bluuebunny/crossref_metadata_2025 huggingface dataset:
Epstein-Barr Virus (EBV)-Specific Cytotoxic T Lymphocytes for the Treatment of Patients With EBV-Positive Relapsed Hodgkin's Disease
Authors: Roskrow Marie A. , Suzuki Nobuhiro , Gan Yan-jun , Sixbey John W. , Ng Catherine Y.C. , Kimbrough Sarah , Hudson Melissa , Brenner Malcolm K. , Heslop Helen E. , Rooney Cliona M.
Published: October 2019

Adoptive transfer of Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) is effective prophylaxis and treatment of EBV-positive immunoblastic lymphoma in immunocompromised patients. In 50% of patients with Hodgkin's disease, the tumor cells are EBV antigen-positive and may therefore also be suitable targets for treatment with virus-specific CTLs. However, Hodgkin's disease may produce several inhibitory effects on immune induction and effector function in vivo, which may preclude the generation or effector function of CTLs reactive against EBV viral proteins, including those expressed by the tumor cells. We have investigated whether EBV-specific CTLs could be generated ex vivo from 13 patients with Hodgkin's disease: nine with active relapsed disease and four who were in clinical remission after a first or subsequent relapse. CTL lines were successfully generated from nine of 13 patients (five active disease, four remission). Although these lines had an abnormal pattern of expansion comparable to EBV-specific CTLs generated from normal donors, their phenotype was normal except for reduced expression of the zeta chain of the T-cell receptor (TCR). Their cytotoxicity was also compared to EBV-specific lines generated from normal donors and included activity against LMP2a, one of the three weakly immunogenic viral antigens expressed by Hodgkin's tumor cells. To assess the activity of the CTLs in vivo, they were gene-marked and infused into three patients with multiply relapsed disease. The CTLs persisted for more than 13 weeks postinfusion and retained their potent antiviral effects in vivo, thereby enhancing the patient immune response to EBV. This approach may therefore have value in the treatment of EBV-positive Hodgkin's disease.
Cytokine-Based Tumor Cell Vaccine Is Equally Effective Against Parental and Isogenic Multidrug-Resistant Myeloma Cells: The Role of Cytotoxic T Lymphocytes
Authors: Shtil Alexander A. , Turner Joel G. , Durfee John , Dalton William S. , Yu Hua
Published: October 2019

Tumor cells that survive initial courses of chemotherapy may do so by acquiring a multidrug-resistant phenotype. This particular mechanism of drug resistance may also confer resistance to physiological effectors of apoptosis that could potentially reduce the efficacy of immune therapies that use these pathways of cell death. We have previously demonstrated high efficacy for a cytokine-based tumor cell vaccine in a murine MPC11 myeloma model. In the present study, the effects of this vaccination were compared in MPC11 cells and their isogenic sublines selected for mdr1/P-glycoprotein (Pgp)-mediated multidrug resistance (MDR). Immunization with MPC11 cells expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-12 (IL-12) led to long-lasting protection of mice against subcutaneous (sc) challenge with both parental cells or their MDR variants. Similarly, immunization with GM-CSF/IL-12–transfected MDR sublines caused rejection of transplantation of both parental cells and the MDR sublines. Whereas MPC11 cells and their MDR variants were resistant to APO-1/CD95/Fas ligand, the immunization generated potent granzyme B/perforin-secreting cytotoxic T lymphocytes (CTLs) that were similarly effective against both parental and isogenic MDR cells. We conclude that MDR mediated bymdr1/Pgp did not interfere with lysis by pore-forming CTLs. Immunotherapy based on pore-forming CTLs may be an attractive approach to the treatment of drug-resistant myeloma.
Conformational Changes in the A3 Domain of von Willebrand Factor Modulate the Interaction of the A1 Domain With Platelet Glycoprotein Ib
Authors: Obert Bernadette , Houllier Anne , Meyer Dominique , Girma Jean-Pierre
Published: October 2019

Bitiscetin has recently been shown to induce von Willebrand factor (vWF)-dependent aggregation of fixed platelets (Hamako J, et al,Biochem Biophys Res Commun 226:273, 1996). We have purified bitiscetin from Bitis arietans venom and investigated the mechanism whereby it promotes a form of vWF that is reactive with platelets. In the presence of bitiscetin, vWF binds to platelets in a dose-dependent and saturable manner. The binding of vWF to platelets involves glycoprotein (GP) Ib because it was totally blocked by monoclonal antibody (MoAb) 6D1 directed towards the vWF-binding site of GPIb. The binding also involves the GPIb-binding site of vWF located on the A1 domain because it was inhibited by MoAb to vWF whose epitopes are within this domain and that block binding of vWF to platelets induced by ristocetin or botrocetin. However, in contrast to ristocetin or botrocetin, the binding site of bitiscetin does not reside within the A1 domain but within the A3 domain of vWF. Thus, among a series of vWF fragments, 125I-bitiscetin only binds to those that overlap the A3 domain, ie, SpIII (amino acid [aa] 1-1365), SpI (aa 911-1365), and rvWF-A3 domain (aa 920-1111). It does not bind to SpII corresponding to the C-terminal part of vWF subunit (aa 1366-2050) nor to the 39/34/kD dispase species (aa 480-718) or T116 (aa 449-728) overlapping the A1 domain. In addition, bitiscetin that does not bind to DeltaA3-rvWF (deleted between aa 910-1113) has no binding site ouside the A3 domain. The localization of the binding site of bitiscetin within the A3 domain was further supported by showing that MoAb to vWF, which are specific for this domain and block the interaction between vWF and collagen, are potent inhibitors of the binding of bitiscetin to vWF and consequently of the bitiscetin-induced binding of vWF to platelets. Thus, our data support the hypothesis that an interaction between the A1 and A3 domains exists that may play a role in the function of vWF by regulating the ability of the A1 domain to bind to platelet GPIb.
Cloning and Characterization of a Lymphoid-Specific, Inducible Human Protein Tyrosine Phosphatase, Lyp
Authors: Cohen Shai , Dadi Harjit , Shaoul Ester , Sharfe Nigel , Roifman Chaim M.
Published: October 2019

