bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of drug sensitivity by ribosomal protein S3a
Authors: Hu Z.-B. , Minden M. D. , McCulloch E. A.
Published: October 2019

When bcl-2 is immunoprecipitated from 32P-labeled cell extracts of all-trans retinoic acid (ATRA)-treated acute myeloblastic leukemia (AML) blasts, a phosphorylated protein of approximately 30 kd is coprecipitated. This protein has been identified as ribosomal protein S3a. The biologic effects of S3a include favoring apoptosis and enhancing the malignant phenotype. We sought to determine whether S3a, like bcl-2, influenced the response of cells to chemotherapeutic drugs and ATRA. Cell lines were studied in which S3a was genetically increased or disrupted; increased S3a was regularly associated with increased plating efficiency and increased sensitivity to either cytosine arabinoside (ara-C) or doxorubicin (DNR). S3a did not affect the sensitivity of cells to paclitaxel. Pulse exposures to either 3HTdR or ara-C showed a greater percentage of clonogenic cells in the S phase of the cell cycle in cells with increased S3a than in controls. Cells with increased S3a responded to ATRA by increased ara-C or DNR sensitivity, whereas cells with reduced S3a protein were either protected by ATRA or not affected. We studied cryopreserved blast cells from patients with AML or chronic myelomonocytic leukemia (CMML). S3a protein levels were heterogeneous in these populations. In 32 cryopreserved blast populations, S3a levels were significantly correlated with both bcl-2 and with cell growth in culture. As in cell lines, high S3a in cryopreserved blasts was associated with ATRA-induced sensitization to ara-C. No significant association was seen between S3a levels and response to treatment.
Novel SH3 protein encoded by the AF3p21 gene is fused to the mixed lineage leukemia protein in a therapy-related leukemia with t(3;11) (p21;q23)
Authors: Sano Kimihiko , Hayakawa Akira , Piao Jin-Hua , Kosaka Yoshiyuki , Nakamura Hajime
Published: October 2019

The mixed lineage leukemia (MLL) gene located at chromosome band 11q23 is frequently rearranged in patients with therapy-related acute monocytic leukemia who received topoisomerase II inhibitors. We have identified a novel fusion partner of MLL(FAB M5b) in a patient who developed t-AML 9 years after treatment for acute lymphoblastic leukemia (ALL). The leukemic cells had a sole karyotypic abnormality of t(3;11) (p21;q23). Screening of a genomic DNA library, prepared from leukemic cell DNA, identified rearranged clones composed of MLL and a novel gene on chromosome 3p21 (AF3p21). The AF3p21 gene encodes a protein of 722 amino acids, which contains an Src homology 3 (SH3) domain, a proline-rich domain, and a bipartite nuclear localizing signal (NLS). RNA analysis demonstrated that exon 6 of the MLLgene fused to exon 2 of the AF3p21 gene. The resulting chimeric protein consists of AT-hooks, methyltransferase, and transcription repressor domains of MLL in addition to the AF3p21 proline-rich domain and NLS but not the AF3p21 SH3 domain.
Phosphatidylinositide 3-kinase localizes to cytoplasmic lipid bodies in human polymorphonuclear leukocytes and other myeloid-derived cells
Authors: Yu Wengui , Cassara Jessica , Weller Peter F.
Published: October 2019

Phosphatidylinositide 3-kinase (PI3K) is a key enzyme implicated in intracellular signaling of diverse cellular responses including receptor-mediated responses and neutrophil activation. Several PI3K subunits have been cloned and shown to be localized to plasma membrane receptors, the cytosol, or intracellular vesicles or caveolae. We report the localization of PI3K to a distinct intracellular site, cytoplasmic lipid bodies, in leukocytes. In U937 monocyte cells, PI3K p85 regulatory and p110β catalytic subunits were localized to lipid bodies by immunocytochemistry and/or immunoblotting and enzyme assays of subcellular fractions. In RAW murine macrophages, p55, p85, and p85β PI3K subunits were present at isolated lipid bodies. PI3K p85 was also shown to colocalize and, by co-immunoprecipitation, to be physically associated with phosphorylated Lyn kinase in lipid bodies induced to form in human polymorphonuclear leukocytes. These findings, therefore, indicate a novel site for PI3K compartmentalization and suggest that PI3K-mediated signaling is active within cytoplasmic lipid bodies in leukocytes.
Cloning of the cellular receptor for feline leukemia virus subgroup C (FeLV-C), a retrovirus that induces red cell aplasia
Authors: Quigley John G. , Burns Cara C. , Anderson Maria M. , Lynch Eric D. , Sabo Kathleen M. , Overbaugh Julie , Abkowitz Janis L.
Published: October 2019

Feline leukemia virus-C (FeLV-C) causes red cell aplasia in cats, likely through its interaction with its cell surface receptor. We identified this receptor by the functional screening of a library of complementary DNAs (cDNA) from feline T cells. The library, which was cloned into a retroviral vector, was introduced into FeLV-C–resistant murine (NIH 3T3) cells. The gene conferring susceptibility to FeLV-C was isolated and reintroduced into the same cell type, as well as into FeLV-C–resistant rat (NRK 52E) cells, to verify its role in viral infection. The receptor cDNA is predicted to encode a protein of 560 amino acids with 12 membrane-spanning domains, termed FLVCR. FLVCR has significant amino acid sequence homology with members of the major facilitator superfamily and especially D-glucarate transporters described in bacteria and in C. elegans. As FeLV-C impairs the in vivo differentiation of burst-forming unit–erythroid to colony-forming unit–erythroid, we hypothesize that this transporter system could have an essential role in early erythropoiesis. In further studies, a 6-kb fragment of the human FLVCR gene was amplified by polymerase chain reaction from genomic DNA, using homologous cDNA sequences identified in the human Expressed Sequence Tags database. By radiation hybrid mapping, the human gene was localized to a 0.5-centiMorgan region on the long arm of chromosome 1 at q31.3.
Stromal cell-derived factor-1 (SDF-1) acts together with thrombopoietin to enhance the development of megakaryocytic progenitor cells (CFU-MK)
Authors: Hodohara Keiko , Fujii Nobutaka , Yamamoto Naoki , Kaushansky Kenneth
Published: October 2019

