Hygienic food to reduce pathogen risk to bumblebees.

Bumblebees are ecologically and economically important pollinators, and the value of bumblebees for crop pollination has led to the commercial production and exportation/importation of colonies on a global scale. Commercially produced bumblebee colonies can carry with them infectious parasites, which can both reduce the health of the colonies and spillover to wild bees, with potentially serious consequences. The presence of parasites in commercially produced bumblebee colonies is in part because colonies are reared on pollen collected from honey bees, which often contains a diversity of microbial parasites. In response to this threat, part of the industry has started to irradiate pollen used for bumblebee rearing. However, to date there is limited data published on the efficacy of this treatment. Here we examine the effect of gamma irradiation and an experimental ozone treatment on the presence and viability of parasites in honey bee pollen. While untreated pollen contained numerous viable parasites, we find that gamma irradiation reduced the viability of parasites in pollen, but did not eliminate parasites entirely. Ozone treatment appeared to be less effective than gamma irradiation, while an artificial pollen substitute was, as expected, entirely free of parasites. The results suggest that the irradiation of pollen before using it to rear bumblebee colonies is a sensible method which will help reduce the incidence of parasite infections in commercially produced bumblebee colonies, but that further optimisation, or the use of a nutritionally equivalent artificial pollen substitute, may be needed to fully eliminate this route of disease entry into factories.

Presentation of exogenous antigen with class I major histocompatibility complex molecules.

Soluble antigens (Ags) in the extracellular fluids are excluded from the class I major histocompatibility complex (MHC)-restricted pathway of Ag presentation in most cells. However, an exogenous Ag can be internalized, processed, and presented in association with class I MHC molecules on specialized Ag-presenting cells (APCs). These APCs express class II molecules and can simultaneously present exogenous Ags to both class I and class II MHC-restricted T cells. These APCs may be important participants in the regulation of host immune responses. This APC activity may explain several phenomena of cytotoxic T lymphocyte (CTL) priming in vivo and might be exploited for eliciting CTL responses to protein vaccines.

Effects of monoclonal antibody and complement treatment of human marrow on hematopoiesis in continuous bone marrow culture.

Long-term bone marrow cultures were established from single-cell suspensions of human bone marrow that had been treated with monoclonal antibodies and complement. Each treated cell suspension was evaluated for production of hematopoietic stem cells over 20 weeks. Treatment with antibody to HLA-DR (Ia), B1, J2, or J5 did not remove adherent cells including those differentiating to adipocytes in 17-hydroxycorticosteroid. In contrast, treatment with monoclonal antibody directed against human beta 2-microglobulin reduced adipocyte numbers by 100-fold and reduced the total adherent cell density over 70%. Cumulative total nonadherent cell and granulocyte-macrophage colony-forming units (GM-CFUc) production over 20 weeks was not significantly altered by one cycle of anti-Ia plus complement or up to three cycles of treatment with complement and anti-J2, -J5, or -B1. However, one cycle of treatment with anti-beta 2-micro-globulin depressed production of both GM-CFUc and nonadherent cells by over 100-fold compared to other treatment groups. While one cycle of treatment of anti-Ia and complement killed all detectable cells forming CFU-erythroid-granulocyte-megakaryocyte-macrophage, blast-forming units (erythroid), and GM-CFUc, GM cluster-forming cells survived. Treatment of marrow with three cycles of anti-Ia and complement removed all detectable GM colony- and GM cluster-forming cells; however, this marrow produced fewer cumulative Ia-positive GM-CFUc. Long-term bone marrow cultures may prove to be an interesting system for in vitro analysis of the effects of new immunotherapeutic agents including other monoclonal antibodies prior to clinical use.

Elimination of clonogenic tumor cells from human bone marrow using a combination of monoclonal antibody:ricin A chain conjugates.

