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[reaction: see text] The synthesis of the title compound 7 from ethyl glyoxylate and dimethyl and diethyl beta-methylglutaconate is described along with its physical properties that suggest its inability to assume a cis-dienoid structure due to steric hindrance between the methyl and carboxyl groups.

Publisher Summary This chapter focuses on the fluorescence methods for monitoring phagosome- lysosome fusion in human macrophages.This chapter presents an alternative assay based on the use of sulforhodamine or rhodamine (R)-labeled dextran. The assay is based on the initial uptake of sulforhodamine or R-dextran into secondary lysosomes of macrophages, and the subsequent colocalization of the rhodamine label and phagocytosed fluorescein-labeled yeast cells on the fusion of phagosomes with secondary lysosomes. The macrophage pathogen Mycobacterium avium is thought to persist in phagosomes by preventing the fusion of the phagosomes with lysosomes. These observations support the hypothesis that live M, avium inhibits phagosomelysosome fusion, and that M-CSF can activate macrophages to overcome the inhibition of phagosome-lysosome fusion. The molecular mechanisms of the inhibition of fusion, and of the reversal of this process by macrophage colony-stimulating factor(M-CSF), remain to be investigated.

The chemistry, immunochemistry, and immunology of the mycobacterial fatty acid esters of sugars and of sulfated sugars have found their principal focus in the simple nonreducing disaccharide α,α-trehalose, i.e., α-D-glucopyranosyl-α-D-glucopyranoside (1).

Trehalose dimycolate, a glycolipid component of the cell walls of mycobacteria, induces interstitial pneumonitis and alveolar hemorrhages in C57BL/6 and C57BL/10 mice. Homozygous nude (nu/nu) mice of these backgrounds are not susceptible to this form of pulmonary injury. However, after administration of T-lymphocyte-enriched spleen cell preparations from syngeneic donors, homozygous nude mice become susceptible to trehalose dimycolate. The observations suggest that production of pulmonary lesions by this mycobacterial component is dependent on T lymphocytes. While the mechanisms are still under study, we propose that trehalose dimycolate can function as an activator of T lymphocytes and that products of activated T cells are responsible for production of the pulmonary lesions.

The simple apolar C-mycosides, i.e., structurally well-defined hydrophobic glycopeptidolipids of several Mycobacterium species (see diagram below), were earlier shown to behave as receptors for adsorption of mycobacteriophage D4. This phage is usually virulent for Mycobacterium smegmatis. More complex, polar C-mycosides with additional carbohydrate substituents attached solely to the deoxytalose have recently been described. They are the highly specific serotyping antigens discovered by W. B. Schaefer--lipids which characterize members of the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum (MAIS) complex. Both kinds are depicted in the structure below: (Formula: see text) where X equals H (for simple, apolar C-mycosides) and X equals small oligosaccharides (for antigenic forms; more complex, polar C-mycosides). The present investigations showed that the purified polar antigenic lipids exhibit considerably less adsorptive activity for D4 than do the apolar C-mycosides. Thus, the haptenic oligosaccharides are believed to shield the site in the molecule that the phage recognizes, and the blocking is reinforced by the specific antibodies that the antigens elicit. Although the MAIS serovars usually also produce the phage-reactive apolar C-mycosides, they are not permissive hosts for D4, nor do whole cells adsorb the phage. We suggest that in these species the apolar forms are probably "covered" at the cell surface by the antigenic lipids. Therefore, these antigenic mycosides may play a putative role in virulence of the MAIS members by protecting these mycobacteria from their own potential pathogen. The results of chemical transformations at specific sites of the mycoside core coupled with studies of simple synthetic lipid glycosides indicated that the principal phage receptor activity resides in the terminal methylated rhamnose (see diagram). It is this sugar which is evidently masked by the (seemingly remote) haptenic oligosaccharides.

