Mycobacterium Bovis Strain BCG Research Articles

Antileishmanial activities of macrophages from C3H/HeN and C3H/HeJ mice treated with Mycobacterium bovis strain BCG

C3H/HeN and C3H/HeJ mice were infected ip with viable BCG, a macrophage-activating agent, and their peritoneal exudate macrophages exposed to Leishmania tropica amastigotes. Macrophages from BCG-infected C3H/HeJ mice had both leishmanicidal activities described for lymphokine activation of C3H/HeJ macrophages in vitro: increased resistance to L. tropica infection, followed by intracellular killing of the parasite. Macrophages from BCG-infected C3H/HeJ mice were also activated to kill tumor cells in vitro. In contrast, macrophages from BCG-treated C3H/HeJ mice were not resistant to L. tropica infection, did not kill intracellular amastigotes over 72 hr in culture, and were not cytotoxic to tumor cells.

Mycolic acid patterns of four vaccine strains of Mycobacterium bovis BCG.

Thin-layer chromatography of methanolysates of four widely used vaccine strains of Mycobacterium bovis BCG showed that only one organism had the expected pattern of mycolic acid methyl esters characteristic of Mycobacterium bovis and Mycobacterium tuberculosis. The remaining three BCG strains lacked methoxy mycolic acid.

Intracellular destruction of Leishmania tropica by macrophages activated in vivo with Mycobacterium bovis strain bcg.

Leishmania tropica, an intracellular protozoan parasite, infects and replicates in macrophages of C3H/HeN and C3H/HeJ mice in vitro (1). Exposure of resident peritoneal macrophages to amastigotes for 1 hr results in 20–25% infected macrophages, Intracellular parasites increase from 1.7 to >3.5 per macrophage over 72 hr.

Effect of interferon on murine cytochrome P-450: effect of partially purified antigen-specific interferon-gamma.

Gamma interferon (IFN-gamma), induced by sensitization of mice with Mycobacterium bovis strain BCG followed by challenge with tuberculin, was partially purified by adsorption on silicic acid. Administration of the partially purified IFN-gamma to mice significantly depressed levels of liver microsomal cytochrome P-450 to an equivalent extent as crude IFN-gamma.

Effects of passive transfer and induction of gamma (type II immune) interferon preparations on the metabolism of diphenylhydantoin by murine cytochrome P-450.

Induction of gamma (Type II) interferon results in depression of the cytochrome P-450 system of mice. The gamma interferon is induced by sensitization of mice with Mycobacterium bovis strain BCG followed by challenge with tuberculin. The degree of depression of the cytochrome P-450 system correlates with the levels of interferon induced. passive transfer of exogenous gamma interferon also results in depression of the murine cytochrome P-450 system. In the present study the metabolism of diphenylhydantoin, a drug metabolized by the cytochrome P-450 system, was examined in mice in which gamma interferon was induced. The metabolism of diphenylhydantoin was severely inhibited in mice in which interferon was induced, and the level of inhibition correlated with the liter of gamma interferon induced. Passive transfer of gamma interferon also depressed the metabolism of diphenylhydantoin by the murine cytochrome P-450 system.

Lung injury induced by mycobacterial cell walls: Effects on connective tissue

Intravenous injection of cell walls from Mycobacterium bovis strain BCG results in increased lung mass in the rat 4 months after injection. Histologically, there is a simultaneous increase in interstitial material and the formation of granulomata. The granulomata do not show increased staining for collagen by a trichrome stain. Prominent reticulin is present in the granulomata as shown by a silver stain. Lung dry weight is significantly increased 4 months after the injection of 300 μg cell walls; the total amount of collagen is significantly increased, but its concentration (amount per mg dry weight) is similar to that of controls. At 6 months, lung collagen amounts in BCG-treated animals are not significantly different from those of controls. By 10 months the granulomata largely disappear, lung architecture returns to normal, and lung collagen amounts are at control levels. In this model of lung injury, increases in lung connective tissue proteins are not selective and they do not persist as the lung returns to normal histologically.

