The effect of hydrocortisone and cyclosporin A on bacillus Calmette-Guérin epitheloid cell granulomas

The injection of bacillus Calmette-Guérin (BCG) intradermally into the ear of guinea pigs leads to the formation in the draining lymph node of granulomas containing epithelioid cells with rough endoplasmic reticulum (RER) and an absence of phagocytosed material. BCG granulomas in hydrocortisone- or cyclosporin A (CsA)-treated animals contain mononuclear phagocytes with no RER. In CsA-treated animals, these cells contain fragments of phagocytosed organisms. CsA was given at two doses, 25 mg/kg orally and 50 mg/kg ip. The higher dose completely suppressed the delayed hypersensitivity (DH) response to purified protein derivative (PPD) but the lower dose did not affect the level of the DH response, but had a profound effect on epithelioid cell formation. The role of lymphokines in the maturation of the monocyte/macrophage to epithelioid cells is discussed.     

Eradication of microscopic lymph node metastases of the guinea pig line-10 tumor after intradermal injection of endotoxin plus mycobacterial components

Summary Non-viable microbial agents were used to treat lymph node metastases of the line-10 hepatocarcinoma in strain two guinea pigs. Oil droplet vaccines were administered by intradermal injection adjacent to the site of dermal tumors. The primary tumors were removed surgically before or after immunotherapy. Control animals, treated with surgery alone, died of metastatic tumor growth. The mycobacterial glycolipid, P₃, plus polysaccharide deficient endotoxin (Re Et) eliminated lymph node metastases when the primary tumors were excised 7 days or 1 day after immunotherapy. The combination of P₃, BCG cell wall skeleton and Re Et was also effective when there was an interval of 1 or 7 days between immunotherapy and surgery. In addition, this combination retarded, and in some experiments, eliminated metastatic tumor growth in animals given immunotherapy immediately prior to surgery and in animals given immunotherapy 2 days after surgery.     

The immunoproliferative response of mice to intravenously or subcutaneously injected BCG

Summary Intravenous injection of BCG caused (1) a transient thymic epithelial hyperplasia with increase of PAS-positive cells in the cortex and medulla which showed the pronounced secretory activity of a substance which could be histochemically identified as an acid mucopolysaccharide; (2) an equally transient increase in the number of pyroninophilic lymphocytes with increased polyribosome content of the cells and mitoses in the thymic cortex; this reached a peak on day 6 following the injection but was unassociated with an increase in thymic weight; and (3) a systemic granulomatous histiocytic reaction in the liver, spleen, lungs, and lymph nodes, but not in the thymus, bone marrow, or Peyer's patches. The significance of the thymic epithelial changes is not clear but it did coincide with increased pyroninophilia and mitotic activity of the thymic cortical cells, suggesting a possible interaction between this secretory product and the thymic cortex. Comparing the thymic changes with the thymus of other animals of the same species injected with i.v. or i.p. LPS, i.v. or s.c. HIU II fraction of BCG, i.v. pertussis vaccine, i.p. complete or incomplete Freund's adjuvant, and killed at the same planned intervals after the injection of the adjuvants, BCG proved to have a unique action on the thymus with regard to both lymphocytic and epithelial changes. Hepatic, pulmonary, and splenic histiocytic granulomas were observed only in those animals injected intravenously with BCG.     

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

Summary 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. Abbreviations used in this paper: BCG, Bacillus Calmette-Guérin; i.a., intraarterially; i.d., intradermally; i.v., intravenously; SDA, superficial distal axillary.     

The safety of a new chemical typhoid preparation containing a complex of Vi and K antigens in tests of its acute and chronic toxicity]

Acute and chronic toxicity of a new chemical typhoid preparation containing the complex of surface Vi- and K-antigens has been studied. The study has revealed that the preparation, when introduced subcutaneously and intravenously in immunizing doses in a single injection or in multiple injections, produces no toxic effect on the organs and tissues of experimental animals. In experiments of chronic toxicity the microscopic study has shown pronounced hyperplasia of the lymphoid system with enhanced macrophage reaction in the spleen, thymus, mesenteric lymph nodes and Peyer's patches in the small intestine.     