The gross structural features of five families of multiacylated trehalose 2-sulfates elaborated by Mycobacterium tuberculosis strain H37Rv are described. The principal sufatide SL-I is a 2,3,6,6'-tetraacyl-alpha,alphs'-D-trehalose 2'-sulfate, whose component carboxylate substituents (and homolgy) were previously established. In the present study the specific locations of the acyl substituents were assigned. The desulfated glycolipid (SL-I-CF) was methanolyzed on a column of diethylaminoethylcellulose (free base form), affording tri-, di-, and monoacylated trehalose mixtures. The most abundant diacyltrehalose generated was identified as 6,6'-bis-(2,4,6,8,10,12,14,16-octamethyl-17-hydroxydotriaconta-noyl)trehalose (6,6'-bis(C40-hydroxyphthioceranoyl)trehalose), along with lower and higher homologues.A small amount (about 15%) of the unhydroxylated analogue (phthioceranate) was also recognized. From the monoacylated carbohydrate mixture (chiefly 6-(C40-hydroxyphthioceranoyl)trehalose) surviving trehalose monopalmitate(s) were isolated by preparative gas chromatography of the trimethylsilylated products. Trehalose 2-palmitate was identified as the principal component. Small amounts of the 3 isomer may also be present, but no 6-palmitate was detectable. Gentle acidic solvolysis, which minimizes the possibility of acyl migrations, afforded a different diacyltrehalose, identified by mass spectrometry of the permethylated derivative as principally 2-palmitoyl(stearoyl)-3-phthioceranoyltrehalose. A variant in which hydroxyphthioceranate substitutes at the 3 position was also recognized. The results indicate that the biological acylation processes at the trehalose core are not entirely specific, but instead yield an SL-I family, for the chief member of which a logical structural expression is deduced.

Tritylation of 2,3,2',3'-tetra-O-benzyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside) (4) (A. Liav, H.M. Flowers and M.B. Goren (1984) Carbohydr. Res. 133, 53-58) followed by benzylation and acid hydrolysis gave 2,3,4,2',3',4'-hexa-O-benzyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside) (6). Triflation of 6 with triflic anhydride gave the ditriflate 7. Treatment of 7 with potassium mycolate or potassium corynomycolate in toluene, followed by catalytic hydrogenolysis afforded the respective cord-factor analogs 6,6'-di-O-mycoloyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside) (10) and 6,6'-di-O-corynomycoloyl (alpha-D galactopyranosyl alpha-D-galactopyranoside) (11). An alternative approach, based on the debenzylation of 2,3,2',3'-tetra-O-benzyl-6,6'-di-O-p-tolylsulfonyl- (alpha-D-galactopyranosyl alpha-D-galactopyranoside) (1) and conversion of the latter into the corresponding 3,4,3',4'-diisopropylidene derivative 3 failed to yield satisfactory results.

It has previously been inferred that the fusion of a macrophage secondary lysosome with a phagosome delivers the entire lysosomal contents uniformly to the phagosome. We found, however, that different fluorescent lysosomal probes can enter phagosomes at remarkably different rates, even though they are initially sequestered together in the same organelles. Thus, sulforhodamine is almost exclusively delivered to yeast-containing phagosomes within 2 h of phagocytosis. But fluoresceinated, high molecular weight dextran accumulates in the same phagosomes only over a period of approximately 24 h. We postulate that the delivery of lysosomal contents may involve an intermittent and incremental process in which individual components can be selectively and sequentially transferred.

Treatment of 6,6′-di- O -trityl-trehalose (1) [2] with benzyl chloride in dioxane followed by acid hydrolysis and chromatography gave the chromatographically pure 2,3,4,2′,3′,4′-hexa- O -benzyl trehalose (2). Compound 2 was converted into the corresponding 6,6′-di- O -methane-sulphonyl derivative 3 in quantitative yield. Treatment of the latter compound with the potassium salts of 4-[ p -(hexadecyloxy)-phenyl]butyric acid, corynomycolic acid and mycolic acid from Mycobacterium bovis afforded the corresponding benzylated-6,6′-di- O -acyl esters 4, 5 and 6 respectively. Catalytic hydrogenolysis of 4, 5, and 6 yielded 6,6′-di- O -4-[ p -(hexadecyloxy)-phenyl] butyryl-trehalose 7; 6,6′-di- O -corynomycolyl-trehalose 8; and 6,6′-di- O -bovi-mycolyl-trehalose 9 respectively.