Mechanisms in the in vivo release of lymphokines : I. Comparative kinetics in the release of six lymphokines in inbred strains of mice

Lymphokines were detected in the sera of 16 strains of inbred mice sensitized intravenously with cell walls of Mycobacterium bovis strain BCG and challenged subsequently intravenously with old tuberculin (OT). Variations occurred between the strains in the types and quantities of six lymphokines studied, namely, chemotactic factor (CF), type II interferon (IF II), lymphotoxin (LT), migration inhibitory factor (MIF), mitogenic factor (MF), and skin reactive factor (SRF). The times for maximum release of the lymphokines in the different strains were similar for MIF, IF II, SRF, and MF, but differed for CF and LT. The degree of activity of MIF and IF II generally paralleled one another in the different strains but such parallelism did not occur for the other four lymphokines. Each of the 16 strains was a high responder for at least one of the lymphokines, indicating that in sensitized mice the release or presence in the circulation of lymphokines in response to a specific antigen is a selective process. Thus, each strain may have an individual combination of lymphokines, interactions of which may determine the types of pathway utilized in an immunological response.

Treatment of NZB/NZW F1 hybrid mice with Mycobacterium bovis strain BCG or type II interferon preparations accelerates autoimmune disease.

NZB/NZW F1 hybrid mice develop a spontaneous autoimmune disease characterized by the appearance of antinuclear antibodies and premature death due to immune complex glomerulonephritis. To investigate the possible effects of cellular immune stimulation on this disorder, groups of female NZB/NZW mice, aged 2, 5, and 7 months, were treated either with the nonspecific immunostimulatory agent Mycobacterium bovis strain BCG or with saline. Mice treated with BCG at ages 5 and 7 months died sooner than age-matched controls, and death was associated with severe glomerulonephritis, suggesting that BCG may have accelerated autoimmunity in these mice. Since BCG is known to stimulate the production of type II (or gamma) interferon, a substance with potent immunoregulatory effects, a second study was carried out to assess the effects of type II interferon on NZB/NZW disease. A greater number of type II interferon-treated mice died by 9 months of age when compared to controls, and the increased death rate was associated with a more rapid development of antinuclear antibodies and histologically confirmed glomerulonephritis. These data, together with a recent report of increases in the level of serum type II interferon in patients with active systemic lupus erythematosus, suggest that type II interferon may play a role in the pathogenesis of autoimmune disease.