Effective autologous bone marrow transplantation for leukemia and lymphoma is likely to depend upon the selective removal in vitro of malignant cells from normal human bone marrow precursors. Highly specific cytotoxic conjugates formed by coupling ricin A chain to monoclonal antibodies might prove useful for the selective elimination of malignant cells. Consequently, ricin A chain conjugates have been prepared with several different murine monoclonal antibodies and tested for their ability to eliminate clonogenic Burkitt's lymphoma cells from an excess of human bone marrow. The most active reagents included an antibody:A chain conjugate which bound to the nonpolymorphic chain of the la molecule and another which reacted with the mu heavy chain of cell surface immunoglobulin. Conjugates formed with anti-common acute lymphoblastic leukemia antigen, anti-Mr 26,000 glycoprotein, and anti-B1 were much less active on these Burkitt's cells, contrasting with results of complement-dependent tumor cell lysis. Tumor cell kill was partially inhibited by the addition of greater than 2 X 10(6) human bone marrow cells/ml but could be potentiated by increasing the concentration of conjugate or by the addition of 10 mM ammonium chloride. In the presence of ammonium chloride, at least 4 logs of clonogenic tumor cells could be eliminated within 24 h from a 20-fold excess of bone marrow using 10(-7) M ricin A chain linked to one or two different antibodies. Similar treatment of normal human bone marrow temporarily inhibited granulocyte-macrophage colony-forming units (cell) formation but did not compromise establishment of continuous bone marrow cultures. The degree of selective elimination of tumor cells with A chain antibody conjugates was comparable to that achieved with 4-hydroperoxycyclophosphamide or with multiple monoclonal antibodies and complement.

Expression of a selectable gene transferred by a retroviral vector to hematopoietic stem cells and stromal cells in murine continuous bone marrow cultures.

Retroviral-mediated gene transfer to multipotent and committed hematopoietic stem cells and marrow stromal cells was evaluated in long-term bone marrow cultures (LTBMCs). The retroviral vector pZIP-Neo(SV)(X) carrying the bacterial neomycin resistance (neor) gene that confers resistance to the neomycin analog G418 in mammalian cells was packaged in a Moloney envelope either as a replication-competent or replication-defective virus. Virus was introduced by infection of long-term marrow cultures at day 7. During a period of 12 weeks in culture, 10%-50% of harvested hematopoietic progenitor cells that formed differentiated CFU-GEMM colonies in response to pokeweed mitogen-containing spleen cell-conditioned medium (SCCM) and erythropoietin expressed the neor gene. In contrast, 1%-10% of hematopoietic progenitor cells that formed colonies in agar in response to WEHI-3B- or L-cell-conditioned medium expressed resistance to G418. The percentage of resistant progenitors was not detectably enhanced when replication-competent Moloney murine leukemia virus (M-MuLV) was present as helper virus, even though M-MuLV infected greater than 90% of cells in the long-term marrow cultures. In a separate CFU-F assay, 12%-17% of the adherent stromal cells in LTBMCs were found to express the neor gene. Thus gene transfer is limited by the fraction of progenitor cells that can integrate and express the transferred genetic sequences, rather than by the fraction of cells that are initially infected by the vector.

Effect of X-irradiation dose rate on the clonagenic survival of human and experimental animal hematopoietic tumor cell lines: evidence for heterogeneity.

It is a generally accepted principle of radiation biology that hematopoietic progenitor cells demonstrate dose rate independent killing by x-irradiation over the clinically relevant range for total body irradiation (TBI) (5-25 rad/min). To determine whether low dose rate (5 rad/min, or 20 rad/min) compared to conventional dose rate (200 rad/min) x-irradiation altered the clonagenic survival of leukemia and lymphoma cell lines, several permanent cell lines were studied. These included: bg/bg cl 1, mouse basophillic leukemia; LW12, [W/fu rat acute myelogenous leukemia (AML)]; and human cell lines: JY and Daudi (B-cell lymphomas); K45, (T-cell leukemia); K562, (erythroleukemia); HL60 and KG1 (monomyeloid leukemias), and U937 (human histiocytic/monocytic lymphoma). Dose rate independent killing was demonstrated at several plating densities with mouse and rat leukemia lines and all human leukemia lines tested except lines HL60 and U937. With HL60, increased plating density increased the D0 at each dose rate. This effect was not attributable to an increased plating efficiency. With line U937 there was a clear dose-rate effect with increase in D0 from 88 rad, n 4.6 at 200 rad/min, to D0 = 166, n 2.3 at 5 rad/min. The data demonstrate that some human hematopoietic tumor derived cell lines of myeloid/monocyte/macrophage lineage can exhibit atypical repair of irradiation damage in vitro. This repair may be enhanced by conditions relevant to clinical TBI including low irradiation dose-rate and cell to cell interactions by tumor cells in close proximity.

Radiosensitivity of human bone marrow granulocyte-macrophage progenitor cells and stromal colony-forming cells: effect of dose rate.

