Histological evaluation of immunologically mediated tumor regression of the line 10 guinea pig hepatocarcinoma

The histology of immunologically mediated tumor regression was studied in the syngenic strain 2 guinea pig/line 10 hepatocellular carcinoma tumor system. Tumor regression was induced non-specifically by the intralesional injection of living Bacillus Calmette-Guérin (BCG) in 7-day-old established tumors (diameter 8-10 mm). In untreated line 10 tumors at day 7 a mild to moderate inflammatory reaction was present, which consisted mainly of small mononuclear cells; in addition large mononuclear cells and basophils were present. Intratumoral BCG-treatment induced a prominent increase in the inflammatory reaction due to an influx of small and large mononuclear cells and neutrophils. Small mononuclear cells were identified mainly as lymphocytes whereas large mononuclear cells belonged mainly to the macrophage line. Intratumoral administration of BCG resulted in a granulomatous reaction. A time-related decrease in the number of tumor cells and an increase in inflammation, associated with purulent lysis of the granulomatous tissue, was observed. Specific immune-mediated tumor rejection occurred in animals both after active immunization and after adoptive transfer of immune spleen cells. In actively immunized animals the tumor cells were rapidly rejected and from day 4 onwards no tumor cells could be detected at the injection site. Lymphocytes were the major component of the inflammatory reaction; large mononuclear cells were present to a lesser extent and basophilic granulocytes were regularly observed. After adoptive transfer of immunity with immune spleen cells given simultaneously with an intradermal innoculation of tumor cells, an essentially similar rejection reaction was found, although tumor cell rejection was delayed. Lymphocytes and large mononuclear cells were found in equal proportions, whereas basophilic granulocytes were always present in smaller numbers. After BCG-induced regression and in adoptively transferred immune rejection, a fibroblast component was more prominent than in untreated control tumors. This reaction tended to isolate smaller tumor cell areas into islets of decreasing sizes. In contrast with the fibroblast component of growing tumors, the proliferative pre-existing fibrous tissue in tumors undergoing regression or rejection showed a loosely arranged architecture and contained a marked cellular infiltrate. From the results of the present study it was concluded that the morphological expression of line 10 tumor rejection varies. Without immune cells, BCG is needed for the induction of a local inflammatory reaction, which was granulomatous in type and eventually led to complete tumor cell eradication.(ABSTRACT TRUNCATED AT 400 WORDS)     

Low-density lipoprotein modification by normal,myeloperoxidase-deficient and NADPH oxidase-deficient granulocytes and the impact of redox active transition metal ions

The modification of low-density lipoprotein (LDL) by normal, myeloperoxidase (MPO)-deficient and NADPH oxidase-deficient granulocytes was investigated using the monoclonal antibody (mAb) OB/04, which was originally generated against copper-oxidized LDL. Incubation of LDL with normal granulocytes increased the reactivity of LDL with mAb OB/04. These effects were even more pronounced using MPO-deficient granulocytes. Inhibitors of oxidative reactions (the NADPH oxidase inhibitor diphenyleneiodonium chloride [DPI], catalase, superoxide dismutase [SOD]) did not significantly reduce LDL oxidation by normal granulocytes. Furthermore, granulocytes of a patient with NADPH oxidase deficiency were almost equally effective as normal granulocytes, indicating that oxidative burst-derived reactive oxygen species are of only minor importance in the generation of mAb OB/04-detectable new epitopes on LDL in vitro. In contrast, incubation of LDL with iron and copper prior to and during incubation with normal granulocytes markedly enhanced the generation of OB/04-detectable epitopes. It is supposed that, besides superoxide (in normal and MPO-deficient granulocytes) or instead of superoxide (in NADPH oxidase-deficient granulocytes), lytic enzymes released by activated granulocytes may enhance the availability of transition metals for oxidation of LDL. Our results support the concept that transition-metal-dependent pathways of LDL oxidation in combination with degranulation products of granulocytes are important.     

