bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of Rantes and MCP-1 Chemokines on In Vivo Basophilic Cell Recruitment in Rat Skin Injection Model and Their Role in Modifying the Protein and mRNA Levels for Histidine Decarboxylase
Authors: Conti Pio , Pang Xinzhu , Boucher William , Letourneau Richard , Reale Marcella , Barbacane Renato C. , Thibault Jean , Theoharides Theoharis C.
Published: October 2019

RANTES and related molecules, constitute the C-C class of chemokine supergene family and a group of cytokines produced by hematopoietic cells constitute the MCP-1 or C-X-C class. The roles of most of these chemokines are not well known, although members of the C-X-C family are inflammatory agents. Here, we report that intradermal injection of RANTES 10 ng/50 μL subcutaneously in the abdominal skin produced a strong inflammatory reaction, as evidenced by Evans blue dye, greater than FMLP (10−6 mol/L) (∼57%); while MCP-1, 10 ng/50 μL was less effective than FMLP (10−6 mol/L) (∼54%). Moreover, the histologic analysis of the cells stained with Toluidine blue (0.1%) were analyzed at a magnification of ×40). RANTES 10 ng/50 μL and LPS produced higher numbers (142 ± 11 and 193 ± 21 of cells/200 mm2, respectively) of basophilic cell accumulation in the skin injection sites compared with FMLP (10−6 mol/L) (127 ± 14/200 mm2), while MCP-1 10 ng/50 μL was less effective (88 ± 10/200 mm2). Electron microscopy (×13,800) studies of skin injection sites revealed that RANTES was chemoattractant for mast cells. In a Northern blot analysis from homogeneous tissue biopsy from the intradermal injection sites, RANTES was more potent than MCP-1 in increasing histidine decarboxylase (HDC) mRNA, the sole enzyme responsible for the production of histamine from histidine. Since PGD2 is formed by mast cells on cell activation, we also studied the effect of RANTES and MCP-1 on PGD2 production in inflamed tissue in vivo. RANTES (20, 10, and 5 ng) and MCP-1 (20, 10, and 5 ng) strongly stimulated PGD2 , in a dose-dependent manner, with a potency rank order of RANTES (10 ng/mL) approximately two times greater than MCP-1 (10 ng/mL).
The Type 2 CD10/Neutral Endopeptidase 24.11 Promoter: Functional Characterization and Tissue-Specific Regulation by CBF/NF-Y Isoforms
Authors: Ishimaru Fumihiko , Mari Bernard , Shipp Margaret A.
Published: October 2019

The cell surface zinc metalloproteinase CD10/neutral endopeptidase 24.11 ([NEP] neprilysin) functions as part of a regulatory loop to control local concentrations of peptide substrates and associated peptide-mediated signal transduction. The physiologic role of the enzyme depends on available substrates in specific organs and cell types. Although CD10/NEP is expressed on a restricted subset of normal and malignant lymphoid progenitors, the enzyme is also expressed by a variety of epithelial cells. To explore the mechanism of tissue-specific expression of this regulatory enzyme, we characterized the major (type 2) CD10/NEP promoter and identified three functionally active transcription factor binding sites (regions I to III). CBF/NF-Y binds to the inverted CCAAT box in region I, whereas a second positive and a third negative factor bind to regions II and III, respectively. Although region I is required for maximal CD10/NEP-driven luciferase activity in the examined epithelial cell lines, this region is not required for maximal activity in the evaluated lymphoid cell lines. The apparent tissue-specific differences in requirements for region I (and CBF/NF-Y) are of particular interest because lymphoid and epithelial cells express alternatively spliced versions of CBF/NF-Y that differ in biologic activity.
Recombinant Sickle Hemoglobin Containing a Lysine Substitution at Asp-85(α): Expression in Yeast, Functional Properties, and Participation in Gel Formation
Authors: Himanen Juha-Pekka , Popowicz Anthony M. , Manning James M.
Published: October 2019

Clinical modalities based on inhibition of gelation of HbS are hindered by the lack of quantitative information on the extent of participation of different amino acid residues in the aggregation process. One such site is Asp-85(α), which is involved in a parallel interdouble strand ionic interaction with Lys-144(β) according to the crystal structure of HbS, but electron microscopy does not specifically show Asp-85(α) as a contact site for fiber formation. Using a yeast recombinant system, we have substituted this site by Lys to abolish ion pairing and to make a quantitative determination of its participation in aggregation. The purified double mutant was shown to have the expected pI, the calculated molecular weight, correct amino acid composition, and peptide map. The recombinant double mutant has an oxygen affinity of 10 mm Hg, which is identical to that for HbA and HbS under the same conditions; it also has high cooperativity with an average n value of 2.7. The change in P50 in response to chloride ions was about 25% less than that for HbA or HbS and is ascribed to the introduction of a new positive charge near one of the major oxygen-linked chloride binding sites of hemoglobin. The gelation concentration of the double mutant was measured by a new procedure (Bookchin et al, 1994); the maximal amount of soluble hemoglobin (Csat ) in the presence of dextran indicated a decreased tendency for gelation with a Csat of 53 mg/mL compared with 34 mg/mL for HbS. This inhibitory effect is smaller than that of the E6V(β)/L88A(β) (Csat , 67 mg/mL) and the E6V(β)/K95I(β) (Csat , 90 mg/mL) recombinant hemoglobins. Thus, we would classify Asp-85(α) as a moderate contributor to the strength of the HbS aggregate. This wide range of gelation values demonstrates that some sites are more important than others in promoting HbS aggregation.
Frame Shift Mutation, Exon Skipping, and a Two-Codon Deletion Caused by Splice Site Mutations Account for Pyruvate Kinase Deficiency
Authors: Kanno Hitoshi , Fujii Hisaichi , Wei David C. , Chan L.C. , Hirono Akira , Tsukimoto Ichiro , Miwa Shiro
Published: October 2019

