bluuebunny/crossref_metadata_2025 huggingface dataset:
Prospective Evaluation of the Thrombotic Risk in Children With Acute Lymphoblastic Leukemia Carrying the MTHFR TT 677 Genotype, the Prothrombin G20210A Variant, and Further Prothrombotic Risk Factors
Authors: Nowak-Göttl Ulrike , Wermes Cornelia , Junker Ralf , Koch Hans-Georg , Schobess Rosmarie , Fleischhack Gudrun , Schwabe Dirk , Ehrenforth Silke
Published: October 2019

The reported incidence of thromboembolism in children with acute lymphoblastic leukemia (ALL) treated with L-asparaginase, vincristine, and prednisone varies from 2.4% to 11.5%. The present study was designed to prospectively evaluate the role of the TT677 methylenetetrahydrofolate reductase (MTHFR) genotype, the prothrombin G20210A mutation, the factor V G1691A mutation, deficiencies of protein C, protein S, antithrombin, and increased lipoprotein (a) concentrations in leukemic children treated according to the ALL-Berlin-Frankfurt-Muenster (BFM) 90/95 study protocols with respect to the onset of vascular events. Three hundred and one consecutive leukemic children were enrolled in this study. Fifty-five of these 301 subjects investigated had one established single prothrombotic risk factor: 20 children showed the TT677 MTHFR genotype; 5 showed the heterozygous prothrombin G20210A variant; 11 were carriers of the factor V G1691A mutation (heterozygous, n = 10; homozygous, n = 1); 4 showed familial protein C, 4 protein S, and 2 antithrombin type I deficiency; 9 patients were suffering from familially increased lipoprotein (a) [Lp(a)] concentrations (>30 mg/dL). In addition, combined prothrombotic defects were found in a further 10 patients: the FV mutation was combined with the prothrombin G20210A variant (n = 1), increased Lp(a) (n = 3), protein C deficiency (n = 1), and homozygosity for the C677T MTHFR gene mutation (n = 1). Lp(a) was combined with protein C deficiency (n = 2) and the MTHFR TT 677 genotype (n = 2). Two hundred eighty-nine of the 301 patients were available for thrombosis-free survival analysis. In 32 (11%) of these 289 patients venous thromboembolism occurred. The overall thrombosis-free survival in patients with at least one prothrombotic defect was significantly reduced compared with patients without a prothrombotic defect within the hemostatic system (P < .0001). In addition, a clear-cut positive correlation (P < .0001) was found between thrombosis and the use of central lines. However, because the prothrombotic defects diagnosed in the total childhood population studied were all found within the prevalences reported for healthy Caucasian individuals, the interaction between prothrombotic risk factors, ALL treatment, and further environmental factors is likely to cause thrombotic manifestations.
Labeling of the Internal Pool of GP IIb-IIIa in Platelets by c7E3 Fab Fragments (abciximab): Flow and Endocytic Mechanisms Contribute to the Transport
Authors: Nurden Paquita , Poujol Christel , Durrieu-Jais Catherine , Winckler Joëlle , Combrié Robert , Macchi Laurent , Bihour Claude , Wagner Carrie , Jordan Robert , Nurden Alan T.
Published: October 2019

Abciximab is a new antiplatelet therapeutic in ischemic cardiovascular disease. The drug, chimeric Fab fragments of a murine monoclonal antibody (MoAb) (c7E3), blocks GP IIb-IIIa function. However, its capacity to reach all receptor pools in platelets is unknown. Electron microscopy and immunogold labeling were used to localize abciximab in platelets of patients receiving the drug for up to 24 hours. Studies on frozen-thin sections showed that c7E3 Fab, in addition to the surface pool, also labeled the surface-connected canalicular system (SCCS) and -granules. Analysis of gold particle distribution showed that intraplatelet labeling was not accumulative and in equilibrium with the surface pool. After short-term incubations of platelets with c7E3 Fab in vitro, gold particles were often seen in lines within thin elements of the SCCS, some of which appeared in contact with -granules. Little labeling was associated with Glanzmann’s thrombasthenia platelets, confirming that the channels contained bound and not free c7E3 Fab. Endocytosis of abciximab in clathrin-containing vesicles was visualized by double staining and constitutes an alternative mechanism of transport. The remaining free pool of GP IIb-IIIa was evaluated with the MoAb AP-2; flow cytometry showed it to be about 9% on the surface of nonstimulated platelets but 33% on thrombin-activated platelets. The ability of drugs to block all pools of GP IIb-IIIa and then to be associated with secretion-dependent residual aggregation must be considered when evaluating their efficiency in a clinical context.
Clonality of Isolated Eosinophils in the Hypereosinophilic Syndrome
Authors: Chang Hsiao-Wen , Leong Kah-Hoo , Koh Dow-Rhoon , Lee Szu-Hee
Published: October 2019