Protein tyrosine phosphatases act in conjunction with protein kinases to regulate the tyrosine phosphorylation events that control cell activation and differentiation. We have isolated a previously undescribed human phosphatase, Lyp, that encodes an intracellular 105-kD protein containing a single tyrosine phosphatase catalytic domain. The noncatalytic domain contains four proline-rich potential SH3 domain binding sites and an NXXY motif that, if phosphorylated, may be recognized by phosphotyrosine binding (PTB) domains. Comparison of the Lyp amino acid sequence with other known proteins shows 70% identity with the murine phosphatase PEP. The human Lyp gene was localized to chromosome 1p13 by fluorescence in situ hybridization analysis. We also identified an alternative spliced form of Lyp RNA, Lyp2. This isoform encodes a smaller 85-kD protein with an alternative C-terminus. The lyp phosphatases are predominantly expressed in lymphoid tissues and cells, with Lyp1 being highly expressed in thymocytes and both mature B and T cells. Increased Lyp1 expression can be induced by activation of resting peripheral T lymphocytes with phytohemagglutinin or anti-CD3. Lyp1 was found to be constitutively associated with the proto-oncogene c-Cbl in thymocytes and T cells. Overexpression of lyp1 reduces Cbl tyrosine phosphorylation, suggesting that it may be a substrate of the phosphatase. Thus, Lyp may play a role in regulating the function of Cbl and its associated protein kinases.
Tandem Amino Acid Repeats From Trypanosoma cruzi Shed Antigens Increase the Half-Life of Proteins in Blood
Authors: Buscaglia Carlos A. , Alfonso Julieta , Campetella Oscar , Frasch Alberto C.C.
Published: October 2019

Proteins containing amino acid repeats are widespread among protozoan parasites. It has been suggested that these repetitive structures act as immunomodulators, but other functional aspects may be of primary importance. We have recently suggested that tandem repeats present in Trypanosoma cruzi trans-sialidase stabilize the catalytic activity in blood. Because the parasite releasestrans-sialidase, this delayed clearance of the enzyme might have implications in vivo. In the present work, the ability of repetitive units from different T. cruzi molecules in stabilizing trans-sialidase activity in blood was evaluated. It is shown that repeats present on T. cruzi shed proteins (antigens 13 and Shed-Acute-Phase-Antigen [SAPA]) increase trans-sialidase half-life in blood from 7 to almost 35 hours. Conversely, those repeats present in intracellular T. cruzi proteins only increase the enzyme half-life in blood up to 15 hours. Despite these results, comparative analysis of structural and catalytic properties of both groups of chimeric enzymes show no substantial differences. Interestingly, antigens 13 and SAPA also increase the persistence in blood of chimeric glutathione S-transferases, thus suggesting that this effect is inherent to these repeats and independent of the carrier protein. Although the molecular basis of this phenomenon is still uncertain, its biotechnological potential can be envisaged.
The Intracellular Serpin Proteinase Inhibitor 6 Is Expressed in Monocytes and Granulocytes and Is a Potent Inhibitor of the Azurophilic Granule Protease, Cathepsin G
Authors: Scott Fiona L. , Hirst Claire E. , Sun Jiuru , Bird Catherina H. , Bottomley Stephen P. , Bird Phillip I.
Published: October 2019

The monocyte and granulocyte azurophilic granule proteinases elastase, proteinase 3, and cathepsin G are implicated in acute and chronic diseases thought to result from an imbalance between the secreted proteinase(s) and circulating serpins such as 1-proteinase inhibitor and 1-antichymotrypsin. We show here that the intracellular serpin, proteinase inhibitor 6 (PI-6), is present in monocytes, granulocytes, and myelomonocytic cell lines. In extracts from these cells, PI-6 bound an endogenous membrane-associated serine proteinase to form an sodium dodecyl sulfate (SDS)-stable complex. Using antibodies to urokinase, elastase, proteinase 3, or cathepsin G, we demonstrated that the complex contains cathepsin G. Native cathepsin G and recombinant PI-6 formed an SDS-stable complex in vitro similar in size to that observed in the extracts. Further kinetic analysis demonstrated that cathepsin G and PI-6 rapidly form a tight 1:1 complex (ka = 6.8 ± 0.2 × 106mol/L−1s−1 at 17°C;Ki = 9.2 ± 0.04 × 10−10 mol/L). We propose that PI-6 complements 1-proteinase inhibitor and 1-antichymotrypsin (which control extracellular proteolysis) by neutralizing cathepsin G that leaks into the cytoplasm of monocytes or granulocytes during biosynthesis or phagocytosis. Control of intracellular cathepsin G may be particularly important, because it has recently been shown to activate the proapoptotic proteinase, caspase-7.
Mice Homozygous for a Truncated Form of CREB-Binding Protein Exhibit Defects in Hematopoiesis and Vasculo-angiogenesis
Authors: Oike Yuichi , Takakura Nobuyuki , Hata Akira , Kaname Tadashi , Akizuki Miwa , Yamaguchi Yuji , Yasue Hirofumi , Araki Kimi , Yamamura Ken-ichi , Suda Toshio
Published: October 2019

