Interferon-gamma and mycobacterial infections.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
A J Crowle
Publications and source records attributed to A J Crowle.
Effects of isoniazid and of ceforanide against virulent tubercle bacilli in cultured human macrophages.
Isoniazid (INH) is said to inhibit tubercle bacilli equally well in vivo and in vitro, and to be mycobactericidal. Ceforanide (CEF) can inhibit tubercle bacilli in vitro but has been found ineffective clinically. These two drugs were tested against virulent tubercle bacilli in cultured human macrophages (MP), partly to compare the results with clinical experience, and partly for a better understanding of antituberculosis activities of these drugs in human beings. INH had the same minimal inhibitory concentration (MIC) against tubercle bacilli in MP as in 7H9 broth cultures. It killed multiplying bacilli in MP but not nonmultiplying bacilli, even at 100 times MIC. It killed both multiplying and nonmultiplying bacilli in broth cultures. It interfered with its own effectiveness against intra-MP bacilli by preventing nonmultiplying bacilli from beginning to multiply and thus become susceptible to killing. These findings help explain why this demonstrably mycobactericidal drug produces relapses of tuberculosis when used alone. It was confirmed that CEF is able to inhibit growth in broth cultures (MIC = 10 micrograms/ml). However, it was not effective against either multiplying or nonmultiplying bacilli in MP at concentrations up to 50 micrograms/ml. These results with the drugs INH and CEF support the good record of correlation between the human MP model of tuberculosis and clinical experience in antituberculosis chemotherapy.
Adult↗
Immunization against tuberculosis: what kind of vaccine?
Explore the source record for details and available documents.
Adjuvants, Immunologic↗
Effectiveness of ofloxacin against Mycobacterium tuberculosis and Mycobacterium avium, and rifampin against M. tuberculosis in cultured human macrophages.
Ofloxacin (OFL) is a new broad-spectrum drug with potentially valuable antimycobacterial activity. It was tested for ability to inhibit virulent tubercle bacilli (TB) and virulent Mycobacterium avium in cultured human macrophages (MP). The first-line antituberculosis drug rifampin (RMP) also was tested against TB in MP. The drugs were added to the MP cultures immediately after infection or 2 days later. Antimicrobial inhibition was measured by colony-forming-unit (cfu) counts of bacilli from lysed samples of the infected MP taken at zero, 4, and 7 days after infection. Drug inhibition of the bacteria in vitro in 7H9 broth also was measured. OFL showed the same minimal inhibitory concentration (MIC) of 1.25 micrograms/ml against TB in vitro and in MP. At 2 micrograms/ml or more it killed TB in vitro and in MP. It was equally effective in MP against initially nonmultiplying TB and MP-established TB, which were multiplying exponentially in the MP. OFL had the same MIC in vitro against M. avium but was ineffective against both M. avium in MP and M. avium isolated directly from MP at as much as 8 micrograms/ml. The MIC for RMP against TB in MP was 0.1 microgram/ml, and TB in vitro 0.02 microgram/ml. At 0.5 microgram/ml or greater, it killed TB in MP. RMP killed TB in MP rapidly, whereas OFL killed them slowly. These results confirm initial clinical evidence, as published by others, that OFL should be a useful antituberculosis drug. However, they suggest that it may not be very effective against MA infections.(ABSTRACT TRUNCATED AT 250 WORDS)
Cells, Cultured↗
Inhibition by 1,25(OH)2-vitamin D3 of the multiplication of virulent tubercle bacilli in cultured human macrophages.
