Pulmonary metastases in guinea pigs as a consequence of dermal implantation of line-10 tumor cells

Summary Metastases to the lungs of guinea pigs occurred at high frequency as a consequence of intradermal implantation of tumor cells derived from the syngeneic hepatocellular carcinoma line-10. Surgery had a major influence on the proportion of guinea pigs found to have pulmonary metastases at necropsy. Without surgery all guinea pigs died with extensive lymph node metastases; macroscopic pulmonary metastases were present in a minority of the animals. Animals treated by excision of dermal tumors survived longer than untreated animals, and macroscopic pulmonary metastases were present in the majority of the animals. Animals treated by excision of dermal tumor and regional lymph nodes were rendered tumor-free. The data suggest that lymph node metastases were the most likely source of the tumor cells that spread to the lungs in animals from whom the dermal tumor transplant had been removed.     

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.     

Antigenic heterogeneity of the guinea pig line 10 hepatocarcinoma. Implications for active specific immunotherapy

Summary Late metastatic disease was studied in L10 tumor-bearing guinea pigs which had shown an initial therapeutic response to a vaccine of x-irradiated L10 tumor cells plus BCG. A single metastatic lesion was isolated from two different animals showing evidence of tumor recurrence on days 134 and 212 after tumor implantation. These putative variants of the L10 parent were designated L10 variant 1 (L10-1) and L10 variant 2 (L10-2), respectively. Comparisons of the antigenic properties of the L10 parent and the two L10 variants showed that the earlier occurring metastasis (L10-1) was not distinguishable from the L10 parent in the ability of the tumor cells to immunize normal animals and elicit delayed-type hypersensitivity (DTH) responses in immunized animals. In contrast, the later occurring metastasis (L10-2) showed a decrease in antigen expression compared with the L10-parent. Although it has been postulated that the antigenic heterogenity of primary or early-passage tumors is lost upon repeated in vivo passage, the present studies show that such heterogeneity does exist or can be induced in a transplantable guinea pig tumor of long duration. Despite the presence of antigenic heterogeneity, active specific immunotherapy of L10 tumor-bearing animals was successful under defined conditions of treatment.     

Specific tumor memory induced by polyethylene-glycol-modified interleukin-2 requires both helper and cytotoxic T cells

Local polyethylene-glycol (PEG)-modified interleukin-2 (IL-2) immunotherapy of the guinea pig Line 10 (L10) tumor was previously demonstrated to evoke long-lasting systemic immunity after cure of the tumor and metastases. T cells, most likely the helper T cell subpopulation, were demonstrated to be crucial to the antitumor effects. Here we show that systemic immunity is induced within 7 days after the start of PEG-IL-2 therapy, indicating a rapid systemic priming of L10-specific T cells. No in vitro cytotoxic activity was detected in cell suspensions obtained from the primary tumor site, the regional lymph node or the spleen when isolated during (days 21 and 28) intratumoral treatment with 200 000 IU PEG-IL-2. These data confirm our carlier results obtained with 60 000 IU PEG-IL-2. Moreover, no cytolytic activity was observed in the chromium-release assay after in vitro restimulation with irradiated tumor cells. Specific L10 immunity can be transferred using spleen cell suspensions. Depletion of such a suspension of helper T cells resulted in rejection of the primary tumor in two out of four animals, but all the guinea pigs developed lymph node metastases. Removal of the cytotoxic/suppressor phenotype caused rejection of the dermal tumor in four of eight guinea pigs, but the capacity to prevent lymph node metastases was retained in all animals. Thus, depletion of either subtype reduces, but does not abrogate, the capacity to transfer L10 immunity with spleen cells. In conclusion, our data suggest that tumor cell killing through direct T cell cytotoxicity is not the main mode of action in PEG-IL-2-induced L10 tumor regression, PEG-IL-2 therapy induces early systemic immunity, resulting in rejection of a distant tumor, and the transfer of this immunity depends mainly on the presence of helper T cells, although cytotoxic T cells may also play a role.     

Complete local tumor regression with antibody to fibrin Fragment E.

