Immunotherapy with allogeneic immune peritoneal cells activated by BCG or pyran copolymer

Summary Both pyran copolymer and BCG augmented specific macrophage-mediated cytotoxicity against allogeneic MBL-2 leukemia cells in a synergistic fashion. Similarly, the local passive transfer of peritoneal exudates (PE) from MBL-2 alloimmune mice treated with pyran or BCG protected against MBL-2 tumor development in the syngeneic host, whereas exudates from immunized or adjuvant-treated animals alone were ineffective. Tumor neutralization by alloimmune-activated PE was specific for the immunizing cell type and required intimate cell contact with target cells. Both adherent and nonadherent PE cells were required for optimal therapeutic effect. Long-term survivors demonstrated resistance to subsequent tumor challenge.     

Inhibition of pulmonary metastases of B16 melanoma with irradiated tumor cells and BCG

Summary When the tumor-bearing leg of C57BL/6J mice was amputated 16 days after SC inoculation of 10⁶B16 melanoma cells, all the amputated mice died of pulmonary metastases. Transfer of lungs from the amputated to normal syngeneic mice revealed tumor cells in the lungs just after amputation. Repeated weekly injections of BCG and irradiated tumor cells, beginning 24 h after amputation of the tumor-bearing limb, prolonged the survival only of mice presensitized to BCG. Injections of BCG or irradiated melanoma cells alone, of neuraminidase- and mitomycin C-treated tumor cells or of Levamisole had no effect, but injections of ConA-coated tumor cells slightly prolonged the survival of the amputated mice. Both BCG and B16 cells induced humoral and cell-mediated immunity but there was no cross-reactivity between BCG and B16 cells. Abbreviations used: ConA, concanavalin A; SC, subcutaneous; IP, intraperitoneal; IV, intravenous; ID, intradermal; IT, intratumoral; PBS, phosphate-buffered saline (0.01 M sodium phosphate, pH 7.1); VCN, Vibrio cholerae neuraminidase; HBSS, Hank's balanced salt solution; RPMIM, Roswell Park Memorial Institute medium     

Antitumor activity of two BCG vaccine preparations against the lewis lung carcinoma in mice

Summary Two BCG vaccine preparations were prepared following different production methods. Immuno-BCG Pasteur F was produced by surface culture on Sauton medium; BCG-RIV was a homogenous stirred deep culture.The antitumor effects of the two BCG vaccines were investigated on the Lewis lung carcinoma (3LL) in C57Bl/6 mice. A direct relationship exists in this tumor model between the log₁₀ dose of single-cell suspension inoculated subcutaneously in the hind footpad of mice and the onset and the degree of local tumor growth and the time of death, which is directly related to the lung metastases. No significant difference from control mice was observed in the two groups of BCG-immunized mice when 3LL tumor cells were injected 2 weeks after BCG immunization. When varying numbers of viable units of the two BCG vaccines were injected together with 10⁵ tumor cells in separate groups of normal mice, a dose-dependent local reaction was observed with Immuno-BCG Pasteur F, which was associated with a delay in the onset and development of tumor growth and an increase in the mean survival time. The local inflammatory reaction produced with BCG-RIV was of lower magnitude, and only the highest concentration (1.8×10⁶ viable units) led to some delay in tumor occurrence and mortality. The antitumor effect of a specific local delayed-type hypersensitivity (DTH) elicited by varying amounts of the two BCG preparations injected together with 10⁵ tumor cells in separate groups of normal or BCG-immunized mice showed that the challenge injection of Immuno-BCG Pasteur F was in all cases more effective than the BCG-RIV, but these two vaccines were more effective in BCG-RIV-immunized mice than in Immuno-BCG F Pasteur-immunized mice.When the same number of viable units within each BCG vaccine was used as a criterion of comparison, Immuno-BCG Pasteur F produced a higher specific and nonspecific local inflammatory reaction (which was associated with a local antitumor effect) than BCG-RIV. But within 2 weeks, the latter was much better able to sensitize the mice to mycobacterial antigens. This was confirmed by the evaluation of local granuloma formation and tuberculin hypersensitivity. BCG vaccines prepared as surface-grown pellets and mechanically dispersed always sensitized mice to a lesser degree and after a much longer period of time than did the well-dispersed deep-cultured vaccine.     