Stromal cell-derived factor-1 (SDF-1) is a CXC chemokine that acts as a stimulator of pre-B lymphocyte cell growth and as a chemoattractant for T cells, monocytes, and hematopoietic stem cells. More recent studies also suggest that megakaryocytes migrate in response to SDF-1. Because genetic elimination of SDF-1 or its receptor lead to marrow aplasia, we investigated the effect of SDF-1 on megakaryocyte progenitors (colony-forming units-megakaryocyte [CFU-MK]). We report that SDF-1 augments the growth of CFU-MK from whole murine bone marrow cells when combined with thrombopoietin (TPO). The addition of SDF-1 to interleukin-3 (IL-3) or stem cell factor (SCF) had no effect. Specific antagonists for CXCR4 (the sole receptor for SDF-1), T22, and 1-9 (P2G) SDF-1 reduced megakaryocyte colony growth induced by TPO alone, suggesting that many culture systems contain endogenous levels of the chemokine that contributes to the TPO effect. To examine whether SDF-1 has direct effects on CFU-MK, we developed a new protocol to purify megakaryocyte progenitors. CFU-MK were highly enriched in CD41high c-kithigh cells generated from lineage-depleted TPO-primed marrow cells. Because the growth-promoting effects of SDF-1 were also observed when highly purified populations of CFU-MK were tested in serum-free cultures, these results suggest that SDF-1 directly promotes the proliferation of megakaryocytic progenitors in the presence of TPO, and in this way contributes to the favorable effects of the bone marrow microenvironment on megakaryocyte development.
Sustained high-level expression of full-length human factor VIII and restoration of clotting activity in hemophilic mice using a minimal adenovirus vector
Authors: Balagué Cristina , Zhou Jiemin , Dai Yifan , Alemany Ramón , Josephs Steven F. , Andreason Grai , Hariharan Mangala , Sethi Erica , Prokopenko Elena , Jan Hsing-yi , Lou Yan-Chun , Hubert-Leslie Debbie , Ruiz Lulio , Zhang Wei-Wei
Published: October 2019

The successful prophylactic treatment of hemophilia A by frequent infusions of plasma concentrates or recombinant factor VIII (hFVIII) indicates that gene therapy may be a potential alternative for the treatment of the disease. For efficient delivery and long-term expression of the hFVIII gene, a novel minimal adenovirus (mini-Ad) vector, MiniAdFVIII, has been developed. The vector is devoid of all viral genes and carries the full-length hFVIII cDNA under the control of the human 12.5-kb albumin promoter. The MiniAdFVIII vector was propagated with the assistance of an ancillary vector in 293 cells and was purified by CsCl banding. Sustained expression of hFVIII at physiologic levels (100-800 ng/mL) was achieved in mice after a single intravenous injection of MiniAdFVIII. The expressed hFVIII had a structure identical to that of recombinant hFVIII, as determined by Western blot analysis. The functionality of the protein was confirmed by the restoration of blood coagulation capacity in MiniAdFVIII-treated hemophilic mice, as determined by tail clipping observations. Although antivector or antihuman FVIII antibodies at various levels were detected, long-term expression of the transgene was observed in the mice that did not generate antibodies against the transgene product. The vector DNA persisted in the liver tissues of the mice with long-term expression. No significant histopathologic findings or toxicities were observed to be associated with the vector in the MiniAdFVIII-treated C57BL/6 mice. These results support the further development of MiniAdFVIII for clinical trials toward the treatment of hemophilia A.
Characterization of hematopoietic lineage-specific gene expression by ES cell in vitro differentiation induction system
Authors: Era Takumi , Takagi Toshiaki , Takahashi Tomomi , Bories Jean-Christophe , Nakano Toru
Published: October 2019

The continuous generation of mature blood cells from hematopoietic progenitor cells requires a highly complex series of molecular events. To examine lineage-specific gene expression during the differentiation process, we developed a novel method combiningLacZ reporter gene analysis with in vitro hematopoietic differentiation induction from mouse embryonic stem cells. For a model system using this method, we chose the erythroid and megakaryocytic differentiation pathways. Although erythroid and megakaryocytic cells possess distinct functional and morphologic features, these 2 lineages originate from bipotential erythro-megakaryocytic progenitors and share common lineage-restricted transcription factors. A portion of the 5′ flanking region of the human glycoprotein IIb (IIb) integrin gene extending from base −598 to base +33 was examined in detail. As reported previously, this region is sufficient for megakaryocyte-specific gene expression. However, previous reports that used human erythro-megakaryocytic cell lines suggested that one or more negative regulatory regions were necessary for megakaryocyte-specific gene expression. Our data clearly showed that an approximately 200-base enhancer region extending from −598 to −400 was sufficient for megakaryocyte-specific gene expression. This experimental system has advantages over those using erythro-megakaryocytic cell lines because it recapitulates normal hematopoietic cell development and differentiation. Furthermore, this system is more efficient than transgenic analysis and can easily examine gene expression with null mutations of specific genes.
Sequential binding of CD11a/CD18 and CD11b/CD18 defines neutrophil capture and stable adhesion to intercellular adhesion molecule–1
Authors: Hentzen Eric R. , Neelamegham Sriram , Kansas Geoffrey S. , Benanti Jennifer A. , McIntire Larry V. , Smith C. Wayne , Simon Scott I.
Published: October 2019