From examination of some 40 patient strains of Mycobacterium tuberculosis , a statistically very significant correlation (Spearman's rho) can be drawn between the root index of virulence for the guinea pig (D. A. Mitchison) and the ability of the individual strains to elaborate strongly acidic lipids (SAL) in culture. These include both sulfatides (SL) and phospholipids (PL). Since essentially all, if indeed not all, of the virulent and only few attenuated strains are prolific in elaborating SAL, this criterion may be a necessary requirement for the expression of virulence in M. tuberculosis. Tested by chi-square, this premise is seen to be statistically and pragmatically highly significant. We speculate that SL may contribute to the pathogenesis of tuberculosis because of a demonstrable activity directed against host liver mitochondrial membranes (manuscript in preparation) and its synergistic potentiation of the specific toxicity of trehalose dimycolate (cord factor). The activity may also be expressed against phagosomal and lysosomal membranes within macrophages. Because of their strongly anionic character, SL and PL may interact with cationic sites on lysosomal hydrolases with resultant immobilization and/or inactivation of the enzymes. By a similar mechanism, these ionic lipids may alter the activity of bactericidal basic proteins, previously recognized in the lysosomal armamentarium. Since a minor but significant fraction of demonstrably attenuated strains is nevertheless prolific in SL or SAL elaboration, this facility alone is evidently not a sufficient criterion for expression of virulence.

The mechanism of a synergistic toxicity of 6,6′-dimycoloyl-α,α′- d -trehalose (cord factor) and 2,3,6,6′-tetraacyl-α,α′- d -trehalose 2′-sulfate (sulfolipid I) for mice was studied. Sulfolipid I was entirely nontoxic, but it markedly accelerated the lethal toxicity of cord factor for mice. In vivo, sulfolipid I affected neither respiration nor accompanying phosphorylation of mouse liver mitochondria, whereas in vitro, it induced a swelling and disruption of mitochondrial membranes and strongly inhibited mitochondrial oxidative phosphorylation. The effect of sulfolipid I on mitochondrial structure and function in vitro was neutralized by bovine serum albumin and various animal sera, whereas that of cord factor and cord factor plus sulfolipid I was not prevented by bovine serum albumin. The simultaneous injection of cord factor and sulfolipid I caused an intensive fragmentation of mitochondria and a marked decrease in respiratory and phosphorylative activity in mitochondria. These data indicate that sulfolipid I can achieve an effective attack on mitochondria in combination with cord factor in vivo and induces heavier damage in mitochondrial structure and function than that produced by cord factor alone.

A single intraperitoneal injection of 10 micrograms of trehalose dimycolate (TDM) produced interstitial and hemorrhagic pneumonitis in C57BL/6 mice. As a part of an investigation of a possible role for cell-mediated immunity in the pathogenesis of this disorder, we found that reserpine, 3 mg/kg, given before, at the same time, or on Day 5 after administration of TDM, significantly reduced development of interstitial pneumonitis by Day 7. Smaller doses were less effective. Administration of reserpine, 3 or 2 mg/kg, 1 to 3 days after administration of TDM was lethal to most mice. Reserpine has been shown to inhibit expression of cell-mediated immune responses in mice, probably by causing intercellular release and degradation of vasoactive amines. Inhibition of pulmonary lesions by reserpine in TDM-treated mice suggests that a similar mechanism may be involved in the pathogenesis of TDM-induced lung injury.

