Identification and purification of human erythroid progenitor cells by monoclonal antibody to the transferrin receptor (TÜ 67)

Summary Anti-TÜ 67 is a murine monoclonal antibody that recognizes the transferrin receptor. With respect to hematopoietic cells TÜ 67 is expressed by human multipotent colony-forming cells (CFU-Mix), erythroid progenitor cells (BFU-E and CFU-E) and a fraction of granulocyte/monocyte colony forming cells, but is not expressed by mature hematopoietic cells including erythrocytes, platelets, lymphocytes, and peripheral blood myeloid cells. The TÜ 67-positive fraction of normal bone marrow, separated by fluorescence-activated cell sorting (FACS) or immune rosettes, contained 87% of the erythroid progenitor cells. Erythroid progenitor cells were enriched up to 50-fold by using a combination of monoclonal antibodies to deplete mature hematopoietic cells, followed by positive selection of BFU-E and CFU-E by TÜ 67 antibody.Supported by the Deutsche Forschungsgemeinschaft (He 1380-2/1)     

BCL-2 Induction is Part of the Strategy of Epstein-Barr Virus

Epstein Barr virus can infect B lymphocytes and epithelial cells. Epithelial cells present the natural reservoir for the virus in man. In vitro, infected cells harbor the virus predominantly in a latent state with the expression of a set of nuclear (EBNA 1-6) and latent membrane genes (LMP 1-2) and virus-transformed B cells grow as permanently immortalized lymphoblastoid cell lines, that show increased resistance to various growth inhibiting factors. Here we show that the lymphoma-associated oncogene BCL-2 is upregulated by different latent Epstein-Barr virus genes in B lymphocytes as well as keratinocyte cell lines. Thus, the induction of BCL-2 gene expression seems to be part of the survival strategy of the virus independently of the host cell infected.     

Prognosis of older patients with acute myeloid leukemia receiving either induction or noncurative treatment: a single-center retrospective study

Since few studies focus on prognostic factors in unselected elderly acute myeloid leukemia (AML) patients, a retrospective analysis of 138 consecutive patients aged >55 years (median age: 67, range: 56-89) with AML diagnosed at a single center over an 8-year period was performed: 69% had de novo AML and 31% secondary (s) AML; 67% of the patients were karyotyped. Of the patients, 73 (53%) were treated with standard induction therapy protocols and 65 (47%) received palliative treatment only. Univariate and multivariate analyses of the effects of the following factors on overall survival (OS) were performed: sex, age > or = vs <65 years, de novo vs sAML, serum (s) lactate dehydrogenase (LDH) > or = vs <400 U/l, leukocytes > or = vs <50,000/ microl, induction therapy, and karyotype. Additionally, in patients receiving induction therapy, complete remission (CR) rates and survival from CR were analyzed. CR rate was 47% [95% confidence interval (35%, 59%)], 53% (39%, 66%) in de novo AML, and 21% (5%, 51%) in sAML. After a median follow-up of 4 years, 130 deaths were observed (94%). In a univariate analysis, significant factors for longer OS were induction therapy, age <65 years, sLDH <400 U/l, and de novo AML. In a multivariate analysis, significant factors for longer OS were sLDH <400 U/l and induction therapy. However, the difference between treatment outcome may also be due to selection criteria not captured, such as performance status, comorbid conditions, wish of the patient, etc. The effects of intensive and nonintensive treatment in this patient group need to be investigated in prospective, randomized trials in which these clinical parameters of high relevance for treatment decisions in older patients are also considered.     

Transforming growth factor-beta 1 delays formation of granulocyte-macrophage colony-forming cells, but spares more primitive progenitors during ex vivo expansion of CD34+ haemopoietic progenitor cells

Ex vivo culture and expansion of autologous haemopoietic transplants has been developed to improve tumour cell purging and accelerate haemopoietic reconstitution by transplantation of increased progenitor cell numbers. We studied the effect of the negative haemopoietic regulator, transforming growth factor beta-1 (TGF-beta 1) on primitive precursors during ex vivo expansion of CD34⁺ cells. When added directly to methylcellulose colony-forming assays, TGF-beta 1 potently suppressed the development of granulocyte-macrophage colonies from CD34⁺ enriched peripheral blood progenitor cells (80–90% inhibition). In contrast, expansion of total nucleated cells and granulocyte-macrophage colony-forming cells (GM-CFC) from CD34⁺ progenitors in liquid culture in the presence of stem cell factor (SCF), interleukin (IL)-1 beta, IL-3, IL-6 and erythropoietin (EPO) was inhibited to 32–65% of control culture levels within 14 d when TGF-beta 1 was added, and still produced an average 3.3-fold absolute amplification of GM-CFC. The inhibitory effect of TGF-beta 1 on GM-CFC generation was reversed when it was washed out on day 6 of ex vivo expansion cultures, and total numbers of GM-CFC generated from expansion cultures then reached levels of untreated controls by day 16. Long-term bone marrow culture-initiating cell (LTCIC) numbers were preserved, at least at input levels, over a culture period of 14 d both in control and TGF-beta-1-treated expansion cultures. These findings suggest that TGF-beta 1, a cytokine which induces apoptosis or terminal differentiation in a number of malignant cell types, may be added to ex vivo expansion cultures without loss of primitive cells from autologous haemopoietic transplants.     

Evidence for distinct lymphocytic and monocytic populations in a patient with terminal transferase--positive acute leukemia.

Two distinct cell populations with lymphoblastic and monocytic characteristics were separated and characterized by multiple cell markers in a patient with terminal transferase-positive acute acute leukemia. The clinical course and sequential cell marker studies were consistent with the interpretation of a defect at the level of a common stem cell giving rise to a terminal transferase--positive lymphoblastic cell population at diagnosis and, following initial therapy, a terminal transferase--negative monocytic population.     