The relative contributions of CD11a/CD18 and CD11b/CD18 to the dynamics and strength of neutrophil adhesion to intercellular adhesion molecule (ICAM)-1–transfected cells were examined over the time course of chemotactic stimulation. Suspensions of neutrophils and transfectants were sheared in a cone-plate viscometer, and formation of heterotypic aggregates was measured by 2-color flow cytometry. The 2-body collision theory was used to compute adhesion efficiency, defined as the proportion of collisions between neutrophils and target cells that resulted in capture. ICAM-1 surface density and shear rate both regulated adhesion efficiency. Target cells expressing approximately 1000 ICAM-1 sites/μm2 (Ilow) were captured with an efficiency of 0.15 at 100 s−1, which decreased to zero at 300 s−1. At 8-fold higher ICAM-1 expression (Ihigh) corresponding to levels measured on interleukin-1–stimulated endothelium, efficiency was 0.3 at 100 s−1 and remained above background to 900 s−1. Shear alone was sufficient for CD11a/CD18-mediated adhesion to ICAM-1, and stimulation with formyl-methionyl-leucyl-phenylalanine boosted capture efficiency through CD11a/CD18 by 4-fold. In comparison, CD11b/CD18 supported one third of this efficiency, but was necessary for aggregate stability over several minutes of shear and at shear stresses exceeding 5 dyne/cm2. Hydrodynamics influenced capture efficiency predominantly through the collisional contact duration, predicted to be approximately 9 milliseconds for successful capture of Ilow and 4 milliseconds for Ihigh. The implication is that an increase in ICAM-1 from resting levels to those on inflamed endothelium effectively increases the permissible shear in which capture through β2-integrins may occur. Neutrophil adhesion to ICAM-1 appears to be a cooperative and sequential process of CD11a-dependent capture followed by CD11b-mediated stabilization.
Immunostimulatory CpG-oligonucleotides cause proliferation, cytokine production, and an immunogenic phenotype in chronic lymphocytic leukemia B cells
Authors: Decker Thomas , Schneller Folker , Sparwasser Tim , Tretter Theresa , Lipford Grayson B. , Wagner Hermann , Peschel Christian
Published: October 2019

Bacterial DNA and synthetic CpG-oligodeoxynucleotides (ODNs) derived thereof have attracted attention because they activate cells of the immune system in a sequence-dependent manner. Here we investigated the potential of CpG-ODNs to cause proliferation, cytokine production, and regulation of surface molecules in human B-chronic lymphocytic leukemia (CLL) cells. CpG-ODN induced proliferation in both B-CLL cells and normal B cells; however, only B-CLL cells increased proliferative responses when CpG-ODN was added to co-cultures of CD40-ligand transfected mouse fibroblasts (CD40LF) and B cells. Production of interleukin-6 and tumor necrosis factor  was detectable at borderline levels, using CpG-ODN as the only stimulus. In contrast, when CpG-ODN was added to co-cultures of B cells and CD40LF, a strong increase in cytokine production occurred in B-CLL cells as well as in normal B cells. The surface molecules CD40, CD58, CD80, CD86, CD54, and MHC class I molecules were up-regulated in B-CLL cells, whereas CD95 expression was not influenced by CpG-ODN stimulation. The same pattern of surface molecule regulation was observed in normal B cells, but up-regulation of CD40 was significantly stronger in B-CLL cells. Costimulation with CpG-ODN and CD40LF resulted in further up-regulation of CD58, CD80, CD86, and MHC class I molecules. In contrast, CD95 expression induced by CD40-ligation was inhibited by CpG-ODN. CpG-ODN activated B-CLL cells acquired a strong stimulatory capacity toward T cells in allogeneic mixed lymphocyte reaction. This effect was completely inhibited by a combination of anti-CD80 and anti-CD86 monoclonal antibody. Taken together, these findings suggest the possible use of CpG-ODN for immunotherapeutic strategies in patients with B-CLL.
Recombinant human antithrombin III improves survival and attenuates inflammatory responses in baboons lethally challenged with Escherichia coli
Authors: Minnema M. C. , Chang A. C. K. , Jansen P. M. , Lubbers Y. T. P. , Pratt B. M. , Whittaker B. G. , Taylor F. B. , Hack C. E. , Friedman B.
Published: October 2019

Plasma-derived antithrombin III (ATIII) prevents the lethal effects of Escherichia coli infusion in baboons, but the mechanisms behind this effect are not clear. In the present study, we evaluated the effects of recombinant human ATIII (rhATIII) on the clinical course and the inflammatory cytokine and coagulation responses in baboons challenged with lethal dose of E coli. Animals in the treatment group (n = 5) received high doses of rhATIII starting 1 hour before an E colichallenge. Those in the control group were administered saline. Survival was significantly improved in the treatment group (P = .002). Both groups had similar hemodynamic responses to E coli challenge but different coagulation and inflammatory responses. The rhATIII group had an accelerated increase of thrombin-ATIII complexes and significantly less fibrinogen consumption compared to controls. In addition, the rhATIII group had much less severe thrombotic pathology on autopsy and virtually no fibrinolytic response to E coli challenge. Furthermore, the rhATIII group had a significantly attenuated inflammatory response as evidenced by marked reduction of the release of various cytokines. We conclude that the early administration of high doses of rhATIII improves the outcome in baboons lethally challenged with E coli, probably due to the combined anticoagulation and anti-inflammatory effects of this therapy.
Polymorphisms within glutathione S-transferase genes (GSTM1, GSTT1, GSTP1) and risk of relapse in childhood B-cell precursor acute lymphoblastic leukemia: a case-control study
Authors: Stanulla Martin , Schrappe Martin , Brechlin Annette Müller , Zimmermann Martin , Welte Karl
Published: October 2019

