bluuebunny/crossref_metadata_2025 huggingface dataset:
Spectrum of Antiphospholipid Antibodies and Associated Thrombotic and Obstetric Events among Patients with Antiphospholipid Syndrome
Authors: Patnaik Mrinal M , Wudhikarn Kitsada , Shroff Monal M , Killeen Anthony A , Reding Mark T. , Kasthuri Raj S.
Published: April 2020

Introduction: Antiphospholipid antibody syndrome (APS) is characterized by arterial or venous thrombosis, and/or adverse obstetric outcomes in the presence of persistent antiphospholipid antibodies (APab). Between 2–10% of all patients with APS are reported to present solely with anti beta-2 glycoprotein 1 antibodies (b2GP1 abs) (ref 1). In 2006 the Sapporo criteria were revised to include b2GP1 abs to the panel of diagnostic tests. While presence of a lupus anticoagulant (LA) has been found to correlate well with occurrence of thromboses, the prognostic and predictive roles of b2GP1 abs and anticardiolipin antibodies (ACLA) need better definition. Previous studies suggest both the severity of the initial event and the risk for recurrent events is greater among patients demonstrating presence of more than one APab on laboratory testing (ref 2 and 3), but this aspect needs further confirmation.

Objectives:

To identify the spectrum of clinical thrombotic and obstetric manifestations associated with the different APab profiles; To determine the prevalence and clinical significance of the b2GP1 ab positive and ACLA/LA negative phenotype; and To evaluate the significance of IgM b2GP1 ab and IgM ACLA.

Methods: Medical records of patients tested for all three antiphospholipid antibodies at the University of Minnesota Medical Center from Dec 2000 to Dec 2005 were reviewed. Patients that met the modified criteria for APS were included. IgG and IgM ACLA were measured using the Diamedix kit (VAX diagnostics) and IgG and IgM b2GP1 abs using the Inovalite kit (NOVA diagnostics). LA was determined using standard protocol.

Results: Over the 5 year period, all three laboratory assays were obtained in 560 individuals, but only 76 (13.5%) patients who met both the clinical and laboratory criteria for APS were included in this study. Seven of the 76 patients (9.2%) had isolated, persistently positive b2GP1 abs. LA alone was present in 21 patients (27.6%). Twenty nine patients (38%) had all three APabs, and had a higher incidence of thrombosis at multiple sites at the time of diagnosis as well as increased rate of recurrent events, in comparison to those with two or less than two APab. Isolated ACLA was present in only 2 patients (2.6%). Interestingly, both IgM (47%) and IgG (50%) b2GP1 abs were associated with thrombosis, whereas only IgG ACLA was found to be associated with thrombosis.

Conclusion: The presence of b2GP1 abs as the sole laboratory anomaly is uncommon, but is noted in patients with APS, thus warranting it’s testing at the time of clinical suspicion. Amongst the different antibody types LA seems to be the strongest predictor for thrombotic events and ACLA seems to have a weaker link, consistent with the results of previous studies (ref 4). The presence of all three AP antibodies does increase the risk for recurrent and multiple thromboses.
Hyperthyroidism as a Risk Factor for Venous Thromboembolism: A Case-Control Study
Authors: van Zaane Bregje , Squizzato Alessandro , Huijgen Roeland , van Zanten Anton P , Fliers Eric , Buller Harry R , Gerdes Victor E.A. , Brandjes Dees P.M.
Published: April 2020

<>BACKGROUND: It has been hypothesized that overt hyperthyroidism may increase venous thromboembolic risk given substantiated evidence of a thyroid-induced hemostatic imbalance favoring a hypercoagulable state. Aim of this study was to investigate whether or not overt hyperthyroidism functions as a risk factor for venous thrombosis.

<>METHODS: We performed laboratory measurement of thyroid function in 190 patients with objectively confirmed venous thrombosis of the lower extremities and 375 sex-matched controls drawn from a prospective study on general risk factors for venous thrombosis.

<>FINDINGS: Of the 190 included cases, 155 patients were diagnosed with deep venous thrombosis (DVT), 12 patients with calfvein thrombosis and 23 with superficial thrombophlebitis. Undetected overt primary hyperthyroidism was found present in 3 DVT patients at the time of the event, whereas none was seen in controls (p=0.037, all thrombosis patients combined; p=0.024, DVT patients only). We found slightly higher levels of serum free thyroxine (fT4) in patients with venous thrombosis as compared to controls, and analysis of the proportion of cases with a higher fT4, above 16 pmol/L (75th percentile of values in controls), yielded an odds ratio of 1.7 for DVT to develop (95% CI 1.12–2.45 when adjusted for sex, age and known thyroid dysfunction). No such association was found for serum levels of thyrotropin or thyroidperoxidase antibodies.

<>INTERPRETATION: Our data suggest overt hyperthyroidism to function as a risk factor for venous thrombosis. In addition, higher levels of fT4, even within the normal range, are associated with increased VTE risk.
Hypercoagulation in Malignancy: Localized by Thromboelastography to the Humoral Phase of Coagulation
Authors: Nagpal Sunil , Schwartz Dianne E , Faber Edward , Cziffer Andrea , Puttarajappa Chethan , Estrada Dexter Toribio , Schwartz Kenneth A.
Published: April 2020

