bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro infection of megakaryocytes and their precursors by human cytomegalovirus
Authors: Crapnell Kirsten , Zanjani Esmail D. , Chaudhuri Aniruddho , Ascensao Joao L. , St. Jeor Stephen , Maciejewski Jaroslaw P.
Published: October 2019

Apart from congenital human cytomegalovirus (HCMV) infection, manifest HCMV disease occurs primarily in immunocompromised patients. In allogeneic bone marrow transplantation, HCMV is frequently associated with graft failure and cytopenias involving all hematopoietic lineages, but thrombocytopenia is the most commonly reported hematologic complication. The authors hypothesized that megakaryocytes (MK) may be a specific target for HCMV. Although the susceptibility of immature hematopoietic progenitors cells to HCMV has been established, a productive viral life cycle has only been linked to myelomonocytic maturation. The authors investigated whether HCMV can also infect MK and impair their function. They demonstrated that HCMV did not affect the thrombopoietin (TPO)-driven proliferation of CD34+ cells until MK maturation occurred. MK challenged with HCMV showed a 50% more rapid loss of viability than mock-infected cells. MK and their early precursors were clearly shown to be susceptible to HCMV in vitro, as evidenced by the presence of HCMV in magnetic column-purified CD42+ MK and 2-color fluorescent staining with antibodies directed against CD42a and HCMV pp65 antigen. These findings were confirmed by the infection of MK with a laboratory strain of HCMV containing the β-galactosidase (β-gal) gene. Using chromogenic β-gal substrates, HCMV was detected during MK differentiation of infected CD34+ cells and after infection of fully differentiated MK. Production of infectious virus was observed in cultures infected MK, suggesting that HCMV can complete its life cycle. These results demonstrate that MK are susceptible to HCMV infection and that direct infection of these cells in vivo may contribute to the thrombocytopenia observed in patients infected with HCMV.
Adenosine receptor occupancy suppresses chemoattractant-induced phospholipase D activity by diminishing membrane recruitment of small GTPases
Authors: Thibault Nathalie , Harbour Danielle , Borgeat Pierre , Naccache Paul H. , Bourgoin Sylvain G.
Published: October 2019

Adenosine (Ado) is an important autocrine modulator of neutrophil functions. In this study, we determined the effects of endogenous Ado on fMet-Leu-Phe (fMLP)–induced phospholipase D (PLD) activity in neutrophils. The removal of extracellular Ado by Ado deaminase (ADA) or the blockade of its action by the A2a receptor antagonists 8-(3-chlorostyryl) caffeine (CSC) or CGS15943 markedly increased fMLP-induced PLD activation. The concentration-dependent stimulatory effects of CSC and CGS15943 were abolished by a pretreatment of neutrophil suspensionswith ADA. In contrast, the selective A2a receptor agonist CGS21680 suppressed fMLP-induced PLD activation. Furthermore, inhibition by CGS21680 of fMLP-induced PLD activity was reversed by CSC or CGS15943. The removal of Ado by ADA or the blockade of its action by CSC or CGS15943, markedly increased the membrane recruitment of cytosolic protein kinase C (PKC), RhoA, and ADP-ribosylation factor (ARF) in response to fMLP. As shown for PLD activity, the stimulatory effect of Ado receptor antagonists on PLD cofactors translocation was abolished by a pretreatment of the cells with ADA. Moreover, the membrane translocation of both PKC, RhoA, and ARF in response to fMLP was attenuated by CGS21680 and this effect of the A2a receptor agonist was antagonized by CSC or CGS15943. These data demonstrate that Ado released by neutrophils in the extracellular milieu inhibits PLD activation by blocking membrane association of ARF, RhoA, and PKC through Ado A2a receptor occupancy.
Reduction of the antigenicity of factor VIII toward complex inhibitory antibody plasmas using multiply-substituted hybrid human/porcine factor VIII molecules
Authors: Barrow Rachel T. , Healey John F. , Gailani David , Scandella Dorothea , Lollar Pete
Published: October 2019

Factor VIII (fVIII) circulates as a heavy chain/light chain (A1-A2-B/ap-A3-C1-C2) heterodimer. The 41-residue light chain activation peptide, ap, is cleaved from fVIII during proteolytic activation by thrombin or factor Xa. We constructed 7 active recombinant hybrid B-domainless human/porcine fVIII molecules that contained combinations of porcine sequence replacements within the A2, ap-A3, and C2 domains. The cross-reactivity of 23 high-titer inhibitory antibodies between human fVIII and the hybrids was inversely related to the degree of porcine substitution. In all plasmas, the substitution of all 3 regions yielded cross-reactivities that were not significantly different from those of porcine fVIII. To differentiate between inhibitor binding to the ap region and the A3 domain, we constructed 2 additional hybrids that contained porcine A2 and C2 domain substitutions and either porcine A3 or porcineap substitutions. The porcine ap segment was less antigenic than the human ap segment in several plasmas that had activity against the ap-A3 region. This indicates that some inhibitor plasmas contain antibodies directed against the fVIIIap segment in addition to A2, A3, and C2 domain epitopes identified in previous studies. Substitution of porcine sequences within the A2, A3, C2, and ap regions of human fVIII is necessary and sufficient to achieve a maximal reduction in antigenicity relative to porcine fVIII with respect to most inhibitory antibody plasmas.
Increased proteasome degradation of cyclin-dependent kinase inhibitor p27 is associated with a decreased overall survival in mantle cell lymphoma
Authors: Chiarle Roberto , Budel Leo M. , Skolnik Jeffrey , Frizzera Glauco , Chilosi Marco , Corato Alessandra , Pizzolo Gianni , Magidson Jory , Montagnoli Alessia , Pagano Michele , Maes Brigitte , De Wolf-Peeters Christine , Inghirami Giorgio
Published: October 2019