Glutathione S-transferases (GSTs) have been associated with outcome in human cancers treated with cytotoxic chemotherapy. In a case-control study, we investigated the association between polymorphisms within theGSTM1, GSTT1, and GSTP1 genes and risk of relapse in childhood acute lymphoblastic leukemia (ALL). Cases were relapsed patients. Controls were successfully treated patients with a minimum follow-up of 5 years. The null genotype (absence of both alleles) for GSTM1 or GSTT1 conferred a 2-fold (OR = 0.5, 95% CI = 0.23-1.07, P = .078) and 2.8-fold (OR = 0.36, 95% CI = 0.13-0.99, P = .048) reduction in risk of relapse, respectively, relative to the presence of the GSTM1 or GSTT1 gene. The GSTP1Val105/Val105 genotype showed a 3-fold decrease in risk of relapse (OR = 0.33, 95% CI = 0.09-1.23,P = .099) in comparison to the combined category of Ile105/Val105 and Ile105/Ile105 genotypes. No particular associations with relapse were observed for the GSTP1polymorphism at codon 114. The risk of relapse when having 1 of the low-risk genotypes (GSTM1 null, GSTT1 null,GSTP1 Val105/Val105) decreased 1.9-fold (OR = 0.53, 95% CI = 0.24-1.19, P = .123), and the risk when having 2 or 3 low-risk genotypes 3.5-fold (OR = 0.29, 95% CI = 0.06-1.37, P = .118), compared with individuals having no low-risk genotype (P for trend = .005). Our results suggest that polymorphisms within genes of the GST superfamily may be associated with risk of relapse in childhood ALL.
An oral CD40 ligand gene therapy against lymphoma using attenuated Salmonella typhimurium
Authors: Urashima Mitsuyoshi , Suzuki Hideaki , Yuza Youki , Akiyama Masaharu , Ohno Noriko , Eto Yoshikatsu
Published: October 2019

CD40 ligand (CD40L) has a great potential as a novel treatment for B-cell lymphoma (BCL). It has previously been demonstrated that a nonvirulent strain of Salmonella typhimurium mutant (ST) can be used not only as a vehicle in oral genetic immunization via the intestinal mucosa, but also as an enhancer of interferon γ– and tumor necrosis factor –mediated immunity. After confirming that human CD40L can up-regulate expression of Fas, B7-1, and B7-2 molecules on murine BCL cells in vitro, we transfected the human CD40L gene intoS typhimurium mutant (ST40L), which was administrated orally to determine whether it was able to prevent the growth of BCL in mice. Expression of human CD40L was confirmed immunohistochemically with protein being detected in the Peyer's patches of mice immunized with ST40L. Moreover, human soluble CD40L had been detectable until 7 to 8 weeks after oral administration of ST40L. Although ST alone exhibited some protective effects, ST40L demonstrated a significantly greater protection against the development of CD40 positive BCL compared with the control. In the surviving mice that had been treated with ST40L, a small and hard nodule was formed at the injection site, which was found to be composed of infiltrating lymphocytes expressing Fas ligand. These results have the potential to be a simple, effective, and above all, safe immune-gene therapy against BCL.
Prevention and treatment of factor VIII inhibitors in murine hemophilia A
Authors: Qian Jiahua , Collins Mary , Sharpe Arlene H. , Hoyer Leon W.
Published: October 2019

Inhibitory antibody formation is a major complication of factor VIII replacement therapy in patients with hemophilia A. To better understand the pathogenesis of this immunologic reaction, we evaluated the role of T-cell costimulatory signals for antifactor VIII antibody formation in a murine model of hemophilia A. Repeated intravenous injections of factor VIII in these factor VIII–deficient mice induced an antifactor VIII inhibitor antibody response. This response was shown to be T-cell dependent by its absence in hemophilic mice also deficient for the T-cell costimulatory ligand B7-2. In separate experiments, injection of murine CTLA4-Ig completely blocked the primary response to factor VIII in hemophilic mice with intact B7 function. This reagent also prevented or diminished further increases in antifactor VIII when given to hemophilic mice with low antifactor VIII antibody titers. These studies suggest that strategies targeting the B7-CD28 pathway are potential therapies to prevent and treat inhibitory antifactor VIII antibodies. Moreover, because the development of antibodies to replaced proteins may limit the success of many human gene therapy approaches, our results may be broadly applicable.
Gene expressions of lipopolysaccharide receptors, toll-like receptors 2 and 4, are differently regulated in mouse T lymphocytes
Authors: Matsuguchi Tetsuya , Takagi Kensuke , Musikacharoen Tipayaratn , Yoshikai Yasunobu
Published: October 2019