CREB-binding protein (CBP) and the closely related adenovirus E1A-associated 300-kD protein (p300) function as coactivators of transcription factors such as CREB, c-Fos, c-Jun, c-Myb, and several nuclear receptors. To study the roles of CBP in embryonic development, we generated CBP homozygous mutant mouse embryos that expressed a truncated form of CBP protein (1-1084 out of 2441 residues). The embryos died between embryonic days 9.5 (E9.5) and E10.5 and exhibited a defect in neural tube closure. They appeared pale and showed decreases in erythroid cells and colony-forming cells (CFCs) in the yolk sac, suggesting defects in primitive hematopoiesis. Immunohistochemistry with an anti-PECAM antibody showed a lack of vascular network formation. Organ culture of para-aortic splanchnopleural mesoderm (P-Sp) with stromal cells (OP9) showed an autonomous abnormality of putative endothelial precursors, which may induce the microenvironmental defect in hematopoiesis. In addition, these defects were partially rescued by the addition of VEGF to this culture. Our analyses demonstrate that CBP plays an essential role in hematopoiesis and vasculo-angiogenesis.
Promoter Element for Transcription of Unrearranged T-Cell Receptor β-Chain Gene in Pro-T Cells
Authors: Doty Raymond T. , Xia Dong , Nguyen Suzanne P. , Hathaway Tanya R. , Willerford Dennis M.
Published: October 2019

The hallmark of T- and B-lymphocyte development is the rearrangement of variable (V), diversity (D), and joining (J) segments of T-cell receptor (TCR) and immunoglobulin (Ig) genes to generate a diverse repertoire of antigen receptor specificities in the immune system. The process of V(D)J recombination is shared in the rearrangement of all seven antigen receptor genes and is controlled by changes in chromatin structure, which regulate accessibility to the recombinase apparatus in a lineage- and stage-specific manner. These chromatin changes are linked to transcription of the locus in its unrearranged (germline) configuration. To understand how germline transcription of the TCRβ-chain gene is regulated, we determined the structure of germline transcripts initiating near the Dβ1 segment and identified a promoter within this region. The Dβ1 promoter is active in the presence of the TCRβ enhancer (Eβ), and in this context, exhibits preferential activity in pro-T versus mature T-cell lines, as well as T- versus B-lineage specificity. These studies provide insight into the developmental regulation of TCRβ germline transcription, one of the earliest steps in T-cell differentiation.
CCAAT/Enhancer Binding Protein ɛ Is Critical for Effective Neutrophil-Mediated Response to Inflammatory Challenge
Authors: Lekstrom-Himes Julie , Xanthopoulos Kleanthis G.
Published: October 2019

Targeted mutation of CCAAT/enhancer binding protein (C/EBP) ɛ in mice results in early death, primarily due to spontaneous infection with Pseudomonas aeruginosa. Functional analysis of C/EBPɛ-deficient neutrophils, in an in vivo model of peritoneal inflammation, shows multiple defects. Reduction of phagocytotic killing by C/EBPɛ-deficient neutrophils is a result of decreased uptake of opsonized bacteria as well as little to no expression of secondary granule proteins. Abnormalities in neutrophil migration detected in a chemical peritonitis model are likely secondary to abnormal CD11b integrin and L-selectin expression on C/EBPɛ-deficient neutrophils. Alterations in neutrophil cytokine expression in response to inflammation show decreased levels of interleukin-1 receptor antagonist (IL-1Ra) and increased levels of tumor necrosis factor- (TNF-) expression by C/EBPɛ-deficient neutrophils. Additionally, TNF- expression is increased in nonactivated, circulating C/EBPɛ-deficient neutrophils. Overall, C/EBPɛ-deficient neutrophils are severely functionally impaired, evoking an abnormal microenvironment, which may contribute to the loss of normal responses to inflammatory stimuli. Similarities between the C/EBPɛ-deficient mouse model and the human disease, specific granule deficiency, will be discussed.
Survival of Donor Leukocyte Subpopulations in Immunocompetent Transfusion Recipients: Frequent Long-Term Microchimerism in Severe Trauma Patients
Authors: Lee Tzong-Hae , Paglieroni Teresa , Ohto Hitoshi , Holland Paul V. , Busch Michael P.
Published: October 2019

We recently reported detection of a transient increase in circulating donor leukocytes (WBCs) in immunocompetent recipients 3 to 5 days posttransfusion (tx) (Blood 85:1207, 1995). We have now characterized survival kinetics of specific donor WBC subsets in additional tx populations. Eight female elective surgery patients (pts) were sampled pre-tx and on days 1, 3, 5, 7, and 14 post-tx. Ten female trauma pts transfused with a total of 4 to 18 U of relatively fresh red blood cells were sampled up to 1.5 years post-tx. WBC subsets from frozen whole blood were isolated using CD4, CD8 (T cell), CD15 (myeloid), and CD19 (B cell) antibody-coated magnetic beads. Donor WBCs were counted by quantitative polymerase chain reaction (PCR) of male-specific sex determining region (SRY) sequences. PCR HLA typing and mixed leukocyte reaction (MLR) between recipient and donor WBCs were performed on two of the trauma tx recipients who had long-term chimerism of donor cells post-tx. In 6 of 8 female surgery pts, circulating CD4+ male donor cells peaked at day 3 or 5 (0.01 to 1 cell/μL), followed by clearance by day 14. In 7 of 10 female trauma pts, we observed multilineage persistence of male donor WBCs (CD4, CD8, CD15, CD19) for 6 months to 1.5 years post-tx at concentrations of 10 to 100 cells/μL. In 2 trauma recipients studied, MLR showed no, or very low, response to WBC of the single donor implicated as the source of microchimerism by HLA typing. Establishment of long-term multilineage chimerism in trauma recipients is probably caused by engraftment of donor stem cells and mutual tolerance between recipient and donor leukocytes. A better understanding of factors determining clearance versus chimerism of transfused leukocytes is critical to prevention of alloimmunization and transfusion-induced graft-versus-host disease, and, potentially, to induction of tolerance for transplantation.
Abnormal Expression and Subcellular Distribution of Subunit Proteins of the AP-3 Adaptor Complex Lead to Platelet Storage Pool Deficiency in the Pearl Mouse
Authors: Zhen Lijie , Jiang Shelley , Feng Lijun , Bright Nicholas A. , Peden Andrew A. , Seymour Albert B. , Novak Edward K. , Elliott Rosemary , Gorin Michael B. , Robinson Margaret S. , Swank Richard T.
Published: October 2019

