bluuebunny/crossref_metadata_2025 huggingface dataset:
U.S. Factor XI Patients: Is There a Safer Treatment? Yes - Hemoleven.
Authors: Aledort Louis M. , Goudemand Jenny
Published: October 2019

Despite donor screening and newer PCR testing, human-derived fresh frozen plasma (FFP) is not without the potential for viral transmission. Factor XI-deficient patients are plentiful in the U.S. (estimate ~1500–2000). Although there is discordance between phenotype and genotype, with substantially fewer “bleeders” than those with deficiency, and spontaneous bleeding is rare, surgical intervention frequently demands replacement therapy. The English F XI concentrate, virally inactivated, had thrombosis as a complication, and heparin was added, but is no longer available in the U.S. An LFB factor XI concentrate (Hemoleven), doubly viral-inactivated, with heparin and ATIII is available internationally, but not in the U.S. A review of 56 cases treated in France, Spain, Switzerland, Canada, and Israel reveal that for minor and major surgery it is both safe and effective. Between 1992 and 2004, data for the use of Hemoleven for either prophylaxis, minor and major surgery was collected. In all, hemostasis was excellent. In three, concentrate was used because of anaphylaxis with FFP. Three had laboratory evidence of DIC and, of these, one had a PE. In addition, these three cases received excessive dosage. Twelve patients had recovery and half-life studies for this concentrate. T1/2 was 45.5 ± 7.9h and recovery was 1.85 ± 0.38%/u/kg. These data are evidence that a safer and effective therapy for F XI patients exist outside of the U.S. We should not have to wait for a new infectious agent transmitted by FFP before we make it available in the U.S.. We should have learned something from HIV, when DDAVP was used everywhere, but not licensed here.
Experience with the “Off Label” Use of Recombinant Factor VIIa in a University Hospital.
Authors: Escobar Miguel A. , Chong Kenneth , Hoots Keith W.
Published: October 2019

Recombinant factor VIIa (rFVIIa), NovoSeven@ (Denmark), is a bypass agent approved in the United States for patients with hemophilia and inhibitors. The off-label use of this medication (non approved use) is becoming common in large institutions. Its main use seems to be for excessive bleeding due to trauma and surgery. Here we report our 2 year experience with the off-label use of this product and describe a protocol that is followed at our teaching hospital. Administration of rFVIIa was based on the described protocol. The medication is controlled by the hospital pharmacy and authorization for its use has to be given by the hematologists in collaboration with the clinical pharmacist. To be eligible for the use of rFVIIa the patient has to have received at least 10 units of red pack cells and fresh frozen plasma. In addition, the patient should have fibrinogen level greater than 100 mg/dl, platelet count greater than 70,000/mm3 and fibrin split products less than 80 mcg/ml. A total of 25 patients where treated off-label for excessive bleeding and 4 patients where given rFVIIa preoperative for prevention of bleeding. The doses ranged from 35 to 120 micrograms/kg of body weight with an average of 2 doses given per patient. The use of Factor VIIa was more common in the neurosurgical (NS) setting (7), followed by cardiovascular (CV) (6), gastrointestinal (GI) surgery (6), trauma (6) and others (4). Overall 62% of the patients had adequate hemostasis after the administration of rFVIIa. Successful hemostasis with rFVIIa by diagnosis was 100% for preoperative prophylaxis and GI, for NS 57%, for trauma 50% and for CV 17%. All the patients in the CV group where admitted with acute dissecting aortic aneurysms. No evidence of thromboembolic events where described. The “off-label” use of rFVIIa continues to be widely used with variable results. Until further randomized or controlled, double blind, clinical trials are done, no definitive recommendations can be given. Although this report includes a small cohort of patients, we will consider revising our protocol for bleeding following cardiovascular surgery and trauma.
Identification of a Novel Factor V A2 Domain Tyrosine Deletion Mutation in a Patient with Factor V Deficiency and Bleeding.
Authors: Jones Carol D. , Negro Fernando , Darnell Katherine , Zehnder James L.
Published: October 2019

The gene for coagulation Factor V (FV) is located on chromosome 1q23. FV deficiency shows an autosomal recessive mode of inheritance; heterozygotes are generally not clinically affected. The homozygous clinical phenotype occurs in approximately 1 per million individuals with variable severity of bleeding. Thus, genotype-phenotype correlations are likely to shed light on functionally important residues of FV. Here we describe a case of FV deficiency with a severe bleeding phenotype. The proband is a male infant from Argentina. His parents are unrelated. He was born healthy with no bleeding from the umbilical stump or other symptoms. He presented at eight months with a CNS hemorrhage, then suffered a second massive subdural bleed at nine months of age. Both episodes required surgical drainage and treatment with fresh frozen plasma He continues to receive prophylactic FFP infusions and has some residual neurologic impairment. The proband’s FV activity ranges from 2–14%. Two siblings are unaffected. His father’s FV activity is 50% and his mother’s is 70%. We performed DNA sequencing spanning the entire coding region of the proband’s FV gene and found two heterozygous mutations: a heterozygous single base pair deletion, del 2952T in exon 13, located in the B-domain of the FV protein, causing a frameshift mutation followed by a premature termination codon 3 amino acids downstream; and a novel 3-bp deletion in exon 10. This deletion is in-frame and results in the deletion of Y478. The del 2952T frameshift mutation was present in the father, while the del Y478 mutation was present in the mother. Y478 is in the A2 domain of FV and adjacent to another tyrosine, Y477. Evidence suggests that these tyrosine residues are important for co-factor function. Tyrosine residue sulfation has been shown to be required for full activity of the homologous co-factor, FVIII, as well as for hirudin. These sulfated tyrosines and surrounding acidic amino acids have been proposed to be important in interactions with the thrombin anion binding exosite; in the case of hirudin, sulfation of a carboxy-terminal tyrosine increases the affinity for thrombin 10-fold. The homologous tyrosines, Y718 and Y719 appear to be sulfated in FVIII. FV has been shown to be sulfated, but the precise location of the FV sulfation sites has not yet been determined. One of this patient’s FV alleles is nonfunctional due to a frameshift and a premature trancation of translation. With respect to the other allele, we hypothesize that, like FVIII, one or both of FV tyrosines 477 and 478 is sulfated, and that deletion of Y478 may result in disruption of FV co-factor function. In vitro mutagenesis and expression studies to characterize the functional consequences of the del Y478 and/or del Y477 are in progress.
DIC with Ulcerative Colitis: The Combined Effects of Prothrombin G20210A, APC Resistance, and Endotoxemia⇒.
Authors: Aue Georg , Carroll Nancy , Horne McDonald
Published: October 2019

