Antitumor activity and other biological properties of two phenotypes isolated from BCG

Summary Two phenotypes isolated from the BCG strain of Mycobacterium tuberculosis were found to differ from each other and from the parental strain by their chemical and antigenic compositions and by some of their biological properties. When used as immunotherapeutic agents in admixture with cancerous cells, one of these phenotypes enhanced whereas the other inhibited tumor growth. As immunoprophylactic agents, both phenotypes exerted, but at different degrees, antitumor protection in mice.     

Anti-tumour activity of Lactobacillus casei on lewis lung carcinoma and line-10 hepatoma in syngeneic mice and guinea pigs

Summary The anti-tumour activity of Lactobacillus casei YIT 9018 (LC 9018) on Lewis lung carcinoma (3LL) in C57BL/6 mice and line-10 hepatoma in strain-2 guinea pigs was examined. Intravenous injection of LC 9018 was effective for inhibition of pulmonary metastases in C57BL/6 mice after s.c. inoculation with 3LL tumours. Intralesional (i.l.) injection of LC 9018 was also effective for both prolongation of the survival period and inhibition of pulmonary metastases in 3LL tumour-bearing mice. The combination treatment of i.l. and i.v. injections of LC 9018 before or after surgical excision of the primary tumour remarkably inhibited the pulmonary metastases after inoculation with 3LL tumour. Intralesional injection of LC 9018 was effective for regression of the established tumours of line-10 hepatoma inoculated i.d. and for induction of systemic tumour immunity in strain-2 guinea pigs.     

immunocytochemical localization of urokinase-type plasminogen activator in lewis lung carcinoma

The invasively growing and metasizing Lewis lung carcinoma consistently contained urokinase-type plasminogen activator (u-PA) enzyme activity. When investigated immunocytochemically with antibodies against u-PA, different parts of individual tumors showed a pronounced heterogeneity in staining intensity. Strong staining was found in areas with invasive growth and degradation of surrounding normal tissue, while other areas were completely devoid of staining. Immunoreactivity occurred both with a perinuclear cytoplasmic localization in tumor cells and associated with apparently extracellular material. SDS PAGE of tumor extracts, under both reducing and nonreducing conditions, followed by immunoblotting, showed only one immunocytochemically stainable band with an electrophoretic mobility corresponding to that of purified proenzyme to u-PA, while no two-chain u-PA was detected. This indicates that the major part of the activator in Lewis lung carcinoma is present as one-chain pro-u-PA.     

Antitumor activities of iodoacetate and dimethylsulphoxide against solid Ehrlich carcinoma growth in mice

Treatment of tumor-bearing mice with LD12.5 values of iodoacetate; IAA (1.84 mg/100 g b.w.) and/or dimethylsulphoxide; DMSO (350 mg/100 g b.w.) significantly increased the cumulative mean survival time and percentage of survivors and reduced the mean tumor weight, compared to tumor-bearing controls, however, a more pronounced effect is recorded in the combined treatment. Also, an increase in the life span (ILS%) and tumor growth inhibition ratio (T/C%) are reported and amounted to 145.78 and 43.80%, 195.54 and 61.30% and 220.77 and 78.40% in IAA, DMSO and combined-treated groups, respectively. Results obtained from biochemical studies reveal that a single IAA treatment of tumor-bearing mice significantly increased the levels of plasma lactate dehydrogenase (LDH) activity, while it also significantly decreased the levels of plasma glucose and liver total protein, RNA and DNA, compared to normal controls. On the other hand, a single DMSO treatment significantly elevated the activities of blood antioxidant enzymes, i.e. glutathione peroxidase (GPx) and glucose-6-phosphate dehydrogenase (G6PDH) and decreased the liver RNA and DNA levels. Combined treatment increased significantly the levels of plasma LDH and erythrocytes G6PDH activities, as well as liver glycogen, and in contrast it decreased the levels of liver total protein, RNA and DNA, compared to normal controls.     