Toll-like receptors (TLRs) are a family of mammalian proteins homologous to Drosophila Toll. Human TLR2 was shown to mediate the responsiveness to lipopolysaccharide (LPS). On the other hand, gene mutations of mouse TLR4 (mTLR4) in LPS-hyporesponsive strains have suggested that mTLR4 is essential for LPS-signaling in mice, but the role of mTLR2 has not been explored. This report describes molecular cloning of the mTLR2 cDNA. Overexpression of mTLR2 and mouse CD14 conferred LPS-inducibility of c-Jun N-terminal kinase phosphorylation and nuclear factor-κB activation to COS7 cells, suggesting that mTLR2 is a signaling receptor for LPS. BothmTLR2 and mTLR4 genes were expressed in T cells. Treatment with anti-CD3ɛ, PMA plus ionomycin, or interleukin-2 (IL-2)/IL-15 increased mTLR2 but not mTLR4 messenger RNA (mRNA) in some T cell lines. Specific inhibitors of mitogen-activated extracellular signal-regulated kinase and fusion protein 38 (p38) kinase inhibited mTLR2 mRNA up-regulation by PMA plus ionomycin. This suggests that extracellular signal-regulated kinase and p38 kinase pathways were involved. Additionally, LPS treatment of EL-4 cell line decreasedIL-4 gene expression. Our results indicate that both mTLR2 and mTLR4 are involved in LPS signaling, but their expressions are regulated differently in T cells, and that LPS may directly affect T-cell functions by binding to TLRs.
Red cell surface changes and erythrophagocytosis in children with severe Plasmodium falciparum anemia
Authors: Waitumbi John N. , Opollo Malachi O. , Muga Richard O. , Misore Ambrose O. , Stoute José A.
Published: October 2019

Severe anemia is one of the most lethal complications in children infected with Plasmodium falciparum. The pathogenesis of this anemia is not completely understood. Experimental data from malaria-infected humans and animal models suggest that uninfected red cells have a shortened life span. This study looked for changes in the red cell surfaces of children with severe malarial anemia that could explain this accelerated destruction. A prospective case-control study was conducted of children with severe P falciparum anemia (hemoglobin of 5 g/dL or lower) admitted to a large general hospital in western Kenya. Children with severe anemia were compared with children who had symptoms of uncomplicated malaria and with asymptomatic children. Cytofluorometry was used to quantify in vitro erythrophagocytosis and to measure red cell surface immunoglobulin G (IgG) and the complement regulatory proteins CR1, CD55, and CD59. Red cells from patients with severe anemia were more susceptible to phagocytosis and also showed increased surface IgG and deficiencies in CR1 and CD55 compared with controls. Red cell surface CD59 was elevated in cases of severe anemia compared with asymptomatic controls but not as compared with symptomatic controls. The surface of red cells of children with severe P falciparum anemia is modified by the deposition of IgG and alterations in the levels of complement regulatory proteins. These changes could contribute to the accelerated destruction of red cells in these patients by mechanisms such as phagocytosis or complement-mediated lysis.
The endothelial cell protein C receptor aids in host defense against Escherichia coli sepsis
Authors: Taylor F. B. , Stearns-Kurosawa D. J. , Kurosawa S. , Ferrell G. , Chang A. C. K. , Laszik Z. , Kosanke S. , Peer G. , Esmon C. T.
Published: October 2019

The influence of the endothelial protein C receptor (EPCR) on the host response to Escherichia coli was studied. Animals were treated with 4 separate protocols for survival studies and analysis of physiologic and biochemical parameters: (1) monoclonal antibody (mAb) that blocks protein C/activated protein C binding to EPCR plus sublethal numbers of E coli (SLEC) (n = 4); (2) mAb to EPCR that does not block binding plus SLEC (n = 3); (3) SLEC alone (n = 4); and (4) blocking mAB alone (n = 1). Those animals receiving blocking mAb to EPCR plus sublethal E coli died 7 to 54 hours after challenge, whereas all animals treated with the other protocols were permanent survivors. Histopathologic studies of tissues from animals receiving blocking mAb plus SLEC removed at postmortem were compared with those animals receiving SLEC alone killed at T+24 hours. The animals receiving the blocking mAb exhibited consumption of fibrinogen, microvascular thrombosis with hemorrhage of both the adrenal and renal cortex, and an intense influx of neutrophils into the adrenal, renal, and hepatic microvasculature, whereas the tissues from animals receiving only sublethal E coli exhibited none of these abnormal histopathologic changes. Compared with the control animals, the animals receiving the blocking mAb exhibited significantly elevated serum glutamic pyruvic transaminase, anion gap, thrombin-antithrombin complex, IL-6, IL-8, and soluble thrombomodulin. The levels of circulating activated protein C varied too widely to allow a clear determination of whether the extent of protein C activation was altered in vivo by blocking protein C binding to EPCR. We conclude that protein C/activated protein C binding to EPCR contributes to the negative regulation of the coagulopathic and inflammatory response to E coli and that EPCR provides an additional critical step in the host defense against E coli.
Initiation of antiretroviral therapy during primary HIV-1 infection induces rapid stabilization of the T-cell receptor β chain repertoire and reduces the level of T-cell oligoclonality
Authors: Soudeyns Hugo , Campi Gabriele , Rizzardi G. Paolo , Lenge Caterina , Demarest James F. , Tambussi Giuseppe , Lazzarin Adriano , Kaufmann Daniel , Casorati Giulia , Corey Lawrence , Pantaleo Giuseppe
Published: October 2019