Rabbit antibody to fibrin fragment E (FFE) was used in an immunotherapy model for the treatment of the line-10 ascites variant of a diethylnitrosamine-induced hepatoma in strain 2 guinea pigs. When 0.75 or 1.0 mg of an IgG preparation containing anti-FFE antibody was injected s.c. 6 and 16 days after the injection of a uniformly lethal dose of line-10 tumor cells, complete regression of the i.d. growing tumor was observed in all 18 strain 2 guinea pigs treated. Thus, this therapy appears to be more effective than any BCG or other immunotherapeutic regimen thus far reported for this tumor. No significant anti-tumor effect was noted when normal rabbit IgG or smaller doses (0.25 or 0.50 mg) of the anti-FFE IgG preparation were used. The injection sites exhibited an inflammatory response for 7 to 10 days characterized by erythema and hemorrhage. Since all animals were treated after the metastatic progression of the tumor is known to frequently occur, the long-term tumor-free survival of these animals as well as their resistance to subsequent tumor challenge indicate that the anti-FFE antibody therapy led to systemic tumor immunity.     

Differential responsiveness of various substrains of inbred strain 2 guinea pigs to immunotherapy with the methanol extraction residue (MER) of BCG

Summary The immunotherapeutic effects of the methanol extraction residue (MER) of BCG were investigated in strain 2 guinea pigs bearing the transplantable line 10 hepatocarcinoma, a tumor originally induced in guinea pigs at the National Institutes of Health (NIH) by ingestion of the carcinogen diethylnitrosamine. MER was more effective in mediating tumor regression in guinea pigs obtained from the Weizmann Institute of Science (WI), Rehovot, Israel, than in animals obtained from the National Institutes of Health (NIH). These differences indicate the dramatic effects which minor histoincompatibilities between cancer cells and animal substrains may have on experimental results, and highlight the need for immunotherapy experiments to be conducted on laboratory tumors grown in their autochthonous hosts. MER was effective only when injected directly into growing tumor nodules and had no effect on tumor development when administered distally. In contrast, all animals which received both MER and tumor cells developed specific cell-mediated anti-tumor immune responsiveness at higher levels than did non-MER-treated tumor-bearing controls as measured by delayed cutaneous hypersensitivity and in vitro lymphocyte reactivity experiments. Furthermore, the results of the latter but not the former studies suggested that guinea pigs which received MER were able to mount such an immune response more rapidly than their non-treated counterparts. This apparent stimulation of anti-tumor immunity was observed in treated animals regardless of substrain or site of MER injection, and could not be correlated with the outcome of immunotherapy.     

Efficacy of commercial vaccines for protecting guinea pigs against Bordetella bronchiseptica pneumonia

Autogenous bacterins are recommended to protect guinea pigs (Cavia porcellus) against pneumonia due to Bordetella bronchiseptica. Bordetella vaccines are available commercially for several other animal species. The substantial antigenic cross-reactivity among Bordetella isolates from various animal species suggests that immunity resulting from use of these vaccines might protect guinea pigs. Groups of ten individually housed Hartley guinea pigs from a colony free of Bordetella were vaccinated with one of two commercial porcine B. bronchiseptica vaccines, a human DPT vaccine (which includes a Bordetella pertussis component), or an autogenous B. bronchiseptica bacterin. Twenty-one days following vaccination, the animals were challenged with an intranasal dose of 10(6) virulent B. bronchiseptica cells. The animals were euthanized and necropsied 15 days after challenge. The nares, nasopharynx, distal trachea and lungs were cultured. All nonvaccinated control animals developed acute signs of pneumonia, while none of the vaccinated animals developed clinical signs of disease or gross lesions. The frequency of B. bronchiseptica isolation from the lungs of animals in each vaccine group was reduced. However, approximately 70% of all animals in each vaccine group harbored B. bronchiseptica in the trachea, and almost all harbored B bronchiseptica in the nares and nasopharynx. The porcine vaccines appeared to afford protection against acute pulmonary disease in the guinea pig.     

Cellular mechanisms of the antitumor activity of recombinant IL-6 in mice.

The systemic administration of human rIL-6 to mice resulted in the regression of established, 3-day pulmonary micrometastases from two weakly immunogenic tumors, but not from a nonimmunogenic tumor, in the absence of observable toxicity. Although IL-6 alone failed to have a significant therapeutic impact on advanced, 10-day pulmonary macrometastases from weakly immunogenic tumors, substantial cure rates of mice could be achieved when this cytokine was combined with cyclophosphamide. Histologic analysis of the lungs of mice receiving IL-6 revealed infiltration with lymphoid cells during the regression of pulmonary nodules from a weakly immunogenic tumor. IL-6-mediated tumor regression could be abrogated after selective in vivo depletion of either CD4 or CD8 T cell subsets by the systemic administration of specific mAb. In vivo generation of tumor-specific CTL, but not of lymphokine-activated killer cells, was detected in the lungs of IL-6-treated mice during regression of pulmonary metastases. Collectively, these findings demonstrate a role for IL-6 in the treatment of established solid tumors that have the capacity to elicit T cell responses in the host. Differences in host cellular mechanisms involved in tumor regression mediated by immunotherapy using IL-6 vs IL-2 are discussed.     