Host defenses to intracellular pathogens

and Historical Perspective.- I. Microbicidal Mechanisms of Leukocytes in Host Defenses.- Oxygen Independent Microbicidal Mechanisms of Human Polymorphonuclear Leukocytes.- Oxidative Metabolism of Leukocytes and Its Relationship to Bactericidal Activity.- Some Paradoxes of Macrophage Function.- Genetic Disorders of Granulocyte Function: What They Tell Us About Normal Mechanisms.- II. The Immune System and Phagocytic Cell Function.- Modulation of Effector Lymphokines.- Stimulation of Host Resistance to Metastatic Tumors by Macrophage Activating Agents Encapsulated in Liposomes.- Effect of Prostaglandins on the Production of Interleukin-2.- Interaction of Mycobacteria with Normal and Immunologically Activated Alveolar Macrophages.- Guinea Pig Alveolar Macrophages Probably Kill M. tuberculosis H37Rv and H37Ra in vivo by Producing Hydrogen Peroxide.- Interferon and Host Defense Systems.- Mediator Interactions Regulating Macrophage Secretion of Interleukin 1 and Interferon.- Macrophage Oxygen-Dependent Killing of Intracellular Parasites: Toxoplasma and Leishmania.- Immunologic Lesions During Toxoplasma gondii Infection.- Immunodepression in BALB/c Mice Infected with Leishmania tropica.- III. Host Defenses to Intracellular Bacteria.- Cellular Mechanisms of Anti-Mycobacterial Immunity.- Host Response to Infection with Mycobacterium bovis (BCG) in Mice: Genetic Study of Natural Resistance.- Cell Mediated Lysis of Lymphocytes Expressing Bacterial Antigens.- Improvement of Abnormal Lymphocyte Responses in Atypical Mycobacteriosis with Indomethacin.- Immunoregulatory Defects in Leprosy.- Cellular Mechanisms of Resistance to Listeria monocytogenes.- Effect of Interferon Inducers and Purified Mouse Interferon on the Susceptibility of Mice to Infection with Listeria monocytogenes.- Natural Resistance to Listeria monocytogenes as a Function of Macrophage Inflammatory Response.- Effect of Acute Nutritional Deprivation on Host Defenses Against Listeria monocytogenes - Macrophage Function.- Pilus-Mediated Clearance of Salmonella typhimurium by the Perfused Mouse Liver.- Immunity to Salmonella Infection.- Strain Dependent Variation of Delayed-Type Hypersensitivity in Salmonella typhimurium Infected Mice.- Monoclonal Antibodies to Salmonella typhimurium and Escherichia coli Lipopolysaccharides.- Characterization of Monoclonal Antibodies Which Recognize Specific Cell Surface Determinants on Salmonella typhimurium.- Monoclonal Antibodies as Probes for Antigens of Mycoplasma pulmonis.- Electron Microscopic Examination of the Inflammatory Response of Guinea Pig Neutrophils and Macrophages to Legionella pneumophila.- IV. Immunity to Rickettsia, Parasites and Fungi.- Activation of Macrophages for Killing of Rickettsiae: Analysis of Macrophage Effector Function After Rickettsial Inoculation of Inbred Mouse Strains.- Parameters of Cellular Immunity in Acute and Chronic Rickettsia tsutsugamushi Infections of Inbred Mice.- Lymphokine Stimulated Macrophages Inhibit Intracellular Chlamydia psittaci Replication by Mechanisms Distinct from Intracellular Inhibition of Toxoplasma gondii Replication.- Natural and Acquired Resistance to Trypanosoma cruzi.- Immunity to Fungal Infections.- Antibody-Independent Mechanisms in the Development of Acquired Immunity to Malaria.- Intracellular Destruction of Leishmania tropica by Macrophages Activated In Vivo with Mycobacterium bovis Strain BCG.- In Vitro Macrophage Antimicrobial Activities and In Vivo Susceptibility to Leishmania tropica Infection.- V. Mechanisms of Antiviral Immunity.- Virus-Immune T Cells and Monoclonal Antibodies in the Mouse Influenza Model.- Escape from Immune Surveillance During Persistent Virus Infection.- Influence of Viruses on Cells of the Immune Response System.- Genetically Controlled Resistance to Viruses.- Macrophage Oxidative Metabolism: A Defense Mechanism Against virus Infection?.- Interferon-Induced Augmentation of Natural Killer Cell Activity by Splenocytes from Leukemia Virus Immunosuppressed Mice.- Immunological Comparison of Ocular Disease Induced by Two Strains of Herpes Virus of Different Virulence.- Cellular Processing of the Large Glycoprotein of Lacrosse Virus (Family Bunyaviridae) Implications for Virion Assembly and Host Defense.- CMV and Renal Allograft Survival.- Martyrs, A Poem by Dr. L. J. Berry.- Contributors.- Author Index.

Mechanism of Action of BCG-Tumor Cell Vaccines in the Generation of Systemic Tumor Immunity. I. Synergism Between BCG and Line 10 Tumor Cells in the Induction of an Inflammatory Response<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

The intradermal injection of a vaccine composed of 10(7) X-irradiated, syngeneic hepatocarcinoma line 10 (L10) cells admixed with 10(8) Mycobacterium bovis strain BCG into inbred Sewall Wright strain 2 guinea pigs induced a local acute and then chronic inflammatory response. Cellular analysis of enzymatically dispersed dermal vaccination sites and regional lymph nodes revealed quantitative differences between the cellular infiltrate induced by a mixed BCG-tumor cell vaccine and the inflammation induced by either 10(8) BCG alone or 10(7) tumor cells alone. Analysis of dermal sites from days 1 through 4 following vaccination showed that sites receiving the BCG-tumor cell vaccine contained twofold to fourfold more cells than did those induced by BCG alone and more than fourfold the number of cells induced by L10 cells alone. These results indicated that there was a synergistic interaction between BCG and L10 cells in the induction of an inflammatory response at the site of vaccination. Analysis of the superficial distal axillary lymph nodes draining the site of vaccination revealed a similar synergistic interaction between BCG and L10, such that lymph nodes draining vaccination sites had a greater total cellular content than that induced by either BCG or L10 cells administered separately. When two other tumors, also syngeneic to strain 2 guinea pigs, were tested for their ability to interact synergistically with BCG to elicit an inflammatory response, it was found that the antigenically distinct hepatocarcinoma line 1 tumor also shared this characteristic with L10, whereas the L2C B-cell leukemia did not.

