bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Analysis of Leukemia-Associated PTPN11 Mutations in Primary Hematopoietic Cells.
Authors: Schubbert Suzanne , Lieuw Kenneth , Rowe Sara L. , Lee Connie M. , Li XiaXin , Loh Mignon , Clapp D. Wade , Shannon Kevin
Published: October 2019

The PTPN11 gene encodes SHP-2, a non-receptor protein tyrosine phosphatase (PTPase) that relays signals from activated growth factor receptors to p21ras (Ras), Src family kinases, and other signaling molecules. Germ-line, missense mutations in PTPN11 account for approximately 50% of cases of the human developmental disorder Noonan Syndrome (NS). More recently, PTPN11 mutations have been identified in approximately 35% of children with juvenile myelomonocytic leukemia (JMML) without NS and have also been detected in other lymphoid and myeloid malignancies. Interestingly, almost all of these leukemia-associated mutations introduce amino acid substitutions within the N-SH2 domain of SHP-2 that are largely distinct from those found in NS. We have assessed the functional consequences of leukemia-associated PTPN11 mutations in primary hematopoietic cells. Expression of an E76K mutant SHP-2 in murine fetal liver and bone marrow cells confers a hypersensitive pattern of colony-forming unit granulocyte-macrophage (CFU-GM) colony growth in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3). Specifically, cells expressing E76K mutant SHP-2 display enhanced colony growth at low concentrations of growth factor compared to cells expressing wild-type (WT) SHP-2 protein. Mutant colonies are significantly larger with an abnormal, spreading morphology. E76K-expressing cells also form CFU-GM colonies in the absence of growth factor. The catalytic activity of the E76K mutant is required for aberrant colony growth as expressing the E76K mutation in the context of defective phosphatase activity (C463S) abolishes hypersensitive CFU-GM growth. Mutant E76K expression also enhances the growth of immature progenitor cells with high repopulating potential (HPP-CFC and LPP-CFC) in response to GM-CSF and IL-3 and perturbs erythroid progenitor colony growth. In addition, expressing the E76K mutation results in more pronounced growth factor hypersensitivity than another leukemia-associated SHP-2 mutation (D61Y), and both of these mutations confer a stronger hematopoietic phenotype than the common N308S substitution found in patients with NS.
Selective Rapid B-Cell Depletion In Vivo by Pharmacologic IKK2 Inhibition.
Authors: Fraser Christopher C. , Nagashima Kumiko , Sasseville Vito , Deeds Jim , McDonald Alice , Simpson Chris , Xu Yajun
Published: October 2019

Studies using genetically deficient mice have revealed that members of the NF-kB family play key roles in B-cell development. IKK2 which activates NF-kB by targeting degradation of IkB, is also required for B-cell development. We have studied the role of IKK2 in hematopoiesis using a chemical specific inhibitor (ML120B). Mice given daily oral dosing of ML120B for 4 days had severe B-cell depletion in spleen and bone marrow. B-cells at all stages (pro-B, pre-B, immature and mature B) were depleted 10 fold in the bone marrow while granulocyte numbers were largely unaffected. IKK2 inhibition in vivo showed selective sensitivity of B-cell progenitors (4 fold decrease) in the marrow compared to myeloid progenitors which were unaffected at an equivalent dose. Foci of cells with an apoptotic morphology were visible in bone marrow and spleen within 6 hours of a single oral dose. Apoptotic cells detected by labeling fragmented DNA were increased within splenic follicles (6 fold) and bone marrow. Also an increase in B220+ / annexin V+ cells and a decrease in pre-B (B220+/IgM−) cells in the marrow were observed. RNA expression studies in the marrow 6 hours after a single oral dose revealed a decrease in IL-7 and increased GM-CSF expression. Image analysis of B220 in spleens within 18 hours of a single dose of an IKK inhibitor revealed decreased follicle size. In order to evaluate hematopoietic progenitor sensitivity to NF-kB inhibition, dose responses to ML120B, panepoxydone (PPD) and proteasome inhibitor Lactacystin (Lcyst) were evaluated in B-cell and myeloid bone marrow colony assays. Inhibitors PPD and Lcyst were more effective at inhibiting B-cell colony growth than myeloid colony growth. In summary, pharmacologic inhibition of IKK2 results in a rapid induction of apoptosis with preferential depletion of B-cells and retention of myeloid cells and progenitors within the bone marrow.
Genetic Modeling of Chemical Antagonists: Genome Scale Validation of Hematopoietic Disease Drug Targets by In Vivo Functional Analysis.
Authors: Oravecz Tamas , Turner Alex , Brown Philip , Zambrowicz Brian , Blobel Gerd
Published: October 2019

