bluuebunny/crossref_metadata_2025 huggingface dataset:
Fas-mediated apoptosis in cultured human eosinophils
Authors: Druilhe A , Cai Z , Haile S , Chouaib S , Pretolani M
Published: October 2019

Previous studies have shown that cytokine-dependent eosinophils undergo apoptosis, yet the mechanisms governing this phenomenon remain obscure. Fas antigen is a transmembrane glycoprotein belonging to the tumor necrosis factor receptor family. Cross-linking of Fas antigen in numerous cell types leads to apoptosis. In the present study, we examined the potential role of Fas antigen in the apoptosis of purified blood eosinophils from healthy donors. Cytokine-deprived eosinophils exhibited a time-dependent loss in viability, accompanied by an increase in the number of apoptotic nuclei and in the expression of Fas antigen and its mRNA, as shown by flow cytometry and reverse transcriptase-polymerase chain reaction, respectively. Cross-linking of Fas antigen with an agonistic anti-Fas monoclonal antibody (MoAb) induced a dose- and time-dependent increase in the number of apoptotic nuclei. Furthermore, using an in vitro coculture system, we showed engulfment of anti-Fas MoAb-treated eosinophils by monocyte-derived macrophages. Finally, incubation of eosinophils with the corticosteroid, dexamethasone, induced apoptosis and augmented that triggered by anti-Fas MoAb. Together, these observations suggest that Fas antigen expression and activation is involved in the apoptosis of human eosinophils and may contribute to the resolution of inflammatory allergic reactions in which eosinophil accumulation is a prominent feature.
Involvement of peripheral benzodiazepine receptors in the protection of hematopoietic cells against oxygen radical damage
Authors: Carayon P , Portier M , Dussossoy D , Bord A , Petitpretre G , Canat X , Le Fur G , Casellas P
Published: October 2019

Several putative functions have been attributed to the peripheral benzodiazepine receptor (PBR), but its precise physiologic role has not been elucidated. In the present study, we investigated PBR function by quantifying this receptor in leukocyte subsets from healthy donors and in leukemic blasts from lymphoid and myeloid lineages. Using a monoclonal antibody (MoAb) directed against the human PBR and a quantitative flow cytometric assay, we found that phagocytic cells from healthy donors displayed a higher level of PBRs than lymphocytes or natural killer (NK) cells. Among the lymphoid lineage, thymocytes and IgD-negative B cells expressed the lowest levels. However, because of the wide heterogeneity of PBR levels among 42 acute or chronic lymphoid and myeloid leukemias, it was not possible to assign PBR expression to a stage of maturation or a cell lineage. Although the PBR displayed a mitochondrial subcellular localization, its expression was not correlated with the mitochondrial content, suggesting a modulation of PBR density at the level of the mitochondria. This modulation was confirmed when we studied in detail the PBR expression during T-cell development by both flow cytometry and confocal microscopy. We found that the PBR was expressed with a bimodal profile during T-cell development, identical to the one observed with the proto-oncogene, Bcl- 2. The high similarity in the expression of both the PBR and the Bcl-2 proto-oncogene in T-cell and B-cell subsets, their common mitochondrial localization, and the observation of high quantities of PBR in phagocytic cells, which are known to produce high levels of radical oxygen species, suggested that PBRs may participate in an antioxidant pathway. Indeed, a strong correlation was established between the ability of hematopoietic cell lines to resist H202 cytotoxicity and their level of PBR expression. Demonstration of the role of PBR in the protection against H202 was obtained by transfecting JURKAT cells with the human PBR cDNA. Transfected cells exhibited increased resistance to H202 compared with wild-type cells, suggesting that PBR may prevent mitochondria from radical damages and thereby modulate apoptosis in the hematopoietic system.
Stem cell factor enhances interleukin-3 dependent induction of 68-kD calmodulin-binding protein and thymidine kinase activity in NFS-60 cells
Authors: Reddy GP , Quesenberry PJ
Published: October 2019

Stem cell factor (SCF) is known to act synergistically with other hematopoietic factors in increasing the colony formation of hematopoietic progenitor cells. We have shown that interleukin-3 (IL-3)- dependent proliferation of NFS-60 cells is associated with the induction of a specific calmodulin-binding protein of about 68 kD (CaM- BP68). To evaluate the relationship between proliferative stimulation and the induction of CaM-BP68 by cytokines, we examined whether the increased proliferative potential of NFS-60 cells in response to SCF is reflected in an increased induction of the CaM-BP68. We observed that SCF alone has a limited effect on proliferative stimulation and on the induction of CaM-BP68 in factor-deprived NFS-60 cells. However, when combined with IL-3, granulocyte colony-stimulating factor (G-CSF), or IL-6, it caused a significant increase in cytokine-dependent proliferative stimulation, as well as in the induction of CaM-BP68. Furthermore, an increase in IL-3-dependent induction of CaM-BP68 in the presence of SCF coincided with a corresponding increase in thymidine kinase activity, whose expression is linked to G1/S transition of the cells. At low concentrations SCF caused a synergistic increase in IL-3- dependent induction of both CaM-BP68 and thymidine kinase activity. In contrast to the changes in CaM-BP68 and thymidine kinase activity, no significant changes in DNA polymerase alpha were observed in factor- deprived NFS-60 cells in response to IL-3 and/or SCF. These observations suggest an increased expression of CaM-BP68 and thymidine kinase are associated with the synergistic effect of SCF on factor- dependent proliferation of hematopoietic progenitor cells.
Expression of the interleukin-2 receptor gamma chain on cord blood mononuclear cells
Authors: Saito S , Morii T , Umekage H , Makita K , Nishikawa K , Narita N , Ichijo M , Morikawa H , Ishii N , Nakamura M , Sugamura K
Published: October 2019