Antitumor activity of eosinophils activated by IL-5 and eotaxin against hepatocellular carcinoma

We examined the antitumor effects of eosinophils to explore the potential of eosinophils as effector cells in tumor cytotoxicity. We expressed eotaxin in hepatocellular carcinoma cells, MH134, and injected them into either normal or IL-5 TG mice intradermally and monitored cell growth. In normal mice, growth of MH134 cells containing the expression plasmid pCXN2-eotaxin was similar to that of vector-transfected MH134 cells for a period of 2 weeks, suggesting that expression of eotaxin does not change the growth rate of tumor cells. In IL-5 TG mice, however, the growth of eotaxin expressing MH134 cells was significantly suppressed. LPS induced eosinophils to produce TNF-alpha to kill MH134 cells in vitro. Intratumor injection of LPS is effective to kill MH134-pCXN2 and MH134-pCXN2-eotaxin only in normal mice. Administration of anti-CD4 or anti-CD8 antibodies suppressed growth of MH134-pCXN2-eotaxin cells compared with control antibodies, suggesting that T cells may interfere with immunity against MH134. Administration of anti-IL-5Ralpha and anti-asialo GM1 antibodies enhanced growth of MH134-pCXN2-eotaxin cells, suggesting involvement of eosinophils and NK cells in suppression of tumor cell growth. Although we cannot exclude the possibility that NK cells participate in tumor cell killing in vivo, the presence of NK markers such as DX5, asialo GM1, Ly49, and CD94, and NKG2D on large numbers of eosinophils activated by eotaxin suggests that eosinophils function in such suppression of tumor cell growth. Furthermore, we showed that anti-NKG2D antibodies could significantly inhibit the LPS-induced cytotoxicity against MH134 by highly enriched fraction of eosinophils.     

Antitumor and antimetastatic activity of the immunoadjuvant peptidoglycan monomer PGM in mice bearing MCa mammary carcinoma

Summary The antitumor and antimetastatic activities of the water-soluble peptidoglycan monomer GlnNAc-Mur-NAc-L-Ala-D-iso-Gln-meso-diamminopimelic acid (-NH₂)-D-Ala-D-Ala (PGM), which has immunostimulant effects, have been evaluated in CBA mice bearing MCa mammary carcinoma. The antineoplastic effects of PGM depend strictly on the dosage and treatment schedule used. Though a significant inhibition of the primary tumor growth is observed over a wide range of dosage, only the IV administration of daily doses of 50 mg/kg/day on days 1, 5, 10, 15 inhibits spontaneous lung metastasis formation and in parallel prolongs the survival time of the treated mice. The magnitude of the antimetastatic effects of PGM depends on the degree of dissemination of the tumor, and is greater when the number of metastatic foci is low. Examination of the therapeutic potential of PGM in combination with surgery has further indicated that the timing of administration plays an important role in the overall effectiveness of this substance. A 5-day interval is necessary between two consecutive injections for the induction of significant increases of the survival times.     

Experimental screening of two BCG preparations produced according to different principles

Summary Two BCG preparations produced according to different principles for use as an adjunct to surgery in a comparative clinical trial in melanoma patients have been examined for immunostimulating, toxic, and antitumor activity in various murine and guinea pig models and for immunostimulation in vitro of human lymphocytes. One BCG preparation was prepared in the classic way, i.e., grown as a surface culture and subsequently homogenized in a ball mill, while the other preparation was grown as a dispersed culture.In general, a comparable effect on spleen weight of the proliferation of lymph node cells and activation of spleen macrophages was observed when a similar amount of culturable particles of either BCG preparation were administered. In parallel with these effects, an increase of the serum glutamic pyruvic transaminase was detected. Activation of natural killer cells and the induction of an immune reaction in lymph nodes appeared to be a function of the total amount of injected viable and dead micro-organisms.The synergistic effect of the BCG preparation grown as a dispersed culture on the stimulation of human lymphocytes by phytohaemagglutinin, lacking in the BCG preparation grown as surface culture and subsequently homogenized in a ball mill, was due to the stabilizers present in the former material.The effect of BCG treatment on the growth of an originally chemically induced murine fibrosarcoma depended on the treatment schedule. Growth enhancement, growth retardation, or no effect was observed, depending on the regimen used. Although differences between the two BCG preparations could be demonstrated in the various models the clinical relevance of these reported results can only be evaluated in conjunction with the outcome of the comparative clinical trial.Now Fellow of the Koningin Wilhelmina Fonds of the National Cancer League of The NederlandsNow Secretary of the Health Council, The Hague, The Netherlands     