Three novel splice site mutations and two novel missense mutations were identified by molecular analysis of pyruvate kinase (PK) deficiency associated with hereditary nonspherocytic hemolytic anemia. A Nepalese PK variant, PK Kowloon, was found to have a homozygous transversion at the 5′-splice site of the seventh intervening sequence (IVS) of the L-type PK gene (Ivs7[+1]gt → tt). Using a reverse transcription polymerase chain reaction (RT-PCR) assay, we showed that the R-type PK mRNA in the proband's reticulocytes included the seventh IVS between the seventh and eighth exon, introducing a stop codon 3 nucleotides downstream of the mutated site. Consequently, the translational product may lack 44% of the R-PK polypeptide. A transition at the last nucleotide of exon 9 (1269GCG → GCA) was found in a Japanese PK variant, PK ‘Kamata.’ The mutation did not alter the amino acid sequence, but caused skipping of the ninth exonic sequence in the R-PK transcripts. As a result, the affected R-type PK lost 51 amino acid residues (373Met-423Ala del). A transversion at the splice acceptor site of the third IVS (Ivs 3[-2]ag → tg) was identified in PK ‘Aomori.’ The mutation resulted in aberrant splicing at a cryptic splice site within exon 4, causing deletion of two codons in the aberrant R-PK transcript (95 Gly-96 Pro → del). Both PK ‘Kamata’ and PK ‘Aomori’ had a missense mutation on the other allele, 1044AAG → AAT (348Lys → Asn) and 1075CGC → TGC (359Arg → Cys), respectively. Although both 348Lys and 359Arg were located in the sixth loop of A domain (β/α)8 barrel, which has been shown to contain the substrate and cation binding sites, the degree of anemia was much more severe in PK ‘Kamata’ than PK ‘Aomori,’ possibly because the 51 amino acid deletion of PK ‘Kamata’ but the 2 amino-acid deletion of PK ‘Aomori’ may abolish PK catalytic activity.
Bcl-2 Does Not Protect Burkitt's Lymphoma Cells From Oxidant-Induced Cell Death
Authors: Lee Yang-ja , Shacter Emily
Published: October 2019

Bcl-2 is an oncogene that confers deregulated growth potential to B lymphocytes through its ability to inhibit apoptotic cell death. A specific molecular activity for the Bcl-2 protein has not been identified, but several lines of evidence have supported a role in protection of cells from oxidative stress. We investigated whether there is a correlation between expression of high levels of Bcl-2 and susceptibility of human Burkitt's lymphoma cell lines to H2O2 -induced killing. The amount of H2O2 required to kill 50% of cells in 24 hours varied widely in the seven different lymphoma cell lines that were tested, ranging from 35 to 500 μmol/L H2O2 . However, expression of high levels of endogenous Bcl-2 did not protect the cells from H2O2 -induced killing, even though it was effective in protecting the cells from apoptosis induced by agents such as A23187. Thus, Bcl-2 was functional in preventing apoptosis but did not act in an antioxidant capacity. The results were confirmed using a Burkitt's lymphoma cell line overexpressing transfected bcl-2. The results may be explained by the observation that H2O2 was inefficient at inducing apoptosis in these mature B-cell lines. Nonapoptotic death induced by H2O2 was not prevented by Bcl-2.
Evaluation of the Revised European-American Lymphoma Classification Confirms the Clinical Relevance of Immunophenotype in 560 Cases of Aggressive Non-Hodgkin's Lymphoma
Authors: Melnyk A. , Rodriguez A. , Pugh W.C. , Cabannillas F.
Published: October 2019

The Revised European-American Lymphoma (REAL) classification has been criticized for its emphasis on the unproven clinical relevance of immunophenotype. A worse prognosis for peripheral T-cell non-Hodgkin's lymphomas (PTCLs) has been inconsistently reported in part because the definition of PTCL has been imprecise (eg, T-cell–rich B-cell non-Hodgkin's lymphomas [TCRBCLs] have been misdiagnosed as PTCLs in the past) and because its correlation with other known prognostic factors has not been studied by multivariate analysis. We analyzed six protocols from 1984 to 1995 with Working Formulation intermediate grade and immunoblastic lymphomas (exclusive of mantle cell) and selected only those cases in which immunophenotyping was performed and was conclusive. Of a total of 560 evaluable patients, 68 were PTCLs (12%) and the remaining 492 (88%) were B-cell non-Hodgkin's lymphomas, including 16 TCRBCLs (3% of total). The 5-year failure-free survival (FFS) for PTCLs and B-cell large-cell lymphomas (BCLCLs) is 38% and 55%, respectively (P < .0001) and the 5-year overall survival (OS) is 39% and 262%, respectively (P < .001). The M.D. Anderson prognostic tumor score (MDATS) and International Prognostic Index (IPI) for all patients was calculated. With MDATS of less than 3 (good prognosis), the 5-year FFS for PTCL and BCLCL is 56% and 69%, respectively (P = .01), and the 5-year OS is 64% and 77%, respectively (P = .06). With MDATS of greater than 2 (poor prognosis), 5-year FFS for PTCL and BCLCL is 26% and 38%, respectively (P = .03), and the 5-year OS is 24% and 41%, respectively (P = .02). With an IPI of less than 3 (good prognosis), the 5-year FFS for PTCL and BCLCL is 49% and 64%, respectively (P = .001), and the 5-year OS is 55% and 71%, respectively (P = .013). With an IPI greater than 2 (poor prognosis), the 5-year FFS for PTCL and BCLCL is 11% and 35%, respectively (P = .044), and the 5-year OS is 10% and 40%, respectively (P = .011). Multivariate analysis shows that MDATS, IPI, and T-cell phenotype are totally independent and are the most significant predictors of FFS and OS. The 68 PTCLs include 45 PTCLs unspecified, 10 Ki-1 anaplastic (ALCL), 8 angioimmunoblastic, and 5 angiocentric lymphomas. Angiocentrics were usually refractory (1 of 5 remissions only). ALCL rarely relapsed late. We conclude that the immunophenotypic basis of the REAL classification is clinically relevant and that, although other prognostic features also influence outcome, the T-cell phenotype still remains an independent and significant prognostic factor.
Isolation and Characterization of Transformed Human T-Cell Lines Infected by Epstein-Barr Virus
Authors: Groux Hervé , Cottrez Françoise , Montpellier Claire , Quatannens Brigitte , Coll Jean , Stehelin Dominique , Auriault Claude
Published: October 2019

