The liposome of trehalose dimycolate extracted from M. bovis BCG induces antitumor immunity via the activation of dendritic cells and CD8+ T cells.

Intravesical Bovis bacillus Calmette-Guérin (BCG) therapy is the most effective immunotherapy for bladder cancer, but it sometime causes serious side effects because of its inclusion of live bacteria. It is necessary to develop a more active but less toxic immunotherapeutic agent. Trehalose 6,6'-dimycolate (TDM), the most abundant hydrophobic glycolipid of the BCG cell wall, has been reported to show various immunostimulatory activities such as granulomagenesis and adjuvant activity. Here, we developed cationic liposomes incorporating TDM purified from Mycobacterium bovis BCG Connaught, and we investigated the antitumor effect of the cationic liposome TDM (Lip-TDM). Lip-TDM exerted an antitumor effect in bladder cancer, colon cancer, and melanoma-bearing mouse models that was comparable or even superior to that of BCG, with no body weight loss or granuloma formation. The antitumor effect of Lip-TDM disappeared in two types of mice: those with depletion of CD8+ T cells, and those with knockout of macrophage-inducible C-type lectin (Mincle) which recognize TDM. Lip-TDM treatment enhanced the maturation and migration of dendritic cells in the tumor microenvironment in a Mincle-dependent manner. Our results elucidate mechanisms that underlie Lip-TDM treatment and suggest that Lip-TDM has potential as a safe and effective treatment for various cancers.

Isolation, structural elucidation and biosynthesis of 3-hydroxy-6-dimethylallylindolin-2-one, a novel prenylated indole derivative from Actinoplanes missouriensis.

Many prenylated indole derivatives are widely distributed in nature. Recently, two Streptomyces prenyltransferases, IptA and its homolog SCO7467, were identified in the biosynthetic pathways for 6-dimethylallylindole (DMAI)-3-carbaldehyde and 5-DMAI-3-acetonitrile, respectively. Here, we isolated a novel prenylated indole derivative, 3-hydroxy-6-dimethylallylindolin (DMAIN)-2-one, based on systematic purification of metabolites from a rare actinomycete, Actinoplanes missouriensis NBRC 102363. The structure of 3-hydroxy-6-DMAIN-2-one was determined by HR-MS and NMR analyses. We found that A. missouriensis produced not only 3-hydroxy-6-DMAIN-2-one but also 6-dimethylallyltryptophan (DMAT) and 6-DMAI when grown in PYM (peptone-yeast extract-MgSO4) medium. We searched the complete genome of A. missouriensis for biosynthesis genes of these compounds and found a gene cluster composed of an iptA homolog (AMIS_22580, named iptA-Am) and a putative tryptophanase gene (AMIS_22590, named tnaA-Am). We constructed a tnaA-Am-deleted (ΔtnaA-Am) strain and found that it produced 6-DMAT but did not produce 6-DMAI or 3-hydroxy-6-DMAIN-2-one. Exogenous addition of 6-DMAI to mutant ΔtnaA-Am resulted in the production of 3-hydroxy-6-DMAIN-2-one. Furthermore, in vitro enzyme assays using recombinant proteins produced by Escherichia coli demonstrated that 6-DMAI was synthesized from tryptophan and dimethylallyl pyrophosphate in the presence of both IptA-Am and TnaA-Am, and that IptA-Am preferred tryptophan to indole as the substrate. From these results, we concluded that the iptA-Am-tnaA-Am gene cluster is responsible for the biosynthesis of 3-hydroxy-6-DMAIN-2-one. Presumably, tryptophan is converted into 6-DMAT by IptA-Am and 6-DMAT is then converted into 6-DMAI by TnaA-Am. 6-DMAI appears to be converted into 3-hydroxy-6-DMAIN-2-one by the function of some unknown oxidases in A. missouriensis.

Cord factor (trehalose 6,6'-dimycolate) forms fully stable and non-permeable lipid bilayers required for a functional outer membrane.

