Actinomyces viscosus T14V and Streptococcus sanguis 34 coaggregate by a mechanism which is not inhibited by 1 M NaCl, is dextran independent, requires calcium, is pH dependent with an optimum at pH 8.0 to 8.5, and appears to require the interaction of a protein or glycoprotein on A. viscosus with a carbohydrate on S. sanguis. The coaggregation is inhibited more than 80% by 0.01 M lactose, 0.02 M /l-methyl-D-galactoside, or 0.05 M D-galactose; inhibition of coaggregation was less than 10% in 0.1 M a-methyl-D-galactoside, melibiose, maltose, cellobiose, sucrose, and a number of monosaccharides. At very high concentrations of enzyme, protease from S. griseus destroyed the reactive site on A. viscosus but not on S. sanguis. Both were totally resistant to dextranase. Periodate (0.01 M; pH 4) inactivated both bacteria. The ability of S. sanguis to coaggregate with A. viscosus was not destroyed by phenol-water extraction at 65°C for 15 min. When the bacteria were cultured under specified conditions, the coaggregation was highly reproducible. Under the same conditions, T14AV, the avirulent mutant of A. viscosus T14V, did not coaggregate with S. sanguis 34. Electron microscopic studies of coaggregates, labeled immunochemically with antibody to A. viscosus, indicated that fibrils on A. viscosus may be involved in the coaggregation.
Recombinant human gamma interferon (rIFN-y) was examined for its ability to activate human peripheral blood monocyte-derived macrophages to kill tumor cells and to affect the replication of two phylogenetically distinct intracellular pathogens, Mycobacterium tuberculosis and Leishmania donovani. Macrophages preincubated overnight with doses of rIFN-y from 5 to 500 U/ml killed [3H]thymidine-labeled mouse L929 tumor targets, as measured by the release of [3H]thymidine into the supernatant after 48 h. Counts of macrophages initially infected with leishmania promastigotes showed that rIFN-y-pretreated macrophages could both inhibit the replication of and kill the resulting intramacrophage amastigotes over a 7-day period. However, rIFN-y pretreatment of macrophages actually enhanced mycobacterial replication over a 5-to 7-day period, as assessed by (i) counting acid-fast bacilli or (ii) lysing macrophages to release bacteria and determining the numbers of viable units. Mycobacterial growth was not affected by rIFN-y in the absence of macrophages. rIFN-y
Concomitant with glucose-induced insulin release, there occurred an increase of ATP from 4.40 plus or minus 0.21 to 23.16 plus or minus 0.52 pmol/100 islets/min (P less than 0.001) in the effluent from perifused rat islets. There is a linear relationship between circulating ATP and insulin levels both in the stimulated and basal state (r = 0.689, P less than 0.01). Islets incubated with labeled adenine for a short period of time (37.5 min) showed no release of radioactivity upon subsequent glucose-induced insulin release. Islets incubated for a prolonged interval with labeled adenine (150 min) showed an increase in acid soluble radioactivity in the effluent during glucose-induced insulin release. Following incubation of the islets with labeled adenine for 150 min, approximately 5% of the homogenate radioactivity was found in the secretory granules. Using column chromatography to separate the adenine nucleotides, the distribution of radioactivity among the various nucleotides in the secretory granule fraction was found to be: AMP 54.42 plus or minus 4.96%, ADP 14.20 plus or minus 1.63%, ATP 15.39 plus or minus 3.84%, and cAMP 16.07 plus or minus 2.11%. The distribution of radioactivity in the effluent adenine nucleotides after glucose-induced insulin release was: AMP 32.83 plus or minus 4.62%, ADP 24.52 plus or minus 2.77%, ATP 28.13 plus or minus 5.45%, and cAMP 26.01 plus or minus 3.34%. The absolute levels of adenine nucleotides in the secretory granules were ATP 4.19 plus or minus 0.88, ATP madp 7.94 plus or minus 2.20 and cAMP 4.46 plus or minus 1.74 pmol/ug prot. The levels in the islet effluent were ATP, 15.30 plus or minus 2.70, ATP qDP, 29.43 plus or minus 3.49 and cAMP 7.66 plus or minus 1.93 pmol/100 islets/min for the first ten min of glucose-stimulated insulin release. Thereafter there was a rapid decline in effluent cAMP while ATP and ADP remained in essentially equivalent amounts. The distribution of radioactivity and absolute levels of the adenine nucleotides in the effluent reflects that found in the secretory granules, confirming previous observations that insulin release is occurring by exocytosis.
Mycobacterium avium is a cause of nontuberculous chronic granulomatous infections which is attracting increased attention as a frequent opportunistic pathogen in acquired immunodeficiency syndrome. Some important aspects of its human pathogenicity were investigated by using cultured human macrophages infected with it. The uptake and replication of various strains of M. avium in the macrophages could be measured by CFU counts of the bacteria in samples of lysed, sonicated macrophages. Microscopic counts of acid-fast bacilli were not useful because the bacteria multiplying in the macrophages were usually not acid fast. Electron microscopy showed the intracellular bacilli to multiply by transverse fission, to be surrounded in individual vacuoles by a broad electronlucent zone, and to have thinner cell walls than extracellularly grown M. avium. Fifteen strains, including examples of serovars 1, 2, 4, 8, and 9, were studied for uptake and rate of replication in cultured macrophages from three normal subjects. The strains were isolates from patients with nontuberculous granulomatous infection, acquired immunodeficiency syndrome, or unrelated problems, or they were laboratory reference cultures. There were no differences among them in phagocytosis, but there were differences in intracellular replication. Laboratory strains tended to be avirulent, that is, they did not replicate in the macrophages. Patient isolates usually were virulent and could be compared for virulence by intracellular replication rates. Virulence correlated with flat, transparent bacterial colony morphology on nutrient agar but not with serovar or kind of patient from whom the bacteria were isolated. However, among strains of transparent colony morphology there were wide differences in virulence. Avirulent bacilli generally produced domed, opalescent colonies on nutrient agar. Avirulent bacilli predominated in populations of M. avium conditioned to growth in bacteriologic culture medium. Bacilli of virulent colony morphology predominated in populations passaged through cultured macrophages. The model described here presents a new approach to the investigation of the pathogenicity of M. avium for human subjects and may be more patient relevant than animal models. Mycobacterium avium is similar to the closely related bacterium Mycobacterium intracellulare and, probably, to Mycobacterium lepraemurium and Mycobacterium paratuberculosis (20, 41, 42). These bacteria are opportunistic pathogens for people (16, 19, 26, 33, 48) and frequently cause disseminated, fatal infections in acquired immunodeficiency syndrome (
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