Lymphocytes in umbilical cord blood and neonatal peripheral blood have been shown to have less ability in an immune reaction. In our present experimental approach to address this issue, we made use of the cord blood of full-term birth infants to investigate the expression of the interleukin- 2 receptor gamma (IL-2Rgamma) chain that is shared with receptors for IL-4, IL-7, IL-9, and IL-15 as well as IL-2. The gamma chain expression in cord blood lymphocytes was about one-third that in the lymphocytes of adults, whereas no significant difference between cord blood and adult monocytes was observed. A reduced expression of the gamma chain was observed in all of the CD4+ T cells, CD8+ T cells, gamma-delta T cells, B cells, CD16+ natural killer (NK) cells, and CD56(bright) NK cells of the cord blood lymphocytes. The reduced gamma chain expression reached two-thirds of that in adults after 3 days of culture in vitro and in infants 3 days after birth, thus implying that the increase in the gamma chain may significantly contribute to the prevention of neonatal infection.
Radioimmunotherapy of relapsed B-cell lymphoma with yttrium 90 anti- idiotype monoclonal antibodies
Authors: White CA , Halpern SE , Parker BA , Miller RA , Hupf HB , Shawler DL , Collins HA , Royston I
Published: October 2019

Tumor-specific anti-idiotype (anti-Id) monoclonal antibodies (MoAbs) to B-cell lymphomas have been administered to patients, resulting in significant clinical responses. However, clinical responses have been limited by the emergence of Id-negative lymphoma. To overcome the problem of tumor heterogeneity, we conducted a pilot evaluation of the safety and effectiveness of yttrium 90 (90Y)-labeled anti-Id and shared Id (sId) MoAbs in non-Hodgkin's B-cell lymphoma. Nine patients with relapsed B-cell lymphoma in whom tumor was successfully targeted with 111In-labeled anti-Id MoAb were treated with 90Y-labeled anti-Id MoAb. A total of 19 courses (one to four per patient) were administered using 1,000 to 2,320 mg unlabeled clearing MoAb and 10 to 54 mCi 90Y MoAb per patient. Two of nine patients had a complete response, one a partial response, three stable disease, and three disease progression. Time to progression varied from 1 to 12 months. Toxicities were predominately hematologic, and only one patient developed infection and required transfusion. At progression, three of five assessable patients had Id- positive lymphoma and two had Id-negative lymphoma. Human antimouse antibodies (HAMA) did not develop in the patients after treatment. 90Y anti-Id MoAbs demonstrated excellent in vivo stability, produced significantly tumor regression in three of nine patients, exhibited acceptable toxicities, and elicited no HAMA formation. Further investigation of repetitive, low-dose 90Y anti-Id and MoAb therapy is warranted; however, the advantages of a pan B MoAb may prove the latter to be the agent of choice for the radio immunotherapy of B-cell lymphoma.
Studies of the activation of factor VII bound to tissue factor [see comments]
Authors: Rao LV , Williams T , Rapaport SI
Published: October 2019

Experiments were performed to evaluate activation of factor VII bound to relipidated tissue factor (TF) in suspension and to TF constitutively expressed on the surface of an ovarian carcinoma cell line (OC-2008). Activation was assessed by measuring cleavage of 125I- factor VII and by the ability of unlabeled factor VII to catalyze activation of a variant factor IX molecule that, after activation, cannot back-activate factor VII. Factor Xa was found to effectively activate factor VII bound to TF relipidated in either acidic or neutral phospholipid vesicles. Autoactivation of factor VII bound to TF in suspension was dependent on the preparation of TF apoprotein used and the technique of its relipidation. This highlights the need for caution in extrapolating data from TF in suspension to the activation of factor VII bound to cell surfaces during hemostasis. A relatively slow activation of factor VII bound to OC-2008 monolayers in the absence of added protease was observed consistently. Antithrombin in the presence or absence of heparin prevented this basal activation, whereas TF pathway inhibitor (TFPI/factor Xa complexes had only a limited inhibitory effect. Adding a substrate concentration of factor X markedly enhanced basal activation of factor VII, but both TFPI/factor Xa and antithrombin/heparin abolished this enhancement. Overall, our data are compatible with the hypothesis that not all factor VII/TF complexes formed at a site of tissue injury are readily activated to factor VIIa (VIIa)/TF complexes during hemostasis. The clinical significance of this is discussed.
Inhibition of angiogenesis by interleukin-12 is mediated by the interferon-inducible protein 10
Authors: Sgadari C , Angiolillo AL , Tosato G
Published: October 2019

Interleukin 12 (IL-12), a multifunctional cytokine produced by macrophages and B-cell lines, induces interferon-gamma (IFN-gamma) production, stimulates growth of both T and natural killer cells, promotes Th1-type helper T-cell responses, and inhibits neovascularization. Because the human interferon-inducible protein 10 (IP-10) can also inhibit neovascularization, we tested whether IP-10, induced by IL-12 through the intermediate IFN-gamma, might be a mediator of IL-12 angiogenesis inhibition. We report here that murine IL-12 profoundly inhibited basic fibroblast growth factor (bFGF)- induced Matrigel neovascularization in vivo, and that this effect of IL- 12 was neutralized by systemic administration of antibodies to either murine IFN-gamma or IP-10. Murine IL-12 induced murine IP-10 expression in mouse splenocytes, and human IFN-gamma induced human IP-10 expression in purified human endothelial cells, suggesting that IL-12 can induce IP-10 expression in certain cells. These results document the important role of IP-10 as a mediator of angiogenesis inhibition by IL-12, and raise the possibility that IP-10 may also contribute to the antitumor effect of IL-12.
Cure of multidrug-resistant human B-cell lymphoma xenografts by combinations of anti-B4-blocked ricin and chemotherapeutic drugs
Authors: Liu C , Lambert JM , Teicher BA , Blattler WA , O'Connor R
Published: October 2019