The pearl mouse is a model for Hermansky Pudlak Syndrome (HPS), whose symptoms include hypopigmentation, lysosomal abnormalities, and prolonged bleeding due to platelet storage pool deficiency (SPD). The gene for pearl has recently been identified as the beta3A subunit of the AP-3 adaptor complex. The objective of these experiments was to determine if the expression and subcellular distribution of the AP-3 complex were altered in pearl platelets and other tissues. The beta3A subunit was undetectable in all pearl cells and tissues. Also, expression of other subunit proteins of the AP-3 complex was decreased. The subcellular distribution of the remaining AP-3 subunits in platelets, macrophages, and a melanocyte-derived cell line of pearl mice was changed from the normal punctate, probably endosomal, pattern to a diffuse cytoplasmic pattern. Ultrastructural abnormalities in mutant lysosomes were likewise apparent in mutant kidney and a cultured mutant cell line. Genetically distinct mouse HPS models had normal expression of AP-3 subunits. These and related experiments strongly suggest that the AP-3 complex regulates the biogenesis/function of organelles of platelets and other cells and that abrogation of expression of the AP-3 complex leads to platelet SPD.
A High-Affinity Human Antibody That Targets Tumoral Blood Vessels
Authors: Tarli Lorenzo , Balza Enrica , Viti Francesca , Borsi Laura , Castellani Patrizia , Berndorff Dietmar , Dinkelborg Ludger , Neri Dario , Zardi Luciano
Published: October 2019

Angiogenesis is a characteristic feature of many aggressive tumors and of other relevant disorders. Molecules capable of specifically binding to new-forming blood vessels, but not to mature vessels, could be used as selective vehicles and would, therefore, open diagnostic and therapeutic opportunities. We have studied the distribution of the ED-B oncofetal domain of fibronectin, a marker of angiogenesis, in four different tumor animal models: the F9 murine teratocarcinoma, SKMEL-28 human melanoma, N592 human small cell lung carcinoma, and C51 human colon carcinoma. In all of these experimental models we observed accumulation of the fibronectin isoform containing the ED-B domain around neovascular structures when the tumors were in the exponentially growing phase, but not in the slow-growing phase. Then we performed biodistribution studies in mice bearing a subcutaneously implanted F9 murine teratocarcinoma, using a high-affinity human antibody fragment (L19) directed against the ED-B domain of fibronectin. Radiolabeled L19, but not an irrelevant anti-lysozyme antibody fragment (D1.3), efficiently localizes in the tumoral vessels. The maximal dose of L19 accumulated in the tumor was observed 3 hours after injection (8.2% injected dose per gram). By virtue of the rapid clearance of the antibody fragment from the circulation, tumor-to-blood ratios of 1.9, 3.7, and 11.8 were obtained at 3, 5, and 24 hours, respectively. The tumor-targeting performance of L19 was not dose-dependent in the 0.7 to 10 μg range of injected antibody. The integral of the radioactivity localized in tumoral vessels over 24 hours was greater than 70-fold higher than the integral of the radioactivity in blood over the same time period, normalized per gram of tissue or fluid. These findings quantitatively show that new-forming blood vessels can selectively be targeted in vivo using specific antibodies, and suggest that L19 may be of clinical utility for the immunoscintigraphic detection of angiogenesis in patients.
A Clinical Trial of Retroviral-Mediated Transfer of arev-Responsive Element Decoy Gene Into CD34+Cells From the Bone Marrow of Human Immunodeficiency Virus-1–Infected Children
Authors: Kohn Donald B. , Bauer Gerhard , Rice C. Robert , Rothschild J.C. , Carbonaro Denise A. , Valdez Penelope , Hao Qian-lin , Zhou Chen , Bahner Ingrid , Kearns Karen , Brody Kate , Fox Sarah , Haden Elizabeth , Wilson Kathy , Salata Cathy , Dolan Cathy , Wetter Charles , Aguilar-Cordova Estuardo , Church Joseph
Published: October 2019

Genetic modification of hematopoietic stem cells with genes that inhibit replication of human immunodeficiency virus-1 (HIV-1) could lead to development of T lymphocytes and monocytic cells resistant to HIV-1 infection after transplantation. We performed a clinical trial to evaluate the safety and feasibility of this procedure, using bone marrow from four HIV-1–infected pediatric subjects (ages 8 to 17 years). We obtained bone marrow, isolated CD34+ cells, performed in vitro transduction with a retroviral vector carrying arev-responsive element (RRE) decoy gene, and reinfused the cells into these subjects with no evidence of adverse effects. The levels of gene-containing leukocytes in peripheral blood samples in the 1 year after gene transfer/cell infusion have been extremely low. These observations support the potential of performing gene therapy for HIV-1 using hematopoietic cells, but emphasize the need for improved gene transfer techniques.
Engraftment of MDR1 and NeoR Gene-Transduced Hematopoietic Cells After Breast Cancer Chemotherapy
Authors: Moscow Jeffrey A. , Huang Hui , Carter Charles , Hines Kenneth , Zujewski JoAnne , Cusack Georgie , Chow Cathy , Venzon David , Sorrentino Brian , Chiang Yawen , Goldspiel Barry , Leitman Susan , Read Elizabeth J. , Abati Andrea , Gottesman Michael M. , Pastan Ira , Sellers Stephanie , Dunbar Cynthia , Cowan Kenneth H.
Published: October 2019

