bluuebunny/crossref_metadata_2025 huggingface dataset:
A Hypomorphic Cbfb Allele Reveals a Critical Dosage-Sensitive Function of Core Binding Factors at the Earliest Stages of T Cell Development.
Authors: Maillard Ivan , Talebian Laleh , Li Zhe , Guo Yalin , Sugiyama Daisuke , Speck Maren E. , Pear Warren S. , Speck Nancy A.
Published: April 2020

The family of core binding factors includes the DNA-binding subunits Runx1-3 and the common non-DNA binding partner CBFβ. Runx1 and CBFβ are essential for the emergence of hematopoietic stem cells during fetal development, but not for stem cell maintenance during later ontogeny. Runx1 is also required for megakaryocyte differentiation, B cell development, and for the DN2 to DN3 transition in thymocyte development. Runx2/CBFβ are critical for normal osteogenesis, and Runx3 for CD4 silencing in CD8+ T cells, but their contribution to other steps of hematopoietic development is unknown. To examine the collective role of core binding factors in hematopoiesis, we generated a hypomorphic Cbfb allele (Cbfbrss). CBFβ protein levels were reduced by approximately 2–3 fold in fetuses homozygous for the Cbfbrss allele (Cbfbrss/rss), and 3–4 fold in fetuses carrying one hypomorphic and one knockout allele (Cbfbrss/−). Cbfbrss/rss and Cbfbrss/− fetuses had normal erythroid and B cell development, and relatively mild abnormalities in megakaryocyte and granulocyte differentiation. In contrast, T cell development was very sensitive to an incremental reduction of CBFβ levels: mature thymocytes were decreased in Cbfbrss/rss fetuses, and virtually absent in Cbfbrss/−fetuses. We next assessed the development of Cbfbrss/rss and Cbfbrss/− fetal liver progenitors after transplantation to irradiated adult recipients, in competition with wild-type (wt) bone marrow cells. Wt, Cbfbrss/rss and Cbfbrss/− fetal progenitors replenished the erythroid, myeloid and B cell compartments equally well. The overall development of Cbfbrss/rss T cells was preserved, although CD4 expression was derepressed in double negative thymocytes. In Cbfbrss/− chimeras, mature thymocytes were entirely derived from competitor cells. Furthermore, the developmental block in Cbfbrss/− progenitors was present at the earliest stages of T cell development within the DN1 (ETP) and DN2 subsets. Our data define a critical CBFβ threshold for normal T cell development, and they situate an essential role of core binding factors during the earliest stages of T cell development. In addition, early thymopoiesis appeared more severely affected by reduced CBFβ dosage than by the lack of Runx1 (Ichikawa et al., Nat Med 2004; Growney et al., Blood 2005), suggesting that Runx2/3 may contribute to core binding factor activity in the T cell lineage.
Newly Developed ADAMTS13 Antigen Assay Reveals Marked Ethnic Differences and Depletion in Acquired Thrombotic Thrombocytopenic Purpura.
Authors: Feys Hendrik B. , Liu Fang , Dong Ningzheng , Pareyn Inge , Vauterin Stephan , Vandeputte Nele , Ruan Changgeng , Hans Deckmyn , Karen Vanhoorelbeke
Published: April 2020

ADAMTS13 is a metalloprotease that cleaves the A2-domain of von Willebrand factor, resulting in a normal processed protein. A defective or blocked enzyme causes a severe disorder, called thrombotic thrombocytopenic purpura (TTP). The need for research tools, both clinical and fundamental, hence is urgent. To bypass the need for substantial amounts of highly purified antigen in order to produce monoclonal antibodies, we used genetic immunisation and an ADAMTS13 specific screening assay. Using this assay, high affinity hybridoma clones were selected and allowed us to set up a sensitive and reproducible sandwich ELISA. This antigen test is specific, as (i) plasma from a patient with acquired TTP but presenting without inhibitor, did not contain antigen and (ii) the binding of recombinant ADAMTS13 was inhibited by increasing amounts of ADAMTS13 in normal plasma. Antigen levels were measured relative to a standard of pooled normal human Caucasian plasma (n=20) that was arbitrarily set to 1 U/mL. Antigen levels in congenital TTP samples (34 ± 21 mU/mL, n =2) (mean ± SD) as well as in samples from patients with acquired TTP (231 ± 287 mU/mL, n=11) were clearly reduced when compared to normal Caucasian donors (951 ± 206 mU/mL, n=16). The samples from the above mentioned acquired TTP patients were taken before plasma exchange (PE). We found that antigen levels (513 ± 223, n = 11) raised significantly after PE therapy.

Figure Figure

Hence, ADAMTS13 antigen is severely reduced (< 0.23 U/mL) at presentation in most (81%) of the investigated acquired TTP cases. Therefore, ADAMTS13 antigen is an important parameter in both congenital and acquired TTP. We also showed that the antigen assay could distinguish mutation carriers (341 ± 125 mU/mL, n = 9) from non-carriers (668 ± 208 mU/mL, n = 11) within a genotyped family of a congenital Chinese TTP patient. We also found that normal Chinese donors have a significantly lower antigen titre (601 ± 129 mU/mL, n=15) than Caucasian normals. The consequences of this finding, on the molecular level, are however not clear.