Mantle cell lymphoma (MCL) is an aggressive neoplasm characterized by the deregulated expression of cyclin D1 by t(11;14). The molecular mechanisms responsible for MCL's clinical behavior remain unclear. The authors have investigated the expression of p53, E2F-1, and the CDK inhibitors p27 and p21 in 110 MCLs, relating their expression to proliferative activity (Ki-67). For comparison, they have similarly analyzed low-grade (12 MALT, 16 CLL/SLL) and high-grade (19 DLCL) lymphomas. p53 was detected more frequently in large-cell MCL (l-MCL; 5 of 7) than in classical MCL (s-MCL; 13 of 103) and DLCL (8 of 19). In MCL and DLCL, the percentage of E2F-1+ nuclei was high, correlating with high Ki-67 expression. Most MCLs (91 of 112) and DLCLs (12 of 19) showed a loss of p27; MALT and CLL/SLL, however, were p27 positive. Reverse transcription–polymerase chain reaction and in vitro protein degradation assays demonstrated that MCLs have normal p27 mRNA expression but increased p27 protein degradation activity via the proteasome pathway. Correlation of MCL p53 and p27 expression with clinical data showed an association between reduced overall survival rates and the overexpression of p53 (P = .001), the loss of p27 (P = .002), or both. Loss of p27 identified patients with a worse clinical outcome among p53 negative cases (P = .002). These findings demonstrated that MCL has a distinct cell cycle protein expression similar to that of high-grade lymphoma. The loss of p27 and the overexpression of p53 in MCL are prognostic markers that identify patients at high risk. The demonstration that low levels of p27 in MCL result from enhanced proteasome-mediated degradation should encourage additional clinical trials. (Blood. 2000;95:619-626)
Loxosceles intermedia spider envenomation induces activation of an endogenous metalloproteinase, resulting in cleavage of glycophorins from the erythrocyte surface and facilitating complement-mediated lysis
Authors: Tambourgi Denise V. , Morgan B. Paul , de Andrade Rute M. G. , Magnoli Fábio C. , van den Berg Carmen W.
Published: October 2019

Loxosceles is the most venomous spider in Brazil, and envenomation causes dermonecrosis and complement (C)-dependent intravascular hemolysis. The authors studied the mechanism of induction of C-induced hemolysis. Purified Loxosceles toxins rendered human erythrocytes susceptible to lysis by human C but did not have an effect on the E-bound C-regulators DAF, CR1, or CD59. However, incubation with venom toxins caused cleavage of glycophorin from the erythrocyte (E) surface, facilitating C activation and hemolysis. The results suggest that glycophorin is an important factor in the protection of E against homologous C. Cleavage of glycophorin (GP) A, GPB, and GPC occurred at sites close to the membrane but could not be accomplished using purified GPA and purified toxins, demonstrating that cleavage was not an effect of a direct proteolytic action of theLoxosceles toxins on the glycophorins. Inhibition of the cleavage of glycophorins induced by Loxosceles venom was achieved with 1,10-phenanthroline. The authors propose that the sphingomyelinase activity of the toxins induces activation of an endogenous metalloproteinase, which then cleaves glycophorins. They observed the transfer of C-dependent hemolysis to other cells, suggesting that the Loxosceles toxins can act on multiple cells. This observation can explain the extent of hemolysis observed in patients after envenomation. Identification of the mechanism of induction of susceptibility to C-mediated lysis afterLoxosceles envenomation opens up the possibility of the development of an effective therapeutic strategy.
PG27, an extract of Tripterygium wilfordii hook f, induces antigen-specific tolerance in bone marrow transplantation in mice
Authors: Chen Yanfei , Zeng Defu , Schlegel Paul G. , Fidler John , Chao Nelson J.
Published: October 2019

PG27, an active fraction purified from an extract of a Chinese herb,Tripterygium wilfordii hook f, was used to prevent graft-versus-host disease (GVHD) in a murine model. Lethally irradiated BALB/c (H-2d) recipients of B10.D2 (H-2d) donor grafts were given daily intraperitoneal injections of PG27 (40 mg/kg per day) for the first 35 days after transplantation. Control mice were given daily injections of solvent vehicle (Ethanol and Cremophor EL). All the control recipients (15/15) died of GVHD within 90 days, but all the recipients given prophylactic treatment with PG27 (15/15) survived beyond 100 days without any signs of GVHD. Furthermore, the GVHD-free recipients were used as donors, and their bone marrow and spleen cells were transplanted into lethally irradiated normal BALB/c (same party) or lethally irradiated normal C3H (H-2k, third party) mice. Although 10 of 10 same-party recipients survived more than 100 days without any signs of GVHD, 10 of 10 third-party C3H recipients died of GVHD within 40 days. Further studies of PG27 in the murine BCL1 leukemia/lymphoma model demonstrated that animals treated with PG27 partially retained the graft-versus-leukemia (GVL) effect of the graft without GVHD. These results suggest that treatment with PG27 induces host-specific tolerance and retains the GVL effect of allogeneic marrow grafts.
Detection of aberrant isotype switch recombination in low-grade and high-grade gastric MALT lymphomas
Authors: Nardini Elena , Aiello Antonella , Giardini Roberto , Colnaghi Maria Ines , Ménard Sylvie , Balsari Andrea
Published: October 2019