To determine whether the multidrug resistance gene MDR1could act as a selectable marker in human subjects, we studied engraftment of peripheral blood progenitor cells (PBPCs) transduced with either MDR1 or the bacterial NeoR gene in six breast cancer patients. This study differed from previous MDR1 gene therapy studies in that patients received only PBPCs incubated in retroviral supernatants (no nonmanipulated PBPCs were infused), transduction of PBPCs was supported with autologous bone marrow stroma without additional cytokines, and a control gene (NeoR) was used for comparison with MDR1. Transduced PBPCs were infused after high-dose alkylating agent therapy and before chemotherapy with MDR-substrate drugs. We found that hematopoietic reconstitution can occur using only PBPCs incubated ex vivo, that theMDR1 gene product may play a role in engraftment, and that chemotherapy may selectively expand MDR1 gene-transduced hematopoietic cells relative to NeoR transduced cells in some patients.
Treatment and Clinical Management of Primary Mediastinal Large B-Cell Lymphoma With Sclerosis: MACOP-B Regimen and Mediastinal Radiotherapy Monitored by 67Gallium Scan in 50 Patients
Authors: Zinzani Pier Luigi , Martelli Maurizio , Magagnoli Massimo , Pescarmona Edoardo , Scaramucci Laura , Palombi Francesca , Bendandi Maurizio , Martelli Maria Paola , Ascani Stefano , Orcioni Giulio Fraternali , Pileri Stefano A. , Mandelli Franco , Tura Sante
Published: October 2019

To evaluate the efficacy of a combined modality treatment (MACOP-B plus mediastinal radiotherapy) and the advantages of Gallium-67-citrate single-photon emission (67GaSPECT) over computed tomography (CT) for restaging in patients with primary mediastinal large B-cell lymphoma (PMLBCL) with sclerosis. Between 1989 and 1998, 50 previously untreated patients with PMLBCL with sclerosis (70% with bulky mass) were treated with MACOP-B regimen plus mediastinal radiotherapy. The radiologic clinical stage with evaluation of tumor size included CT and 67GaSPECT at diagnosis, after chemotherapy, and after radiotherapy. Forty-three patients (86%) achieved a complete response and 7 were nonresponders to treatment. For the imaging evaluation, only 47 patients were evaluable because 3 had disease progression during chemotherapy. After treatment, 3/5 (60%) patients with positive 67GaSPECT and negative CT scan relapsed, as against 0/21 (0%) with negative 67GaSPECT and CT scan. Twenty-one patients had a positive CT scan: of these, the 4 with positive 67GaSPECT all progressed, whereas there were no relapses among the 17 with negative 67GaSPECT. After radiotherapy, there was a decrease of positive CT (from 33 to 21 cases) and of positive 67GaSPECT (from 31 to 9 cases). Relapse-free survival rate was 93% at 96 months (median 39 months). In patients with PMLBCL with sclerosis, MACOP-B plus radiation therapy is a very useful first-line treatment and radiation therapy may play an important role. As regards restaging, 67GaSPECT should be considered the imaging technique of choice at least in patients who show CT positivity.
Functional Characterization of the Human Platelet Glycoprotein V Gene Promoter: A Specific Marker of Late Megakaryocytic Differentiation
Authors: Lepage Adeline , Uzan Georges , Touche Nadège , Morales Martine , Cazenave Jean-Pierre , Lanza François , de la Salle Corinne
Published: October 2019

Glycoprotein V (GPV), a subunit of the platelet GPIb-V-IX receptor for von Willebrand factor and thrombin, is specifically found in platelets and mature megakaryocytes. Studies of the GPV gene can therefore provide insight into the mechanisms governing megakaryocyte differentiation. The human GPV promoter was isolated, and elements important for its tissue specific transcriptional activity were localized using systematic DNase I protection and reporter deletion assays. A −1413/+25 fragment inserted into a luciferase reporter construct displayed promoter activity in Dami and HEL but not in K562, HL60, or HeLa cells. Progressive 5′ to 3′ deletion showed a putative enhancer region in the −1413/−903 segment that contained closely spaced GATA and Ets sites protected from DNase I digestion in Dami extracts. Regions similar to a GPIIb gene repressor were found at −816 and −610, with the first exhibiting repressor activity in Dami and HEL cells and the second protected from DNAse I. Deletions from −362 to −103, an area containing protected sites for Sp1, STAT, and GATA, induced a progressive decrease in activity. The −103/+1 fragment, bearing a proximal Ets footprinted site and a GATA/Ets tandem footprint, displayed 75% activity relative to the full-length promoter and retained cell specificity. In summary, this work defines several regions of the GPV gene promoter important for its activity. It contains megakaryocyte-specific signals, including erythro-megakaryocytic GATA, and Ets cis-acting elements, GPIIb-like repressor domains, and binding sites for ubiquitous factors such as Sp1, ETF, and STAT.
Regulation of p21rac Activation in Human Neutrophils
Authors: Geijsen Niels , van Delft Sanne , Raaijmakers Jan A.M. , Lammers Jan-Willem J. , Collard John G. , Koenderman Leo , Coffer Paul J.
Published: October 2019

