Characterization of in vitro reactivity by BCG-treated guinea pigs to syngeneic line-10 hepatocarcinoma

Summary The purpose of this study was to characterize in vitro the systemic tumor immunity induced by a BCG-intratumoral injection in line-10 hepatocarcinoma established in the skin of inbred guinea pigs (strain 2). Macrophages from BCG-tumor-cured guinea pigs at effector to target cell ratios of 10:1 and 100:1 were cytotoxic in vitro to line-10 tumor cells, and this cytotoxicity was potentiated by autologous serum. Significant cytotoxicity of lymphocytes from BCG-tumor-cured guinea pigs could only be achieved at ratios of 10,000:1, and no effect of autologous serum could be demonstrated. Lymphocytes from both normal and BCG-tumor-cured (line-10 immune) guinea pigs had a significant cytotoxic effect on the highly antigenic line-1 cells at ratios of 1:10,000. Macrophages from both normal and line-10 immune guinea pigs were cytotoxic to line-1 target cells at ratios of 1:100. With respect to specific cytotoxicity (cytotoxicity above and beyond levels achieved with effector cells from normal animals), the only significant difference was demonstrated when line-10 served as target cells and the effector cells were isolated from BCG-tumor-cured (line-10 immune) guinea pigs.Abbreviations used in this paper: BCG, Bacillus Calmette-Guérin; CMEM, complete minimum essential medium; cpm, counts per minute; HBSS, Hanks' balanced salt solution; i.d. intradermally; i.p., intraperitoneally; PEC, peritoneal exudate cells; SDA, superficial distal axillary; ¹²⁵IdUrd, [¹²⁵I]iododeoxyuridine.     

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.     

Hormonal control of implantation in guinea pigs

In the guinea pig, for which implantation is supposedly progesterone-dependent, actual hormonal requirements were assessed by measuring the levels of circulating estradiol and progesterone and correlating them with their content in the ovaries and uterus, and uterine concentrations of their receptors prior to, during, and immediately after implantation. Ovarian and uterine content and plasma levels of estradiol and progesterone, as well as uterine cytosolic receptors of these two hormones, were high at proestrus. Up to day 3 of pregnancy, estradiol remained high in peripheral plasma, ovarian and uterine tissues, but reached low levels at the time of implantation. The levels of progesterone showed a gradual increase in plasma and ovaries till the time of implantation, with the embryonic site of the uterus accumulating more of progesterone compared to estradiol. As pregnancy progressed, a gradual translocation of cytosolic to nuclear receptors occurred, both with estradiol and progesterone receptors. Comparing the receptor values for estradiol at each uterine site showed no significant alterations between embryonic and interembryonic cytosolic receptors. While significantly high levels of nuclear estradiol receptor were found at the inter-embryonic site on day 9 of pregnancy, the cytosolic and nuclear progesterone receptor concentrations were greater at the embryonic site on the same day. These findings demonstrated that the uterus is adequately exposed to estradiol and progesterone prior to ovulation and again in early pregnancy (day 1-3), thus facilitating implantation in the guinea pig (on days 7-8).     

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

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

Peptides as requirement for immunotherapy of the guinea-pig line-10 tumor with endotoxins