Gastric mucosa-associated lymphoid tissue (MALT) lymphoma originates from reactive lymphocytic infiltrates during chronic gastritis, closely associated with Helicobacter pylori infection. MALT lymphomas may be either “low grade” or “high grade,” and transformation from low grade to high grade can occur. To obtain information on the maturational state of MALT lymphoma cells, we investigated their ability to undergo isotype switch recombination, which together with immunoglobulin variable gene somatic mutation, contributes to normal B-cell maturation. Using specific probes for the immunoglobulin heavy-chain (IgH) switch regions, we found by Southern blot that 3 out of 5 low-grade cases and 2 out of 2 high-grade cases showed rearrangements within IgH switch regions, which appeared aberrant in 4 of the 5 cases. The cloning of two rearranged fragments from one low-grade and one high-grade case confirmed the aberrant nature of the rearranged fragments. A deletion from the switch μ region (Sμ) to the first constant μ exon (Cμ 1) and a second deletion from the second constant μ exon (Cμ 2) to the gamma 3 region (γ 3) was detected in the low-grade case. In the high-grade case, there was a deletion of the IgH intronic enhancer (Eμ) and a 336–base pair (bp) insertion into the Sμ region of a gene (KIAA0307) normally located at 15q24. These data demonstrate for the first time the ability of MALT lymphoma cells to undergo aberrant isotype switch recombinations, which might be directly involved in the development or progression of malignancy.
Myeloma progenitors in the blood of patients with aggressive or minimal disease: engraftment and self-renewal of primary human myeloma in the bone marrow of NOD SCID mice
Authors: Pilarski Linda M. , Hipperson Gail , Seeberger Karen , Pruski Eva , Coupland Robert W. , Belch Andrew R.
Published: October 2019

The myelomagenic capacity of clonotypic myeloma cells in G-CSF mobilized blood was tested by xenotransplant. Intracardiac (IC) injection of NOD SCID mice with peripheral cells from 5 patients who had aggressive myeloma led to lytic bone lesions, human Ig in the serum, human plasma cells, and a high frequency of clonotypic cells in the murine bone marrow (BM). Human B and plasma cells were detected in BM, spleen, and blood. Injection of ex vivo multiple myeloma cells directly into the murine sternal BM (intraosseus injection [IO]) leads to lytic bone lesions, BM plasma cells, and a high frequency of clonotypic cells in the femoral BM. This shows that myeloma has spread from the primary injection site to distant BM locations. By using a cellular limiting dilution PCR assay to quantify clonotypic B lineage cells, we confirmed that peripheral myeloma cells homed to the murine BM after IC and IO injection. The myeloma progenitor undergoes self-renewal in murine BM, as demonstrated by the transfer of human myeloma to a secondary recipient mouse. For 6 of 7 patients, G-CSF mobilized cells from patients who have minimal disease, taken at the time of mobilization or after cryopreservation, included myeloma progenitors as identified by engraftment of clonotypic cells and/or lytic bone disease in mice. This indicates that myeloma progenitors are mobilized into the blood by cyclophosphamide/G-CSF. Their ability to generate myeloma in a xenotransplant model implies that such progenitors are also myelomagenic when reinfused into patients, and suggests the need for an effective strategy to purge them before transplant.
Thrombopoietin induces rapid resolution of thrombocytopenia after orthotopic liver transplantation through increased platelet production
Authors: Peck-Radosavljevic Markus , Wichlas Martina , Zacherl Johannes , Stiegler Gabriele , Stohlawetz Petra , Fuchsjäger Michael , Kreil Anna , Metz-Schimmerl Sylvia , Panzer Simon , Steininger Rudolf , Mühlbacher Ferdinand , Ferenci Peter , Pidlich Johann , Gangl Alfred
Published: October 2019

Thrombopoietin (TPO) deficiency has been proposed as an important etiologic factor for thrombocytopenia in advanced-stage liver disease. To clarify the contributions of platelet production, platelet consumption, coagulation activation, and splenic sequestration to thrombocytopenia in liver disease, we studied TPO serum levels and markers of platelet production, platelet activation, and coagulation activation before and 14 days after orthotopic liver transplantation (OLT) in 18 patients with advanced liver cirrhosis. Thrombocytopenia before transplantation occurred with low-normal serum levels of TPO, normal levels of platelet and coagulation activation markers, and no increase in bone marrow production of platelets. TPO serum levels increased significantly on the first day after OLT, preceding the increase of reticulated platelets by 3 days and peripheral platelets by 5 days. Normalization of the peripheral platelet count occurred in most patients within 14 days of OLT, irrespective of the change in spleen size assessed by computed tomography volumetry. Normalization of platelet counts was not hampered by a certain degree of platelet activation observed during the steepest increase in the peripheral platelet count. Bone marrow production of platelets increased significantly within 2 weeks of transplantation. Low TPO serum levels with low platelet counts and without platelet consumption suggests low TPO production in end-stage liver disease. The rapid increase in TPO serum levels after transplantation induces an increase in the bone marrow production of platelets. Decreased TPO production in the cirrhotic liver is an important etiologic factor for thrombocytopenia in liver disease that is rapidly reversed by transplantation.
Opposing effects of engagement of integrins and stimulation of cytokine receptors on cell cycle progression of normal human hematopoietic progenitors
Authors: Jiang Yuehua , Prosper Felipe , Verfaillie Catherine M.
Published: October 2019