Protection of cotton rats against experimental Echinococcus multilocularis infections with BCG cell walls

Previous works has indicated that cotton rats treated with Mycobacterium bovis strain BCG are effectively protected against an infection with the metastatically proliferating metacestodes of Echinococcus multilocularis. In an attempt to induce a similar protection in the absence of tubercular granulomatous lesions, cotton rats were treated with BCG cell walls. A single injection of 150 micrograms of cell walls, emulsified in mineral oil-Tween-saline, 2 weeks before the inoculation of the parasite completely protected the animals against infection with E. multilocularis. This protection was correlated with an increase in the numbers of monocytes and, as judged by acid phosphatase activity, an activation of these cells. This study shows that BCG cell walls are as effective in protecting animals against E. multilocularis as the viable organism.

A biological study on hot-water extract from delipidated Mycobacterium bovis strain BCG.

The biological activity of a hot-water extract from delipidated BCG, designated as HSA (Hot-water Soluble Adjuvant), was investigated. The HSA did not induce hyperreactivity to bacterial endotoxin. The hot-water extract from which nucleic acids had been removed by streptomycin (SM-HSA) was found to enhance the delayed-type hypersensitivity as evidenced by the results of footpad reaction of mice. The HSA and SM-HSA could be injected to mice by an intraperitoneal route for 20 consecutive days without undesirable side effects. A comparative study was made on the effects of HSA in relation to the duration and doses of treatment with HSA using the ddI mice inoculated with Sarcoma-180. The most remarkable effect was observed when 0.25 mg of HSA had been injected for 20 consecutive days. Also SM-HSA was found to exert antitumor activity when applied in the same manner as above. These results suggest that the presence of nucleic acids is not related to the biological and antitumor activities of the hot-water extract.

Synergistic tumor regressive activity observed following treatment of line-10 hepatocellular carcinomas with deproteinized BCG cell walls and mutant Salmonella typhimurium glycolipid

Synergistic tumor-regressive activity was observed when the water-soluble portion of a phenol-water extract from mutant Salmonella typhimurium whole cells was combined with deproteinized cell walls from Mycobacterium bovis strain BCG. As little as 50 μg deproteinized cell walls combined with 50 μg water-soluble extract from the mutant salmonella produced 89–100% cures of line-10 dermal tumors in treated strain 2 guinea-pigs. However, none of the animals was cured following treatment with 50 μg of deproteinized cell walls alone. Only 17% of treated animals were cured following treatment with 50 μg of the water-soluble extract from the mutant salmonella. The deproteinized cell walls and water-soluble extract were suspended in oil-in-water emulsions and injected directly into 10 mm tumors. The deproteinized cell walls were prepared by treating BCG cell walls with proteolytic enzymes and denaturing agents (KCl, urea, Triton X-100, and guanidine hydrochloride). Urea or a combination of denaturing agents reduced the protein content of protease-treated cell walls from approximately 2% (w/w) protein to 0.7% protein. The antigenicity of the effectively deproteinized cell walls, as measured by skin testing in presensitized guinea-pigs, was reduced approximately ten-fold compared with untreated cell walls. Injection to mice of 500 μg deproteinized cell walls in combination with 500 μg water-soluble extract from the mutant salmonella produced transient, clinical signs of toxicity (malaise, conjunctivitis, diarrhea, and rough hair coats) lasting approximately 5 days. However, no deaths were observed. The synergistic antitumor effect of combining deproteinized BCG cell walls with the water-soluble extract from mutant salmonella may be useful for treatment of certain cases of spontaneous neoplastic disease.

Type I and II interferons and migration inhibitory factor: production in Mycobacterium bovis BCG-infected mice desensitized with old tuberculin or lipopolysaccharide

Mice with delayed hypersensitivity induced by infection with Mycobacterium bovis strain BCG were desensitized by a single large dose of specific antigen (old tuberculin, OT) or a nonspecific interferon stimulus (bacterial lipopolysaccharide, LPS). Subsequent challenge of the desensitized animals revealed only a homologous hyporeactivity, that is, mice desensitized with OT showed decreased type II and migration inhibitory factor (MIF) responses to the specific antigen, which were unaffected by desensitization with LPS. Conversely, mice desensitized with LPS showed a decreased type I interferon and MIF response to LPS, which was unaffected by desensitization with OT. These results suggest that type I interferon and its accompanying low-titered MIF activity are produced by cell populations different from those that produce type II interferon and its accompanying high-titered MIF activity.