Clinical management remains elusive for a large number of patients with hematological and other disorders necessitating the development of new, more effective pharmaceutical agents. While the sequencing of the human genome has provided a list of all drug targets that will ever exist, in order to improve the speed and cost of drug development there is a critical need to identify the small subset of these targets whose modulation will lead to therapeutic outcomes. As a genetic model of a perfectly selective and potent chemical antagonist, the analysis of the phenotypes of knockout mice allows one to predict the mechanism based efficacy and side effect profile of modulators of a particular target in a physiologic mammalian setting without having to first develop a candidate drug with which to validate the target. The power of this approach has been demonstrated by studies examining the targets of the best-selling drugs and current pharmaceutical pipelines. These analyses have confirmed a strong (~85%) correlation between the target’s knockout phenotype and the efficacy and/or side effects of drugs that modulate them. We have implemented this target validation approach by developing systems and infrastructure to generate and comprehensively phenotype mutant mice at a rate of more than fifteen novel lines per week. The battery of tests encompassed by our analysis have been specifically selected to reveal those genes that encode key control points in mammalian physiology whose modulation may be used to treat major disease processes including hematologic disorders. Tests to detect hematologic phenotypes include complete blood count; immunophenotyping; blood chemistry; analysis of soluble factors (immunoglobulins, cytokines and inflammatory mediators); full histopathologic examination, and various in vivo and in vitro immune challenge and cell activation assays. In addition, selected lines are subjected to more extensive analyses including additional challenge assays, gene expression profiling, and bone marrow transplantation. Data will be presented describing one of the lines analyzed in this screen, the Abl2 knockout, which has an increased mean total white blood count relative to wild-type littermates, as well as smaller body size (decreased length and weight). In parallel with the identification of therapeutically relevant proteins, we are actively exploring pharmacologic methods, both with small molecules and therapeutic antibodies, to modify the function of these novel gene targets, and ultimately to treat human disease.
Association between Statin and Fibrate Use and Venous Thromboembolism: A Case-Control Study.
Authors: Lacut Karine , Le Gal Gregoire , Oger Emmanuel , Mottier Dominique
Published: October 2019

Background:

Previous studies of selected patients have suggested a reduction in the risk of venous thromboembolism (VTE) with the use of statins, and no effect of fibrates.

Objective:

To evaluate the influence of statin and fibrate use on the risk of venous thromboembolic events.

Design:

Case-control study (EDITH) designed to investigate genetic and environmental risk factors of VTE.

Setting:

Brest University Hospital.

Participants:

857 patients consecutively hospitalized for a documented venous thromboembolic event were included between May 2000 and May 2004. Controls were matched on age, sex and the main risk factors of VTE (cancer, surgery, pregnancy…).

Results:

The mean age of patients was 67.7 year. No significant difference was found between cases and controls concerning the main characteristics, except for smocking and body mass index. Controls had more often previous vascular events (coronary heart disease, stroke or arteriopathy of the lower limbs) than cases but the difference was no significant. Statin use was associated with a significant decreased risk of VTE (odds ratio (OR) = 0.58; 95% confidence interval (CI), 0.41–0.82), whereas fibrate use was associated with a significant increased risk of VTE (OR = 1.60; 95% CI, 1.09–2.34). After adjustment on the main confounding factors including aspirin use and cardiovascular disease, these associations remained significant. Among pleiotropic effects of statins, some antithrombotic mechanisms could be proposed to explain their possible protective effect. Concerning the possible negative effect of fibrates, some authors found that the most prescribed fibrates, but not statins, caused hyperhomocysteinemia. In our study, analyses of homocysteinemia are ongoing.

Conclusion:

In this case-control study of hospitalized patients, statin use was associated with a significant decreased risk of VTE, whereas fibrate use was associated with a significant increased risk. Homocysteinemia may be involved in the difference between the effects of these two categories of lipid-lowering drugs on VTE. Because our study was observational, the protective effect of statins as regards the risk of VTE remains questionable and further prospective studies are needed.
The Risk of Venous Thromboembolism (VTE) among Cancer Patients by Tumor Site: A Population-Based Study.
Authors: Heit John A. , Petterson Tanya M. , Marks Randolph S. , Bailey Kent R. , Melton L. Joseph
Published: October 2019

Active cancer is an independent VTE risk factor (overall 6- to 9-fold increased risk) and accounts for almost 20% of all VTE in the community, but which cancer patients are at risk for VTE is largely unknown. Reportedly, VTE risk varies by tumor site, and cancer of the ovary, pancreas, colon, stomach, lung, prostate, and kidney convey particularly high VTE risk.

Objective: To estimate VTE risk by tumor site.

Methods: We enumerated observed cancers by tumor site for Olmsted County, MN active cancer patients with incident VTE over the seven-year period, 1991–1997 (n=152). We used 1991–1997 State Surveillance, Epidemiology, and End Results (SEER) data for Iowa to estimate the expected age-specific prevalence of cancer by tumor site in Olmsted County. VTE risk ratios (RR) for each tumor site were estimated by dividing the observed number of cancers by the expected number (calculated as the product of the SEER prevalence and the number of incident VTE cases in the age stratum).

Results: For our population of 1991–1997 VTE cases, all tumor sites had RR > 5.0 (range 5.2 to 37.3, all p-values<0.05). Compared to published overall VTE odds ratios of 6–9 for active cancer compared to no cancer, the RR for some tumor sites were particularly increased. A Chi-squared test of heterogeneity of the RR across sites was highly significant (p-value<0.001). Three rare cancer sites - pancreatic cancer, lymphoma, and brain cancer - had unusually high RR (all RR>25). The high number of VTE cases with lymphoma was not due to catheter-related arm vein thrombosis. Liver, leukemia, other gastrointestinal (esophagus, small intestine, gallbladder, other biliary) and other gynecologic (primarily cervical) cancers had over twice the baseline risk (i.e., RR>17.0). On the other hand, the RR for many common cancers (breast, colorectal, ovary, lung, prostate) were essentially the same as the overall baseline risk (all had 9.5<RR<12.0).