Cord factor (trehalose 6,6'-dimycolate, TDM) is the major lipid in the outer membrane of Corynebacteria and Mycobacteria. Although its role is well recognized in the immune response phenomena, its membrane biophysical properties remained largely unexplored and TDM has often been described as a detergent. We purified the main components of the outer membrane from Corynebacterium glutamicum and analyzed their membrane forming properties. In mixture with endogenous cardiolipin, but not alone, the spontaneous hydration of TDM produces liposomes. As a pure component, TDM formed vesicles only by the detergent dialysis method. Perdeuterated cardiolipin-TDM mixtures were shown by deuterium nuclear magnetic resonance (NMR) to exhibit a gel to liquid crystalline phase transition over a 273-295K temperature range, for cells grown at 303K, and thus to be in a liquid crystalline state at physiological temperature. Molecular dynamics simulations of hydrated TDM bilayers provided the trehalose average orientation and conformation, the chain order parameters, the area per lipid and the bilayer thickness which was confirmed by electron microscopy. Finally the Porin A-Porin H ion channel from the Corynebacterial outer membrane was reconstituted in TDM liposomes. With properly mycoloylated proteins, it manifested the typical voltage dependent ion channel properties of an outer membrane porin.

Comparable studies of immunostimulating activities in vitro among Mycobacterium bovis bacillus Calmette-Guérin (BCG) substrains.

During the serial passage of Mycobacterium bovis bacillus Calmette-Guérin (BCG) in different countries after initial seed distribution from the Pasteur Institute, specific insertions and deletions in the genome among BCG substrains were observed and speculated to result in differences in immunological activities. 'Early-shared strains' of BCG (Russia, Moreau, Japan, Sweden, Birkhaug), distributed by the Pasteur Institute, which conserve three types of mycolate (alpha, methoxy, keto) in cell wall, exhibited stronger activities of induction of nitric oxide, interleukin-1beta (IL-1beta), IL-6, IL-8, IL-12 and tumor necrosis factor (TNF)-alpha, from human epithelial cell line A549, human myelomonocytic cell line THP-1 and mouse bone marrow cells in the presence of interferon-gamma (IFN-gamma) than did 'late-shared strains' of BCG (Danish, Glaxo, Mexico, Tice, Connaught, Montreal, Phipps, Australia, Pasteur). The stronger induction of IL-12 and TNF-alpha in the presence of IFN-gamma was also observed by trehalose 6,6'-dimycolate (TDM) extracted from BCG-Japan than by TDM from BCG-Connaught, which lacks the methoxymycolate residue. These results suggest that 'early-shared strains' are more potent immunostimulating agents than 'late-shared strains', which could be attributed partially to methoxymycolate. Our study provides the basic information for immunological characterization of various BCG strains and may contribute to a re-evaluation of them as a reference strain for vaccination against tuberculosis.

Mycobacterial glycolipid trehalose 6,6'-dimycolate-induced hypersensitive granulomas: contribution of CD4+ lymphocytes.

The granulomatous response is a characteristic histological feature of Mycobacterium tuberculosis infection responsible for organism containment. The development of cell-mediated immunity is essential for protection against disease, as well as being required for maintenance of the sequestering granulomatous response. Trehalose 6,6'-dimycolate (TDM; cord factor), a glycolipid associated with the cell wall of mycobacteria, is implicated as a key immunogenic component in M. tuberculosis infection. Models of TDM-induced hypersensitive granulomatous response have similar pathologies to that of active tuberculosis infection. Prior immunization (sensitization) of mice with TDM results in exacerbated histological damage, inflammation and lymphocytic infiltration upon subsequent TDM challenge. Adoptive transfer experiments were performed to ascertain the cell phenotype governing this response; CD4(+) cells were identified as critical for development of related pathology. Mice receiving CD4(+) cells from donor TDM-immunized mice demonstrated significantly increased production of Th1-type cytokines IFN-gamma and IL-12 within the lung upon subsequent TDM challenge. Control groups receiving naïve CD4(+) cells, or CD8(+) or CD19(+) cells isolated from TDM-immunized donors, did not exhibit an exacerbated response. The identified CD4(+) cells isolated from TDM-immunized mice produced significant amounts of IFN-gamma and IL-2 when exposed to TDM-pulsed macrophages in vitro. These experiments provide further evidence for involvement of a cell-mediated response in TDM-induced granuloma formation, which mimics pathological damage elicited during M. tuberculosis infection.

Identification of trehalose dimycolate (cord factor) in Mycobacterium leprae.

Glycolipids of Mycobacterium leprae obtained from armadillo tissue nodules infected with the bacteria were analyzed. Mass spectrometric analysis of the glycolipids indicated the presence of trehalose 6,6'-dimycolate (TDM) together with trehalose 6-monomycolate (TMM) and phenolic glycolipid-I (PGL-I). The analysis showed that M. leprae-derived TDM and TMM possessed both alpha- and keto-mycolates centering at C78 in the former and at C81 or 83 in the latter subclasses, respectively. For the first time, MALDI-TOF mass analyses showed the presence of TDM in M. leprae.