Figure Figure
Sensitivity and Efficacy in Measurement of Anti-Platelet Medications.
Authors: Blain Rachelle , Nakagawa Peggy , Belvedere Melissa , Scherer Monika , Nugent Diane
Published: April 2020

INTRODUCTION: Large prospective studies must be performed to measure the true effect of clopidogrel (Plavix) or aspirin (ASA) in prevention of vascular disease. An easy and accurate point of service instrument will facilitate these studies and confirm effectiveness and compliance with medications. With such a variety of equipment available for analysis of platelet dysfunction, it is important to understand the sensitivity and efficacy of each assay when evaluating patients. We have found significant differences in our comparative studies of the PFA-100, optical aggregation, and Thrombelastograph (TEG) platelet coagulation in normal individuals using Plavix or ASA.

METHODS: We have compared these three techniques to evaluate their effectiveness in assessing ASA and Plavix inhibition of platelet function. Eighteen normal individuals, who had been examined previously for ASA effect on these instruments, were studied at baseline and after three days of Plavix 75mg daily. All samples were drawn and run simultaneously. The TEG platelet coagulation assay used whole blood in a sodium heparin tube instead of a 3.2 % citrate tube which was used for aggregation and the PFA-100. The PFA-100 whole blood assays included both collagen-epinephrine (EPI) and collagen-adenosine diphosphate (ADP) cartridges. The TEG platelet coagulation assays were run with arachidonic acid (AA) and adenosine diphosphate (ADP) as agonists. The optical aggregation used platelet rich plasma with AA and ADP agonists.

RESULTS: We found significant differences between the PFA-100, TEG platelet function, and standard optical aggregation in measurement of Plavix effect. Optical aggregation and the TEG platelet assay did show the effects of Plavix, with the aggregometer being more sensitive than the TEG platelet assay although much more time consuming. In additional studies, the Plavix inhibition became more striking in the TEG by extending treatment to 6 days, suggesting that future studies should be done after a week (rather than 3 days) to see full effect. In contrast, Plavix effect was absent on the PFA-100. All three assays did show the effects of ASA in previously identified aspirin- responsive individuals. Ideally, one instrument could be used to assess response to both agents. However, the sensitivity of the instrument and the pharmacology of these and future medications must play an important role in the selection of which assay will be most informative for these large population studies in the prevention of vascular disease.

Figure Figure
Incident TTP: Plasmapheresis Therapy-Related Complications Are Common.
Authors: Zakarija Anaadriana , McCaffrey Kathryn R. , Bandarenko Nicholas , Pandey Dilip , Cursio John , Tevar Amul , Kwaan Hau , Yarnold Paul R. , Burton Maddie , Bennett Charles L.
Published: April 2020

Background: Plasmapheresis is the mainstay of therapy for thrombotic thrombocytopenic purpura (TTP). Due to the significant mortality associated with TTP if left untreated and the effectiveness of therapy, plasmapheresis may be instituted before the diagnosis is clearly established. Our case-control study of incident TTP allowed us to evaluate the incidence of complications in cases treated at multiple sites across the United States.

Methods: The SERF-TTP study is a NHLBI-funded 15-site study to identify 300 cases of incident TTP and 600 age and gender-matched community controls. Each case of incident TTP is identified upon referral for therapeutic plasma exchange (TPE). Exclusion criteria include solid organ or allogeneic stem cell transplant, anti-neoplastic therapy, or metastatic adenocarcinoma. Epidemiologic information, laboratory data, and plasma samples are collected from both patients and controls. In addition, information is collected about volume of TPE, frequency, type of replacement fluid, duration of TPE, adverse reactions associated with TPE, and concurrent therapy.

Results: To date, 11 sites across the US have IRB approval, and 50 cases have been identified. Data are available for the first 32 patients, whose mean age is 41.9 years. All patients underwent TPE, a number of which received more than one type of replacement fluid. The majority of patients received fresh frozen plasma (FFP): 60.0% FFP, 46.7% cyroprecipitate poor plasma, and 16.7% albumin. Additionally, 63.3% of patients received concurrent corticosteroid therapy. The mean number of treatment days prior to the achievement of platelet count > 150,000 per microliter was 9.7 (range 0–43 days). Overall survival was 90.0%. Adverse reactions were observed in 53.3% of patients (16/30). The most frequently observed toxicity was an allergic reaction in 40% (12/30) of patients, followed by citrate-related toxicity in 30% (9/30) of patients. No patients experienced an anaphylactic reaction. 16.7% of cases (5/30) had venous access complications, most frequently a catheter-associated thrombosis (3/5). In addition, one patient had a significant hemorrhage associated with venous access, which required treatment in intensive care.

Conclusions: Preliminary data from this cohort of patients suggest that adverse reactions are commonly seen in incident TTP patients undergoing TPE; the most frequent are allergic reactions to plasma. This highlights the need for development of reliable diagnostic criteria for TTP so patients are not exposed to the risks of TPE unnecessarily.
Assessment of Bleeding in Thrombocytopenic Patients (pts) with Immune Thrombocytopenic Purpura (ITP).
Authors: Page Lemke K. , Elish Andrew S. , Provan Drew , Myrint Steven , Hamilton Michael , Jenkins Julian , Bussel James B.
Published: April 2020

Introduction: Assessment of bleeding in ITP is difficult. There is no agreed upon methodology; the WHO bleeding score does not seem useful for ITP. We report an ITP bleeding score including the relationship between site-specific bleeding severity (BLS), platelet count (plt ct), and plt size.

Methods: A bleeding score was created which has 11 sites, 9 by history (Hx) of the previous week: skin, oral, epistaxis, GI, GU, GYN, hemoptysis, subconjunctival (SC), and intracranial hemorrhage (ICH); and 2 by Exam (PE): skin and oral. The BLS grades (gr) pts for each site on a defined scale from no bleeding (0) to “major” bleeding (2). Plt cts and large plts (LP) were measured on the Bayer Advia 120. No pt had > 2 study visits.