We evaluated the effect of β1-integrin receptor engagement on the expression and activity of cell cycle regulatory proteins in CD34+ cells under conditions that mimic the steady-state marrow microenvironment and in the presence of supraphysiological concentrations of interleukin-3 (IL3) and stem cell factor (SCF). Adhesion of CD34+ progenitors to fibronectin (FN) was similar whether IL3 or SCF was present or absent. Engagement of β1-integrins blocked S-phase entry of CD34+ cells in the absence of IL3 or SCF, whereas addition of 10 ng/mL IL3 or SCF prevented such a block in S-phase entry. In the absence of IL3 or SCF, cyclin-E levels were significantly lower and p27KIP1 levels significantly higher in FN-adherent than in FN-nonadherent cells, or than in poly-L-lysine (PLL)–adherent or (PLL)–nonadherent cells. Cyclin-dependent-kinase (cdk)-2 activity was decreased and levels of cyclin-E–cdk2 complexes were lower in FN-adherent than in PLL-adherent cells. In contrast, cyclin-E and p27KIP1 protein levels and cdk2 activity in cells adherent to FN in the presence of IL3 or SCF were similar to those in PLL-adherent and FN-nonadherent or PLL-nonadherent cells. In conclusion, under physiological cytokine conditions, integrin engagement prevents S-phase entrance of CD34+ cells, which is associated with elevated levels of the contact-dependent cyclin kinase inhibitor p27KIP1. Supraphysiological concentrations of IL3 or SCF prevent p27KIP1 elevation and override the integrin-mediated inhibition of entry into S phase.
PU.1 is required for myeloid-derived but not lymphoid-derived dendritic cells
Authors: Guerriero Anastasia , Langmuir Peter B. , Spain Lisa M. , Scott Edward W.
Published: October 2019

The ets-family transcription factor PU.1 is required for the proper development of both myeloid and lymphoid progenitors. We used PU.1-deficient animals to examine the role of PU.1 during dendritic cell development. PU.1−/−animals produce lymphoid-derived dendritic cells (DC): low-density class II major histocompatibility complex [MHC-II+] CD11c+ CD8+DEC-205+. But they lack myeloid-derived DC: low-density MHC-II+ CD11c+ CD8−DEC-205−. PU.1−/− embryos also lack progenitors capable of differentiating into myeloid DC in response to granulocyte-macrophage colony-stimulating factor plus interleukin-4. The appearance of lymphoid DC in developing PU.1−/−thymus was initially delayed, but this population recovered to wild type (WT) levels upon organ culture of isolated thymic lobes. PU.1−/−lymphoid DC were functionally equivalent to WT DC for stimulating T-cell proliferation in mixed lymphocyte reactions. These results demonstrate that PU.1 is required for the development of myeloid DC but not lymphoid DC.
Molecular mechanisms of platelet exocytosis: role of SNAP-23 and syntaxin 2 in dense core granule release
Authors: Chen Dong , Bernstein Audrey M. , Lemons Paula P. , Whiteheart Sidney W.
Published: October 2019

To characterize the molecular mechanisms of platelet secretion, we focused on the calcium-induced exocytosis of dense core granules. Platelets contain several known t-SNAREs (soluble N-ethylmaleimide sensitive factor [NSF] attachment protein receptors) such as syntaxins 2, 4, and 7 and SNAP-23 (synaptosomal associated protein 23). By using an in vitro exocytosis assay, we have been able to assign roles for some of these t-SNAREs in dense core granule release. This calcium-induced secretion relies on the SNARE proteins because it is stimulated by the addition of recombinant -SNAP and inhibited by a dominant negative -SNAP–L294A mutant or by anti–-SNAP and anti-NSF antibodies. SNAP-23 antibodies and an inhibitory C-terminal SNAP-23 peptide both blocked dense core granule release, demonstrating a role for SNAP-23. Unlike other cell types, platelets contain a significant pool of soluble SNAP-23, which does not partition into Triton X-114. Of the anti-syntaxin antibodies tested, only anti–syntaxin 2 antibody inhibited dense core granule release. Immunoprecipitation studies showed that the 2 t-SNAREs syntaxin 2 and SNAP-23 do form a complex in vivo. These data clearly show that SNAPs, NSF, and specific t-SNAREs are used for dense core granule release; these data provide a greater understanding of regulated exocytosis in platelets.
Proteolytic processing of human coagulation factor IX by plasmin
Authors: Samis John A. , Ramsey Gillian D. , Walker John B. , Nesheim Michael E. , Giles Alan R.
Published: October 2019

Previous studies have shown that thrombin generation in vivo caused a 92% decrease in factor IX (F.IX) activity and the appearance of a cleavage product after immunoblotting that comigrated with activated F.IX (F.IXa). Under these conditions, the fibrinolytic system was clearly activated, suggesting plasmin may have altered F.IX. Thus, the effect(s) of plasmin on human F.IX was determined in vitro. Plasmin (50 nM) decreased the 1-stage clotting activity of F.IX (4 μM) by 80% and the activity of F.IXa (4 μM) by 50% after 30 minutes at 37°C. Plasmin hydrolysis of F.IX yields products of 45, 30, 20, and 14 kd on reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and 2 products of 52 and 14 kd under nonreducing conditions. Plasmin-treated F.IX did not bind the active site probe, p-aminobenzamidine, or form an SDS-stable complex with antithrombin. It only marginally activated human factor X in the presence of phospholipid and activated factor VIII. Although dansyl-Glu-Gly-Arg-chloromethyl ketone inactivated–F.IXa inhibited the clotting activity of F.IXa, plasmin-treated F.IX did not. Plasmin cleaves F.IX after Lys43, Arg145, Arg180, Lys316, and Arg318, but F.IXa is not appreciably generated despite cleavage at the 2 normal activation sites (Arg145 and Arg180). Tissue plasminogen activator–catalyzed lysis of fibrin formed in human plasma results in generation of the 45- and 30-kd fragments of F.IX and decreased F.IX clotting activity. Collectively, the results suggest that plasmin is able to down-regulate coagulation by inactivating F.IX.
The receptor tyrosine kinase c-kit provides a critical signal for survival, expansion, and maturation of mouse natural killer cells
Authors: Colucci Francesco , Di Santo James P.
Published: October 2019