The idiopathic hypereosinophilic syndrome (IHES) is a rare disorder characterized by unexplained, persistent eosinophilia associated with multiple organ dysfunction due to eosinophilic tissue infiltration. In the absence of karyotypic abnormalities, there is no specific test to detect clonal eosinophilia in IHES. Analysis of X-chromosome inactivation patterns can be used to determine whether proliferative disorders are clonal in origin. Methylation of HpaII andHha I sites near the polymorphic trinucleotide repeat of the human androgen receptor gene (HUMARA) has been shown to correlate with X-inactivation. In this study, we have used the polymerase chain reaction (PCR) with nested primers to analyze X-inactivation patterns of the HUMARA loci in purified eosinophils from female patients with eosinophilia. Peripheral blood eosinophils were isolated by their autofluoresence using flow cytometric sorting. Eosinophils purified from a female patient presenting with IHES were found to show a clonal pattern of X-inactivation. Eosinophil-depleted leukocytes from this patient were polyclonal by HUMARA analysis, thus excluding skewedness of random X-inactivation. After corticosteroid suppression of her blood eosinophilia, a clonal population of eosinophils could no longer be detected in purified eosinophils. In contrast, eosinophils purified from a patient with Churg-Strauss syndrome and from six patients with reactive eosinophilias attributed to allergy, parasitic infection, or drug reaction showed a polyclonal pattern of X-inactivation by HUMARA analysis. The finding of clonal eosinophilia in a patient presenting with IHES indicates that such patients may have, in reality, a low-grade clonal disorder that can be distinguished from reactive eosinophilias by HUMARA analysis. Further, the method described can be used to monitor disease progression.
TEL/PDGFβR Induces Hematologic Malignancies in Mice That Respond to a Specific Tyrosine Kinase Inhibitor
Authors: Tomasson Michael H. , Williams Ifor R. , Hasserjian Robert , Udomsakdi Chirayu , McGrath Shannon M. , Schwaller Juerg , Druker Brian , Gilliland D. Gary
Published: October 2019

The TEL/PDGFβR fusion protein is expressed as the consequence of a recurring t(5;12) translocation associated with chronic myelomonocytic leukemia (CMML). Unlike other activated protein tyrosine kinases associated with hematopoietic malignancies, TEL/PDGFβR is invariably associated with a myeloid leukemia phenotype in humans. To test the transforming properties of TEL/PDGFβR in vivo, and to analyze the basis for myeloid lineage specificity in humans, we constructed transgenic mice with TEL/PDGFβR expression driven by a lymphoid-specific immunoglobulin enhancer-promoter cassette. These mice developed lymphoblastic lymphomas of both T and B lineage, demonstrating that TEL/PDGFβR is a transforming protein in vivo, and that the transforming ability of this fusion is not inherently restricted to the myeloid lineage. Treatment of TEL/PDGFβR transgenic animals with a protein tyrosine kinase inhibitor with in vitro activity against PDGFβR (CGP57148) resulted in suppression of disease and a prolongation of survival. A therapeutic benefit was apparent both in animals treated before the development of overt clonal disease and in animals transplanted with clonal tumor cells. These results suggest that small-molecule tyrosine kinase inhibitors may be effective treatment for activated tyrosine kinase–mediated malignancies both early in the course of disease and after the development of additional transforming mutations.
The Somatostatin Analog Octreotide Inhibits Growth of Interleukin-6 (IL-6)–Dependent and IL-6–Independent Human Multiple Myeloma Cell Lines
Authors: Georgii-Hemming Patrik , Strömberg Thomas , Janson Eva Tiensuu , Stridsberg Mats , Wiklund Helena Jernberg , Nilsson Kenneth
Published: October 2019

Somatostatin and its analogs can inhibit growth in several cell types, in part by interfering with insulin-like growth factor-I (IGF-I) signaling. Our previous studies point to the importance of paracrine and autocrine IGF-I in the support of growth and survival of human multiple myeloma (MM) cell lines. In this report, we have investigated the potential role of a somatostatin analog, octreotide, in regulating growth and/or survival in MM. The results show that all MM cell lines express functional somatostatin receptors (sst). The MM cell lines express the subtypes sst2, sst3, and predominantly sst5 as determined by reverse-transcriptase polymerase chain reaction and fluorescence-activated cell sorter analysis. Octreotide inhibited the growth of both the interleukin-6 (IL-6)–dependent and the IL-6–independent MM cell lines. The effect is mainly cytostatic, resulting in 25% to 45% growth inhibition, and in three of eight of the MM cell lines a weak induction of apoptosis was recorded. Our results also show that octreotide may act as an inducer of apoptosis in primary B-B4+ plasma cells isolated from bone marrow of MM patients. In conclusion, the results show a novel pathway for growth inhibition of MM cells: the activation of somatostatin receptor signaling.
Adverse Effects of Activated Cytotoxic T Lymphocytes on the Clinical Outcome of Nodal Anaplastic Large Cell Lymphoma
Authors: ten Berge Rosita L. , Dukers Danny F. , Oudejans Joost J. , Pulford Karen , Ossenkoppele Gert J. , de Jong Daphne , Miseré Jo F.M.M. , Meijer Chris J.L.M.
Published: October 2019

Systemic (nodal) anaplastic large cell lymphoma (ALCL) is a subgroup of T-cell non-Hodgkin’s lymphomas with a relatively favorable clinical outcome. Part of systemic ALCLs harbor a genetic aberration (usually the t(2;5)(p23;q35) translocation) containing the anaplastic lymphoma kinase (ALK) gene at 2p23, which results in aberrant expression of the ALK protein. Recently, we have shown that the presence of high percentages of activated cytotoxic T lymphocytes (CTLs) in tumor biopsy specimens of Hodgkin’s disease (HD) is associated with a poor prognosis. In the present study, we investigated the prognostic value of percentages of activated CTLs in combination with ALK expression in primary nodal ALCL. Primary nodal biopsies of 42 patients with ALCL were investigated for the percentage of activated CTLs (quantified using Q-PRODIT) and the expression of ALK by immunohistochemistry using monoclonal antibodies (MoAbs) directed against T-cell antigen granzyme B (GrB) and ALK, respectively. These parameters were evaluated for their predictive value regarding progression-free and overall survival time. The presence of a high percentage of activated CTLs (ie, ≥15%) was found to be an unfavorable prognostic marker. In combination with a lack of ALK expression, it was possible to identify a group of patients with a very poor prognosis. In this group, 13 of 16 patients died within 2 years as a result of the disease. Of the remaining 26 patients, only three (all ALK negative) died (P < .0001). Furthermore, the percentage of activated CTLs combined with ALK status appeared to be of stronger prognostic value than the International Prognostic Index (IPI). We conclude that a high percentage of activated CTLs present in biopsy material of patients with primary nodal ALCL is a strong indicator for an unfavorable clinical outcome. The combination of ALK expression and percentage of activated CTLs appears to be more sensitive than the IPI in identifying a group of patients with a highly unfavorable clinical outcome who may be eligible for alternative (high dose) therapy schemes.
Whole-Body Positron Emission Tomography Using18F-Fluorodeoxyglucose for Posttreatment Evaluation in Hodgkin’s Disease and Non-Hodgkin’s Lymphoma Has Higher Diagnostic and Prognostic Value Than Classical Computed Tomography Scan Imaging
Authors: Jerusalem G. , Beguin Y. , Fassotte M.F. , Najjar F. , Paulus P. , Rigo P. , Fillet G.
Published: October 2019