Patients with cancer are considered hypercoagulable since their risk of thromboembolic events is significantly higher than patients without cancer. Thromboelastography (TEG) was used to investigate hypercoagulation in cancer patients. TEG continually measures over time the physical visco-elastic properties of blood as it transforms from a free flowing fluid to a gel-like clot. Our hypothesis was that blood from cancer patients would form a clot at a faster rate than blood from non-cancer patients hospitalized for thrombo-embolic disease. We studied 75 patients mean age was 57±14 composed of 46 females and 29 males. The patients were divided into 3 different groups: 14 patients with malignancy and thrombosis, 47 malignancy without thrombosis and 14 thrombosis without malignancy. TEG was performed using whole blood stimulated with two agonists: tissue factor and kaolin. Rate of clot development was increased in the cancer patients with and without thrombosis when compared to non-cancer thrombosis group and was localized to the humoral phase of coagulation (p<0.05). Both tissue factor and kaolin stimulated TEGs showed shortening of the “r” time interval in the cancer patients (reflective of a more rapid rate of activation of humoral coagulation factors) and a larger α=alpha angle (reflective of fibrinogen activity). No difference was observed in the platelet phase of coagulation (MA). Cancer patients treated with erythropoietin did not differ from patients who were not treated. Class of chemotherapeutic agent (alkylating agent, antimetabolite, both or neither) was not correlated with TEG. Tissue type of the patients’ malignancy (adenocarcinoma, lymphoproliferative disease, squamos cell carcinoma or other) did not influence the TEG. We conclude that Cancer patients with and without thrombosis differ when compared non-cancer patients with thrombosis by demonstrating a more rapid rate of clot development in the humoral phase of coagulation.
Efalizumab-Induced Autoimmune Hemolytic Anemia
Authors: Lee Patrick , Schwarsin Alexandra , Ascensao Joao L. , Schechter Geraldine P.
Published: April 2020

Efalizumab (Raptiva; Genentech Inc.) is a humanized, recombinant, T-cell targeted monoclonal antibody, which binds to CD11a which combined with CD18 forms lymphocyte function-associated antigen 1 (LFA-1) and is approved for use in adults with moderate to severe plaque psoriasis. Previous publications have described autoimmune thrombocytopenia, hemolytic anemia as well as neutropenia with use up to 8 months after initiating treatment. We now report two patients who developed refractory autoimmune hemolytic anemia nine and twenty-six months after successful treatment of their psoriasis with efalizumab. The first patient is a 56 year old African-American man who presented with severe Coombs positive (IgG+, C3d −}anemia, with a hemoglobin of 4.1 g/dL, reticulocyte count of 25% after 26 months of treatment with efalizumab. Over the next two weeks the anemia was refractory to high dose prednisone, a total dose of 100 gms of intravenous immune globulin (IVIG), mycophenolate, danazol and 2 doses of rituximab and 10 units of blood, hemoglobin remained at 5.9 g/dLand .splenectomy was performed two weeks after presentation. Hemoglobin 2 weeks post splenectomy improved to 10.4 g/dL with near normalization one month later. The second patient a 61 year old woman, presented to an outside hospital with severe anemia. Upon transfer 1 week later, she was found to be Coombs positive, with a hemoglobin of 6.1 g/dL, reticulocytes of 1 %., and underwent a bone marrow aspirate and biopsy with noted erythroid hyperplasia, this after nine months of treatment with efalizumab. The patient underwent splenectomy three weeks after high dose prednisone, intravenous cyclophosphamide, cyclosporine, danazol, IVIG, rituximab and plasmapheresis failed to induce a remission. After splenectomy, the patient remained refractory to treatment and eventually succumbed to a combination of arterial and venous thrombi, bowel ischemia and bacterial sepsis six weeks after presentation. Little is known regarding the mechanism behind the immune mediated cytopenias following efalizumab therapy. In addition to lymphocytes, CD11a is expressed on several other cell lines, however, CD11a is not expressed on mature erythrocytes making a direct antibody mediated response against an efalizumab/LFA-1 complex less likely. The development of anti-efalizumab antibody occurs in 5% of patients but is not associated with autoimmune phenomena. More likely mechanisms may include dysregulation of autoreactive T cells from either defective T-cell function, loss of regulatory T cells or aberrant antigen presentation. These two cases highlight the difficulty and relative resistance to treatment in patients who develop autoimmune hemolytic anemia secondary to efalizumab and stress the importance of continued close monitoring of complete blood counts throughout the duration of active treatment.
Therapeutic Plasma Exchange (TPE, plasmapheresis) for Treatment of Refractory Autoimmune Hemolytic Anemia (AIHA)
Authors: Sandilya Vijay , Chen Lingyi , Anand Rachna , Choi Eugene , Chopra Akhil , Styler Michael , Crilley Pamela , Topolsky David , Ward Kristine
Published: April 2020