Epstein-Barr virus (EBV) is a human lymphotropic virus whose main targets have traditionally been described as B lymphocytes and epithelial cells. Here we report the isolation and characterization of largely monoclonal transformed human T-cell lines infected by EBV. The transformed T cells expressed CD2, CD3, and either CD4 or CD8 surface molecules and more generally displayed the phenotype of naive T cells with a complete and clonal rearrangement of the T-cell receptor. None of the cell lines expressed B cells, natural killer, or myeloid antigens or had immunoglobulins genes rearrangement. They grew in the absence of growth factor; however, they all secreted interleukin-2 after mitogenic activation. Polymerase chain reaction (PCR) analysis showed the presence of EBV DNA in all these cell lines. Moreover, Southern blot analysis of one of these cell lines shows the presence of circular episomic EBV DNA, and by Northern blot or reverse transcriptase-PCR analysis, only the expression of Epstein-Barr nuclear antigen-1 (EBNA-1) and latent membrane protein-1 (LMP-1) genes was detected. Finally, the complete transformed phenotype of this T-cell line was shown by its injection into nude or recombination activating gene 2 (RAG2)-deficient mice that led to the formation of solid tumors.
Isolation and Characterization of Complement Receptor Type 1 (CR1) Storage Vesicles From Human Neutrophils Using Antibodies to the Cytoplasmic Tail of CR1
Authors: Kumar Anoopa , Wetzler Erica , Berger Melvin
Published: October 2019

Neutrophil (PMN) activation is associated with increased surface expression of several membrane proteins that are translocated from intracellular pools. Indirect evidence suggests that the intracellular storage pools of complement receptor type 1 (CR1) in resting PMN are distinct from traditional granules and may be the secretory vesicles in which albumin is also stored, but it is not known if this compartment is homogeneous or heterogeneous. To isolate and characterize the CR1-containing vesicles, we used antibodies against unique sequences in the cytoplasmic tail of CR1. Affinity-purified IgG was used to adsorb CR1 storage vesicles from the light membrane fraction (γ-band) of nitrogen cavitates of resting PMN. The immunoadsorbent could quantitatively remove the CR1-containing vesicles, whereas control adsorbents with nonimmune IgG showed no specific binding of CR1. Immunoblots of specifically isolated vesicles also showed enrichment of albumin, decay-accelerating factor, FcγRIII, and CR3; whereas HLA class I was not detectable in these vesicles. Enzyme assay of specifically isolated vesicles after treatment with Triton X-100 showed that these vesicles contained most of the cells' latent alkaline phosphatase. An additional population of vesicles containing albumin, but not CR1, and that did not bind to anti-CR1 adsorbent was also identified. Immunoelectron microscopy showed that the specifically isolated vesicles had mean diameters of 0.086 to 0.1 μm and stained positive for CR1 and albumin. These results indicate that CR1 storage vesicles can be isolated with antibodies against the cytoplasmic tail of CR1 and show that these vesicles also contain albumin as well as glycosyl-phosphatidyl inositol-anchored proteins. These results are most compatible with the hypothesis that CR1-containing vesicles have arisen by endocytic retrieval of proteins that had been on the plasma membrane.
FcγRIIA H/R131 Polymorphism, Subclass-Specific IgG Anti-Heparin/Platelet Factor 4 Antibodies and Clinical Course in Patients With Heparin-Induced Thrombocytopenia and Thrombosis
Authors: Arepally Gowthami , McKenzie Steven E. , Jiang Xiao-Ming , Poncz Mortimer , Cines Douglas B.
Published: October 2019

The explanation why only a subset of patients with heparin-induced thrombocytopenia (HIT) develop clinically apparent thromboses (HITT) remains uncertain. It has been proposed that platelet activation induced by cross-linking of FcγRIIA by anti-heparin/platelet factor 4 (PF4) antibodies is central to the pathogenesis of thrombosis. The observation that a common functional polymorphism of FcγRIIA, involving either an arginine (R) or histidine (H) at amino acid 131, may underlie disease susceptibility prompted us to investigate the prevalence of receptor isoforms in patients with HIT and HITT. Furthermore, because these isoforms reportedly differ in their avidity for immune complexes containing human IgG2 , we also analyzed sera from patients with HIT and HITT for the prevalence of various subclass-specific IgG anti-heparin/PF4 antibodies. No difference in the allele frequency of FcγRIIA-H131 or R131 was identified among 13 patients with HIT or 23 with HITT compared with 102 controls (χ2 = 1.21, P = .8). Furthermore, although most patients had IgG2 antibodies (62%), IgG1 was the predominant subclass in 30 of the 34 patients with IgG anti-heparin/PF4 antibodies and in 12 was the exclusive subclass found. Also, there was no association between the concordance of IgG2 anti-heparin/PF4 antibodies and the expression of FcγRIIA-H131 in patients with HITT compared with patients with thrombocytopenia alone. These results make it unlikely that the FcγRIIA-H131 isoform or IgG2 anti-heparin/PF4 antibodies are required to develop HITT, suggesting that factors in addition to cross-linking of FcγRIIA receptors contribute to the pathogenesis of thrombosis in patients with heparin-dependent antiplatelet antibodies.
Differential Activation of the Tyrosine Kinases ZAP-70 and Syk After FcγRI Stimulation
Authors: Taylor Naomi , Jahn Thomas , Smith Susan , Lamkin Thomas , Uribe Lisa , Liu Yenbou , Durden Donald L. , Weinberg Kenneth
Published: October 2019