A residual mass after treatment of lymphoma is a clinical challenge, because it may represent vital tumor as well as tissue fibrosis. Metabolic imaging by 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) offers the advantage of functional tissue characterization that is largely independent of morphologic criteria. We compared18F-FDG PET to computed tomography (CT) in the posttreatment evaluation of 54 patients with Hodgkin’s disease (HD) or intermediate/high-grade non-Hodgkin’s lymphoma (NHL). Residual masses on CT were observed in 13 of 19 patients with HD and 11 of 35 patients with NHL. Five of 24 patients with residual masses on CT versus 1 of 30 patients without residual masses presented a positive18F-FDG PET study. Relapse occurred in all 6 patients (100%) with a positive 18F-FDG PET, 5 of 19 patients (26%) with residual masses on CT but negative 18F-FDG PET, and 3 of 29 patients (10%) with negative CT scan and18F-FDG PET studies (P ≤ .0001). We observed a higher relapse and death rate in patients with residual masses at CT compared with patients without residual masses at CT (progression-free survival at 1 year: 62 ± 10 v88 ± 7%, P = .0045; overall survival at 1 year: 77 ± 5 v 95 ± 5%, P = .0038). A positive18F-FDG PET study was even more consistently associated with poorer survival: compared with patients with a negative18F-FDG PET study, the 1-year progression-free survival was 0% versus 86% ± 5% (P < .0001) and the 1-year overall survival was 50% ± 20% versus 92% ± 4% (P < .0001). The detection of vital tumor by 18F-FDG PET after the end of treatment has a higher predictive value for relapse than classical CT scan imaging (positive predictive value: 100% v42%). This could help identify patients requiring intensification immediately after completion of chemotherapy. However,18F-FDG PET mainly predicts for early progression but cannot exclude the presence of minimal residual disease, possibly leading to a later relapse.
The Significance of Hepatitis G Virus in Serum of Patients With Sporadic Fulminant and Subfulminant Hepatitis of Unknown Etiology
Authors: Muñoz Santiago J. , Alter Harvey J. , Nakatsuji Yoshiyuki , Shih James W.-K. , Reddy Rajender K. , Jeffers Lennox , Schiff Eugene R. , Reid Andrea E. , Marrone Aldo , Rothstein Kenneth , Manzarbeitia Cosme , Liang T. Jake
Published: October 2019

Excluding acute hepatic failure caused by drugs, the etiology of fulminant hepatitis (FH) remains unknown in many patients. There are conflicting data about a possible pathogenic role for the hepatitis G virus (HGV) in patients with cryptogenic fulminant hepatitis (non–A-E FH). We investigated the presence of circulating HGV in 36 patients with well-documented non–A-E fulminant and 5 patients with subfulminant hepatitis from 3 geographic locations in the United States. Serum HGV RNA was determined by reverse transcriptase-polymerase chain reaction using primers from the NS5 region of the HGV genome. HGV RNA was also measured before and after liver transplantation in 5 patients and at different time points in 7 patients. Serum samples were recoded and reanalyzed for HGV RNA using different primer sets to assess the validity of the HGV RNA assay. HGV was present in serum of 14 of the 36 patients (38.8%) with non–A-E fulminant hepatitis. Twenty percent of patients from the Northeast, 11% of the patients from the Southeast, and 50% from the Mid-Atlantic regions of the United States had circulating HGV RNA. The use of therapeutic blood products was significantly associated with the presence of serum HGV RNA (P < .02). Retesting for HGV RNA with different primers was positive in all but 1 case. HGV RNA is not causally related to non–A-E fulminant hepatitis. The finding of HGV RNA in serum from these patients is likely related to the administration of blood product transfusion after the onset of fulminant hepatitis.
Evidence for Extracellular Processing of Pro-von Willebrand Factor After Infusion in Animals With and Without Severe von Willebrand Disease
Authors: Turecek P.L. , Pichler L. , Auer W. , Eder G. , Varadi K. , Mitterer A. , Mundt W. , Schlokat U. , Dorner F. , Drouet L.O. , Roussi J. , van Mourik J.A. , Schwarz H.P.
Published: October 2019