Thrombosis is a recognized complication of inflammatory bowel disease (IBD). In contrast disseminated intravascular coagulation (DIC) has rarely been described with IBD, and then only during periods of intense disease activity or during colectomy. However, we recently encountered a young woman with a 3-year history of ulcerative colitis (UC) and a 10-year history of recurrent thrombosis who presented with DIC when her disease appeared to be well controlled. For the previous two months she had been forced to discontinue her usual warfarin because of extreme over anticoagulation and extensive ecchymoses. Otherwise she felt well and was working full time. When she presented to us, the following laboratory data were obtained: pro time 18.7s, aPTT 43.6s, fibrinogen 56 mg/dL, D-dimer 8–16 μg/mL, platelet count 123K/μL, thrombin-antithrombin complexes 47 μg/mL (nl, 1.3–4.0), plasma endotoxin 6.3 EU/mL (control 0.1), positive prothrombin G20210A heterozygosity, negative FVLeiden, but positive APC resistance. Antithrombin, protein C, protein S, and homocysteine were within normal limits. Extensive imaging studies failed to reveal evidence of malignancy or venous malformations. The patient was treated with 1 mg/kg/d enoxaparin with resolution of her ecchymoses and normalization of her laboratory values. Subsequently we exposed cultured human umbilical vein endothelial cells (HUVECs) to our patient’s serum and assayed HUVEC lysates for tissue factor. After a 2 hour incubation a 20% dilution of her serum increased the tissue factor in the HUVECs to a level twice that observed with serum from a normal donor. Furthermore, the effect of her serum was negated by the addition of 1 μg/mL polymyxin-B, which specifically inhibits endotoxin.

Discussion: Prothrombin G20210A, APC resistance, and UC are individually associated with an increase risk of thrombosis. However, the combination of all 3 factors in our patient apparently led to an intense degree of hypercoagulability that provoked not only recurrent thrombosis but DIC. Furthermore, the contribution of UC to her hypercoagulability may have been mediated by endotoxin diffusing from her inflamed bowel and upregulating tissue factor in her endothelium and/or monocytes. Because this patient’s recurrent thromboses predated her diagnosis of UC by 7 years, we suggest that occult IBD should be considered in the evaluation of idiopathic thrombophilia.
Elevated Factor VIII: C Level Is a Risk Factor for Cerebral Infarction.
Authors: Hua Baolai , Pan Jiraqi , Zhu Yicheng , Liu Xiaoli , Fan Liankai , Cun Liying , Zhao Yongqiang , Shen Ti
Published: October 2019

To explore the association between the risk of cerebral infarction (CI) and markers of coagulation and fibrinolytic systems. A prospective case-control study was carried out in PUMCH, in which 124 unselected patients with CI as well as 91 control patients were studied. Blood samples in acute phase of CI were obtained within 10 days from onset, and the convalescent smples were obtained at least 6 months after onset. Factor VII, FVIII:C, FX, vWF, AT-III, PC, PS, FPS,protein Z( PZ), D-dimer and TAT levels were measured. Associtions were assessed by multiple logistic regression analyses. Compared with control group, levels of FVIII:C, vWF, FVII, DD, TAT in patients with acute CI were marked elevated (FVIII:C194.6±48.6% vs 91.1±26.9%, p=0.000; vWF 123.2±28.8% vs 90.0±20.5%, p=0.003; FVII 98.7±25.2% vs 82.3±25.3%, p=0.002; DD 0.41±0.61 mg/l vs 0.12±0.10 mg/l, p=0.008; TAT 40.9±73.3 ng/ml vs 12.3±11.5 ng/ml, p=0.028, whereas level of FX was not changed significantly. Levels of anticoagulation proteins were decreased. Levels of PZ was 0.41μg/ml lower than cortrol (1.32±0.83μg/ml vs 1.73 ±0.55μg/ml, p=0.004) The result of logistic regression analysis showed levels of FVIII:C and vWF in acute phase were related with CI, with OR 1.143, 95%CI 1.099~1.189, P<0.01 and OR 1.040, 95%CI 1.003~1.082, P<0.05 for FVIII:C and vWF respectively. Compared with acute phase, levels of FVIII:C, DD and TAT in convalescent phase were reduced (FVIII:C 144.3±37.2% vs 193.5±46.2%, P=0.000; DD 0.21±0.20 mg/l vs 0.41±0.78 mg/l,p=0.025; TAT 15.2±10.3 ng/ml vs 42.9±72.6 ng/ml, p=0.030). However, the levels of FVIII:C, vWF and FVII were still higer than that of control(FVIII: C 144.3±47.2%, vs 91.1±26.9%, vWF 110.7±23.2% vs 90.0±20.5%, FVII 93.6±25.5% vs 82.3±25.3%, P<0.05). Level of PZ was elevated by 0.33μg/ml. The result of multiple logistic regression analyses indicated FVIII:C, vWF were the independent risk factors for CI
A Novel Flow Cytometric Annexin A5 Competition Assay for the Diagnosis of Antiphospholipid Syndrome.
Authors: Senzel Lisa , Wu Xiao-Xuan , Rand Jacob
Published: October 2019