The CD-19-directed immunotoxin anti-B4-blocked ricin (anti-B4-bR) is currently in clinical trials for the treatment of B-cell malignancies. To explore the potential of using anti-B4-bR with chemotherapy protocols we tested the in vivo efficacy of the immunotoxin in combination with two multi-drug chemotherapeutic regimens in severe combined immunodeficient (SCID) mice bearing disseminated tumors of the multidrug-resistant human B-cell lymphoma Namalwa/mdr-1. In cytotoxicity studies in vitro, combinations of the immunotoxin with cisplatin produced supra-additive killing effects on both Namalwa and Namalwa/mdr-1 cells, whereas anti-B4-bR combined with 4-hydroperoxy- cyclophosphamide caused additive killing of both cell lines. In vivo cyclophosphamide, cisplatin, vincristine, doxorubicin, and etoposide as single agents, were effective in prolonging the survival of SCID mice burdened with the Namalwa tumor, whereas only cyclophosphamide and cisplatin were effective on Namalwa/mdr-1 tumors. Treatment of Namalwa/mdr-1-bearing mice with anti-B4-bR alone or with the drug combination CHOE (consisting of cyclophosphamide, vincristine, doxorubicin, and etoposide) alone increased the lifespan of the tumor- burdened mice by 58% and 73%, respectively. However, treatment with five daily bolus intravenous injections of anti-B4-bR followed by CHOE increased the lifespan by 173%, and 20% of the mice were cured. The drug combination CCE (cyclophosphamide, cisplatin, and etoposide) alone could increase the lifespan of the Namalwa/mdr-1 tumor-burdened mice by 129% compared with untreated controls. Combination therapy with anti-B4- bR and CCE produced long-term cures in 50% of the tumor-burdened mice. These results suggest that anti-B4-bR in combination with current multidrug regimens may constitute a highly efficacious modality for the treatment of drug-resistant B-cell malignancies.
Arachidonic acid mediates interleukin-1 and tumor necrosis factor-alpha- induced activation of the c-jun amino-terminal kinases in stromal cells
Authors: Rizzo MT , Carlo-Stella C
Published: October 2019

We have previously shown that arachidonic acid mediates interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha)-induced transcription of c-jun. The signaling pathway of arachidonic acid-induced c-jun transcription was independent of protein kinase C activation and involved a tyrosine kinase-dependent process. The present study was undertaken to further elucidate the signal transduction pathway of arachidonate-induced c-jun transcription. We used a glutathione-S-transferase-c-jun fusion protein containing the aminoterminal domain of c-jun (residues 5 to 89) to explore the hypothesis that arachidonic acid stimulates c-jun amino-terminal kinase (JNK) activity in the murine stromal cell line +/+.1 LDA 11. Extracts from arachidonic acid-treated cells catalyzed phosphorylation of the c- jun fusion protein, indicating stimulation of JNK activity. Similar results were obtained when cells were challenged with IL-1 and TNF- alpha. The effect of arachidonic acid was specific, because extracts from stimulated cells failed to phosphorylate a mutated fusion protein in which serine 63 and 73 of c-jun were each substituted with leucine. Arachidonic acid induced JNK activation in a time- and dose-dependent manner that was not mimicked by saturated fatty acids such as palmitic acid or other unsaturated fatty acids from the n-3, n-6, or n-9 series. Furthermore, other lipids, such as diacylglycerol, phosphatidic acid, and C2-ceramide, failed to induce a significant increase in JNK activity. Treatment of stromal cells with propyl gallate, a dual inhibitor of lipoxygenase and cyclooxygenase enzymes, did not affect the ability of arachidonic acid to induce JNK activation. Moreover, ETYA (5,8,11,14-eicosate-traynoic acid), a nonmetabolizable arachidonate analogue, also induced JNK activation. These results are consistent with the hypothesis that the signal transduction pathway by which arachidonate stimulates c-jun transcription involves activation of the JNK cascade. Furthermore, arachidonic acid itself and not its cyclooxygenase or lipoxygenase metabolites is involved in stimulating JNK activity. Thus, arachidonic acid may act as a second messenger in mediating the effects of IL-1 and TNF-alpha in the activation of c-jun.
The c-kit ligand potentiates the allogeneic mixed lymphocyte reaction
Authors: Bluman EM , Schnier GS , Avalos BR , Strout MP , Sultan H , Jacobson FW , Williams DE , Carson WE , Caligiuri MA
Published: October 2019