Although proteolytic processing of pro-von Willebrand factor (pro-vWF) resulting in free propeptide and mature vWF is known to be initiated intracellularly, vWF released from endothelial cells may contain a high proportion of incompletely processed pro-vWF. Because pro-vWF is only rarely detectable in normal human plasma, we investigated whether extracellular processing of pro-vWF is possible. A recombinant preparation (rpvWF) containing both pro-vWF and mature vWF subunits was infused into 2 pigs and 1 dog with severe von Willebrand disease, 2 mice with a targeted disruption of the vWF gene, and 2 healthy baboons. Total vWF antigen (vWF:Ag), free propeptide, and pro-vWF were measured using enzyme-linked immunosorbent assay techniques in blood samples drawn before and after infusion. vWF:Ag increased promptly. No pro-vWF could be detected when the first postinfusion sample was drawn after 30 minutes (pigs) or 60 minutes (mice), but pro-vWF was detectable for short periods when postinfusion samples were drawn after 15 minutes (dog) or 5 minutes (baboons). In contrast, free propeptide was increased at the first timepoint measured, suggesting that it was generated from the pro-vWF in the rpvWF preparation. vWF multimers were analyzed in the rpvWF preparation and in plasma samples drawn before and after infusion of rpvWF using ultra-high resolution 3% agarose gels to allow separation of homo- and hetero-forms of the vWF polymers. Within 30 minutes after infusion in the pigs, 1 hour in the dog and the mice, and within 2 hours in the baboons, the multimer pattern had changed to that typically seen in mature vWF. These data indicate that propeptide cleavage from unprocessed vWF can occur extracellularly in the circulation. The enzyme or enzymes responsible for this cleavage in plasma remain to be identified.
Fusion of a Novel Gene, BTL, to ETV6 in Acute Myeloid Leukemias With a t(4;12)(q11-q12;p13)
Authors: Cools Jan , Bilhou-Nabera Chrystèle , Wlodarska Iwona , Cabrol Christine , Talmant Pascaline , Bernard Philippe , Hagemeijer Anne , Marynen Peter
Published: October 2019

The ETV6 gene (also known as TEL) is the main target of chromosomal translocations affecting chromosome band 12p13. The rearrangements fuse ETV6 to a wide variety of partner genes in both myeloid and lymphoid malignancies. We report here 4 new cases of acute myeloid leukemia (AML) with very immature myeloblasts (French-American-British [FAB]-M0) and with a t(4;12)(q11-q12;p13). In all cases, ETV6 was found recombined to a new gene, homologous to the mouse Brx gene. The gene was named BTL (Brx-likeTranslocated in Leukemia). Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments indicate that the expression of the BTL-ETV6 transcript, but not of the reciprocal ETV6-BTL transcript, is a common finding in these leukemias. In contrast to the majority of other ETV6 fusions, both the complete helix-loop-helix (HLH) and ETS DNA binding domains of ETV6 are present in the predicted BTL-ETV6 fusion protein, and the chimeric gene is transcribed from theBTL promoter.
Circumvention of Methotrexate Resistance in Childhood Leukemia Subtypes by Rationally Designed Antifolates
Authors: Rots Marianne G. , Pieters Rob , Peters Godefridus J. , van Zantwijk Christina H. , Mauritz Rob , Noordhuis Paul , Willey James C. , Hählen Karel , Creutzig Ursula , Janka-Schaub Gritta , Kaspers Gertjan J.L. , Veerman Anjo J.P. , Jansen Gerrit
Published: October 2019

Cellular methotrexate (MTX) resistance may cause treatment failure in childhood common/preB-acute lymphoblastic leukemia (c/preB-ALL), T-lineage ALL (T-ALL), and acute myeloid leukemia (AML). The ex vivo potency of several antifolates (MTX, trimetrexate [TMQ], GW1843U89, multitargeted antifolate [MTA], Raltitrexed, and ZD9331) was studied via in situ inhibition of thymidylate synthase (TS). After short-term exposure, relapsed c/preB-ALL (rALL, n = 21), T-ALL (n = 22), and AML (n = 22) were 3-fold, 10-fold, and 6-fold less sensitive to MTX (P ≤ .01) compared with initial c/preB-ALL (n = 43). This difference in resistance was not observed for TMQ. Also for GW1843U89 and MTA, no resistance was observed in rALL and AML compared with c/preB-ALL. T-ALL compared with c/preB-ALL tended to be less resistant to GW1843U89 (3-fold) and MTA (6-fold) than to MTX (10-fold) (P= .06). Raltitrexed was more active against c/preB-ALL compared with the other subtypes. After 21 hours continuous incubation, T-ALL and AML samples were equally sensitive as c/preB-ALL to MTX, but rALL was 3-fold resistant to MTX compared with initial c/preB-ALL (P = .003). The resistance of rALL was circumvented by TMQ (1-fold; P = .03) and GW1843U89 (1.4-fold; P= .004). Novel antifolates, except MTA, displayed a more potent TS inhibition than MTX during continuous exposure. These results suggest that MTX resistance in AML and T-ALL can be circumvented by continuous exposure, and that novel antifolates should be explored further for MTX-resistant T-ALL, rALL, and AML cells.
Membrane-Bound Fas (Apo-1/CD95) Ligand on Leukemic Cells: A Mechanism of Tumor Immune Escape in Leukemia Patients
Authors: Buzyn Agnès , Petit Frederic , Ostankovitch Marina , Figueiredo Suzanne , Varet Bruno , Guillet Jean-Gérard , Ameisen Jean-Claude , Estaquier Jérôme
Published: October 2019

There is evidence from bone marrow transplantation that T cells may be involved in the immunologic control of leukemia. But many patients relapse despite a potent graft-versus-leukemia effect mediated by allogeneic T cells. The expression of the FasL protein has been suggested as a mechanism of tumor immune escape. We, therefore, evaluated the capacity of leukemic cells from patients with acute or chronic myelogenous leukemia to escape the allogeneic or autologous immune response by bearing the FasL molecule. Although almost all leukemic cells express the 37-kD form of FasL, only 54% of acute myeloblastic leukemia and 27% of chronic myeloid leukemia (CML) cells bore a FasL with killing properties, as assessed by the ability of leukemic cells to cause the apoptosis of a Fas-sensitive target cell line or autologous activated T cells in 3 tested leukemic cases. Experiments with a recombinant Fas-Fc molecule confirmed the role of Fas/FasL in leukemic-mediated cell death. Only CML leukemic cells from certain individuals contained the 26-kD truncated form of FasL. Thus, myeloid leukemic cells from some, but not all patients can set up a mechanism of immune escape involving the Fas/FasL pathway. This leukemic escape may have implications for patients eligible for adoptive cellular immunotherapy.
Effects of megakaryocyte growth and development factor on platelet production, platelet life span, and platelet function in healthy human volunteers
Authors: Harker Laurence A. , Roskos Lorin K. , Marzec Ulla M. , Carter Richard A. , Cherry Judith K. , Sundell Birgitta , Cheung Ellen N. , Terry Dixon , Sheridan William
Published: October 2019