Management strategies for AIHA include corticosteroids, intravenous immunoglobulin, immunosuppressive agents, cytotoxic agents, monoclonal antibodies (Rituximab) and in severe and or refractory cases splenectomy. Our patient is a 49 year-old Jamaican man with past medical history of HIV infection and recently diagnosed stage IV Hodgkin lymphoma with marrow and organ involvement who presented to the ER with severe fatigue, chills and high-grade fever. CBC testing revealed hemoglobin of 3.2gm/dl with a normal leukocyte and platelet count. Further testing revealed strongly positive direct coombs test, undetectable haptoglobin, elevated unconjugated bilirubin, elevated LDH and plasma free hemoglobin confirming the diagnosis of AIHA. Laboratory parameters for disseminated intravascular coagulation (DIC) were negative. Infection was ruled out with cultures and radiological studies. Severe ongoing hemolysis led to an acute change in mental status, hypotension and acute renal failure requiring the patient to be transferred to the ICU for hemolytic shock. Patient failed to respond to conventional treatments including high dose steroids and IVIG. A single dose of rituximab was also tried without any clinical improvement. Patient’s clinical status deteriorated rapidly and within 48 hours, he developed multi-organ failure requiring continuous venovenous hemodialysis (CVVHD), hemodynamic support with pressors, and aggressive hematological support with multiple blood transfusions. At this point a decision was made to initiate TPE. Within hours of the first TPE, the patient’s clinical status improved. His mental status improved dramatically from a stupor to a fully conscious state. Daily TPE was continued for four days, which resulted in stabilization of his hematocrit, discontinuation of pressors as well as CVVHD. Laboratory parameters of hemolysis normalized. He was eventually able to receive definitive chemotherapy for Hodgkin lymphoma. He is currently three months status post last TPE and undergoing the third cycle of chemotherapy with no recurrence of AIHA. Our experience indicates that TPE can be an effective and rapid treatment modality for patients with severe AIHA secondary to IgG antibodies refractory to conventional means of therapy.
Human and Simian Immunodeficiency Viruses De-Regulate Early Hematopoiesis through a Novel Nef/PPAR-γ/STAT5 Signaling Pathway
Authors: Prost Stéphane , le Dantec Mikael , Augé Sylvie , Le GRAND Roger , Derdouch Sonia , Auregan Gwenaelle , Déglon Nicole , Relouzat Francis , Aubertin Anne-Marie , Maillere Bernard , Dusanter-fourt Isabelle , Kirszenbaum Marek
Published: April 2020

Infection of primates by HIV-1 and SIV induces multiple hematological abnormalities of central hematopoietic origin. Although these defects greatly contribute to the pathophysiology of HIV-1 infection, the molecular basis for altered BM function remains unknown. Here we show that when cynomolgus macaques were infected with SIV, the multipotent potential of their hematopoietic progenitor cells was lost, and this correlated with down regulation of STAT5A and STAT5B expression. However, forced expression of STAT5B entirely rescued the multipotent potential of the hematopoietic progenitor cells. In addition, an accessory viral protein required for efficient SIV and HIV replication and pathogenicity, “Negative factor” (Nef), was essential for <SIV-mediated impairment of the multipotent potential of hematopoietic progenitors ex vivo and in vivo. This newly uncovered property of Nef was both conserved between HIV-1 and SIV strains and entirely dependent upon the presence of PPARγ in targeted cells. Further, PPARγ agonists mimicked Nef activity by inhibiting STAT5A and STAT5B expression and hampering the functionality of hematopoietic progenitors both ex vivo and in vivo. These findings have extended the role of Nef in the pathogenicity of HIV-1 and SIV and reveal a pivotal role for the PPARγ/STAT5 pathway in the regulation of early hematopoiesis. This study may provide a basis for investigating the potential therapeutic benefits of PPARγ antagonists in both patients with AIDS and individuals with hematopoietic disorders.
AKR-501 Activates the Thrombopoietin Receptor through Interaction with the Transmembrane Domain
Authors: Zhang Jie , Alt Jesse , Yang Min , Baranwal Akhil , Luby Thomas M.
Published: April 2020

AKR-501 (1-(3-Chloro-5-{[4-(4-chloro-2-thienyl)-5-(4-cyclohexylpiperazin-1-yl) thiazol-2-yl]carbamoyl}-2-pyridyl)piperidine-4-carboxylic acid, formerly known as YM-477) is an orally-active full agonist of the thrombopoietin (TPO) receptor, c-Mpl. Human c-Mpl consists of an N-terminal extracellular domain containing a membrane-distal cytokine receptor homology region (CRH-1) which is the site of TPO binding, as well as a membrane-proximal CRH region (CRH-2) with yet to be identified functions. The C-terminal end of c-Mpl contains the transmembrane domain (TM), and a cytoplasmic signal transduction domain. It has previously been demonstrated that AKR-501 does not interfere with TPO binding to c-Mpl at the CRH-1 domain, and that AKR-501 is highly species-specific with action only with human and chimpanzee c-Mpl. In the current study, we sought to define the AKR-501 binding domain on c-Mpl by constructing a series of human and mouse chimera c-Mpl receptors in order to evaluate which domain(s) of human c-Mpl confers growth stimulation in response to AKR-501. We found that Ba/F3 cells expressing a chimeric c-Mpl that contained both human CRH-2 and TM responded to AKR-501 and stimulated growth to the same extent as TPO. Other chimeras with only human CRH-2 could not, suggesting that TM is critical for AKR-501 action. A construct was then built to substitute the mouse TM leucine amino acid residue at position 499 with the corresponding human histidine amino acid; this residue is conserved between humans and chimpanzee, but is not found in other species. We found the mouse c-Mpl with a His499 substitution was sufficient to support AKR-501 stimulated growth of Ba/F3 cells. Thus, we concluded that the c-Mpl TM appears essential for AKR-501 activity, and the species specific activity of AKR-501 depends on His499 in the TM.
Characterization of Angiogenic Potential of Late Endothelial Progenitor Cells on Human Cord Blood and Bone Marrow
Authors: Yoo Eun-Sun , Ahn Jee-Young , Kwon KiHwan , Oh Soo-Ah , Choi Moon-Young , Mun Yeung-Chul , Lee Kyoung-Eun , Seong Chu-Myong
Published: April 2020

Background: The identification of circulating endothelial progenitor cells (EPCs) has revolutionized approaches to cell-based therapy for injured and ischemic tissues. Recently, we have demonstrated that there are 2 distinct types of EPCs from UCB having different biologic properties for angiogenic capabilities in vitro and in vivo. In present study, the aim is to directly compare umbilical cord blood (UCB)- and BM-derived late EPC surface phenotypes and in vitro functional capacity.