Intravesical BCG administration in the guinea pig

Intravesical BCG administration is used as an adjuvant therapy after transurethral resection for superficial bladder cancer in man. The mechanisms of its antitumor activity are not known. The aim of this study was to characterize the histomorphological changes in various organs of the guinea pig after intravesical BCG administration. The BCG preparation used was BCG-RIVM, a Dutch BCG preparation. Instillations were performed in previously undamaged bladders weekly for 6 consecutive weeks and lasted 30 min or 1 h. Different doses were used ranging from 10³ culturable particles (c.p.) to 5 × 10⁷ c.p. of BCG. After 6 weeks, the animals were killed and postmortem examination was performed. The bladder wall, retroperitoneal lymph nodes, spleen, liver, lungs and distant lymph nodes were examined histologically. The BCG therapy, with a dose of 10₆ culturable particles and higher, induced an inflammatory reaction consisting of mononuclear infiltrates in the subepithelial tissue of the bladder wall. In approximately 50% of the animals investigated, the infiltrates were accompanied by noncaseating granulomatous lesions indicated by the presence of epithelioid cells. In general, the epithelial layer of the bladder showed no visible alterations. Similarly, a granulomatous inflammatory reaction was observed in the first retroperitoneal (iliac) lymph nodes draining the bladder. Granulomatous lesions were occasionally also present in liver and lung. In three of the 29 animals investigated, lesions were present both in liver and lungs, and in two of these three animals a granulomatous reaction was observed in the spleen and distant lymph nodes indicating a generalized inflammatory response induced by BCG. No microorganisms were detected by Ziehl-Neelsen (ZN) staining or culture in L?wenstein-Jensen medium in the first draining (iliac) lymph nodes of the bladder or in the spleen. In this study we found that BCG could induce inflammatory reactions in the bladder wall after its introduction into the previously undamaged bladder. Ulceration of the epithelium covering the mononuclear infiltrates was not observed. Occasionally a generalized inflammatory response to BCG was present in the animals investigated.     

The effects of intravenous administration of Methanol Extraction Residue (MER) of tubercle bacilli in the dog

Summary The effects of intravenous administration of the insoluble methanol extraction residue (MER) of tubercle bacilli were studied in six adult Beagle dogs. All animals were closely monitored for clinical signs, alterations in serum biochemistry or hematologic values, and for gross and histologic findings at autopsy. Immune responsiveness was assessed by skin reactivity to test antigens and by ability of spleen and lymph node lymphocytes to produce macrophage activating factor (MAF).Regardless of the dose of MER or frequency of administration, there were no clinical or biochemical alterations detected in the animals. Immediately after injection of MER, a massive but transient granulocytopenia appeared. Skin reactivity failed to demonstrate any alterations in immune responsiveness, but lymphocytes from MER-treated dogs released MAF in response to in vitro stimulation with purified protein derivative (PPD) in contrast to those from nontreated control dogs.Histologic findings were associated with the immune response to MER and were most dramatic in the liver and lungs, where formation of a large number of epithelioid granulomas was observed. MER, intravenously administered in relatively high doses, was well tolerated by dogs, in whom it produced little evidence of toxicity, and seemed to increase some systemic immunologic effects.     

Experimental screening of BCG preparations produced for cancer immunotherapy: Safety and immunostimulating and antitumor activity of four consecutively produced batches

Summary Four consecutively produced batches of Bacillus Calmette-Guérin (BCG) especially intended to be used for cancer immunotherapy were investigated for consistency of the vaccine. Each batch was investigated directly after production of the vaccine, so that the four batches were not tested simultaneously. The activity of the four batches was investigated in general safety assays, immunostimulation assays, and two different tumor models.General safety assays showed dose-dependent growth retardation and increased serum glutamic pyruvic transaminase activity in mice, and a long-lasting temperature rise in rabbits after IV administration of the BCG preparations. In a skin reactivity assay, reactions were found acceptable for all preparations when compared with a reference batch.The results of the immunostimulation and antitumor studies can be summarized as follows. All four batches induced a specific delayed-type hypersensitivity reaction to PPD, indicating the induction of cell-mediated immunity. A stimulating effect on lymphoreticular organs was concluded from increased spleen weight and enhanced cell proliferation in draining lymph nodes. Enhanced macrophage function (phagocytosis and killing of bacteria) was demonstrated by an increased resistance to Listeria monocytogenes. YAC lymphoma target cells were killed nonspecifically by BCG-activated peritoneal exudate cells (PEC), indicating the induction of natural killing activity by BCG.Intralesional injection of BCG induced tumor regression in the guinea pig line 10 hepatocellular carcinoma, followed by immunity to the line 10 tumor. In the murine 5D04 squamous cell carcinoma, BCG had no effect on the primary tumor. However, IV-injected BCG resulted in a decreased number of lung metastases.In general, the four consecutively produced batches showed similar safety and activity in the immunostimulation assays and antitumor activity. Since only minor differences between the batches were found, which can also be attributed to the variation in experimental conditions common to biological assays, it is concluded that the vaccine batches produced show an acceptable consistency.Abbreviations used BCG, Bacillus Calmette-Guérin; C. parvum, Corynebacterium parvum; c. p., culturable particles; IU, international unit; PPD, purified protein derivative; PEC, peritoneal exudate cells     