Efficacy of BCG presensitization in combination with cyclophosphamide (CY) on murine tumor growth and immunity

Summary The effect of BCG in combination with cyclophosphamide (CY) was studied in a solid tumor system in syngeneic mice. There was a marked effect of intradermal (ID) presensitization with BCG (10⁷ organisms) followed by a single intraperitoneal (IP) injection of CY (200 mg/kg) one day after the tumor inoculation on tumor suppression and tumor immunity, while there was no effect by either BCG or CY treatment alone. The optimal interval between BCG sensitization and tumor inoculation seemed to be about 3 weeks.Research supported in part by Grant-in-Aid Cancer Research from the Ministry of Health and Welfare, Japan     

Mouse-strain difference in immunoprophylactic and immunotherapeutic effects of BCG on carcinogen-induced autochthonous tumors

The prophylactic and therapeutic effects of BCG on the tumors induced by 3-methylcholanthrene (MCA) were studied comparatively between two inbred mouse strains, SWM/Ms and C3H/He, the first tumor appeared 5 weeks after MCA and the cumulative tumor incidence reached almost 100% within 20 weeks. On the other hand, the first tumor appeared 8 weeks after MCA in SWM/Ms, the number of tumor-bearers increased more slowly than in C3H/He, and the final tumor incidence (at 30 weeks) was about 90-80%. Single subcutaneous injection with BCG 2 weeks prior to MCA significantly protected SWM/Ms from tumor development, but not in C3H/He. These tumors, once appeared grew progressively and killed the hosts equally in both the strains. Intratumor (i.t.) injection with BCG showed more or less therapeutic effects in SWM/Ms; most tumors regressed or retarded after BCG. The time period after tumor-appearance to tumor-death was prolonged in most of SWM/Ms mice given i.t. injection with BCG, except a few mice that died earlier than non-treated controls after BCG. Contrary, no therapeutic effect of i.t. injection with BCG was observed in C3H/He. different host responses to BCG between SWM/Ms and C3H/He were found by the peritoneal macrophage disappearance test and the footpad reaction test; SWM/Ms was a high-responder to BCG and C3H/He was a low-responder. The marked differences between SWM/Ms and C3H/He in prophylactic and therapeutic effects of BCG on the autochthonous tumors were discussed in terms of difference of the host immune response to BCG that is defined genetically.     

A new side effect of BCG immunotherapy — BCG-induced arthritis in man

Summary Ten of 159 patients showed arthritic symptoms during the course of BCG immunotherapy for advanced cancer. The arthritic symptoms occurring after BCG injections had the following characteristics: (1) The incidence of arthritis was closely correlated with the host immunologic responsiveness to BCG; (2) These symptoms usually occurred 1–5 months after the first BCG injection (7/10); (3) The arthritic symptoms usually started with morning stiffness (9/10), which was followed by acute inflammatory signs in the affected joints. They gradually subsided in response to treatment with nonsteroid antiinflammatory drugs, but were not completely cured while the effectiveness of BCG continued; (4) The symptoms were aggravated by additional BCG injections (8/10). (5) This form of arthritis could be differentiated from rheumatoid arthritis, tuberculous, or purulent arthritis by its clinical course and by roentgenograms of the affected joints. It is thought to be induced by the adjuvant effect of BCG, and is a new side effect of BCG immunotherapy.     

Localization and expression of inducible nitric oxide synthase in patients after BCG treatment for bladder cancer

Treatment with Bacillus Calmette Guerin (BCG) bladder instillations is an established treatment modality for superficial urinary bladder cancer and carcinoma in situ (CIS), but the anti-tumor mechanisms following BCG instillations remain largely unknown. Previous data show increased nitric oxide (NO) concentrations in the urinary bladder from patients treated with BCG suggesting that NO-formation may be involved in the BCG mediated effect. In the present study we evaluated 11 patients with urinary bladder cancer who had received BCG treatment and 11 tumor free control subjects. We performed immunohistochemistry, Western blot and real-time polymerase chain reaction (PCR) on bladder biopsies to establish inducible nitric oxide synthase (iNOS) protein levels and localization as well as iNOS mRNA expression. Endogenous NO formation in the bladder was also measured. In patients with bladder cancer who had received BCG treatment iNOS-like immunoreactivity was found in the urothelial cells but also in macrophages in the submucosa. Furthermore, endogenously formed NO was significantly increased (p<0.001) in the BCG treated patients and they had a ten-fold increase in mRNA expression for iNOS compared to healthy controls (p=0.003). In conclusion iNOS was found to be localized to the urothelium and macrophages underlying it. Our study also confirms elevated levels of endogenously formed NO and increased mRNA expression and protein levels for iNOS in patients with BCG treated bladder cancer. These data further support the notion that NO may be involved in the anti-tumor mechanism that BCG exerts on bladder cancer cells.     