Results: The 65 ITP pts on 100 visits over 6 months had a median (mdn) plt ct of 41,500/uL (41.5k). The mdn age was 31.5 yrs; 20 pts were children. 56 pts had chronic ITP, 26 s/p splenectomy, with a mdn duration of ITP of 6 yrs. There were no GU, SC hemorrhage (hem), or ICH. 72% of all gr 1 hem’s were skin whereas 46% of gr 2 hem’s were oral. There was 1 GI hem and 3 hemoptysis visits that were gr 2. 42 women on 63 visits had 5 visits each with GYN BLS gr 1 and gr 2. X2 testing correlated plt ct and BLS for 6 sites: both oral and both skin sites, epistaxis, and GYN (p’s < 0.05). Oral PE correlated best with the plt ct (fig 1). For oral PE, BLS of gr 2 did not occur at plt cts > 20k.

When the BLS was scored independently by two observers (LKP & JB) on 63 of the100 visits, 92% of individual BLS grs were identical (PearsonR=0.75). Skin PE and epistaxis each had 11/63 discrepancies. In addition, 16 BLS for oral (15 for skin) were different comparing Hx and PE (fig 2).

LP did not correlate well with site-specific BLS when only plt cts ≤ 30k were included, suggesting that large, young platelets may not primarily determine bleeding severity in ITP. However for Skin Hx, if the BLS was gr 0, 4/6 had LP ≥ 2 whereas only 7/28 LP were ≥ 2 when the BLS was gr 1.

Conclusions: The Bleeding Score reported here was validated by: a) the relationship of bleeding severity at multiple sites to plt ct including the absence of gr 2 bleeding at plt cts >20k; and b) the high degree of interobserver reliability despite a low but irreducible error rate based on variability in pt recall. It takes ≤ 3–5 minutes to complete. The significance of LP remains unclear. Studies of the BLS in larger numbers of patients will further determine its usefulness.

Figure 1. Bleeding Severity for Oral Cavity (PE) Versus Platelet Count Figure 1. Bleeding Severity for Oral Cavity (PE) Versus Platelet Count

Figure 2. Comparision of Oral PE to Oral Hx Figure 2. Comparision of Oral PE to Oral Hx
Effective Depletion of Alloreactive T Cells by CD95l-Transduced Cells with Preservation of FoxP3 Regulatory T-Cells.
Authors: Jacobsen Eva-Maria , Wenger Till , Herr Ingrid , Strauss Gudrun , Debatin Klaus-Michael , Friedrich Wilhelm , Muller Susanna M.
Published: April 2020

Adoptive transfer of donor T-cells after stem cell transplantation across HLA barriers could bridge the period of prolonged immunodeficiency until new thymic derived T-cells develop. In such an approach, however, donor T-cells reactive to alloantigens of the recipient induce GvHD, necessitating their selective elimination, inactivation or modulation. Depletion of alloreactive cells has been attempted by monoclonal antibodies, but prevention of GvHD was insufficient in clinical application so far. We developed a system allowing efficient selective depletion of alloreactive cells by CD95L-mediated apoptosis, which in addition has the advantage of preserving FoxP3 regulatory T-cells. An apoptosis-resistant B-cell line (C1RA1) was transduced with lentiviral CD95L (C1RA1-CD95L). CD95L was stably expressed by 65–80% of the cells and was fully functional to induce apoptosis in Jurkat cells. MNC of donor A were stimulated with APC of donor B, and, after 5 days of culture, viable responder cells were enriched and cultured with C1RA1-CD95L for another 3 days. T cells obtained by this culture system had lost proliferative activity towards cells of donor B, while proliferation against 3rd-party cells was fully preserved. Likewise, cytotoxic activity against donor B was markedly reduced. Since CD4+CD25+ T-cells were still detectable at levels of 7 to 10% of MNC at the end of the culture despite effective depletion of alloactivated cells, we analyzed for the presence of FoxP3+ regulatory T-cells (Treg). While gene expression of FoxP3 was almost undetectable in MNC before culture, it was present after activation of MNC and was strongly expressed after induction of apoptosis with C1RA1-CD95L at the end of our culture system. Suppression of proliferation of about 50% by these cells was noted in a suppressor assay using fresh MNC from donor A stimulated with cells of donor B. When suppression was analyzed in a stimulation system with 3rd-party stimulatory cells, reduction of proliferation was less prominent, indicating the possibility that the suppressive activity of Treg preserved in our culture system could be alloantigen-specific. In summary, we have developed a culture system, allowing alloantigen-specific depletion of T cells while preserving FoxP3+ regulatory T-cells. This sequential system of alloantigen-specific stimulation followed by CD95L-mediated apoptosis of activated cells by an unrelated, modified cell line offers the advantage of broad applicability in clinical transplantation settings.
Long-Term Engraftment and Self-Renewal of AML Stem Cells in the Newborn NOD-scid/IL2rgnull Immunodeficient Mouse Model.
Authors: Yoshida Shuro , Ishikawa Fumihiko , Yasukawa Masaki , Miyamoto Toshihiro , Yoshimoto Goichi , Kawano Noriaki , Shimoda Kazuya , Akashi Koichi , Shultz Leonard D. , Harada Mine
Published: April 2020