The allogeneic mixed lymphocyte reaction (MLR) is a complex in vitro assay of T-cell recognition and responsiveness in which interleukin-2 (IL-2) plays a central role. We have previously demonstrated that c-kit ligand (KL) can enhance IL-2-induced proliferation in a subset of human natural killer cells expressing the c-kit tyrosine kinase receptor. In the present study, we asked whether KL could enhance IL-2-mediated T-cell proliferation in the allogeneic MLR. We demonstrate that the vast majority of activated human T-cell clones express the c-kit mRNA transcript. Binding studies performed on activated T cells with radioiodinated KL were consistent with the expression of a single class of c-kit receptors. The addition of exogenous KL to the MLR led to an increase in tritiated thymidine (3[H]-TdR) incorporation in the absence of other exogenous cytokines, and did so in a dose-dependent fashion. A reproducible increase in 3[H]-TdR incorporation was noted at concentrations of KL, which approximate those normally found in vivo. Antibody blocking of KL binding to c-kit, T-cell depletion and sorting experiments suggest that the action of KL is mediated at least in part by a direct effect on both CD4+ and CD8+ T-cells. KL's enhancement of the MLR also requires the binding of IL-2 to its high-affinity IL-2 receptor. Given the abundance of KL normally found in human serum, these data suggest that this cytokine may have a role during T-cell activation in vivo.
Prognostic implications of ploidy and proliferative activity in the diffuse, aggressive non-Hodgkin's lymphomas
Authors: Winter JN , Andersen J , Variakojis D , Gordon LI , Fisher RI , Oken MM , Neiman RS , Jiang S , Bauer KD
Published: October 2019

The International Index is a powerful predictor of outcome in the aggressive non-Hodgkin's lymphomas that is based solely on clinical features. Proliferative activity (% S-phase) measured by flow cytometry has been reported to have prognostic significance in many series and may represent a biologic correlate of clinical behavior that further defines prognosis. Flow cytometric analysis of cellular DNA content and proliferative activity (% S-phase) was performed on fixed paraffin-embedded biopsy specimens from 242 previously untreated patients with diffuse, aggressive non-Hodgkin's lymphomas entered on phase III intergroup clinical trials. The International Index was calculated for each patient based on stage, lactate dehydrogenase, performance status, number of extranodal sites, and age, as previously reported. The International Index consistently predicted response to therapy (P = .027) and survival (P = .007) in this series. DNA aneuploidy was shown in 57% of cases, but was not predictive of clinical outcome. The median % S-phase was 9.9 (median coefficient of variation, 3.6%), which was highly correlated with mitotic index (P = .0001). Although a trend associating low proliferative activity with good early survival and very high S-phase with a shortened survival was shown, International Index risk was the only significant predictor of survival in the multivariate analysis. Although proliferative activity quantitated by flow cytometric analysis of nuclei extracted from paraffin-embedded specimens is probably predictive of survival, it is a less powerful prognostic indicator than clinical parameters represented by the International Index and provides no additional prognostic information.
Anaplastic large-cell lymphomas of T-cell and null-cell phenotype express cytotoxic molecules
Authors: Foss HD , Anagnostopoulos I , Araujo I , Assaf C , Demel G , Kummer JA , Hummel M , Stein H
Published: October 2019

To further specify the cellular origin and nature of anaplastic large- cell lymphoma (ALCL) and its relationship to other lymphoid neoplasms, particularly Hodgkin's disease (HD), we investigated the presence of cytotoxic molecules in a large well-characterized series of these tumors. For expression of the cytotoxic molecules perforin and granzyme B, in situ hybridization (ISH) and immunohistology were used, respectively. Overall, 23 of 25 ALCLs of T/null phenotype and five (three mixed cellularity and two nodular sclerosis) of 57 HD cases showed the presence of perforin transcripts and/or granzyme B molecules in neoplastic cells. Polymerase chain reaction (PCR) analysis of ALCLs showed that most (10 of 11) cases of null-cell ALCL (null-ALCL) contained a clonal rearrangement of T-cell receptor beta-chain genes, as did T-cell ALCL (T-ALCL; 9 of 10 cases). However, both cytotoxic molecules and clonally rearranged T-cell receptor beta-chain genes were absent in seven of seven and eight of nine cases of B-cell ALCL (B-ALCL), respectively. These data show that all or nearly all T-ALCLs, irrespective of the clinical subform or the lack of T-cell-associated molecules, are derived from activated cytotoxic T cells. The same appears to be true for the neoplastic cells of rare HD cases. These findings indicate that T-ALCLs are different from B-ALCLs and the majority of HD cases, and suggest that some HD cases, especially those with T-cell antigen-positive tumor cells, may be closely related to T- ALCL, at least in terms of cellular origin.
p21/WAF1 cyclin-kinase inhibitor expression in non-Hodgkin's lymphomas: a potential marker of p53 tumor-suppressor gene function
Authors: Chilosi M , Doglioni C , Magalini A , Inghirami G , Krampera M , Nadali G , Rahal D , Pedron S , Benedetti A , Scardoni M , Macri E , Lestani M , Menestrina F , Pizzolo G , Scarpa A
Published: October 2019

p21WAF1 (wild-type p53-activated fragment 1) is involved in the control of mammalian cell cycle through the binding and inhibition of cyclin-dependent kinases (Cdk). Because the product of WAF1 gene is a potent downstream effector of the p53 tumor-suppressor gene function, its pattern of cellular expression might correlate with nuclear accumulation of p53-encoded protein and/or p53 gene mutations occurring in malignant lymphomas. To investigate this issue, we analyzed immunohistochemically the expression of p53 and p21WAF1 proteins in tissue involved by non-Hodgkin's lymphomas (NHLs;253 cases) of various histologic types. In a proportion of them (80 cases), we also investigated the possible presence of p53 gene mutations using single- strand conformation polymorphism analysis and direct DNA sequencing. The absence of both p21WAF1 and p53 proteins was observed in 147 of 217 cases (67.7%) among CD30-NHL and in only 8 of 36 (22.2%) CD30+cases, which were mostly anaplastic large-cell lymphomas. A consistent number (> 10%) of p21WAF1-expressing cells was shown in 48 of 253 (18.9%) NHL cases, with a higher incidence in CD30+cases (25/36 [69.4%]), which mostly (21/36) coexpressed p53. These latter cases were characterized by a germline configuration of the p53 gene. In 50 of 253 NHL samples (19.7%), 47 of which (21.6%) belong to the CD30-group, neoplastic cells were p53+/p21-. In all of these cases, the p53+cells accounted for more than 50% of neoplastic cells, up to 100%. Point mutations of p53 gene were solely observed in all investigated cases with this latter phenotype. Our findings strongly suggest that the combined immunohistochemical evaluation of p53 and p21WAF1 is a valuable means of assessing the functional status of the p53 tumor-suppressor gene product in NHL with potential application in the monitorage and prognostication of individual cases.
A combination of the effects of rare genotypes at the XK and KEL blood group loci results in absence of Kell system antigens from the red blood cells
Authors: Daniels GL , Weinauer F , Stone C , Ho M , Green CA , Jahn-Jochem H , Offner R , Monaco AP
Published: October 2019