Summary The transplantable line-10 hepatocellular carcinoma of guinea-pigs has been used as a model for the study of immunotherapy of malignant tumors. Cure rates of up to 100% have been obtained with ReGl-CM from 0 antigen-deficient (Re) mutant strains of Enterobacteriaceae, provided they were combined with mycobacterial trehalose dimycolate (cord factor, P3). Whereas highly endotoxic LPS extracts from all wild-type strains so far tested have failed to cause tumor regression, acid hydrolysis of such LPS samples led to residual fractions (RESI) that cross-reacted serologically with ReGl-CM samples (Chang and Nowotny, 1975) and provided cure rates up to 100%. RESI from Serratia marcescens was essentially nonpyrogenic and 100 times less lethal for chick embryos than potent endotoxins. Antigenic material associated with endotoxic extracts appears to be cryptic or sterically hindered from being effective in wild-type LPS but is exposed in ReGl and RESI samples.Reduction of the aminoacid content of ReGl-CM by microparticulate silica gel chromatography or by treatment with Triton X-100 significantly lowered the ability to bring about tumor regression without affecting endotoxicity. Antitumor activity could be restored by the addition of synthetic N-acetyl-muramyl-l-seryl-d-isoglutamine (MDP) or a nontoxic lipoid side fraction recovered during the isolation of ReGl-CM, which contained a small amount of peptidic substances. It is concluded that the addition of peptidic material, which may act as an adjuvant, to endotoxins is required to make them useful for immunotherapy of the weakly immunogenic line-10 tumor.Chemical procedures known to detoxify endotoxins while retaining adjuvanticity, such as succinylation and phthalylation, resulted in complete loss of endotoxicity and tumor-regressive potency of ReGl-CM. Transesterification with sodium methoxide led to a water-soluble phase, which cured 50% of tumor-bearing animals even though lethality and pyrogenicity were reduced by 100 times and 50 times, respectively. Thus there was no direct correlation between endotoxic potency and tumor-regressive activity. In addition, our findings indicate that a low level of toxicity may be required to obtain optimal levels of tumoricidal action.Abbreviations P3 trehalose dimycolate isolated by microparticulate silica gel chromatography (Ribi et al., 1974) - LPS lipopolysaccharide from wild-type gram-negative bacteria - ReGl endotoxic glycolipids from Re mutant gram-negative bacteria - CM chloroformmethanol - PW phenol-water - PCP phenol-chloroform-petroleum ether - ReGl-CM ReGl-PW, ReGl-PCP refer to ReGl extracted with CM, PW, or PCP, respectively - ACP acetone-precipitated by-product of ReGl-CM - B1, B2, B4 chromatographic fractions of ReGl-CM - lipid A hydrochloric acid hydrolyzate of LPS or ReGl - RESI organic solvent-insoluble fraction of lipid A (Chang and Nowotny, 1975) - KDO keto-3-deoxyoctonate - BSA bovine serum albumin - CWS cell wall skeleton of BCG (Bacillus Calmette-Guérin) - PPD purified protein derivative (tuberculoprotein) - TAP tuberculin-active peptide - MDP N-acetyl-muramyl-l-seryl-d-isoglutamine     

Further studies on the structural requirements of agents for immunotherapy of the guinea pig line-10 tumor

Summary We made a comparative study of the in vivo binding of immunoglobulins (Ig) to a polyoma virus-induced ascitic tumor propagated in syngeneic or allogeneic mice. The Ig coat was found to appear more rapidly and to be denser in H 2-incompatible than in H 2-compatible mice. This suggests that antibodies were fixed specifically on strong normal transplantation antigens (H-2) recognized as non-self by allogeneic mice. Experiments with mice in which immunosuppression had been achieved by means of X-irradiation confirmed that the Ig fixed on SEWA cells are actively bound antibodies. The only mice that could fix Ig on tumor cells were those that had been specifically immunized against cell surface antigens shared by SEWA cells before irradiation, while mice hyperimmunized against nonrelated antigens could not.In partial fulfilment of doctorate thesis requirements     

Resistance to tumor growth in guinea pigs immunized with lyophilized tumor cells

Summary Living BCG, killed Mycobacterium tuberculosis cells, or BCG cell walls (CW) augmented the immunogenicity of lyophilized syngeneic ascites hepatoma (line 10) of strain-2 guinea pigs. Effective vaccine contained living BCG and lyophilized line-10 cells, or mycobacterial cells or CW attached to oil droplets and lyophilized line-10 cells. Protection against the challenge tumor was evident 14 or 21 days after one administration of either vaccine.     

Suppression of growth of guinea pig line-10 hepatocarcinoma. I. Effect of simultaneous administration of tumor cells and antibodies from normal rabbits.

Suppression of growth of the line-10 hepatocarcinoma in strain-2 guinea pigs occurred when line-10 cells were injected intradermally together with sera or immunoglobulins derived from normal rabbits. A significant number of animals were resistant to subsequent rechallenge with tumor cells. This immunity was specific, depended on contact of immunoglobulins with tumor cells and on the concentration of immunoglobulins. Repeated injections acted as potent vaccines and resulted in the development of immunity in 84.6% of recipients. Fc receptors were not detected on line-10 cells. Antibodies reacting with line-10 cell unique antigens as well as with antigens common to line-10, line-1 and normal guinea pig spleen cells were found in NRS. Injection of line-10 cells together with rabbit immunoglobulins from which antibodies reacting with antigens derived from line-10 cells had been removed did not result in tumor suppression. The specific antigen(s) recognized by antibodies that suppressed growth of the line-10 tumor in vivo was not determined.     