Historically, sunlight has seemed to fortify antituberculosis resistance. Evidence is presented here suggesting a role for vitamin D in this effect. The active metabolite of this photosynthesized vitamin, 1,25-dihydroxy-vitamin D3 (1,25D), promotes maturation and activation of human monocytes and macrophages (MPs). Therefore, it was tested for ability to protect MPs against virulent tubercle bacilli. MPs were derived by 7-day culture from blood monocytes, infected with the bacilli, and exposed to 1,25D in several regimens. Their inhibition of bacilli was measured by lysing samples of the cultures at 0, 4, and 7 days after infection and making bacillary CFU counts from serial dilutions of the lysates. 1,25D enabled MPs to slow or stop bacillary replication. Autologous serum supported the 1,25D-induced protection because the vitamin was not effective in medium supplemented with a serum substitute and was less effective in a heterologous AB serum than in autologous serum. The protection developed rapidly and could be induced even when 1,25D was added 3 days after infection. A concentration on the order of 4 micrograms/ml was needed for protection by the regimens used in these experiments. That is considerably higher than normal circulating concentrations of 1,25D but could be reached in infectious granulomas, because MPs can make 1,25D from precursor 25-hydroxyvitamin D3. The precursor circulates at levels 10(3) higher than those of 1,25D and is directly influenced by dietary intake or photosynthetic production of vitamin D. These results identify 1,25D as an immunomodulator which can reproducibly activate human MPs to express tuberculoimmunity. They connect vitamin D, sunlight, and tuberculoimmunity and suggest that vitamin D should be considered a vital factor in the practical control of tuberculosis.
Calcitriol↗
Protein abnormalities in adult respiratory distress syndrome, tuberculosis, and cystic fibrosis sera.
Crossed immunoelectrophoresis (X-IEP) revealed several abnormalities in serum proteins from patients with adult respiratory distress syndrome (ARDS), tuberculosis (TB), and cystic fibrosis (CF). The two quite different kinds of pulmonary disease, one acute (ARDS) and the other chronic (TB and CF) exhibited serum changes specific for each disease and abnormalities associated with inflammation and pathogenesis, in general. In ARDS sera, most proteins were extremely low, presumably due to leakage into the lungs through damaged tissue, while the acute-phase proteins, orosomucoid, alpha 1-antitrypsin, alpha 1-antichymotrypsin, and haptoglobin, were markedly high when compared to the overall protein pattern. The extremely high alpha 1-antichymotrypsin values were not seen in corresponding TB and CF sera. Numerous TB patients had elevated alpha 1-antitrypsin, alpha 1-antichymotrypsin, and haptoglobin, but only the alpha 1-antitrypsin population mean was significantly different from normal. Gc-globulin, ceruloplasmin, and beta-lipoprotein were higher and alpha 1-lipoprotein and inter-alpha-trypsin inhibitor lower than normal. All other quantitative serum changes were not statistically significant. Surprisingly, all TB patients belonged to the Gc-1-1 genotype in contrast to the Gc-1-1, Gc-1-2, Gc-2-2 polymorphisms of the other populations. CF homozygote sera revealed statistically significant increases in the acute-phase proteins, alpha 1-antitrypsin, alpha 1-antichymotrypsin, and haptoglobin, while orosomucoid, transferrin, IgA, and IgG tended to be higher than normal. The tendency for higher levels of transferrin indicated possible iron deficiency in some patients. In contrast, prealbumin, alpha 1-lipoprotein, and inter-alpha-trypsin inhibitor were significantly depressed in CF patients. CF heterozygotes shared the decrease of alpha 1-lipoprotein with the patients while exhibiting small but significant depressions of alpha 2-macroglobulin and IgG. Though not statistically significant, lowered concentrations of alpha 1-antitrypsin were evident for the heterozygotes.
Acute-Phase Proteins↗
Crossed immunoelectrophoretic analysis of ARDS lavage proteins.
Serum proteins in normal and ARDS bronchoalveolar lavages were analyzed using crossed immunoelectrophoresis. Normal lavages demonstrated relatively few proteins (albumin, alpha 1-antitrypsin, transferrin, and haptoglobin) in low concentrations. In contrast, substantial amounts of all identifiable serum proteins were detected in ARDS lavages. IgA was apparently locally produced. Two of the largest proteins, beta-lipoprotein (mol wt greater than 2 million) and IgM (mol wt approximately 900,000) were found to be complexed as evidenced by their coprecipitation in a single spike in ARDS lavage. Electrophoretic modifications of ARDS albumin and alpha 1-antitrypsin precipitation peaks and partial identity spurring of the alpha 1-lipoprotein peak with other precipitation loops indicated possible complex formation between these proteins and other possibly pathogenic lung fluid constituents. Similarly, modifications of orosomucoid and Gc-globulin peaks indicated possible molecular alterations resulting from interactions with other components. The relatively few protein modifications exhibited in ARDS lavages together with alpha 1-antitrypsin-protease complex formation confirm the relative absence of substantial proteolytic activity in ARDS edema fluids obtained within 12 hr of the onset of the syndrome demonstrated in previous studies.