The effects of thrombopoietic stimulation on megakaryocytopoiesis, platelet production, and platelet viability and function were examined in normal volunteers randomized to receive single bolus subcutaneous injections of 3 μg/kg pegylated recombinant megakaryocyte growth and development factor (PEG-rHuMGDF) or placebo in a 3:1 ratio. PEG-rHuMGDF transiently doubled circulating platelet counts, from 237 ± 41 × 103/μL to 522 ± 90 × 103/μL (P< .0001), peaking on day 12. Baseline and day-12 samples showed no differences in responsiveness of platelets to adenosine diphosphate or thrombin receptor agonist peptide (P > .4 in all cases); expression of platelet ligand-induced binding sites or annexin V binding sites (P > .6 in both cases); or density of platelet TPO-receptors (P > .5). Platelet counts normalized by day 28. The life span of autologous 111In-labeled platelets increased from 205 ± 18 hours (baseline) to 226 ± 22 hours (P < .01) on day 8. Platelet life span decreased from 226 ± 22 hours (day 8) to 178 ± 53 hours (P < .05) on day 18. The theoretical basis for senescent changes in mean platelet life span was illustrated by biomathematical modeling. Platelet turnover increased from 43.9 ± 11.9 × 103 platelets/μL/d (baseline) to 101 ± 27.6 × 103 platelets/μL/d (P = .0009), and marrow megakaryocyte mass expanded from 37.4 ± 18.5 fL/kg to 62 ± 17 × 1010 fL/kg (P = .015). Although PEG-rHuMGDF initially increased megakaryocyte volume and ploidy, subsequently ploidy showed a transient reciprocal decrease when the platelet counts exceeded placebo values. In healthy human volunteers PEG-rHuMGDF transiently increases megakaryocytopoiesis 2-fold. Additionally, peripheral platelets expand correspondingly and exhibit normal function and viability during the ensuing 10 days. The induced perturbation in steady state thrombopoiesis resolves by 4 weeks.
Many multipotential gene-marked progenitor or stem cell clones contribute to hematopoiesis in nonhuman primates
Authors: Kim Hyeoung Joon , Tisdale John F. , Wu Tong , Takatoku Masaaki , Sellers Stephanie E. , Zickler Philipp , Metzger Mark E. , Agricola Brian A. , Malley James D. , Kato Ikunoshin , Donahue Robert E. , Brown Kevin E. , Dunbar Cynthia E.
Published: October 2019

Retroviral insertion site analysis was used to track the contribution of retrovirally transduced primitive progenitors to hematopoiesis after autologous transplantation in the rhesus macaque model. CD34-enriched mobilized peripheral blood cells were transduced with retroviral marking vectors containing the neo gene and were reinfused after total body irradiation. High-level gene transfer efficiency allowed insertion site analysis of individual myeloid and erythroid colony-forming units (CFU) and of highly purified B- and T-lymphoid populations in 2 animals. At multiple time points up to 1 year after transplantation, retroviral insertion sites were identified by performing inverse polymerase chain reaction and sequencing vector-containing CFU or more than 99% pure T- and B-cell populations. Forty-eight unique insertion sequences were detected in the first animal and also in the second animal, and multiple clones contributed to hematopoiesis at 2 or more time points. Multipotential clones contributing to myeloid and lymphoid lineages were identified. These results support the concept that hematopoiesis in large animals is polyclonal and that individual multipotential stem or progenitor cells can contribute to hematopoiesis for prolonged periods. Gene transfer to long-lived, multipotent clones is shown and is encouraging for human gene therapy applications.
Heparin-induced thrombocytopenia: new evidence for the dynamic binding of purified anti-PF4–heparin antibodies to platelets and the resultant platelet activation
Authors: Newman Peter M. , Chong Beng H.
Published: October 2019

Immune heparin-induced thrombocytopenia (HIT) is associated with antibodies directed against a complex of platelet factor 4 (PF4) and heparin. We were able to affinity purify anti-PF4–heparin IgG (HIT IgG) from the plasma of 2 patients with HIT. Under conditions that were more physiological and sensitive than those in previous studies, we observed that this HIT IgG caused platelet aggregation on the addition of heparin. Platelets activated with HIT IgG increased their release and surface expression of PF4. We quantitated, for the first time, the binding of affinity-purified HIT iodine 125–IgG to platelets as they activated in a plasma milieu. Binding of the HIT IgG was dependent on heparin and required some degree of platelet activation. Blocking the platelet FcγRII with the monoclonal antibody IV.3 did not prevent HIT IgG binding to activated platelets. We concluded that anti-PF4–heparin IgG is the component in these HIT plasmas that induces platelet aggregation. The Fab region of HIT IgG binds to PF4–heparin on the surface of activated platelets. We propose that only then does the Fc portion of the bound IgG further activate the same or adjacent platelets through the Fc receptor. Our data support a dynamic model of platelet activation in which released PF4 enhances further antibody binding and more release.
Human endothelial cells express CCR2 and respond to MCP-1: direct role of MCP-1 in angiogenesis and tumor progression
Authors: Salcedo Rosalba , Ponce Maria Lourdes , Young Howard A. , Wasserman Ken , Ward Jerrold M. , Kleinman Hynda K. , Oppenheim Joost J. , Murphy William J.
Published: October 2019