Intact molecular characterization of cord factor (trehalose 6,6'-dimycolate) from nine species of mycobacteria by MALDI-TOF mass spectrometry.

Cord factor (trehalose 6,6'-dimycolate, TDM) is an unique glycolipid with a trehalose and two molecules of mycolic acids in the mycobacterial cell envelope. Since TDM consists of two molecules of very long branched-chain 3-hydroxy fatty acids, the molecular mass ranges widely and in a complex manner. To characterize the molecular structure of TDM precisely and simply, an attempt was made to determine the mycolic acid subclasses of TDM and the molecular species composition of intact TDM by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the first time. The results showed that less than 1 microg mycolic acid methyl ester of TDM from nine representative species of mycobacteria and TDM from the same species was sufficient to obtain well-resolved mass spectra composed of pseudomolecular ions [M+Na]+. Although the mass ion distribution was extremely diverse, the molecular species of each TDM was identified clearly by constructing a molecular ion matrix consisting of the combination of two molecules of mycolic acids. The results showed a marked difference in the molecular structure of TDM among mycobacterial species and subspecies. TDM from Mycobacterium tuberculosis (H37Rv and Aoyama B) showed a distinctive mass pattern and consisted of over 60 molecular ions with alpha-, methoxy- and ketomycolate. TDM from Mycobacterium bovis BCG Tokyo 172 similarly showed over 35 molecular ions, but that from M. bovis BCG Connaught showed simpler molecular ion clusters consisting of less than 35 molecular species due to a complete lack of methoxymycolate. Mass ions due to TDM from M. bovis BCG Connaught and Mycobacterium kansasii showed a biphasic distribution, but the two major peaks of TDM from M. kansasii were shifted up two or three carbon units higher compared with M. bovis BCG Connaught. Within the rapid grower group, in TDM consisting of alpha-, keto- and wax ester mycolate from Mycobacterium phlei and Mycobacterium flavescens, the mass ion distribution due to polar mycolates was shifted lower than that from the Mycobacterium avium-intracellulare group. Since the physico-chemical properties and antigenic structure of mycolic acid of TDM affect the host immune responses profoundly, the molecular characterization of TDM by MALDI-TOF mass analysis may give very useful information on the relationship of glycolipid structure to its biological activity.

Mycobacterium tuberculosis controls host innate immune activation through cyclopropane modification of a glycolipid effector molecule.

Mycobacterium tuberculosis (Mtb) infection remains a global health crisis. Recent genetic evidence implicates specific cell envelope lipids in Mtb pathogenesis, but it is unclear whether these cell envelope compounds affect pathogenesis through a structural role in the cell wall or as pathogenesis effectors that interact directly with host cells. Here we show that cyclopropane modification of the Mtb cell envelope glycolipid trehalose dimycolate (TDM) is critical for Mtb growth during the first week of infection in mice. In addition, TDM modification by the cyclopropane synthase pcaA was both necessary and sufficient for proinflammatory activation of macrophages during early infection. Purified TDM isolated from a cyclopropane-deficient pcaA mutant was hypoinflammatory for macrophages and induced less severe granulomatous inflammation in mice, demonstrating that the fine structure of this glycolipid was critical to its proinflammatory activity. These results established the fine structure of lipids contained in the Mtb cell envelope as direct effectors of pathogenesis and identified temporal control of host immune activation through cyclopropane modification of TDM as a critical pathogenic strategy of Mtb.

Cord factor trehalose 6,6'-dimycolate (TDM) mediates trafficking events during mycobacterial infection of murine macrophages.

The persistence of tuberculosis within pulmonary granulomatous lesions is a complex phenomenon, with bacterial survival occurring in a focal region of high immune activity. In part, the survival of the organism may be linked to the ability of the surface glycolipid trehalose 6,6'-dimycolate (TDM; cord factor) to inhibit fusion events between phospholipid vesicles inside the host macrophage. At the same time, TDM contributes to macrophage activation and a cascade of events required for initiation and maintenance of granulomatous responses. This allows increased sequestration of organisms and further survival and persistence within host tissues. Bacterial viability, macrophage cytokine and chemokine response, and intracellular trafficking were investigated in Mycobacterium tuberculosis from which TDM had been removed. Removal of surface lipids led to enhanced trafficking of organisms to acidic compartments; reconstitution of delipidated organisms with either pure TDM or the petroleum ether extract containing crude surface lipids restored normal responses. Use of TDM-coated polystyrene beads demonstrated that TDM can mediate intracellular trafficking events, as well as influence macrophage production of pro-inflammatory molecules. Thus, the presence of TDM may be an important determinant for successful infection and survival of M. tuberculosis within macrophages.