For preparing pseudo cord-factors, similar in gross structure to α,α-trehalose 6,6'-dimycolate, we have synthesized (α- d -glucopyranosyluronic acid) (α- d -glucopyranosiduronic acid) (“trehalose dicarboxylic acid”) by catalytic oxidation of trehalose. Such simple derivatives as the dimethyl ester and the hexa- O -acetyl diacid chloride are useful for the attachment of lipid substituents to the carbohydrate core to yield the desired pseudo cord-factors. Some of the glycolipids have toxic and antitumor properties resembling those of the natural product.

Lipid compositions of 40 patient isolates of Mycobacterium tuberculosis derived from Madras, Burma, Rangoon, and East Africa were studied, and two major populations of tubercle bacilli were distinguished. Nearly all of the strains previously designated as attenuated for the guinea pig (D. A. Mitchison) are characterized by a specific phenolic phthiocerol diester which is identified with the aglycone moieties of mycosides A and B (and probably G). This lipid (AI) was not seen in any of the more virulent strains. Thus, presence of AI may be taken as definitive for attenuation ( P ≪ 0.001). Phthiocerol dimycoserosate (DIM), a companion substance, is ubiquitous for the series of 40 strains. However, a dramatic attenuation found in a DIM-less H37Rv mutant may support a role for this substance in the virulent state. Since mycosides A, G, and B seem to be restricted to certain chromogenic and bovine species, respectively, we speculate that lysogenization or transduction of fully virulent M. tuberculosis may have provided the genetic determinants for attenuation and AI synthesis, and thus led to the two classes of tubercle bacilli.

Acylation of 2,3,4,2′,3′,4′-hexa-O-benzyl-6,6′-di-O-methanesulphonyl-α-α-trehalose (1) with a reduced amount of potassium corynomycolate yielded a mixture which consisted mainly of 2,3,4,2′,3′,4′-hexa-O-benzyl-6-O-corynomycoloyl-6′-O-methanesulphonyl-α,α-trehalose (2). Catalytic hydrogenolysis of 2 gave the mono-mesylate 4 which was converted into 3′,6′-anhydro-6-O-corynomycoloyl-α,α-trehalose (5) but treatment with sodium hydride. The structure of 5 was studied by mass-spectroscopy. Compound 5 was found to be identical with the byproduct obtained in the acylation of 6,6′-di-O-p-toluenesulphonyl-α,α-trehalose with potassium corynomycolate.

Harvests of Mycobacterium lepraemurium obtained from livers of moribund infected mice yielded M. lepraemurium cell walls that were extracted with solvent to provide crude M. lepraemurium cell wall lipids. By solvent fractionation and chromatography on DEAE cellulose and cellulose, a cord factor-like glycolipid contaminated with mycoside C was obtained. Additional solvent treatment provided the purified glycolipid, which was identified as 6,6'-trehalose dimycolate, by infrared and Chromatographic comparison with authentic samples from M. tuberculosis , by identification of trehalose and specific mycolates of M. lepraemurium , and by permethylation analysis. This constitutes the first unequivocal identification of cord factor as a product of in vivo-derived mycobacteria.