Differential endocytosis of fluorescein iso-thiocyanate-concanavalin A by normal and chronic myeloid leukemic granulocytes

Isolated granulocytes from normal individuals and patients suffering from chronic myeloid leukemia (CML) displayed different fluorescent patterns on treatment with fluorescein isothiocyanate concanavalin A (Fl-Con A). The ligand was internalized by 86% of the normal granulocytes, while 80% of the leukemic granulocytes exhibited Fl-Con A localized on the cell periphery. In further experiments, pretreatment of the normal granulocytes with cytochalasin B, iodoacetamide, 2-deoxyglucose and sodium fluoride (but not with sodium azide or dinitrophenol) was found to drastically inhibit internalization of the ligand. However, pretreatment of granulocytes from CML patients with cytochalasin B and 2-deoxyglucose, caused only a little alteration in the pattern of Fl-Con A labelling relative to untreated cells. These results indicate that CML granulocytes are defective in their ability to endocytose Fl-Con A. We suggest that this differential interaction between Fl-Con A and normal and leukemic granulocytes is a convenient system to study the initial steps in receptor mediated endocytosis of Concanavalin A.     

3-Aminobenzamide inhibits cytotoxicity and adhesion of phorbol-ester-stimulated granulocytes to fibroblast monolayer cultures

Damage of 3T3 fibroblasts as induced by short-term co-cultivation with O2(-)-producing granulocytes, stimulated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), was compared with that induced by treatment with enzymically generated O2- and with the alkylating agent dimethyl sulfate. The action of stimulated granulocytes was different in several aspects: (a) DNA fragmented by the products of TPA-stimulated granulocytes showed a biphasic alkaline elution pattern while fragmentation induced by alkylation or by enzymically produced O2- was monophasic. (b) Poly(ADP-ribosyl)ation of nuclear proteins after treatment with TPA-stimulated granulocytes exhibited a lag phase and was, in most experiments, less pronounced than after equitoxic dimethyl sulfate treatment. (c) 3-Aminobenzamide, the most widely used inhibitor of ADP-ribosylation, partially protected target cells from the cytotoxic effects of TPA-stimulated granulocytes, while it enhanced alkylation-induced and O2(-)-induced cytotoxicity. Protection by 3-aminobenzamide in the granulocyte system was apparently not mediated by an inhibition of nuclear poly(ADP-ribosyl)ation. Other inhibitors, like benzamide and nicotinamide, augmented cytotoxicity of TPA-stimulated granulocytes. The unique effect of 3-aminobenzamide in this system appeared to relate to TPA-induced adhesion of the neutrophils to surfaces. In the presence of 1 mM 3-aminobenzamide, but not of benzamide, the adhesion of stimulated granulocytes to 3T3 monolayer cultures was markedly reduced or even abolished. This effect was also seen in granulocyte preparations depleted of monocytes. Since 3-aminobenzamide at the doses applied does not inhibit TPA-induced superoxide production in isolated granulocytes, its specific anticytotoxic effect appears to result from a 'dilution' of granulocyte-derived damaging agents into the medium. Our data suggest that prevention of granulocyte adhesion is likely to reduce tissue damage and carcinogenesis in areas of chronic inflammation.     

Effects of BCG (Bacillus Calmette–Guérin) Vaccines on Immune Responses in Mice

The effects of killed and living BCG on antibody production against hamster erythrocytes (HRBC) and the 2, 4, 6-trinitrophenyl (TNP) group were studied in SL mice. Killed and living BCG, each in doses of 0.008 mg, 0.08 mg, 0.8 mg and 8 mg per mouse, were intravenously inoculated 7 days prior to primary immunization with HRBC. Secondary immunization was carried out 28 days later with TNP-HRBC. Anti-HRBC and anti-TNP antibodies were estimated by a hemagglutination test. The results showed that pretreatment with killed or living BCG enhanced the antibody production against both HRBC and TNP. Comparing the effects of these two BCG preparations, it was noted that killed BCG augmented the anti-HRBC antibody production more effectively than living BCG. In regard to the anti-TNP antibody production, living BCG exhibited a greater augmenting effect than killed BCG. This difference in the modes of action of killed and living BCG was remarkable when two groups given 8 mg of killed and living BCG were compared. In addition, it was shown that living BCG at a dose as high as 8 mg was able to augment the anti-TNP antibody production, even in the absence of preceding immunization with HRBC.     