Although several CXC chemokines have been shown to induce angiogenesis and play roles in tumor growth, to date, no member of the CC chemokine family has been reported to play a direct role in angiogenesis. Here we report that the CC chemokine, monocyte chemotactic protein 1 (MCP-1), induced chemotaxis of human endothelial cells at nanomolar concentrations. This chemotactic response was inhibited by a monoclonal antibody to MCP-1. MCP-1 also induced the formation of blood vessels in vivo as assessed by the chick chorioallantoic membrane and the matrigel plug assays. As expected, the angiogenic response induced by MCP-1 was accompanied by an inflammatory response. With the use of a rat aortic sprouting assay in the absence of leukocytic infiltrates, we ruled out the possibility that the angiogenic effect of MCP-1 depended on leukocyte products. Moreover, the direct effect of MCP-1 on angiogenesis was consistent with the expression of CCR2, the receptor for MCP-1, on endothelial cells. Assessment of supernatant from a human breast carcinoma cell line demonstrated the production of MCP-1. Treatment of immunodeficient mice bearing human breast carcinoma cells with a neutralizing antibody to MCP-1 resulted in significant increases in survival and inhibition of the growth of lung micrometastases. Taken together, our data indicate that MCP-1 can act as a direct mediator of angiogenesis. As a chemokine that is abundantly produced by some tumors, it can also directly contribute to tumor progression. Therefore, therapy employing antagonists of MCP-1 in combination with other inhibitors of angiogenesis may achieve more comprehensive inhibition of tumor growth.
Expression of Kell blood group protein in nonerythroid tissues
Authors: Russo David , Wu Xu , Redman Colvin M. , Lee Soohee
Published: October 2019

The Kell blood group protein is a zinc endopeptidase that yields endothelin-3, a potent bioactive peptide, by cleavage of big endothelin-3, a larger intermediate precursor. On red cells, Kell protein is linked by a single disulfide bond to XK, a protein that traverses the membrane 10 times and whose absence, as occurs in the McLeod phenotype, is associated with a set of clinical symptoms that include nerve and muscle disorders and red cell acanthocytosis. Previous studies indicated that Kell is primarily expressed in erythroid tissues, whereas XK has a wider tissue distribution. The tissue distribution of Kell protein has been further investigated by Northern blot analysis, PCR-screening of tissue complementary DNAs (cDNAs), and Western immunoblots. Screening of an RNA dot-blot panel confirmed that Kell is primarily expressed in erythroid tissues but is also expressed in a near equal amount in testis, with weaker expression in a large number of other tissues. PCR-screening of cDNAs from different tissues and DNA sequencing of the products gave similar results. In 2 of the nonerythroid tissues tested, testis and skeletal muscle, Kell protein was detected by Western immunoblotting. In skeletal muscle, isolation of XK with a specific antibody coisolated Kell protein. These studies demonstrate that Kell is expressed in both erythroid and nonerythroid tissues and is associated with XK.
Risk for subsequent venous thromboembolic complications in carriers of the prothrombin or the factor V gene mutation with a first episode of deep-vein thrombosis
Authors: Simioni Paolo , Prandoni Paolo , Lensing Anthonie W. A. , Manfrin Davide , Tormene Daniela , Gavasso Sabrina , Girolami Bruno , Sardella Corrado , Prins Martin , Girolami Antonio
Published: October 2019

Carriers of a mutation in the prothrombin (clotting factor II) or factor V gene have a 2- to 4-fold greater risk for venous thromboembolism than subjects without the mutations. Whether both mutations also predispose to recurrent venous thromboembolism is unclear. Outpatients who had a first episode of proven symptomatic deep-vein thrombosis and a long-term follow-up were studied. The outcome measure was the cumulative incidence of confirmed venous thromboembolic complications. Two hundred fifty-one patients were enrolled in the study. Mean duration of follow-up was 8.3 years. The prothrombin gene mutation was demonstrated in 27 patients (prevalence, 10.8%; 95% CI, 6.9 to 14.6), and the factor V gene mutation was demonstrated in 41 patients (prevalence, 16.3%; 95% CI, 11.8 to 20.9). The cumulative incidence of venous thromboembolic complications after 10 years was 61.3% (95% CI, 35.7 to 87.9), and the hazard ratio was 2.4 (95% CI, 1.3 to 4.7; P = .004) in patients with the prothrombin gene mutation); the cumulative incidence of venous thromboembolic complications after 10 years was 55.2% (95% CI, 36.4 to 74.0), and the hazard ratio was 2.4 (95% CI, 1.4 to 4.1;P = .001) in patients with the factor V gene mutation. In comparison, the cumulative incidence of venous thromboembolic complications after 10 years was 23.1% (95% CI, 16.2 to 30.1) in patients without the mutations. Prothrombin and factor V gene mutations occur frequently in patients with venous thrombosis and are associated with an increased risk for recurrent venous thromboembolic complications
Down-regulation of L-selectin expression in neutrophils by nonsteroidal anti-inflammatory drugs: role of intracellular ATP concentration
Authors: Gómez-Gaviro Maria Victoria , Domı́nguez-Jiménez Carmen , Carretero Jorge Moreno , Sabando Pedro , González-Alvaro Isidoro , Sánchez-Madrid Francisco , Dı́az-González Federico
Published: October 2019

