P. D'Arcy Hart

Manipulations of the phagosome-lysosome fusion response in cultured macrophages. Enhancement of fusion by chloroquine and other amines

Abstract The fusion of secondary lysosomes of cultured normal mouse peritoneal macrophages with phagosomes containing ingested Saccharomyces cerevisiae is inhibited by polyanions previously incorporated in the medium. In contrast, this fusion process can be accelerated by chloroquine and some other secondary and tertiary amines; and these compounds can reverse the inhibition induced by a polyanion. The non-fusion pattern usually associated with intracellular Mycobacterium tuberculosis can also be reversed by chloroquine. Our observations offer a possible new approach to the study of subcellular membrane fusion, and of factors influencing the course of experimental intracellular infections.

Mycobacterium tuberculosis in macrophages: effect of certain surfactants and other membrane-active compounds.

Some compounds, not directly inhibitory or enhancing, nevertheless influence growth of tubercle bacilli in macrophages in cell culture. They include certain surfactants whose effects can be varied by their structural design. The compounds are probably stored in cell lysosomes. They can interact with various membranes to affect permeability. The anti- and protuberculous surfactants differ in such interaction and also in effect on lysosomal enzyme activity in infected macrophages. A link between the effect on lysosomal membranes and on tuberculous infection is suggested.

The effect of inhibitors and enhancers of phagosome--lysosome fusion in cultured macrophages on the phagosome membranes of ingested yeasts.

Abstract For some hours after ingestion of Saccharomyces cerevisiae by cultured macrophages, the phagosome membranes almost all appeared to be applied closely to the cell walls of the enclosed yeasts; most of these “tight” phagosomes showed evidence of having fused with ferritin-labelled secondary lysosomes. If the macrophages were pretreated with any of several polyanionic inhibitors of phagosome-lysosome (P-LF) (e.g. poly- d -glutamic acid) (PGA), and were fixed for transmission electron microscopy (EM) 1 h or more after ingestion of the yeasts, the phagosome membrane frequently appeared to be separated from the yeast cell wall by a wide electron-lucent zone. These “loose”, unfused, phagosomes in PGA-pretreated macrophages developed from tight phagosomes (also unfused), formed immediately after ingestion. The development of loose phagosome membranes could be prevented or rapidly reversed in PGA-treated macrophages by exposing them to chloroquine, one of a number of lipophilic secondary and tertiary amines that enhance P-LF; this exposure also partly reversed the PGA-induced inhibition of P-LF. The evidence suggests that the inhibitors of P-LF evoke loose membrane formation through their effect on the fusion process. On the other hand, reversal of this inhibition of fusion appears to follow the resumption of tightness brought about by chloroquine. The polyanionic inhibitors accumulate in secondary lysosomes, through which their effect on P-LF is presumably mediated. The phenomenon of loose phagosome formation, however, during the inhibition of fusion indicates that other cytoplasmic elements must be involved. The possibility that the depletion of the intracellular free calcium level, by complexing with polyanions, is a relevant factor, is briefly discussed.

Site of action of a polyanion inhibitor of phagosome-lysosome fusion in cultured macrophages

Abstract The subcellular site of action of a new polyanionic inhibitor of phagosome-secondary lysosome fusion (P-LF) in cultured macrophages is described. This semi-synthetic compound chlorite-oxidized amylose (COAM) prepared from amylose starch differs from previously reported inhibitors by its rapidity in suppressing P-LF, and shows low toxicity and a wide range of effective dose. The inhibitor was conjugated to a fluorescent label or to an electron-opaque label and its subcellular location directly determined in living macrophages by fluorescence microscopy and in thin sections by electron microscopy. The fluorescent polyanion was seen only in secondary lysosomes. In cells containing both the fluorescent inhibitor and phagocytosed yeast cells there was no transfer of fluorescence to phagosomes, indicating inhibition of P-LF. The inhibition of fusion was reversed by low levels of lipophilic amines. The possibility that polyanions inhibit P-LF by acting on the lysosomal membrane is discussed.

Chemotherapy of Tuberculosis—Part I

Viewed historically, the conception of treatment in tuberculosis has been as complex and as changing as in any branch of medicine. Emphasis on it as a social disease, a hereditary stigma, a contagion, an inflammation, or a bacterial infection has determined the approach to treatment and prevention. In the present lecture I shall attempt to trace the historical development of the chemotherapeutic aspect of tuberculosis treatment, so that the physician, more particularly the non-specialist, may be helped to see in perspective the successes which, if not already in the foreground of the future, are perhaps no farther away than its middle distance. I shall aim to present this development in relation, on the one hand, to the tremendous advances in general chemotherapy, and, on the other, to the advances in the broad knowledge of tuberculosis and of its management and treatment; and I shall attempt to weave into the complex historical pattern the development of the use of antibiotic substances.

Incidence of Intrathoracic Sarcoidosis Among Young Adults Participating in a Trial of Tuberculosis Vaccines

In the course of the Medical Research Councils clinical trial of tuberculosis vaccines (Medical Research Council, 1956, 1959, 1963) records have been kept of the cases of definite or possible sarcoidosis which developed among the participants. These findings have now been analysed from several points of view; (1) The effect of tuberculin status on entry to the trial, and of B.C.G. and vole-bacillus vaccination, on the subsequent incidence of sarcoidosis. (2) The attack rate of intrathoracic sarcoidosis in the total participants, originally living in urban or suburban areas in England, and observed between the ages of about 15 and 25 years. (3) The relation between sarcoidosis and tuberculin sensitivity, as shown by the periodic tuberculin tests made during the trial.