Trehalose dimycolate is a glycolipid component of the cell walls of mycobacteria, nocardia, and corynebacteria. When trehalose dimycolate is injected into certain strains of mice, they develop interstitial pneumonitis that is characterized by mononuclear cell infiltration of the alveolar walls, intra-alveolar hemorrhages, and in some animals, granuloma formation. The disorder is seldom fatal, and in approximately 4 weeks, the lungs are normal. There is strong evidence that T lymphocytes are essential for production of interstitial pneumonitis by trehalose dimycolate, but little is known about the mechanisms of lung injury in this model. The experiments described in this report were conducted to identify the roles of the various cells that accumulate in the lungs of mice with this form of interstitial pneumonitis. We found that Mac3+ macrophages were the first cells to appear in the alveolar walls. Increases in the number of L3T4+ T lymphocytes, Lyt2+ T lymphocytes, and surface-immunoglobulin-positive lymphocytes followed, but significant increases in the number of lymphoid cells were not observed until day 7, when the pulmonary lesions were well developed. Treatment of the mice with cyclophosphamide or anti-T-cell sera significantly reduced the number of lymphoid cells in the alveolar walls but did not affect the number of Mac3+ cells and did not affect development of intra-alveolar hemorrhages. Treatment with poly(I.C) significantly decreased the number of Mac3+ cells in the lungs, and these mice did not develop pulmonary hemorrhages. We conclude that although development of pulmonary lesions in trehalose dimycolate-treated mice is a T-cell-dependent process, macrophages are also essential and are more directly involved in production of the lung injury. We postulate that the lung lesions are the direct effect of macrophage-produced cytokines, such as tumor necrosis factor.

Selective triflation of 4,6:4',6'-di-O-benzylidene-alpha,alpha-trehalose gave 4,6:4',6'-di-O-benzylidene-2,2'-di-O-triflyl-alpha,alpha-trehalose , the structure of which was confirmed by the 1H-n.m.r. spectrum of its 3,3'-di-O-acetyl derivative (4). Treatment of 4 with sodium nitrite in hexamethylphosphoric triamide, followed by benzylation, afforded 2,3,2',3'-tetra-O-benzyl-4,6:4',6'-di-O-benzylidene-(alpha-D-mannopyrano syl alpha-D-mannopyranoside (7). Removal of the two benzylidene groups from 7, and selective tosylation of the product, gave a mixture of the 6,6'-ditosylate (11) and the 6-monotosylate (12), which were separated by chromatography. Treatment of 11 with potassium corynomycolate or potassium mycolate afforded the corresponding 6,6'-diesters, 14 and 15, respectively. Treatment of the monotosylate 12 with potassium corynomycolate gave the 6-monoester 18. Catalytic hydrogenolysis of 14, 15, and 18 gave the respective cord-factor analogs.

Interpretation of an earlier published infrared spectrum of Mycobacterium smegmatis lipids with receptor site activity for D4 phage led us to the inference that the active substance is very likely a mycoside C. This hypothesis was confirmed: the well-characterized mycosides C s and C 1217 elaborated by the heterologous strains M. scrofulaceum and Mycobacterium species 1217, respectively, are essentially indistinguishable from the smegmatis lipids in their behavior toward D4. Minute quantities adsorb and extensively inactivate the phage on appropriate incubations. In accord with derivative expectations, Mycobacterium species 1217 is a permissive host, attacked and lysed by D4. However, our current strains of M. butyricum, M. avium , and M. scrofulaceum , which reputedly produce various related mycosides C, are neither lysed by nor do they significantly adsorb the phage. Implications of these observations are discussed.

A nonprotective hyperimmune state characterized by high serum titers specific for cryptococcal capsular substance can be stimulated in mice by injections of soluble cryptococcal polysaccharide coupled to bovine gamma globulin. When the nonprotective quality of the antibody became apparent, we sought to learn whether the humoral anticryptococcal polysaccharide was reactive in vivo for cryptococcal capsules after an experimental infection. For this purpose homogenates of organs from infected mice were examined by immunofluorescence techniques for detecting mouse immunoglobulins on the capsules of tissue fungi. In vivo specific and pseudo-immune reactions between humoral constituents and the capsular substance were observed and differen

