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.

Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules.

Reagents that inhibit the ubiquitin-proteasome proteolytic pathway in cells have not been available. Peptide aldehydes that inhibit major peptidase activities of the 20S and 26S proteasomes are shown to reduce the degradation of protein and ubiquitinated protein substrates by 26S particles. Unlike inhibitors of lysosomal proteolysis, these compounds inhibit the degradation of not only abnormal and short-lived polypeptides but also long-lived proteins in intact cells. We used these agents to test the importance of the proteasome in antigen presentation. When ovalbumin is introduced into the cytosol of lymphoblasts, these inhibitors block the presentation on MHC class I molecules of an ovalbumin-derived peptide by preventing its proteolytic generation. By preventing peptide production from cell proteins, these inhibitors block the assembly of class I molecules. Therefore, the proteasome catalyzes the degradation of the vast majority of cell proteins and generates most peptides presented on MHC class I molecules.

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.

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.

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.

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.

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.

Chemical cross-linking of class I molecules on cells creates receptive peptide binding sites.

Class I heterodimers on the surface of cells are generally unreceptive to binding peptides in the absence of exogenous beta 2-microglobulin. Paraformaldehyde covalently cross-links beta 2-microglobulin to class I heavy chains in situ and stabilizes empty class I heterodimers. Functionally, this cross-linking creates receptive class I peptide binding sites by acting on beta 2-microglobulin-associated molecules. The presentation of preexisting peptide-class I complexes is also enhanced. These findings support a model whereby a structural alteration, the dissociation of beta 2-microglobulin, limits the existence of receptive class I molecules on normal cells and may control the half-life of active class I molecules.

Dissociation of beta 2-microglobulin leads to the accumulation of a substantial pool of inactive class I MHC heavy chains on the cell surface.

A large pool of free class I heavy chains is detected in situ on the plasma membrane of living cells. These chains are present on cells of different MHC genotypes and appear to exist under physiological conditions in vivo. These molecules arise from the dissociation of previously assembled class I heterodimers at the cell surface. The ratio of intact to dissociated heterodimers is strongly affected by the occupancy of the peptide-binding site of the class I molecule. Upon dissociation of the heterodimer, the class I molecule is functionally inactive. These findings may help to explain why class I molecules on the cell surface are unreceptive to binding peptides yet readily associate with peptides in the presence of exogenous beta 2-microglobulin. These results have implications for understanding the distinct functions of class I versus class II molecules and how the immunological identity of cells is preserved.

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.

Reassociation with beta 2-microglobulin is necessary for Kb class I major histocompatibility complex binding of exogenous peptides.

T lymphocytes recognize endogenously produced antigenic peptides in association with major histocompatibility complex (MHC)-encoded molecules. Peptides from the extracellular fluid can be displayed in association with class I and class II MHC molecules. Here we report that mature Kb class I MHC molecules bind peptides upon dissociation and reassociation of their light chain. Intact Kb heterodimers, unlike class II MHC molecules, are relatively unreceptive to binding peptides. This property may maintain segregation of class I and class II MHC-restricted peptides and has implications for the use of peptides as vaccines.

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.

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.

Infection of hematopoietic and stromal cells in human continuous bone marrow cultures by a retroviral vector containing the neomycin resistance gene.

Stability and expression of the bacterial neomycin resistance gene (neor) transferred to human continuous marrow cultures by a retroviral vector [pZIP-NeoSV(X)] was evaluated over 4 weeks. Following infection of long-term human marrow cultures with pZIP-NeoSV(X), 10-15% of the stromal cells demonstrated high replating efficiency in a dose of the neomycin analogue G418 that was toxic to stromal cells from uninfected cultures. In contrast, G418 resistance was detected in less than or equal to 1% of GM-CFUc and CFU-GEMM derived from the same virus-infected compared to control cultures. Infection of human CFU-GEMM enriched 100 X by monoclonal antibody selection with pZIP-NeoSV(X) did not increase the percentage of neor progenitors. Marrow cells from cultures infected with pZIP-NeoSV(X) and a replication competent amphotropic virus transferred the vector and G418 resistance to HeLa cells at a frequency of 1/10(5) for nonadherent and 1/10(4) for adherent cells. Two established human hematopoietic (HL60 and K562) and one stromal cell line (KM101) stably expressed the neor gene. Thus, a higher efficiency of infection and expression of a gene transferred by pZIP-NeoSV(X) to permanent human hematopoietic tumor cell lines and fresh marrow stromal cells contrasts with a lower level of expression in fresh CSF-dependent human hematopoietic stem cells.

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.

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.

Carcinoma of the large intestine and nontraumatic, metastatic, clostridial myonecrosis.

Traumatic, clostridial myonecrosis is a rare and serious complication of wounds. Nontraumatic, metastatic, clostridial myonecrosis may be caused by carcinoma of the large intestine. Nontraumatic myonecrosis becomes evident with localized pain, generalized toxicity, local signs of inflammation, and crepitation. Serum creatine kinase determinations may be of help in diagnosing patients suspected of having acute myonecrosis. Immediate heroic surgical intervention, usually with demonstration of Clostridium septicum, is mandatory to control the myonecrosis. Appropriate antibiotic therapy is a valuable adjunct to surgical intervention, and penicillin in massive doses appears to be the agent of choice for the clostridia. Hyperbaric oxygen therapy may help in the optimal control. General supportive measures, including frequent blood transfusions, are most important. To save the life of the patient with nontraumatic, metastatic, clostridial myonecrosis, it is necessary, as soon as the patient's general condition permits, to diagnose and eliminate the cause of the myonecrosis. In addition to the case reported, 16 cases have been reported in the literature, making a total of 17. Five patients have survived (survival rate, 29 percent).

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.

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.