Methods: Mononuclear cells from UCB and BM cultured using EGM-2 medium with VEGF, IGF-1 and FGF for 21 days. Late outgrowing endothelail cells(late OECs) which were in peak growth at third weeks of culture were analyzed for expression of various surface markers by flow cytometry/RT-PCR/IF, tube formation in Matrigel plates, proliferation assay, endothelial colony assay and the role of SDF-1/VEGF on migration.

Results: The adherent cells after culture of 7 days exhibited a fibroblast like shape in BM and a cobblestone shaped cells in UCB. Although two sources of OECs were comparable in expression of endothelial and various adhesion molecule markers, BM-derived OECs contained higher proportion of cells expressing smooth muscle cell markers(SMMHC), several adhesion molecule(CD49d, CD62L and VCAM-1), whereas the expression of CXCR-4, PECAM-1 and CD62E and expression of mRNA on endothelial marker genes were higher in UCB-derived OECs. UCB-OECS stained positive for uptake of acetylated low-density lipoprotein and had more migratory ability in the presence of SDF-1 and VEGF compared with BM-OECs. Both sources OECs effectively formed capillary tubes in Matrigel plates.

Conclusion: We directly compared OECs derived from UCB and BM and two source of OECs differ in aspect of several adhesion molecule and angiogenic potential in vitro. These difference of UCB render it potentially advantageous for human therapeutic OECs applications for potential applications for a “cell therapy” in the situations on vascular injuries when compared with patients-derived BM.
The Role of HDAC10 in γ-Globin Gene Regulation
Authors: Nimer Sadeieh , Muralidhar Shalini A , Pace Betty
Published: April 2020

HDACs (histone deacetylases) are enzymes that cause chromatin modifications through deacetylation of histones and the recruitment of repressor complexes to mediate gene silencing. To explore this mechanism further, experiments were performed to determine if HDACs are involved in drug-mediated γ-globin gene induction. When human K562 cells were treated with the γ-globin inducer sodium butyrate, we observed 1.8-fold increase in HDAC10 transcription compared to untreated cells. This provided indirect evidence that HDAC10 may be involved in γ-globin gene regulation. To further understand the mechanism, enforced expression experiments using 10–50 μg of the expression plasmid pCMX-HDAC10 and the pCMX empty plasmid were performed by transient transfection of K562 cells via electroporation. Total RNA was isolated and subjected to reverse transcription followed by real time quantitative PCR using gene-specific primers to measure endogenous γ-globin gene levels. Enforced expression of HDAC10 resulted in dose-dependent silencing of γ-globin gene expression. To gain further evidence for a role of HDAC10 in regulating γ-globin gene expression, we performed siRNA knockdowns using SMARTpool-siHDAC10 (Dharmacon) at four concentrations (80nM–320nM) using Oligofectamine (Invitrogen). The γ-globin gene levels were not changed significantly by the siRNA treatment. We next performed enforced expression of HDAC10 in a K562 stable line established using the pGL4.17-Luc2-neo as a base vector in which the expression of luciferase reporter was driven by the Gγ-globin promoter (−1500 to +36). Control stable lines were also established with the empty vector. Preliminary studies of HDAC10 siRNA treatment of the KGγ-CRE stable lines produced a 1.2 fold increase in γ-globin gene activity. These results suggest that HDAC10 may play a role in γ-globin gene regulation during the adult development. Understanding novel mechanisms of γ-gene regulation will expand capabilities to develop therapeutics for sickle cell patients.
Dichotomy in NF-κb Signaling and Chemoresistance in IgVH Mutated Versus Unmutated CLL Cells Revealed by CD40Ligand/CpG Stimulation
Authors: Tromp Jacqueline , Tonino Sanne H. , Van Oers Marinus H.J. , Eldering Eric
Published: April 2020

Introduction and aim IgVH mutated (M) and unmutated (UM) Chronic Lymphocytic Leukemia (CLL) cells differ in their response to the TLR-9 ligand CpG. Whereas CpG induces proliferation in UM CLL it induces apoptosis in M CLL cells obtained from peripheral blood (PB). Yet, PB CLL cells are generally not representative of the cycling fraction which resides in lymph node (LN) proliferation centers. In vitro CD40 stimulation of CLL cells enhances NF-κB activity, upregulates anti-apoptotic proteins Bcl-XL and Bfl-1 and results in chemoresistance. These characteristics are highly similar to, and provide a model for, the situation in LN. In the present study we analyzed the effect of CD40 ligation on CpG responses of both IgVH M and UM CLL cells.

Methods An in vitro co-culture system of PB CLL cells with 3T3 fibroblasts expressing human CD40L was employed to which soluble CpG was added. Cells, RNA and protein fractions were analyzed with respect to:

apoptosis, proliferation and drug sensitivity and NF-κB signaling and downstream targets.

Results In both prognostic subgroups 72 hours of CD40 stimulation results in chemoresistance to different classes of drugs (fludarabine, velcade and roscovitine), as we reported before. However, an important difference in chemoresistance was seen when soluble CpG was added to the culture; UM CLL cells remained drug resistant, but M CLL cells now became sensitive. A corresponding dichotomy in NF-κB signaling occurred upon stimulation with CD40L/CpG compared to CD40L alone. M CLL cells showed gradually declining classical and alternative NF-κB activation and the downstream target Bcl-XL, whereas in UM CLL cells NF-κB and Bcl-XL levels remained equal. Interestingly, after treatment with ABT-737, a BH3 mimetic which is very effective against Bcl-XL, also UM CLL cells became sensitive for cytostatic drugs underscoring the importance of anti-apoptotic Bcl-XL. Induction of TNF-α secretion, also a downstream effect of NF-κB activation, took place similarly in both subgroups upon stimulation with CD40L and CpG. We hypothesize that the source of the dichotomy NF-κB activation lays in upstream signaling determinants such as cIAP1/2, NIK and TRAF family members.