Intravesical BCG administration in the guinea pig. A histomorphological study

Intravesical BCG administration is used as an adjuvant therapy after transurethral resection for superficial bladder cancer in man. The mechanisms of its antitumor activity are not known. The aim of this study was to characterize the histomorphological changes in various organs of the guinea pig after intravesical BCG administration. The BCG preparation used was BCG-RIVM, a Dutch BCG preparation. Instillations were performed in previously undamaged bladders weekly for 6 consecutive weeks and lasted 30 min or 1 h. Different doses were used ranging from 10(3) culturable particles (c.p.) to 5 x 10(7) c.p. of BCG. After 6 weeks, the animals were killed and postmortem examination was performed. The bladder wall, retroperitoneal lymph nodes, spleen, liver, lungs and distant lymph nodes were examined histologically. The BCG therapy, with a dose of 10(6) culturable particles and higher, induced an inflammatory reaction consisting of mononuclear infiltrates in the subepithelial tissue of the bladder wall. In approximately 50% of the animals investigated, the infiltrates were accompanied by non-caseating granulomatous lesions indicated by the presence of epithelioid cells. In general, the epithelial layer of the bladder showed no visible alterations. Similarly, a granulomatous inflammatory reaction was observed in the first retroperitoneal (iliac) lymph nodes draining the bladder. Granulomatous lesions were occasionally also present in liver and lung. In three of the 29 animals investigated, lesions were present both in liver and lungs, and in two of these three animals a granulomatous reaction was observed in the spleen and distant lymph nodes indicating a generalized inflammatory response induced by BCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

BCG preparations,cultured homogeneously dispersed or as a surface pellicle,elicit different immunopotentiating effects but have similar antitumor activity in a murine fibrosarcoma

Summary Four preparations of Bacillus Calmette-Guérin (BCG), cultured either homogeneously dispersed or as a surface pellicle, were compared with reference to their immunomodulating capacities and antitumor effects. The BCG preparations included two vaccines that originated from the same seed strain, but one had subsequently been produced in each way. The immunological assays included in vivo stimulation of lymph nodes in a mouse model, in vitro stimulation of murine spleen lymphocytes, and in vivo stimulation of macrophages in a Listeria monocytogenes clearance model in the mouse. The antitumor effect was determined in a non-immunogenic, non-metastasizing murine fibrosarcoma. The results indicated that vaccines produced as a homogeneous culture in general induced a higher lymphocyte stimulation both in vivo and in vitro. In the Listeria clearance model a markedly enhanced clearance was established with three of the four preparations, the phenomenon being related to the number of culturable particles administered. This difference was not attributed to the production method, but to other factors, including the actual composition of the vaccine.The results found in the immunological assays were not connected to the observed antitumor activities, as for each preparation a combination of route, dose, and time interval for tumor regression was found. Prophylactic administration of BCG had no effect at all; enhancement was observed after intratumoral administration of the two preparations prepared as surface pellicles.It is concluded that a protocol for the quality control of bacterial vaccines used for cancer immunotherapy should include both immunological assays and a range of different animal tumor models.Fellow of the Koningin Wilhelmina Fonds of the National Cancer League of the Netherlands The Abbreviations used are: BCG, Bacillus Calmette-Guérine; c.p., culturable particles; Con A, Concanavalin A; MEM, Minimum Essential Medium; FCS, fetal calf serum; cpm, counts per minute; IT, intratumoral     

Induction of experimental diabetes in frog (author's transl)]

The effect of diabetogenic agents, alloxan and streptozotocin, in frogs has been studied. These drugs were administered to the animals by injection into the dorsal lymph sac. Alloxan did not exert any effect at non-lethal doses. At 300 mg/kg alloxan caused death of most of the animals in an hyperglycemic state in less than 72 hours. Streptozotocin at doses lower than 1 g/kg was ineffective. At 1.5 g/kg, a non-lethal dose, about half of the animals became diabetic.     