Annexin A5 (A5) is a potent anticoagulant protein that crystallizes over phospholipid surfaces, shielding them from availability for coagulation enzyme reactions. The antiphospholipid antibody (aPL) syndrome (APS) is an autoimmune thrombophilia that is marked by the presence of antibodies against phospholipid-binding proteins and the paradoxical lupus anticoagulant phenomenon. aPL antibodies can promote coagulation by interfering with the crystallization of A5, thereby increasing the exposure of blood to thrombogenic anionic phospholipids. Since a flow cytometric assay for aPL measuring A5 binding to frozen thawed platelets was previously reported, we investigated whether phosphatidylserine-bound polystyrene beads could provide a robust platform for the assay. Beads were incubated with sera from patients with the aPL syndrome and from non-aPL controls. Fluorescence-tagged A5 was added and samples were analyzed by flow cytometry. Similarly treated beads were also used in coagulation assays to determine whether A5 anticoagulant function was also inhibited. Control sera permitted fluorescent labeling of nearly all beads.

In contrast, sera from aPL syndrome patients produced a major population of unlabeled beads, sometimes accompanied by a minor population of labeled beads.

7 of 13 confirmed aPL syndrome patients, and 7 of 12 anticardiolipin antibody positive patients, demonstrated reduced A5 binding in this assay, while 10 of 10 healthy blood donor controls did not. Dilution of the aPL sera resulted in progressive increase of A5 binding. Reduction of A5 binding appeared to correlate with reduced A5 anticoagulant activity (r=0.42, n=33, p=.01). This assay shows promise for investigating the effects of aPL antibodies on A5 binding and for the clinical diagnosis of the aPL syndrome.

Figure Figure Figure Figure
Cyp33 Immortalizes Mouse Bone Marrow Progenitor Cells.
Authors: Chen Jing , Diaz Manuel O.
Published: October 2019

MLL is a gene involved in translocations associated with leukemia. It encodes a protein with PHD fingers and SET domains that are conserved in the related Drosophila protein trithorax. Cyp33, a nuclear cyclophilin with an RNA binding domain, interacts with the MLL PHD finger domain. Overexpression of Cyp33 represses the Hoxc8 gene in human leukemia cells in an MLL dependent manner. This close relationship between Cyp33 and the genes involved in hematopoiesis and leukemogenesis inspires us to further clarify the function of Cyp33 in primary hematopoietic cells. Murine bone marrow progenitor cells (BMPC) were retrovirally transduced to express Cyp33 and their replicative potential was then investigated by repeatedly plating on methylcellulose medium supplemented with GM-CSF. Upon over-expression of Cyp33, p27 is down-regulated and the progenitor cells keep proliferating after the fourth plating while the control cells (infected with empty retroviral vector only) differentiate and stop forming colonies after the third plating. Furthermore, after the third plating, Cyp33 over-expressing cells exhibit primitive cell morphology after Wright staining, while the control cells show mature macrophage/granulocyte characteristics. We also generated a Cyp33-BMPC line by growing the Cyp33 over-expressing cells in liquid culture containing GM-CSF, after the fourth plating. Regardless of the presence of GM-CSF, the Cyp33-BMPC cell line cycles and grows exponentially maintaining stem cell surface markers: Sca-1+, c-Kit+, CD34+ and Lin− (B220−, CD3e−, Mac-1−, Gr-1−). Morphologically, Cyp33-BMPCs are like immature progenitor cells. In addition, these cells can again form compact colonies on the same methylcellulose media as described before. These data suggest that Cyp33 may immortalize mouse bone marrow progenitor cells and arrest them at the primitive cell stage.
Induction of Transient Cytopenia to Detect Hematopoietic Defects in the Mouse.
Authors: Anderson Nicole M. , Adamson Lee , Barber Dwayne L. , Stanford William L.
Published: October 2019

In an attempt to generate new mouse models of human hematopoietic diseases and increase our knowledge of the genetic networks that orchestrate hematopoiesis, we are developing a sensitized screen that will more effectively identify hematopoietic in heterozygous mutants. The chemical mutagen ethylnitrososurea (ENU) is a highly efficient mutagen in the mouse and other model organisms, generating primarily point mutations. As a part of the Centre for Modeling Human Disease (CMHD), we have used simple clinical assays such as blood morphology and automated blood cell counts to test saphenous vein collected peripheral blood from approximately 6500 offspring of ENU mutagenized mice to identify clinically relevant dominantly inherited hematopoietic disease models. Phenodeviant G1 animals are bred to wild type mice to generate G2 mice, and G2 strains demonstrating high penetrance undergo a genome scan. This strategy has yielded 14 heritable dominant mutants to date. However, many genes and pathways will not express phenotypes in a dominant manner due to the homeostatic balance maintained by complex genetic networks, possessing excess capacity or redundancy, a problem exacerbated by using a viable peripheral blood assay. For example, only one of the 14 heritable hematopoietic mutants demonstrates anemia. Because environmental sensitization can exacerbate latent phenotypes, such as the effect of a high salt diet in an individual with prehypertension, sensitized screens should identify subtle and additional phenotypes of dominant mutations. Thus, we are developing a sensitized screen to uncover subtle dominant phenotypes by inducing transient cytopenia and following the kinetics and magnitude of the recovery. We are using a variety of drugs (5-fluorouracil, phenylhydrazine, and hydroxyurea) to induce cytopenia and recruit hematopoietic stem cells and progenitors to repopulate the blood with differentiated cells. We hypothesize that animals with defects in blood development will demonstrate altered recovery kinetics. This strategy will allow us to determine if there is a defect in blood cell progenitors, while keeping the animals viable for further screening and breeding. We are testing these drugs on known dominant and recessive mutants to validate the assay, determine the best dosages, and compare the effects of each drug. However, we have already begun to use 5-fluoruracil on a cohort of G1 animals and have identified two G1s that display latent recovery in at least 1 blood lineage. The two G1s are confirmed heritable and are currently undergoing a genome scan to map position the mutation. We believe this strategy will further our understanding of the molecular mechanisms that regulate hematopoietic development, as well as generate new mouse models of human hematopoietic diseases and identify the underlying molecular defects of diseases whose etiological origins are currently unknown.
Expression of CXCR4 on Intrahepatic Stem Cells from Organ Donors and Patients with Chronic Liver Disease.
Authors: Porretti Laura , Lopa Raffaella , Gramignoli Roberto , Ambrosone Antonella , Mosca Annamaria , Cattaneo Alessandra , Scalamogna Mario , Gatti Stefano , Rossi Giorgio , Colli Agostino , Prati Daniele
Published: October 2019