Engagement of the high-affinity IgG Fc receptor (FcγRI) activates a signal transduction pathway involving tyrosine phosphorylation of associated kinases. We compared the activation of the related protein tyrosine kinases (PTKs), Syk and ZAP-70, in FcγRI-mediated signaling. Cross-linking of the FcγRI multimeric receptor in monocytic cells results in tyrosine phosphorylation of the FcεRIγ subunit and association of Syk with this complex. We stably introduced ZAP-70 via a retroviral vector into two monocytic cell lines, U937 and THP-1, which normally do not express ZAP-70. Neither Syk nor MAP kinase activation was affected by the presence of ZAP-70. Although transduced ZAP-70 had in vitro kinase activity and associated with FcεRIγ after receptor aggregation, it was not tyrosine phosphorylated. In contrast, both ZAP-70 and Syk were phosphorylated in a T-cell line in which their respective levels of expression were similar to those detected in U937/ZAP-70 cells. Therefore, these results suggest that requirements for Syk and ZAP-70 phosphorylation are distinct in a monocytic cell context.
Ectopic Expression of the Erythropoietin Receptor in a Murine Interleukin-6–Dependent Plasmacytoma Cell Line (TEPC-2027) Confers Proliferative Responsiveness to Erythropoietin
Authors: Féger Frédéric , Dubart Anne , Lacout Catherine , Dusanter-Fourt Isabelle , Mayeux Patrick , Vainchenker William , Duménil Dominique
Published: October 2019

To compare the signal transduction pathways used by erythropoietin (Epo) and interleukin-6 (IL-6), the cDNA for the murine Epo receptor (Epo-R) was introduced into an IL-6–responsive plasmacytoma cell line (TEPC-2027) by retrovirally mediated gene transfer. G418-resistant clones were amplified in IL-6 and studied for their ability to grow and differentiate in response to Epo. Epo-R synthesized from the viral gene showed the same affinity for Epo as did the receptor on erythroid cells; however, the numbers of Epo receptors expressed on the cell membrane varied among clones. After a delay of 3 to 5 days in the presence of Epo, all the clones studied proliferated as well in response to Epo as in response to IL-6. In response to IL-6, Stat3 was activated and JunB mRNA was accumulated, whereas in response to Epo, Jak2 and Stat5 were activated and JunB mRNA was not accumulated in Epo-R–expressing TEPC (Epo-R/TEPC) cells. These results suggest that Epo and IL-6 transduced their proliferative signals through different pathways. Further studies showed that, in Epo-R/TEPC cells, Epo neither induces the synthesis of erythroid-specific mRNA nor modifies the synthesis of γ1 Ig heavy chain, suggesting that ectopic expression of the Epo-R in plasmacytoma cells does not modify their differentiative potential. The data show that Epo induces a proliferative response without differentiation providing a new cellular model for evaluating molecular events specific for proliferation.
Relationship of p53, bcl-2, and Tumor Proliferation to Clinical Drug Resistance in Non-Hodgkin's Lymphomas
Authors: Wilson Wyndham H. , Teruya-Feldstein Julie , Fest Thierry , Harris Cynthia , Steinberg Seth M. , Jaffe Elaine S. , Raffeld Mark
Published: October 2019

Although the cause(s) of clinical drug resistance in non-Hodgkin's lymphomas (NHL) is unknown, in vitro studies suggest that abnormalities of the p53 gene, bcl-2 overexpression, and low tumor proliferation rates may increase chemotherapy resistance. We analyzed tumor tissue from 75 patients with relapsed/refractory NHL (Working Formulation A through H) for p53 mutation/overexpression (abnormality), bcl-2 expression, and tumor proliferation and correlated them with multiple clinical characteristics, response to therapy, disease-free survival, and overall survival (OS). All tumor biopsy specimens were obtained within 6 weeks of treatment with EPOCH (infusional etoposide, vincristine, and doxorubicin and bolus prednisone and cyclophosphamide) chemotherapy. Overall, 16 (21%) tumors had a p53 abnormality. Of 13 tumors with overexpression, mutations were confirmed by sequence analysis in 11, and, in 44 tumors without overexpression, 3 showed mutations. A multivariate analysis showed that tumors with a p53 abnormality were more likely to be drug resistant than tumors with normal p53 (56% v 17%; P2 = .008) and to have a shorter median progression-free survival (PFS; 2.1 v 8.2 months; P2 = .008) and OS (11.7 v 21.5 months; P2 = .038), respectively. The presence of a p53 abnormality did not correlate with any clinical characteristic, bcl-2 expression, or tumor proliferation. A significant correlation was found between low tumor proliferation and drug resistance in a univariate (P2 < .006) but not multivariate analysis. Patients with tumor proliferation of less than 80% were significantly more likely to have no response to therapy (31% v 6%) or to fail to achieve a complete response (16% v 44%) and tended to have shorter PFS and OS than did patients with higher proliferation. No significant association was found between bcl-2 expression and drug resistance, PFS or OS, although patients with intermediate-grade histologies and high bcl-2 expression tended to be drug resistant as compared with low level expressors (P2 < .065). Of interest was the finding of a significant association between high bcl-2 and low tumor proliferation (P2 = .0045). In studies that have found an association between high bcl-2 expression and short PFS, bcl-2 may have been a surrogate for low tumor proliferation. Further studies are warranted to examine this question. These results suggest that p53 mutation and low tumor proliferation, but not bcl-2, may be important causes of clinical drug resistance in NHL.
Evidence for the Participation of Interleukin-2 (IL-2) and IL-4 in the Regulation of Autonomous Growth and Tumorigenesis of Transformed Cells of Lymphoid Origin
Authors: Hassuneh Mona R. , Nagarkatti Prakash S. , Nagarkatti Mitzi
Published: October 2019