The immunopathological effects of intracolonic injection of Mycobacterium bovis BCG cell wall emulsions in guinea pigs

Summary The immunological and pathological responses of guinea pigs to an intramural colonic injection of emulsions containing cell wall (CW) extracts of Mycobacterium bovis BCG and mineral oil were studied from week 1 to week 36 post-inoculation. The emulsions contained variable concentrations of BCG CW attached to (lipid-phase), or separate from (aqueous-phase), the mineral oil. Delayed cutaneous hypersensitivity to PPD was present throughout the course of the study in a variable percentage of guinea pigs inoculated with either type of emulsion. PPD-induced blast transformation of peripheral blood lymphocytes, studied in guinea pigs which received lipid-phase emulsion, was also detectable throughout the course of the study, with maximal response seen 2 weeks post-inoculation. The intracolonic inoculations were well tolerated, with the exception of the most concentrated lipid-phase emulsion (3 mg/ml BCG CW and 5% oil), after which one of eight guinea pigs died due to a colonic impaction and rupture at the site of inoculation. The pathological response to either type of emulsion was a focal granulomatous colitis, which tended to be more severe as the concentration of BCG CW and oil increased. Extracolonic lesions were usually limited to a granulomatous lymphadenitis of lymph nodes draining the injection site; however, the most concentrated lipid-phase emulsion occasionally produced granulomatous inflammatory foci in the liver and lungs. In general, the lesions induced by the lipid-phase emulsions were more severe than those induced by aqueous-phase emulsions, but the intensity of both types of lesions peaked at 2 or 4 weeks post-inoculation. It was concluded that the guinea pig may serve as a useful model to study BCG immunotherapy of colonic neoplasms, since intracolonic injection of BCG CW resulted in systemic immunity toward mycobacterial antigens and a localized accumulation of macrophages without untoward complications. The abbreviations used in this paper are: BCG CW, bacillus Calmette-Guérin cell walls; PPD, purified protein derivative; PBL, peripheral blood lymphocytes; cpm, counts per minute; SI, stimulation index; DCH, delayed cutaneous hypersensitivity     

Studies on the mechanisms of action of BCG

Summary Bacillus Calmette-Guérin was given intravenously (i.v.) or subcutaneously (s.c.), 14 days before antigenic or mitogenic stimulation.When given i.v., BCG increased plaque-forming cell production against thymus-dependent antigens (sheep red blood cells and a hapten coupled to hemocyanin) and against a thymus-independent antigen (the same hapten coupled to polymerized flagellin). BCG given s.c. was generally ineffective in potentiating humoral responses.In contrast, a marked depression of spleen cell reactivity in vitro to a T-cell mitogen, concanavalin A (Con A) but an increased response to a B-cell mitogen, dextran sulfate, were observed after BCG i.v., and to a lesser extent after BCG s.c. This inhibitory effect was reversed after removal of cells adherent to nylon or plastic. Normal spleen cell reactivity to Con A was strongly depressed when the cells were cultivated in presence of unfractionated or plastic-adherent BCG-treated spleen cells, suggesting that BCG induces suppressor cells which inhibit T-cell responses in vitro. Similar impairment of T-cell functions in vivo was not observed since BCG given i.v. significantly increased the intensity of delayed-type hypersensitivity to picryl chloride.BCG also induced macrophage activation, measured by macrophage tumoricidal activity in vitro, more strongly and more rapidly when given i.v. than s.c.Modifications of the distribution of lymphocytes from the circulation to the organs were observed. An early increase of lymphocyte migration to the spleen and a long-lasting depression of cell migration to mesenteric lymph nodes and bone marrow were seen after BCG given i.v. When given s.c., BCG induced a progressive increase of cell migration to the draining lymph nodes concomitant with a depression of migration to mesenteric nodes.These results demonstrate the complexity of the mode of action of BCG, but many of its effects on immune responses may result from a primary action on macrophages.     

Degree of chromatin activity and the RNA level in lymphocytes of popliteal lymph nodes of dogs after administration of an antigen]

In the experiment performed on 127 dogs by means of cytospectrofluorometric analysis, using fluorochrome acridine orange in dynamics up to 1 year, changes in the level of chromatin activation and RNA content have been studied in lymphocytes of the germinative centers and the crown of lymphoid nodules, in the paracortical zone and medullary cords of the regional and contralateral popliteal lymph nodes, after subcutaneous injection of antigen (BCG vaccine, 0.2 mg/kg) into the lateral area of the foot of the left pelvic extremity. The immune response is accompanied with a periodical increase in the level of chromatin activation and RNA content in populations of lymphocytes in the regional and contralateral popliteal lymph nodes with maximum in 6 h, 3-7 days, 1-3 months after the antigen injection. The intensity of these processes has an unequal level in lymphoid cells of various structural components; it is higher in lymphocytes of the contralateral lymph node.     