Conclusions: In contrast to previous reports, pancreas, lymphoma, brain, liver, leukemia, other gastrointestinal, and other gynecologic cancers have the highest VTE risk. Prior estimates of VTE risk by tumor site may have been biased by studies of prevalent cancers among patients hospitalized in tertiary care centers.
Thrombohemorrhagic Defects and Recurrent Miscarriage Syndrome.
Authors: Bick Rodger L. , Hoppensteadt Deborah.
Published: October 2019

We have assessed 351 women referred for thrombosis & hemostasis evaluation after suffering recurrent miscariages. All patients were referred by a high-risk obstetrician or reproductive medicine specialist after anatomic, hormonal or chromosomal defects had been ruled out. These patients were assessed over a three year period. The mean patient age at referral was 34 yrs. and the mean number of miscarriages was 3.4 (2–9). All patients underwent a thorough evaluation for thrombophilia and, when indicated, a hemorrhagic disorder. Of the 351 patients, 29 (8%) had no defect. Of the remaining 322 patients, 7 (2%) had a bleeding disorder: 3 with platelet dysfunction, 1 with Factor XIII deficiency, 3 with von Willebrands and 3 with Osler-Weber-Rendu. The remainder of the patients had a thrombophilia as follows: 195 (60%) had antiphospholipid syndrome, 64 (20%) had Sticky Platelet Syndrome, 38 (12%) had MTHFR mutation, 23 (7.1%) had PAI-1 polymorphism, 12 (3.7%) had Protein S deficiency, 12 (3.7%) had Factor V Leiden, 3 (1%), had AT deficiency, 3 (1%) had Heparin-Cofactor II deficiency, 3 (1%) had TPA deficiency, and 6 (2%) had Protein C deficiency. There were a total of 364 defects found in the 312 patients harboring thrombophilia; thus, several harbored two and a few harbored three separate defects.

All patients with thrombophilia were treated with preconception ASA at 81 mg. / day with the immediate post-conception addition of heparin or LMW heparin (Dalteparin). Both ASA and heparin / LMW heparin were used to term. The first 120 patients were treated with unfractionated heparin at 5,000 U every 12 hours, subcutaneously and the last 192 have been treated with Dalteparin at 5,000 U / day subcutaneously. The patients with MTHFR were also treated with folate at 5 mg. / day + pyridoxine at 50 mg. / day. All patients were carefully monitored with CBC & platelet counts, anti-Xa levels, frequent ultrasounds and physical exams. Only 2 of the thrombophilia patients suffered another miscarriage; all others had a normal term delivery. There were no pregnancy-related thromboses, no delivery complications and no episodes of post-partum thrombosis. The only bleeding consisted of 1 – 4 cm bruises at injection sites. No episodes of thrombocytopenia (HIT) were noted.

In our experience, thrombophilia is a common cause of recurrent miscariage and all patients with no anatomical, hormonal or chromosomal defect should be evaluated for thrombophilia or a bleeding disorder. The success rate of normal term delivery in these 312 patients was 94% using ASA + heparin or Dalteparin. In addition, side effects of therapy were minimal.
A Novel Mechanism of Plasmin-Induced Mitogenesis in Fibroblasts.
Authors: Mandal Samir K. , Rao L. Vijaya Mohan , Tran Thuy Tien T. , Pendurthi Usha
Published: October 2019

The plasminogen activator/plasmin system plays an important role, in addition to fibrinolysis, in many pathophysiological processes. Our recent studies showed that plasmin, similar to factor VIIa/tissue factor and thrombin, upregulates Cyr61, a growth factor like immediate early response gene, in fibroblasts via protease-activated receptor-mediated signaling. In preliminary studies we found that when fibroblasts were treated with VIIa (100 nM), thrombin (10 nM) or plasmin (50 nM), in concentrations that were shown to induce Cyr61 expression maximally, VIIa and thrombin increased the DNA synthesis by only about 20–30% whereas plasmin increased the DNA synthesis by 200 to 500%. The present study was carried out to investigate potential mechanism(s) by which plasmin promotes the DNA synthesis in fibroblasts and the role of protease-activated receptors and Cyr61 in plasmin-induced DNA synthesis. The plasmin-induced DNA synthesis in fibroblasts was dependent on its protease activity as the addition of active site-inhibited plasmin had no effect; and tissue factor pathway inhibitor-2 (TFPI-2) completely abrogated the plasmin-induced response. Neutralizing antibodies against PAR-1 and not PAR-2 attenuated the plasmin-induced DNA synthesis. Cyr61 antibodies completely blocked the plasmin-induced DNA synthesis suggesting that Cyr61 acts as a down-stream mediator. Surprisingly, we found no detectable increase in Cyr61 protein in plasmin-treated cell lysates whereas Cyr61 protein levels were clearly increased in thrombin-treated cells. These data suggest that plasmin might be cleaving newly synthesized Cyr61, which accumulates in ECM. Studies performed with purified recombinant Cyr61 and tumor cells that constitutively express abundant Cyr61 provided support for this notion. To test the hypothesis that plasmin release of Cyr61 from ECM plays a role in promoting DNA synthesis, we investigated the effect of conditioned media from control, plasmin-, and thrombin-treated fibroblasts in promoting DNA synthesis. The data revealed that the conditioned media of plasmin-treated cells (the protease activity in the media was inactivated) and not others significantly increased 3H-thymidine incorporation in fibroblasts. Addition of Cyr61 antibodies to the conditioned media of plasmin-treated cells attenuated the enhancing effect of the conditioned media. In summary, the data presented herein provide a novel mechanism by which plasmin promotes cell proliferation. In this mechanism, plasmin first induces the expression of Cyr61 via activation of PAR-1 mediated signaling. Next, plasmin releases/cleaves newly synthesized Cyr61 from the ECM, making it accessible to cells. The ability of plasmin to induce mitogenesis, in addition to its pericellular proteolysis, may provide a coordinated spatiotemporal effect that is required for would healing and tissue remodeling.
Interim Analysis of a Phase II Study of Denileukin Diftitox (Ontak) for Relapsed/Refractory T-Cell Non-Hodgkin’s Lymphoma.
Authors: Dang Nam H. , Pro Barbara , Hagemeister Fredrick B. , Jones Dan , Samuels Barry , Rodriguez Maria A. , Goy Andre , Romaguera Jorge E. , Walker Pamela L. , Kwak Larry , Fayad Luis
Published: October 2019