The 22 antigens of the Kell blood group system are located on a red blood cell (RBC) membrane glycoprotein that shows sequence homology with a family of metalloendopeptidases. Expression of the Kell system antigens is partially governed by XK, an X-linked gene that encodes the Kx protein; absence of Kx results in reduced Kell antigen expression. Almost total absence of Kell antigens from the RBCs of a German man with no symptoms of neuroacanthocytosis could not be due to the Kell- null phenotype, Ko, because his RBCs had very weak expression of Kx antigen and his three children were Kp(a + b+). Kell antigens were normal on the RBCs of his son but weak on those of his two daughters. An Nla III restriction fragment-length polymorphism within the KEL gene showed the Kpa/Kpa genotype in the propositus. Sequencing of his XK gene showed a single base change within the donor splice consensus sequence of intron 2. A BsaAl restriction fragment-length polymorphism showed the mutation in both of his daughters but not in his son. The extreme depression of the Kell antigens of the propositus must be due to a combination of effects, ie, homozygosity for Kpa and deficiency of Kx protein, each of which is capable of causing some degree of weakening of Kell antigens.
Characterization of autoantigenic epitopes on platelet glycoprotein IIb/IIIa using random peptide libraries
Authors: Bowditch RD , Tani P , Fong KC , McMillan R
Published: October 2019

Most patients with chronic immune thrombocytopenic purpura (ITP) have autoantibodies directed against the glycoprotein (GP) IIb/IIIa complex. We have used a filamentous phage library that displays random linear hexapeptides to identify peptide sequences recognized by these autoantibodies. Plasma antibody eluates from two patients were used to select for phage displaying autoantibody-reactive peptides. From patient ITP-1 (known to have two distinct autoantibodies), we identified anti-GPIIb/IIIa antibody-specific phage encoding the peptide sequences Arg-Glu-Lys-Ala-Lys-Trp (REKAKW) and Pro-Val-Val-Trp-Lys-Asn (PVVWKN). Patient ITP-2 bound phage encoding the hexapeptide sequence Arg-Glu-Leu-Leu-Lys-Met. Each phage showed saturable dose-dependent binding to immobilized autoantibody, and binding could be blocked with purified GPIIb/IIIa. Patient ITP-1 autoantibody recognition of phage encoding REKAKW could be blocked with a synthetic peptide derived from the GPIIIa cytoplasmic tail; however, the PVVWKN was not. Using sequential overlapping peptides from the GPIIIa cytoplasmic region, an epitope for ITP-1 was localized to the sequence Arg-Ala-Arg-Ala-Lys-Trp (GPIIIa 734–739). Inhibition studies using synthetic peptides showed that phage REKAKW and PVVWKN were recognized by distinct autoantibodies from patient ITP-1. To determine whether individual patients with ITP possessed autoantibodies that recognize similar antigenic determinants on GPIIb/IIIa, the three phage were tested for binding to five other ITP patient autoantibodies. The phage encoding the peptide PVVWKN was found to bind ITP-1 and one other patient autoantibody. This result suggests that ITP patients recognize a limited number of shared epitopes.
CD38-mediated growth suppression of B-cell progenitors requires activation of phosphatidylinositol 3-kinase and involves its association with the protein product of the c-cbl proto-oncogene
Authors: Kitanaka A , Ito C , Nishigaki H , Campana D
Published: October 2019

The signalling pathways that arrest the cell cycle and trigger cell death are only partially known. Dimerization of CD38, a 45-kD transmembrane type II glycoprotein highly expressed in immature B cells, inhibits cell growth and causes apoptosis in normal and leukemic B-cell progenitors, but the molecular mechanisms underlying these cellular responses are unknown. In the present study, we found that CD38 ligation in the immature B-cell lines 380, REH, and RS4;11 caused rapid tyrosine phosphorylation of the protein product of the proto- oncogene c-cbl. Dimerization of CD38 was accompanied by the association of cbl with the p85 subunit of phosphatidylinositol 3-kinase (Pl 3-K), resulting in markedly increased Pl 3-K activity in antiphosphotyrosine and anti-cbl immunoprecipitates. Wortmannin and LY294002, two potent inhibitors of Pl 3-K, rescued immature B cells from CD38-mediated growth suppression. This effect was observed not only in model B-cell lines, but also in cultures of leukemic lymphoblasts from patients, and in normal bone marrow B-cell progenitors as well. Concentrations of inhibitors that reversed cellular responses to CD38 significantly decreased Pl 3-K activity. By contrast, rapamycin, a p70 S6-kinase inhibitor, did not rescue immature B cells from CD38-mediated suppression. These results suggest that Pl 3-K activity is essential for CD38-mediated inhibition of lymphopoiesis and that cbl and Pl 3-K are regulatory molecules whose activation can result in suppression of cell proliferation and apoptosis in immature lymphoid cells.
Glutamine- and phosphate-containing hypotonic storage media better maintain erythrocyte membrane physical properties
Authors: Dumaswala UJ , Wilson MJ , Jose T , Daleke DL
Published: October 2019