Study of the radiation biology of human bone marrow hematopoietic cells has been difficult since unseparated bone marrow cell preparations also contain other nonhematopoietic stromal cells. We tested the clonogenic survival after 0.05 or 2 Gy/min X irradiation using as target cells either fresh human bone marrow or nonadherent hematopoietic cells separated from stromal cells by the method of long-term bone marrow culture (LTBMC). Sequential nonadherent cell populations removed from LTBMC were enriched for hematopoietic progenitors forming granulocyte-macrophage colony-forming unit culture (GM-CFUc) that form colonies at Day 7, termed GM-CFUc7, or Day 14 termed GM-CFUc14. The results demonstrated no effect of dose rate on the D0 or n of fresh marrow GM-CFUc (colonies greater than or equal to 50 cells) after plating in a source of their obligatory growth factor, colony-stimulating factor (CSF) (GM-CFUc7 irradiated at 2 Gy/min, D0 = 1.02 +/- 0.05, n = 1.59 +/- 0.21; at 0.05 Gy/min, D0 = 1.07 +/- 0.03, n = 1.50 +/- 0.04; GM-CFUc14 at 2 Gy/min, D0 = 1.13 +/- 0.03, n = 1.43 +/- 0.03; at 0.05 Gy/min, D0 = 1.16 +/- 0.04, n = 1.34 +/- 0.05). There was a decrease in the radiosensitivity of GM-CFUc7 and GM-CFUc14 derived from nonadherent cells of long-term bone marrow cultures compared to fresh marrow that was observed at both dose rates. In contrast, adherent stromal cells irradiated at low compared to high dose rate showed a significantly greater radioresistance (Day 19 colonies of greater than or equal to 50 cells; at 2 Gy/min, D0 = 0.99 Gy, n = 1.03; at 0.05 Gy/min D0 = 1.46 Gy, n = 2.00). These data provide strong evidence for a difference in the radiosensitivity of human marrow hematopoietic progenitor compared to adherent stromal cells.

Subcortical connections of area 17 in the tree shrew: an autoradiographic analysis.

The present anterograde autoradiographic study reveals several targets of the striate cortex (area 17) of the tree shrew which were not previously observed in studies which used anterograde degeneration methods; our data also confirm several previous findings. The results are discussed in the context of these projections modulating ascending visual information (claustrum, lateral intermediate nucleus, pulvinar, dorsal lateral geniculate, cells of the external medullary lamina, reticular nucleus of the thalamus, superficial collicular layers, and the anterior and posterior pretectal nuclei) or visuomotor information (putamen, caudate, ventral lateral geniculate, pontine gray, and the anterior and posterior pretectal nuclei).

Activated human N-ras oncogene enhances x-irradiation repair of mammalian cells in vitro less effectively at low dose rate. Implications for increased therapeutic ratio of low dose rate irradiation.

The effect of x-irradiation dose rate on the clonagenic survival of mouse embryo fibroblast cell line NIH/3T3 and its N-ras human oncogene transformed subline was studied. Both control and N-ras transformed cell lines were maintained in Dulbecco's modified Eagle's medium at 37 degrees C with 5% CO2. These cell lines were passaged twice weekly and the cells were irradiated in log phase on a 250 kVp orthovoltage unit at 5 or 200 rad/min, adjusting filtration and FSD to account for each dose rate. After irradiation, the cells were replated and colonies of greater than or equal to 50 cells were scored on day 7. D0 and n were calculated via linear regression analysis. There was a significant increase in saturation density and plating efficiency of N-ras transformed cells with loss of contact inhibition. There was no significant difference in radiosensitivity between the two cell lines at 5 rad/min. For NIH/3T3 D0 = 336, n = 2.19; for N-ras transformant D0 = 314, n = 2.35 (p = 0.65); however, irradiation at 200 rad/min revealed a significant survival advantage for the transformed line. For NIH/3T3 D0 = 145, n = 9.1 and for the N-ras transformed line D0 = 208, n = 4.05 (p = 0.0018). The data provide evidence that repair factors which govern irradiation survival may differ for high and low dose rate irradiation and that repair of high dose rate irradiation damage is enhanced directly or indirectly by expression of the N-ras oncogene. The data support hyperfractionated (low dose rate) irradiation for improving the therapeutic ratio during control of rapidly proliferating tumors expressing an activated N-ras oncogene.

Bone-resorbing activity is expressed by rat macrophages in response to arthropathic streptococcal cell wall polymers.