In the current study, we investigated the role of interleukin-2 (IL-2) and IL-4 as autocrine growth factors responsible for autonomous growth of four murine tumor cell lines: LSA, a radiation leukemia virus-induced T-cell lymphoma; EL-4, a chemically triggered T-cell lymphoma; PE-3T, a T-cell line that underwent spontaneous transformation ex vivo; and P815, a mastocytoma. All tumor cell lines screened constitutively expressed IL-2 receptor (IL-2R) and IL-4R genes. However, only LSA and PE-3T cells expressed IL-2 and IL-4 genes constitutively, whereas EL-4 and P815 tumor cells expressed only IL-4 but not IL-2. Monoclonal antibodies (MoAbs) against IL-2, IL-4, or a combination of these, as well as MoAbs against IL-2R significantly inhibited the proliferation of LSA but not that of other tumor cell lines ex vivo. To exclude the possibility that, in other tumor cell lines, the autocrine growth factor may interact with its receptor within the cell, the ability of antisense phosphorothioate oligonucleotides to inhibit the growth of the tumor cells was tested. The antisense phosphorothioate oligonucleotides specific for IL-2, IL-4, IL-2Rβ, or IL-2Rγ chains, added in culture, could markedly inhibit the growth of LSA but not that of the other tumor cell lines screened. Inasmuch as IL-2Rβ and IL-2Rγ subunits also serve as a component of the receptors for IL-4, IL-7, IL-9, and IL-15, the above data suggested that such cytokine redundancy was not responsible for autonomous growth of the other tumor cell lines. Addition of exogenous IL-2 or IL-4 to the tumor cell cultures caused significant enhancement in the proliferation of PE-3T cells, whereas other cell lines were either not significantly affected or slightly inhibited from growing. Interestingly, the LSA tumor growth in nude mice was significantly inhibited after treatment of these mice with a combination of MoAbs against IL-2 and IL-4. Together, our studies show for the first time that IL-2 and IL-4 may serve as autocrine growth factors in the autonomous proliferation of tumor cells, particularly those that are retrovirally induced. Second, some tumor cell lines, despite expressing certain cytokines and their receptors constitutively, may not depend exclusively on such factors for autocrine growth.
The Amino Terminal Lectin-Like Domain of Thrombomodulin Is Required for Constitutive Endocytosis
Authors: Conway Edward M. , Pollefeyt Saskia , Collen Desiré , Steiner-Mosonyi Marta
Published: October 2019

Thrombomodulin (TM) is a multidomain protein that serves as a cofactor in a major natural anticoagulant system. To further characterize the structure-function of TM, we have transfected COS cells with different truncated forms of TM. In the first form, COS cells expressing TM that lacks the putative signal peptide (17 residues); the lectin-like, hydrophobic N-terminal domain (226 residues); and 12 residues of the first epidermal growth factor (EGF )-like repeat (COSdel.238 cells) were found to function normally with respect to TM transport to the cell surface and thrombin-dependent protein C activation. However, in contrast to wild-type TM, as visually studied by immunofluorescence and immunogold electron microscopy, the COSdel.238 cells did not constitutively internalize anti-TM–TM or thrombin-TM complexes. To identify the region responsible for mediating the endocytic process, deletant forms of TM lacking either the lectin-like region (residues 2-155) or the hydrophobic region of the N-terminal domain (residues 161-202) were expressed in COS cells (COSdel.2-155 and COSdel.161-202, respectively). Protein C cofactor activity was maintained in both cells. Although the COSdel.161-202 cells behaved similarly to wild-type TM-transfected cells, visual studies showed a lack of constitutive internalization of thrombin-TM or anti-TM–TM complexes in the COSdel.2-155 cells. We conclude that the lectin-like domain of human TM serves to regulate cell surface expression of TM via the endocytic route and therefore may also play a major physiologic role in controlling intracellular and extracellular accumulation of thrombin in a variety of biologic systems.
Globin Gene Switching in Transgenic Mice Carrying HS2-Globin Gene Constructs
Authors: Roberts N.A. , Sloane-Stanley J.A. , Sharpe J.A. , Stanworth S.J. , Wood W.G.
Published: October 2019

We have examined the pattern of human globin gene switching in transgenic mice containing three different γ and β gene constructs (HS2GγAγδβ, HS2Aγβneo, and HS2Aγenβ) and compared the results with previously described transgenics (HS2Aγβ, HS2GγAγ-117δβ, and LCRεGγAγδβ). Developmental regulation was observed in all cases with identical patterns in lines bearing the same construct. Three different patterns of switching were observed: LCRεGγAγδβ and HS2Aγβneo mice switched rapidly, HS2GγAγδβ and HS2GγAγ-117δβ at an intermediate rate, and HS2Aγβ and HS2Aγenβ mice showed delayed switching, with a plateau in late fetal-early neonatal life and readily detectable levels of γ mRNA in adults. No difference was observed in the time of switching of the HS2GγAγδβ mice compared with those with the Aγ-117 hereditary persistence of fetal hemoglobin mutation, but adult levels of γ mRNA were significantly higher (≈5%) in lines carrying the mutation than in those without (≈1%). Reversion to the rapid switch of the LCRεGγAγδβ mice was observed in three lines with the HS2Aγβ neo construct in which expression of the tk-neo gene was approximately equal to that of the globin genes. The inclusion of the Aγ enhancer in HS2Aγβ mice did not alter the pattern of switching, or reduce the relatively high levels of γ mRNA in these lines. However, unlike other HS2 mice, the combination of HS2 and the Aγ enhancer resulted in copy number-dependent expression in HS2Aγenβ lines, with intrauterine death at ≈12.5 days gestation at high copy numbers. These results demonstrate that numerous elements throughout the β globin gene cluster interact to produce the correct pattern of developmental regulation of these genes. Furthermore, extinction of γ gene expression in adult life is not completely autonomous and is incomplete when HS2 is the only LCR element present.
Defective Transport Is a Common Mechanism of Acquired Methotrexate Resistance in Acute Lymphocytic Leukemia and Is Associated With Decreased Reduced Folate Carrier Expression
Authors: Gorlick Richard , Goker Erdem , Trippett Tanya , Steinherz Peter , Elisseyeff Yaroslav , Mazumdar Madhu , Flintoff Wayne F. , Bertino Joseph R.
Published: October 2019