Thymic and splenic changes following injection of living Brucella and BCG in the Guinea pig

Summary The thymic and splenic reactions, following injections of BCG and living Brucella M. in the guinea pigs, were studied. Both microorganisms injected intravenously produced thymic involution, maximal after BCG inoculation, followed by regeneration that was complete by day 10 in Brucella M.-treated guinea pigs, and by day 15, in BCG injected guinea pigs. Increase of mitotic index was more accentuated and more persistent after Brucella M. than after BCG treatment in the thymus. After a short involution period the spleen of injected animals increased in size in both groups. The splenic enlargement was dramatic and occurred at an accelerated rate in animals given BCG. It appeared to be the result of a conspicuous involvement of the red pulp by multiple granulomas. In Brucella M. treated guinea pigs the splenic enlargement was less obvious, but the splenic white pulp was more abundant in BCG-treated guinea pigs. Granulomas were observed only in the periarterial sheaths of the white pulp.These observations provide evidence for the hypothesis that injections of both BCG and Brucella M. provoke a proliferation of B and T lymphocytes, a migration of T lymphocytes from the thymus to the T-dependent area of the spleen which seems, perhaps, more marked after injection of Brucella M., and a strong granulomatous histiocytic reaction which is more conspicuous in animals given BCG.     

Equine sarcoid: BCG immunotherapy compared to cryosurgery in a prospective randomised clinical trial

Summary A total of 30 horses with single or multiple sarcoid tumors of the skin were randomly divided into three treatment groups: (i) cryosurgical treatment, (ii) intralesional immunotherapy with a live BCG vaccine, (iii) intralesional immunotherapy with a BCG cell wall preparation. Complete tumour regression was obtained in all 10 crysurgically treated horses, in 6 of 10 live BCG treated horses, and in 7 of 10 BCG cell wall treated horses. One live BCG and 2 BCG cell wall treated horses showed partial tumour regression of more than 50% of the tumour area. Eleven horses with sarcoid tumours were not eligible for random allocation in the trial because unfavourable site or size of the tumour precluded cryosurgical treatment. These animals were treated with BCG cell wall vaccine except for 1 animal, which was treated with live BCG. In 4 cases this treatment was combined with cytoreductive surgery of the tumour. In this prognostically unfavourable group 8 animals showed complete tumour regression and 3 animals did not respond.Regression after BCG immunotherapy appeared to correlate with size (larger tumours worse response) and localization of the sarcoid (less favourable results in the limb), and increase in peripheral blood leucocytes after the first injection. Horses with a positive delayed type hypersensitivity reaction to PPD before the start of treatment showed a tendency to more favourable prognosis than PPD negative horses. No correlation was present between regression and single or multiple presence of sarcoids, increase in body temperature after injection of BCG and the formation of specific antibodies to BCG. None of the cured animals have shown tumour recurrence 3 to 40 months following treatment.Animals were maintained under the guidelines prescribed by the Faculty of Veterinary Medicine, State University Utrecht, The NetherlandsGrant recipient of the Koningin Wilhelmina Fonds (Netherlands Cancer Foundation)     

Lethal infection with BCG in thymectomized mice]

Investigations carried out on CBA mice demonstrated that adult mice to which mycobacteria BCG were injected intravenously 12 months after thymectomy (in a dose of 2 mg for a period of 2 1/2 months) died of disseminated BCG infection against the background of depression of hypersensitivity of delayed type. The usual vaccine process developed in the sham-operated animals.     

Human toxicology of BCG applied in cancer immunotherapy

Summary Seventy-five patients were treated for short periods with BCG either per os (7), by aerosols (2), i.d. with needles, i.d. with heaf-gun, on one or four scarification areas, i.v., or intratumorally. Two hundred and seventy-seven were treated for more than 3 years by BCG applied scarifications.The local reactions after application on scarification are negligible, the most frequent being pruritus and adenopathies.The systemic reactions are due to the BCG septicemia which is induced and which has been proved by the search for liver granulomas. Some reactions are of an allergic nature (e.g., choroiditis), but most are direct manifestations of the septicemia (fever, hepatomegaly, splenomegaly). One death was observed after tumoral injection in a terminal patient, otherwise there were no deaths, even in the patients under long-term treatment with the other metabolites.Deterioration of immune reactions may be induced either by the method of BCG application which is followed by a (probably) very small penetration (on scarified area in allergics), or by the penetration of high doses (four scarified areas in anergics and intravenous injections in anergics and in allergics). Reprint requests should be addressed to: G. Mathé, 14-16, avenue Paul-Vaillant-Couturier, F-94800 Villejuif (France)     

