Adenovirus-mediated cytotoxicity of chronic lymphocytic leukemia cells.

We have studied adenovirus-mediated cytotoxicity after infection of malignant cells obtained from patients with chronic lymphocytic leukemia (CLL). Our studies indicate that adenoviruses can infect primary CLL cells and that infection of CLL cells with a replication-competent strain of human adenovirus 5 (Ad5dl309) results in cytotoxicity. Adenovirus-mediated cytotoxicity was also seen after infection of CLL cells with a variety of viruses attenuated by mutations in the adenovirus early region 1 (E1) or early region 2 (E2). Even viruses attenuated by deletion of the entire E1 region resulted in cytotoxicity after infection of the CLL cells obtained from some patients. Although there was variability in the degree of cytotoxicity induced by different viruses in different patients cells, a virus with a mutation in the E1B 19K gene resulted in the greatest degree of cytotoxicity in most of the CLL samples tested. These studies demonstrate that infection of CLL cells by attenuated adenoviruses with specific mutations in the E1 or E2 region results in cell death. Attenuated adenoviruses should be developed further as therapeutic agents for patients with CLL.

Recombinant human interleukin-3 (rhIL-3) enhances the mobilization of peripheral blood progenitor cells by recombinant human granulocyte colony-stimulating factor (rhG-CSF) in normal volunteers.

To identify a precisely timed and safe protocol for progenitor cell mobilization, we studied the effects of rhIL-3 and rhG-CSF administration to normal volunteers. rhG-CSF 5 micrograms/kg/d was administered subcutaneously (s.c.) for 7 consecutive days either alone or preceded by rhIL-3 5 micrograms/kg/d s.c. for 4 consecutive days in sequential or partially overlapping schedules. The combined cytokines were well-tolerated--adverse effects were similar to those of the individual agents. Total white blood cell (WBC) and neutrophil counts rose briskly in response to rhG-CSF, and peak mean values were similar between treatment cohorts. Mean platelet counts were modestly elevated during rhG-CSF treatment only in the cohorts receiving rhIL-3 and rhG-CSF. Mean circulating CD34+ cells peaked on day 5 in the rhG-CSF group (38.9+/-14.3/microliter), day 6 in the sequential rhIL-3/rhG-CSF group (56.4+/-12.4/microliter), and day 6 in the partial overlap group (46.1+/-10.9/microliter). On day 3, mean CD34+ cell counts of the subjects who received sequential treatment were markedly higher than observed in the other groups (p<0.05) and were estimated to have been sufficient for collection of adequate grafts by single 10-L leukapheresis procedures in 60% of subjects. Circulating clonogenic cells (CFU-GM and/or BFU-E) were substantially higher in the sequential group than the rhG-CSF group on days 3-6 but were only minimally elevated above baseline in the partial overlap group. The numbers of circulating CD34+/Lin-/Thy-1+ cells (putative stem cells) were increased substantially, especially in the sequential group. On the basis of this pilot trial, we conclude that priming with rhIL-3 is a safe and well-tolerated method for enhancing the mobilization of human blood progenitors and stem cells by rhG-CSF.

Concurrent presentation of erythrocytic and megakaryocytic aplasia.

A case of a patient presenting with idiopathic concurrent erythrocytic and megakaryocytic aplasia is reported. The patient's response to immunosuppressive therapy and her bone marrow pathology clearly suggest an immune mechanism. Based on the lack of suppression of erythroid colony growth, several mechanisms are postulated. Well-established molecular and genetic evidence, along with clinical observations, suggests that a relationship exists between the erythrocytic and megakaryocytic cell lines. This may be related to a common bipotential stem cell or common cell surface markers. This case provides strong clinical evidence to support this relationship.

Cell targeting with retroviral vector particles containing antibody-envelope fusion proteins.

Retroviral vectors are the most efficient tool to introduce genes into vertebrate cells. However, their use is limited by the host range of the retrovirus from which they were derived. To alter the host range of the vector particle, we developed a method to substitute the receptor-binding domain of the envelope protein of a retrovirus with an antigen-binding site of an antibody. To test whether such particles are competent for infection, we established a model system using an antigen-binding site of an antibody against the hapten dinitrophenol (DNP). Retroviral vector particles containing such chimeric envelope proteins were able to bind to and infect cells that were not infectable with wild-type virus after DNP was conjugated to the cell surface. They did not infect such cells without DNP conjugation. Control experiments with chimeric envelope proteins of ecotropic murine leukemia virus (eco-MLV) and spleen necrosis virus (SNV) indicate that the pathway of virus entry of scA-env-containing virus particles was different from that of wild-type virus.

Downstream insertion of the adenovirus tripartite leader sequence enhances expression in universal eukaryotic vectors.

A series of universal eukaryotic gene expression vectors was constructed. All vectors contain a viral promoter and enhancer, a polylinker for insertion of the gene of interest and a polyadenylation sequence. To enhance translation, we inserted the tripartite leader sequence of an adenovirus downstream of the promoter. Using the chloramphenicol acetyl transferase (cat) gene as a marker, we show that the strength of various promoters/enhancers in different cell lines differed by two orders of magnitude. The presence of the tripartite leader increased the efficiency of gene expression up to 18-fold. The level of increase is promoter specific and is most likely influenced by additional sequences flanking the tripartite leader sequence.