Degradative studies have led to an elucidation of the core structure of the principal sulfolipid class (Sulfolipid I) elaborated by Mycobacterium tuberculosis, Strain H37Rv. In the intermediate stages of alkali-catalyzed ethanolysis of sodium sulfolipid I, a complex mixture of substances is produced which appears to contain the entire gamut of sulfolipids seen in the original crude extracts. At least four carboxylic esters, readily separatable in thin-layer chromatography but not yet characterized, were recovered. From the reaction mixture a semi-crystalline substance was obtained and identified as α,α-trehalose 2-sulfate. Alkaline ethanolysis of desulfated ammonium sulfolipid I affords crystalline α,α-trehalose directly. Permethylation of the desulfated lipid followed by saponification gave a tetramethyl trehalose; on acidic cleavage this afforded, among traces of other substances, essentially equal quantities of 4-O- methyl - d - glucose and 2,3,4-tri- O-methyl- d -glucose . Thus the principal sulfolipid produced by Strain H37Rv is a 2,3,6,6'-tetraacyl-α,α-trehalose 2'-sulfate. The relation of this structure to the chemical and physiological properties of ammonium sulfolipid I is discussed.

A sulfur-containing lipid extracted from virulent human strains of Mycobactenum tuberculosis and previously described by Middlebrook and co-workers has been shown to consist of several families of sulfolipids, many of which are structurally related. The most abundant class, sulfolipid I, is obtained by DEAE-cellulose column Chromatographic procedures. This substance is a complex glycolipid ester of average empirical formula C145H275O20NS, molecular weight 2400; the carbohydrate moiety bears a single sulfuric acid half ester substituent, in a secondary equatorial position. The ammonium salt undergoes spontaneous desulfation under such innocuous circumstances as mere dissolution in ether. Strains of Mycobacterium bovis which have been examined elaborate only minute amounts of similar substances; none appear to be identical with the principal sulfolipid of human strains.

Summary Soluble polysaccharide from culture filtrates of Cryptococcus neoformans can be made potently antigenic in mice if it is coupled to a heterologous protein by a reaction sequence that permits retention of the ester functions (specifically identified as acetate) in the native polysaccharide. The key step in the reaction sequence which was developed rests on the utility of dimethylsulfoxide (DMSO) as an aprotic homogeneous reaction solvent for the polysaccharide. This allows controlled nitrocarbanilation of the carbohydrate without cleavage of the acetyl functions. The aminocarbanilate derived from the nitro compound was diazotized and coupled to bovine γ-globulin (BGG) to yield gelled protein-azocarbanilate-polysaccharide conjugates. Homogenized conjugates dispersed in Freund's adjuvant were potent antigens in mice and stimulated production of humoral anti-BGG and antipolysaccharide. De-acetylated polysaccharide coupled to BGG and polysaccharide-mouse globulin conjugate were essentially inert. The polysaccharide and its derivatives were subject to ready insolubilization by what is judged to be an intermolecular crosslinking. It is suggested that the physical integrity of the cryptococcal capsule may be a consequence of a similar behavior in vivo. In addition, DMSO is an effective decapsulating agent for this yeast.

Lysosomotropic fluorescent aminoacridines such as acridine orange and quin- acrine have achieved prominence as markers for studying lysosome-phago- some fusion, especially in macrophages. Experiments described demonstrate that because the aminoacridines traverse biological membranes with facility, they diffuse throughout the system, and ultimately accumulate intra- or extra- cellularly where they are most efficiently bound. Their presence or absence in phagosomes is therefore not unequivocally indicative of fusion or nonfusion. Alternative fluorescent lysosomal markers are described, and systems defined for which the aminoacridines may probably be used with confidence.

Unter den angegebenen Reaktionsbedingungen entsteht aus dem Ditosylat (Ia) die im Titel genannte Dianhydroverbindung (Ha), die auch als Acetat (IIb) charakterisiert wird.