Transplantation of human leukemic cells into severe combined immunodeficiency (SCID) mice has been used to analyze developmental mechanisms of human leukemogenesis. Previous models, however, were limited in efficient or long-term engraftment of leukemia initiating cells. Here we report a new SCID model that supports highly efficient long-term engraftment of primary human acute myelogenous leukemia (AML) cells. We have established a novel immune-compromised mouse by backcrossing a complete null mutation of the common cytokine receptor g chain onto NOD-scid mice (NOD/SCID/IL2rgnull mice), and reported that normal human cord blood-derived hematopoietic stem cells efficiently engrafted in newborn NOD/SCID/IL2rgnull mice as compared to NOD/SCID/b2mnull mice (Ishikawa et al, Blood in press). Injection of 5x106 total bone marrow mononuclear cells from primary AML patients (FAB subtypes: M1, M2, M3, M4 and M7) into sublethally-irradiated newborn NOD/SCID/IL2rgnull mice, however, did not result in efficient engraftment of AML cells, while predominant proliferation of human CD4+ and CD8+ T cells was seen. These human T cells expressed CD45RO, and levels of human IFN-g in sera of the recipients significantly elevated, suggesting that human T cells were activated and inhibited the engraftment of human AML cells in the xenogeneic setting. We thus transplanted AML cells after T cell depletion. Strikingly, transplantation of 4x106 T cell-depleted AML bone marrow cells into neonatal NOD/SCID/IL2rgnull mice resulted in the efficient AML engraftment, whose levels were significantly higher than those in transplantation of the same number of T cell-depleted AML cells into NOD/SCID/b2mnull newborns or NOD/SCID/IL2rgnull adults. We also transplanted 103–104 hCD34+hCD38− bone marrow cells purified from AML patients. These low-doses of hCD34+hCD38− cells also successfully engrafted, progressively giving rise to hCD34+hCD38+ and hCD34− leukemic cells over 16 weeks. hCD34+hCD38− cells purified from the bone marrow of primary NOD/SCID/IL2rgnull recipients again reconstituted AML in secondary recipients, indicating that this system supports self-renewal capacity of AML stem cells within the hCD34+hCD38− fraction. Thus, the NOD/SCID/IL2rgnull newborn system provides a powerful model to study human leukemogenesis as well as the interaction between human T cells and AML cells in vivo.
Divergent Roles of Histone Deacetylases in Human Adult Eythropoiesis.
Authors: Ohishi Kohshi , Katayama Naoyuki , Yamamura Kentaro , Heike Yuji , Takaue Yoichi , Shiku Hiroshi
Published: April 2020

Histone deacetylases (HDACs) plays a regulatory role in the expression and activity of transcription factors. Several studies suggested that HDACs influence the proliferation and differentiation of hematopoietic progenitors. Here, we studied the role of HDACs in human adult erythropoiesis, using a HDAC inhibitor FK228 [depsipeptide]. We first examined the effect of FK228 on the growth of erythroid progenitors by incubating G-CSF-mobilized peripheral blood CD34+ cells (500 cells/well) in semisolid serum-free cultures containing IL-3, SCF, or IL-3+SCF in the presence or absence of FK228. Addition of FK228 (0.5 ng/ml) to the cultures with IL-3 and SCF+IL-3 resulted in 32-fold and 12-fold increases, respectively, in the number of erythroid colonies that consist of CD36+ glycophorin A (GPA)low/negative cells, compared with the cultures without FK228. FK228 also increased the number of cells comprising erythroid colonies in the cultures with IL-3 and SCF+IL-3. No colony growth was detected with SCF and SCF+FK228. We next examined the effect of FK228 on BFU-E colony formation by incubating CD34+ cells with EPO or SCF+EPO in the presence or absence of FK228. While BFU-E colony formation was observed with SCF+EPO but not with EPO alone, addition of FK228 to the cultures containing SCF+EPO almost completely inhibited BFU-E colony formation. To further elucidate the effect of FK228 on BFU-E progenitors that grow in an EPO-dependent fashion, CD34+ cells were cultured for 7 days with SCF, Flt3L, TPO, and IL-3, and CD36+ cells were purified. Although BFU-E colony formation was induced by incubation of the CD36+ cells with EPO, it was strongly repressed by the presence of FK228. Suspension cultures of CD36+ cells revealed that viable cell numbers in the cultures with EPO+FK228 rapidly declined from 2 days of cultures, in contrast to an increase in the number of erythroid cells in the cultures with EPO. Annexin V/propidium iodide assay showed that, upon exposure to FK228, a remarkably higher fraction of the CD36+ erythroid progenitors expressed an annexin V+PI− phenotype, indicative of an apoptotic cell death, compared with the control cultures (71 ± 8% vs 4 ± 1%). On the other hand, even though FK228 blocked the BFU-E colony formation from CD34+ cells in the presence of SCF+EPO, FK228 did not significantly increase the population of cells that undergo an apoptotic cell death in CD34+ cells. These findings suggest that HDACs, while playing an essential role in SCF/EPO- and EPO-dependent erythropoiesis, negatively regulate the action of IL-3 and IL-3+SCF on the growth of early erythroid progenitors. Our results indicate that HDACs have diverse functions in human adult erythropoiesis.
Conversion of Alloantigen-Specific CD8+ T-Cell Anergy to T-Cell Priming through In Vivo Ligation of Glucocorticoid-Induced Tumor Necrosis Factor Receptor.
Authors: Kim Hawk , Kwon Byungsuk , Kim Juyang , Choi Woon S. , Kim Hye J. , Suh Jae-Hee , Sakaguchi Shimon
Published: April 2020