Rat peritoneal macrophages stimulated in vivo by group A streptococcal peptidoglycan-polysaccharide (PG-APS) resorb bone as measured by solubilization of 45Ca from radiolabeled, devitalized bone chips. Activity was strain-dependent and correlated with the susceptibility of rat strains to PG-APS-induced arthritis. PG-APS-stimulated macrophages from the resistant Buf rat strain were not induced to resorb bone, but ingested equivalent concentrations of PG-APS compared to bone-resorbing macrophages from the arthritis-susceptible Lew strain. Resorptive activity peaked at three to five days and decreased to background levels by 10 days after injection. PG-APS-stimulated macrophages from congenitally athymic Lew rats were as effective as macrophages from heterozygous littermates at resorbing bone. Lew macrophages were also responsive to small, nonarthropathic PG-APS polymers generated by mutanolysin digestion. Resident peritoneal macrophages did not respond to stimulation by PG-APS in vitro. Indomethacin at a concentration of 10 micrograms/ml was an effective blockade against PG-APS-induced macrophage bone resorption in vitro, but catalase was ineffective. These results indicate that expression of rat macrophage bone-resorbing activity reflects genetic regulation of the response to PG-APS rather than a defect in ingestion of these polymers and imply that PG-APS-stimulated, bone-resorbing macrophages may contribute to early, initial bone destruction that occurs in inflammatory arthritis.

Alkalinization of mepivacaine does not alter onset of caudal anesthesia.

STUDY OBJECTIVE: To determine the effect of alkalinization of mepivacaine on onset of caudal anesthesia. DESIGN: Randomized, blind study. SETTING: Colon-Rectal Surgery Service of the Tertiary Center at Cleveland Clinic Foundation. PATIENTS: Young, healthy adults undergoing anal surgery. INTERVENTIONS: Addition of bicarbonate (study group) or saline (control group) to mepivacaine. MEASUREMENTS AND MAIN RESULTS: At the onset of sacral anesthesia, demographics were measured. A slightly faster onset was found in the study group (4.28 vs. 6.08 minutes), but this was not statistically significant. CONCLUSIONS: Alkalinization of mepivacaine does not significantly accelerate the onset of caudal anesthesia.

Memory scanning for words in visual images.

This study attempts to replicate an experiment reported by Seamon (1972). In the present investigation, as in the study by Seamon, the scanning of short-term memory was compared when its contents were rehearsed words vs. a mental image. Memory sets were composed of either one, two, or three words. In the relational imagery group, subjects were required to form a single interactive mental scene of the entities which the memory set words represent. Nonimagery subjects were given instructions to covertly rehearse the memory set. In both groups, the usual memory set size (m) effect was obtained, i.e., reaction time (RT) increased linearly with m. Moreover, the set size effect was the same for both groups. This latter finding stands in marked contrast to the result obtained by Seamon; he found no effect of set size when subjects were given interactive imagery instructions. Because of the failure to replicate Seamon, an additional group of subjects were given imagery instructions. For this latter group, some of the procedural discrepancies between the relational imagery group of the present study and the corresponding group in Seamon's study were resolved. Also, in this additional group, the set size effect was examined as a function of the subjects' ratings of the quality of the images which they had formed. The same set size effect was found for this additional group, and the effect was independent of image quality.

Analysis of the association of peptides of optimal length to class I molecules on the surface of cells.

The association of major histocompatibility complex (MHC) class I molecules on the surface of cells with synthetic antigenic peptides of eight or nine amino acid residues was examined. Peptides were synthesized that correspond to the antigenic sequences from ovalbumin and influenza nucleoprotein believed to be naturally processed and presented by cells with Kb and Db MHC class I molecules, respectively. Consistent with the results of others, these peptides were 10(3)-10(5) times more active in stimulating specific T cells as compared to peptides of longer sequences. When cells are incubated with these peptides at less than 0.01-0.1 microM, the association of the peptides with class I molecules is dependent on (i) the reassociation of free beta 2-microglobulin from the extracellular fluids, (ii) a process that requires cells to be metabolically active, or (iii) stabilization of class I heterodimers by chemical crosslinking. In contrast, when cells are incubated with these peptides at greater than 0.1-1.0 microM, the peptides associate with class I molecules in the absence of exogenous beta 2-microglobulin, energy, or chemical crosslinking. Antigen competition experiments suggest that the class I molecules that bind peptides offered at high concentration become only transiently receptive to binding peptide. The concentration of peptides required for presentation to T cells under these conditions corresponds to those that stabilize Kb molecules on the surface of RMA-S mutant cells in the absence of exogenous beta 2-microglobulin. These results support the concept that the receptivity of class I molecules on cells is determined by the dissociation of beta 2-microglobulin from MHC class I that lacks bound peptides.