6,6'-Dimycoloyl trehalose from a rapidly growing Mycobacterium: an alternative antigen for tuberculosis serodiagnosis.

Mycobacterial O-acyltrehaloses have been described as highly specific and sensitive reagents for tuberculosis immunodiagnosis. An O-acyltrehalose-containing lipid fraction from the rapidly growing Mycobacterium fortuitum was found to include additional antigens, which presented high cross-reactivity with sera from tuberculosis-infected patients. Based on a combination of selective chemical degradations, thin-layer-chromatography analyses and (1)H-nuclear magnetic resonance spectroscopy, the antigenic by-product was identified as 6,6'-dimycoloyl trehalose, the so-called cord factor. The lipid was purified and tested in ELISA for pulmonary tuberculosis serodiagnosis. Sensitivity and specificity of the test were found to be 66.6-74.1% and 95.2-99.0%, respectively, showing a slightly higher efficiency as compared to the ELISA performed using 6,6'-dimycoloyl trehalose from Mycobacterium tuberculosis. No cross-reactivity was found with sera from Nocardia-infected individuals.

Priming and activation of mouse macrophages by trehalose 6,6'-dicorynomycolate vesicles from Corynebacterium glutamicum.

Vesicles consisting of pure trehalose dicorynomycolate (TDCM), the corynebacterial analog of the most studied mycobacterial glycolipid 'cord factor', were isolated from Corynebacterium glutamicum cells by mild detergent treatment; these induced in vivo a macrophage priming similar to that obtained with mycobacterial-derived trehalose dimycolate. In vitro, both TDCM and bacterial lipopolysaccharide (LPS) induced in macrophages the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha), endotoxin tolerance, and were primed for an enhanced secondary NO response to LPS. Interferon-gamma pretreatment did not influence the LPS-induced TNF-alpha response, but considerably increased the TDCM-induced response.

[The 72nd Annual Meeting Education Lecture. Cord factor].

Mycobacterial cell wall contains various lipids (glycolipids and phospholipids) to contribute to its hydrophobic property or acid-fastness and these surface molecules contact with host cells in the early step of infection. Among them, cord factor (trehalose 6,6'-dimycolate, TDM or CF) is the most ubiquitous component, which may be a key molecule for pathogenesis and immunity. Initially, cord factor was isolated from a highly virulent strain of M. tuberculosis which grows in the form of serpentine cords, and showed a marked toxicity for mice when it was administrated intravenously. These observations led to the early hypothesis that cell wall components are related to virulence. However, later studies revealed that cord factors were also found in other non-cord-forming mycobacterial species and other mycolic acid-containing bacteria. Structural studies demonstrated that there were various mycoloyl glycolipids differing in carbohydrate moiety such as glucose mycolate, mannose mycolate, arabinose mycolate and fructose mycolate besides trehalose mycolate in acid-fast bacteria. Therefore, the interest has been focused to the structure-activity relationships of mycoloyl glycolipid and to the mechanism of virulence for host animals. So far, it has been demonstrated that cord factor showed lethal toxicity, granuloma forming activity, adjuvant activity, tumor regressing activity and non-specific infection prevention activity in experimental animals. We have extended investigations further on the structure analysis and immunomodifying activities of cord factor and related mycoloyl glycolipids from various species of mycobacteria, nocardia, rhodococci and gordona, and demonstrated that the most activities were shown in trehalose or glucose esters, but not in mannose, arabinose or fructose esters. Furthermore, it was shown that the longer chain-mycoloyl glycolipids showed the higher toxicity and immunomodifying activities. In mice, in vivo, cytokine inducing activities such as IL-1, IFN-gamma, TNF-alpha, GM-CSF and chemotactic factor were observed and in vitro, TNF-alpha, GM-CSF, chemotactic factor, complement, NO, PGE2 inductions and protein kinase C activation were demonstrated. Furthermore, recently, we have demonstrated that cord factor induced a marked thymic atrophy due to the cortical lymphocyte apoptosis before granuloma formation in mice. It was also established that cord factor showed antigenicity in mice and rabbits and human tuberculous patient sera contained specific antibody (IgG) reactive against cord factor. From above results, cord factor seems to be one of the most potent immunomodulators in the mycobacterial cell wall components pathologically and beneficially.

