Detection of mutations in transgenic fish carrying a bacteriophage lambda cII transgene target.

To address the dual needs for improved methods to assess potential health risks associated with chemical exposure in aquatic environments and for new models for in vivo mutagenesis studies, we developed transgenic fish that carry multiple copies of a bacteriophage lambda vector that harbors the cII gene as a mutational target. We adapted a forward mutation assay, originally developed for lambda transgenic rodents, to recover cII mutants efficiently from fish genomic DNA by lambda in vitro packaging. After infecting and plating phage on a hfl- bacterial host, cII mutants were detected under selective conditions. We demonstrated that many fundamental features of mutation analyses based on lambda transgenic rodents are shared by transgenic fish. Spontaneous mutant frequencies, ranging from 4.3 x 10(-5) in liver, 2.9 x 10(-5) in whole fish, to 1.8 x 10(-5) in testes, were comparable to ranges in lambda transgenic rodents. Treatment with ethylnitrosourea resulted in concentration-dependent, tissue-specific, and time-dependent mutation inductions consistent with known mechanisms of action. Frequencies of mutants in liver increased insignificantly 5 days after ethylnitrosourea exposure, but increased 3.5-, 5.7- and 6. 7-fold above background at 15, 20, and 30 days, respectively. Mutants were induced 5-fold in testes at 5 days, attaining a peak 10-fold induction 15 days after treatment. Spontaneous and induced mutational spectra in the fish were also consistent with those of lambda transgenic rodent models. Our results demonstrate the feasibility of in vivo mutation analyses using transgenic fish and illustrate the potential value of fish as important comparative animal models.

Differential release of interleukin (IL)-1 alpha, IL-1 beta, and IL-6 from normal human monocytes stimulated with a virulent and an avirulent isogenic variant of Mycobacterium avium-intracellulare complex.

Members of the Mycobacterium avium-intracellulare complex (MAC) can exist in a transparent or opaque colonial morphology when cultured on synthetic medium. An opaque variant was developed from a transparent strain of a clinical MAC isolate. Comparison of the two variants showed a greater ability of the transparent colonial variant to infect normal human monocytes as measured by growth in monocyte-bacteria cocultures. Further analyses indicated diminished ability of the transparent variant to induce extracellular secretion of interleukin (IL)-1 and IL-6, as well as membrane-associated IL-1 when compared with the opaque isotype. At the molecular level, induction of specific IL-1 alpha, IL-1 beta, and IL-6 mRNAs was consistent with the protein results. These results suggest that the virulent transparent MAC, as opposed to the avirulent opaque type, may escape host defenses by failing to induce IL-1 and IL-6, key factors in the initiation of a normal immune response.

Involvement of HLA-DR+ large granular lymphocytes in the induction of tumor necrosis factor by Mycobacterium avium-intracellulare complex.

This study shows that normal human large granular lymphocytes (LGL) secrete tumor necrosis factor (TNF) in response to Mycobacterium avium-intracellulare complex (MAI). Percoll density gradient fractionation of peripheral mononuclear cells showed TNF activity in the fractions corresponding to LGL and not T cells, even when 5% monocytes were added to the T lymphocytes for accessory function. TNF release was not abrogated by treatment of the crude LGL preparations with anti-Leu M3, -CD4, and -CD8 antibodies (Ab) plus complement (C), but was abrogated by anti-CD16 and -CD2 Ab, as expected. Interestingly, anti-HLA-DR monoclonal antibody (mAb) treatment significantly diminished TNF activity from LGL, but maintained natural killer (NK) cell function unmodified as opposed to CD2+ and CD16+ cell depletion. Panning studies demonstrated that TNF secretion upon MAI stimulation resided only in the HLA-DR+ LGL and not the DR- LGL population. These results indicate that normal fresh HLA-DR+ LGL, as well as monocytes, are also responsible for rapid TNF secretion during early MAI infection. These DR+ cells appear to be distinct from those expressing NK function.

Interferon decreases the growth inhibition of Mycobacterium avium-intracellulare complex by fresh human monocytes but not by culture-derived macrophages.

Using a rapid radiolabel assay, monocytes derived from the peripheral blood of normal donors were found to kill 40%-92% of inoculated Mycobacterium avium-intracellulare complex (MAC), an opportunistic pathogen commonly found in AIDS patients. However, bactericidal activity was significantly lower in 4-day culture-derived macrophages compared with matched monocyte cultures. The addition of interferon-gamma (IFN-gamma) to monocytes was found to inhibit the bactericidal activity of fresh monocytes. The number of bacteria recovered from fresh monocytes exposed to IFN-gamma was significantly higher than that in control cultures with MAC alone, suggesting that intracellular MAC growth could be stimulated by IFN-gamma. This enhancement of MAC survival and growth by IFN-gamma was not observed when culture-derived macrophages were used. Similar results were obtained with IFN-alpha/A2. These results indicate, therefore, that the innate efficiency of mycobacterial killing by monocytes can be down-regulated by IFN, but macrophages are not significantly affected.