Cytochemical and biological properties ofMycobacterium bovis BCG

It was the aim of the present communication to find a simple test for a reliable discrimination of Mycobacterium bovis BCG from Mycobacterium tuberculosis. A total of 26 BCG strains, out of them 10 Czechoslovak strains (2 lyophilized cultures of BCG of different batch, 6 strains isolated from abscesses of children after BCG-vaccination and 2 strains from fatal cases after BCG-vaccination) and 16 strains obtained from foreign laboratories, were used. Of the tested characteristics a combination of 3 tests, sensitivity to 1 microgram of 2-thiophene carbonylhydrazide (TCH), activity of 3 acylamidases (urease, nicotinamidase and pyrazinamidase) and a quantitative nitrate test, was found to be most advantageous. The Czechoslovak strains of Mycobacterium bovis BCG were fully sensitive to TCH, of the 3 acylamidases mentioned above only urease was positive and nitrate was reduced only little or not at all. On the other hand, strains of Mycobacterium tuberculosis were always resistant to TCH, had positive urease, nicotinamidase and pyrazinamidase and reduced nitrate very intensively.

Mitogenic Activity of Cell‐Wall Components in Mouse Spleen Cells

In the previous paper (5), the authors examined the mitogenic activity of the cell walls of mycobacteria, nocardia and corynebacteria, and showed that these cell walls were active as mitogens in normal spleen cells, anti-0 sera-treated spleen cells, macrophage-depleted spleen cells and cortisone-resistant thymocytes of C57BL/6J mice. It was also demonstrated that these cell walls were mitogenic in the spleen cells and macrophage-depleted spleen cells of congenitally athymic nude (BALB/c -nu/nu) mice. From the above results, we have concluded that the cell walls of mycobacteria, nocardia and corynebacteria act as mitogens on both thymus-derived lymphocytes (T cells) and bone marrow-derived lymphocytes (B cells). This note deals with the chemical constituents which are responsible for mitogenic activity of the mycobacterial cell wall. It has been established that the chemical structure of mycobacterial cell wall is a acid-arabinogalactan-mucopeptide complex and the detailed chemical properties of each component were described previously (2, 3). The chemical components of mycobacterial cell walls were prepared by the following procedure and their mitogenic activity in the spleen cells of C57BL/6J and congenitally athymic nude mice, and cortisone-resistant thymocytes of C57BL/6J mice was examined. Mycolic acid, known to be a-branched i3-hydroxy fatty (1), was prepared from the cell wall of Mycobacterium tuberculosis train Aoyama B by alkaline hydrolysis using 2.5% KOH in benzene-methanol (1 :1), and mycolic acid methyl ester was prepared by refluxing mycolic acid with 3% HC1-methanol for 3 hr. D-Arabinose5-mycolate was purified from the firmly bounded lipids of M. tuberculosis Aoyama B as described previously (6). Arabinogalactan, the neutral polysaccharide of mycobacterial cell walls, was purified from the cell wall of Mycobacterium bovis strain BCG according to the method of Misaki et al (11). Mycoloyl-arabinogalactan (D-CMI-A) was prepared by further fractionation of so-called wax D which was extracted by the same procedure as described previously (3, 7) from the cells of M. bovis strain Ushi 10 which were cultured on Sauton's synthetic medium at 37 C for 8 weeks. The mycoloyl-arabinogalactan designated as D-CMI-A of M. bovis Ushi 10 is composed of arabinogalactan (46%) and mycolic acid (51 %) but lacks mucopeptide moiety. The purification and porperties of the cell wall of M. bovis BCG were described pre-

Failure of BCG to protect calves against Babesia divergens infection.

THE possible use of nonspecific immunity to control Babesia infections has not been much examined. Cattle treated with Escherichia coli lipopolysaccharide (1.25 µg per kg live weight) immediately before infection with B. argentina ( =B. bovis) were slightly more resistant than controls1. An interferon inducer, a double stranded RNA, slightly delayed death in mice infected with B. rodhaini2. The question has been reopened by some work with B. microti and B. rodhaini in which mice injected intravenously (i.v.) with 2×107 Mycobacterium bovis strain BCG (Glaxo strain) became completely immune to intraperitoneal challenge with 106 Babesia parasites3. These remarkable results suggested that the method should be examined in domestic animals. We decided to assess it in calves and report here that BCG fails to protect against infection with B. divergens, the common cause of redwater in the UK.