Purification and biochemical characterization of human pluripotent hematopoietic colony-stimulating factor.

Pluripotent hematopoietic colony-stimulating factor (pluripotent CSF), a protein that is constitutively produced by the human bladder carcinoma cell line 5637, has been purified from low serum (0.2% fetal calf serum)-containing conditioned medium. The purification involved sequential ammonium sulfate precipitation, ion-exchange chromatography, gel filtration, and reversed-phase high-performance liquid chromatography. The purified protein has a molecular weight of 18,000 in NaDodSO4/polyacrylamide gel electrophoresis, both by the silver staining technique and by elution of biological activity from a corresponding gel slice, and has an isoelectric point of 5.5. Pluripotent CSF supports the growth of human mixed colonies, granulocyte-macrophage colonies, and early erythroid colonies and induces differentiation of the human promyelocytic leukemic cell line HL-60 and the murine myelomonocytic leukemic cell line WEHI-3B (D+). The specific activity of the purified pluripotent CSF in the granulocyte-macrophage colony assay is 1.5 X 10(8) units/mg of protein.     

Identification of their epitope reveals the structural basis for the mechanism of action of the immunosuppressive antibodies basiliximab and daclizumab

Interleukin-2 (IL-2) and its receptor (IL-2R) play a major role in cellular immunity. The monoclonal antibodies basiliximab and daclizumab directed against the IL-2R subunit CD25 are widely used to prevent graft or host rejection after allogeneic tissue transplantation. Although these antibodies have been used for this purpose for many years, their common epitope within the CD25 protein is unknown. We screened a random phage display library to isolate peptides specifically binding to basiliximab. A striking amino acid sequence motif was enriched. This motif is homologous to the peptide ERIYHFV comprising amino acid positions 116 to 122 within the extracellular domain of CD25, suggesting that this is the basiliximab epitope. Basiliximab and daclizumab binding of selected phage was specific, as no binding was observed to isotype antibody controls. Phage binding could be inhibited by the cognate peptide. In cells expressing mutant CD25, binding of basiliximab was abolished when two or more amino acids of the suspected epitope were changed. In contrast, basiliximab binding remained unaffected in cells expressing CD25 versions with mutations outside this epitope. We therefore conclude that the (116)ERIYHFV(122) string within CD25 is the epitope recognized by basiliximab and daclizumab. This epitope overlaps with the interaction site of CD25 and IL-2, thus revealing the structural basis for the inhibition of IL-2R binding by this class of immunosuppressive antibodies.     

Dose-intense therapy with etoposide, ifosfamide, cisplatin, and epirubicin (VIP-E) in 100 consecutive patients with limited- and extensive-disease small-cell lung cancer

Background: We conducted a phase I/II trial to assess the feasibilityand activity of VIP-E chemotherapy in small-cell lung cancer. End-points weretreatment-related morbidity and mortality, response to treatment, duration ofresponse, and survival.Patients and methods: Two cycles of combination chemotherapy followedby granulocyte colony-stimulating factor (G-CSF) were given at a dose ofetoposide (500 mg/m²), ifosfamide (4000mg/m²), cisplatin (50 mg/m²), and epirubicin(50 mg/m²) to 100 consecutive patients with SCLC. Thirtypatients (19 with LD, and 11 with ED SCLC) proceeded to VIC-E high-dosechemotherapy with autologous peripheral blood stem cell transplantation(PBSCT) at a cumulative dose of etoposide 1500 mg/m²,ifosfamide 12,000 mg/m², carboplatin 750 mg/m²and epirubicin 150 mg/m² (VIC-E). Surgical resection ofprimary tumor was attempted at the earliest feasible point. Thoracicirradiation was given after completion of chemotherapy.Results of conventional-dose VIP-E: 97 patients were evaluable forresponse. Objective response rate was 81% in LD-SCLC (33% CR,48% PR; excluding patients in surgical CR) and 77% in ED-SCLC(18% CR, 58% PR). Treatment mortality was 2%. Mediansurvival was 19 months in LD-SCLC and 6 months in ED-SCLC. Two-year survivalwas 36% in LD and 0% in ED SCLC.Results of high-dose VIC-E: All 30 patients improved on or maintainedprior responses. Four patients (13%) died of treatment-relatedcomplications. Median survival was 26 months in LD-SCLC and 8 months inED-SCLC. Two-year survival was 53% in LD and 9% in ED SCLC.Conclusion: VIP-E chemotherapy is an effective induction therapy forSCLC. Compared with traditional protocols such as ACO orcarboplatin/etoposide, response rates are slightly improved, while survivalis not different. In the LD SCLC subgroup, high-dose chemotherapy improvedresponse rates and survival, especially for patients in surgical CR prior tohigh-dose therapy. In ED SCLC, however, higher response-rates did nottranslate into improved survival. Selected LD-SCLC patients with good partialor complete remissions after prior therapy may benefit from HDC and PBSCT.     

NTS influence on transgene expression from mono- and bicistronic plasmid vectors after lipofection in a human fibroblast cell line

 We evaluated the survival, transgene production, and copy numbers of integrated plasmid units per host genome after lipofection with mono- and bicistronic plasmid vectors in different cell lines and under various conditions. The addition of an integration enhancing murine sequence nontranscribed spacer (NTS) to the plasmids increased transfection efficiency, survival, and transgene expression. However, in human fibroblast cells this sequence had only marginal effects on overall plasmid copy number in bulk cultures. Clones producing the highest amounts of the transgene contained only one or two copies of plasmid per genome, independent of cell type and plasmid design. Received: 1 October 1996 / Accepted: 27 November 1996