We developed a rapid, quantitative culture method to estimate the replication of Mycobacterium avium-intracellulare complex (MAIC) in human peripheral blood mononuclear cells. Mononuclear cells were plated in a 96-well tray, infected with clinically isolated strains of MAIC in the presence of autologous plasma, and further cultivated for 1 to 2 weeks in a tissue culture medium. No MAIC cells proliferated extracellularly, since human plasma inhibited extracellular growth of the mycobacteria. The mononuclear cells were lysed through a brief treatment with alkali, and surviving intracellular mycobacteria were diluted and plated with tissue culture medium in a 96-well tray. Mycobacterial colonies were counted under a microscope after a 5-day incubation. The number of viable MAIC cells continuously increased, reaching 10 times the number of inoculated cells in a week. Thus, mononuclear phagocytes were the permissive site for the replication of MAIC. Intra-and extracellular susceptibilities of seven MAIC strains to four aminoglycoside antibiotics were then studied. The mycobacteria were most susceptible in vitro to dibekacin (MICs, 3.13 to 12.5 ,ug/ml). Dibekacin at 12.5 ,ug/ml was bacteriostatic to five of seven strains in the monocytes. Also, intracellular replication of the other two strains was greatly suppressed by that concentration of dibekacin.Mycobacterium avium-intracellulare complex (MAIC) frequently causes pulmonary inflammatory diseases in middleaged men who have some sort of chronic lung disease (10). Also, cases of disseminated MAIC infection have been reported in immunodeficient patients (1-3, 6, 8-11). Since MAIC is resistant to most antitubercle drugs (7), the current therapy for this infection is disappointing (3). Previously, we reported a method for rapidly determining the drug susceptibilities of mycobacteria in tissue culture medium (5). In that study, 7 of 55 clinically isolated mycobacterial strains were MAIC. In this report, susceptibilities of these MAIC strains to the aminoglycoside antibiotics streptomycin (SM), kanamycin (KM), amikacin (AMK), and dibekacin (DKB) were studied. Since mycobacteria are an intracellular parasite, it is essential to estimate the drug susceptibilities of intracellularly replicating mycobacteria to predict the chemotherapeutic effectiveness of a drug. We attempted to develop a simple, quantitative method to determine the replication of MAIC in human peripheral blood mononuclear phagocytes. Intracellular susceptibilities of the MAIC strains to the aminoglycoside antibiotics were then studied. MATERIALS AND METHODSMycobacteria and culture. MAIC strains were isolated and identified in Tranomon Hospital, Tokyo, Japan, and Juntendo University Hospital, Tokyo. The mycobacteria were precultured in F12 medium (Nissui Seiyaku Ltd., Tokyo) supplemented with 5% heat-inactivated fetal bovine serum (General Scientific Laboratories, Los Angeles, Calif.) and 0.05% Tween 80 (Wako Pure Chemical Industries, Osaka, Japan) (FST medium).Preparation of plasma and mononuclear cells. A ...

Intra-and extracellular susceptibility of 35 clinically isolated Mycobacterium avium-intracellulare strains to cefotaxime (CTX), ceftizoxime (CZX), and cefoperazone was studied. MICs for 50% of the isolates in vitro were 6.25 ,Ig/mI for CTX and CZX and 25 ,ug/ml for cefoperazone. A strain susceptible to CTX (MIC, 0.78 ,ug/ml) and CZX (MIC, 1.56 ,ug/ml) infected human peripheral blood mononuclear cells in the presence of 20% autologous plasma. The mycobacteria replicated exclusively in monocytes under the above culture condition. Concentrations of CZX 1-to 16-fold higher than its in vitro MIC had little effect on intracellular replication of the strain. A concentration of CTX 16-fold higher than its in vitro MIC was bacteriostatic to the mycobacteria, but CTX of lower concentrations showed no effect on intracellular replication. Thus, ineffectiveness of the cephems on the therapy of M. avium-intracellulare infection was suggested.Since Mycobacterium avium-intracellulare is resistant to most antitubercle agents, the current therapy for this infection is disappointing (2-4, 9-11). We look for agents effective against this organism among nonantitubercle drugs. Thirdgeneration cephem antibiotics were studied in this report. The susceptibility of 35 clinically isolated M. avium-intracellulare strains to cefoperazone (CPZ), cefotaxime (CTX), and ceftizoxime (CZX) was tested in FST medium (8) (Fig. 1). MICs of CPZ, CTX, and CZX for 90% of the isolates were 25, 6.25, and 6.25 pug/ml, respectively. It was reported that five strains of M. avium-intracellulare isolated from acquired immune deficiency syndrome patients were all resistant to 10 ,ug of CPZ per ml (3), coinciding with our result. Latamoxef, another third-generation cephem, was found to be the least effective (MIC, 200 ,ug/ml) compared with the three cephems and was omitted from Fig. 1

NADPH oxidase was induced in HL-60 human promyelocytic leukemia cells when these cells were treated with 10(-7) M 1,25-dihydroxyvitamin D3 (VD3) for 4 days. The treated cells were disrupted by sonication, and the postnuclear fraction was separated into 48,000 X g supernatant (cytosol) and precipitate (membrane) fractions. Membrane-bound NADPH oxidase was activated in vitro with SDS and cytosol. However, the cytosol from untreated HL-60 cells could not activate NADPH oxidase. The cytosolic activity was induced 2 days after VD3 treatment and fully expressed on day 4. The activity was heat-sensitive and destroyed by trypsin. The possibility that the cytosolic activation factor is a protein kinase C (PKC) was then tested. Ca2+- and phospholipid-dependent PKC activity was low in the cytosol of untreated HL-60 cells but increased in the cytosols of VD3-treated cells 4 and 11 times, respectively, 2 days and 4 days after treatment. H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride], inhibited PKC activity in a dose-dependent manner at 1-100 microM. Cytosolic activity of NADPH oxidase was not inhibited at all at those concentrations. Furthermore, PKC activity was lost when Ca2+ was omitted from the assay mixture, but NADPH oxidase was activated in the presence of EGTA. These results indicated that the cytosolic factor is not a PKC, and that NADPH oxidase in this cell-free system is activated by a mechanism that does not involve PKC.

Human promyelocytic leukemia (HL-60) cells were induced to differentiate into macrophage-like cells by treatment with 10(-7) mol/L 1,25-dihydroxyvitamin D3 (VD3). A monoclonal antibody (MoAb, 60B8), reactive with the particulate of the differentiated cells but not of the untreated cells, was isolated. The antigen recognized by the MoAb became apparent two days after VD3 treatment, and its concentration increased and peaked on day 6. Human neutrophils, followed by monocytes and differentiated HL-60 cells, showed the greatest abundance of the antigen. Monocytes cultured for eight days in vitro lost the antigen. No 60B8 antigen was seen in other blood cells. The MoAb precipitated two polypeptides with an apparent molecular weight (mol wt) of 15,000 (15 k) and 13 k in the detergent-solubilized, 35S-methionine-labeled lysate of the differentiated HL-60 cells. Double-sandwich type enzyme- linked immunosorbent assay (ELISA) devised for the quantitative assay of 60B8 antigen indicated that some 2% to 5% of neutrophil protein was 60B8 antigen. This antigen was not exposed on the neutrophil cell surface, since the cells were not stained immunofluorescently with either mono- or polyclonal antibody, unless they had become permeable. The neutrophil membrane and the granules were separated on the Percoll density gradient, and the antigen was found localized in the plasma membrane-rich fraction. These findings suggested that 60B8 antigen is a novel differentiation antigen for phagocytic cells.