Inhibition of class I and class II MHC-restricted antigen presentation by cytotoxic T lymphocytes specific for an exogenous antigen.

Ag in the extracellular fluids can be internalized, processed, and presented in association with class I MHC molecules on specialized APC in normal spleen. We examine the fate of these APC after they present Ag to a CTL. When splenocytes present exogenous OVA to CTL, their ability to subsequently present native Ag in association with both class I and class II molecules is inhibited. CTL do not inhibit the ability of splenocytes to present processing independent peptides with class I or class II molecules. Inhibition of Ag presentation is only observed in the presence of the specific Ag recognized by the CTL. This inhibition is MHC-restricted. In the presence of specific Ag, CTL inhibit the ability of APC to present unrelated Ag. However, bystander APC are not affected by activated CTL. Taken together these results indicate that when APC present exogenous Ag to CTL, they are inhibited or killed. The CTL that mediates this activity has a conventional CD4-CD8+ phenotype and utilizes a TCR-alpha beta. The potential significance of these findings and their possible relationship to phenomena associated with Ts cells are discussed.

Reassociation with beta 2-microglobulin is necessary for Db class I major histocompatibility complex binding of an exogenous influenza peptide.

A synthetic peptide corresponding to residues 365-380 of the influenza nucleoprotein (NP365-380) has been previously shown to associate with class I major histocompatibility complex-encoded molecules and to stimulate cytotoxic T lymphocytes [Townsend, A. R. M., Rothbard, J., Gotch, F. M., Bahadur, G., Wraith, D. & McMichael, A. J. (1986) Cell 44, 959-968]. We find that intact Db class I heterodimers on the cell surface are unreceptive to binding this antigen. However, NP365-380 readily associates with Db molecules on the plasma membrane in the presence of exogenous beta 2-microglobulin. In addition, there is a second pathway through which this peptide associates with class I molecules that requires energy and de novo protein synthesis. These findings have implications for maintaining the immunological identity of cells and for the use of peptides as vaccines for priming cytolytic T-cell immunity.

Characterization of antigen-presenting cells that present exogenous antigens in association with class I MHC molecules.

Exogenous Ag in the extracellular fluids do not gain access to the class I Ag-presenting pathway in most cells. However, there is an APC resident in spleen that can process and present exogenous Ag in association with class I molecules. We characterize the phenotype of this cell. This APC is of low buoyant density, is adherent to Sepharose and glass, and expresses both class II molecules and FcR. This phenotype identifies this APC as a macrophage. Resident, peptone- and thioglycolate-induced peritoneal macrophages also display this Ag-presenting activity. Analysis with CTL clones suggest that this Ag-presenting pathway may be active in only a subset of macrophages. A similar Ag-presenting activity is also present in dendritic cell-enriched populations from spleen although we cannot rule out the possible involvement of contaminating macrophages. In contrast, B and T cells that are resident in spleen and LPS blasts are unable to present exogenous Ag in association with class I molecules. The presentation of exogenous OVA with class I molecules is not inhibited by the inhibitors of thiol proteases, leupeptin, and antipain. The presence of gelonin, a ribosomal inactivating protein, in the extracellular fluids inhibits the ability of these APC to present exogenous OVA. Under identical conditions, gelonin does not inhibit Con A-stimulated T cell proliferation, or LPS-stimulated B cell proliferation and Ag presentation. These results are discussed in relation to the potential pathways through which an Ag in the extracellular fluids is presented with MHC class I molecules.

Generation of class I MHC-restricted T-T hybridomas.