Major T-cell receptor β chain variable region (TCRBV) repertoire perturbations are temporally associated with the down-regulation of viremia during primary human immunodeficiency virus (HIV) infection and with oligoclonal expansion and clonal exhaustion of HIV-specific cytotoxic T lymphocytes (CTLs). To determine whether initiation of antiretroviral therapy (ART) or highly active antiretroviral therapy (HAART) during primary infection influences the dynamics of T-cell–mediated immune responses, the TCRBV repertoire was analyzed by semiquantitative polymerase chain reaction in serial blood samples obtained from 11 untreated and 11 ART-treated patients. Repertoire variations were evaluated longitudinally. Stabilization of the TCRBV repertoire was more consistently observed in treated as compared with untreated patients. Furthermore, the extent and the rapidity of stabilization were significantly different in treated versus untreated patients. TCRBV repertoire stabilization was positively correlated with the slope of HIV viremia in the treated group, suggesting an association between repertoire stabilization and virologic response to treatment. To test whether stabilization was associated with variations in the clonal complexity of T-cell populations, T-cell receptor (TCR) heteroduplex mobility shift assays (HMAs) were performed on sequential samples from 4 HAART-treated subjects. Densitometric analysis of HMA profiles showed a reduction in the number of TCR clonotypes in most TCRBV families and a significant decrease in the total number of clonotypes following 7 months of HAART. Furthermore, a biphasic decline in HIV-specific but not heterologous CTL clones was observed. This indicates that ART leads to a global reduction of CD8+T-cell oligoclonality and significantly modulates the mobilization of HIV-specific CTL during primary infection.
Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification
Authors: le Coutre Philipp , Tassi Elena , Varella-Garcia Marileila , Barni Rossella , Mologni Luca , Cabrita Gonçalo , Marchesi Edoardo , Supino Rosanna , Gambacorti-Passerini Carlo
Published: October 2019

The 2-phenylaminopyrimidine derivative STI571 has been shown to selectively inhibit the tyrosine kinase domain of the oncogenicbcr/abl fusion protein. The activity of this inhibitor has been demonstrated so far both in vitro with bcr/abl expressing cells derived from leukemic patients, and in vivo on nude mice inoculated with bcr/abl positive cells. Yet, no information is available on whether leukemic cells can develop resistance to bcr/ablinhibition. The human bcr/abl expressing cell line LAMA84 was cultured with increasing concentrations of STI571. After approximately 6 months of culture, a new cell line was obtained and named LAMA84R. This newly selected cell line showed an IC50 for the STI571 (1.0 μM) 10-fold higher than the IC50 (0.1 μM) of the parental sensitive cell line. Treatment with STI571 was shown to increase both the early and late apoptotic fraction in LAMA84 but not in LAMA84R. The induction of apoptosis in LAMA84 was associated with the activation of caspase 3–like activity, which did not develop in the resistant LAMA84R cell line. LAMA84R cells showed increased levels of bcr/abl protein and mRNA when compared to LAMA84 cells. FISH analysis with BCR- and ABL-specific probes in LAMA84R cells revealed the presence of a marker chromosome containing approximately 13 to 14 copies of the BCR/ABL gene. Thus, overexpression of the Bcr/Abl protein mediated through gene amplification is associated with and probably determines resistance of human leukemic cells to STI571 in vitro.
MSH2-deficient murine lymphomas harbor insertion/deletion mutations in the transforming growth factor beta receptor type 2 gene and display low not high frequency microsatellite instability
Authors: Lowsky Robert , Magliocco Anthony , Ichinohasama Ryo , Reitmair Armin , Scott Stuart , Henry Michele , Kadin Marshall E. , DeCoteau John F.
Published: October 2019

High-frequency microsatellite instability (MSI), defined as more than 20% unstable loci, is an inconsistent finding in hematologic malignancies; consequently, the significance of deficient DNA mismatch repair (MMR) to their pathogenesis has been questioned. To further investigate the relationship between MMR deficiency and genomic instability in hematologic malignancies, this study evaluatedMSH2−/− murine lymphomas for insertion/deletion (ID) mutations within the transforming growth factor (TGF)-beta receptor type II (TβR-II) gene and MSI at 10 neutral microsatellites. The lymphomas displayed ID mutations within short mononucleotide runs of TβR-II at a high frequency, whereas nonmalignant tissue from corresponding animals lacked mutations. Loss ofTβR-II transcripts and protein was seen in 6 of 7 murine lymphomas harboring acquired TβR-II mutations. In the analysis of paired nonmalignant and tumor DNA samples, low-frequency but not high-frequency MSI was found. Low-frequency MSI occurred in 8 of 20 lymphomas and 12 displayed microsatellite stability. MSI was even less frequent in nonmalignant tissue as only 3 of 20 samples displayed low-frequency MSI and 17 displayed stability. Evaluation of 20 single cell clones from the MSH2−/− lymphoma cell lines R25 and L15 identified high-frequency MSI in 4 and 2 clones, respectively. The remaining clones showed low-frequency MSI or stability. These findings suggest that acquired TβR-IImutations represent important inactivating events in tumor pathogenesis following MSH2 deficiency. Furthermore, for some hematolymphoid malignancies, the evaluation of cancer-associated genes for ID mutations may represent a more sensitive marker of MMR deficiency than evaluation of neutral microsatellites for high-frequency MSI.
The synthetic chemoattractant Trp-Lys-Tyr-Met-Val-DMet activates neutrophils preferentially through the lipoxin A4 receptor
Authors: Dahlgren Claes , Christophe Thierry , Boulay Francois , Madianos Phoebus N. , Rabiet Marie J. , Karlsson Anna
Published: October 2019