Denileukin diftitox (Ontak) is a fusion protein combining the enzymatically active domain of diphtheria toxin and the full-length sequence of interleukin-2 (IL-2) that targets tumor cells expressing the IL-2 receptor (IL-2R). The drug has established efficacy in cutaneous T-cell lymphoma (CTCL), and we have recently demonstrated its single-agent activity in B-cell non Hodgkin’s lymphoma (NHL) (Dang et al. Journal of Clinical Oncology. In Press). We initiated a phase II study to evaluate its efficacy in relapsed/refractory T-cell NHL, excluding CTCL. Denileukin diftitox was administered at a dose schedule of 18 μg/kg/day by IV infusion once daily for 5 days every three weeks, for up to 8 cycles. Premedications in the form of corticosteroids, antihistamines and fluids were given prior to each drug infusion to reduce the incidence and severity of acute hypersensitivity. 14 patients are currently evaluable for response. Median age was 57 (range 26–80), and mean number of previous treatments was 2.2 (range 1–4). Tumor CD25 status was determined by immunohistochemistry and/or flow cytometry, with CD25-positivity being defined as 10% or more of tumor cells expressing detectable CD25. Of the 7 patients with CD25+ T-NHL, there were 2 CR (1 case of Alk-1 negative ALCL and 1 case of PTCL), 3 PR (1 case of PTCL and 2 cases of angioimmunoblastic lymphoma), 1 SD (1 case of PTCL) and 1 PD (1 case of PTCL). Of the 7 patients with CD25− T-NHL, there were 2 PR (1 case of PTCL and 1 case of T/NK-lymphoma), 4 SD (3 cases of PTCL and 1 case of Sezary syndrome), and 1 PD (1 case of angioimmunoblastic lymphoma). Overall response rate (CR+ PR) was 50%, with 2 of 14 patients having CR (14%) and 5 of 14 patients having PR (36%). One patient with Alk-1negative ALCL still has an ongoing CR at 15+ months. Treatment was well-tolerated, with the majority of toxicity being grade 1 or 2 and transient. Denileukin diftitox appears to have activity in relapsed/refractory T-cell NHL, and is well-tolerated at the dosing schedule tested. Additional patients are being studied to further evaluate the relationship between detectable CD25 expression and tumor response to denileukin diftitox.
IL-2 Ameliorates Chronic GVHD by Inducing Antigen-Specific Regulatory T Cells.
Authors: Lohr Jens G. , Knoechel Birgit , Kahn Estelle C. , Abbas Abul K.
Published: October 2019

We have developed a mouse model in which a GvHD-like syndrome develops in response to a defined soluble self-antigen. This phenotype is caused after transfer of CD4+ T cells that have a single specificity and are reactive to the self-antigen into a lymphopenic host that expresses the cognate antigen. By using a clonotypic antibody we are able to identify these cells and can therefore follow their migration, kinetics and functional characteristics. At least two distinct phases can be identified by clinical picture and correlated with accumulation of T cells - an early phase, resembling acute GvHD, leading to wasting and death coinciding with rapid accumulation of T cells, and a late phase in which a stable number of T cells is maintained clinically reminiscent of chronic GvHD. We show here that a fraction of the naïve T cells that encounter the self-antigen after transfer develop into CD4+CD25+ regulatory T cells (Treg) in the periphery. This population controls T cell homeostasis, activation and severe immune pathology. The development of CD4+CD25+ Treg critically depends on IL-2 produced by the T cells. Therefore, in the absence of IL-2, T cell homeostasis cannot be maintained and massive accumulation of CD4+ T cells leads to severe inflammation of the skin. Importantly, only IL-2 that is produced by the T cells themselves, but not from peripheral tissues, leads to efficient generation of Treg and T cell homeostasis. We suggest that Treg-development is a differentiation step of T cells that encounter self-antigen in the periphery, and is essential for maintaining homeostasis even in the presence of self-recognition. Our data provide mechanistic insight into the re-establishment of homeostasis after cell transfer into a lymphopenic host and have important implications for the use and timing of therapeutic approaches targeting the IL-2 pathway.
Sphingosine 1-Phosphate (S1P) Is a Chemotactic Factor for CD34+ Progenitors and CD34+ Cell Lines Expressing the S1P1 Receptor: Possible Contribution to Progenitor Cell Trafficking.
Authors: Seitz Gabriele , Boehmler Andreas M. , Wiesner Tina , Kanz Lothar , Möhle Robert
Published: October 2019