Presence of activated lymphocytes in the urine of patients with superficial bladder cancer after intravesical immunotherapy with bacillus Calmette-Guérin

Summary To study the mode of action of intravesical bacillus Calmette-Guérin (BCG) immunotherapy in the prevention and cure of superficial bladder cancer, flow-cytofluorometric analysis of the cellular immunological reaction in the urine of patients was performed. Fresh urinederived leucocytes were obtained from eight patients before (t ₀) and 24 h (t ₂₄) and 48 h (t ₄₈) after repeated intravesical BCG instillations (at least 5 instillations). For two patients urine-derived leucocytes were investigated at the first BCG instillation. The number of leucocytes in the urine was markedly increased 24 h after repeated BCG instillations, indicating a local cellular immunological reaction induced by BCG. The mean number of cells per milliliter of urine at that time was 2.9×10⁶±3.6×10⁶ (n = 8). These leucocytes consisted mainly of granulocytes (75±11%,n = 8). In addition monocytes/macrophages (4±2%,n = 8) and T lymphocytes were present (1±1%,n = 5). The relative increase of monocytes/macrophages in the urine after BCG application tended to be higher compared to the other leucocyte subtypes. As T lymphocytes may play an important role in the BCG-mediated antitumour activity, subsets of lymphocytes were further characterized att ₀,t ₂₄, andt ₄₈ after repeated BCG instillations. The lymphocyte population consisted mainly of T cells (86% CD3⁺,t ₀). Most of the T cells were CD4⁺ (helper/inducer) and were significantly decreased at 48 h (62±9% att ₀ vs 49±6% att ₄₈). Lymphocytes partly expressed HLA-DR antigens (44%,t ₀). The percentage of lymphocytes with interleukin-2 (IL-2) receptors (CD25⁺) was significantly increased at 24 h and 48 h, compared to pre-instillation values (19±11% and 10±4% vs 3±3% respectively). Natural killer cells (CD 16⁺ and/or CD56⁺) and B cells (CD 19⁺) were less numerous (10% and 19% att ₀ respectively). After the first BCG instillation the increase in the number of leucocytes in urine seemed to be less compared to the numbers after repeated BCG instillations. Lymphocytes could not be detected in the urine collected before or after the first BCG instillation. In conclusion, we demonstrated the presence of considerable numbers of leucocytes in the urine 24 h after repeated BCG instillations, i.e. shortly after immunological activation. The antigen expression of the lymphocytes suggested that they may represent the lymphocytes in the bladder wall. Expression of HLA-DR and IL-2 receptors on lymphocytes indicated activation of T cells by the intravesical BCG treatment. These leucocytes may be useful for functional studies, which are essential to elucidate the actual effector mechanism(s) in the mode of action of BCG against superficial bladder cancer in man.     

Glycosphingolipid patterns of peripheral blood lymphocytes, monocytes, and granulocytes are cell specific

We analyzed the amounts and types of glycosphingolipids (GSLs) from peripheral blood lymphocytes, monocytes, and granulocytes isolated by counter-current elutriation. The three cell types contained different amounts of neutral and acidic GSLs. The highest amount of neutral GSLs (109 micrograms/10(8) cells) was found in granulocytes, with considerably less found in monocytes (11 micrograms/10(8) cells) and lymphocytes (4 micrograms/10(8) cells). The neutral GSLs were composed of four types of lipids, GL1 through GL4 (mono-, di-, tri-, and tetraosylceramide). The highest percentage of GL1 was detected in lymphocytes and the lowest percentage in granulocytes, with the reverse order observed for GL2. GL3 and GL4, which were minor components of the neutral GSLs, were highly cell specific, with lymphocytes containing GL3 and GL4 of the globo series, granulocytes containing GL3 and GL4 of the lacto or neolacto series, and monocytes containing GL3 and GL4 of both types. The acidic GSL, sialosyl hexaosylceramide (lacto-series), was abundant in granulocytes but not in monocytes or lymphocytes. Another ganglioside, GM3, although present in all three cell types, was most abundant in monocytes and lymphocytes, whereas sialosyl paragloboside was higher in granulocytes than in lymphocytes and monocytes. These results indicate that peripheral blood lymphocytes, monocytes, and granulocytes have distinct "GSL fingerprints."     