Augmentation of the therapeutic efficacy of adoptive tumor immunotherapy by in vivo administration of slowly released recombinant interleukin 2

Summary Immunization of C57BL/6 mice with MMC-treated syngeneic lymphoma cells, MBL-2, caused the generation of antitumor effector cells in vivo and the immunized mice permanently rejected viable MBL-2 lymphoma cells. Both plastic nonadherent T cells and plastic adherent MØ obtained from MBL-2 immunized mouse peritoneal exudate cells revealed strong cytotoxic activity against MBL-2 lymphoma cells, whereas immune spleen cells were not highly active against MBL-2 lymphoma cells in vitro. However, systemic adoptive transfer of immune spleen cells into the MBL-2-bearing mice by i.v. infusion in conjunction with i.p. cyclophosphamide (100 mg/kg) treatment cured the mice of tumor. This therapeutic efficacy of immune spleen cells was reflected by the number of transferred effector cells and over 5×10⁷ immune spleen cells were required to cure the mice completely. The cells mediating in vivo rejection of MBL-2 lymphoma cells were Thy 1.2⁺ T cells. This ACIT was specific against MBL-2 lymphoma cells and had no effect on the growth of other syngeneic tumors, B16 melanoma or BMC6A fibrosarcoma. In vivo administration of recombinant interleukin 2 (r-IL 2) combined with ACIT greatly modulated the cure rate of tumor-bearing mice. In addition, we found that slowly released r-IL 2 administratered from an ALZET miniosmotic pump was more effective in augmenting the therapeutic efficacy of immune spleen cells in ACIT than a single injection of the same total dose of r-IL 2.     

Effects of Toxoplasma gondii and Toxocara canis Antigens on WEHI-164 Fibrosarcoma Growth in a Mouse Model

Cancer is the main cause of death in developed countries. However, in underdeveloped countries infections and parasitic diseases are the main causes of death. There are raising scientific evidences indicating that parasitic infections induce antitumor activity against certain types of cancers. In this study, the effects of Toxoplasma gondii and Toxocara canis egg antigens in comparison with Bacillus Calmette Guerin (BCG) (known to have anticancer distinctive) on WEHI-164 fibosarcoma transplanted to BALB/c mice was investigated. Groups of 6 male BALB/c mice injected with T. gondii antigen, BCG, or T. canis egg antigen as case groups and alum alone as control groups. All mice were then challenged with WEHI-164 fibrosarcoma cells. The mice were examined for growth of the solid tumor and the tumor sizes were measured every other day up to 4 wk. The mean tumor area in T. gondii, BCG, or alum alone injected mice in 4 different days of measurements was 25 mm², 23 mm², and 186 mm² respectively. Also the mean tumor area in T. canis injected mice in 4 different days was 25.5 mm² compared to the control group (alum treated) which was 155 mm². T. gondii parasites and T. canis egg antigens induced inhibition of the tumor growth in the fibrosarcoma mouse model. We need further study to clarify the mechanisms of anti-cancer effects.     

Curative effects of combination therapy with lentinan and interleukin-2 against established murine tumors,and the role of CD8-positive T cells

The antitumor activity of a combination of an antitumor polysaccharide, lentinan (a 1–3 glucan with 1–6 branches), and interleukin-2 (IL-2) was evaluated against established MBL-2 lymphoma and S908.D2 sarcoma at i.d. sites. Treatment of the MBL-2-tumor-bearing BDF1 mice with lentinan and IL-2 induced complete regression of tumor in 87.5% of mice treated. In contrast, treatments using either lentinan or IL-2 alone failed to induce complete regression of tumor, although temporal growth inhibition of tumor was observed about in half of the mice treated. Improvements of antitumor effects by the combination of lentinan and IL-2 were also observed in the MBL-2/B6 and S908.D2/B10.D2 systems. Expression of the antitumor effects of lentinan/IL-2 treatments required the intact T cell compartment, because the effects were not observed when nude mice were used. In the MBL-2/B6 system, the antitumor action of lentinan/IL-2 treatment was abolished in mice treated with antibody to CD8 antigen, whereas antibodies to CD4 or NK1.1 were ineffective. Furthermore, augmented tumor-specific cytotoxic T lymphocyte (CTL) activity was observed in regional lymph node cells of the mice after lentinan and IL-2 administration. These data indicate that the antitumor effects of lentinan/IL-2 are mediated by CD8⁺ CTL but not by CD4⁺ T cells or NK1.1⁺ NK/LAK cells, and suggest that this combined therapy may be effective against even established tumors that are resistant to IL-2 therapy.Abbreviations B6 C57BL/6 - BDF1 C57BL/6 × DBA/2 F1 - Lyt2 murine CD8, Lyt2.1. allele of murine CD8 - Lyt2.2 allele of murine CD8 - Lyt3 murine CD8 - L3T4 murine CD4     