Trafficking of hematopoietic stem and progenitor cells (HPC) is controlled by G protein coupled receptors (GPRs), particularly by the chemokine receptor CXCR4. However, homing of HPC does not exclusively depend on CXCR4 and its ligand SDF-1. In addition to chemokine receptors, also GPRs for non-peptide lipid mediators may be involved in HPC migration. In the present study, we demonstrate that sphingosine 1-phosphate (S1P) is chemotactic for human CD34+ HPC (optimum dose 5 μM), as measured in a modified Boyden chamber system. In CD34+ cell lines, the chemotactic response was even stronger and occurred at lower S1P concentrations (e.g., optimum dose 100 nM for Jurkat cells, 2-fold stronger migration compared to HPC). By RT-PCR, we measured mRNA expression of the 5 different S1P receptors (S1P1,2,3,4,5). All CD34+ hematopoietic cell lines analyzed (KG1, KG1a, Jurkat) expressed S1P1, a S1P receptor with known chemotactic activity, while CD34- cell lines (HL-60, THP-1) were negative for S1P1. In HL-60 cells, chemotaxis in response to S1P was even reduced compared to spontaneous migration, which might be due to the expression of S1P receptors other than S1P1 with known inhibitory effects, particularly S1P2. In mobilized peripheral blood CD34+ progenitor cells from different donors, S1P1 was consistently expressed in both CD34+CD38+ and more primitive CD34+CD38- HPC, while expression of S1P2,3,4,5 was variable. S1P induced also other typical responses of GPR-mediated signaling in CD34+ cell lines and HPC, such as polymerization of filamentous actin, as measured by flow cytometry after labeling of the cells with FITC-phalloidin. We conclude that S1P is a chemotactic factor for CD34+ HPC and CD34+ cell lines due to expression of the GPR S1P1. As megakaryocytes and platelets represent an abundant source of S1P in the bone marrow, our results suggest that S1P and its receptor S1P1 may contribute to trafficking and spatial distribution of HPC in the hematopoietic microenvironment.
EPCs Derived from Ex Vivo Expansion of Unmobilized Human Peripheral Blood Co-Express Hematopoietic and Endothelial-Specific Markers and Can Be Derived from Culture of Highly Purified Populations of CD14-Positive Monocytes.
Authors: Sharpe Emerson E. , Teleron Amylynn A. , Li Bin , Young Pampee P.
Published: October 2019

An increasing amount of data has suggested a more dynamic role of vasculogenesis, whereby bone marrow (BM)-derived circulating endothelial progenitor cells (EPCs) home and contribute to new blood vessel formation during tumor growth, ischemic injury, and wound healing. EPCs can be obtained by isolating hematopoietic progenitor cells from BM or cord blood. Alternatively, ex vivo expansion of unmobilized human peripheral blood (PB) can generate adherent cells, PB-EPCs, that express endothelial markers and also, upon administration, incorporate into developing neovasculature. The relative ease of obtaining unmobilized human PB has made PB-EPCs an attractive candidate with which to develop cell based therapy to treat ischemia. In parallel with clinical trials designed to understand their therapeutic potential, there is a continued effort to better characterize the PB-EPC and understand its biology. It is currently thought that EPCs are directly derived from a CD34+/lin- faction of hematopoietic stem cells (HSCs). However, in our current study, we have confirmed prior reports that ex vivo expansion of human PB generates similar numbers of EPCs as compared to plating unfractionated human BM, which contains >50-fold higher CD34+/lin- content, suggesting that PB-EPCs may not be derived from the CD34+/lin- population. We used immunofluorescence and FACs analysis to further show that PB-EPCs not only express endothelial markers such as vWF, Vascular Endothelial Growth Factor Receptor 1 and 2 (also known as flt-1 and flk-1, respectively), VE-cadherin, UEA-1 lectin, Tie-1 and Tie-2 but also hematopoietic markers such as CD45 and CD14, a marker enriched on monocytes. To test if PB-derived CD14-positive cells can give rise to PB-EPCs, we isolated them from human PB to >98% purity and plated them on fibronectin-coated coverslips. In vitro culture of CD14-positive cells generated adherent clusters of spindle shaped cells morphologically similar to EPCs. Culture of the CD14-negative fraction failed to yield any adherent cells. After ten days, the coverslips were removed and the cells were stained with various endothelial (flk-1, vWF, uptake of DiI-AcLDL, and UEA-1 lectin) and monocyte/hematopoietic (CD14 and CD45) cell markers. In analysis of these slides, the EPCs derived from the purified CD14 fraction stained positive for all six markers. These observations suggest that PB-EPCs can differentiate from cells of the monocytic lineage in vitro without the necessity of interaction from cells contained in the CD14-negative population. Further experiments will test the possibility that monocytes may be an intermediate in the differentiation of EPCs in vivo.
Hematopoietic Stem Cells Can Give Rise to Satellite-Like Cells in Skeletal Muscles.
Authors: Yoshimoto Momoko , Heike Toshio , Shiota Mitsutaka , Kobayashi Hirohiko , Umeda Katsutsugu , Nakahata Tatsutoshi
Published: October 2019

Recent studies have indicated that bone marrow cells can regenerate damaged muscles, but also that they can adopt phenotype of other cells by cell fusion. It has also been reported that single hematopoietic stem cells (HSCs) can regenerate skeletal muscle although it is still controversial whether HSCs differentiate into satellite cells in muscle or not. In order to investigate the roles of HSCs in muscle regeneration and whether they can generate satellite cells or not, we purified and injected CD45+Lin−Sca-1+c-kit+(CD45+KSL) HSCs labeled by green fluorescent protein (GFP) into mice with or without irradiation. We examined time-course behavior of HSCs in recipient muscles with a fluorescent stereomicroscope and then immunohitochemical staining during the early and late phase after transplantation. Our direct visualization system gave evidence of massive GFP signals in all the muscles of only irradiated mice in early phase after transplantation. Transverse cryostat sections showed GFP+ Myosin+ muscle fibers along with numerous GFP+ hematopoietic cells in damaged muscle. We also found myogenin+GFP+ cells like myoblasts in very low number. These phenomena were temporal and GFP signals had dramatically reduced 30 days after transplantation. FISH analysis confirmed the GFP-DNAs in the nuclei of muscle fibers. These results suggested that most of GFP+HSCs fused with myofibers and participated in regeneration of damaged muscles, and a very few HSCs can differentiate into myoblast like cells expressing myogenin. After 6 months, GFP+ fibers could be hardly detected but GFP+c-Met+ mononuclear cells were located beneath the laminin+ basal lamina. Single fiber cultures from these mice showed proliferation of GFP+ fibers. These results suggested that HSC-derived cells settled beneath the basal lamina like satellite cells and might acquired the satellite cell activity.
Rhnull Syndrome: Identification of a Novel Mutation in RHce Gene.
Authors: Reid Marion E. , Rosa Karina A. , Powell Vivien I. , Lomas-Francis Christine , Costa Fernando F. , Stinghen Servio T. , Watanabe Alexandra M. , Carboni Edna K. , Baldon Joseani P. , Jucksch Maria Marta F. , Castilho Lilian
Published: October 2019