L-selectin is an adhesion molecule that plays an essential role in the early events of the inflammatory response. Our group has recently described that several nonsteroidal anti-inflammatory drugs (NSAIDs) are able to induce both in vivo and in vitro the shedding of L-selectin in neutrophils through an unknown mechanism. In this work, we have studied potential mechanisms involved in the shedding of L-selectin induced by NSAIDs. This effect of NSAIDs did not involve any detectable intracellular calcium flux. Pretreatment of neutrophils either with Ro 31-8220 and H7, 2 specific inhibitors of protein kinase C (PKC), or with inhibitors of protein tyrosine kinases such as tyrphostin A25 or herbimycin A did not prevent the NSAID-mediated L-selectin shedding. However, the KD-IX-73-4, an inhibitor of L-selectin proteolysis was able to block the effect of NSAIDs on L-selectin expression. Remarkably, NSAIDs caused a variable reduction in the neutrophil intracellular ATP concentration that highly correlated with the differential ability of NSAIDs to trigger L-selectin shedding (r = 0.8, P < .01). In agreement with this finding, azide plus 2-deoxy-D-glucose, 2 metabolic blockers, also induced a rapid L-selectin shedding (65% ± 8%) without affecting the neutrophil viability, activation, or expression level of other surface molecules with soluble isoforms such as CD16 and CD59. These data indicate that the maintenance of L-selectin on the neutrophil surface requires energy consumption, which suggests that L-selectin is shed in neutrophils by default. Interestingly, NSAIDs seem to cause the shedding of L-selectin, at least in part, through the reduction of the intracellular ATP concentration.
Evaluation of Apaf-1 and procaspases-2, -3, -7, -8, and -9 as potential prognostic markers in acute leukemia
Authors: Svingen Phyllis A. , Karp Judith E. , Krajewski Stan , Mesner Peter W. , Gore Steven D. , Burke Philip J. , Reed John C. , Lazebnik Yuri A. , Kaufmann Scott H.
Published: October 2019

Recent studies have suggested that variations in levels of caspases, a family of intracellular cysteine proteases, can profoundly affect the ability of cells to undergo apoptosis. In this study, immunoblotting was used to examine levels of apoptotic protease activating factor-1 (Apaf-1) and procaspases-2, -3, -7, -8, and -9 in bone marrow samples (at least 80% leukemia) harvested before chemotherapy from adults with newly diagnosed acute myelogenous leukemia (AML, 42 patients) and acute lymphocytic leukemia (ALL, 18 patients). Levels of each of these polypeptides varied over a more than 10-fold range between specimens. In AML samples, expression of procaspase-2 correlated with levels of Apaf-1 (Rs = 0.52, P < .02), procaspase-3 (Rs = 0.56,P < .006) and procaspase-8 (Rs = 0.64, P < .002). In ALL samples, expression of procaspases-7 and -9 was highly correlated (Rs = 0.90,P < .003). Levels of these polypeptides did not correlate with prognostic factors or response to induction chemotherapy. In further studies, 16 paired samples (13 AML, 3 ALL), the first harvested before induction therapy and the second harvested at the time of leukemia regrowth, were also examined. There were no systematic alterations in levels of Apaf-1 or procaspases at relapse compared with diagnosis. These results indicate that levels of initiator caspases vary widely among different leukemia specimens but cast doubt on the hypothesis that this variation is a major determinant of drug sensitivity for acute leukemia in the clinical setting.
Evolving characteristics of AIDS-related lymphoma
Authors: Levine Alexandra M. , Seneviratne Lasika , Espina Byron M. , Wohl Amy Rock , Tulpule Anil , Nathwani Bharat N. , Gill Parkash S.
Published: October 2019

Over time, the epidemiologic and demographic characteristics of AIDS have changed in the United States, while the use of highly active antiretroviral therapy has changed the natural history of the disease. The goal of the study was to ascertain any changes in the epidemiologic, immunologic, pathologic, or clinical characteristics of AIDS-related lymphoma (ARL) over the course of the AIDS epidemic. Records of 369 patients with ARL diagnosed or treated at a single institution from 1982 through 1998 were reviewed. Single institutional data were compared to population-based data from the County of Los Angeles. Significant changes in the demographic profile of patients with newly diagnosed ARL have occurred, with the later time intervals associated with a higher prevalence in women (P = .25), in Latino/Hispanic individuals (P < .0001), and in those who acquired human immunodeficiency virus (HIV) heterosexually (P = .01). These changes were similar in both countywide, population-based analyses and in those from the single institution. The median CD4+ lymphocyte count at lymphoma diagnosis has decreased significantly over the years, from 177/dL in the earliest time period (1982-1986), to 53/dL in the last time period from 1995 to 1998 (P = .0006). The pathologic spectrum of disease has also changed, with a decrease in the prevalence of small noncleaved lymphoma (P = .0005) and an increase in diffuse large cell lymphoma (P < .0001). Despite changes in the use of antiretroviral or chemotherapy regimens, the median survival has not significantly changed.
Functional characterization of an IL-7–dependent CD4+CD8αα+ Th3-type malignant cell line derived from a patient with a cutaneous T-cell lymphoma
Authors: Poszepczynska Eva , Bagot Martine , Echchakir Hamid , Martinvalet Denis , Ramez Mohamed , Charue Dominique , Boumsell Laurence , Bensussan Armand
Published: October 2019