Antibodies to melanoma cell and BCG antigens in sera from tumour-free individuals and from melanoma patients.

THE Mycobacterium bovis strain BCG shares antigenic components with several neoplasms, including human malignant melanoma1–5. Other species of bacteria also have antigenic similarities with a guinea pig hepatoma6. Antibodies to mycobacteria and other bacterial antigens are universally present in sera from normal humans7,8. Since there is a broad range of shared or cross-reactive antigens among mycobacteria and unrelated microorganisms9, it is conceivable that an immune response by normal humans to mycobacteria has been induced by exposure to taxonomically unrelated microorganisms ubiquitous in the environment. Cytotoxic reactions by lymphoid cells from normal animals and humans to many kinds of tumour cells have been reported10–15. Antibodies in sera from patients with various neoplasms have often been observed that react with antigens derived from their own tumours or from those of the same histological type16–18.Humoral responses to tumour-associated antigens have also been noted in sera from normal humans, gibbons and mice19–21. We have investigated whether sera from normal, tumour-free humans contain antibodies which can react with melanoma-associated as well as with BCG antigens. If this were the case, perhaps immunological reactions by normal humans to certain tumours are widespread and reflect previous sensitisation by microorganisms.

The efficacy of BCG-induced tumor immunity in guinea pigs with regional and systemic malignancy

It has been previously demonstrated that transplanted syngeneic line-10 hepatocarcinoma established in the skin of inbred guinea pigs (strain 2) regressed and regional lymph node metastases were eliminated after intratumoral injection of viable Mycobacterium bovis strain BCG. During the course of this reaction there is the development of systemic tumor immunity. The purpose of this study was to determine the relative efficacy of the induced tumor immunity to eliminate regional as well as systemic tumor burden. The approach to evaluate the efficacy of BCG-induced systemic tumor immunity in vivo, for regional as well as systemic tumor, was to develop a competition assay using increasing doses of intravascular disseminated line-10 tumor cells in animals with established regional tumors. The results clearly show that the efficacy of intratumoral BCG injection in producing regression of regional tumor is abrogated by initial intravascular doses of 103–106 line-10 cells. That the vascular systemic tumor burden diminished the effective systemic tumor immunity was demonstrated by the inability of animals with systemic tumor burdens to reject contralateral challenge of line-10 tumor cells. The capability of BCG-treated animals to reject contralateral line-10 challenge was inversely proportional to the initial intravascular tumor dose. Survival studies clearly demonstrate that a significant therapeutic effect could be achieved in guinea pigs with regional skin tumors and limited vascular metastases when the modality of therapy included BCG-intratumoral injection, followed 6 weeks later by surgery of the established skin tumor and regional lymph node. These results suggest that the development of tumor immunity after BCG-intratumoral injection is not impaired by the systemic tumor burden, but rather that it is preempted at distant sites.

Evaluation of BCG Administered by Scarification for Immunotherapy of Metastatic Hepatocarcinoma in the Guinea Pig2

In inbred guinea pigs, administration of Mycobacterium bovis strain BCG by scarification at a site distant from an excised skin tumor, but in the regional lymph node drainage, was evaluated for its immunotherapeutic effect on the development of lymph node metastases. Scarification was performed after surgical excision of intradermally transplanted syngeneic (line-10) hepatocarcinoma at a time when microscopic foci of tumor cells were present in regional lymph nodes. Various strains of BCG were evaluated for their immunotherapeutic potential: fresh-frozen Phipps, Pasteur, and Tice; and lyophilized Pasteur, Tice, and Connaught. Scarification commenced 3 days after surgical removal of the tumor and continued once a week for 5 weeks. Only lymph nodes from fresh-frozen Phipps- and Pasteur-scarified animals were significantly smaller than those in the control groups. Differences in lymph node weight correlated histologically with less detectable metastases. This cytostatic effect was short lived; eventually, the metastatic tumor growth was not significantly different from that of control animals. No significant differences were observed in mean survival time: All animals died as a result of metastases 3 months after tumor inoculation. These results demonstrated that limited scarification with BCG of certain strains temporarily inhibits the growth and proliferation of metastases in regional lymph nodes after removal of the primary tumor.