Methotrexate (MTX) transport was examined in 27 patients with untreated acute lymphocytic leukemia (ALL) and 31 patients with relapsed ALL using a previously described fluorescent MTX analog (PT430) displacement assay (Blood 80:1158, 1992). Only 13% of untreated patients were considered to have impaired MTX transport, whereas more than 70% of relapsed patients had evidence of impaired MTX transport. To further characterize the basis for this defect, Northern analyses for the reduced folate carrier (RFC) were performed on the RNA available from the leukemic blasts of 24 patients in whom MTX transport had been measured. Six of nine samples with impaired MTX transport had decreased RFC expression (one had no detectable RFC expression), while three had no decrease in RFC expression. None of 15 samples with normal MTX transport had decreased RFC expression. A reverse-transcriptase polymerase chain reaction (RT-PCR) assay was developed to quantitate RFC mRNA expression more accurately. Decreased RFC expression was demonstrated in six of the nine samples with impaired MTX transport, confirming the results obtained by Northern blot. These data indicate decreased RFC expression associated with impaired MTX transport is observed in relapsed ALL following treatment with MTX-containing therapy.
A Novel Specific Immunoassay for Plasma Two-Chain Factor VIIa: Investigation of FVIIa Levels in Normal Individuals and in Patients With Acute Coronary Syndromes
Authors: Philippou Helen , Adami Antonella , Amersey Rajiv A. , Stubbs Peter J. , Lane David A.
Published: October 2019

We report the development of an enzyme-linked immunosorbent assay (ELISA) that is specific for factor VIIa (FVIIa). This assay uses a neoantigen specific capture antibody directed to the amino acid peptide sequence N terminal to the FVII cleavage activation site. The antibody exhibits ∼3,000-fold greater reactivity to FVIIa than FVII on a molar basis. Experiments using plasma with added (exogenous) human FVIIa gave quantitative recovery in the ELISA over a range of 0.20 to 3.2 ng/mL of FVIIa. The intra- and inter-assay coefficient of variation (CVs) of the ELISA are 4.5% and 9.8%, respectively. The ELISA shows excellent correlation (r = .99) with a functional assay (using recombinant soluble tissue factor) in detecting FVIIa added to plasma over the range 0.05 to 18.0 ng/mL. However, a major discrepancy exists between the two assays when normal endogenous plasma concentrations of FVIIa are measured. Using normal plasma (n = 14) the functional assay reported 3.10 ± 0.30 ng/mL (mean ± SE) whereas only 0.025 ± 0.010 ng/mL was detected in the same samples by the immunoassay. Patients (n = 43) presenting with acute coronary syndromes (myocardial infarction and unstable angina) exhibited elevations (P < .05) in immunologically detected FVIIa, 0.093 ± 0.013 ng/mL (mean ± SE) compared to patient controls (n = 20) contemporaneously admitted with noncardiac chest pain, 0.048 ± 0.007 ng/mL (mean ± SE). These elevations in the acute coronary syndromes were accompanied by increased (P < .05) and correlating prothrombin fragment F1 + 2 levels (Spearman correlation coefficient rs = .4, P < .01), demonstrating that thrombin generation is certainly associated with, and may even be caused by, extrinsic pathway activation.
Superiority of Tandem Autologous Transplantation Over Standard Therapy for Previously Untreated Multiple Myeloma
Authors: Barlogie Bart , Jagannath Sundar , Vesole David H. , Naucke Sally , Cheson Bruce , Mattox Sandy , Bracy Dwayne , Salmon Sydney , Jacobson Joth , Crowley John , Tricot Guido
Published: October 2019

Virtually no progress has been made during more than 2 decades of clinical trials for multiple myeloma (MM) involving standard therapy (ST). Recent studies suggest that dose intensification requiring hematopoietic stem cell support results in higher complete response (CR) rates and extended disease control. “Total Therapy” (TT) consisting of non–cross-resistant induction regimens, followed by a double autotransplant (AT) procedure, was administered to 123 untreated patients with symptomatic MM. Upon hematologic recovery, interferon (IFN) maintenance (3 million units [MU]/m2 subcutaneously thrice weekly) was given until disease recurrence/progression. Results were compared with the outcome of untreated patients receiving ST according to Southwest Oncology Group (SWOG) trials. One hundred sixteen pair mates were selected from both TT and among 1,123 patients to match for the major prognostic features. TT induced CR in 40% of all 123 patients (intent-to-treat). By 12 months, 7% had died, including 4% from treatment-related complications. With a median follow-up of 31 months, median durations of event-free survival (EFS) and overall survival (OS) are 49 and 62+ months, respectively. Abnormalities of chromosomes 11q and 13 were associated with inferior outcome, whereas CR within 6 months after induction was a favorable prognostic feature for both EFS and OS. In comparison to ST, TT induced higher PR rates (85% v 52%, P < .0001) (CR rates not available on SWOG trials) and extended EFS (49 v 22 months, P = .0001) and OS (62+ v 48 months, P = .01). Compared to ST, dose intensification with double AT markedly augments tumor cytoreduction, effecting not only higher CR rates but also significantly extending EFS and OS in previously untreated patients with MM.
Comparison Between Conventional Salvage Therapy and High-Dose Therapy With Autografting for Recurrent or Refractory Hodgkin's Disease
Authors: Yuen Alan R. , Rosenberg Saul A. , Hoppe Richard T. , Halpern Jerry D. , Horning Sandra J.
Published: October 2019