We have shown that red blood cell (RBC) adenosine-5′-triphosphate (ATP) is better maintained and that there is less hemolysis and K+ leakage in hypotonic experimental additive solutions (EASs) containing glutamine and glutamine plus phosphate (Pi) than in the conventional additive solution Adsol during blood bank storage. The objective of this study was to determine if the beneficial effect produced in these media correlates with better preservation of RBC membrane properties including lipid content, phospholipid organization, aminophospholipid transport (flippase), and prothrombin converting activity. Aliquots of packed RBCs were stored in EASs containing adenine, glucose, sodium chloride, and mannitol, with 10 mmol/L glutamine (EAS 44) or with 10 mmol/L glutamine and 20 mmol/L Pi(EAS 45), or in Adsol. RBC membranes were studied after 0, 28, 42, and 84 days of storage, and vesicle membranes were studied after 84 days. RBC cholesterol and phospholipid content remained significantly greater (P < .01) in EASs than in Adsol. The degree of membrane vesiculation was more than 50% lower in EASs than in Adsol (P < .01). After 42 days of storage, the accessibility of phosphatidylethanolamine to phospholipases was approximately 1.5 times greater for Adsol and EAS 44 samples than for EAS 45 samples (43.5% v 28%). The rates of phosphatidylserine transport were 43% to 70% lower for stored cells but were not dependent on storage media. The amounts of bands 3 and 4.1 in the microvesicle membranes were not statistically different in any of the preparations. These results suggest that storage of RBCs in glutamine and Pi-medium better maintains ATP, lipid content, and phospholipid asymmetry and results in decreased vesiculation.
Electroporation of synthetic oligodeoxynucleotides: a novel technique for ex vivo bone marrow purging
Authors: Bergan R , Hakim F , Schwartz GN , Kyle E , Cepada R , Szabo JM , Fowler D , Gress R , Neckers L
Published: October 2019

Recent data suggest that tumor cells contaminating reinfused bone marrow may contribute to relapse in patients undergoing autologous bone marrow transplantation. Purging strategies that are able to remove these contaminating tumor cells need to be developed. This study describes how electroporation (EP) can be used to improve intracellular delivery of synthetic antisense oligodeoxynucleotides (ODNs), thereby enhancing their ability to suppress a target protein. Antisense ODNs that were introduced into cells by EP led to immediate suppression of targeted c-myc protein; this was associated with rapid cell death in the diffuse histiocytic lymphoma, U937; Burkitt's lymphoma, ST486; breast carcinoma, MCF-7; and Ewing's sarcoma, CHP-100, cell lines. Electroporation was found to have little or no detrimental effect on cells responsible for murine hematopoietic long-term reconstitution as determined from in vivo competitive repopulation studies. Using human c-myc-directed antisense ODNs as a model for the application of this approach to bone marrow purging, selective killing of human lymphoma U937 cells relative to normal human bone marrow cells was shown in cell mixing studies. In vivo studies were performed in which a survival advantage was shown for athymic mice that were inoculated with antisense-treated U937 cells as opposed to control cells. These studies suggest that EP of bone marrow may be of use in enhancing intracellular delivery of a variety of molecular/pharmaceutical agents. Taken together, these data suggest that the use of electroporation to enhance delivery of antisense ODNs is a promising new approach towards ex vivo bone marrow purging.
p120c-cbl is present in human blood platelets and is differentially involved in signaling by thrombopoietin and thrombin
Authors: Oda A , Ozaki K , Druker BJ , Miyakawa Y , Miyazaki H , Handa M , Morita H , Ohashi H , Ikeda Y
Published: October 2019

To investigate the signaling processes induced by recombinant thrombopoietin, we used human platelets to recently show that thrombopoietin induces rapid tyrosine phosphorylation of Jak2, Tyk2, Shc, Stat3, Stat5, and other proteins in human platelets. Because the apparent molecular weight of a major tyrosine-phosphorylated protein in platelets stimulated by thrombopoietin is approximately 120 kD, we examined the possibility that this could be p120c-cbl, a protein known to be involved in signaling by many growth factors. Specific antisera against p120c-cbl recognized the same 120-kD protein in lysates of Jurkat cells, which are known to express p120c-cbl, and platelets, indicating that platelets have p120c-cbl. Thrombopoietin induced rapid tyrosine phosphorylation of p120c-cbl in platelets. Thrombopoietin also induced tyrosine phosphorylation of p120c-cbl in FDCP cells genetically engineered to express the thrombopoietin receptor, c-Mpl. Interestingly, FDCP cells, expressing a truncated c-Mpl devoid of the box-2 domain, proliferate in response to thrombopoietin. However, no increase in tyrosine phosphorylation of p120c-cbl was observed upon treatment of these cells with thrombopoietin, indicating that in this system tyrosine phosphorylation of p120c-cbl may not be essential for cell proliferation. This suggests that tyrosine phosphorylation of p120c-cbl may be required for nonmitogenic responses induced by thrombopoietin in postmitotic cells such as platelets. On the other hand, p120c-cbl was not significantly tyrosine-phosphorylated upon treatment of platelets with thrombin. However, it became incorporated into the Triton X-100-insoluble, 10,000g-sedimentable residue in an aggregation-dependent manner, suggesting that it may have a regulatory role in platelet cytoskeletal processes. p120c-cbl was constitutively associated with a 28-kD adapter protein, Grb2, that was also incorporated into the Triton X-100-insoluble, sedimentable residue dependent on aggregation. Further, we found that p120c-cbl is an endogenous substrate for calpain, a protease that may play a role in postaggregation signaling processes. Our data suggest that p120c-cbl may be involved in signal transduction following ligand binding to c- Mpl through its inducible tyrosine phosphorylation, and it may also be involved in signaling during platelet aggregation by its redistribution to the cytoskeleton.
Opposite sorting of tissue factor in human umbilical vein endothelial cells and Madin-Darby canine kidney epithelial cells
Authors: Camerer E , Pringle S , Skartlien AH , Wiiger M , Prydz K , Kolsto AB , Prydz H
Published: October 2019