Albumins↗
Comparison of 15 laboratory and patient-derived strains of Mycobacterium avium for ability to infect and multiply in cultured human macrophages.
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. A virulent bacilli generally produced domed, opalescent colonies on nutrient agar. A virulent 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.
Acquired Immunodeficiency Syndrome↗
Natural mycobacteriostatic activity in human monocyte-derived adherent cells.
The effects of human monocyte maturity on the replication of virulent Mycobacterium tuberculosis were examined. Mycobacteria grew readily in freshly isolated, adherent peripheral blood monocytes and in monocyte-derived macrophages obtained after 7 days in culture, as measured by counts of acid-fast bacilli and colony-forming units. Monocytes cultured for only 3 days before infection, however, were less permissive for the mycobacteria than either uncultured or 7-day cells. The association between the low permissiveness of 3-day cells and superoxide production was examined. Mycobacteria induced only a slight increase in superoxide production during the first 60 min of infection in uncultured and in 3-day cells, and no increase in cells cultured for 7 days before infection. Freshly isolated adherent cells produced small amounts of superoxide in response to phorbol myristate acetate (PMA) stimulation, but PMA-induced superoxide production increased steadily for 7 days. Mycobacteria had no effect on superoxide production by PMA-stimulated adherent cells. These results suggest that the suppressive activity of the 3-day cells is not associated with the production of increased amounts of reactive oxygen species.
Cell Survival↗
Inhibition by pyrazinamide of tubercle bacilli within cultured human macrophages.
Pyrazinamide (PZA) is a unique antituberculosis drug because it is effective in vivo but not in mediums commonly used to culture tubercle bacilli. Consequently, it was employed to test the validity of an in vitro macrophage model of human tuberculosis for value as a correlate of clinical events. The drug was as active in the macrophage model as it was clinically, inhibiting virulent tubercle bacilli at concentrations at 20 micrograms/ml or higher. By contrast, it was ineffective in 7H9 bacteriologic culture medium, even at concentrations as high as 2,560 micrograms/ml. It could be either bacteriostatic or bactericidal against intramacrophage tubercle bacilli, depending on its concentration, the donor of the macrophages, and the length of exposure of the infected macrophages to the drug. The data presented suggest that the clinical effectiveness of PZA is determined by a complicated self-modulating sequence of interactions between it, tubercle bacilli, and host macrophages. Specific evidence was found that tubercle bacilli may replicate within human macrophages in non-acid-fast form, thus indicating that colony-forming unit counts are inherently more accurate than acid-fast bacilli counts in these experiments, and also suggesting that important changes in bacillary cell wall composition may occur among tubercle bacilli within infected human macrophages.
Adult↗
Pyrazinamide is not active in vitro against Mycobacterium avium complex.
Strains of M. tuberculosis that elaborate pyrazinamidase are typically susceptible in vitro and in vivo to pyrazinamide (PZA). However, we found that 33 strains of M. avium complex (MAC), all of which were pyrazinamidase-positive, were resistant in vitro to a high concentration (100 micrograms/ml) of PZA when tested at low pH in 7H12 broth by radiometric (BACTEC) method. The drug was equally ineffective against these bacteria within cultured normal human macrophages. We conclude that the pyrazinamidase test is not suitable for susceptibility studies against M. avium complex. On the basis of our in vitro studies, which support earlier data, we believe that PZA is not appropriate for therapy of MAC disease.
Amidohydrolases↗
Studies of antituberculosis chemotherapy with an in vitro model of human tuberculosis.