Production of granulocyte-macrophage colony-stimulating factor (GM-CSF) by monocytes and large granular lymphocytes stimulated with Mycobacterium avium-M. intracellulare: activation of bactericidal activity by GM-CSF.

Treatment of monocytes with recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) was shown to enhance their antimycobacterial activity in an in vitro assay. Furthermore, Mycobacterium avium-M. intracellulare was found to induce the production of this hemopoietic growth factor. Human peripheral blood mononuclear cells were fractionated by plastic adherence and Percoll density centrifugation, and each population of cells was stimulated with mycobacteria. GM-CSF was produced by both monocytes and large granular lymphocytes (LGL) but not T lymphocytes. The phenotype of the GM-CSF-producing LGL was found to be CD2+, CD16+, and HLA-DR+ but negative for T-cell and monocyte markers. Kinetic studies demonstrated that GM-CSF appeared in the supernatant fluids within 2 days of culture of either monocytes or LGL and continued to be produced up to 7 days of incubation. Northern (RNA) blot analysis of RNA from both cell types demonstrated the expression of GM-CSF message within 24 h of stimulation. From these studies, LGL and monocytes are capable of responding to M. avium-M. intracellulare by producing factors that augment normal immune functions, including the antibacterial capability of monocytes.

Mycobacterium avium-intracellulare induces interleukin-6 from human monocytes and large granular lymphocytes.

Mycobacterium avium-intracellulare (MAI) is an opportunistic pathogen commonly found in acquired immunodeficiency syndrome patients, whose immune systems are severely compromised. However, normal responses to this bacterium are apparently sufficient to prevent disseminated infection because disease is rarely found unless an immunocompromised state is present. Because interleukin-6 (IL-6) is an inflammatory cytokine with a multitude of activities, we investigated the potential of MAI to induce IL-6 from normal human leukocytes. Peripheral blood mononuclear cells were fractionated into monocytes (Mo), large granular lymphocytes (LGL), and T cells and stimulated with bacteria. Culture supernatants were collected and assayed for IL-6 activity by bioassay. Mo and LGL, but not T cells, were found to release IL-6 within 12 hours of stimulation, with optimal production occurring by 2 days of culture. Production of IL-6 from human leukocyte subsets was confirmed by Northern blot analysis and by neutralization of biologic function of the culture supernatants with specific antisera. Taken together, these results indicate that production of IL-6 is a key response of Mo and LGL to MAI. The role of IL-6 in MAI infection, therefore, needs to be further investigated.

Production of granulocyte-macrophage colony-stimulating factor by large granular lymphocytes stimulated with Candida albicans: role in activation of human neutrophil function.

In the present study, culture supernatants from larger granular lymphocytes (LGL) that were activated with Candida albicans antigens were shown to stimulate the ability of neutrophils to inhibit fungal growth. Identification of the activation factors showed that granulocyte-macrophage colony-stimulating factor (GM-CSF), a hematopoietic growth factor, was involved. Human peripheral blood mononuclear cells were fractionated by Percoll density centrifugation and each subpopulation of cells was stimulated with C albicans yeast cells. GM-CSF was produced in those fractions enriched for LGL, but not T lymphocytes or adherent monocytes. Additionally, the phenotype of the GM-CSF-producing cell was found to be CD2+, CD16+, HLA-DR+, and negative for CD4, CD8, and CD15. Kinetic studies demonstrated that GM-CSF appeared in the supernatants within 2 days of culture and continued to be produced up to 7 days. Optimal stimulation of LGL was seen at a ratio of 3 heat-killed C albicans yeast cells per LGL. Thus, LGL play an important role in host defense against this opportunistic pathogen by producing cytokines, including GM-CSF, which in turn activates the fungicidal activity of neutrophils.

A rapid [3H]glycerol radioassay for determination of monocyte-mediated growth inhibition of Mycobacterium avium.