Sixty patients with Hodgkin's disease, refractory to or at first recurrence after chemotherapy, received cytoreductive therapy followed by high-dose etoposide, cyclophosphamide and either total body irradiation or carmustine and autografting (median follow-up, 3.6 years; range, 1.1 to 7.5 years). A matched conventional salvage group of 103 patients was selected from patients treated at Stanford University Medical Center between January 1976 and January 1989 (median follow-up, 10.3 years; range, 3.0 to 15.7 years). Overall survival (OS), event-free survival (EFS), and freedom from progression (FFP) at 4 years follow-up favored patients who received high-dose therapy compared with conventional salvage treatment (OS: 54% v 47%, P = .25; EFS: 53% v 27%, P < .01; FFP: 62% v 32%, P < .01). In Cox regression analysis, response to cytoreductive or salvage therapy and B symptoms at relapse were the most important predictors of OS. The use of high-dose therapy at relapse, a longer duration of remission, and favorable response to cytoreductive or salvage therapy were most predictive of superior FFP and EFS. These data from a single institution comparing conventional and high-dose therapy in matched patients demonstrate an advantage for high-dose therapy and autografting in the sustained control of Hodgkin's disease. As with primary therapy, it is difficult to demonstrate a statistically significant survival advantage, despite an apparently superior cure rate. However, patients failing induction therapy or relapsing within 1 year benefited significantly from high-dose therapy by all outcome measures (OS, EFS, FFP). As the transplant-related mortality rates decline in Hodgkin's disease, it is predicted that cure rates and late effects will become ultimate determinants of the success of high-dose therapy and autografting.
Missense Mutations in the Fas Gene Resulting in Autoimmune Lymphoproliferative Syndrome: A Molecular and Immunological Analysis
Authors: Bettinardi Alessandra , Brugnoni Duilio , Quiròs-Roldan Eugenia , Malagoli Alberto , La Grutta Stefania , Correra Antonio , Notarangelo Luigi D.
Published: October 2019

Programmed cell death (or apoptosis) is a physiological process essential to the normal development and homeostatic maintenance of the immune system. The Fas/Apo-1 receptor plays a crucial role in the regulation of apoptosis, as demonstrated by lymphoproliferation in MRL-lpr/lpr mice and by the recently described autoimmune lymphoproliferative syndrome (ALPS) in humans, both of which are due to mutations in the Fas gene. We describe a novel family with ALPS in which three affected siblings carry two distinct missense mutations on both the Fas gene alleles and show lack of Fas-induced apoptosis. The children share common clinical features including splenomegaly and lymphadenopathy, but only one developed severe autoimmune manifestations. In all three siblings, we demonstrated the presence of anergic CD3+CD4−CD8− (double negative, [DN]) T cells; moreover, a chronic lymphocyte activation was found, as demonstrated by the presence of high levels of HLA-DR expression on peripheral CD3+ cells and by the presence of high levels of serum activation markers such as soluble interleukin-2 receptor (sIL-2R) and soluble CD30 (sCD30).
Integration Patterns of HTLV-I Provirus in Relation to the Clinical Course of ATL: Frequent Clonal Change at Crisis From Indolent Disease
Authors: Tsukasaki Kunihiro , Tsushima Hideki , Yamamura Masaomi , Hata Tomoko , Murata Ken , Maeda Takahiro , Atogami Sunao , Sohda Hisashi , Momita Saburo , Ideda Shuichi , Katamine Sigeru , Yamada Yasuaki , Kamihira Shimeru , Tomonaga Masao
Published: October 2019

We examined human T-lymphotropic virus type I (HTLV-I) DNA integration in 68 patients with adult T-cell leukemia/lymphoma (ATL) by Southern blotting using EcoRI, which does not cut within the 9 kb of the genome and probes for pX and gag-pol region of HTLV-I. We detected defective proviral integration as a monoclonal band of various sizes with the pX but not with the gag-pol probe, or a monoclonal band of less than 9 kb with the pX probe, in 20 patients (29.4%). These were designated defective (D) type. With both probes, a single band greater than 9 kb was detected in 34 (50.0%), designated complete (C) type, and two or more bands greater than 9 kb, were designated multiple (M) type, in 14 (20.6%). Advanced age, a high LDH value, and hypercalcemia were more frequent in D type patients. The median survival time (MST) was 6.8, 24.4, and 33.3 months, for D, C, and M types, respectively (log rank P = .006). Among 52 sequentially examined patients, the HTLV-I integration patterns changed in 4 (7.5%). In three of these four, the rearrangements of the T-cell receptor (TCR)b gene concomitantly changed, suggesting the appearance of a new ATL clone. Another patient had the same rearrangement of the TCRb gene, indicating clonal evolution. The HTLV-I integration pattern changed at crisis from indolent to aggressive ATL in three patients. These findings suggested that the HTLV-I integration patterns have clinical implications in ATL pathophysiology. In contrast to the clonal evolution characteristic of the multistep carcinogenesis of most human malignancies, the frequent clonal change of ATL at crisis is a peculiar phenomenon, probably reflecting the emergence of multiple premalignant clones in viral leukemogenesis as suggested in Epstein-Barr virus associated lymphomagenesis in the immunocompromised host.
Augmented Production of Interleukin-6 by Normal Human Osteoblasts in Response to CD34+ Hematopoietic Bone Marrow Cells In Vitro
Authors: Taichman Russell S. , Reilly Marcelle J. , Verma Rama S. , Emerson Stephen G.
Published: October 2019