Tissue factor (TF) is a 48-kD transmembrane glycoprotein that triggers the extrinsic pathway of blood coagulation by interacting with the plasma coagulation factor VII (FVII). TF is also a true receptor in that a cellular signal is generated when activated FVII (FVIIa) binds to TF. For both of these functions, the cellular surface distribution of TF is important, since FVII is primarily available on the apical side of vascular endothelial cells and on the basolateral side of epithelial cells lining the internal and external surfaces. We show that in endothelial cells, TF (both antigen and procoagulant activity) is sorted to the apical surface, whereas in wild-type and stably transfected Madin-Darby canine kidney epithelial cells (MDCK), which form tight junctions and express TF constitutively, TF antigen is on the basolateral surface. No significant clotting activity is detectable on this surface. Truncated TF (cytoplasmic tail residues 246 to 263 deleted) is sorted as wild-type in MDCK cells.
Reactivity of murine cytokine fusion toxin, diphtheria toxin390-murine interleukin-3 (DT390-mIL-3), with bone marrow progenitor cells
Authors: Chan CH , Blazar BR , Greenfield L , Kreitman RJ , Vallera DA
Published: October 2019

Myeloid leukemias can express interleukin-3 receptors (IL-3R). Therefore, as an antileukemia drug, a fusion immunotoxin was synthesized consisting of the murine IL-3 (mIL-3) gene spliced to a truncated form of the diphtheria toxin (DT390) gene coding for a molecule that retained full enzymatic activity, but excluded the native binding domain. The DT390-mIL-3 hybrid gene was cloned into a vector under the control of an inducible promote. The fusion protein was expressed in Escherichia coli and then purified from inclusion bodies. The fusion toxin was potent because it inhibited FDC-P1, an IL-3R- expressing murine myelomonocytic tumor line (IC50 = 0.025 nmol/L or 1.5 ng/mL). Kinetics were rapid and cell-free studies showed that DT390-mIL- 3 was as toxic as native DT. DT390-mIL-3 was selective because anti-mIL- 3 monoclonal antibody, but not irrelevant antibody, inhibited its ability to kill. Cell lines not expressing IL-3R were not inhibited by the fusion protein. Because the use of DT390-mIL-3 as an antileukemia agent could be restricted by its reactivity with committed and/or primitive progenitor cells, bone marrow (BM) progenitor assays were performed. DT390-mIL-3 selectively inhibited committed BM progenitor cells as measured by in vitro colony-forming unit-granulocyte- macrophage and in vivo colony-forming unit-spleen colony assays. To determine if this fusion protein was reactive against BM progenitor cells required to rescue lethally irradiated recipients, adoptive transfer experiments were performed. Eight million DT390-mIL-3-treated C57BL/6 Ly5.2 BM cells, but not 4 million, were able to rescue lethally irradiated congenic C57BL/6 Ly5.1 recipients, suggesting that progenitor cells might be heterogenous in their expression of IL-3R. This idea was supported in competitive repopulation experiments in which DT390-mIL-3 treated C57BL/6 Ly5.2 BM cells were mixed with nontreated C57BL/6 Ly5.1 BM cells and used to reconstitute C57BL/6 Ly5.1 mice. A significant reduction, but not elimination, of Ly5.2- expressing cells 95 days post-BM transplantation and secondary transfer experiments indicated that IL-3R is not uniformly expressed on all primitive progenitor cells. The fact that some early progenitor cells survived DT390-mIL-3 treatment indicates that this fusion toxin may be useful in the treatment of myeloid leukemias that express the IL-3R.
Theophylline synergizes with chlorambucil in inducing apoptosis of B- chronic lymphocytic leukemia cells
Authors: Mentz F , Mossalayi MD , Ouaaz F , Baudet S , Issaly F , Ktorza S , Semichon M , Binet JL , Merle-Beral H
Published: October 2019

We tested the effects of theophylline, a phosphodiesterase inhibitor inducing intracellular accumulation of cyclic adenosine monophosphate (cAMP), on malignant B cells from 15 patients with B-chronic lymphocytic leukemia (B-CLL). We observed a large increase in apoptotic cell numbers (mean, 90% v 20% in medium alone) in the presence of theophylline (100 micrograms/mL) or chlorambucil (10 mumol/L) after 72 hours of incubation. Maximal apoptosis (90%) was reached after 36 hours when the two drugs were used together at fourfold lower concentrations, indicating a synergistic effect; no effect was observed with normal B cells, suggesting that the combination might have therapeutic interest. Chlorambucil induced intracellular Ca+2 influx, pointing to the involvement of two signaling pathways that might explain its synergy with theophylline through their effects on oncogenes. The expression of bcl-2 protein, a proto-oncogene inhibiting apoptosis, decreased after incubation with the drugs, while c-myc, recently described as having a potent role in apoptosis, was overexpressed. For p53 we observed an overexpression in the presence of chlorambucil or both theophylline- chlorambucil and a decrease after theophylline incubation. Chlorambucil- and theophylline-induced apoptosis was partially inhibited by interleukin-4 (IL-4), which also abrogated the effects on oncogene expression. These results provide insight into the mechanisms underlying B-CLL apoptosis and suggest that the theophylline- chlorambucil combination may be of therapeutic value in this setting.
Biosynthesis of prothrombin: intracellular localization of the vitamin K-dependent carboxylase and the sites of gamma-carboxylation
Authors: Bristol JA , Ratcliffe JV , Roth DA , Jacobs MA , Furie BC , Furie B
Published: October 2019