Fetal liver kinase ligands (flk2L/flt3L) and stem cell factor (SCF) have been shown to promote natural killer (NK) cell differentiation from hematopoietic stem cell (HSC) precursors in vitro. However, the contribution of signaling through the receptors for these growth factors for in vivo NK cell development remains ill-defined. We have analyzed the role of the SCF receptor c-kit in NK cell differentiation by reconstituting NK-deficient mice with fetal liver (FL) HSCs of c-kit−/− (W/W) mice. Although c-kit−/−NK cells were generated inW/W chimeras, they were reduced in number, contained a lower percentage of CD45R (B220)+ cells, and were poorly cytolytic. In vitro experiments showed that generation of NK cells from FL precursors was reduced in the absence of c-kit signaling and that SCF promoted the survival of peripheral c-kit+ NK cells. We conclude that c-kit/SCF interactions in vivo are dispensable for the commitment of HSC to the NK lineage, but they provide essential signals for generating normal numbers of fully mature NK cells.
Sickle cell anemia day hospital: an approach for the management of uncomplicated painful crises
Authors: Benjamin Lennette J. , Swinson Gwendolyn I. , Nagel Ronald L.
Published: October 2019

Painful crisis episodes are poorly treated in sickle cell anemia, both in timeliness and appropriateness of care. Delayed treatment in Emergency Departments, unrelieved pain, frequent admissions, and prolonged hospitalizations are common. We established a Day Hospital (DH) to determine if an alternative care delivery system could improve pain relief and reduce unnecessary hospital admissions for patients with uncomplicated painful crises. Trained DH staff delivered prompt titration for pain relief based on each patient's analgesic history and qualitative and quantitative assessments. Response to therapy and comorbidities commanded disposition. During the first 5 years of DH operation there were 2554 visits; 60% of the patients had severe pain. During an average visit of 4.5 hours, 84% of the patients were titrated to relief; 90% had pain relief within 2 to 4 hours. Overall, 81% of the patients were discharged home (70% initially and 90% to 94% in the last 3 years). During the first 5 years of the DH, there were 2612 emergency department (ED) visits that averaged 13 hours each. The combined ED and DH admissions during this time represented a 40% decrease in the baseline ED admission rate of 92%, (1 year pre-DH). Patients with uncomplicated painful crisis were admitted 5 times less often from the DH (8.3%) than from the ED (42.7%). The length of stay (LOS) for inpatients followed by the DH staff decreased by 1.5 days, while the LOS for patients followed by non-DH staff remained unchanged. Reduction of admissions and LOS represented a savings of approximately $1.7 million. We conclude that a dedicated facility provides the kingpin for effective and rapid painful crisis management, reduces hospitalizations, and facilitates integration of the approach into other areas of care.
Quantitative assessment of retroviral transfer of the human multidrug resistance 1 gene to human mobilized peripheral blood progenitor cells engrafted in nonobese diabetic/severe combined immunodeficient mice
Authors: Schiedlmeier B. , Kühlcke K. , Eckert H. G. , Baum C. , Zeller W. J. , Fruehauf S.
Published: October 2019

Mobilized peripheral blood progenitor cells (PBPC) are a potential target for the retrovirus-mediated transfer of cytostatic drug-resistance genes. We analyzed nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse-repopulating CD34+ PBPC from patients with cancer after retroviral transduction in various cytokine combinations with the hybrid vector SF-MDR, which is based on the Friend mink cell focus-forming/murine embryonic stem-cell virus and carries the human multidrug resistance 1 (MDR1) gene. Five to 13 weeks after transplantation of CD34+ PBPC into NOD/SCID mice (n = 84), a cell dose-dependent multilineage engraftment of human leukocytes up to an average of 33% was observed. The SF-MDR provirus was detected in the bone marrow (BM) and in its granulocyte fractions in 96% and 72%, respectively, of chimeric NOD/SCID mice. SF-MDR provirus integration assessed by quantitative real-time polymerase chain reaction (PCR) was optimal in the presence of Flt-3 ligand/thrombopoietin/stem-cell factor, resulting in a 6-fold (24% ± 5% [mean ± SE]) higher average proportion of gene-marked human cells in NOD/SCID mice than that achieved with IL-3 alone (P < .01). A population of clearly rhodamine-123dull human myeloid progeny cells could be isolated from BM samples from chimeric NOD/SCID mice. On the basis of PCR and rhodamine-123 efflux data, up to 18% ± 4% of transduced cells were calculated to express the transgene. Our data suggest that the NOD/SCID model provides a valid assay for estimating the gene-transfer efficiency to repopulating human PBPC that may be achievable in clinical autologous transplantation. P-glycoprotein expression sufficient to prevent marrow aplasia in vivo may be obtained with this SF-MDR vector and an optimized transduction protocol.
GATA-1 blocks IL-6-induced macrophage differentiation and apoptosis through the sustained expression of cyclin D1 and Bcl-2 in a murine myeloid cell line M1
Authors: Tanaka Hirokazu , Matsumura Itaru , Nakajima Koichi , Daino Hanako , Sonoyama Junko , Yoshida Hitoshi , Oritani Kenji , Machii Takashi , Yamamoto Masayuki , Hirano Toshio , Kanakura Yuzuru
Published: October 2019