Replication of mature hepatocytes can ensure hepatic tissue renewal in normal liver. However, in certain physiopathologic conditions, cells with stem-like properties can proliferate and/or be recruited from extra-hepatic compartments. For a better understanding of the mechanisms related to liver regeneration, more data on the morphological and differentiative profiles of liver stem cells are needed. This study was aimed at the immunophenotypic characterization of circulating and intrahepatic stem cells in organ donors and in patients with chronic liver disease.

We performed a four-colors flow cytometric evaluation of liver mononuclear cell suspensions (L-MNC) obtained after enzymatic digestion of liver biopsy specimens from 8 patients and 6 multi-organ donors. Paired peripheral blood mononuclear cells (PB-MNC) from the same subjects were also examined. L-MNC and PB-MNC were incubated with antibodies to the following markers: CD34, CD133, CD45, CD117 (c-kit) and CXCR4 (Stromal derived factor-1 receptor). Cell viability was performed using 7-aminoActinomycin D to exclude dead cells.

In patients, the stem cell marker CD34 was detected on the surface of 0.6±0.9% of the L-MNC, a proportion similar to that observed in donors (0.6±0.5%). In both patients and donors, the majority of CD34+ cells co-expressed CD45 (86±9% and 60±31% respectively). The subset of CD34+/CD45- cells expressing c-kit was increased in L-MNC from patients as compared to donors (13.8±12% vs.1.5±1% of the total CD34+ cells; p<0.05 by Student t test), while it was scarcely represented or not detectable in PB-MNC. The proportion of CD34+ cells with a definite hematopoietic profile (CD45+/c-kit+) did not differ between patients and donors, both in L-MNC (50±32% vs. 57±13%) and in PB-MNC (66±20% vs. 45±23%). However, in the latter compartment we identified a subset of cells expressing both CD133 and CXCR4. This subset of CD34+ cells was more frequently found in diseased (74±25%) and normal liver (50±18%) as compared to peripheral blood from both patients and donors (24±22% and 30±30% respectively), (p<0.01; paired-t test).

Flow cytometry analysis of intrahepatic CD34+ stem cells allows the identification of at least two phenotipically distinct populations. The first population co-expresses c-kit, is increased in the presence of chronic hepatocellular damage and is likely resident in the liver, being rarely detectable in peripheral blood. The second population resembles the classical hematopoietic stem cell and harbors CD133 and the chemokine receptor CXCR4 for trafficking to the liver in response to liver injury.
Erythropoietin Augments Antibody Response to DNP in Immunized Mice.
Authors: Yeyni Lidor , Katz Odelia , Prutchi-Sagiv Sari , Skaat Alon , Barzilay Eran , Mittelman Moshe , Neumann Drorit
Published: October 2019

Recombinant human erythropoietin (rHuEpo), the main growth regulator of red blood cells, has been used in clinical practice for the treatment of anemia of various etiologies. Over the last decade several reports have indicated that the action of Epo is not restricted to the erythroid lineage. Studies indicate that rHuEpo treatment may have an immunomodulatory effect on hemodialysis patients. Here we address the effects of rHuEpo on the humoral immune response of mice. Naïve BALB/c mice (two groups of 16 mice each) were immunized with 2mg antigen dinitrophenylated keyhole limpet hemocyanin (DNP-KLH). Concomitant to antigen injection, the mice were injected three times weekly with rHuEpo (180U per injection) or with diluent (control). Blood samples were taken 2 and 4 weeks after immunization and anti-DNP antibody levels were measured by ELISA. Production of anti DNP antibodies increased significantly (1.5–2 fold) in the initial phase of the response (2 weeks following DNP-KLH injection) in mice injected with rHuEpo compared to their counterpart control antigen-injected mice, that received diluent only.

These data indicate that erythropoietin may have a role in stimulating the humoral arm of the immune system, suggesting an immunomodulatory and/or immunostimulatory role for rHuEpo and may open new avenues in improving the immune response of immunocompromised patients.
Bcr-Abl Protection of Fas-Induced Apoptosis.
Authors: Brumatti Gabriela , Castro Fabiola A. , Bueno-da-Silva Ana Elisa B. , Vaccarezza Gabriela , Amarante-Mendes Gustavo P.
Published: October 2019