Testing new drugs or drug combinations for activity against tuberculosis is highly problematic: Clinical therapy trials are expensive and time-consuming; animal trial results may not be applicable to humans; and simple in vitro testing on culture medium excludes a vital component of the natural infection, namely the macrophage. Described here is a technique to treat with chemotherapy human macrophages that have been infected ex vivo with tubercle bacilli. Briefly discussed are the results of treating such infected phagocytes with a variety of agents: Streptomycin, ethambutol, pyrazinamide, isoniazid, and ceforanide. Interesting parallels between the macrophage-model results and observed clinical phenomena are noted. This model appears to have considerable potential for evaluating drug activity against tubercle bacilli, nontuberculous mycobacteria, and, perhaps, other intracellular parasites.
Antitubercular Agents↗
Differential serum protein binding of benzidine- and benzidine-congener based dyes and their derivatives.
Environmental dyes and their derivatives, some of which are genotoxic, must be transported within the body to the tissues which they affect. One mechanism for this can be observed directly by crossed immunoelectrophoresis (X-IEP). Binding of these chemicals to certain serum proteins changes electrophoretic and immunoprecipitation morphology in X-IEP patterns. This is demonstrated here for four azo dyes derived from benzidine, 3,3'-dimethylbenzidine, and 3,3'-dimethoxybenzidine, and their parent aromatic amines. Direct Red 2 (a 3,3'-dimethylbenzidine-based dye), Direct Blue 15 (a 3,3'-dimethoxybenzidine-based dye), Direct Black 38 (a benzidine-based dye), and Evans Blue (a 3,3'-dimethylbenzidine-based dye) all bound to albumin, alpha 1-lipoprotein, beta-lipoprotein, and hemopexin. Direct Red 2 only slightly affected the mobilities of these proteins. Direct Blue 15 bound also to prealbumin and alpha 1-antichymotrypsin, and degraded C3 globulin. Direct Black 38 and Evans Blue bound to numerous additional proteins. Evans Blue bound variably to proteins of sera from different individuals, suggesting that there are individual differences in serum protein binding capabilities for these chemicals. Of the three derivatives of the benzidine dyes, only 3,3'-dimethylbenzidine caused changes in X-IEP patterns, indicating its binding to the serum proteins. This chemical differentially affected sub-populations of alpha 1-lipoprotein, either by altering its electrophoretic mobility or inhibiting its recognition by antibodies. Autoradiographic analyses demonstrated the binding of benzidine and 3,3'-dimethylbenzidine to both alpha 1- and beta-lipoproteins.
Azo Compounds↗
Gamma interferon activates human macrophages to become tumoricidal and leishmanicidal but enhances replication of macrophage-associated mycobacteria.
Recombinant human gamma interferon (rIFN-gamma) 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-gamma 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-gamma-pretreated macrophages could both inhibit the replication of and kill the resulting intramacrophage amastigotes over a 7-day period. However, rIFN-gamma 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-gamma in the absence of macrophages. rIFN-gamma pretreatment had opposite effects on the uptake of mycobacteria and leishmania. As many as 80% fewer activated macrophages ingested mycobacteria compared with controls, whereas 50% more activated macrophages were infected with leishmania. These results suggest that rIFN-gamma may interfere with the immune destruction of intracellular tubercle bacilli and that the mechanisms of immunity against mycobacteria and leishmania may differ.
Cytotoxicity, Immunologic↗
The effect of ethambutol on tubercle bacilli within cultured human macrophages.
Ethambutol (EMB) generally is believed to be clinically mycobacteriostatic. This concept was reexamined in an in vitro model of human tuberculosis that has direct relevance to in vivo effects in human subjects. Cultured human monocyte-derived macrophages infected with virulent Erdman tubercle bacilli were treated with EMB, and the resulting antimycobacterial consequences were measured by counts of acid-fast bacilli and bacterial colony-forming units. When added immediately after infection of the macrophages, EMB inhibited and killed tubercle bacilli within the cells at the same concentrations as it did in bacteriologic culture medium. When added 2 days after infection, it first appeared to increase the viable bacillary count above control culture levels, then killed intramacrophage bacilli at lower concentrations than in bacteriologic culture medium. We speculate that this occurs because macrophages enhance EMB effectiveness by killing tubercle bacilli, which have defective cell walls due to the effects of the drug. The concentrations of EMB that proved mycobactericidal in human macrophages are readily achieved clinically. The purported lesser antituberculosis effectiveness of EMB when compared to other bactericidal agents may be due to its less direct and efficient mode of killing tubercle bacilli, and the necessity therefore, for stricter maintenance of effective drug concentrations in vivo.