Induction of potent immune responses by cationic microparticles with adsorbed human immunodeficiency virus DNA vaccines

The effectiveness of cationic microparticles with adsorbed DNA at inducing immune responses was investigated in mice, guinea pigs, and rhesus macaques. Plasmid DNA vaccines encoding human immunodeficiency virus (HIV) Gag and Env adsorbed onto the surface of cationic poly(lactide-coglycolide) (PLG) microparticles were shown to be substantially more potent than corresponding naked DNA vaccines. In mice immunized with HIV gag DNA, adsorption onto PLG increased CD8(+) T-cell and antibody responses by approximately 100- and approximately 1,000-fold, respectively. In guinea pigs immunized with HIV env DNA adsorbed onto PLG, antibody responses showed a more rapid onset and achieved markedly higher enzyme-linked immunosorbent assay and neutralizing titers than in animals immunized with naked DNA. Further enhancement of antibody responses was observed in animals vaccinated with PLG/DNA microparticles formulated with aluminum phosphate. The magnitude of anti-Env antibody responses induced by PLG/DNA particles was equivalent to that induced by recombinant gp120 protein formulated with a strong adjuvant, MF-59. In guinea pigs immunized with a combination vaccine containing HIV env and HIV gag DNA plasmids on PLG microparticles, substantially superior antibody responses were induced against both components, as measured by onset, duration, and titer. Furthermore, PLG formulation overcame an apparent hyporesponsiveness of the env DNA component in the combination vaccine. Finally, preliminary data in rhesus macaques demonstrated a substantial enhancement of immune responses afforded by PLG/DNA. Therefore, formulation of DNA vaccines by adsorption onto PLG microparticles is a powerful means of increasing vaccine potency.     

Comparison of Immunity in Mice Cured of Primary/Metastatic Growth of EMT6 or 4THM Breast Cancer by Chemotherapy or Immunotherapy

Purpose

We have compared cure from local/metastatic tumor growth in BALB/c mice receiving EMT6 or the poorly immunogenic, highly metastatic 4THM, breast cancer cells following manipulation of immunosuppressive CD200:CD200R interactions or conventional chemotherapy.

Methods

We reported previously that EMT6 tumors are cured in CD200R1KO mice following surgical resection and immunization with irradiated EMT6 cells and CpG oligodeoxynucleotide (CpG), while wild-type (WT) animals developed pulmonary and liver metastases within 30 days of surgery. We report growth and metastasis of both EMT6 and a highly metastatic 4THM tumor in WT mice receiving iv infusions of Fab anti-CD200R1 along with CpG/tumor cell immunization. Metastasis was followed both macroscopically (lung/liver nodules) and microscopically by cloning tumor cells at limiting dilution in vitro from draining lymph nodes (DLN) harvested at surgery. We compared these results with local/metastatic tumor growth in mice receiving 4 courses of combination treatment with anti-VEGF and paclitaxel.

Results

In WT mice receiving Fab anti-CD200R, no tumor cells are detectable following immunotherapy, and CD4+ cells produced increased TNFα/IL-2/IFNγ on stimulation with EMT6 in vitro. No long-term cure was seen following surgery/immunotherapy of 4THM, with both microscopic (tumors in DLN at limiting dilution) and macroscopic metastases present within 14 d of surgery. Chemotherapy attenuated growth/metastases in 4THM tumor-bearers and produced a decline in lung/liver metastases, with no detectable DLN metastases in EMT6 tumor-bearing mice-these latter mice nevertheless showed no significantly increased cytokine production after restimulation with EMT6 in vitro. EMT6 mice receiving immunotherapy were resistant to subsequent re-challenge with EMT6 tumor cells, but not those receiving curative chemotherapy. Anti-CD4 treatment caused tumor recurrence after immunotherapy, but produced no apparent effect in either EMT6 or 4THM tumor bearers after chemotherapy treatment.

Conclusion

Immunotherapy, but not chemotherapy, enhances CD4⁺ immunity and affords long-term control of breast cancer growth and resistance to new tumor foci.     