The Bcr-Abl tyrosine kinase found in Philadelphia chromosome (Ph1)-positive leukaemia is the result of a reciprocal translocation between chromosomes 9 and 22. Depending on the breakpoint of the bcr gene, different isoforms of Bcr-Abl appear and are associated with specific types of leukemias; in chronic myelogenous leukaemia (CML), a 210kDa protein is responsible for a deregulated proliferation and consequently accumulation of myeloid cells and their precursors, whereas in acute lymphocytic leukaemia (ALL) a 185kDa Bcr-Abl protein is the oncogenic counterpart. This chimeric protein not only induces transformation of cell lines, but also regulates cell proliferation and is one of the most potent anti-apoptotic molecules responsible for resistance to a variety of apoptosis-inducing agents. The signalling pathway of apoptosis which may be regulated by Bcr-Abl is still unknown, but our latest findings suggested that Bcr-Abl act at the mitochondrial level, preserving the mitochondria integrity during several apoptogenic stimuli thereby preventing release of cytochrome c and the consequent activation of effector caspases. To investigate the mechanisms whereby Bcr-Abl interferes with the cell apoptotic machinery, we asked where in Fas signalling cascade that Bcr-Abl blocks the apoptogenic signal. For this purpose, HL-60.neo, HL-60.Bcr-Abl, HL-60.Bcl-2 and HL-60.Bcl-xL cells were stimulated with anti-Fas antibodies (CH11), in the presence or not of cycloheximide, and the levels of apoptosis and activation of caspases where compared between wild-type and transfected cells with anti-apoptotic molecules. Our results demonstrate that Bcr-Abl blocks both mitochondria dependent and independent Fas pathway, suggesting that Bcr-Abl has a bigger spectrum of action than Bcl-2 and Bcl-xL.

CNPq, FAPESP and IEP-HIAE
Mutations Innate Immune System NOD/CARD15 Gene and Toll-Like-Receptor-4 Gene Are Associated with Increased Risk for Severe Acute Gvhd in Patients Who Underwent Allogeneic Transplantation.
Authors: Elmaagacli Ahmet H. , Koldehoff Michael , Steckel Nina K. , Hindahl Heidrun , Trenschel Rudolf , Ottinger Hellmut , Beelen Dietrich W.
Published: October 2019

The innate immune system detects invading pathogens through several pattern-recognition receptors and represents the first line of mucosal host defense. Mutations of the NOD2/CARD15 and Toll like receptor −2, −4, −9 genes have been associated with an increased incidence of inflammatory bowel disease due to a diminished response to bacterial cell wall products. Moreover, it was reported that mutations of NOD2 gene loci might be associated with an increased risk for TRM and severe GVHD in patients who underwent allogeneic blood stem cell transplantation. Here we analyzed 201 patients and their respective donors for NOD2, TLR-2, TLR-4 and TLR-9 mutations and correlated the results with the incidence of overall acute GVHD and intestinal GVHD. Further, we evaluated if the occurrence of NOD2/CARD15 and TLR allele mutations are accompanied with an increased risk for transplant-related mortality and overall survival.

Mutated alleles of the NOD2 gene were observed in 17% of the patients and 16% of the donors. NOD2 gene mutations in patients and donors together were associated with an increased incidence of severe acute GVHD grade III-IV (6/11 patients; 55% versus 35/145 patients; 24%). If a mutated allele of the NOD2 gene was found either in patients or in donors only no correlation with increased incidence of severe acute GVHD was seen in this study.

Patients with mutated alleles in TLR-4 genes (Asp299Gly and Thr399Ile) had a higher incidence of intestinal GVHD (p<0.02), whereas mutations of donor alleles only had no influence on the occurrence of intestinal GVHD in this analysis.

No correlation with intestinal GVHD or overall acute GVHD was seen in patients with mutated alleles for TLR-2 and TLR-9. Overall survival, TRM and relapse risks was not influenced in none of the patients with mutated alleles of the innate immune system genes. Multivariate analysis including all potential factors, confirmed that that mutations of NOD2/CARD15 in patients and donors together influenced the occurrence of severe acute GVHD, whereas the occurrence of intestinal GVHD was influenced in patients by mutated alleles of TLR-4 gene. But mutations in alleles of the NOD2 or TLR-2,−4,−9 had no influence on the outcome of allogeneic transplant (TRM and overall survival) in the multivariate analysis.

Since all patients in our institution had received an intestinal bacterial decontamination using metronidazole and ciprofloxacin after allogeneic stem cell transplantation, it might be speculative, if the reduction of concentrations of anaerobic bacteria in the intestinum might have protected patients from the occurrence of increased TRM. Decontamination of anaerobic bacteria is associated with a reduced risk for severe acute GVHD as reported earlier (Blood1999; 93:3267–75).
CD133 Positive Mesenchymal Stem Cells in Mobilized Peripheral Blood and Cord Blood: Proliferation, Oct4 Expression and Plasticity.
Authors: Tondreau Tatiana , Meuleman Nathalie , Dejeneffe Marielle , Delforge Alain , Bron Dominique , Lagneaux Laurence
Published: October 2019

Background: Mesenchymal Stem Cells (MSC) can be isolated from bone marrow, adipose and fetal tissues, but their presence in Mobilized Peripheral Blood (MPB) and in Umbilical Cord Blood (UCB) remains controversial.

Methods: In this study we evaluated whether MPB (n=6) and UCB (n=8) could be two other sources of MSC beside Bone Marrow (BM). CD133 positive cell fraction was isolated through immunomagnetic system and MNC were seeded in medium supplemented or not with 5% of conditioned medium durong the first 48 hours of adhesion. MSC derived from MPB or UCB were identified by their expression of mesenchymal (SH2, SH3) and hematopoietic markers (CD14, CD34, CD45 and CD62-E). MPB and CB-MSC were also tested for their capacity to generate CFU-F (Fibroblast colony-forming units) and to differentiate into adipocytes, osteocytes, chondrocytes and neuronal/glial cells after specific induction. Finally we tested through RT-PCR the gene expression of Oct4, a transcriptional binding factor present in undifferentiated cells with high proliferative capacity.