Conclusions Our data uncover a novel distinction in NF-κB activation and drug susceptibility in mutated versus unmutated CLL cells in a relevant model for LN proliferation centers. Together, they provide a rationale for the development of novel drug regimens targeting the NF-κB pathway to treat UM CLL patients.
Cardiac and Liver Magnetic Resonance Characterization of Thalassemia Intermedia Patients: A Comparative Multicenter Study Versus Thalassemia Major Patients
Authors: Pepe Alessia , Favilli Brunella , Ramazzotti Anna , Positano Vincenzo , Borgna-Pignatti Caterina , Capra Marcello , Lai Maria-Eliana , Spasiano Anna , Pitrolo Lorella , Filosa Aldo , Cianciulli Paolo , Mascolino Antonietta , Giuffrida Gaetano , Forni Gianluca , Maggio Aurelio , Lombardi Massimo
Published: April 2020

Little is known about cardiac involvement in Thalassemia Intermedia (TI) using magnetic resonance imaging (MRI). Thus, we investigated myocardial and liver iron overload, myocardial fibrosis, biventricular function parameters and their inter-relationship in 60 TI using MRI. Then, we compared the data of 35 selected TI with age < 39 years with that of 60 Thalassemia Major (TM) patients matched for age. Myocardial Iron Overload (MIO) was assessed using a multislice multiecho T2* approach. Biventricular function parameters were measured quantitatively by cine images. Myocardial fibrosis was evaluated by late gadolinium-enhanced. MIO was present in the 22% of TI patients, more frequently (92%) with an heterogeneous distribution. TI patients with myocardial fibrosis (32%) showed significantly reduced left ventricular ejection fraction (LVEF) (P=0.01). No significant correlation was found between myocardial fibrosis and MIO. Figure shows a TI patient with no myocardial iron overload (all 16 segments and the mid-ventricular septum with T2* values > 20 ms) (A) and late enhancement (white arrows) in the antero-septal junction, in the infero-septal junction and in the mid-ventricular septum (B). In TI patients MIO was significantly lower respect to TM patients; conversely volumes, cardiac indexes, LVEF, bi-atrial areas, and liver iron overload were significantly higher. Myocardial fibrosis was comparable between the 2 groups. TI patients showed lower myocardial iron burden and more pronounced high cardiac output findings with myocardial fibrosis significantly associated to impaired heart function. These data seem to suggest using MRI also for TI patients and could place the basis to reconsider the current cardiological and haematological management in this population.

Figure Figure
Hematopoietic Stem Cells Mediate the Repair of Damaged Endothelium
Authors: Pfaffle Dana L. , Jiang Shuguang , Goldman Devorah C. , Fleming William H.
Published: April 2020

Recent studies indicate that vascular endothelium is an important component of the hematopoietic niche. As endothelial cells (ECs) are sensitive to radiation-induced damage, we evaluated the potential role of hematopoietic stem cells to enhance EC proliferation and repair. To test this hypothesis, lethally irradiated mice were transplanted with either 200–500 c-kit+, Sca+, lineage- (KSL) cells or an equivalent dose of unfractionated bone marrow (BM) cells (1×106 cells). Control groups included irradiated, non-transplanted, and non-irradiated, non-transplanted mice. Immediately after irradiation, all recipients were maintained on 0.8mg/ml Bromodeoxyuridine (BrdU) -containing water. Eleven days following irradiation, liver tissue was harvested and the fraction of proliferating BrdU+ ECs in the portal vein was assessed by immunostaining using both light and fluorescence microscopy. In irradiated, non-transplanted mice, 0.95% ± 0.17 SEM of portal vein ECs demonstrated the incorporation of BrdU. Transplantation of KSL cells increased the frequency of proliferating endothelial cells 2.5-fold to 2.5% ± 0.20 (p<0.0006). The transplantation of an equivalent number of unfractionated BM cells further increased the frequency of proliferating ECs by more than 3.5-fold (3.75% ± 0.21; p<0.0005). In non-transplanted, non-irradiated mice, BrdU+ ECs were detected at an intermediate level (2.30% ± 0.24) that is significantly higher than irradiated nontransplant recipients (p<0.006). To gain a better understanding of how hematopoietic stem cells (HSCs) influence the label retention capacity of ECs, we performed a BrdU pulse-chase experiment. Lethally irradiated mice were transplanted with 200 KSL cells, allowed 4 weeks for recovery, and then maintained on BrdU drinking water for 4 weeks. Consistent with our findings from the short term experiment described above, significantly more BrdU+ ECs were detected in the portal veins of KSL transplanted mice (15.36% ± 2.07) compared to those in non-transplanted, non-irradiated mice (8.68% ± 0.54; p<0.04) at the start of the chase period. During the first 24 weeks of the washout phase, BrdU+ ECs declined at a greater rate in the KSL recipients than in controls, indicating increased EC turnover. Interestingly, however, in both experimental groups, BrdU retention plateaued at 24 weeks and remained constant through 36 weeks. Taken together, our results indicate that radiation damage suppresses the incorporation of BrdU into ECs compared to steady state conditions and that this suppression can be reversed by the transplantation of either hematopoietic stem cells or unfractionated bone marrow. The extent to which BM derived ECs contribute to the proliferating EC pool will be addressed in future studies.
Detection of Functional Hematopoietic Stem Cell Niche Using Real-Time Imaging
Authors: Yin Tong , Xie Yucai , Wiegraebe Winfried , He Xi , Li Linheng
Published: April 2020