Cytokines exert pleiotropic effects on target cells in a manner dependent on the cell type or stage of differentiation. To determine how instinctive cell properties affect biological effects of cytokine, we introduced an erythroid/megakaryocyte lineage-specific transcription factor, GATA-1, into a murine myeloid cell line M1, which is known to undergo macrophage differentiation in response to interleukin 6 (IL-6). Overexpression of GATA-1 changed the phenotype of M1 cells from myeloid to megakaryocytic lineage. Furthermore, GATA-1 blocked both IL-6-induced macrophage differentiation and apoptosis of M1 cells. Although STAT3 is essential for IL-6-induced macrophage differentiation of M1 cells, GATA-1 had little or no effect on tyrosine phosphorylation, DNA binding, and transcriptional activities of STAT3 in Western blot analysis, electropholic mobility shift assay (EMSA), and luciferase assays. During IL-6-induced macrophage differentiation of M1 cells, IL-6 down-regulated cyclin D1 expression and induced p19INK4D expression, leading to reduction in cdk4 activities. In contrast, sustained expression of cyclin D1 and a significantly lesser amount of p19INK4D induction were observed in IL-6-treated M1 cells overexpressing GATA-1. Furthermore, although bcl-2 expression was severely reduced by IL-6 in M1 cells, it was sustained in GATA-1-introduced M1 cells during the culture with IL-6. Both IL-6-induced macrophage differentiation and apoptosis were significantly abrogated by coexpression of cyclin D1 and bcl-2, whereas overexpressions of cyclin D1 or bcl-2 inhibited only differentiation or apoptosis, respectively. These results suggested that GATA-1 may not only reprogram the lineage phenotype of M1 cells but also disrupt the biologic effects of IL-6 through the sustained expression of cyclin D1 and bcl-2.
Migration of activated CD8+ T lymphocytes to sites of viral infection does not require endothelial selectins
Authors: Bartholdy Christina , Marker Ole , Thomsen Allan Randrup
Published: October 2019

Using mice deficient of E-selectin and E/P-selectin, we have studied the requirement for endothelial selectins in extravasation of leukocytes at sites of viral infection, with major emphasis on the recruitment of virus-specific TC1 cells. Lymphocytic choriomeningitis virus (LCMV)–induced meningitis was used as our primary experimental model. Additionally, localized subdermal inflammation and virus clearance in internal organs were analyzed during LCMV infection. The generation of CD8+ effector T cells in infected mutants was unimpaired. Quantitative and qualitative analysis of the inflammatory exudate cells in intracerebrally infected mice gave identical results in all strains of mice. Expression of endothelial selectin was also found to be redundant regarding the ability of effector cells to eliminate virus in nonlymphoid organs. Concerning LCMV-induced footpad swelling, absent or marginal reduction was found in E/P-sel −/− mice, compared with wild-type mice after local challenge with virus or immunodominant viral MHC class I restricted peptide, respectively. Similar results were obtained after adoptive transfer of wild-type effector cells into E/P-sel −/− recipients, whereas footpad swelling was markedly decreased in P-sel/ICAM-1 −/− and ICAM-1 −/− recipients. LCMV-induced footpad swelling was completely inhibited in ICAM-deficient mice transfused with donor cell preincubated with soluble VCAM-1-Ig chimeric protein. Taken together, the current findings strongly indicate that the migration of TC1 effector cells to sites of viral infection can proceed in the absence of endothelial selectins, whereas ligands of the Ig superfamily are critically involved in this process.
Both Stat5a and Stat5b are required for antigen-induced eosinophil and T-cell recruitment into the tissue
Authors: Kagami Shin-ichiro , Nakajima Hiroshi , Kumano Kotaro , Suzuki Kotaro , Suto Akira , Imada Kazunori , Davey Helen W. , Saito Yasushi , Takatsu Kiyoshi , Leonard Warren J. , Iwamoto Itsuo
Published: October 2019

Antigen-induced eosinophil recruitment into the airways of sensitized mice is mediated by CD4+ T cells and their cytokines, especially IL-5. In this study, we found that the antigen-induced airway eosinophilia was diminished in Stat5a-deficient (Stat5a−/−) mice and Stat5b-deficient (Stat5b−/−) mice. We also found that antigen-induced CD4+ T-cell infiltration and IL-5 production in the airways were diminished in Stat5a−/− mice and Stat5b−/− mice. Moreover, antigen-induced proliferation of splenocytes was diminished in Stat5a−/− mice and Stat5b−/− mice, suggesting that the generation of antigen-primed T cells may be compromised in Stat5a−/−mice and Stat5b−/− mice and this defect may account for the diminished antigen-induced T-cell infiltration into the airways. Interestingly, IL-4 and IL-5 production from anti-CD3–stimulated splenocytes was diminished in Stat5a−/− mice and Stat5b−/− mice. However, antigen-specific IgE and IgG1 production was diminished in Stat5a−/− mice but not in Stat5b−/− mice, whereas antigen-specific IgG2a production was increased in Stat5a−/− mice, suggesting the enhanced Th1 responses in Stat5a−/− mice. Finally, we found that eosinophilopoiesis induced by the administration of recombinant IL-5 was also diminished in Stat5a−/− mice and Stat5b−/− mice. Together, these results indicate that both Stat5a and Stat5b are essential for induction of antigen-induced eosinophil recruitment into the airways and that the defects in antigen-induced eosinophil recruitment in Stat5a−/− mice and Stat5b−/− mice result from both impaired IL-5 production in the airways and diminished IL-5 responsiveness of eosinophils.
Mutation analysis of the 5′ noncoding regulatory region of the BCL-6 gene in non-Hodgkin lymphoma: evidence for recurrent mutations and intraclonal heterogeneity
Authors: Lossos Izidore S. , Levy Ronald
Published: October 2019