In this study, we investigated the effect of an agonistic mAb (DTA-1) against glucocorticoid-induced tumor necrosis factor receptor (GITR) in a murine model of systemic lupus erythematosus (SLE)-like chronic graft-versus-host disease (cGVHD). A single dose of DTA-1 inhibited the production of anti-DNA IgG1 autoantibody and the development of glomerulonephritis, typical symptoms of cGVHD. DTA-1-treated mice showed clinical and pathological signs of acute GVHD (aGVHD), such as lymphopenia, loss of body weight, increase of donor cell engraftment, and intestinal damage, indicating that DTA-1 shifted cGVHD towards aGVHD. The conversion of cGVHD to aGVHD occurred because DTA-1 prevented donor CD8+ T cells from being anergic. Functionally active donor CD8+ T cells produced high levels of IFN-γ and had an elevated CTL activity against host antigens. In in vitro MLR, anergic responder CD8+ T cells were generated and DTA-1 stimulated the activation of responder CD8+ T cells that were fated to be anergic. We further confirmed in vivo that donor CD8+ T cells, but not donor CD4+ T cells, are responsible for the DTA-1-mediated conversion of cGVHD to aGVHD in a CD4+CD25+ regulatory T cell-independent manner. These results indicate that donor CD8+ T-cell anergy is a restriction factor in the development of aGVHD and that GITR stimulation prevent CD8+ T-cell anergy by activating CD8+ T cells prone to anergy. In sum, our data suggest GITR as an important costimulatory molecule regulating cGVHD versus aGVHD and as a target for therapeutic intervention in a variety of related diseases.
Toll like Receptor 4 Is Involved in In VIvo Antiphospholipid-Mediated Thrombosis and Endothelial Cell Activation.
Authors: Pierangeli Silvia S. , Vega-Ostertag Mariano E. , Raschi Elena , Liu Xiaowei , Borghi Maria O. , Meroni Pier Luigi
Published: April 2020

Background: Antiphospholipid antibodies (aPL) are associated with thrombosis and pregnancy loss in patients with Antiphospholipid Syndrome (APS). aPL and bacterial lipopolysaccharide (LPS) bind to and activate endothelial cells (EC) through NFκB and p38 MAPK pathways. Recent studies suggest that aPL might interact with toll-like receptor-4 (TLR-4), the receptor for LPS.

Aim: to investigate the role of TLR-4 in antiphospholipid syndrome (APS).

Methods: we examined: i) the aPL effects on thrombosis and EC activation in LPS non-responsive (LPS−/−) mice that display a spontaneous mutation of TLR-4 vs LPS-responsive (LPS+/+) mice displaying wild type TLR-4, ii) the prevalence of TLR-4 Asp299gly and Thr399Ile polymorphisms - both associated with decreased response to LPS - in 110 APS patients (with arterial and/or venous thrombosis) vs 220 controls (of same ethnic background). IgGs were purified from a patient with APS (IgG-APS) and from control serum (IgG-NHS). LPS −/− and LPS +/+ mice, in groups of nine, were treated with IgG-APS or with IgG-NHS twice intraperitoneally. Size of induced thrombi and # of leukocyte (WBC) adhering to endothelial cells in the microcirculation of endothelium of the cremaster muscle of mice (as a means to measure endothelial cell activation) were determined in vivo, seventy-two hours after the first injection. TLR-4 Asp299gly & Thr399Ile polymorphisms were evaluated by Allele-Specific PCR.

Results: LPS +/+ mice treated with IgG-APS produced significantly larger thrombi when compared to mice treated with IgG-NHS (2166 ± 1419 μm2 vs 1176 ± 841 μm2) and significantly larger number of WBC adherence to ECs (4.5 ± 1.9 vs 2.2 ±1.1). Thrombus size and number of adhering WBC to ECs were significantly lower in LPS −/− mice treated with IgG-APS compared to LPS +/+ mice treated with IgG-APS [thrombus size: 779 ± 628 μm2 vs 2166 ± 1419 μm2 (p<0.05) and number of adherent WBC to EC: 1.0 ± 0.5 vs 4.5 1.9 (p<0.05)], respectively. The titer of anticardiolipin antibodies in the sera of mice injected with aPL was 48.2 ± 17.1 GPL (for LPS −/− mice) and 50.8 ± 11.2 GPL (for LPS +/+ mice), respectively (NS). A significant reduction in TLR-4 Asp 299gly & Thr399Ile polymorphisms was observed in APS patients (5%) compared to controls (11.4%) (p<0.05).

Conclusions: These findings strongly suggest that TLR-4 is involved in aPL interaction with endothelial cells and mediates their pathogenic effects.
A t(8;21) Fusion Protein Disrupts the Spindle Checkpoint and Promotes Aneuploidy.
Authors: Boyapati Anita , Peterson Luke F. , Yan Ming , Le Beau Michelle M. , Zhang Dong-Er
Published: April 2020

The 8;21 translocation occurs in approximately 8% of patients with acute myeloid leukemia. This chromosomal lesion results in the fusion of the AML1 (RUNX1) gene on chromosome 21 to the ETO (MTG8) gene on chromosome 8 and generates the AML1-ETO fusion protein. We identified a truncated form of AML1-ETO which lacks approximately 200 amino acids at the C-terminus of full length AML1-ETO and strongly induces leukemia development (Yan et al, PNAS101(49): 17186–91, 2004). This protein is almost identical to a natural splice form of AML1-ETO designated AML1-ETO9a, which is expressed in t(8;21) patients. To study the leukemogenic properties of the truncated form of AML1-ETO (AEtr), we examined cell cycle profiles to compare it to full length AML1-ETO. In contrast to full length AML1-ETO which induced growth arrest of K562 cells, AEtr did not inhibit cell proliferation. Interestingly, AEtr expressing cells continued to proliferate in the presence of microtubule toxins that normally activate the spindle checkpoint and arrest cells in prometaphase of mitosis. Evaluation of critical components of the spindle checkpoint revealed a significant decrease in BubR1 protein in AEtr cells compared to vector control cells. BubR1 is a potent inhibitor of the anaphase promoting complex and reduction of BubR1 by either siRNA approaches or gene deletion in mice results in aneuploidy. Cytogenetic analyses of leukemic splenocytes from mice transplanted with AEtr cells identified multiple chromosomal abnormalities, including chromosome loss and gain as well as translocations. Observations of chromosomal alterations in t(8;21) patients have been well documented and include loss of either sex chromosome in 70% of patients, deletions of chromosome 9, and trisomy 8. These results indicate that inactivation of the spindle checkpoint through BubR1 reduction may be an important event in the development of t(8;21) leukemias.
Inactivation of NIPA Results in Premature Mitotic Entry and Subsequent Mitotic Catastrophe.
Authors: Bassermann Florian C. , Muench Silvia , von Klitzing Christine , Morris Stephan W. , Peschel Christian , Duyster Justus
Published: April 2020

