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.     

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.     

Tumour rejection after adoptive transfer of line-10-immune spleen cells is mediated by two T cell subpopulations

Summary The growth of line-10 tumours in naive guinea pigs is prevented by adoptive transfer of spleen cells that are hyperimmune to this hepatocellular carcinoma. To study the T cell subpopulations responsible for the adoptive transfer of immunity, various cell populations were removed from immune spleen cells using monoclonal antibodies (mAbs) and magnetic microspheres. Spleen cell subpopulations were identified by mAb after flow cytometry and rosette formation with the magnetic microspheres. mAb CT5 was confirmed to be a pan T cell marker, while the CT6 (anti-T-suppressor/cytotoxic) and CT7 (anti-T-cell) markers were present on two different T cell subpopulations. So our results show that CT7 mAb cannot be used as a pan T cell marker as was published previously. Moreover, the mAb H155 (anti-T-helper/inducer) reacted with the same T cell subpopulation recognized by CT7. So we designated this H155/CT7-positive subpopulation as T helper/inducer cells. Removal of the CT6-, CT7-, or the H155-positive T cells from the immune spleen cells resulted in loss of the in vitro poliferative response to line-10 tumour protein and tuberculin purified protein derivative (PPD). The H155/CT7(anti-T-helper/inducer)-positive spleen cells did not express MHC class II antigens as determined by mAb 25E3. In most experiments, elimination of MHC-class-II-positive cells did not change the in vitro proliferative response to line-10 protein, whereas the response to tuberculin PPD was completely abrogated. Immune spleen cells after depletion of CT6-, CT7- or H155-positive cells, failed to transfer immunity. However, after depletion of MHC-class-II-antigen-positive cells the line-10 immunity was still present, whereas the immune response to tuberculin PPD was lost. In conclusion, our data indicate that immunity to the line-10 tumour is the result of a cooperation between at least two different T cell subpopulations, the T helper/inducer (CT7/H155) cells and the T suppressor/cytotoxic cells (CT6). If this is a common feature, then the therapeutic approach of in vitro expanded TIL cells should take into consideration the requirement of two T cell subsets.     

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.     

Analysis of the intralesional adriamycin-induced regression of primary and metastatic growth of line-10 guinea-pig hepatoma

Summary A single intralesional injection of 2.4 mg adriamycin/kg into 7-day-old intradermal strain-2 guinea-pig hepatoma, line-10, caused regression of the tumor and prevented the growth of regional lymph node metastasis. Cured animals were resistant to challenge with the same tumor. Intratumoral injection of 20 mg DTIC/kg was not effective in causing tumor regression or preventing growth of regional lymph node metastasis. Comparative histological examinations performed on the tumor site and the draining lymph node showed that both chemotherapeutic drugs caused extensive necrosis at the injection site and that within 7 days only adriamycin eradicated tumor cells from the draining lymph nodes.The number of host lymphocytes and monocytes in the adriamycin-treated tumor sites was less than that seen in the saline- or DTIC-injected animals at all time intervals examined. Minimal peripheral effects, as measured by total and differential analysis of the blood, were noted in drug-treated normal and tumor-bearing animals. In addition, the concentration of drug used did not interfere with the development of immunity.The results suggest that the effect(s) of intralesional adriamycin treatment is probably caused by a combination of cytotoxic and cytostatic actions of the drug and the development of tumor-specific immunity. Abbreviations used: DTIC, 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboximide; BCNU, 1,3-bis-(2-chloroethyl)-1-nitrosourea; SDA, superficial distal axillary lymph node     

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     

Antibodies to bacterial and tumor-derived antigens in sera from normal guinea pigs.

Antibodies that react with radiolabeled antigens derived from guinea pig line-10 tumor cells and Mycobacterium bovis (BCG) were detected in sera from normal tumor-free strain-2 guinea pigs (NGPS). Binding by NGPS to the two antigens was inhibited by extracts of either line-10 cells or BCG. Binding by NGPS to the line-10 antigen was inhibited by a number of other bacterial extracts. NGPS was tested after absorption with a variety of cells including line-10, line-1, normal guinea pig spleen, normal adult and fetal liver cells. Results indicated that some of the antibodies in NGPS were directed to line-10-specific determinants. The specific stimulating antigen for these antibodies was not identified but because of the antigenic relationship between BCG, line-10 cells and other bacteria, antibodies to line-10-associated antigens might have been induced by exposure to environmental microorganisms.     

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.     

Naturally soluble tumor antigens from guinea pig hepatomas: isolation and partial characterization.