A D-methionine–containing peptide, Trp-Lys-Tyr-Met-Val-D-Met-NH2 (WKYMVm), featuring a unique receptor specificity was investigated with respect to its ability to activate neutrophil effector functions. The peptide was found to be more potent than the N-formylated peptide N-formyl-Met-Leu-Phe (fMLF) at inducing neutrophil chemotaxis, mobilization of neutrophil complement receptor 3 (CR3), and activation of the neutrophil NADPH-oxidase. The fact that binding of fML[3H]F was inhibited by both fMLF and WKYMVm suggests that N-formyl peptide receptor (FPR) is shared by these peptides. However, the neutrophil response induced by the WKYMVm peptide was insensitive to the fMLF antagonists, cyclosporin H, and Boc-FLFLF that specifically block the function of the FPR. These results suggest that even though WKYMVm may bind FPR the cells are activated preferentially through a receptor distinct from the FPR. Using transfected HL-60 cells expressing either the FPR or its neutrophil homologue FPRL1, also referred to as LXA4R because it has been shown to bind lipoxin A4, we show that WKYMVm is about 300-fold more active at mobilizing intracellular calcium through FPRL1 than through FPR. The WKYMVm activates FPRL1-expressing cells in a cyclosporin H-independent manner with an EC50 of around 75 pmol/L, whereas it activates FPR-expressing cells with an EC50 of around 25 nmol/L. The observation that exudated cells are primed in their response to WKYMVm suggests that FPRL1/LXA4R like FPR is stored in mobilizable organelles.
A study of the coregulation and tissue specificity of XGand MIC2 gene expression in eukaryotic cells
Authors: Fouchet Claude , Gane Pierre , Huet Martine , Fellous Marc , Rouger Philippe , Banting George , Cartron Jean-Pierre , Lopez Claude
Published: October 2019

CD99, the product of the MIC2 gene, exhibits an erythroid-specific quantitative polymorphism coregulated with the polymorphism of the XG blood group gene. As a preliminary study of this phenomenon, human XG and CD99 recombinant proteins were expressed in murine RAG cells and analyzed by flow cytometry. Both proteins were expressed independently and at a similar level in single and double transfectants. Immunoprecipitation and Western blot analysis, using the murine monoclonal antibodies NBL-1 and 12E7, revealed species of 26 kd (XG) and 32 kd (CD99), respectively. A putative 28-kd intracellular precursor of CD99 was also detected, as was a 26-kd species after neuraminidase treatment of CD99-expressing cells. No evidence of association or complex formation between XG and CD99 proteins could be proven, either on transfected RAG cells or on human erythrocytes. These results were confirmed using somatic hybrids between single transfectants. These findings suggest that the phenotypic relationship between XG and CD99 is mostly regulated at the transcriptional level, but they do not formally exclude some posttranscriptional effect. Studies on the tissue specificity of XG expression showed that surface expression of the XG protein could not be restored in somatic hybrids between B-lymphoblastoid cell lines from Xg(a+) persons and fibroblasts (RAG) or erythroid (MEL) cells. RT-PCR analysis of the transcripts revealed the existence of an XG mRNA in each cell line, suggesting that the tissue-specific regulation of cell surface XG expression occurs either at a quantitative transcriptional level or is a posttranscriptional event. By Northern blot analysis,XG transcripts were detected in erythroid tissues and several nonerythroid tissues.
Prognostic implications of differences in telomere length between normal and malignant cells from patients with chronic myeloid leukemia measured by flow cytometry
Authors: Brümmendorf Tim H. , Holyoake Tessa L. , Rufer Nathalie , Barnett Michael J. , Schulzer Michael , Eaves Connie J. , Eaves Allen C. , Lansdorp Peter M.
Published: October 2019

Chronic myeloid leukemia (CML) is a clonal, multilineage myeloproliferative disorder characterized by the Philadelphia chromosome (Ph) and a marked expansion of myeloid cells. Previous studies have indicated that the telomere length in blood cells may indicate their replicative history. However, the large variation in telomere length between individuals complicates the use of this parameter in CML and other hematologic disorders. To circumvent this problem, we compared the telomere length in peripheral blood or bone marrow cells with purified normal (Ph−) T lymphocytes from the same CML patient using fluorescence in situ hybridization and flow cytometry. Overall telomere fluorescence was significantly reduced in Ph+ cells from patients with CML compared to blood leukocytes from normal individuals (P < 0.001) or normal (Ph−) T lymphocytes from the same individuals (n = 51, P < 0.001). Cells from patients in accelerated phase or blast phase (AP/BP) showed significantly shorter average telomere length than cells from patients in chronic phase (CP,P = 0.02) or cytogenetic remission (CR,P = 0.03). Patients in CP who subsequently developed BP within 2 years had significantly shorter telomeres than those who did not develop BP for at least 2 years (P < 0.05). Accelerated replication-dependent telomere shortening in Ph+ versus Ph− leukocytes supports previous evidence that Ph+ stem cells cycle more actively than their counterparts in normal individuals. Our data further suggest that telomere shortening may serve as a surrogate marker of disease progression in patients with CP CML, supporting a mechanistic link between CML stem cell turnover, genetic instability, and malignant evolution in this disease. (Blood. 2000;95:1883-1890)
Engagement of the α2β1 integrin inhibits Fas ligand expression and activation-induced cell death in T cells in a focal adhesion kinase-dependent manner
Authors: Aoudjit Fawzi , Vuori Kristiina
Published: October 2019