Passive transfer of tumor immunity with spleen cells from guinea pigs cured of hepatocarcinoma by nonspecific immunotherapy

Summary The purpose of this study was to evaluate cell-mediated tumor immunity in strain-2 guinea pigs cured of line-10 hepatocarcinoma by oil-in-water emulsions containing phenol-water extracts from either BCG or the Re mutant of Salmonella typhimurium (Re ET) admixed with mycobacteria glycolipid (P3). Treatment with these emulsions produced complete regression of established tumor nodules and prevented the growth of lymph node metastases in 25 of the 28 animals inoculated intradermally (ID) with 10⁶ line-10 cells and given intralesional immunotherapy 6 days later. No tumor regression was observed in animals given phenol-water extracts alone. Spleen cells, taken from guinea pigs cured of line-10 by BCG extract + P3 or Re ET + P3, were tested for their influence on tumor growth by means of an in vivo adoptive neutralization test (Winn test). Cell transfer was accomplished by the subcutanous injection of various concentrations of spleen cells admixed with 10⁵ viable line-10 cells. The results showed that as few as 10⁷ immune spleen cells completely inhibited the growth of 10⁵ tumor cells in 46–54% of the animals. The best tumor growth inhibition (77–85%) was observed in animals given 5 × 10⁷ immune cells admixed with 10⁵ tumor cells. The onset of transferrable tumor immunity was earlier in animals treated with the BCG extract + P3 than in those given the Re ET + P3. However, the duration of detectable tumor immunity was longer in the latter group. In contrast, no inhibition of tumor growth was observed in animals given spleen cells from normal or tumor-bearing guinea pigs. Moreover, spleen cells obtained from guinea pigs immunized with BCG extract + P3 or Re ET + P3 emulsions only and admixed with line-10 cells failed to transfer tumor immunity to normal animals. Thus, results from this study clearly demonstrated that cell-mediated tumor immunity was elicited in animals cured of line-10 tumor with combinations of P3 and phenol-water extracts of either BCG or Re mutant of S. typhimurium and that sensitized spleen cells effectively transferred systemic tumor immunity to normal recipients.     

Blastogenic responsiveness of spleen cells fromLegionella pneumophila-infected mice immunosuppressed with cyclophosphamide

Although guinea pigs are highly susceptible to experimental infection withLegionella pneumophila, mice are considered resistant. In the present study it was found that, although untreated mice resisted lethal infection with up to 10⁷ L. pneumophila, mice treated with three divided doses of cyclophosphamide became 10–100 times more susceptible. Injection of mice with 150 mg cyclophosphamide/kg body weight 96 and 48h prior to and on the same day as intraperitoneal challenge with graded dose ofL. pneumophila resulted in markedly increased lethality. Approximately half of the mice pretreated with cyclophosphamide succumbed to 10⁶ legionellae within 4–10 days after infection, and all treated animals given 10⁷ bacteria died. Legionellae were readily recovered from spleen, lymph nodes, and liver of surviving mice 4–10 days after infection, but not thereafter. Sensitization of mice with Legionella antigen was evident by the lymphocyte blastogenic test in vitro, by use of spleen cells at various times after infection. Mice given graded doses ofL. pneumophila evinced enhanced responsiveness to either formalin-killed whole cell vaccine, cell-free sonicate, or purified outer membrane antigen when tested in vitro on days 3 and 5. Peak responses generally occurred 20–35 days after infection. Mice given none or one dose of cyclophosphamide and injected with legionellae showed enhanced responses on day 5 of culture in vitro, a time when spleen cells from control nonsensitized animals showed much lower responses. Surviving mice given three doses of cyclophosphamide had lower blastogenic responses, generally as low as that occurring with spleen cells from nonsensitized animals. Thus suppression of immune responses of mice by cyclophosphamide substantially increased susceptibility toL. pneumophila and depressed blastogenic responsiveness.     

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.     

C1s-induced vascular permeability in C2-deficient guinea pigs

Normal guinea pigs that have been intradermally injected with C1s exhibit increased vascular permeability at the injection site. Guinea pigs that are genetically deficient in complement component C2 do not exhibit increased vascular permeability when given a similar injection. The C2-deficient guinea pigs respond normally to injections of bradykinin and kallikrein, suggesting that these animals can respond to kinins and have a normal kininogen pathway. When the C2-deficient guinea pigs are given guinea pig C2 before C1s injection, increased vascular permeability is observed. These results demonstrate a definite requirement for complement component C2 in the generation of C1s-induced vascular permeability.     