Effects of non‐ionic surfactants that modify experimental tuberculosis on lipase activity of macrophages

1 Six series of non‐ionic surface active polyethylene glycol ethers, whose effects on experimental tuberculosis have previously been correlated with their polyoxyethylene chain lengths, were examined for their influence on the activity of a lipase present in homogenates of normal mouse peritoneal macrophages. The surfactants are concentrated in the lysosomes of macrophages—a cell type in which the host‐parasite confrontation takes place. A preparation of soy bean oil was used as triglyceride substrate; and hydrolysis at pH 4·5 was compared in the presence and absence of surfactant, the products of hydrolysis being assayed by photodensitometry of thin‐layer chromatograms. 2 The compounds with short polyoxyethylene chains inhibited the release of fatty acid, compared with surfactant‐free standard, more than did those with long chains; and some of the latter showed actual enhancement of release. Accumulation of monoglyceride was observed in the presence of six of the seven long‐chained compounds, but with none of the seven short‐chained compounds. 3 The similarity between this correlation of chain length of the surfactants with their effect on macrophage lipase activity, and the known correlation of their chain length with their effect on experimental tuberculosis, suggests a possible connection. How this connection might relate to the mechanism of the varying effects on tuberculosis is briefly discussed.

The lethal effect of cotton-wool lipid on tubercle bacilli in acid conditions and its prevention by surface-active agents.

Long-chain fatty acids are not appreciably bactericidal to tubercle bacilli at neutral pH, but become so even in slightly acid solution. In this way tubercle bacilli, normally resistant to 0·1 N hydrochloric acid (pH 1·0), are rapidly killed at this pH if traces of fatty acid are present, such as can be found inside test tubes that have been plugged with cotton wool and sterilized by dry heat. The lethal effect is largely prevented by non-ionic, cationic, and to a lesser extent anionic detergents, which probably take up the fatty acid into the micelles so that it can no longer attack the bacilli. The various fatty acids identified in the deposit from heated cotton wool were tested individually and found to be bactericidal. They comprised the usual C 12 to C 18 saturated acids and also oleic acid. The latter, however, showed no particular lethal toxicity in excess of that shown by, for example, palmitic acid. Shorter chained fatty acids than those represented in the deposit were less lethal. We wish to thank Dr A. T. James for the chromatographic analyses and for advice; Dr R. J. W. Rees for carrying out counts of total acid-fast bacilli in two experiments; Messrs. Robinson & Sons Ltd, Chesterfield, for information on the industrial processes involved in the preparation of cotton wool; Mr E. J. Latham for advice on preparing glassware; and Dr J. Marks for helpful criticism of the manuscript.

Enhancement of experimental tuberculosis in the mouse by suramin

Summary Because of the favourable effect of suramin, a powerful trypanocidal agent, which had been reported in patients with pulmonary tuberculosis, its action on the tubercle bacillus in vitro and on experimental tuberculosis in the mouse was investigated. Suramin was found to have no growth-inhibiting or promoting action on cultures in vitro , nor to have a therapeutic effect in the mouse. On the contrary, it increased the susceptibility of mice to both heavy and light tuberculous infections and caused the breakdown of established pulmonary tuberculosis. Furthermore, BCG multiplied more freely in the tissues of mice receiving suramin than in untreated animals. The possible mode of action of suramin in experimental tuberculosis is discussed, and it is suggested that it may alter the lipid surface of the tubercle bacilli in vivo so that they are less susceptible to destruction by the host defence system. This hypothesis is strengthened by the finding that suramin modifies the surface lipids of red blood cells.

Tubular lysosomes and their drug reactivity in cultured resident macrophages and in cell-free medium

Lysosomes were assessed in normal living resident mouse peritoneal macrophages, using mainly phase-contrast microscopy (PCM), darkfield microscopy (DFM), and fluorescence microscopy (FM) after terminal acridine orange (AO) staining; these procedures avoided dyes during experimentation. After a few hours of culture a variable proportion of the normal spherical lysosomes began to assemble in a linear fashion. Fully formed tubular structures, with appearances generally recognized as characteristic of tubular lysosomes (TL), could be seen by PCM or, after labeling, by FM, at 2-5 days (best usually at 4-5 days). This peak was followed by a reduction, and at 8-10 days most of the TL had disappeared, leaving only spherical lysosomes. Renewal of the medium at this stage was followed by a temporary reappearance of TL, suggesting that the medium was a major factor in their initial development also. Formation of TL was enhanced by chloroquine (Cq), though to a lesser degree than by phorbol ester (PMA); in contrast NH4Cl (like Cq a weakly basic amine) caused their disassembly into spherical lysosomes. Manual disruption of the monolayer macrophages enabled TL to be transferred to a cell-free medium, in which they remained apparently stable for several hours. Two known microtubule depolymerizers caused disassembly of TL in the intact cells, reinforcing the idea that the TL are associated with the cytoplasmic microtubule (MT) system; but these agents were inactive in vitro, suggesting that disorganization of the system was responsible for this change.(ABSTRACT TRUNCATED AT 250 WORDS)