Hematopoietic stem cell (HSC) niches play an essential role in regulating adult stem cell self-renewal and differentiation. HSC niches have only recently been identified based on genetic models that showed a parallel increase in the numbers of both HSCs and osteoblasts, or by static immunolabeling of putative HSCs and the adjacent locations. However, osteoblasts and vasculature were viewed as separate niches with distinctive roles, their interrelationships and interactions with HSCs in vivo remain largely undefined. To study the dynamic interaction between the HSC and its niche, we developed a method of ex vivo imaging stem cells (EVISC) using two-photon microscopy. This method combined the technology of real-time imaging and the functional feature that Flk2−LSK HSCs can be prospectively isolated with high purity and can home to their niche upon transplantation into irradiated recipient mice. Using EVISC in combination with immunoassaying, we found that transplanted HSCs tended to home to the endosteal region predominantly in the trabecular bone area (TBA) compared to compact bone area (CBA) under irradiated conditions, but were randomly distributed and unstable under non-irradiated conditions. Mechanistically, we found that the ratio of expression level of SDF-1 between TBA and CBA was significantly increased in response to irradiation. By monitoring individual HSC behavior using EVISC ranging from minutes to 16-hours, we found that a small portion of the homed HSCs underwent active division in the irradiated mice, coinciding with their initial expansion as measured by flow assay and immunoassay. Our findings suggest that the endosteal region in the trabecular bone area formed a special zone, which normally maintains HSCs but promotes their expansion in response to irradiation. This zone includes both osteoblastic and vascular components and signals, which both underwent dynamic changes from homeostatic to stressed conditions.
RCHOP and RDHAP Followed by Autologous Stem Cell Transplantation (ASCT) in Mantle Cell Lymphoma (MCL) : Final Results of a Phase II Study from the GELA
Authors: Delarue Richard , Haioun Corinne , Ribrag Vincent , Brice Pauline , Delmer Alain , Tilly Herve , Salles Gilles , Van Hoof Achiel , Casasnovas Olivier , Brousse Nicole , Lefrere Francois , Hermine Olivier
Published: April 2020

Introduction: Treatment of mantle cell lymphoma (MCL) in younger patients (pts) is still a challenge, with questions about best induction regimen before autologous stem cell transplantation (ASCT) and impact of Rituximab. We report here the final results with extended follow-up of a prospective phase II trial.

Methods: Patients under 66 years with histologically proven, stage III-IV, MCL were included. Treatment consisted of three courses of CHOP with Rituximab at the third one and three courses of RDHAP. Peripheral blood stem cells harvest was performed and responding pts were eligible for an ASCT after high dose radio-chemotherapy with TAM6 (TBI 10 Gy, Aracytine 6 g/m², Melphalan 140 mg/m²) or BEAM if TBI could not be performed.

Results: From May 2000 to September 2003, 60 pts were included. Median age was 57 years. Characteristics of patients were as follow : bone marrow involvement 85%, leukemic disease 48%, gastrointestinal involvment 52%, PS>1 6%, LDH > 1N 38%. Overall response rate was high with 93% after (R)CHOP and 95% after RDHAP. Interestingly, CR was uncommon after (R)CHOP (12%), whereas high proportion of patients (61%) were in CR after RDHAP, suggesting higher efficacy of high dose AraC. Forty-nine pts were autografted (41 with TAM6) : all patients but two (96%) were in CR. With a median follow-up of 67 months, median EFS was 83 months and median OS was not reached. Five years OS was 75%. Neither toxic death nor unexpected toxicities were observed. The comparison with our previous French oligocentric study using the same regimen but without Rituximab (Lefrere, Hematologica 2007) suggests a better outcome when Rituximab is added (median EFS : 51 months).

Conclusion: This study confirms that regimens containing Aracytine and Rituximab are safe and prolong survival and may even induce cure in MCL patients. Thus, they should be used in induction treatment before ASCT. This regimen is currently compared with the classical RCHOP induction in a multicentric European protocol within the EMCL network.
Molecular Remission after Combined Immunochemotherapy Is of Prognostic Relevance in Patients with MCL: Results of the Randomized Intergroup Trials of the European MCL Network
Authors: Pott Christiane , Hoster Eva , Böttcher Sebastian , Siebert Reiner , Klapper Wolfram , Delfau Marie Helene , Unterhalt Michael , Hiddemann Wolfgang , Kneba Michael , Dreyling Martin
Published: April 2020

Two randomized intergroup trials of the European MCL Network investigating the impact of different combined immunochemotherapy protocols for patients with stage II-IV mantle cell lymphoma (MCL) followed by autologous stem cell transplantation for patients <65 yrs and a rituximab or interferon maintenance treatment for patients > 65 yrs are currently performed.

Patients and methods: 182 German patients with evidence for peripheral blood (PB) involvement as demonstrated by consensus IGH-PCR at diagnosis were analyzed for quantitative PB involvement and molecular response (MR) by Real Time Quantitative (RQ) IGH-PCR. The results were evaluated according to ESG criteria (van der Velden, Leukemia 2007) and were compared to clinical parameters at diagnosis and outcome.