Results: Through CFU-F, we observed that the selection of CD133 positive cells allows to obtain a great amount of MSC in comparison with the MNC fraction seeded (MPB-MSC, 139 versus 72 and UCB-MSC 165 versus 8 after the primoculture). After four passages, more than 1.108 cells were obtained by the cell expansion assays from 1.106 cells seeded in primoculture. During the first culture, whatever the medium used or cell fraction seeded, the cell population was composed of osteoblasts, osteoclasts, marcophages and hematopoietic cells with less than 5%SH3 positive cells. After two passages, cells derived from MPB or UCB expressed mesenchymal markers (SH3 and SH2) and Oct4. Under appropriate conditions these cells were able to differentiate into adipocytes, osteocytes, chondrocytes and neuronal/glial cells.

Conclusion: These results indicate that MSC are present in MPB and UCB, with similar characteristics to bone marrow (self-renewal capacity and multi-diffentiation potential), and may have a major clinical importance due to their accessibility. They could be an attractive target for cellular and gene therapies

Mesenchymal marker expression (%)

T0 (%) second passage (%) MPB SH2 2,7±1,1 96,1±2,9 SH3 4,9±1,5 95,7±3,3 UCB SH2 1,4±0,6 98±0,7 SH3 5,2±1,8 96,5±2,5
Establishment and Characterization of Human Fetal Liver Derived Stem Cell Line.
Authors: Chung Hyung-Min , Rhee Yun-Hee , Cha Kwang-Yul , Baek Jin-Young
Published: October 2019

Mesenchymal stem cells (MSC) have been derived from different sources, including bone marrow and liver. To further support the hypothesis that MSC may also exist in most postnatal tissues, we isolated a clonogenic, multipotent, rapidly proliferating population of cells from a fetal liver and defined them as mesenchymal-like stem cells (FL-MLSC) derived from human fetal liver. In this study, FACS analysis showed that FL-MLSC are positive for CD105 (endoglin/SH-2), CD73 (5′ terminal nucleotidase/SH-3), CD166 (ALCAM-1), CD44 (the hyaluronate receptor), HLA class-I(HLA-ABC), CDw90 (thy-1), CD13, and CD106 but negative for CD34 and HLA class-II(HLA-DR). Cell-cycle analysis showed that more than 81.9% cells were arrested in G0 and G1 phases, whereas an only small subpopulation of cells was actively engaged in proliferation (S + G2 + M = 18%) and doubling in about 48 hours. FL-MLSC retained normal diploid karyotypes and growth characteristics over the successive culture. In addition, under differentiation culture conditions, these cells showed the capability of differentiation into various cell types including chondrogenic, adipogenic, osteogenic, neuronal and endocrine lineages. These results demonstrated that FL-MLSC could be isolated from human fetal livers by means of their adherent ability and suggested that they were capable of self-renewal and multipotent differentiation.
Analyzing the MLL Recombinome.
Authors: Meyer Claus , Schneider Bjoern , Reichel Martin , Angermuller Sieglinde , Schnittger Susanne , Schoch Claudia , Jansen Mieke W.J.C. , van Dongen Jacques J.. M. , Pieters Rob , Haas Oskar , Strehl Sabine , Dingermann Theo , Klingebiel Thomas , Marschalek Rolf
Published: October 2019

Acute leukemias are frequently associated with specific chromosomal translocations of the human MLL gene. In general, MLL translocations define a distinct disease entity that needs to be diagnosed with precision to facilitate rapid clinical decisions. Here we present data about a new PCR based method that uses patient genomic DNA to identify any MLL fusion. Fourty different MLL translocations were successfully analyzed. We will present three novel MLL translocation partner genes and a new MLL deletion. The benefits of this novel technique for diagnosis and MRD analyses will be discussed. Supported by grant 2001.061.1 from the Wilhelm Sander foundation.
Long Range PCR Analysis for the t (8;14)(q24;q32) in Burkitts Lymphomas.
Authors: Mahlangu Johnny N. , Stevens Wendy S. , Stevens Gwyneth
Published: October 2019

Background: The breakpoints in the cytogenetic lesion in Burkitts lymphoma are very far apart such that the t(8,14)9q24;q32) is not amenable to standard polymerase chain reaction analysis. Long range polymerase chain reaction (LD-PCR) with its enzyme mix, appears to be capable of amplifying the t (8;14)(q24;32) in the majority of published sporadic Burkitts lymphoma analyses. The utility of t(8;14)(q24;q32) LD-PCR for routine use in the diagnosis of African and AIDS-related Burkitts lymphoma has not been studied. This study aims to analyze bone marrow of known African and AIDS-related Burkitts Lymphomas using LD-PCR and to establish if this procedure is suitable for routine diagnostic use.

Materials and methods: High molecular weight DNA was extracted from stored unstained bone marrow slides of previously diagnosed Burkitts lymphoma. Three hundred nanograms of patient and control DNA were amplified as per published LD-PCR methods. Each DNA sample was amplified with up to three pairs of MYC/IgH primer sets. The resulting amplicons were separated on a 0.8% agarose gel and their size compared to that of positive t(8;14)(q24;q32) controls, tPa controls and molecular weight markers.

Results: DNA was extracted from 74 Burkitts lymphoma bone marrow slides. Only 41 of these were amplifiable with the tPa primers and therefore suitable for further analysis. A t(8;14)(q24;q32) specific product was demonstrable in only 6 of 41 patients.