Based on anatomic and developmental findings characterizing hematopoietic cells in close approximation with endosteal cells, we have begun an analysis of osteoblast/hematopoietic cell interactions. We explore here the functional interdependence between these two cell types from the standpoint of de novo cytokine secretion. We determined that, over a 96-hour period, CD34+ bone marrow cells had no significant effect on osteoblast secretion of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, or transforming growth factor-β1 , but in some experiments minor increases in leukemia inhibitory factor levels were observed. However, when CD34+ bone marrow cells were cocultured in direct contact with osteoblasts, a 222% ± 55% (range, 153% to 288%) augmentation in interleukin-6 (IL-6) synthesis was observed. The accumulation of IL-6 protein was most rapid during the initial 24-hour period, accounting for nearly 55% of the total IL-6 produced by osteoblasts in the absence of blood cells and 77% of the total in the presence of the CD34+ cells. Cell-to-cell contact does not appear to be required for this activity, as determined by coculturing the two cell types separated by porous micromembranes. The identity of the soluble activity produced by the CD34+ cells remains unknown, but is not likely due to IL-1β or tumor necrosis factor-α, as determined with neutralizing antibodies. To our knowledge, these data represent the first demonstration that early hematopoietic cells induce the production of molecules required for the function of normal bone marrow microenvironments, in this case through the induction of hematopoietic cytokine (IL-6) secretion by osteoblasts.
A Collagen-Like Peptide Stimulates Tyrosine Phosphorylation of syk and Phospholipase Cγ2 in Platelets Independent of the Integrin α2β1
Authors: Asselin Judith , Gibbins Jonathan M. , Achison Marcus , Lee Young Han , Morton Laurence F. , Farndale Richard W. , Barnes Michael J. , Watson Steve P.
Published: October 2019

Activation of platelets by collagen is mediated through a tyrosine kinase-dependent pathway that is associated with phosphorylation of the Fc receptor γ chain, the tyrosine kinase syk, and phospholipase Cγ2 (PLCγ2). We recently described a collagen-related triple-helical peptide (CRP) with the sequence GCP*(GPP*)GCP*G (single letter amino acid code: P* = hydroxyproline; Morton et al, Biochem J 306:337, 1995). The cross-linked peptide is a potent stimulus of platelet activation but, unlike collagen, does not support α2β1-mediated, Mg2+-dependent adhesion, suggesting that its action is independent of the integrin α2β1 . This finding suggests the existence of a platelet receptor other than α2β1 that underlies activation. In the present study, we show that CRP stimulates tyrosine phosphorylation of the same pattern of proteins in platelets as collagen, including syk and PLCγ2. Protein tyrosine phosphorylation induced by CRP is not altered in the absence of Mg2+ or the presence of monoclonal antibodies (MoAbs) to the integrin α2β1 (MoAb 6F1 and MoAb 13), conditions that prevent the interaction of collagen with the integrin. In contrast, phosphorylation of syk and PLCγ2 by collagen is partially reduced by MoAb 6F1 and MoAb 13 or by removal of Mg2+. This may reflect a direct role of α2β1 in collagen-induced signaling events or an indirect role in which the integrin facilitates the binding of collagen to its signaling receptor. The results show an α2β1-independent pathway of platelet activation by CRP that involves phosphorylation of syk and PLCγ2. This pathway appears to contribute to platelet activation by collagen.
Thymidine Kinase (TK) Gene-Transduced Human Lymphocytes Can Be Highly Purified, Remain Fully Functional, and Are Killed Efficiently With Ganciclovir
Authors: Munshi N.C. , Govindarajan R. , Drake R. , Ding L.M. , Iyer R. , Saylors R. , Kornbluth J. , Marcus S. , Chiang Y. , Ennist D. , Kwak L. , Reynolds C. , Tricot G. , Barlogie B.
Published: October 2019

A graft-versus-leukemia (GVL) effect has been considered a major factor responsible for cures in patients with hematologic malignancies undergoing allogeneic bone marrow transplantation; however, associated graft-versus-host disease (GVHD) results in significant morbidity and mortality. T-cell depletion reduces the incidence and severity of GVHD but eliminates, at least partially, the GVL effect. Reinfusion of donor T lymphocytes at relapse posttransplantation can induce a potent antitumor response, but GVHD still occurs in the majority of patients. Prior transduction of T lymphocytes with the suicide gene, the viral thymidine kinase (TK), permits specific cell kill on administration of ganciclovir (GCV). Therefore, infusion of TK-transduced T lymphocytes may induce GVL effect and allow for their subsequent selective elimination in case GVHD develops. To evaluate the efficacy and feasibility of this promising approach, anti-CD3–stimulated primary human lymphocytes cultured in interleukin-2 were TK-transduced by a retroviral vector carrying both TK and neomycin-resistance genes. After selection in G418, more than 90% of the cells contained the TK gene as shown by a semiquantitative polymerase chain reaction. In addition, 1 to 5 days of GCV exposure, at clinically achievable concentrations of 20 to 50 μmol/L, induced ≥90% killing of G418-selected cells without affecting nontransduced cells. Correlation of the extent of T-cell kill and the proportion of TK-gene–transduced cells is consistent with the absence of a bystander effect. Transduced cells were CD3+ and either CD8+ or CD4+ and retained functional properties of untransduced cells. In vivo administration of GCV prevented tumor development after subcutaneous injection of TK-transduced murine myeloma cells (MOPC-11), whereas such an effect was not observed on injection of untransduced cells into the opposite flank. Our studies provide critical information that (1) adequate numbers of TK-transduced lymphocytes can be selected efficiently with ≥90% purity, (2) selected cells remain functional, (3) 24 hours of exposure to GCV at clinically achievable concentration effects ≥90% killing of selected cells, and (4) GCV is effective in vivo in killing TK-transduced cells. Based on these data, a clinical study has been initiated in patients with multiple myeloma with persistent or relapsing disease after T-cell–depleted allogeneic transplants.