Ubiquitin-mediated destruction of regulatory proteins marks a vital means of controlling cell cycle progresssion. The E3 ubiquitin-ligases are prominent in this process as they determine specificity of the ubiquitination process and thus regulate proteasomal degradation of target proteins. Recently, a class of E3 ligases referred to as SCF complexes has been identified. The substrate binding specifity within this class of E3 ligases is mediated by a class of molecules termed F-box proteins. We previously reported the cloning of NIPA (nuclear interaction partner of ALK) in complex with constitutively-active oncogenic fusions of ALK, which contribute to the development of certain lymphomas and sarcomas. Subsequently we characterized NIPA as a human F-box protein that defines an oscillating ubiquitin ligase (SCF-NIPA) which targets nuclear cyclin B1 in interphase. We have now determined the consequence of inactivating NIPA with regard to cell cycle regulation using an RNAi approach. Kinetic analysis of cell cycle phase transition times revealed a premature onset of Cdk1/cyclin B1 kinase activity and early mitotic entry in cells treated with NIPA siRNA. Cyclin B1 was shown to accumulate within the nucleus in these cells correlative to a reduced ubiquitination activity of the SCF-NIPA complex. Subsequent to premature mitotic entry, NIPA inactivated cells arrested in prometaphase and mitotic catastrophe was observed thereafter. We searched for relevant proteins involved in this process and found Survivin, a member of the inhibitor of apoptosis proteins (IAP) to be strongly downregulated in NIPA siRNA treated cells. Survivin has been shown to function as a mitotic checkpoint molecule that induces mitotic catastrophe subsequent to aberant mitosis when inactivated or downregulated. While the precise functional relationship between Survivin and NIPA is still under investigation, the current data distinguishes NIPA as a central molecule in timing mitotic entry. Given this function, NIPA directly influences the fidelity of DNA replication and segregation. Interference with NIPA function may therefore be an oncogenic principle that favours genomic instability in tumor cells.
Elevation of Kinase Interacting with Stathmin (KIS) and Promotion of Cellcycle Progression by KIS in Leukemia Cells.
Authors: Nakamura Satoki , Ono Takaaki , Sugimoto Yuya , Kobayashi Miki , Sahara Naohi , Shinjo Kaori , Shigeno Kazuyuki , Ohnishi Kazunori
Published: April 2020

Purpose: The protein p27 is an important regulator of cell cycle. An increase in p27 causes proliferation of cells, while a decrease in p27 induces quiescence of cells. p27 is regulated by transcriptional, translational and proteolytic mechanisms. Among them, an importnant mechanism in the regulation of p27 is proteolysis. Kinase interacting with stathmin, KIS, is a serine / threonine kinase, and regulates cell cycle progression through the phosphorylation of p27 on serine 10. The S10 phosphorylation on p27 plays an important role in p27 degradation. KIS that phosphorylates p27 on S10 and its role in the regulation of cell cycle progression have not been defined in leukemia cells. In this study, we investigated the role of KIS in leukemia cells.

Materials and Methods: We examined the biological significance of KIS expression in K562, NB4, U937, CEM, MOLT4, and SUP-B15 leukemia cells and relationship with the G1 regulaters, such as p27. Moreover, we generated the lentivirus vector inserted with the dsDNA of KIS small interfering RNA (siRNA), and effects of down-regulation of KIS by siRNA transfection were investigated in leukemia cells.

Results: RT-PCR and western blot analysis showed high KIS expression in all leukemia cells. The p27 phosphorylation on S10 was significantly lower in the leukemia cells transduced with KIS siRNA and depletion of KIS enhanced growth arrest. The down-regulation of KIS induced G1 arrest in cell cycle, but not apoptosis. In cell cycle analysis, control leukemia cells showed 42.3 ± 1.8 %, but leukemia cells transfected with KIS siRNA showed 67,1 ± 2.1 % in G1 fraction. Moreover, these cells significantly showed small population in S ansd G2 fractions compared compared with controls.