The BCL-6 proto-oncogene is involved in the genesis of non-Hodgkin lymphoma (NHL). Rearrangements due to chromosomal translocations and somatic mutations of the 5′ noncoding regulatory region of the BCL-6 gene are potential mechanisms for altering its expression in NHL. To further elucidate the nature of the somatic mutations in the regulatory region of this gene, we have studied 10 healthy donors and 11 NHL biopsy samples by extensive molecular cloning and sequencing. In addition, we analyzed the BCL-6 genes of tumor and nontumor cells from 2 of the cases. The germ line sequence of this region was defined, which differs in 7 positions from that previously reported. In addition, 1 polymorphic variation at position 397(G or C) was identified. Deletions, insertions, and repeated substitution mutations were detected among the molecular isolates in 8 tumor specimens, with a mutational incidence ranging from 1.3 × 10−3 to 1.3 × 10−2/bp (base pair). A total of 20 distinct substitution mutations, 1 insertion and 3 deletions were observed. One of these deletion mutations and 2 of the substitutions were observed in more than 1 tumor specimen from different individuals. In 3 tumor samples, identical mutations affecting both alleles were observed. These findings suggest the presence of mutational hot spots and hot specific events, a finding supported by our compilation of previously published data. In 6 samples, the nucleotide sequences showed evidence of intraclonal heterogeneity, consistent with a stepwise ongoing mutational process affecting the BCL-6 gene in the tumor cells. These mutations accumulating in the regulatory region of the BCL-6 gene could play a role in lymphoma progression and in the transformation of follicular lymphomas to more aggressive large cell lymphomas.
Three differentially expressed survivin cDNA variants encode proteins with distinct antiapoptotic functions
Authors: Conway Edward M. , Pollefeyt Saskia , Cornelissen Jan , DeBaere Inky , Steiner-Mosonyi Marta , Ong Kelly , Baens Mathijs , Collen Désiré , Schuh Andre C.
Published: October 2019

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is believed to play a role in oncogenesis. To elucidate further its physiologic role(s), we have characterized the murinesurvivin gene and complementary DNA (cDNA). The structural organization of the survivin gene, located on chromosome 11E2, is similar to that of its human counterpart, both containing 4 exons. Surprisingly, 3 full-length murine survivin cDNA clones were isolated, predicting the existence of 3 distinct survivin proteins. The longest open reading frame, derived from all 4 exons, predicts a 140-amino acid residue protein, survivin140, similar to human survivin, which contains a single IAP repeat and a COOH-terminal coiled-coil domain that links its function to the cell cycle. A second cDNA, which retains intron 3, predicts the existence of a 121-amino acid protein, survivin121 that lacks the coiled-coil domain. Removal of exon 2-derived sequences by alternative pre-messenger RNA (mRNA) splicing results in a third 40-amino acid residue protein, survivin40, lacking the IAP repeat and coiled-coil structure. Predictably, only recombinant survivin140 and survivin121 inhibited caspase-3 activity. All 3 mRNA species were variably expressed during development from 7.5 days postcoitum. Of the adult tissues surveyed, thymus and testis accumulated high levels of survivin140 mRNA, whereas survivin121-specific transcripts were detected in all tissues, while those representing survivin40 were absent. Human counterparts to the 3 survivin mRNA transcripts were identified in a study of human cells and tissues. The presence of distinct isoforms of survivin that are expressed differentially suggests that survivin plays a complex role in regulating apoptosis.
Hematopoietic-specific expression of MEFV, the gene mutated in familial Mediterranean fever, and subcellular localization of its corresponding protein, pyrin
Authors: Tidow Nicola , Chen Xiaoguang , Müller Carsten , Kawano Seiji , Gombart Adrian F. , Fischel-Ghodsian Nathan , Koeffler H. Phillip
Published: October 2019

Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by recurrent, self-limited attacks of fever and serositis and by infiltration of affected tissues by large numbers of neutrophils. A candidate gene for FMF was identified by positional cloning and named “MEFV.” The corresponding protein was named “pyrin.” To elucidate the currently unknown function of pyrin, we characterized its tissue distribution, regulation of expression during hematopoietic differentiation, and subcellular localization. Reverse transcription-polymerase chain reaction analysis, followed by hybridization with an internal oligonucleotide, demonstrated expression of MEFV in different populations of peripheral blood cells. Among hematopoietic cell lines, MEFVwas almost exclusively expressed in cells of the myeloid lineage. Furthermore, MEFV messenger RNA was strongly expressed within 24 hours of dimethyl sulfoxide–induced granulocytic differentiation of HL-60 cells. Analysis of complementary DNA from human solid tumor–derived cell lines revealed expression of MEFV in several cell lines derived from colon and prostate cancers. Expression of MEFV fused to enhanced green fluorescent protein showed that pyrin localized in distinct patches in the cytoplasm, forming a perinuclear cap. Taken together, MEFV is predominantly expressed in myeloid cells and upregulated during myeloid differentiation, and the corresponding protein, pyrin, is expressed in the cytoplasm.
Tropomyosin isoform 5b is expressed in human erythrocytes: implications of tropomodulin-TM5 or tropomodulin-TM5b complexes in the protofilament and hexagonal organization of membrane skeletons
Authors: Sung Lanping Amy , Gao Ke-Ming , Yee Leland J. , Temm-Grove Constance J. , Helfman David M. , Lin Jim J.-C. , Mehrpouryan Majid
Published: October 2019