Shared antigens between human malignant melanoma cells and Mycobacterium bovis (BCG).

This study was undertaken to investigate the antigenic relationships between human malignant melanoma cells and Mycobacterium bovis (BCG). Rabbits were immunized with sonicates of BCG or with malignant melanoma cells from different patients and the resulting antisera were tested for their capacity to bind radiolabeled soluble extracts prepared from BCG and melanoma cells. The binding of antibodies to radiolabeled antigens was studied by precipitation of radiolabeled antigen-antibody complexes by anti-rabbit immunoglobulin. Antibodies in sera from rabbits immunized with either BCG (anti-BCG) or melanoma cells (anti-melanoma) bound both the labeled BCG and melanoma antigens. Control antisera, from rabbits immunized with human acute or chronic lymphatic leukemia cells or with normal human spleen cells, did not bind significant amounts of radiolabeled BCG. Antibodies in sera from rabbits immunized with normal spleen cells bound small but significant amounts of radiolabeled melanoma antigens. Binding by anti-BCG and anti-melanoma to the radiolabeled antigens was studied before and after absorption of antisera with cells from human melanoma, leukemia, guinea pig hepatoma, and normal human spleen cells. Inhibition studies using unlabeled BCG extracts also were carried out. The absorption and inhibition studies confirmed that the binding reactions were specific and that antigens from five melanoma patients shared antigenic determinants with BCG.     

The enzyme cytochemistry and composition of elasmobranch granulocytes

Peroxidase, alkaline phosphatase, acid phosphatase, β-glucuronidase, α-naphthyl acetate esterase (ANAE), α-naphthyl butyrate esterase, naphthol AS-D chloroacetate esterase, acetyl-L-tyrosine-α-naphthyl esterase (ATNE), tosyl-L-lysine-α-naphthyl esterase (TLNE) and periodic acid-Schiff (PAS) were studied in 17 species of elasmobranchs in which granulocytes had previously been identified at the ultrastructural level.
Eosinophils, eosinophilic and neutrophilic granulocytes contained variable acid phosphatase, esterases and PAS, but they were strongest in neutrophilic granulocytes; particularly ANAE. Esterases were released into surrounding plasma and therefore probably function as ectoenzymes. In eosinophils and some neutrophilic granulocytes there were indications of weak peroxidase, but this could not be conclusively demonstrated. Alkaline phosphatase was diffuse between granules in some eosinophils of Pavoraja , and (β-glucuronidase was diffuse in neutrophilic granulocytes of Etmopterus baxteri , otherwise granulocytes lacked these enzymes. Neutrophilic granulocytes stained moderately to strongly for ATNE and weakly and inconsistently for TLNE in Squalus acanthias and Dalatias licha . with a similar reaction in granular lymphocytoid and thrombocytoid cells of Galeorhinus ausiralis and Raja nasuta . The enzyme composition of these granulocytes is discussed.     

Clinical latency and reactivation of AIDS-related mycobacterial infections

The immune mechanisms associated with the evolution from latent to clinically active mycobacterial coinfection in human immunodeficiency virus type 1 (HIV-1)-infected humans remain poorly understood. Previous work has demonstrated that macaques infected with simian immunodeficiency virus (SIVmac) can develop persistent Mycobacterium bovis BCG coinfection and a fatal SIV-related tuberculosis-like disease by 4 months after BCG inoculation. In the present study, SIVmac-infected monkeys that developed clinically quiescent mycobacterial infection after BCG inoculation were followed prospectively for the reactivation of the BCG and the development of SIV-related tuberculosis-like disease. The development of clinically latent BCG coinfection in these SIVmac-infected monkeys was characterized by a change from high to undetectable levels of bacterial organisms, with or without measurable BCG mRNA expression in lymph node cells. The reactivation of clinically latent BCG coinfection and development of SIV-related tuberculosis-like disease were then observed in these SIVmac-BCG-coinfected monkeys during a 21-month period of follow-up. The reactivation of SIV-related tuberculosis-like disease in these animals coincided with a severe depletion of CD4 T cells and a loss of BCG-specific T-cell responses. Interestingly, bacterial superantigen challenge of the SIVmac-BCG-coinfected monkeys resulted in an up-regulation of clinically latent BCG coinfection, suggesting that infection with superantigen-producing microbes may increase the susceptibility of individuals to the reactivation of AIDS-related mycobacterial coinfection. Thus, reactivation of latent mycobacterial infections in HIV-1-infected individuals may result from a loss of T-cell immunity or from a superimposed further compromise of the immune system.     