Conclusion: These findings suggest KIS expression promotes cell cycle progression in leukemia cells. Depletion of KIS using siRNA in leukemia cells induced cell cycle arrest in G1 phase compared with control cells. In this study, we showed that KIS might be the target for the molecular therapy.
Human CD34+ Cells Expressing CBFβ-MYH11 Exhibit a Myelomonocytic Phenotype with Greatly Enhanced Proliferative Ability.
Authors: Wunderlich Mark , Krejci Ondrej , Wei Junping , Lu Thomas X. , Mulloy James C.
Published: April 2020

The Core Binding Factor (CBF) transcription factor complex is required for normal hematopoiesis. Knockout of either component of the complex, AML1 or CBFβ, results in embryonic lethality in mice due to a lack of definitive hematopoiesis. Chromosomal rearrangements involving the CBF genes are commonly associated with leukemia. The t(8:21) fuses the AML1 gene to ETO (AE) and is predominantly observed in leukemias of the FAB M2 subtype of acute myeloid leukemia (AML). The t(16:16) and inv(16) are associated with FAB M4Eo leukemias and result in fusion of the CBFβ gene to MYH11 (CM). While knockin of either AE or CM is not sufficient to cause disease, expression of either protein increases the incidence of leukemia in mice when combined with mutagens or specific secondary genetic manipulations. We have previously shown that retroviral transduction of primary human CD34+ cells with AE causes a dramatic accumulation of CD34+ progenitor cells with increased CFU-C and CAFC activity during an increased lifespan in vitro. These cultures retain the ability to differentiate to myeloid, lymphoid, and erythroid lineages and the ability to engraft NOD/SCID mice. Consistent with mouse studies, these cells fail to cause disease in mice or become transformed in vitro. These cultures serve as a preleukemic model for human AML associated with AML1-ETO expression. We show here that retroviral transduction of CM into human CD34+ hematopoietic cells similarly mimics a putative pre-leukemic condition initiated by inv(16). Transduced cells proliferate for up to 7 months in culture with retention of CD34 expression on a subset of cells. Cells from CM cultures display a more mature morphology than AE cells, consistent with the association with M4Eo AMLs, and have a greatly reduced ability to differentiate to the erythroid lineage in methylcellulose assays. Nevertheless, long-term CM cultures retain CFU-C activity, the ability to engraft NOD/SCID-B2M mice, and the potential for B-cell development in vitro, implicating a primitive progenitor as the target cell rather than a committed granulocyte/monocyte progenitor. Microarray analysis of AML leukemias has identified specific genes with altered expression in inv(16) patient samples. We have found that a number of these inv(16)-associated transcripts are likewise altered in CD34+ cells from our CM cultures when compared to control CD34+ cells, while others are not affected. These data suggest that our system will be useful in examining the effects of CBFβ-MYH11 on gene expression in the human preleukemic cell, in characterizing the effect of this oncogene on human stem cell biology and in defining its contribution to the development of leukemia.
Leukemic CD34+CD38- Cells Display Conserved Differential Expression of Many ATP Binding-Cassette Transporter Genes.
Authors: Raaijmakers Marc H.G.P. , de Grouw Elke P.L.M. , van de Locht Louis T.F. , van der Reijden Bert A. , de Witte Theo J.M. , Jansen Joop H. , Raymakers Reinier A.P.
Published: April 2020

In most cases of acute myeloid leukemia (AML) CD34+CD38− cells are considered to be stem cells, responsible for the maintenance and relapse of AML. ATP binding cassette transporters function in the extrusion of xenobiotics and chemotherapeutical compounds, and may be involved in therapy resistance. Elucidation of mechanisms conferring drug resistance to CD34+CD38− cells is essential to provide novel targets for stem cell eradication in AML. We studied gene expression of all 45 transmembrane ABC transporters (the complete ABCA, B, C, D and G family) in human hematopoietic CD34+CD38− cells and more committed CD34+CD38+ progenitor cells, from healthy donors and patients with non-hematological diseases (N=11) and AML patients (N=11). Gene expression was assessed using a novel real-time RT-PCR approach with micro fluidic cards. In normal CD34+CD38− cells 36 ABC transporters were expressed, 22 of these displayed significant higher expression in the CD34+CD38− cell fraction compared to the CD34+CD38+ cell fraction. In addition to the known stem cell transporters (ABCB1, ABCC1 and ABCG2) these differential expressed genes included many members not previously associated with stem cell biology. In AML the ABC transporter expression profile was largely conserved, including expression of all 13 known drug transporters. These data suggest an important role for many ABC transporters in hematopoietic stem cell biology. In addition, the preferential expression of a high number of drug transport related transporters predicts that broad spectrum inhibition of ABC transporters is likely to be required for CD34+38− stem cell eradication in AML. This approach will, apart from affecting the leukemic stem cells, equally affect the normal stem cells.
Phenotyping of Neoplastic (CD34+/CD38−/CD123+) Stem Cells in Myeloid Malignancies Reveals Expression of Multiple Molecular Targets.
Authors: Florian Stefan , Sonneck Karoline , Hauswirth Alexander W. , Krauth Maria-Theresa , Sperr Wolfgang R. , Valent Peter
Published: April 2020