The small guanosine triphosphate (GTPase) p21rac is highly expressed in human neutrophils where it is thought to play a role in cytoskeletal reorganization and superoxide production. Using the p21rac binding domain of PAK (PAK-RBD) as an activation-specific probe, we have investigated agonist-stimulated activation of p21rac. Stimulation of neutrophils with the chemoattractants fMet-Leu-Phe (fMLP) or platelet-activating factor (PAF) induced an extremely rapid and transient p21rac activation, being optimal within 5 seconds. This activation correlates with the rapid changes of intracellular free Ca2+ ([Ca2+]i) stimulated by fMLP; however, changes in [Ca2+]i were neither sufficient nor required for p21rac activation. Furthermore, fMLP-induced p21rac activation was not inhibited by broad tyrosine kinase inhibitors or specific inhibitors of ERK, p38 mitogen activated protein kinase, Src, or phosphatidylinositol 3-kinases. Surprisingly, the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor- did not cause p21rac activation or modulate fMLP-induced p21rac activation. AlF−, a potent activator of heterotrimeric G-protein -subunits, however, was found to activate p21rac. Stimulation of neutrophils with phorbol myristate acetate (PMA) strongly activated the respiratory burst, but did not induce p21rac activation, suggesting that superoxide production per se can occur independently of p21rac activation. These data suggest that in human granulocytes, G-protein coupled receptors, but not cytokine receptors, activate p21rac via a rapid, novel exchange-mechanism independently of changes in [Ca2+]i, tyrosine phosphorylation, or PI3K.
Direct Evidence for Multiple Self-Renewal Divisions of Human In Vivo Repopulating Hematopoietic Cells in Short-Term Culture
Authors: Glimm H. , Eaves C.J.
Published: October 2019

Recently, culture conditions that stimulate the proliferation of primitive hematopoietic cells defined by various phenotypic and functional endpoints in vitro have been identified. However, evidence that they support a high probability of self-renewal leading to a large net expansion in vitro of transplantable cells with lympho-myeloid repopulating ability has been more difficult to obtain. The present study was designed to investigate whether the low overall expansion of human repopulating hematopoietic cells seen in vitro reflects a selective unresponsiveness of these rare cells to the growth factors currently used to stimulate them or, alternatively, whether they do proliferate in vitro but lose engrafting potential. For this, we used a high-resolution procedure for tracking and reisolating cells as a function of their proliferation history based on the loss of cellular fluorescence after staining with (5- and 6-) carboxyfluorescein diacetate succinimidyl ester. The results show that the vast majority of long-term culture-initiating cells and in vivo lympho-myeloid competitive repopulating units present in 5-day suspension cultures initiated with CD34+ human cord blood and fetal liver cells are the progeny of cells that have divided at least once in response to stimulation by interleukin-3, interleukin-6, granulocyte colony-stimulating factor, Steel factor, and Flt3-ligand. Thus, most human repopulating cells from these two sources are stimulated to undergo multiple divisions under currently used short-term suspension culture conditions and a proportion of these retain engraftment potential.
Cytokine-Release Syndrome in Patients With B-Cell Chronic Lymphocytic Leukemia and High Lymphocyte Counts After Treatment With an Anti-CD20 Monoclonal Antibody (Rituximab, IDEC-C2B8)
Authors: Winkler U. , Jensen M. , Manzke O. , Schulz H. , Diehl V. , Engert A.
Published: October 2019

Eleven patients with relapsed fludarabine-resistant B-cell chronic lymphocytic leukemia (CLL) or leukemic variants of low-grade B-cell non-Hodgkin’s lymphoma (NHL) were treated with the chimeric monoclonal anti-CD20 antibody rituximab (IDEC-C2B8). Peripheral lymphocyte counts at baseline varied from 0.2 to 294.3 × 109/L. During the first rituximab infusion, patients with lymphocyte counts exceeding 50.0 × 109/L experienced a severe cytokine-release syndrome. Ninety minutes after onset of the infusion, serum levels of tumor necrosis factor- (TNF-) and interleukin-6 (IL-6) peaked in all patients. Elevated cytokine levels during treatment were associated with clinical symptoms, including fever, chills, nausea, vomiting, hypotension, and dyspnea. Lymphocyte and platelet counts dropped to 50% to 75% of baseline values within 12 hours after the onset of the infusion. Simultaneously, there was a 5-fold to 10-fold increase of liver enzymes, d-dimers, and lactate dehydrogenase (LDH), as well as a prolongation of the prothrombin time. Frequency and severity of first-dose adverse events were dependent on the number of circulating tumor cells at baseline: patients with lymphocyte counts greater than 50.0 × 109/L experienced significantly more adverse events of National Cancer Institute (NCI) grade III/IV toxicity than patients with less than 50.0 × 109/L peripheral tumor cells (P= .0017). Due to massive side effects in the first patient treated with 375 mg/m2 in 1 day, a fractionated dosing schedule was used in all subsequent patients with application of 50 mg rituximab on day 1, 150 mg on day 2, and the rest of the 375 mg/m2 dose on day 3. While the patient with the leukemic variant of the mantle-cell NHL achieved a complete remission (9 months+) after treatment with 4 × 375 mg/m2 rituximab, efficacy in patients with relapsed fludarabine-resistant B-CLL was poor: 1 partial remission, 7 cases of stable disease, and 1 progressive disease were observed in 9 evaluable patients with CLL. On the basis of these data, different infusion schedules and/or combination regimens with chemotherapeutic drugs to reduce tumor burden before treatment with rituximab will have to be evaluated.
Coinheritance of Gilbert Syndrome Increases the Risk for Developing Gallstones in Patients With Hereditary Spherocytosis
Authors: del Giudice Emanuele Miraglia , Perrotta Silverio , Nobili Bruno , Specchia Claudia , d’Urzo Giovanna , Iolascon Achille
Published: October 2019