Prothrombin is a vitamin K-dependent blood coagulation protein that undergoes posttranslational gamma-carboxylation and propeptide cleavage during biosynthesis. The propeptide contains the gamma-carboxylation recognition site that directs gamma-carboxylation. To identify the intracellular sites of carboxylation and propeptide cleavage, we monitored the synthesis of prothrombin in Chinese hamster ovary cells stably transfected with the prothrombin cDNA by immunofluorescent staining. The vitamin K-dependent carboxylase was located in the endoplasmic reticulum and Golgi complex. Antibodies specific to prothrombin processing intermediates were used for immunocytolocalization. Anti-des-gamma-carboxyprothrombin antibodies stained only the endoplasmic reticulum whereas antiproprothrombin antibodies (specific for the propeptide) and antiprothrombin:Mg(II) antibodies (which bind the carboxylated forms of proprothrombin and prothrombin) stained both the endoplasmic reticulum and the Golgi complex. Antiprothrombin:Ca(II)-specific antibodies (which bind only to the carboxylated form of prothrombin lacking the propeptide) stained only the Golgi complex and secretory vesicles, and colocalized with antimannosidase II and anti-p200 in the juxtanuclear Golgi complex. These results indicate that uncarboxylated proprothrombin undergoes complete gamma-carboxylation in the endoplasmic reticulum and that gamma-carboxylation precedes propeptide cleavage during prothrombin biosynthesis.
Inhibition of proliferation and induction of apoptosis in juvenile myelomonocytic leukemic cells by the granulocyte-macrophage colony- stimulating factor analogue E21R
Authors: Iversen PO , Rodwell RL , Pitcher L , Taylor KM , Lopez AF
Published: October 2019

Juvenile myelomonocytic leukemia (JMML) is a malignancy that almost inevitably leads to death before adulthood. Chemotherapy has given disappointing results and a substantial number of patients relapse after bone marrow transplantation. A salient feature of this disease is that the JMML cells produce granulocyte-macrophage colony-stimulating factor (GM-CSF) spontaneously and survive and proliferate without exogeneous GM-CSF. Furthermore, JMML cells are hypersensitive to GM-CSF with addition of this cytokine leading to enhanced proliferation. We have recently generated a human GM-CSF analogue, E21R, that acts as a complete and selective GM-CSF receptor antagonist. We have now tested this molecule as a potential new agent to control the leukemic cell load in JMML with particular emphasis on its role in JMML cell survival. We found that E21R inhibited the spontaneous growth of JMML cells in vitro and caused their apoptosis in a dose- and time-dependent manner in seven of seven cases. In contrast, neither a neutralizing anti-GM-CSF monoclonal antibody (MoAb) nor a selective interleukin-1 (IL-1) receptor antagonist affected JMML cell survival. Furthermore, the apoptotic effect of E21R was seen even in the presence of interleukin-1 beta and tumor necrosis factor-alpha, which have also been implicated in the pathogenesis of JMML. The inhibitory effects of E21R on JMML cell growth and viability offer a novel approach to therapy in this lethal childhood leukemia.
Contribution of both STAT and SRF/TCF to c-fos promoter activation by granulocyte-macrophage colony-stimulating factor
Authors: Rajotte D , Sadowski HB , Haman A , Gopalbhai K , Meloche S , Liu L , Krystal G , Hoang T
Published: October 2019

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that has been shown to support call proliferation in murine fibroblasts engineered to stably express both chains of the human GM-CSF receptor (NIH-GMR). Because the proto-oncogene c-fos is believed to provide a link between short-term signals elicited at the membrane and long-term cellular response, we chose to study the mechanism of GM-CSF-dependent cell regulation using c-fos promoter activity as a molecular marker in both NIH-GMR transfectants and in the CD34+ cell line TF-1. The importance of c-fos and related AP-1 activity in GM-CSF signalling was suggested by a tight correlation between GM-CSF-dependent activation of the c-fos promoter and cell proliferation and by the inhibitory effect of a trans-dominant c-fos mutant on cell growth. To evaluate the contribution of the serum response factor (SRF) associated with the ternary complex factor (TCF) and of STAT proteins to c-fos promoter activation in response to GM-CSF, the SRF binding site (SRE) and/or the STAT binding site (SIE) were inactivated. In serum-free medium, both SRE and SIE are essential to c-fos promoter activation by GM-CSF in NIH-GMR transfectants and in TF-1 cells. No response to GM-CSF was observed when both sites were mutated. The nature of the STAT family member was further investigated by Wester blots and DNA retardation assays using an SIE probe. Our data indicate that GM-CSF induced DNA binding of both STAT1 and STAT3 in NIH-GMR and mainly of STAT3 in TF-1 cells. STAT5 tyrosine phosphorylation was also observed in TF-1 cells. Finally, expression of a dominant negative MAPK mutant, ERK192A, resulted in a decrease of both SRE- and SIE-dependent activation of c-fos promoter by GM-CSF, suggesting that STAT1/3 are regulated not only by tyrosine kinases, but also partially by MAPK.