Conclusion: In this t(8;14)(q24;32) LD-PCR analysis of known Burkitts lymphomas comprising largely of African and AIDS-related variants, the procedure appears to be labour intensive and costly with a low diagnostic yield. These results may reflect the fundamental molecular differences between the sporadic and African or AIDS-related Burkitts lymphomas.
Differential Effects of Three Small Molecules Blocking Phosphatidylinositol-3 Kinase or AKT in Hodgkin Disease Cell Lines: Induction of Apoptosis and Cell Cycle Arrest.
Authors: Georgakis Georgios V. , Li Yang , Rassidakis Georgios Z. , Medeiros Jeffrey L. , Mills Gordon B. , Younes Anas
Published: October 2019

Several types of tumor cells utilize the phosphatidylinositol 3 Kinase (PI3K)/AKT pathway to support their growth and survival, and blockade of this pathway has been shown to have an antiproliferative effect in these cells. The significance of this survival pathway in Hodgkin disease (HD), and in particular, the neoplastic Hodgkin and Reed-Sternberg (H/RS) of Hodgkin disease (HD) is unknown. We studied routinely processed tissue sections of 42 cases of primary HD (n=42, 35 classical HD and 7 nodular lymphocyte predominant) for the expression of the active phosphorylated form of AKT. We also studied AKT expression in a panel of 4 well-characterized HD-derived cell lines (HD-MyZ, HD-LM2, L-428, and KM-H2). 27 of 42 (64.3%) cases of primary HD sections expressed phospho AKT (23/35 classical and 4/7 nodular lymphocyte predominant). Cultured H/RS cells also showed constitutive phosphorylation of AKT on Ser473. CD30 ligand (CD30L), CD40L and RANKL, increased the phosphorylation of AKT and downstream molecules as early as 1 hour. The effect of 3 small molecule inhibitors of PI3K/AKT pathway (PI3K inhibitor LY294002; AKT inhibitors QLT0394 and QLT0395 from QLT Inc, Vancouver, Canada) on cell proliferation and survival of cultured H/RS cells was examined by the MTS assay. The PI3K inhibitor LY294002 showed antiproliferative activity in a time and dose dependent manner in all cell lines tested. This antiproliferative effect was primarily due to cell cycle arrest in G0/G1 phase, as determined by propidium iodide staining, and to a less extent due to induction of apoptosis, as determined by the Annexin-V binding assay. However, when the AKT inhibitors QLT0394 and QLT0395 were used, they primarily induced apoptosis even at nanomolar doses. Apoptosis was mediated by caspase 8 activation as determined by western blot and was partially reversed by the pancaspase inhibitor ZVAD-FMK. Both AKT inhibitors alteredf AKT phosphorylation within 24 hours, and reduced phosphorylation of downstream molecules, such as 4E-BP1. Moreover, the AKT inhibitor QLT0395 showed downregulation of MDM2 and cyclin D1 within 24 hours, and increased ERK1/2 phosphorylation with subsequent decrease of total ERK1/2 levels at the same time frame, but had no effect in BCL-2, cFLIP or Bax. Importantly, both AKT inhibitors (QLT0394 and QLT0395) maintained their killing activity even in the presene of CD30L, CD40L, and RANKL. These preclinical data suggest that small molecules hitting different targets within PI3K/AKT pathway may have different effects on proliferation and apoptosis and that blockade of this pathway may be of therapeutic value in the treatment of patients with HD.
Escherichia Coli Expressing Recombinant WT1 Protein and Listeriolysin O Stimulate Antigen-Specific T Cells Following Vaccination in C57BL/6 Mice.
Authors: Nitcheu Josianne , Ramirez-Jimenez Francisco S. , Stauss Hans J. , Vassaux Georges
Published: October 2019

The Wilm’s Tumor (WT1) gene is expressed at high levels in most types of leukemia, but also in various types of solid tumors. We tested the efficacy of recombinant Escherichia Coli (E. Coli) expressing Listeriolysin O (LLO) as an antigen delivery vector for tumor specific immunotherapy targeting WT1 gene products. C57BL/6 (B6) mice were immunised with E. Coli expressing a form of WT1 protein truncated of 70 amino acids at the N-Terminus portion, and then challenged subcutaneously with 5X106 WT1-expressing TRAMP-C tumor cells. A significant inhibitory effect on TRAMP-C tumor growth was observed in vivo in vaccinated mice, with no signs of toxicity in tissues such as liver and kidney on histopathlogic examination. Furthermore, splenocytes from immunised mice were stimulated in vitro either with peptides that have been shown to contain major histocompatibility (MHC) class I binding anchor motifs (P126: RMFPNAPYL and P330: CNKRYFKL), or with irradiated TRAMP-C tumor cells. Specific CD8-positive cytotoxic T lymphocytes (CTLs) responses against TRAMP-C tumor cells were seen in cultures stimulated with irradiated tumor cells. However, no CTL activity was found in cultures stimulated with the relevant WT1 peptides that specifically lyse TRAMP-C tumor cells or the leukemia cell line RMA-S cells pulsed with each of the peptides. These results suggest that P126 and P330 might not be naturally processed WT1 epitopes for B6 mice and supports the possibility of other B6 CTLs epitopes. We are currently using a WT1 peptide library to screen immunised animals to look for novel peptides. Immunisation with E. Coli expressing WT1 protein but not LLO did not show any effect on TRAMP-C tumor growth in vivo. In this latter group, western blot analysis revealed WT1 specific antibodies directed against the N-terminus portion of the WT1 protein in the sera of immunised mice. Altogether the results presented here showed that E. Coli expressing WT1 could stimulated antigen-specific T cells dependant on LLO co-expression and suggest possible vaccine strategies for generating CD8+ T cell responses against tumors.
C/EBPδ Modulates Cell Growth, Differentiation and Apoptosis of Myeloid Leukemia, Prostate and Breast Cancer Cells.
Authors: Gery Sigal , Stanosaki Sakae , Ikezoe Takayuki , Hofmann Wolf K. , Gombart Adrian F. , Koeffler H. Phillip
Published: October 2019