The precocious formation of bilirubinate gallstones is the most common complication of hereditary spherocytosis (HS), and the prevention of this problem represents a major impetus for splenectomy in many patients with compensated hemolysis. Because Gilbert syndrome has been considered a risk factor for gallstone formation, there are reasons for postulating that the association of this common inherited disorder of hepatic bilirubin metabolism with HS could increase cholelithiasis. To test this hypothesis, 103 children with mild to moderate HS who, from age 1, have undergone a liver and biliary tree ultrasonography every year, were retrospectively examined. The 2-bp (TA) insertion within the promoter of the uridine diphosphate-glucuronosyltransferase gene (UGT1A1), associated with Gilbert syndrome, was screened. The risk of developing gallstones was statistically different among the 3 groups of patients: homozygotes for the normal UGT1A1 allele, heterozygotes, and homozygotes for the allele with the TA insertion. Fitting a Cox regression model, in fact, a statistically significant hazard ratio of 2.19 (95% confidence interval: 1.31 to 3.66) was estimated from one to the next of these genetic classes. The individual proneness to form gallstones from TA insertion in the TATA-box of the UGT1A1 promoter should be considered during the follow-up of patients with HS. Although patients with HS were the only ones studied, extrapolating these data to patients who have different forms of inherited (eg, thalassemia, intraerythrocytic enzymatic deficiency) or acquired (eg, autoimmune hemolytic anemia, hemolysis from mechanical heart valve replacement) chronic hemolysis can be warranted.
Identification of the SH2 Domain Binding Protein of Bruton’s Tyrosine Kinase as BLNK—Functional Significance of Btk-SH2 Domain in B-Cell Antigen Receptor-Coupled Calcium Signaling
Authors: Hashimoto Shoji , Iwamatsu Akihiro , Ishiai Masamichi , Okawa Katsuya , Yamadori Tomoki , Matsushita Masato , Baba Yoshihiro , Kishimoto Tadamitsu , Kurosaki Tomohiro , Tsukada Satoshi
Published: October 2019

Bruton’s tyrosine kinase (Btk) is a critical component in the B-cell antigen receptor (BCR)-coupled signaling pathway. Its deficiency in B cells leads to loss or marked reduction in the BCR-induced calcium signaling. It is known that this BCR-induced calcium signaling depends on the activation of phospholipase Cγ (PLCγ), which is mediated by Btk and another tyrosine kinase Syk and that the SH2 and pleckstrin homology (PH) domains of Btk play important roles in this activation process. Although the importance of the PH domain of Btk has been explained by its role in the membrane targeting of Btk, the functional significance of the SH2 domain in the calcium signaling has remained merely a matter of speculation. In this report, we identify that one of the major Btk-SH2 domain-binding proteins in B cells is BLNK (B-cell linker protein) and present evidences that the interaction of BLNK and the SH2 domain of Btk contributes to the complete tyrosine phosphorylation of PLCγ.
Modulation of T-Cell Functions in KIR2DL3 (CD158b) Transgenic Mice
Authors: Cambiaggi Anna , Darche Sylvie , Guia Sophie , Kourilsky Philippe , Abastado Jean-Pierre , Vivier Eric
Published: October 2019

In humans, a minor subset of T cells express killer cell Ig-like receptors (KIRs) at their surface. In vitro data obtained with KIR+ β and γδ T-cell clones showed that engagement of KIR molecules can extinguish T-cell activation signals induced via the CD3/T-cell receptor (TCR) complex. We analyzed the T-cell compartment in mice transgenic for KIR2DL3 (Tg-KIR2DL3), an inhibitory receptor for HLA-Cw3. As expected, mixed lymphocyte reaction and anti-CD3 monoclonal antibody (MoAb)-redirected cytotoxicity exerted by freshly isolated splenocytes can be inhibited by engagement of transgenic KIR2DL3 molecules. In contrast, antigen and anti-CD3 MoAb-induced cytotoxicity exerted by alloreactive cytotoxic T lymphocytes cannot be inhibited by KIR2DL3 engagement. In double transgenic mice, Tg-KIR2DL3 × Tg-HLA-Cw3, no alteration of thymic differentiation could be documented. Immunization of double transgenic mice with Hen egg white lysozime (HEL) or Pigeon Cytochrome-C (PCC) was indistinguishable from immunization of control mice, as judged by recall antigen-induced in vitro proliferation and TCR repertoire analysis. These results indicate that KIR effect on T cells varies upon cell activation stage and show unexpected complexity in the biological function of KIRs in vivo.
Triggering Noncycling Hematopoietic Progenitors and Leukemic Blasts to Proliferate Increases Anthracycline Retention and Toxicity by Downregulating Multidrug Resistance
Authors: Smeets Mariëlle E.P. , Raymakers Reinier A.P. , Vierwinden Gerty , Pennings Arie H.M. , Wessels Hans , de Witte Theo
Published: October 2019

Expression of the multidrug resistance (MDR) mechanisms P-glycoprotein (Pgp) and MDR-related protein (MRP) decrease cellular retention and consequently cytotoxicity of anthracyclines. MDR is expressed on normal human hematopoietic progenitors and leukemic blasts. Normal CD34+ progenitors showed rhodamine efflux in 20% to 30% of the cells, which could be blocked by verapamil. These cells appeared noncycling, in contrast to the proliferating rhodamine bright (RhoB) cells. We postulated that MDR expression can be downregulated by proliferation induction. Triggering rhodamine dull (RhoD) CD34+ cells to proliferate indeed resulted in a higher rhodamine retention and significantly decreased efflux modulation by verapamil (P = .04). Also in acute myeloid leukemia (AML), the proliferation rate (percentage S/G2+M and Iododeoxyuridine labelings index) was significantly less in the RhoD blasts (P ≤ .008) and proliferation induction of RhoD blasts resulted in increased rhodamine retention. Anthracycline cytotoxicity was less for RhoD than RhoB cells in both normal progenitors and leukemic blasts. Proliferation induction of the RhoD cells resulted in increased anthracycline sensitivity. We conclude that noncycling progenitors, both normal and leukemic, have a relatively high MDR expression. Triggering these cells into proliferation downregulates MDR expression. These findings can be exploited to overcome MDR in the treatment of AML patients.
M-Ras, a Widely Expressed 29-kD Homologue of p21 Ras: Expression of a Constitutively Active Mutant Results in Factor-Independent Growth of an Interleukin-3–Dependent Cell Line
Authors: Ehrhardt Götz R.A. , Leslie Kevin B. , Lee Frances , Wieler James S. , Schrader John W.
Published: October 2019