Recent data suggest that myeloid neoplasms are organized hierarchically in terms of self renewal and maturation of early progenitor cells, similar to normal myelopoiesis. In acute myeloid leukemia (AML), the NOD/SCID mouse-repopulating leukemic stem cells usually co-express CD123 with CD34, but lack CD38. So far, however, little is known about expression of other markers and targets on these progenitors. In the present study, expression of target antigens on CD34+/CD38− cells was analyzed by multicolor flow cytometry in patients with AML (n=18), myelodysplastic syndromes (MDS, n=6), chronic myeloid leukemia (CML, n=8), systemic mastocytosis (SM, n=9), and normal bone marrow (n=5). The IL-3Ra chain (CD123) was found to be expressed on CD34+/CD38− cells in a majority of all patients in all disease-categories. Independent of the type of disease, the vast majority of these stem cells also co-expressed aminopeptidase-N (CD13) and the target receptor CD44 in all patients. CD34+/CD38− progenitor cells expressed variable amounts of the Mylotarg® receptor CD33, KIT (CD117), HLA-DR, and AC133 (CD133). With regard to AC133, two distinct subpopulations of progenitor cells were detected in many cases, namely a CD133+ and a clearly CD133- cell-fraction. In patients with AML, the levels of CD33 varied from patient to patient with a broad range of reactivity, whereas in most patients with MDS, CML, and SM, CD33 was found to be consistently expressed on most progenitors. In most patients, neoplastic stem cells did not express substantial amounts of the GM-CSF receptor alpha chain (CD116), Thy-1 (CD90), E-NPP3 (CD203c), MDR-1 (CD243), or PAR-2. In the normal bone marrow, CD34+/CD38− cells co-expressed CD13, CD44 and CD45, but did not express CD33, CD116, or CD123. In conclusion, neoplastic stem cells in various myeloid neoplasms appear to express a similar phenotype including target receptors such as CD13, CD33, and CD44. These antigens may thus be attractive targets of therapy in AML. However, since many of these targets are not expressed on all stem cells in all patients, the elimination of the entire clone may require combinations of targeted antibodies or use of additional drugs. In other cases (CD13, CD44, CD45), the target antigen is also expressed on normal stem cells, so that targeted therapy is likely to be an ablative maneuver and thus would require a combined stem cell transplantation approach.
The Centromeric Protein, CEN(P)-F, a Marker of Cell Proliferation Is Regulated by Hypoxia in Human Mesenchymal Stem Cells and Their Bone Marrow Stromal Progeny.
Authors: Watt Suzanne M. , Hale Sarah , Baban Dilair , Roubelakis Maria , Moukayed Meis , Lim Jaeseung , Ryan Dacey J. , Davies Kay , Harris Adrian L. , Martin-Rendon Enca
Published: April 2020

Human bone marrow mesenchymal stem cells (MSC) are multipotent progenitors that generate osteoblasts, chondrocytes, adipocytes, myoblasts and the bone marrow stromal cells that support hematopoiesis. Although the bone marrow microenvironment is hypoxic, little is known about the maintenance and response of MSC and their bone marrow stromal progeny to hypoxia. Using cDNA microarray hybridization technologies, we show, for the first time, that a total of 231 mRNAs in cultured MSCs are regulated by short exposures (4–48hrs) to hypoxia. These include known hypoxia-responsive genes, such as BHLHB2, PGK1, GLUT-1 and VEGF. Interestingly, we demonstrate that a significant proportion of genes involved in cell growth, proliferation or survival are also regulated by hypoxia in these cells. Amongst these, we have identified the centromeric protein CENP-F as a novel gene up-regulated in cultured MSCs by hypoxia. This up-regulation results in an increased level of CENP-F protein. The hypoxic stimulus also enhances cell division in the bone marrow MSC-derived stromal cells, resulting in a doubling in cell number within 24hrs. This contrasts with the effects of hypoxia on mature endothelial cells (EC), where recruitment into cell cycle is unchanged. Our demonstration that hypoxia promotes cell division and cell proliferation in the bone marrow stromal progeny of human MSC may have wider implications for the regulated growth and survival of other stem cells in hypoxic microenvironmental niches.
Mesenchymal Stem Cells (MSCs) Suppress T Cell Proliferation by a Novel NO-stat5 Dependent Mechanism.
Authors: Sato Kazuya , Ozaki Katsutoshi , Oh Iekuni , Hatanaka Keiko , Nagai Tadashi , Muroi Kazuo , Ozawa Keiya
Published: April 2020

Mesenchymal stem cells (MSCs) are attractive source for regenerative therapy as they have been shown to be capable of differentiating into adipocytes, chondrocytes, osteoblasts, myocytes, cardiomyocytes, and neural precursors. MSCs have also been shown to suppress T cell proliferation in vitro and were reported to be effective as a treatment for acute graft-versus-host disease (GVHD) but the underlying molecular mechanisms for T cell suppression are uncertain. So far, TGF-β, HGF, and PGE2 were shown to be candidates as molecules causing the suppression. To address the molecular mechanisms, we used primary mouse MSCs derived from bone marrow cells and CFSE (carboxyfluorescein diacetate succinimidyl ester) or thymidine uptake for T cell proliferation assay. Co-culture of MSCs inhibited T cell proliferation induced by PMA plus Ionomycin, suggesting that TCR and signaling molecules interacting with TCR such as Lck and ZAP70 are not involved and that downstream signals of PMA plus Ionomycin are essential for the suppression by MSCs. The proliferation of either purified CD4 or CD8 cells induced by PMA plus Ionomycin was also inhibited by co-culture with MSCs, indicating MSCs suppression is active on both CD4 and CD8 cells. Stat5 phosphorylation in activated T cells was suppressed by co-culture with MSCs. Induction of cell-cycle promoting proteins such as CDK6, Cyclin D2, and Cyclin E by mitogenic stimulation were inhibited and suppression of a cell-cycle inhibitor, Kip1, was abolished. A previous report showed that T cells from stat5 deficient mice failed to induce cell-cycle promoting proteins and were not be able to proliferate on the stimulation through TCR. It was also reported that Nitric Oxide (NO) suppressed stat5 phosphorylation. Taken together with these reports, we hypothesized that NO is another candidate for the cause of suppression. In fact, NO synthase inhibitor (N-nitro-L-arginine methyl ester) recovered T cell proliferation from the suppression by MSCs in a dose-dependent manner. The amount of NO production and the strength of T cell suppression were parallel and dependent on the number of MSCs. MSCs blocked production of IFNγ but induction of T cell activation markers such as CD25 and CD69 and production of IL-2 were unaffected as reported. Our data suggest that MSCs block stat5 phosphorylation by production of NO, resulting in that T cells can neither proliferate nor produce high level of IFNγ. Here we demonstrate a new critical NO-stat5 dependent mechanism for T cell suppression by MSCs.