The human erythrocyte membrane skeleton consists of hexagonal lattices with junctional complexes containing F-actin protofilaments of approximately 33-37 nm in length. We hypothesize that complexes formed by tropomodulin, a globular capping protein at the pointed end of actin filaments, and tropomyosin (TM), a rod-like molecule of approximately 33-35 nm, may contribute to the formation of protofilaments. We have previously cloned the human tropomodulin complementary DNA and identified human TM isoform 5 (hTM5), a product of theγ-TM gene, as one of the major TM isoforms in erythrocytes. We now identify TM5b, a product of the -TM gene, to be the second major TM isoform. TM5a, the alternatively spliced isoform of the-TM gene, which differs by 1 exon and has a weaker actin-binding affinity, however, is not present. TM4, encoded by the δ-TM gene, is not present either. In sodium dodecyl sulfate–polyacrylamide gel electrophoresis, hTM5 comigrated with the slower TM major species in erythrocyte membranes, and hTM5b comigrated with the faster TM major species. TM5b, like TM5, binds strongly to tropomodulin, more so than other TM isoforms. The 2 major TM isoforms, therefore, share several common features: They have 248 residues, are approximately 33-35 nm long, and have high affinities toward F-actin and tropomodulin. These common features may be the key to the mechanism by which protofilaments are formed. Tropomodulin-TM5 or tropomodulin-TM5b complexes may stabilize F-actin in segments of approximately 33-37 nm during erythroid terminal differentiation and may, therefore, function as a molecular ruler. TM5 and TM5b further define the hexagonal geometry of the skeletal network and allow actin-regulatory functions of TMs to be modulated by tropomodulin.
CD20 monoclonal antibody (rituximab) for therapy of Epstein-Barr virus lymphoma after hemopoietic stem-cell transplantation
Authors: Kuehnle Ingrid , Huls M. Helen , Liu Zhensheng , Semmelmann Micah , Krance Robert A. , Brenner Malcolm K. , Rooney Cliona M. , Heslop Helen E.
Published: October 2019

After bone marrow transplantation (BMT) using T-cell–depleted marrow from an unrelated donor or HLA-mismatched related donor, the risk of developing lymphoproliferative disease associated with the Epstein-Barr virus (EBV) ranges from 1% to 25%. We have shown that administration of donor-derived EBV-specific cytotoxic T lymphocytes (CTL) is effective prophylaxis and treatment for this complication, and we routinely generate CTL for high-risk patients. However, EBV lymphoma can occur in recipients of matched-sibling transplants for whom CTL are unavailable or in patients for whom CTL administration is contraindicated. We report on 3 such patients, who were successfully and safely treated with rituximab, a CD20 monoclonal antibody. The patients remain disease free 7, 8, and 9 months, respectively, after therapy. We conclude that CD20 antibody may be a useful alternative treatment strategy in patients with EBV lymphoma after BMT.
Mutations of the E2F4 gene in hematological malignancies having microsatellite instability
Authors: Komatsu Naoki , Takeuchi Seisho , Ikezoe Takayuki , Tasaka Taizo , Hatta Yoshihiro , Machida Hisanori , Williamson Ian K. , Bartram Claus R. , Koeffler H. Phillip , Taguchi Hirokuni
Published: October 2019

Mutations of coding repeats within the E2F4, TGF-βRII, BAX, IGFIIR, and hMSH3 are critical targets of microsatellite instability (MSI) in many kinds of cancers. We analyzed 9 childhood acute lymphoblastic leukemia (ALL) samples, 5 acute myelocytic leukemia (AML) samples, and 10 adult T-cell leukemia (ATL) samples having MSI to determine whether they had mutations of the E2F4, TGF-βRII, BAX, IGFIIR, and hMSH3 genes. Frameshift mutations were found at trinucleotide repeats within a coding exon of the E2F4 gene in 2 of 10 (20%) ATL samples and 1 of 9 (11%) childhood ALL samples. No mutations were found in the TGF-βRII, BAX, IGFIIR, andhMSH3 genes. E2F4 is a transcription factor that influences the cell-cycle progression. These results suggest that mutations of the E2F4 gene, presumably caused by an abnormality of one of the DNA repair genes, may play an important role in development of ATL and childhood ALL.
Leukemia initiated by PMLRARα: the PML domain plays a critical role while retinoic acid–mediated transactivation is dispensable
Authors: Kogan Scott C. , Hong Suk-hyun , Shultz David B. , Privalsky Martin L. , Bishop J. Michael
Published: October 2019

The most common chromosomal translocation in acute promyelocytic leukemia (APL), t15;17(q22;q21), creates PMLRAR andRARPML fusion genes. We previously developed a mouse model of APL by expressing PMLRAR in murine myeloid cells. In order to examine the mechanisms by which PMLRAR can initiate leukemia, we have now generated transgenic mice expressingPMLRARm4 and RARm4, proteins that are unable to activate transcription in response to retinoic acid.PMLRARm4 transgenic mice developed myeloid leukemia, demonstrating that transcriptional activation by PMLRAR is not required for leukemic transformation. The characteristics of the leukemias arising in the PMLRARm4 transgenic mice varied from those previously observed in our PMLRAR transgenic mice, indicating that ligand responsiveness may influence the phenotype of the leukemic cells. The leukemias that arose in PMLRARm4transgenic mice did not differentiate in response to retinoic acid therapy. This result supports the hypothesis that a major therapeutic effect of retinoic acid is mediated directly through thePMLRAR protein. However, a variable effect on survival suggested that this agent may be of some benefit in APL even when leukemic cells are resistant to its differentiative effects. Transgenic mice expressing high levels of RARm4 have not developed leukemia, providing evidence that the PML domain ofPMLRAR plays a specific and critical role in the pathogenesis of APL.