Lack of relationship of transplantation immunogenicity in C3H/He mammary carcinomas,with lymphoid hyperplasia and radiation-induced growth enhancement

Summary The therapeutic use of (a) radiation-inactivated tumor cells, (b) Bacillus Calmette-Guérin (BCG), and (c) heparinized plasma from normal mice to reduce radiation-induced impairment of existing antitumor resistance was investigated in female C3H/He hosts of syngeneic mammary carcinoma implants. The mice, which had been moderately presensitized 50 days before challenge, were given 300 rad whole-body irradiation at various times up to the day of challenge and 3 days after. Irradiated presensitized and irradiated unsensitized animals were maximally immunodepressed 1–2 weeks after exposure. The levels of resistance seen in unirradiated presensitized and in unirradiated unsensitized controls were recovered by irradiated presensitized and by irradiated unsensitized mice in about 3 and 4 weeks, respectively. Repeated injections of radiation-inactivated tumor cells were most effective in supporting the immune status of irradiated mice and in promoting an early recovery. Injections of BCG had only an insignificant effect. Injections of normal plasma was effective in reducing the immune suppression but did not promote an earlier recovery.     

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     

Inhibition of Lewis lung carcinoma in mice by local microwave hyperthermia combined with immunomodulating Propionibacterium granulosum KP-45

Summary C-57 BL/6 mice with Lewis lung carcinoma were treated 2 weeks after tumor implantation with local microwave hyperthermia (2450 MHz, tumor temperature 43.5° C, 30 min) and/or intratumoral or intraperitoneal injection of 1 mg cell walls of Propionibacterium granulosum KP-45. Tumor growth, survival time of the animals, and the delayed skin hypersensitivity to oxazolone were followed up, as well as the ³H-thymidine uptake of tumorous tissues and the number of lung metastases. The combined treatment of microwave hyperthermia with immunomodulating P. granulosum KP-45 resulted in significantly stronger inhibition of tumor growth than with each of these methods alone. The number of lung metastases could be significantly lowered, and the skin reactivity to oxazolone remained enhanced during the whole observation period (over 70 days after tumor implantation). The implications of the test observation are discussed.     

Immunotherapeutic potential in guinea-pig tumor model of deoxyribonucleic acid from Mycobacterium bovis BCG complexed with poly-L-lysine and carboxymethylcellulose.

In vivo antitumor activity of a deoxyribonucleic acid fraction obtained from Mycobacterium bovis BCG (named MY-1) increased when it was complexed with poly-L-lysine (poly LL) solubilized by addition of carboxymethylcellulose (CMC). The complex of MY-1 and poly LL/CMC induced interferon in vivo at a low dose of MY-1 which alone exerted no IFN induction. With Line 10 hepatoma (L10) which is syngeneic with strain 2 guinea pigs, it was demonstrated that repeated intralesional injections of the complex resulted in delay of tumor growth and complete cure of animals from L10 tumor inoculated. Similar treatment of the animals with the same amount of MY-1 or poly LL/CMC alone had little therapeutic effect on the tumor growth.     

Intratumoral BCG and Corynebacterium parvum therapy of canine mammary tumours before radical mastectomy

Summary In two parallel studies, bitches with mammary tumour received single intralesional injections of BCG (1 mg: 10⁷ living bacteria) and Corybacterium parvum (10⁹ killed bacteria) (53 bitches) or C. parvum alone (129 bitches) at the same dosage. Control groups received injections, following the same protocol, of 1 ml BCG suspension medium diluted in saline in the first study (51 bitches) or no injections at all (120 bitches in the second study).A block dissection, including mammary tumours, adjacent mammary glands, and regional lymph nodes, was performed 2 weeks later in all animals. On the basis of histologically confirmed malignant tumours, 48 bitches (25 treated by immunotherapy and 23 controls) in the first study and 67 bitches (30 treated by immunotherapy and 37 controls) in the second study remained for postsurgical follow-up.The clinical tolerance of the treatment was generally good. No significant differences were found in cumulative survival rates between treated and control group in either studies.     