In this report we describe a system for the generation of functional, class I MHC-restricted, T-T hybridomas. The BW5147 cell line was transfected with the CD8 gene. BW5147 transfectants were obtained that stably expressed CD8 and this expression was maintained after somatic cell hybridization with activated T lymphocytes. To determine whether the stable expression of CD8 would facilitate the generation of class I MHC-specific T-T hybridomas, the transfected cells were fused with alloreactive T cells and the resultant hybrids were screened for their ability to produce lymphokines in response to antigenic stimulation. Somatic cell hybridizations with BW5147-CD8 transfectants give rise to a much higher frequency of class I MHC-specific T-T hybridomas relative to parallel fusions with BW5147. To determine whether the BW5147-CD8 transfectants would also support the generation of Ag-specific, class I MHC-restricted T-T hybridomas, they were fused with OVA-specific CTL. Several T-T hybrid clones were identified that produced lymphokines after stimulation with a transfected APC that was synthesizing OVA, or with a tryptic digest of OVA in the presence of syngeneic APC. The stimulation by Ag was MHC-restricted and mapped to the Kb molecule. An anti-CD8 mAb inhibited the stimulation of these hybridomas by Ag plus APC, whereas their stimulation by mitogen was unaffected. Cytolytic activity was not detected when several of the OVA-specific or alloreactive hybridomas were tested for their ability to kill target cells bearing the appropriate Ag. These results demonstrate that the BW5147-CD8 transfectants allow the generation of class I MHC-restricted T-T hybridomas. The potential utility of this system is discussed.

Cerulenin is a potent inhibitor of antigen processing by antigen-presenting cells.

Cerulenin is an antibiotic that inhibits eukaryotic lipid and sterol synthesis and blocks lipid modification of proteins. The effect of cerulenin on the ability of accessory cells to present antigen to T cells was investigated. This antibiotic strongly inhibits the ability of accessory cells to present antigen to murine T-T hybrids. This effect is observed for multiple distinct antigens including L-glutamic acid60-L-alanine30-L-tyrosine10, bovine insulin, L-glutamic acid56-L-lysine35-L-phenylalanine9, and ovalbumen. Presentation by both macrophage and B lymphoblastoid cell lines is inhibited. The ability to effectively pulse these cells with antigen is inhibited but not the ability of these same cells to present antigen that they have previously processed. Furthermore, this inhibition is selective as it can occur without significant inhibition of the antigen-presenting cell protein or DNA synthesis. Cerulenin does not inhibit antigen uptake or catabolism as assessed with labeled antigen. By these criteria this drug is shown to interfere with an antigen-processing step. The ability of cerulenin to block processing was compared with other known inhibitors. Although cerulenin was effective with all antigens tested, at least one inhibitor was not. Taken together, these results suggest that the effect of cerulenin may define a distinct step in antigen processing and provides evidence that some other processing events are not universally required. The ability of cerulenin to interfere with antigen processing is discussed in the context of the known actions of this antibiotic and events of antigen processing and presentation.

Amphotropic retrovirus vector transfer of the v-ras oncogene to human hematopoietic and stromal cells in continuous bone marrow cultures.

Human continuous bone marrow cultures were established from intraoperative marrow specimens and infected with amphotropic murine leukemia virus (Am-MuLV) pseudotypes of Kirsten or Harvey murine sarcoma virus, and the biologic effects were compared with mouse continuous bone marrow cultures. Cultures were tested for production of total nonadherent granulocytes and granulocyte-macrophage progenitor cells (GM-CFUc); virus replication by supernatant reverse transcriptase activity; percentage of adherent and nonadherent cells and GM-CFUc that released virus by infectious center assay; and for synthesis of Harvey ras p21 protein. High-efficiency, stable Am-MuLV infection of over 90% of human marrow-culture nonadherent and adherent cells and both seven- and 14-day GM-CFUc were detected as Kirsten or Harvey pseudotype virus release by infectious center assay. Synthesis of Harvey ras p21 was detected in the adherent and nonadherent cell populations of human as well as mouse continuous marrow cultures infected with Kirsten or Harvey pseudotype virus. In contrast to data with mouse cultures, cumulative production of GM-CFUc and differentiated granulocytes in human cultures was not detectably altered by Harvey or Kirsten virus infection, and all cultures ceased to produce hematopoietic cells by 20 weeks. Of 54 virus-infected cultures in ten separate experiments, 13 produced a second peak of nonadherent cells (greater than 10(5) per flask) after 20 weeks, significantly more frequently than did control uninfected cultures (one of 32). When subcultured, these harvests produced permanent Epstein-Barr virus (EBV)-transformed pre-B cell lines that released the original inoculating pseudotype virus. Thus, Am-MuLV is a potentially valuable vector for inserting genetic sequences by recombinant techniques into human hematopoietic and stromal cells in culture; however, activation of EBV may be a significant complication.