Improved protection against disseminated tuberculosis by Mycobacterium bovis bacillus Calmette-Guerin secreting murine GM-CSF is associated with expansion and activation of APCs

Modulating the host-immune response by the use of recombinant vaccines is a potential strategy to improve protection against microbial pathogens. In this study, we sought to determine whether secretion of murine GM-CSF by the bacillus Calmette-Guérin (BCG) vaccine influenced protective immunity against Mycobacterium tuberculosis. BCG-derived GM-CSF stimulated the in vitro generation of functional APCs from murine bone marrow precursors, as demonstrated by the infection-induced secretion of IL-12 by differentiated APCs, and the ability of these cells to present Ag to mycobacterium-specific T cells. Mice vaccinated with BCG secreting [corrected] murine GM-CSF (BCG:GM-CSF) showed increased numbers of CD11c+MHCII+ and CD11c-CD11b+F480+ cells compared with those vaccinated with control BCG, and this effect was most apparent in the draining lymph nodes at 7 and 14 days postvaccination. Vaccination with BCG:GM-CSF also resulted in enhanced expression of costimulatory molecules on migratory dendritic cells in the draining lymph nodes. The increased APC number was associated with an increase in the frequency of anti-mycobacterial IFN-gamma-secreting T cells generated after BCG:GM-CSF vaccination compared with vaccination with control BCG, and this effect was sustained up to 17 wk in the spleens of immunized mice. Vaccination with BCG:GM-CSF resulted in an approximately 10-fold increase in protection against disseminated M. tuberculosis infection compared with control BCG. This study demonstrates the potential of BCG secreting [corrected] immunostimulatory molecules as vaccines to protect against tuberculosis and suggests BCG:GM-CSF merits further appraisal as a candidate to control M. tuberculosis infection in humans.     

The Use of Directed Evolution to Create a Stable and Immunogenic Recombinant BCG Expressing a Modified HIV-1 Gag Antigen

Numerous features make Mycobacterium bovis BCG an attractive vaccine vector for HIV. It has a good safety profile, it elicits long-lasting cellular immune responses and in addition manufacturing costs are affordable. Despite these advantages it is often difficult to express viral antigens in BCG, which results in genetic instability and low immunogenicity. The aim of this study was to generate stable recombinant BCG (rBCG) that express high levels of HIV antigens, by modification of the HIV genes. A directed evolution process was applied to recombinant mycobacteria that expressed HIV-1 Gag fused to the green fluorescent protein (GFP). Higher growth rates and increased GFP expression were selected for. Through this process a modified Gag antigen was selected. Recombinant BCG that expressed the modified Gag (BCG[pWB106] and BCG[pWB206]) were more stable, produced higher levels of antigen and grew faster than those that expressed the unmodified Gag (BCG[pWB105]). The recombinant BCG that expressed the modified HIV-1 Gag induced 2 to 3 fold higher levels of Gag-specific CD4 T cells than those expressing the unmodified Gag (BCG[pWB105]). Mice primed with 10⁷ CFU BCG[pWB206] and then boosted with MVA-Gag developed Gag-specific CD8 T cells with a frequency of 1343±17 SFU/10⁶ splenocytes, 16 fold greater than the response induced with MVA-Gag alone. Levels of Gag-specific CD4 T cells were approximately 5 fold higher in mice primed with BCG[pWB206] and boosted with MVA-Gag than in those receiving the MVA-Gag boost alone. In addition mice vaccinated with BCG[pWB206] were protected from a surrogate vaccinia virus challenge.     

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.     