不同剂量的塞来昔布对C57BL/6小鼠移植瘤微淋巴管密度抑制作用的研究(英文)

目的:观察不同剂量的塞来昔布对C57BL/6小鼠肺癌移植瘤生长、COX-2表达和微淋巴管密度影响,探讨塞来昔布对C57BL/6小鼠肺癌移植瘤淋巴管生成可能作用机制及量效关系。方法:将Lewis肺癌细胞株接种于C57BL/6小鼠左侧腹股沟皮下建立移植瘤模型,随机分为4组:对照组、塞来昔布低剂量、中剂量、高剂量组。观察荷瘤小鼠生存状态,瘤体积变化,种瘤42天后牺牲小鼠,western blot半定量检测COX-2表达及微淋巴管密度。结果:Western blot半定量显示:塞来昔布高、中剂量组COX-2的表达水平及免疫组织化学染色微淋巴管密度计数均明显减低,差异有统计学意义(P0.05),低剂量组略有减低但差异无统计学意义(P0.05)。抑制程度呈明显的剂量依赖性。结论:塞来昔布抑制Lewis肺癌移植瘤的生长及淋巴转移,可能与下调COX-2的表达,阻遏了淋巴管生成的信号通路,抑制微淋巴管生成有关,该抑制作用呈一定的剂量相关性。     

Effect of glutamine analogue-acivicin on tumor induced angiogenesis in Ehrlich ascites carcinoma

The inhibition of tumor growth and tumor induced angiogenesis by the glutamine antimetabolite acivicin was evaluated in 6-7 weeks old male Swiss albino mice bearing Ehrlich ascites carcinoma (EAC) transplanted by intraperitoneal (ip) injections of EAC cells. Treatment involving ip injections with two different doses of acivicin (0.05 and 0.41microg/g body weight/day) in saline revealed decrease in tumor volumes and reduced number of blood vessels on peritoneal wall after 10 and 15 days of treatment when compared to control (i.e. injected with saline only). Vascular hyperpermeability was found to be lesser in the treated groups of mice than the control as indicated by the FITC- D and colloidal carbon assay. Serum VEGF level was found to decrease in the drug treated groups both after 10 and 15 days of treatment. The results thus suggest that acivicin may suppress tumoral angiogenesis through regulation of VEGF level.     

Lack of cell-cycle-specific effects of recombinant tumor necrosis factor in vivo

Several in vitro studies have demonstrated that tumor cells arrested in the G2 and M phases of the cell cycle expressed an increased sensitivity to the tumor necrosis factor (TNF). The scope of the present study was to investigate whether this cycle dependence of TNF effects also exists in vivo. The experiments were performed by using the Lewis lung carcinoma (LLC), which had been allotransplanted to nude mice. In order to induce delays of the tumor cell cycle in G2, the animals were treated with etoposide (40 mg/kg body weight i.p.) or with local radiation (15 Gy), each increasing the G2 fraction of the LLC from 10% to 35% and 50% respectively. For combination therapy with recombinant (r)TNF, the tumor was transplanted to four groups of six mice each, one of them serving as a control group the others being treated either with a G2 inductor alone, with rTNF alone, or with rTNF and a G2 inductor combined. Administration of rTNF (125 or 250 g/kg body weight i.v.) was always carried out 24 h after therapy with etoposide or radiation when the maximum of G2 accumulation had developed. The growth behavior of the treated tumors revealed that the response of the LLC to rTNF in vivo was not improved by pretreatment with a G2 inductor and, thus, obviously lacked cell-cycle specificity. It is supposed that direct interactions of TNF with the tumor cells, which are a basic requirement for cell-cycle-linked phenomena, play a minor role in the therapeutic outcome of the LLC under in vivo conditions.     

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.     

Inhibition of Lewis lung carcinoma in mice by local microwave hyperthermia combined with immunomodulating Propionibacterium granulosum KP-45

C-57 BL/6 mice with Lewis lung carcinoma were treated 2 weeks after tumor implantation with local microwave hyperthermia (2450 MHz, tumor temperature 43.5 degrees C, 30 min) and/or intratumoral or intraperitoneal injection of 1 mg cell walls of Propionibacterium granulosum KP-45. Tumor growth, survival time of the animals, and the delayed skin hypersensitivity to oxazolone were followed up, as well as the 3H-thymidine uptake of tumorous tissues and the number of lung metastases. The combined treatment of microwave hyperthermia with immunomodulating P. granulosum KP-45 resulted in significantly stronger inhibition of tumor growth than with each of these methods alone. The number of lung metastases could be significantly lowered, and the skin reactivity to oxazolone remained enhanced during the whole observation period (over 70 days after tumor implantation). The implications of the test observation are discussed.     