T-cell receptor (TCR)-mediated apoptosis, also known as activation-induced cell death (AICD), plays an important role in the control of immune response and in the development of T-cell repertoire. Mechanistically, AICD has been largely attributed to the interaction of Fas ligand (Fas-L) with its cell surface receptor Fas in activated T cells. Signal transduction mediated by the integrin family of cell adhesion receptors has been previously shown to modulate apoptosis in a number of different cell types; in T cells, integrin signaling is known to be important in cellular response to antigenic challenge by providing a co-stimulatory signal for TCR. In this study we demonstrate that signaling via the collagen receptor 2β1 integrin specifically inhibits AICD by inhibiting Fas-L expression in activated Jurkat T cells. Engagement of the 2β1 integrin with monoclonal antibodies or with type I collagen, a cognate ligand for 2β1, reduced anti-CD3 and PMA/ionomycin-induced cell death by 30% and 40%, respectively, and the expression of Fas-L mRNA by 50%. Further studies indicated that the 2β1-mediated inhibition of AICD and Fas-L expression required the focal adhesion kinase FAK, a known component in the integrin signaling pathways. These results suggest a role for the 2β1 integrin in the control of homeostasis of immune response and T-cell development.
A monoclonal antibody (MUM1p) detects expression of the MUM1/IRF4 protein in a subset of germinal center B cells, plasma cells, and activated T cells
Authors: Falini Brunangelo , Fizzotti Marco , Pucciarini Alessandra , Bigerna Barbara , Marafioti Teresa , Gambacorta Marcello , Pacini Roberta , Alunni Cristina , Natali–Tanci Laura , Ugolini Barbara , Sebastiani Carla , Cattoretti Giorgio , Pileri Stefano , Dalla-Favera Riccardo , Stein Harald
Published: October 2019

A new monoclonal antibody (MUM1p) was used to study the cell/tissue expression of human MUM1/IRF4 protein, the product of the homologous gene involved in the myeloma-associated t(6;14) (p25;q32). MUM1 was expressed in the nuclei and cytoplasm of plasma cells and a small percentage of germinal center (GC) B cells mainly located in the “light zone.” Its morphologic spectrum ranged from that of centrocyte to that of a plasmablast/plasma cell, and it displayed a phenotype (MUM1+/Bcl-6−/Ki67−) different from that of most GC B cells (MUM1−/Bcl-6+/Ki67+) and mantle B cells (MUM1−/Bcl-6−/Ki67−). Polymerase chain reaction (PCR) analysis of single MUM1+cells isolated from GCs showed that they contained rearranged Ig heavy chain genes with a varying number of VHsomatic mutations. These findings suggest that these cells may represent surviving centrocytes and their progeny committed to exit GC and to differentiate into plasma cells. MUM1 was strongly expressed in lymphoplasmacytoid lymphoma, multiple myeloma, and approximately 75% of diffuse large B-cell lymphomas (DLCL-B). Unlike normal GC B cells, in which the expression of MUM1 and Bcl-6 were mutually exclusive, tumor cells in approximately 50% of MUM1+ DLCL-B coexpressed MUM1 and Bcl-6, suggesting that expression of these proteins may be deregulated. In keeping with their proposed origin from GC B cells, Hodgkin and Reed–Sternberg cells of Hodgkin's disease consistently expressed MUM1. MUM1 was detected in normal and neoplastic activated T cells, and its expression usually paralleled that of CD30. These results suggest that MUM1 is involved in the late stages of B-cell differentiation and in T-cell activation and is deregulated in DLCL-B.
Deletions of chromosome 5q13.3 and 17p loci cooperate in myeloid neoplasms
Authors: Castro Patricia D. , Liang Jan C. , Nagarajan Lalitha
Published: October 2019

Nonrandom interstitial deletions and monosomy of chromosomes 5, 7, and 17 in refractory myelodysplasia (MDS) and acute myelogenous leukemia (AML) suggest a multistep pathway that culminates in aggressive clinical course. Because cytogenetic studies frequently identify chromosome 5 and 17 deletions within a single clone, we searched for allele loss for 5q loci and TP53 gene mutations in the same leukemic samples. Cosegregating deletions of chromosomes 5 and 17 were found to specifically include the 5q13.3 interval between the lociD5S672 and D5S620/D5S626, a locus hypothesized to harbor a tumor suppressor gene1 and the TP53 gene on 17p. A rare patient with secondary refractory MDS and an unbalanced translocation [der(5;17)], which resulted in deletions of the 5q13.3-qter and 17p loci, provided clues on the sequence of genetic alterations. Serial molecular analysis of this patient revealed a dysplastic clone with der(5;17), which gave rise to a leukemic clone on acquiring an inactivating mutation of TP53. Our findings are consistent with functional cooperation between a putative tumor suppressor gene at 5q13.3 that contributes toward the progression of early stages of MDS, and the TP53 gene when mutated, causes transformation to AML.
Recombination events between the p47-phoxgene and its highly homologous pseudogenes are the main cause of autosomal recessive chronic granulomatous disease
Authors: Roesler Joachim , Curnutte John T. , Rae Julie , Barrett David , Patino Pablo , Chanock Stephen J. , Goerlach Agnes
Published: October 2019

Chronic granulomatous disease (CGD) is an inherited disease caused by defects in the superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of phagocytes. Genetic lesions in any of 4 components of this antimicrobial enzyme have been detected. Family-specific mutations are found in 3 of 4 forms of CGD due to deficiencies of the gp91-phox, p22-phox, andp67-phox genes. In p47-phox–deficient CGD (autosomal recessive form A47°) patients, a GT deletion (▵GT) at the beginning of exon 2 of the p47-phox gene has been reported in 19 of 20 alleles. This GT deletion is also characteristic for the recently identified p47-phox pseudogenes. To explore a possible link between these findings, a sequence analysis of 28 unrelated, racially diverse A47° CGD patients and 37 healthy individuals was performed. The GT deletion in exon 2 was present on all alleles in 25 patients. Only 3 patients but all healthy individuals contained the GTGT and ▵GT sequences. A total of 22 patients carried additional pseudogene-specific intronic sequences on all alleles, either only in intron 1 or in intron 1 and intron 2, which lead to different types of chimeric DNA strands. It is concluded that recombination events between the p47-phox gene and its highly homologous pseudogenes result in the incorporation of ▵GT into the p47-phox gene, thereby leading to the high frequency of GT deletion in A47° CGD patients.