Background: Rhnull syndrome, which includes the amorph and regulator types, is a rare genetic disorder characterized by stomatocytosis and chronic mild hemolytic anemia. The suppression of Rh antigen expression for regulator types is attributed to mutations of the RHAG gene. The deficiency of Rh proteins on the red blood cells (RBCs) from the rare individuals of the Rhnull amorph type may be the result of homozygosity for a silent RHCE allele at the RH locus in which the RHD is absent. We studied a Brazilian family transmitting an amorph Rhnull disease gene on a consanguineous background and identified a novel mutation in RHce gene causing the loss of function phenotype.

Methods: RBCs from two Rhnull sisters (G1 and G2) of the amorph type and from family members were analyzed by serology and flow cytometry with monoclonal antibodies specific for Rh (D, C, c, E and e), and LW antigens, for RhAG and CD47 as well as alloantibodies directed against GPB (S, s and U) antigens. Genomic DNA samples and transcripts were tested by PCR and sequence analysis.

Results: RBCs from G1 and G2 did not react with the anti-Rh and anti-LW reagents and reacted weakly with anti-RhAG, anti-CD47, anti-s, and anti-U reagents. RBCs from G1 were S+ and from G2 S-. Molecular analyses showed a deletion of the nucleotide at position 963 in exon 7 of the RHce gene [GGG(Glu321→GG)] in addition to a deletion of the RHD gene. This nucleotide deletion in exon 7 introduced a frameshift after Glu321 and a premature stop codon, resulting in a shorter predicted protein with 359 amino acids, including a new C-terminal sequence that is likely to alter the protein conformation and impair the Rh complex assembly.

Conclusion: We describe a novel mutation of an amorph Rhnull disease gene leading to a loss of function phenotype. Our findings reinforce previous studies suggesting that protein folding and/or protein-protein interaction mediated by the C-terminal domain of the Rh proteins plays a role in the stability of the Rh membrane complex.
Quantum Dots, Efficient Fluorescent Markers for Red Cells.
Authors: Castro Vagner , Farias Patrícia M.A. , Santos Beate S. , Menezes Frederico D. , Ferreira Ricardo C. , Lima Paulo R.M. , Fontes Adriana , Barjas-Castro Maria Lourdes , Cesar Carlos L.
Published: October 2019

Semiconductor quantum dots (QDs) are nanometer-sized crystals with unique photochemical and photophysical properties that are not available from either isolated molecules or bulk solids. In comparison with organic dyes and fluorescent proteins, QDs are emerging as a new class of fluorescent labels with improved brightness, resistance against photobleaching, and multicolor fluorescence emission. These properties could improve the sensitivity of biological detection and imaging by at least 10 to 100 fold. Recent research has shown that QDs can be covalently linked with biorecognition molecules such as peptides, antibodies, nucleic acids and small molecules for use as fluorescent probes. The aim of the present study was to evaluate the application of a Cadmium Sulfide QDs method for the analysis of the expression and distribution of A antigen on living red blood cell surface. Highly luminescent and stable cadmium sulfide colloidal particles were obtained in the nanometer size range. Their surface was functionalized with commercially available monoclonal anti-A antibody via an one-pot glutaraldehyde cross linking procedure, followed by their conjugation to red cells. Samples were obtained from blood bank red cell units, of groups A (n=3), A2 (n=2) and O (n=3). Confocal microscopy (LSM 510 - Carl Zeiss, Jena, Germany) was used and for each cell type, the images were reproduced at least three times. The acquisition confocal image parameters, such as pinhole, filters, beam splitters and photomultiplier gain, were maintained constant to permit a comparative analysis of the luminescence intensity maps. Analyzing the intensity map of group A erythrocytes, a homogeneous emission profile was observed throughout the cell surface, suggesting a homogeneous distribution of A antigen. In contrast, the intensity map analysis of the confocal images showed a lower intensity pattern of the A2 group erythrocytes studied, suggesting different antigen expression on cell surface. The O group cells showed no emission. This result suggests that this new efficient, fast and simple labeling procedure may be employed as a quantitative tool to investigate the expression and distribution of molecules in red blood cells.
High Frequency of Unusual ABO Alleles in Patients with Leukemia.
Authors: Novaretti Marcia C.Z. , Pares Madalena M.N.S. , Domingues Alexandre E. , Dorlhiac-llacer Pedro E. , Chamone Dalton A.F.
Published: October 2019

Background: Mutations critical for ABO group phenotypes which encode the catalytic domain of ABO glycosyltransferases have been studied in healthy blood donors. Weakening of A and B transferases have been reported in patients with leukemia, particularly those in which the myeloid lineage is involved. The purpose of this study was to use sequence specific PCR (PCR-SSP) to perform ABO genotyping of leukemic patients.

Material and Methods: A total of 108 unrelated leukemic patients were initially classified serologically using tube test technique and gel test for ABO typing and subgroup detection. Genomic DNA was prepared from peripheral blood cells. All samples were then screened by 32 different PCR-SSP, each specific for a single nucleotide variation to screen 16 known polymorfic sites of exons 6 and 7 (modified from Seltsam, Transfusion 2003,43(4):428–39.