Immunotherapy of breast cancer,malignant melanoma,and acute leukemia with BCG: Prolongation of disease free interval and survival

Summary Results of immunotherapy with BCG in patients with malignant melanoma, breast cancer, and acute leukemia are described. The first study demonstrated that high doses of living BCG organisms (6×10⁸ viable units) delivered by scarification in the upper arms and legs prolonged the disease-free interval and survival of 52 malignant melanoma patients with regional lymph node metastases compared to 218 comparable surgical control patients. Patients with trunk and extremity, but not head and neck melanoma, benefited from BCG, suggesting the importance of the delivery of BCG into the tumor-involved lymphatics.The second study evaluated the therapeutic efficacy of living BCG organisms by scarification in a group of adult acute leukemia patients after the cessation of chemotherapy. Thirty-seven patients had been in remission on intermittent chemotherapy for 12–24 months. Following late intensive consolidation chemotherapy, 7 consecutive patients received no further therapy and then 30 consecutive patients received BCG. Patients maintained on BCG have had a prolonged disease-free interval compared to those given on no further therapy (P=0.07) or compared to a group of similar patients maintained on chemotherapy alone (P=0.001). Similarly, the survival has been improved for patients maintained on BCG compared to those left unmaintained (P=0.009), or those maintained on chemotherapy (P=0.001).The principles of intermittent chemotherapy combined with BCG immunotherapy, first developed in patients with disseminated melanoma and acute myelogenous leukemia, have been confirmed in a series of patients with disseminated breast cancer. Forty-five patients treated with a combination of 5-FU, adriamycin, and cyclophosphamide (FAC) plus BCG by scarification showed prolongation of remission as well as survival compared to a comparable group of 44 patients treated with FAC chemotherapy without immunotherapy. Thus, 23/44 patients treated with FAC have died (median=14 months) compared to only 5/45 patients on FAC-BCG (median=12⁺ months), P=0.005. The limitations of BCG immunotherapy as well as speculations for future developments of immunotherapy are discussed.This work was supported by Contract No1-CB 33888 from the National Institutes of Health, Public Health Service, Bethesda, Maryland 20014. Drs. Gutterman and Mavligit are the recipients of Career Development Awards (Ca 71007-02 and CA 00130-01, respectively) from the National Institutes of Health, Education, and Welfare, Bethesda, Maryland 20014.     

Induction of delayed-type hypersensitivity and antitumor immunity by systemic BCG

Both delayed-type hypersensitivity (DTH) and antitumor resistance induced in mice by intravenous (i.v.) and local injection of highly immunogenic irradiated Meth A cells were potentiated by prior systemic BCG infection. DTH and antitumor immunity were not elicited by i.v. injection of poorly immunogenic irradiated mastocytoma cells, P 815 (MA), but were induced by the local injection of these cells when animals were systemically infected with BCG. The level of the potentiated response corresponded with the dose of immunogen up to an optimum, beyond which additional immunogen was suppressive. At all dose levels the subcutaneous (s.c.) route of immunogen inoculation was more effective than the i.v. route. Significant DTH was first detected 7 days after the local administration of immunogen and was correlated with antitumor immunity. Systemically administered BCG grew mainly in the liver and spleen until the development of maximal tuberculin sensitivity when the number of organisms decreased. However, the small number of mycobacteria that reached the peripheral lymph nodes remained constant after maximal tuberculin sensitivity but failed to augment the cell proliferation that occurred in these lymph nodes as a result of the local inoculation of irradiated tumor cells. Autoradiographs of such nodes revealed proliferation in the thymus-dependent areas whereas nodes from mice immunized with immunogen alone manifested B- as well as T-cell activity. Local immunization in both BCG-infected and uninfected hosts was also associated with a proliferative response in the red pulp of the spleen but the BCG-infected hosts differed conspicuously by virtue of the presence of tubercles and depletion of lymphoid cells from the periarteriolar sheath. Immunity generated by the local administration of immunogen in systemically infected mice was tumor specific and could be adoptively transferred with spleen cells.