantage for suspended cells compared to naïve, FLT3-wt or GFP-transduced HP. Addition of the CXCR4 chemokine inhibitor AMD3100 to the co-culture decreased the cobble stone formation and growth advantage of FLT3-ITD transduced cord blood HP. Similar results were obtained using FLT3-ITD positive AML cells from 3 patients. Using CFSE to track cell proliferation and PI/Annexin staining to analyze apoptosis, we demonstrate that the addition of AMD3100 had no effect on apoptosis but decreased the proliferation rate of FLT3-ITD positive blast cells. Using primary AML blasts and FLT3-ITD transgene overexpressing cord blood HP, we demonstrate that the robust FLT3-ITD mediated proliferation advantage can be partly inhibited by the CXCR4 antagonist AMD3100.