Results: Patients had a median age of 61 years, an elevated LDH in 37%, B-symptoms in 42% and extranodal involvement in 36%. According to the MIPI prognostic index 20% had an adverse, 37% an intermediate and 43% a good prognosis. Median level of circulating MCL cells in PB at initial diagnosis were 6×10−2 (range 2×10-4 to 8×10-1) and correlated significantly with clinical parameters as advanced stage (p=0.0016), elevated LDH (p= 0.0026), bone marrow infiltration (p= 0.0001) and MIPI prognostic index (p<0.0001). MR after induction was achieved in 55% (67/121) of all evaluable patients and correlated with stage (p=0.058), the presence of B-symptoms (p=0.016) and BM infiltration at diagnosis (p=0.011). Interestingly, the rate of MR was higher in patients receiving the protocol for elderly than for young patients (69% vs. 45% p=0.0006). This effect was especially prominent in the BM with an MRD clearance of 65% in elderly compared to only 35% in younger patients (p<0.0001). BM was more sensitive for MRD detection compared to PB after rituximab containing treatment. Of 62 paired samples only 2 BM samples were BM MRD- with concomitant MRD+ in the PB, whereas 13 MRD- PB samples demonstrated MRD+ in the corresponding BM (p=0.045). Patients achieving a MR in the BM after induction demonstrated a significantly improved remission duration compared to MRD+ patients (100% vs. 66% at 24 months, p=0.0285, median observation time 15 months).

Conclusion: Quantitative detection of circulating MCL cells improves clinical risk assessment and staging accuracy. Furthermore RQ-PCR allows to evaluate the impact of different treatment modalities on tumor clearance in PB and BM. This is the first randomized trial demonstrating that achievement of MR by combined immunochemotherapy might be an early indicator for treatment outcome in MCL.
Onco-Mir-155 Targets SHIP to Regulate TNF-Dependent B-Cell Lymphoma Growth
Authors: Pedersen Irene Munk , Otero Dennis C , Kao Elaine , Sedy Ana , Hother Christoffer , Ralfkiaer Elisabeth , Rickert Robert C , Gronbaek Kirsten , David Michael
Published: April 2020

Diffuse large B-cell lymphoma (DLBCL) accounts for approximately 40% of non-Hodgkin lymphomas, with an overall 3-year relapse-free survival of 53%. DLBCL consists of a germinal center B-cell (GCB-like) subgroup and an activated B-cell (ABC-like) subgroup exist, associated with good and poor prognosis, respectively. In addition to differential expression of coding genes, ABC-type lymphoma cells express high levels of the non-coding microRNA, miR-155, an onco-miR whose ectopic expression in the mouse has been shown to give rise to B-cell malignancies. DLBCL feature a dysregulated PI3 kinase pathway, which can promote proliferation and survival. PI3K is regulated by phosphatases PTEN (phosphatase and tensin homolog deleted on chromosome ten) and SHIP (SH2 domain-containing inositol 5-phosphatase). PTEN is a known tumor suppressor and is mutated in human cancer with a frequency approaching that of p53. cDNA array analyses of DLBCL patient specimens indicate that low mRNA expression level of SHIP can be used as a negative prognostic marker for survival. We have now identified SHIP as the first bona-fide target of miR-155. RT-PCR analysis of micro-dissected lymphoma samples confirmed high levels of miR-155 and low levels of SHIP in poor-prognosis ABC-cells as compared to GCB-cells. We further demonstrate that elevated levels of miR-155, and consequent abrogation of SHIP expression, is not due to point mutations or promoter hypermethylation of SHIP in primary DLBCL cells, but rather the result of a TNFa-mediated autocrine loop, a pro-inflammatory cytokine whose serum levels are elevated in DLBCL patients. Consequently, anti-TNFa regimens (Etenercept (Enbrel®), Infliximab (Remicade®)) are sufficient to reduce miR-155 levels, restore expression of SHIP, decrease p-Akt levels, and slow tumor cell growth of ABC-sub-type DLBCL cell lines. Furthermore, we observe a highly significant decrease in tumor growth when DLBCL-xenograft mice are treated with Etenercept, suggesting a novel approach for restoring tumor suppressor activity and inducing growth inhibition of these aggressive tumors. Our findings offer the first direct evidence that specific miRs can function as a the link between inflammation and cancer. Finally, our hypothesis, that TNFa promotes survival of DLBCL via miR-155, SHIP and pAkt, opens up a novel and immediately accessible (co)treatment for poor prognosis DLBCL.
MTG16, a Target of the T(16;21) in Acute Myeloid Leukemia, Is Required for Hematopoietic Stem and Progenitor Cell Functions
Authors: Steapleton Melissa Ann , Moreno Isabel , Chyla Brenda , Hiebert Scott
Published: April 2020

The t(8;21) and t(16;21) disrupt two closely related Myeloid Translocation Gene family members respectively, MTG8 and MTG16. Whereas the expression of MTG8 is highly regulated, MTG16 is more widely expressed and is the family member most highly expressed in hematopoietic stem cells. Therefore, to address the contribution of MTG16 to HSC functions and hematopoiesis, we created mice lacking this gene. We show that this transcriptional co-repressor is required for hematopoietic stem and progenitor cell functions such as cell fate decisions and early progenitor cell proliferation. Inactivation of Mtg16 skewed early myeloid progenitor cells towards the granulocytic/macrophage lineage, while reducing the numbers of megakaryocyte-erythroid progenitor cells, which was shown using both flow cytometry and methylcellulose colony formation assays. In addition, inactivation of Mtg16 impaired the rapid expansion of long and short-term stem cells, multi-potent progenitor cells and megakaryocyte-erythroid progenitor cells that are required under hematopoietic stress/emergency. Due to this, the Mtg16-null mice could not respond to phenylhydrazine or 5-fluorouracil treatment and were completely defective in the colony forming unit-spleen (CFU-S) assays. Additionally, Mtg16-null bone marrow failed to repopulate the hematopoietic system when it was transplanted into an irradiated recipient mouse and also failed to compete with wild-type bone marrow in a competitive bone marrow transplant. This impairment appeared to be due to a failure to proliferate rather than an induction of cell death, as expression of c-Myc, but not Bcl2, complemented the Mtg16(−/−) defect. Thus, like other key transcriptional co-repressors (e.g., the retinoblastoma protein, pRB, and the nuclear hormone co-repressor, N-CoR) Mtg16 is a key regulator of stem cell functions and lineage commitment in hematopoiesis.
Regulation of Stem Cell Aging by Retinoblastoma Like-1 (p107)
Authors: Oakley Erin J. , Geiger Hartmut , Van Zant Gary
Published: April 2020