Selective localization of tumor-immune spleen cells at the tumor challenge site after adoptive transfer of line 10 tumor immunity in strain 2 guinea pigs

In this study, the distribution of immune spleen cells was investigated after adoptive transfer of immunity in inbred strain 2 guinea pigs. Spleen cells obtained from line 10 immune donor animals became specifically restimulated in vitro with 3 M KCl-extracted line 10 soluble proteins, but not with 3 M KCl-extracted line 1 or liver proteins. After 4 days culture in vitro, these specifically restimulated immune spleen cells retained their antitumor activity in vivo after adoptive transfer. The specifically restimulated immune spleen cells were radiolabeled with [3H]thymidine, 1 X 10(8) viable cells were adoptively transferred in tumor-bearing guinea pigs, and their distribution was investigated. As controls for the specific localization of the immune cells at the line 10 tumor, the presence of labeled cells was studied in the contralateral transplanted line 1 hepatoma as well as in cellular inflammatory reactions elicited by injection with incomplete Freund's adjuvant (IFA) and complete Freund's adjuvant (CFA). A significantly higher localization of the labeled immune spleen cells in the line 10 tumor and the first and second draining lymph nodes of the line 10 challenge site were found when compared to the influx of these cells in the line 1 tumor and the nontumor antigen-related inflammatory reactions. Because our immune donor animals were immunized with a mixture of line 10 cells and BCG, these animals are immune to both. Line 10 immune spleen cells were restimulated in vitro with PPD and were radiolabeled. These PPD-restimulated immune spleen cells showed no preferential localization at the line 10 tumor challenge site but, as expected, a tendency for localization at the CFA (H37Ra) injection site. Furthermore, PPD-reactive spleen cells from BCG-immunized guinea pigs showed a significantly higher accumulation at the CFA injection site compared to the IFA injection site and the line 10 and line 1 tumor challenge site. From the results, it is concluded that line 10 tumor-immune and BCG-immune spleen cells are two distinct cell populations, and that the existence of cross-reacting antigens between BCG and the line 10 hepatocarcinoma are of no importance for the rejection of the line 10 tumor by immune spleen cells.     

Exogenous non-crosslinked collagen enhances granulation tissue formation in dermal excision wounds in guinea pigs

Based on previous observations indicating a role for collagen peptides in eliciting a positive feedback for collagen biosynthesis, this study was initiated to elucidate the effect of non-crosslinked collagen on granulation tissue formation in dermal excision wounds. The wounds were treated with either non-crosslinked or crosslinked native collagen, or left untreated as controls. Granulation tissue was analyzed for collagen type I mRNA, for levels of interstitial collagen and for the number of blood vessels. The results indicated significant increases in procollagen type I mRNA, in interstitial collagen, in the number of blood vessels and in epithelial advance in the non-crosslinked collagen-treated wounds relative to the untreated controls. It is assumed that the presence of non-crosslinked collagen in a healing wound enhances both procollagen type I biosynthesis and the repair process of dermal wounds, due to the more readily released collagen peptides derived from this exogenous collagen dressing.     

Active specific immunotherapy of guinea pigs with visceral tumor implants

Summary Guinea pigs, each with established, 7-day-old, syngeneic visceral micrometastases of line 10 tumor implanted intravenously, were immunized by intradermal inoculations into several sites of a mixture of irradiated line 10 cells and an emulsion containing heat-killed BCG or Mycobacterium phlei bacilli. This treatment led to survival of 72 of 80 treated animals (90%). Therapeutic effectiveness depended on the dose of mycobacteria and on that of irradiated tumor cells. Animals treated by intradermal injection of mycobacteria attached to oil droplets alone or with irradiated tumor cells alone, all died with multiple foci of pulmonary tumor.     

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.