Studies of polymorphic DNA fingerprinting and lipid pattern of Mycobacterium tuberculosis patient isolates in Japan.

Strain differentiation by DNA restriction fragment length polymorphism (RFLP) has been used mainly for the epidemiological purpose of Mycobacterium tuberculosis infection. In this study, we tried to connect the molecular and phenotypic characteristics of M. tuberculosis patient isolates by comparing the DNA fingerprints obtained by RFLP using IS6110 and lipid patterns using two-dimensional thin-layer chromatography (2-D TLC) with silica gel, since M. tuberculosis has a lipid-rich cell envelope which contributes to the virulence and immunomodulatory properties. We found that 66 isolates of M. tuberculosis from tuberculosis patients showed that the occurrence of IS6110 varied from 1 to 24 copies. The IS6110 patterns were highly variable among isolates. Fifty different RFLP patterns were observed, and 12 RFLP patterns were shared by two or more strains. By computerized analysis of the RFLP patterns of M. tuberculosis patient isolates, we found that 95% of the isolates fell into seven clusters, from A to G, with at least two isolates in each (> 30% similarity). Among the cellular lipids, the phospholipid composition did not differ by strain, whereas the glycolipid pattern differed markedly. Especially, the relative concentration of cord factor and sulfolipid, both of which were known as virulent factors, varied by strain. The fingerprints of some strains showed an association between the DNA and glycolipid patterns, even though some of the same DNA fingerprint strains showed differences in lipid patterns. Among the patient isolates, M. tuberculosis strain 249 possessed a specific glycolipid with 2-O-methyl-L-rhamnose and L-rhamnose, which is rarely found in other strains. This glycolipid showed serological activity against the sera of tuberculosis patients, even if the reactivity was not as strong as trehalose dimycolate. It also showed the inhibition of phagosome-lysosome fusion in macrophages, suggesting involvement with virulence. These results suggest that RFLP analysis using IS6110 is useful for clustering the human isolates of M. tuberculosis, however, for further strain differentiation on virulence, a lipid analysis provides more information.

Enzyme immunoassay to detect antituberculous glycolipid antigen (anti-TBGL antigen) antibodies in serum for diagnosis of tuberculosis.

We report the development of an EIA specific for antituberculosis antibody in human serum for the clinical evaluation of tuberculosis. We developed a TLC immunostaining method to detect specific antigens for antibodies in the serum of patients with tuberculosis. The detected specific antigens, TDM and specific gylcolipid fraction, were individually purified from M. tuberculosis H37Rv by column chromatography. The two purified fractions were mixed and the mixture, termed TBGL antigen, was applied to an enzyme immunoassay suitable for the measurement of antituberculosis antibodies in serum. This EIA meets all the requirements of routine clinical assay in terms of sensitivity (detection limit: 0.125 U/ml), reproducibility (total CV : 3.3-6.0%), accuracy (recovery: 96-105%), simplicity and rapidity (< 2.5 h). Clinical validation of the assay was confirmed by the measurement of the anti-tuberculosis antibody in the serum of normal subjects and patients with pulmonary tuberculosis. The EIA tested in this study showed a high serodiagnostic discriminating power (90% sensitivity and 98% specificity).

Clinical evaluation of rapid serodiagnosis of pulmonary tuberculosis by ELISA with cord factor (trehalose-6,6'-dimycolate) as antigen purified from Mycobacterium tuberculosis.

Immunoglobulin G (IgG) antibodies against purified cord factor (trehalose-6,6'-dimycolate) prepared from Mycobacterium tuberculosis H37Rv were determined by enzyme-linked immunosorbent assay (ELISA), and its diagnostic usefulness was evaluated. Serum specimens from 65 patients with active pulmonary tuberculosis, 58 patients with inactive pulmonary tuberculosis, 36 patients with diseases other than tuberculosis, and 66 healthy adults were examined. Patients with active pulmonary tuberculosis showed significantly higher titers of IgG antibodies against cord factor than did other groups (p < 0.001). The antibody titer greater than 0.29 in absorption difference (492 to 630 nm) of 160-times diluted serum was set as positive in ELISA. For patients with active and untreated pulmonary tuberculosis, the ELISA had a sensitivity of 81% and a specificity of 96%. From these results, it was concluded that the detection of IgG antibodies against cord factor is useful for serodiagnosis of active pulmonary tuberculosis. It was also indicated that the anticord factor antibody titers decline to the normal level as a result of antituberculosis chemotherapy.