Differences in distribution of ribonuclease isoenzymes in cytosol and granules in normal human granulocytes and in granulocytes of patients with chronic granulocytic leukemia (CGL)

Distribution of ribonuclease (RNAase), acid phosphatase (acid Ph-ase) and beta glucuronidase (BGU) between the granule, cytosol-soluble and post-granule fractions in normal human granulocytes and in granulocytes of chronic granulocytic leukemia (CGL) was studied. CGL granulocytes were found to display relative RNAase activity 1.2 times higher, relative acid Ph-ase activity 2.5 times higher than normal granulocytes. The granule fraction of CGL granulocytes showed 1.4 times higher relative RNAase activity but 0.87 times lower acid Ph-ase activity and the same BGU activity as normal granulocytes. On the other hand, the supernatant soluble fraction of CGL granulocytes showed 4.4 times higher relative RNAase activity, 1.2 times higher relative acid Ph-ase activity and BGU 2.2 times higher than in cytosol soluble fraction of normal granulocytes. Thus, cytosol soluble fraction of CGL granulocytes show a relative activity of the lysosomal enzymes studied which is remarkably higher than in normal granulocytes. The percentage distribution of RNAase, acid Ph-ase and BGU showed that CGL granulocytes contain only 36% of total RNAase activity versus 46% of that in normal ones. On the other hand, CGL granulocytes in cytosol soluble fraction will contain 48% of total RNAase versus 29% of total RNAase in cytosol of normal granulocytes. The isoenzyme profiles of RNAase of granule fractions were similar in normal and CGL granulocytes, while the RNAase isoenzyme profiles of cytosol fractions were different for normal and CGL granulocytes, indicating that some essential part of CGL granulocyte cytosol RNAase differs from RNAase contained in granules and in cytosol of normal granulocytes.     

Synergistic effect of bacillus calmette guerin and a tuberculosis subunit vaccine in cationic liposomes: increased immunogenicity and protection

In the present work, we evaluated a new TB vaccine approach based on a combination of the Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine and a subunit vaccine consisting of the proteins Ag85B and ESAT-6. We demonstrate that in addition to its vaccine efficacy BCG is an immune modulator that can potentiate a Th1 immune response better than the well-known adjuvant mono phosphoryl lipid A, leading to enhanced recognition of the subunit vaccine Ag85B-ESAT-6. Importantly, adding a vehicle to the vaccine, such as the cationic liposome dimethyl dioctadecyl ammonium bromide (DDA), significantly increased the potentiating effect of BCG. This synergistic effect between BCG and Ag85B-ESAT-6/liposome required drainage to the same lymph node of all vaccine components but did not require direct mixing of the components and was therefore also observed when BCG and Ag85B-ESAT-6/liposome were given as separate injections at sites draining to the same lymph node. The resulting optimized vaccine protocol consisting of BCG and subunit in liposomes (injected side by side) followed by boosting with the subunit in conventional adjuvant resulted in an impressive increase in the protective efficacy of up to 7-fold compared with BCG alone and 3-fold compared with unaugmented BCG boosted by the subunit vaccine. Thus, these studies suggest an immunization strategy where a novel TB subunit vaccine is administered as part of the child vaccination program together with BCG in neonates and followed by subunit boosting.     

The role of the BCG dose and the mouse strain in the inhibition of development of neoplasms susceptible and nonsusceptible to the action of natural cytotoxic cells

The susceptibility of Ehrlich Ascites Carcinoma (EAC) cells to the action of natural cytotoxic cells of DBA/2 and Balb/c mice in vitro was established. Leukaemia L 1210 cells proved insensitive to the in vitro action of natural cytotoxic cells of DBA/2 mice, but not to those of Balb/c ones. BCG, one of the inductors of cytotoxic NK cells, when administered to DBA/2 or Balb/c mice before introduction of EAC cells inhibited the growth of this tumour but did not retard the development of leukaemia L 1210 in DBA/2 mice. The change in the number of peritoneal exsudate cells (PEC) in DBA/2 mice after intraperitoneal injection of BCG was demonstrated to be dependent on the dose and the time elapsed after bacilli introduction. The antitumour action of BCG does not depend on changes in the number of PEC caused by the bacilli. Both large (3.0 mg) and small (0.02 mg) doses of BCG inhibit the development of EAC in Balb/c mice ("sensitive" to BCG), notwithstanding the time of administration of the bacilli. In DBA/2 mice ("resistant" to BCG) development of EAC can be inhibited only by the large dose of BCG since small one is sometimes ineffective.