Adult↗
Crossed immunoelectrophoresis of rat serum.
A crossed immunoelectrophoresis (X-IEP) reference profile of normal (Sprague-Dawley) rat serum antigens was developed. Twenty-three antigens were detected; 13 were identified. These included inter-alpha-trypsin inhibitor, alpha 1-antichymotrypsin, and fibronectin, which had not previously been identified in rat serum. Many of the antigens were similar electrophoretically to their analogs in human and mouse serum, but some were substantially different. Inter-alpha-trypsin inhibitor, fibronectin, and C3 globulin were more anodic, while transferrin was more cathodic. Rat haptoglobin's mobility was between those of human and mouse haptoglobins. As in the mouse, but more so, alpha-macroglobulin in the rat had alpha 1 mobility rather than alpha 2 seen for this protein in human serum. Only rat alpha 1-antichymotrypsin exhibited a double peak. There were variations in haptoglobin and alpha 1-lipoprotein mobilities among different strains of rat. This establishment of a standard X-IEP map of rat serum will simultaneously permit multiple quantitative and qualitative analyses of rat serum antigens for various experimental and clinical purposes.
Alpha-Globulins↗
Inhibition by streptomycin of tubercle bacilli within cultured human macrophages.
The strategy for using streptomycin against tuberculosis assumes that it is not effective intracellularly. But according to animal cell experiments, this is probably incorrect. We retested this assumption with a new experimental model using cultured human macrophages infected with tubercle bacilli so that the results would be directly relevant to human disease. At 5 and 50 micrograms/ml, streptomycin inhibited the bacilli strongly and killed some; at the lowest tested concentration of 0.5 micrograms/ml, it inhibited them weakly. It was acting intracellularly, because it could inhibit even when added 2 days after the macrophages had been infected and washed free of extracellular bacilli, and because in our experimental model the bacilli were shown to be unable to multiply extracellularly. However, as has been reported for animal macrophages, the antibiotic was quantitatively more than 2 orders of magnitude less effective in human macrophages than in simple bacteriologic medium. Probably this is because streptomycin is concentrated within lysosomes where low pH greatly inhibits it. The human macrophage-tubercle bacillus chemotherapeutic bioassay we describe here for the first time could be a superior patient-consonant new method for testing antituberculosis agents and treatment regimens. It retains important in vivo features, the complete host cell-parasite relationship for instance, without giving up the in vitro advantages of rapidity and objectivity.
Cells, Cultured↗
Replication of lyophilized and cultured BCG in human macrophages.
If BCG must replicate well in vivo to immunize, presumably mainly in macrophages, the potency of BCG vaccines in human subjects might be measurable as the ability of the BCG to replicate in cultured human macrophages. We used this assumption to compare 6 different batches of lyophilized BCG with freshly cultured BCG and freshly cultured virulent Erdman tubercle bacilli, and to study how tubercle bacilli infect and multiply in human macrophages. The bacilli were readily phagocytized. They replicated in macrophages in proportion to how many were alive and how virulent they were, more the former than the latter among the lyophilized bacilli. The proportion of culturable bacilli in exponentially growing suspensions of BCG cultures was unexpectedly low. For suspensions of 5 of the 6 batches of lyophilized BCG, the great majority of bacilli were nonculturable. Suspensions made from the lyophilized BCG contained many large clumps of bacteria that resisted dispersion even by ultrasonic agitation. This clumpiness, presumably imparted to the bacilli during freeze-drying, caused the lyophilized BCG to infect macrophages more heavily than freshly cultured bacilli. In these experiments, native differences among macrophages from different subjects in capacity to phagocytize BCG and to inhibit its intracellular replication also were detected. These studies thus suggest that tests measuring responses of cultured human macrophages to infection with tubercle bacilli offer relatively simple, rapid, and human-being-related means for assessing several important aspects of human immunization with BCG.
Adult↗