Immunogenicity of “viable” tumor cells after storage in liquid nitrogen

Summary Injection of vaccines containing BCG and irradiated L10 hepatoma cells into strain 2 guinea pigs led to arrest and regression of viable L10 cells injected contralateral to and simultaneous with the vaccine. If the tumor cells in the vaccine had been stored in LN2, the vaccine was significantly less effective. The diminished immunogenicity of the stored cells could not be attributed to the sequence of freezing and irradiation, nor to the presence of dead cells which had been killed during cryopreservation. We concluded that cells which had been stored in LN2 had undergone changes which decreased their immunogenicity but which did not alter permeability to trypan blue.     

Immunotherapy of established murine B cell lymphoma. Combination of idiotype immunization and cyclophosphamide

A murine B cell lymphoma (38C13) was used to study the efficacy of idiotype immunotherapy against established tumors. Immunization of mice with 38C13 tumor-derived Ig, administered after a lethal tumor inoculation, significantly prolonged survival of animals compared to control groups. The efficacy of active immunotherapy was dramatically enhanced when combined with chemotherapy. Cyclophosphamide (100 mg/kg), administered in combination with idiotype immunization to mice bearing 10-day-old, 1 to 2 cm diameter s.c. tumors, resulted in a significant prolongation of survival as compared with either cyclophosphamide or immunization alone and yielded approximately 50% cures. Additional studies combining active immunotherapy with surgical excision of the primary s.c. tumor nodule were less effective than combination chemoimmunotherapy, indicating that reduction of tumor burden was necessary, but not sufficient for effective treatment of established 38C13 lymphoma.     

减毒单核细胞增生李斯特氏菌作为疫苗载体在肿瘤治疗中的应用

肿瘤免疫疗法已成为继手术、化疗和放疗之后的第四种肿瘤治疗方法,是当今肿瘤治疗的新希望。将细菌疫苗应用于肿瘤治疗的研究已经历了多方面的探索。单核细胞增生李斯特氏菌因其能够趋向肿瘤病灶,在肿瘤微环境保护下激起肿瘤浸润细胞的免疫反应,削弱免疫抑制作用而受到研究者的广泛关注。并且,其在乳腺癌、肝癌、黑素瘤、胰腺癌等癌症中都已具有较为广泛的研究。本文就单核细胞增生李斯特氏菌的免疫应答方式、作为肿瘤载体的优势、减毒策略以及在多种肿瘤免疫治疗中的应用进行综述。     

A specific vaccine effective against stage I and stage II malignant disease in guinea pigs effect of variations in preparations and storage

Summary Guinea pigs, each with an established, syngeneic dermal line 10 tumor and lymph node metastases, were immunized by intradermal injection of a mixture of irradiated line 10 cells and an emulsion containing heat-killed BCG. Immunization eradicated 7- or 10-day-old dermal tumors (about 10 or 12 mm in diameter, respectively) and prevented growth of microscopic lymph node metastases. Fourteen-day-old dermal tumors (about 15 mm in diameter) were not rejected by immunization.Guinea pigs with stage II disease (21-day-old dermal tumors and palpable metastases in the first draining lymph node) were treated by excision of the dermal tumor and the first draining lymph node, and by specific immunization. This treatment eliminated tumor cells remaining in the second draining lymph nodes. The surgical treatment alone was not curative, palpable metastases in the second draining lymph nodes progressed and the animals died (some with visible lung metastases).Emulsions containing killed BCG were good adjuvants even after prolonged storage at 4° C, but lost most of their adjuvant activity after autoclaving or freezing.     

Effect of immunosuppression on experimental Argentine hemorrhagic fever in guinea pigs.

Immunosuppression with cyclosporin A or cyclophosphamide had no apparent effect on the disease course of guinea pigs infected with a virulent strain of Junin virus. Immunosuppression of guinea pigs infected with an attenuated strain of Junin virus led to fulminating Argentine hemorrhagic fever. All immunosuppressed infected animals died. Virus distribution patterns in target organs, as determined by plaque assay and fluorescent antibody procedures, were similar to those from non-immunosuppressed animals infected with a virulent strain. Histopathological lesions in immunosuppressed guinea pigs infected with an attenuated strain of virus were similar to those in non-immunosuppressed guinea pigs infected with a virulent strain. Histological changes attributable to the immunosuppressive drug(s) were regularly observed. Immunosuppressed animals infected with attenuated Junin virus and non-immunosuppressed animals infected with virulent virus failed to develop antibody or responded at a minimal level. Virus-specific cytotoxic spleen cell activity, previously shown to be antibody dependent, failed to develop in the same animals. The presence of a competent immune response, probably serum antibody, determined whether Argentine hemorrhagic fever infection of the guinea pig was lethal or whether recovery ensued; no evidence for harmful effects of the immune response was obtained.