Antitumor activity of Nocardia cell wall skeleton preparations in transplantable tumors in syngeneic mice and patients with malignant pleurisy

Summary The antitumor activity of the cell wall skeleton preparations of four species of Nocardia, N. brasiliensis strain 146, N. coeliaca strain 122, N. polychromogenes strain 6, and N. rubra, which showed potent adjuvant activity on the induction of cell-mediated cytotoxicity in allogeneic mice, was examined with the aid of EL-4 leukemia, melanoma B16, and MH-134 hepatoma in syngeneic mice. Preliminary clinical trials were performed and the results suggest that the cell wall skeleton of N. rubra, upon intrapleural injection, may be useful as an immunotherapeutic agent for patients with malignant pleurisy. The chemical properties of these cell wall skeleton preparations are described.     

Enhanced effect of BCG vaccine against pulmonary Mycobacterium tuberculosis infection in mice with lung Th17 response to mycobacterial heparin‐binding hemagglutinin adhesin antigen

Although the BCG vaccine can prevent tuberculosis (TB) in infants, its ability to prevent adult pulmonary TB is reportedly limited. Therefore, development of a novel effective vaccine against pulmonary TB has become an international research priority. We have previously reported that intranasal vaccination of mice with a mycobacterial heparin‐binding hemagglutinin adhesin (HBHA) plus mucosal adjuvant cholera toxin (CT) enhances production of IFN‐γ and anti‐HBHA antibody and suppresses extrapulmonary bacterial dissemination after intranasal infection with BCG. In the present study, the effects of intranasal HBHA + CT vaccine on murine pulmonary Mycobacterium tuberculosis (Mtb) infection were examined. Intranasal HBHA + CT vaccination alone failed to reduce the bacterial burden in the infected lung. However, a combination vaccine consisting of s.c. BCG priming and an intranasal HBHA + CT booster significantly enhanced protective immunity against pulmonary Mtb infection on day 14 compared with BCG vaccine alone. Further, it was found that intranasal HBHA + CT vaccine enhanced not only IFN‐γ but also IL‐17A production by HBHA‐specific T cells in the lung after pulmonary Mtb infection. Therefore, this combination vaccine may be a good candidate for a new vaccine strategy against pulmonary TB.     

Inhibition of tumor metastasis of lewis lung carcinoma in C57BL/6 mice by intrapleural administration of Lactobacillus casei

Summary The antimetastatic effect of Lactobacillus casei YIT9018 (LC 9018) against Lewis lung carcinoma (3LL) in C57BL/6 mice was determined. Intrapleural (i.pl.) administration of LC 9018 was effective in inhibiting pulmonary metastasis after s.c. inoculation of 3LL tumors into C57BL/6 mice. The combination of i.pl. and intralesional or i.v. injections of LC 9018 also markedly inhibited pulmonary metastasis in 3LL-bearing mice. The i.pl. administration of LC 9018 into mice induced an increase in the number of thoracic exudate cells (TEC) and the cell population in the TEC was mainly polymorphonuclear leukocytes in the early stage, while macrophages were dominant in the late stage. In addition, in vitro cytolytic activity against 3LL cells and natural killer cell activity of TEC were augmented by the i.pl. administration of LC 9018. Furthermore, i.pl. administration of LC 9018 into the mice rendered their lung macrophages tumoricidal for 3LL cells in vitro. These results show that TEC induced by i.pl. administration of LC 9018 played a key role in the inhibtion of metastasis in 3LL-bearing mice.     

Immunotherapy of lewis lung carcinoma with hydrosoluble peptidoglycan monomer (PGM)

The water-soluble peptidoglycan monomer (PGM) isolated from the culture fluid of Brevibacterium divaricatum, which has immunostimulating activity, has been examined for its antitumor effects in C57BL mice bearing Lewis lung carcinoma. The formation of spontaneous lung metastases from SC tumor implants is significantly inhibited. The growth of SC primary tumors, including advanced ones, is also significantly inhibited, though to a less pronounced extent than the growth of metastases. The effects on metastases are evident with all treatment schedules used, whereas those on SC primary tumors are treatment schedule-dependent. The treatment with PGM was found to be therapeutically useful when combined with surgical removal of IM implants; in conditions where a single post-operative treatment was ineffective, combined post-operative and immediately pre-operative administration of PGM significantly increased (by 40%) the survival time of treated animals over that of controls undergoing surgery only.     