The early studies of Lurie1 clearly established that virulent tubercle bacilli are highly infectious for susceptible hosts. For example, he observed repeatedly that one colony-forming unit (CFU) of the highly virulent H37Rv strain of Mycobacterium tuberculosis could develop one tubercle in the lungs of susceptible rabbits. Although not proved at that time, it is likely that even one organism is capable of producing one tubercle in the lungs of NZW rabbits (noninbred). By contrast, the H37Ra strain, an avirulent mutant of the H37Rv strain, is incapable of replicating in the macrophages of normal rabbits. Lurie’s findings were particularly important with respect to the multiplication of the attenuated BCG strain of Mycobacterium bovis. In this case, even though BCG could not produce progressive infection in the rabbit, it was able to multiply in normal alveolar macrophages (AM) essentially at the same rate as the virulent H37Rv strain prior to the time specific cell-mediated immunity was acquired. All these early studies indicated that normal AM are incapable of inhibiting or containing the multiplication of phagocytosed organisms of the virulent H37Rv strain of M. tuberculosis, as well as the attenuated BCG strain during the early intervals after infection. Accordingly, Lurie concluded that all the immunity expressed against M. tuberculosis is acquired after infection. Lurie also demonstrated that macrophages from immune animals were resistant to the replication of virulent mycobacteria. These observations suggested that pathogenic mycobacteria have some virulence mechanism that enables them to multiply in normal macrophages, whereas immunologically activated macrophages are capable of inhibiting the growth of virulent mycobacteria.

Intracellular parasites (e.g., Mycobacterium tuberculosis, Toxoplasma gondii, and some Chlamydiae) may promote their survival within the host by acting from within phagosomes to prevent phagolysosome formation, thus avoiding exposure to the lysosomal hydrolases. The present studies demonstrate that when sulfatides of M. tuberculosis (anionic trehalose glycolipids largely responsible for the neutral red reactivity of virulent strains) are administered to cultured mouse peritoneal macrophages, they accumulate in the secondary lysosomes, which are rendered incompetent for fusion with phagosomes containing suitable target particles such as viable yeasts. This antifusion effect is also exhibited when small amounts of sulfatide are introduced directly into phagosomes by attachment to the target yeasts prior to their ingestion. The sulfatides evidently exert a selective inhibitory influence on membrane fusion, analogous to what occurs typically when macrophage cultures are infected with tubercle bacilli. This effect may be due to ionic interaction between the polyanionic micelles of bacterial sulfatide and organelle membranes, modifying the latter and inducing dysfunction.

We review the experimental difficulties that have been encountered in hydrolyzing mycolic acid esters, their beta-O-substituted analogues, permethylated cord factor, phthiocerol dimycocerosates and similar mycobacterial lipids. Hydrolysis of the beta-O-substituted methyl mycolates is invariably sluggish and is accompanied by considerable beta-elimination to generate mycolenoic acids. Historical evidence for this often undesirable side reaction is presented. Improvements in methodology are described in which hydrolysis is promoted and beta-elimination is minimized. The reaction systems developed were found applicable to hydrolysis of the quite inert phthiocerol dimycocerosates. Permethylation of the recovered phthiocerol provides an excellent derivative for mass spectrometric analysis to define its complete structure.

This paper, dealing with several curious paradoxes in macrophage function, is principally in the nature of an editorial review, although some recent, as yet unpublished, work from our laboratory is described. In its preparation, I was at first dismayed to realize that it raises so many questions, but answers very few. Still the purpose of such an effort is to arouse interest in what I believe are provocative issues, with the hope that their examination and analysis might stimulate some productive research, whether in my own group or by others. Both consequences are manifestations of the creative process that drives us all, and therefore they bring both participatory and vicarious satisfaction.

SUMMARY: Trehalose-6-monomycolate (TMM) was isolated from the lipids of armadillo-derived Mycobacterium leprae. Only meagre amounts of this glycolipid were recovered, but its structure was unequivocally established. Only α-mycolates were detected in the TMM by 252Cf plasma desorption mass spectrometry. Electron impact mass spectrometry showed the alpha branch to be principally C20. Trehalose dimycolate (cord factor) was not detectable. Since we have also found TMM in M. lepraemurium and in every Mycobacterium species so far examined, we suggest that this glycolipid is truly ubiquitous amongst mycobacteria.

alpha,alpha-Trehalose 2-sulfate, the core carbohydrate of sulfatides of Mycobacterium tuberculosis, and the 3-sulfate isomer were synthesized by sulfation of 4,6:4',6'-di-O-benzylidene-alpha,alpha-trehalose with pyridine-sulfur trioxide complex to give the 2- and 3-sulfates, which were separated by column chromatography. The ammonium 2-sulfate salt wa was identical with the natural product obtained from the principal sulfatide (SL-I) of M. tuberculosis.