C/EBPδ belongs to the family of highly conserved CCAAT/enhancer binding protein (C/EBP) transcription factors. Members of this family play a critical role in the regulation of mitotic growth arrest and differentiation in numerous cell types. To examine the consequences of C/EPBδ expression, we transfected C/EPBδ into CML myeloid leukemia (KCL22, K562), prostate (LNCaP, PC3, DU145), and breast (MCF-7, T47D, MDA-MB-231) cancer cell lines. C/EBPδ expression resulted in a proliferative arrest and an increase in apoptosis of the myeloid leukemia cells, as well as the prostate cells LNCaP and PC3, and the breast cells MCF-7 and T47D. In contrast, DU145 prostate and MDA-MB-231 breast cancer cells were not inhibited by C/EBPδ, indicating that the biologically properties of C/EBPδ depend upon its cellular context. We further studied the molecular mechanisms underlying the affect of C/EPBδ expression in CML leukemic cells. Myeloid differentiation of KCL22 and K562 blast cells as shown by morphologic changes and induction of secondary specific granule genes, occurred within 4 days of inducing expression of C/EBPδ. Furthermore, expression of C/EBPδ was associated with downregulation of c-Myc and cyclin E, and upregulation of the forkhead transcription factor FoxO1a (FKHR) and the cyclin-dependent kinase inhibitor p27Kip1. In addition, microarray analysis showed that C/EBPδ mRNA is upregulated during granulocytic differentiation of normal CD34+ bone marrow cells, suggesting that C/EBPδ is involved in lineage-specific differentiation. Taken together, these results show that expression of C/EBPδ in BCR-ABL-positive CML cells in blast crisis, is sufficient for neutrophil differentiation and suggest that ectopic induction of C/EBPδ in the blastic phase of CML, as well as in certain cases of prostate and breast cancers, may hold promising therapeutic potential.
Genetic Identification of Hematopoietic Stem Cell Immortalization Genes.
Authors: Du Yang , Jenkins Nancy A. , Copeland Neal G.
Published: October 2019

Hematopoietic cancer is thought to arise from small populations of immortalized cancer stem cells that give rise to phenotypically diverse cells with less proliferative capacity. Cancer stem cell immortalization genes represent attractive drug targets for treating human cancer. We have found that immortalized cell lines can be generated in vitro following infection of mouse bone marrow cells with replication defective murine stem cell virus (MSCV). Immortalized myeloid progenitors can be selected in the presence of SCF plus IL-3 while immortalized multi-potent early hematopoietic progenitor cells can be selected with SCF and Flt3 ligand. So far, more than 80 such lines have been generated and they exhibit many properties attributed to cancer stem cells; they undergo unlimited self-renewal yet are able to give rise to phenotypically diverse cells with less proliferative capacity and in some cases they are leukemogenic in transplanted hosts. Immortalization often results from the insertional mutagenesis of human leukemia gene orthologues or genes that regulate them. Insertional mutagenesis by MSCV thus produces cells that share many properties attributed to cancer stem cells and may mark a subset of genes important for the immortalization of leukemic stem cells.
Cyclin-C: Analysis of a Candidate Tumour Suppressor Genes on 6q15-q21 in Malignant Lymphoid Cell Lines and Lymphoid Acute Leukemias.
Authors: Sinclair Paul B. , Mitchell Wayne , Kaeda Jaspal , Foroni Letizia
Published: October 2019

Using FISH and LOH in ALL cases with microscopically visible deletions of the long arm of chromosome 6q, we previously defined a region of minimal deletion (RMD) in a spectrum of lymphoid malignancies and with other investigators have suggested that the region may harbour several tumour suppressor genes. (Sinclair et al 2004; Cancer Research, 64: 4089). 15 recognised genes were positioned in the RMD and three were found expressed in bone marrow: GRIK2, C6orf111 and Cyclin-C, the investigation of which is reported in this study.

GRIK2 analysis. Combining published RMD in ALL with our own data, we extensively analysed GRIK2 in ALL patients carrying heterozygous 6q deletion. Although a paternally inherited non-silent mutation of GRIK2 was identified in one of 14 patients, we concluded that loss of one or more other genes in the 6q15-q21 region was likely to contribute to ALL pathogenesis.

Cyclin-C (CCNC) analysis. Lymphoid cell lines without and with del (6q)(Peer, Molt-4 and NKL) were assessed for expression levels of C6orf111 and Cyclin-C and other candidate tumour suppressor genes within the region (FLASH, Q9NR58, Cx43, BACH2, BLIMP1, APG5L, TAK1 and AIM1) using real-time quantitative RT-PCR (RQ-PCR). While easily detectable in all other cell lines, no expression of CCNC was demonstrated in NKL (by RT-PCR and Western blotting), this in spite of the presence of at least one copy of the gene (as demonstrated by FISH using PAC P13743 which contains CCNC) suggesting that expression of the second allele was ablated through either micro-deletion, point mutation or epigenetic silencing. Expression levels of other candidate genes were similar, albeit with minor variations, in all cell lines. We further analysed expression of CCNC in whole PB and BM, a variety of haemopoietic cells isolated from PB and in 66 de novo ALL patients (9 T ALL; 48 B cell ALL (predominantly common ALL) and 9 other ALL). Expression was relatively high in all cell lines, intermediate in normal T-lymphocytes and low in other normal cells. Leukemic cells collected from diagnostic material from adult acute lymphoid malignancies showed a wide variation in levels of CCNC expression. To exclude the possibility that low expression in some cases may be due to the presence of a high proportion of normal cells CCNC levels were normalised against Ki67 expression (as an indicator of mitotic index) by RQ-PCR. At least 27 patients showed a CCNC level below the median value. Twenty-five cases showed value above the median value. Levels of expression of CCNC and Ki67 were correlated with survival, immunophenotype or other clinical parameters, but no statistically significant correlation was demonstrated.