Influence of BCG vaccine strain on the immune response and protection against tuberculosis

The Bacille Calmette–Guérin (BCG) vaccine has been used for more than 80 years to protect against tuberculosis. Worldwide, over 90% of children are immunized with BCG, making it the most commonly administered vaccine, with more than 120 million doses used each year. Although new tuberculosis vaccines are under investigation, BCG will remain the cornerstone of the strategy to fight the worsening tuberculosis pandemic for the foreseeable future. The recent delineation of genetic differences between BCG vaccine strains has renewed interest in the influence of the vaccine strain on the protective efficacy against tuberculosis. This review critically examines the data from animal and human studies comparing BCG vaccine strains. Although there is good evidence to support the notion that the induced immune response and protection afforded against tuberculosis differs between BCG vaccine strains, currently, there are insufficient data to favour or recommend one particular strain. Identifying BCG strains with superior protection would have a dramatic effect on tuberculosis control at a population level: a small increment in protection provided by BCG immunization will prevent large numbers of cases of severe tuberculosis and deaths, particularly in children.     

Effective nonliving vaccine against experimental tuberculosis in mice

Ribi, Edgar (Rocky Mountain Laboratory, Hamilton, Mont.), Carl Larson, William Wicht, Robert List, and Granville Goode. Effective nonliving vaccine against experimental tuberculosis in mice. J. Bacteriol. 91:975-983. 1966.-Antituberculosis vaccines were prepared in one of three manners: lyophilized BCG suspended in light mineral oil was disrupted in a Sorvall pressure cell and the "oil disruption product" was collected by centrifugation; BCG was disrupted in water, lyophilized, and worked into a paste with a small amount of oil (about 0.16 ml per 50 mg); BCG was disrupted in water, and the cell wall fraction was isolated, lyophilized, and prepared in an oil paste. These vaccines were suspended in Tween-saline to a concentration of 5 mg/ml and heated at 65 C for 30 min. In protection tests based on pulmonary infection with Mycobacterium tuberculosis H37Rv, the median number of virulent organisms in lung tissue of mice immunized with a few hundred micrograms of these three vaccines was 3 to 4 logs lower than in unvaccinated control mice. A similar dose of viable BCG standard vaccine reduced the lung count 1 to 2 logs below the controls. Protection afforded by nonviable, whole BCG, with or without oil, was of only borderline significance. Since oil-treated fractions containing cell walls produced effective immunity, while the oil-treated protoplasm or whole cells were not active, the protective antigen appeared to be an inner component of the cell wall, exposed when the cell was disrupted, and activated by oil. Extraction of oil from immunogenic disruption products resulted in loss of ability of the products to confer protection against the aerosol challenge, whereas high protection against the conventional challenge by intravenous infection with up to 1.4 x 10(8) cells of M. tuberculosis H37Rv was retained. Retreatment with oil of these nonimmunogenic products restored the immunogenicity if the oil was applied to dried products. The consistent finding that moisture interferes with the enhancement of the vaccine potency by oil suggested that such enhancement may not be the same as that ordinarily produced by water-in-oil emulsions.     

Effects of pretreatment of mice with BCG cell walls in saline on subsequent vaccination with BCG oil droplet vaccine

Multiple intravenous injections of bacillus Calmette-Guérin cell walls (BCG CW) suspended in Tween-saline into mice produced an unresponsive state to a subsequent vaccination with BCG-CW-oil droplet mixture. This was manifest by loss of delayed hypersensitivity to PPD as measured by footpad swelling, a diminished granulomatous response in pulmonary tissue, and almost complete abrogation of protection to an aerosol challenge with virulent Mycobacterium tuberculosis, strain H37Rv. The unresponsive state disappeared 20 days after stopping pretreatment. There was evidence of immunity early after H37Rv challenge, but by 4 wk it had markedly diminished and by 6 wk there was no difference between nonvaccinated animals and the pretreated vaccinated animals. Unresponsiveness was specific since pretreated mice were capable of developing delayed type reaction to immunization with modified sheep red blood cells in the usual manner. Relationships among delayed hypersensitivity, granulomatous inflammation, and protection against aerosol infection with virulent M. tuberculosis are discussed in the light of these findings.