[3H]glycerol was used to radiolabel Mycobacterium avium (MA) bacteria after interaction with human monocytes in a rapid in vitro assay for determination of the growth inhibition of the mycobacteria by monocytes. Monocytes and MA were co-cultured in 96-well microtiter plates for 1-5 days, and [3H]glycerol was added for an additional 3 days of incubation to radiolabel residual bacteria. The results indicate that monocytes inhibited mycobacterial growth within 24 h of co-culture, an activity which increased during incubation until optimal growth inhibition was noted by 3-4 days. A comparison with conventional plate counting methodology demonstrated similar responses between the two assays except that the conventional assay required 2-3 weeks of culture before visible MA colonies could be detected and enumerated. Thus, the development of a rapid radiolabel assay to quantitate the interaction between monocytes and MA will facilitate the investigation of normal host responses to this opportunistic pathogen.

Lysis of mycobacteria-infected monocytes by IL-2-activated killer cells: role of LFA-1.

Mycobacterium avium-intracellulare (MAI) is a ubiquitous soil contaminant that rarely causes disseminated disease in adults regardless of immunological status. In AIDS patients, however, this organism invades virtually every tissue and organ, and most conventional chemotherapeutic agents are usually ineffective against MAI. We report here that monocytes, in which MAI has established an intracellular parasitic stage, are under the control of natural killer (NK) cells. Autologous large granular lymphocytes (LGL), purified from human peripheral blood leukocytes, were capable of efficiently lysing autologous MAI-infected monocytes in a 5-hr 51Cr release assay. More importantly, interleukin 2 (IL-2) was able to activate the LGL to a higher degree of lysis of infected monocytes. LGL cultured in medium alone could not kill normal monocytes, but showed some degree of lysis of MAI-infected cells. IL-2 activated killer (LAK) cells, on the other hand, lysed normal monocytes to a moderate degree and this activity was makedly enhanced if the monocytes were infected with MAI. The sensitivity of monocytes was directly proportional to the inoculating number of bacteria, indicating that increased bacterial burden would enhance susceptibility to LAK-mediated lysis. Finally, the addition of monoclonal antibodies to LFA-1 (both alpha and beta chains), but not LFA-2 or LFA-3, blocked lysis of both infected and uninfected monocytes when added directly to the cytotoxicity assays, indicating that this adhesion protein is involved in the lysis of autologous, infected monocytes. Thus, NK/LAK cells may be important in containment of infection by lysis of infected monocytes before the bacteria can multiply and spread to other sites.

Human saliva and taste responses to acids varying in anions, titratable acidity, and pH.

Twenty subjects recorded perceived sourness of solutions of citric + fumaric and of citric + tartaric acids, at pH 3.5 and titratable acidity (TiA) of 4.0 g/l on a moving chart, while parotid saliva flow was recorded via a sialometer . Sourness intensity and flow were greater when citric was the minor acid than when it was dominant. Subjects varied widely in calculated volume of saliva reservoir, but not flow rate (time to 2/3 reservoir vol.). In tartaric-fumaric acid mixtures varying in pH (3.0-3.75) at a constant TiA of 4.0 g/l, and varying in TiA (3.7-4.6 g/l) at a constant pH of 3.5, sourness intensity and parotid flow increased with acidity and decreased with pH. However, eight subjects with a high flow (HF = 1.2 +/- 0.28 g/2 min) and nine subjects with a low flow (LF = 0.43 +/- 0.11 g/2 min) differed widely: (a) In response to variation in stimulus pH and TiA, HF demonstrated marked alteration in flow, but little change in sourness ; LF responded at a lower absolute level, but showed marked changes in sourness and little change in flow; (b) Salivary pH was higher and Na+ was three times greater for the HF than for the LF subjects; and (c) Salivary Ca++ showed a direct relationship with flow and pH among the HF, but an inverse relationship for the LF subjects.

Improving the record-keeping performance of direct service personnel.

Record review procedures and contingent performance feedback were used to monitor and improve the record-keeping performance of human service staff in a behavioral residential treatment setting. A multiple baseline design was employed across three groups of B.A.-level human service personnel. The study consisted of five conditions: (1) Baseline 1; (2) Written Instructions (memoranda); (3) Written Performance Feedback; (4) Verbal Performance Feedback; and (5) Baseline 2 (return to the Baseline 1 condition). Dependent measures included highly reliable ratings by independent observers of the (1) essential documents present in case records, (2) documents approximately located/organized in case records, and (3) an overall rating of documents present, correctly organized, signed and dated, and not duplicated in the records. Results indicated that the procedures most frequently used to provide feedback to human services personnel--meetings, policy and procedure manuals, and/or written memoranda--were not as effective as verbal feedback sessions in prompting staff participation in case record maintenance. Findings were interpreted to suggest that, with adequate training, supervision, and performance appraisal, direct service personnel in residential treatment settings can effectively manage clients' case records and become more involved in, and committed to, accountability and quality control.