CONTENTS INTRODUCTION Transition . MICROBICIDAL MECHANISMS IN PHAGOCYTIC CELLS . Metabolism . Reduced pyridine nucleotides in the metabolic burst ••••.• •..•... Superoxide anion . The Peroxidase Paradox . . . . . . . . . . . ..... . ANTAGONISM OF HOST DEFENSES BY MICROORGANISMS .. Mycobacteria . Mycobacterium lepraemurium . Mycobacterium tuberculosis . Chlamydia and Rickettsia . Toxoplasma and Besnoitia .. Aspects of toxoplasma-induced cellular or humoral immunity .. Macrophages in immunity to Toxoplasma and Besnoitia . Miscellaneous Parasitic Agents .. . . Streptococci and a Digression . Streptolysins and leucocidin . +1715

Mycolic acids are among the most persistent contaminants of the toxic mycobacterial glycolipid 6,6′ dimycoloyl trehalose (cord factor). In the past, alternating multiple silicic acid and magnesium silicate column chromatography has been employed, not always successfully, to purify the glycolipid [5]. An ancillary problem which required a rapid method for bulk separation of certain derivatives of mycolic esters from the free acids led us to a disarmingly simple solution to both problems and ultimately to an improved method for isolation and purification of cord factor from wax C. Neutral lipids can be separated from quite large amounts of contaminant free carboxylic acids by adsorption chromatography on the free base form of diethylaminoethylcellulose (DEAE). The present paper describes the rationale and methods developed for cord factor purification.

Publisher Summary This chapter discusses polyanionic agents and the inhibition process of phagosome–lysosome fusion. It is concerned with a hypothesis about the antagonistic effects of endocytosed polyanionic substances on phagosome–lysosome fusion in cultured macrophages. It is observed that no important macrophage function has been discerned so far that is antagonized by lysosomal accumulation of polyanions—particularly of phagocytosis, intracellular digestion, and microbicidal activities. The apparent block to fusion is indifferent to Ca2+ concentrations and cannot be imposed from the phagosomal compartment by particles of cation-exchange resins. If this “phenomenon” depends on perturbations in vesicle membranes, pinosomal membranes would appear to be embarrassing exceptions to the inhibition. They will apparently behave like phagosomal membranes, both allowing fusion with “polyanion” lysosomes. Neutral hydrocolloids mimic the behavior of polyanions with thorotrast markers, but still allow delivery of permeant or nonpermeant mobile lysosomal fluors to phagosomes.

In their heuristic studies, Fell and Mellanby (1950,1952) described the destructive effects of vitamin A acetate on limb-bone rudiments and other tissues from mouse and chick embryos in tissue culture. The dissolution of, e.g., cartilage muco-protein associated with the tissue destruction was ultimately linked to proteolytic (cathepsin) activity released from tissue lysosomes which were rendered fragile and unstable, probably owing to a drug-induced labilization of lysosomal (and other) membranes.

Small quantities of cord factor (trehalose-6,6'-dimycolates) can be readily and almost quantitatively permethylated in anhydrous diethyl ether/dimethylformamide mixtures with CH3I and NaH-oil dispersion in the presence of molecular sieve. Hydrolysis of the permethylated products from cord factor ‘Peurois’ and ‘P3-Aoyama-B’ prove these to be 6,6'-dimycolates. Experiments with a 4-palmitoyl glucose suggest that acyl migration in this system may not occur.