Rapid serodiagnosis of human mycobacteriosis by ELISA using cord factor (trehalose-6,6'-dimycolate) purified from Mycobacterium tuberculosis as antigen.

IgG antibodies against purified cord factor (trehalose-6,6'-dimycolate, TDM) in sera of 99 patients infected with mycobacteria (42 patients with tuberculosis excreting tubercle bacilli in the sputum, 11 patients with non-tuberculous mycobacteriosis excreting acid-fast bacilli in the sputum, and 46 patients without bacilli in the sputum but diagnosed as having pulmonary tuberculosis by chest X-ray films and physical examination), five patients with lung cancer, and 100 healthy controls which included subjects positive and negative for the tuberculin test were tested by the ELISA with TDM purified from Mycobacterium tuberculosis H37Rv as the antigen. Of the 99 cases of mycobacteriosis, 83 patients (83.8%) had positive results (48 samples from 53 patients, or 90.5%, with bacilli in the sputum, and 35 samples from 46 patients (76%) with tuberculosis diagnosed clinically). The sera of the five patients with lung cancer and the 100 controls all gave negative results. Thus, the sensitivity and specificity were 83.8% and 100%, respectively. ELISA with TDM as the antigen is simple, reproducible, and useful for the rapid serodiagnosis of general mycobacterial infections including tuberculosis, because it does not involve the cultivation of bacteria.

Cord factor (alpha,alpha-trehalose 6,6'-dimycolate) inhibits fusion between phospholipid vesicles.

The persistence of numerous pathogenic bacteria important in disease states, such as tuberculosis, in humans and domestic animals has been ascribed to an inhibition of fusion between the phagosomal vesicles containing the bacteria and lysosomes in the host cells [Elsbach, P. & Weiss, J. (1988) Biochim. Biophys. Acta 974, 29-52; Thoen, C. O. (1988) J. Am. Vet. Med. Assoc. 193, 1045-1048]. In tuberculosis this effect has been indirectly attributed to the production of cord factor (alpha,alpha-trehalose 6,6'-dimycolate). We show here that cord factor is extraordinarily effective at inhibiting Ca2(+)-induced fusion between phospholipid vesicles and suggest a mechanism by which cord factor confers this effect. These findings are likely to be important in our understanding of the pathogenesis and treatment of many diseases of bacterial etiology.

Detection of trehalose monomycolate in Mycobacterium leprae grown in armadillo tissues.

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 alpha-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.

Interstitial and hemorrhagic pneumonitis induced by mycobacterial trehalose dimycolate.

Intraperitoneal injections of cord factor (trehalose dimycolate, TDM) provides a model for interstitial and hemorrhagic lung disease that is produced by a chemically defined substance. A single injection of 10 micrograms of TDM, in light mineral oil or hexadecane, into C57BL/6 mice produces interstitial and hemorrhagic pneumonitis. Following injection of TDM the pulmonary lesions increase gradually and become maximal by the seventh to ninth day, at which time 70% of the mice show both gross hemorrhages and dense mononuclear infiltrates; an additional 20% of the mice show only microscopic lesions. From day 14 onward the incidence and severity of the lesions decrease, and by day 28 the lungs are normal by both gross and light-microscopy examination. Only 5% of the mice succumb. Except for peritonitis other organs are not affected. Doses of 3.3 and 10 micrograms of TDM are equally effective in producing the lesions, but a dose of 1.0 microgram of TDM causes only mild interstitial inflammation and lesser doses do not induce lesions. A single subcutaneous injection of 10 micrograms of TDM causes lesions in only 20% of mice. Vehicle-injected mice do not develop lesions. Electron microscopy revealed that the majority of the infiltrating cells are monocytes and macrophages and that extensive interstitial damage is produced. The mechanism of the effects of TDM are unknown and is currently under study. Our preliminary data suggests that the phenomenon is dependent upon T-lymphocytes.