M-Ras, a recently identified homologue of p21 Ras, is widely expressed, with levels of the 29-kD protein in spleen, thymus, and NIH 3T3 fibroblasts equaling or exceeding those of p21 Ras. A G22V mutant of M-Ras was constitutively active and its expression in an interleukin-3 (IL-3)–dependent mast cell/megakaryocyte cell line resulted in increased survival in the absence of IL-3, increased growth in IL-4, and, at high expression levels, in factor-independent growth. Expression of M-Ras G22V, however, had a negative effect on growth in the presence of IL-3, suggesting that M-Ras has both positive and negative effects on growth. Expression of M-Ras G22V in NIH-3T3 fibroblasts resulted in morphological transformation and growth to higher cell densities. M-Ras G22V induced activation of thec-fos promoter, and bound weakly to the Ras-binding domains of Raf-1 and RalGDS. Expression of a mutant of M-Ras G22V that was no longer membrane-bound partially inhibited (40%) activation of thec-fos promoter by N-Ras Q61K, suggesting that M-Ras shared some, but not all, of the effectors of N-Ras. An S27N mutant of M-Ras, like the analogous H-Ras S17N mutant, was a dominant inhibitor of activation of the c-fos promoter by constitutively active Src Y527F, suggesting that M-Ras and p21 Ras shared guanine nucleotide exchange factors and are likely to be activated in parallel. Moreover, M-Ras was recognized by the monoclonal anti-Ras antibody Y13-259, commonly used to study the function and activity of p21 Ras. Mammalian M-Ras and a Caenorhabditis elegans orthologue exhibit conserved structural features, and these are likely to mediate activation of distinctive signaling paths that function in parallel to those downstream of p21 Ras.
Calreticulin and Calreticulin Fragments Are Endothelial Cell Inhibitors That Suppress Tumor Growth
Authors: Pike Sandra E. , Yao Lei , Setsuda Joyce , Jones Karen D. , Cherney Barry , Appella Ettore , Sakaguchi Kazuyasu , Nakhasi Hira , Atreya Chintamani D. , Teruya-Feldstein Julie , Wirth Peter , Gupta Ghanshyam , Tosato Giovanna
Published: October 2019

Several angiogenesis inhibitors are fragments of larger proteins that are themselves not active as angiogenesis inhibitors. Vasostatin, the N-terminal domain of calreticulin inclusive of amino acids 1-180, is an angiogenesis inhibitor that exerts antitumor effects in vivo. In the present study, we examined whether the full-length calreticulin molecule shares the antiangiogenic and antitumor activities of vasostatin. Similar to vasostatin, calreticulin selectively inhibited endothelial cell proliferation in vitro, but not cells of other lineages, and suppressed angiogenesis in vivo. When inoculated into athymic mice, calreticulin inhibited Burkitt tumor growth comparably with vasostatin. Calreticulin lacking the N-terminal 1-120 amino acids inhibited endothelial cell proliferation in vitro and Burkitt tumor growth in vivo comparably with vasostatin. An internal calreticulin fragment encompassing amino acids 120-180 also inhibited endothelial cell proliferation in vitro and angiogenesis in vivo comparably with calreticulin and vasostatin. These results suggest that the antiangiogenic activities of vasostatin reside in a domain that is accessible from the full-length calreticulin molecule and localize to calreticulin N-terminal amino acids 120-180. Thus, calreticulin and calreticulin fragments are inhibitors of angiogenesis that directly target endothelial cells, inhibit angiogenesis, and suppress tumor growth. This information may be critical in designing targeted inhibitors of pathological angiogenesis that underlies cancer and other diseases.
Presence of Proteinase 3 in Secretory Vesicles: Evidence of a Novel, Highly Mobilizable Intracellular Pool Distinct From Azurophil Granules
Authors: Witko-Sarsat Véronique , Cramer Elisabeth M. , Hieblot Corinne , Guichard Josette , Nusbaum Patrick , Lopez Sandra , Lesavre Philippe , Halbwachs-Mecarelli Lise
Published: October 2019

Proteinase 3 (PR3), which is also called myeloblastin, the target autoantigen for antineutrophil cytoplasmic antibodies (ANCA) in Wegener’s granulomatosis, is a serine proteinase stored in azurophil granules of human neutrophils. We have previously shown that, in contrast to elastase or myeloperoxidase, PR3 is also expressed at the plasma membrane of a subset of unactivated neutrophils and that a high proportion of neutrophils expressing membrane PR3 is a risk factor for vasculitis. The present study demonstrates that the association of PR3 with the plasma membrane is not an ionic interaction and seems to be covalent. Fractionation of neutrophils shows that, besides the azurophil granules, PR3 could be detected both in specific granules and in the plasma membrane-enriched fraction containing secretory vesicles, whereas elastase and myeloperoxidase were exclusively located in azurophil granules. Electron microscopy confirms that PR3 is present along with CR1 in secretory vesicles as well as in some specific granules. In neutrophils stimulated with an increasing dose of FMLP, membrane PR3 expression increased with the degranulation of secretory vesicles, followed by specific granules, and culminated after azurophil granules mobilization. The presence of a readily plasma membrane-mobilizable pool of PR3 contained in the secretory vesicles might play a relevant role in the pathophysiological mechanisms of ANCA-associated vasculitis.