It is well documented that both quantitative and qualitative changes in the murine hematopoietic stem cell (HSC) population occur with age. We have previously mapped a quantitative trait locus (QTL) to murine chromosome 2 that is associated with the variation in frequency of HSCs between aged C57BL/6 (B6) and DBA/2 (D2) mice. In B6 mice the HSC population steadily increases with age, whereas in D2 mice, this population declines. A QTL regulating the natural variation in lifespan between the two strains was mapped to the same location on mouse Chr 2, thus leading to the hypothesis that stem cell function affects longevity. B6 alleles of this locus, associated with expansion of the stem cell pool, are also associated with a ~50% increase in lifespan. In the present study, we characterize a congenic mouse model which was generated by introgressing D2 alleles in the QTL onto a B6 background. Using a surrogate assay to mimic aging, we analyzed the cell cycle, apoptotic and self-renewal capabilities of congenic and B6 HSCs and show that D2 alleles in the QTL affect the apoptotic and self-renewal capabilities of HSCs. Next, we used oligonucleotide arrays to compare the differential expression of B6 and congenic cells using a population enriched for primitive stem and progenitor cells. Three variables were examined using Affymetrix M430 arrays:

the effect of strain—congenic versus background; the effect of age—2 months versus 22 months; and the effects of 2 Gy of irradiation because previous studies indicated that congenic animals were highly sensitive to the effects of mild radiation compared to B6 background animals.

Extensive analysis of the expression arrays pointed to a strong candidate, the gene encoding Retinoblastoma like protein 1, otherwise known as p107. The B6 allele is associated with increased p107 expression in old HSCs therefore p107 in this context is a positive regulator of stem cell number in aged mice. Real-time PCR was used to validate the differential expression of p107 in lineage negative and lineage negative Sca-1+, c-kit+ (LSK) cells. Detailed sequence analysis of the gene revealed the presence of 4 non-synonymous, coding region single nucleotide polymorphisms (SNPs) between B6 and D2 mice, which may contribute to the differential expression of the gene and function of the protein. Perhaps most importantly, we show that overexpression of p107 in congenic HSCs increases day 21, day 28, and day 35 CAFC numbers in vivo by 2- to 4-fold, therefore confirming its role as a positive regulator of primitive progenitor populations including HSCs. These studies uncover a novel role for p107 and provide additional clues in the complex regulation of stem cell aging.
Second Primary Malignancy in Diffuse Large B Cell Lymphoma (DLBCL)
Authors: Ghimire Krishna B , Shah Binay K.
Published: April 2020

Introduction:

Patients with non-Hodgkin lymphoma are at significantly increased risk of second primary malignancies (SPM). There are few studies on SPM in diffuse large B-cell lymphoma. We aim to evaluate the risk of SPM after diagnosis of primary DLBCL using Surveillance, Epidemiology and End Results (SEER) database.

Methods:

We used SEER 13 database to select adult patients with primary DLBCL diagnosed between 1992- 2009. We used SEER*Stat’s Multiple Primary - Standardized Incidence Ratios (MP-SIR) to calculate SPM in DLBCL. We analyzed SPM by age (≥20, 20-59 and ≥60 years), sex and latency. Second primary malignancy was defined as a metachronous malignancy developing six months or more after an index DLBCL.

Results:

A total of 28,368 adult DLBCL patients were selected for analysis. Among them, 54.4% were male, 44.1% were <60 years of age. The total number (n) of all site SPMs in DLBCL patients was 2,597, an Observed/ Expected (O/E) ratio of 1.22, P< 0.05, excess risk of 30.93. The risk of following SPMs was significantly higher in the patients with DLBCL: head & neck, anus & colorectal, lung, skin (melanoma), kidney, thyroid, Kaposi sarcoma and hematological malignancies.

In young DLBCL patients (20-59 years) group, 12,509 patients developed 830 all site SPMs with O/E: 1.56, P value < 0.05, excess risk 37.01. SPMs which were significantly increased as compared to general population were: head & neck, anus & colorectal, lung, kidney, thyroid, Kaposi sarcoma and hematological malignancies. Similarly, 15,859 older (≥60 years) DLBCL patients developed 1,767 all site SPMs with O/E: 1.11, P< 0.05, excess risk 24.07. SPMs which were significantly increased in this age group as compared to general population were: head & neck, skin, colon, kidney, thyroid and hematologic malignancies.

There was significantly higher risk of tumors of anus & colorectal, liver, kidney, thyroid, Kaposi sarcoma and hematologic malignancies within 5 years of latency. Malignancies of head & neck, anus &, colorectal, skin, breast, urinary bladder and hematologic malignancies were significantly higher after 5 years of latency.

Conclusion:

Patients with DLBCL were at significantly increased risk of developing SPMs as compared to general population. Survivors of DLBCL need to be closely followed with special attention to SPM.

Disclosures

No relevant conflicts of interest to declare.