Results: The results of PCR screening were conclusive and consistent with the ABO phenotypes in 71 patients (65.07%). Common ABO alleles were found in only 15 (13.8%) of patients studied. Unusual ABO alleles were found in 26 of 28 blood group O patients (ABO*O05, ABO*O21, ABO*O30, ABO*O33, ABO*O35, ABO*O36, ABO*O43 and ABO*O45), in 20 of 43 blood group A patients (ABO*A103, ABO*A104, ABO*A106, ABO*A202, ABO*A203), in 8 of 10 blood group B patients (ABO*B104, ABO*B105) and in 2 of 4 AB blood group patients (ABO*Bx01, ABO*B103). In 37 patients (34.2%), PCR screening revealed allele combinations that were incompatible with known ABO allele combinations or subgroups predicted by serologic analysis.

Conclusion: This is the first report of using PCR-SSP for ABO genotyping of leukemic patients. The pathophysiologic role of unusual ABO allele combinations in patients with leukemia warrants further investigation.
Second Donation of Bone Marrow; Japan Marrow Donor Program (JMDP) Experience.
Authors: Akiyama Hideki , Hara Masamichi , Hino Masayuki , Sao Hiroshi , Hoshi Yasutaka , Ohto Hitoshi , Kai Shunro , Ochiai Ryoichi , Morita Kiyoshi , Nakao Yasuo
Published: October 2019

In Japan, JMDP, founded in 1991, had collected and transplanted more than 5,000 bone marrows by the end of 2003. Meanwhile, total of 137 donors donated their bone marrow twice. The efficiency and the safety of those donations and the efficacy of those cells in the engraftment were evaluated retrospectively. In JMDP, volunteers aged from 20 to 50 are enrolled as the donor. A year after the first donation, the donors are allowed to donate their marrow again, if they wish. Marrow was collected from the posterior iliac crest under general anesthesia and, generally, different patients received the marrow from the single donor. Peripheral blood stem cell harvest is not permitted by the JMDP. Their mean age was 36.6 and 36 of them were female. The volume of the marrow and the nucleated cell number collected in the first and the second donation were 760 ± 290 vs 857 ± 230 ml (p=0.001) and 15.3 ± 6.8 vs 15.6 ± 6.9 x 10E9 cell (p=0.674), respectively. Cell concentration of bone marrow solution and nucleated cell number per recipient’s weight were significantly lower in the second donation (2.2 ± 0.8 vs 1.8 ± 0.7X 10E7 cell/ml (p=0.0004) and 3.3 ± 1.1 vs 2.8 ± 1.1 x 10E8 cell/kg (p<0.0001), respectively) revealing decreased cell dose. The efficacy of those cells was also evaluated comparing the available data from 127 pairs of the patients who received the marrow. Within these 254 recipients, data of 8 patients (2 in the first and 6 in the second donation) are missing and another 19 (8 and 11) were excluded because of early death before day 28. There was no difference between the first and the second donated marrow in the date of neutrophil recovery (p=0.5214) and platelet recovery (p=0.4108). There were two cases each of slow engraftment (up to day 35) and late rejection in 1 and 5 patients respectively (p=0.2132). The adverse effects of those donations were also compared. The incidence of fever more than 38 degrees, the duration of the febrile period and other adverse effects such as urinary pain, sore throat, infectious disease, liver dysfunction and pain on the wound lasting more than 7 days were compared and no significant difference was observed. In conclusion, second donation can be performed safely and the functions of those cells were as good as the ones donated first. However, because the cell dose of the second collection could be lower, it might be necessary to collect more marrow than the expected.
Proteasome Inhibiters Up-Regulate TRAIL/Apo2L and Its Receptors Significantly Contributing to Proteasome Inhibitor-Induced Apoptosis in Primary Chronic Lymphocytic Leukemia (CLL) Cells.
Authors: Kabore Albert F. , McCrea Kristin , Johnston James B. , Gibson Spencer B.
Published: October 2019

The proteasome inhibitor, bortezomib has recently emerged as a new therapeutic treatment for refractory multiple myeloma and is presently being evaluated for other hematological malignancies either alone or in combination with other antitumor agents. Proteasome inhibitors cause the accumulation of many proteins but the precise mechanism responsible for their antitumor effect is unclear. In the present study, we have determined that cytotoxic effect the proteasome inhibitor MG-132 in primary chronic lymphocytic leukemia (CLL) cells is through the activation of the TRAIL (tumor necrosis factor-related apoptosis inducing ligand) apoptotic pathway. MG-132 induced apoptosis in approximately 70% of primary CLL cells as measured by annexin V staining. Addition of DR4:Fc that prevents TRAIL ligation with its receptors decreased the amount of MG-132 induced apoptosis by approximately 40% suggesting MG-132 caused activation of the TRAIL apoptotic pathway. MG-132 also up-regulated both the mRNA and protein levels of TRAIL and protein levels of TRAIL receptors DR4 and DR5. This upregulation correlated with activation of caspase 8 and cleavage of pro-apoptotic Bcl-2 family member Bid. Moreover, MG-132 treatment also induced a substantial reduction in the FLICE-like inhibitory protein (c-FLIP) protein levels. In contrast to CLL cells, proteasome inhibitors failed to activate the TRAIL apoptotic pathway in normal B-cells. This indicates that proteasome inhibitors are inducing apoptosis in primary CLL cells through activation of the TRAIL apoptotic signaling pathway through up-regulation of TRAIL and its cognate receptors and reduced FLIP expression. Thus, proteasome inhibitors may have a therapeutic role in CLL, either when used alone or in combination with TRAIL or antibodies against DR4/DR5.
Ribosomal Protein S19 and Diamond Blackfan Anemia.
Authors: Ellis Steven R. , Arce-Lara Carlos , Caffrey Jacqueline M. , Alvarez-Arias Diana A.
Published: October 2019