Isolation of embryonic stem (ES) cells in media supplemented with recombinant leukemia inhibitory factor (LIF)

The isolation of pluripotent murine embryonic stem (ES) cells has previously been achieved by coculturing the ES cells with fibroblast feeder cells. In this report we demonstrate that ES cell lines can be isolated from murine 129/Sv He blastocysts in the absence of feeder cells in culture medium supplemented with recombinant leukemia inhibitory factor (LIF). Three of the ES cell lines (MBL-1, MBL-2, and MBL-3) were isolated by directly explanting blastocysts, whilst two ES cell lines (MBL-4 and MBL-5) were isolated from blastocysts pretreated by immunosurgery. Three of the ES cell lines contained the Y chromosome (MBL-1, MBL-2, and MBL-5) with a high proportion of the cells displaying a normal diploid karyotype with a modal chromosome number of 40. All of the ES cell lines tested expressed the stem cell markers ECMA-7 and alkaline phosphatase, which were lost on removal of LIF when the ES cells differentiated into a variety of cell types. The full developmental potential of the ES cells was determined by injecting cells from two of the independently derived ES cell lines, MBL-1 and MBL-5, into C57BL/6J blastocysts. A high proportion of the pups born were chimeric as judged by coat pigmentation. Subsequent breeding established that the ES cells had contributed to the germ line. These results demonstrate that feeder cells are not essential for the isolation of pluripotent ES cell lines.     

Effect of BCG on hematopoietic stem cells: Experimental and clinical study

Summary In (DBA/2×C57Bl/6) F1 mice the i.v. injection of 1 mg of living BCG does not increase the total number of CFU/s per femur, but a marked increase in the percentage of CFU/s in S phase is noted as early as the 8th hr. BCG injected i.v. also increases the absolute number of colony-forming units in agar per femur. The effect of BCG appears quite different from the known effect of bacterial endotoxin, and in particular it does not induce a significant increase in the level of CSF. The administration of BCG 24 hrs after treatment with a single dose of 200 mg/kg of cyclophosphamide significantly reduces the time of hematologic restoration, but the same dose of BCG given after a lethal dose of total body irradiation does not increase survival time in mice. These different effects of BCG seem to be related to the role of BCG in stimulating the multiplication maturation pool of the bone marrow without producing any increase in the reserve pool.     

Active specific immunotherapy of residual micrometastasis

Summary A vaccine of Bacillus Calmette-Guérin (BCG) admixed with tumor cells induced systemic immunity and had a therapeutic effect on subclinical, disseminated micrometastasis. Inbred strain-2 guinea-pigs given IV injections of 5×10³ to 10⁶ syngeneic L10 hepatocarcinoma cells were vaccinated after metastatic foci were established in the lung parenchyma. The purpose of this study was to establish the variables that can be manipulated to assure optimal immunotherapy while minimizing deleterious side effects of the BCG. In the present study we examined the variables of source, dose, and ratio of BCG to tumor cells. Four BCG sources (lyophilized Tice and Connaught; fresh-frozen Phipps and Tice) were compared. No significant differences among these BCG preparations could be detected with respect to adjuvant potential when they were admixed with attenuated tumor cells in a vaccine. The dose study clearly demonstrated that a BCG dose dependency exists with relation to induction of effective cell-mediated immunity or survival from disseminated micrometastatic disease. Furthermore, evaluations of dose versus ratio of BCG to tumor cells also supported a BCG dose dependency, with the lowest effective BCG dose being directly influenced by tumor burden of the host. Cutaneous reactivity and hypersensitivity of the primary and secondary immunization sites of tumor-bearing animals treated with effective and ineffective vaccines supported the direct association of reaction to BCG and specific tumor immunity. However, when an in vitro leukocyte migration inhibition assay was used, the degree of reactivity to BCG could not be exploited as a quantitative, diagnostic monitor of effective systemic tumor immunity.