CDR3 of the functional rearranged T-cell receptor variable β region (TCR-Vβ) transcript was sequenced in order to demonstrate for the first time the identity between a long-term cultured T-cell line derived from a cutaneous T-cell lymphoma (CTCL) patient and the malignant T-cell clone present in the blood. The patient's peripheral blood lymphocyte-derived cultured T-cell line had a CD3+Vβ22+CD4+CD8+CD25−phenotype. It was named Pno and had been cultured for more than 1 year. Both fresh and long-term–cultured tumor cells proliferated highly in response to interleukin-7 (IL-7), and exogeneous IL-7 prevented Pno lymphocytes from apoptosis and maintained high levels of Bcl-2 expression. This unique malignant cloned lymphocyte line was further used to carry out functional studies. The results indicated that the CD3/TCR structures expressed by the Pno lymphocytes were functional because an immobilized anti-CD3 monoclonal antibody (mAb) or the combination of a soluble anti-CD3 mAb with submitogenic doses of phorbol 12 β-myristate 13 -acetate induced a proliferative response. Further, the CD2 and CD28 coreceptors were functional because they were able to induce a strong proliferative response upon their specific stimulation. Finally, the Pno T cell line had a Th3-type cytokine profile because it produced high amounts of the immunosuppressor cytokine tumor growth factor–β1 (TGF-β1). This high production of TGF-β1 may inhibit antitumor specific responses in CTCL.
Induction of monocyte– and T-cell–attracting chemokines in the lung during the generation of idiopathic pneumonia syndrome following allogeneic murine bone marrow transplantation
Authors: Panoskaltsis-Mortari Angela , Strieter Robert M. , Hermanson John R. , Fegeding Konstantin V. , Murphy William J. , Farrell Catherine L. , Lacey David L. , Blazar Bruce R.
Published: October 2019

Idiopathic pneumonia syndrome (IPS) is a significant complication following bone marrow transplantation (BMT). We have developed a murine model in which severe IPS is induced by pre-BMT conditioning and allogeneic T cells and is characterized by the recruitment of host monocytes and donor T cells into the lung by day 7 post-BMT. Chemokines regulate cellular recruitment and the migration of cells into inflammatory lesions. In this study, we examined the profiles of chemokines produced locally in the lung (parenchyma and bronchoalveolar lavage fluid) and systemically (serum) during the generation of IPS in the peri-BMT period. Protein and messenger RNA (mRNA) levels of CC chemokines (monocyte/lymphocyte attractants), especially monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1, RANTES (regulated upon activation normal T-cell expressed and secreted), and C10, were preferentially induced in the lung by day 7 postallogeneic BMT. In addition, there was an increase in mRNA for IP-10 (a monocyte and Th1-cell chemoattractant). The CXC chemokines MIP-2 and KC, known neutrophil attractants, were moderately elevated. For the most part, these increases in chemokines were dependent on the coinfusion of allogeneic T cells with the BM inoculum. Ribonuclease protection assay and in situ hybridization analyses post-BMT showed that the lung was a major producer of MCP-1, a potent inducer of monocyte chemotaxis. Increases in MCP-1 levels in the lung preceded host APC influx whereas MIP-1 levels accompanied donor T-cell infiltration. In summary, we have shown that monocyte- and T-cell–attracting chemokines are associated with monocyte and T-cell recruitment during IPS.
LEC induces chemotaxis and adhesion by interacting with CCR1 and CCR8
Authors: Howard O. M. Zack , Dong Hui Fang , Shirakawa Aiko-Konno , Oppenheim Joost J.
Published: October 2019

Liver-expressed chemokine (LEC) is an unusually large CC chemokine, which is also known as LMC, HCC-4, NCC-4, and CCL16. Previously, LEC was shown to induce leukocyte migration but the responsible signaling receptors were not characterized. We report chemotaxis and competitive binding studies that show LEC binds to and activates CCR1 and CCR8 transfected HEK-293 cells. LEC induced maximal migration of CCR1 and CCR8 transfected cells at 89.3 nmol/L and cell adhesion at 5.6 nmol/L. The molar concentration of LEC required to induce maximum cell migration is 20- to 200-fold greater than that required for RANTES or I309, respectively. All 3 chemokines induced maximal static adhesion at 5 to 7 nmol/L. A neutralizing polyclonal antibody to LEC was developed to demonstrate that the unusually high concentration of LEC required to induce chemotaxis was a property of LEC and not as a result of an irrelevant protein contamination. This study suggests that LEC may be a more effective inducer of cell adhesion than cell migration.
Heparin-induced thrombocytopenia with thromboembolic complications: meta-analysis of 2 prospective trials to assess the value of parenteral treatment with lepirudin and its therapeutic aPTT range
Authors: Greinacher Andreas , Eichler Petra , Lubenow Norbert , Kwasny Harald , Luz Matthias
Published: October 2019

This meta-analysis focuses on 2 prospective studies in patients with heparin-induced thrombocytopenia (HIT) and thromboembolic complication (TEC) who were treated with lepirudin (n = 113). Data were compared with those of a historical control group (n = 91). The primary endpoint (combined incidence of death, new TEC, and limb amputation) occurred in 25 lepirudin-treated patients (22.1%; 95% CI, 14.5%-29.8%): 11 died (9.7%; 95% CI, 4.9%-16.8%), 7 underwent limb amputation (6.2%; 95% CI, 2.5%-12.3%), and 12 experienced new TEC (10.6%; 95% CI, 5.8%-18.3%). The risk was highest in the period between diagnosis of HIT and the start of lepirudin therapy (combined event rate per patient day 6.1%). It markedly decreased to 1.3% during lepirudin treatment and to 0.7% in the posttreatment period. From the start of lepirudin therapy to the end of follow-up, lepirudin-treated patients had consistently lower incidences of the combined endpoint than the historical control group (P = .004, log-rank test), primarily because of a reduced risk for new TEC (P = .005). Thrombin–antithrombin levels in the pretreatment period (median, 43.9 μg/L) decreased after the initiation of lepirudin (at 24 hours ± 6 hours; median, 9.18 μg/L.) During treatment with lepirudin, aPTT ratios of 1.5 to 2.5 produced optimal clinical efficacy with a moderate risk for bleeding, aPTT ratios lower than 1.5 were subtherapeutic, and aPTT levels greater than 2.5 were associated with high bleeding risk. Bleeding events requiring transfusion were significantly more frequent in patients taking lepirudin than in historical control patients (P = .02). In conclusion, this meta-analysis provides further evidence that lepirudin is an effective and acceptably safe treatment for patients with HIT.