Diamond Blackfan Anemia (DBA) is one of several bone marrow failures that have been linked to defects in ribosome synthesis. 25% of DBA cases are linked to mutations in ribosomal protein S19 (Rps19). The etiology of the remaining cases is unknown. To gain a better understanding of the function of the Rps19 family of proteins we have characterized members of this protein family in the yeast, Saccharomyces cerevisiae. In yeast, Rps19 is encoded by duplicated genes, RPS19A and RPS19B. Yeast cells lacking both RPS19 genes are not viable, whereas those lacking a single gene are viable but have growth defects. These latter strains are defective in a specific step in rRNA processing that preferentially affects the maturation of 40S ribosomal subunits. We scanned other yeast strains with mutations in genes for 40S subunit proteins for processing phenotypes similar to RPS19 mutants. Several have phenotypes that overlap with RPS19 mutants, but only RPS18 stands out as being virtually identical to RPS19 mutants. The human RPS18 gene is therefore a candidate locus for pathogenic mutations in DBA patients with normal RPS19. We are currently developing strategies to sequence RPS18 genes from DBA patients with normal RPS19 to determine if mutations in RPS18 are associated with DBA. We have also developed a yeast system for the functional testing of mutant alleles of RPS19 found in DBA patients. In general, a mutation is considered pathogenic if it is not found in unaffected family members and in the general population. We have found, however, that several missense mutations classified as pathogenic in DBA patients do not affect Rps19 function in the yeast system. The failure of these mutations to affect Rps19 function in yeast points to a need for functional testing of RPS19 mutant alleles in human cells.
Generation of Dendritic Cells in a Closed System Using GMP Guidelines.
Authors: Giulivi Antonio , Halpenny Mike , Birch Paul , Yang Lyn , Martin Lisa
Published: October 2019

Objective: The application of a dendritic cell (DC) based immunotherapy requires the ability to consistently produce a clinically acceptable product using Good Manufacturing Practice (GMP). We describe our experience using the Nexell SteriCell container to generate monocyte derived DC in a closed system using GMP guidelines.

Design/Materials and Methods: Leukapheresis product was used as source material to first isolate monocytes by adherence to culture bag and then generate DC from monocytes using a serum free media containing GM-CSF and IL-4 and culturing for 7 days at 37°C /5% CO2. DC generation was characterized by phenotyping cultured cells for decreased expression of CD14 and increased appearance of CD80, CD86 and DR. In addition to phenotyping, cultures were tested pre and post culturing for viability and microbial contamination.

Results: Cultured cells expressed a decreased expression of CD14 (mean % decrease 58.8 /range 11.2–94.9) and increased expression of CD 80/86 (mean fold increase 23.6X /range 5.7 – 36.2), and DR (mean fold increase 9.3X /range 3.2 – 16). All microbial cultures were negative and viability ranged from 54% to 90% post incubation.

Conclusions: The Canadian Blood Services cell processing laboratory is a FACT (Foundation for the Accreditation of Cellular Therapy) accredited facility following GMP guidelines. The results obtained from our study demonstrate a system easily adaptable to larger scale production of DC for possible future immunotherapy clinical application.
Upregulation of MT1-MMP Expression by Hyaluronic Acid Enhances Homing-Related Responses of Hematopoietic CD34+ Cells to an SDF-1 Gradient.
Authors: Shirvaikar Neeta , Montano Jencet , Turner Andrew R. , Ratajczak Mariusz Z. , Janowska-Wieczorek Anna
Published: October 2019

Hyaluronic acid (HA), the bone marrow (BM) extracellular matrix microenvironment (ECM) component, not only supports cell adhesion but also promotes migration and homing of hematopoietic stem/progenitor cells (HSPC) by interacting with its cell surface receptor CD44. CD44 has been shown to co-localize with matrix metalloproteinases (MMPs), particularly membrane-type (MT)-1 MMP and MMP-9, at the leading edge of migrating tumor cells, and the cleavage of CD44 by MT1-MMP is critical for tumor cell migration and invasion. MT1-MMP has strong pericellular proteolytic activity and also activates latent forms of MMP-2 and MMP-9. In this study we examined the effect of HA on MT1-MMP expression and migration of BM, peripheral blood and cord blood CD34+ cells. We found that HA upregulates mRNA for MT1-MMP and MMP-9, increases MT1-MMP protein (as evaluated by Western blotting) and stimulates MMP-9 and MMP-2 activity (as determined by zymography) in CD34+ cells. In chemotaxis assays HA alone did not show any chemotactic activity but primed the chemotaxis of CD34+ cells to a low SDF-1 gradient (10 ng/mL) and their trans-Matrigel chemoinvasion to a low SDF-1 gradient. Similarly, SDF-1 besides stimulating MMP-2 and MMP-9 (as we previously described in Exp Hematol2000; 28:1274), also upregulated MT1-MMP in CD34+ cells. In addition, we found that the HA enhanced activation of latent MMP-2 in co-cultures of CD34+ cells with HUVEC. In conclusion, we demonstrate for the first time that (i) HA primes CD34+ cell chemotaxis and chemoinvasion to a low SDF-1 gradient; (ii) that both HA and SDF-1 stimulate MT1-MMP, MMP-2 and MMP-9 in CD34+ cells, and (iii) HA enhances activation of proMMP-2 in the ECM. Hence the interaction of HA and SDF-1 with MT1-MMP could play an important role in HSPC migration and homing and we postulate that pretreatment of HSPC with HA before transplantation could enhance their homing by inducing expression of MMPs that is optimal for stem cell engraftment.