Naturally soluble tumor antigens were detected in the ascites fluid of guinea pigs bearing an ascites tumor and from exhausted tissue culture media of cultured tumor cells. Two antigenically distinct cell lines of diethylnitrosamine-induced strain-2 guinea pig hepatomas (line-10 and line-1) served as the source of tumor antigens. Tumor antigen activity was detected by four different techniques: immunodiffusion, inhibition of complement-mediated cytotoxicity, inhibition of membrane immunofluorescence, and delayed cutaneous hypersensitivity. With syngeneic tumor-specific antiserum, line-10 guinea pig tumor antigens were detected by immunofluorescence in the concentrated ascites and tissue culture fluids. With a xenogenic antiserum, demonstrated to be tumor specific, line-10 tumor antigens were detected not only in the concentrated ascites and tissue culture fluids but also in two of the partially purified fractions of these fluids. When the line-10 concentrated ascites and its fraction I were subjected to ultracentrifugation at 300,000 x G for 1 hr, the antigen activity was retained in the supernatant and thus by this criterion the tumor antigens detected in these samples are soluble. Immunodiffusion data indicate that more than one antigen is present in the line-10 system since three lines of precipitation were detected when line-10 concentrated ascites was reacted with the line-10 tumor-specific antiserum. In contrast to this, the line-10-concentrated tissue culture fluid displayed only one line of precipitation. Although tumor antigens could not be demonstrated in the other antigenically distinct tumor cell line, line-1, by immunodiffusion or inhibition of membrane immunofluorescence, inhibition of complement-mediated cytotoxicity was able to detect tumor antigens in the line-1 concentrated ascites and tissue culture fluids.     

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.     

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.     

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.     

Major histocompatibility complex class II antigen expression during potentiation of line-10 tumor immunity after intralesional administration of bacillus Calmette-Guérin

Summary Intralesional injection of BCG into an established line-10 hepatocellular carcinoma in the strain-2 guinea pig causes regression of the tumor and induction of line-10 immunity. We found that the animals were already protected for a second challenge with line-10 tumor cells 7 days after BCG treatment. We studied whether this early induction of immunity was correlated with the expression of MHC class II antigens on line-10 tumor cells and was correlated with an increased expression of MHC class II antigens on leukocytes in the primary tumor and in the regional lymph node (Ln. axillaris accessorius). The MHC class II antigens and the leukocyte subpopulations were measured with monoclonal antibodies and flow cytofluorometry. In the draining lymph node the number of nucleated cells increased about 10-fold during the first 5 days after intralesional injection of BCG. At this time the MHC class II antigen expression of these cells was increased from 21%–32% in the naive controls to 39%–53% in animals with BCG-treated tumors. This implies that the number of MHC-class-II-positive cells increased about 20-fold in the draining lymph node. Surprisingly, the increase in percentage of MHC-class-II-antigen-positive cells was mainly due to an increase of IgM-positive B cells from 8%–11% to 22%–41% and an increase of IgG-positive B cells from 7%–27% to 25%–44%. In the tumor, BCG treatment induced a small increase of MHC-class-II-antigen-positive cells from 11%–12% to 15%–20%. Probably this increase came not from tumor cells but mainly from a BCG-induced infiltration of mononuclear cells, as an increase of T cells from 14% to 20%, an increase of macrophages from 8% to 18%, and an increase of B cells from 0 to 6% was observed. We conclude that the potentiation of anti-(line-10 tumor cell) immunity correlated with a 20-fold increase of MHC-class-II-antigen-positive cells in the lymph nodes and a small increase in the number of MHC-class-II-antigen-positive tumor-infiltrating cells.     

Stimulation of the synthesis and release of lipids in tumor cells under attack by antibody and C

Antibody-sensitized line-1 or line-10 tumor cells treated with GPC (TAC) incorporated fatty acids into complex cellular lipids and released increased amounts of fatty acids within 5 to 10 min after the addition of GPC as compared to control cells. This effect was dependent on the concentration of GPC used; however, under conditions where the cells were not killed, the enhanced synthesis and release of lipids were not dependent on the antibody concentration used to sensitize the cells. Treatment of the cells with antibody alone, GPC alone, or antibody plus heat-inactivated GPC did not result in enhanced synthesis or release of lipids. No enhancement in DNA, RNA, or protein synthesis in TAC was noted. Line-1 cells, which can be killed by GPC when sensitized with excess anti-Forssman IgM antibody, demonstrated enhanced lipid synthesis within 1 to 3 min after the addition of GPC to the antibody-sensitized cells, before measurable killing of the cells had occurred. This effect persisted in the surviving cells when tested 5 and 10 min after the formation of TAC. Addition of GPC deficient in C4 to antibody-sensitized cells did not result in enhanced lipid synthesis or release. These data suggest that the synthesis of macromolecules of which lipids are a major component is of central importance for the ability of the cells to resist antibody-GPC mediated attack.     

Identification of lipids associated with the ability of tumor cells to resist humoral immune attack.

Certain metabolic inhibitors or chemotherapeutic agents that increase the susceptibility of line-1 or line-10 guinea pig hepatoma cells to humoral immune attack were studied for their effects on the ability of the cells to synthesize lipids. A direct correlation was found between the drug-induced increase in sensitivity to antibody-C mediated killing and the inhibition of the ability of the cells to incorporate acetate, glycerol, and fatty acids into complex cellular lipids. Drug-treated cells recultured in drug-free medium regained their resistance to antibody-C mediated killing; these cells recovered their ability for complex lipid synthesis at this time. Thin layer chromatography of CHCl3:CH3OH lipid extracts from these cells indicated that the drug-induced increase in susceptibility to humoral immune attack correlated with the inhibition of acetate, glycerol, and fatty acid incorporation into cardiolipin and triglyceride in line-10 cells and the inhibition of incorporation of these compounds into cardiolipin alone in line-1 cells. No direct correlation was found between the sensitivity of the cells to humoral immune attack and the ability of the cells to incorporate precursors of lipid synthesis into other lipid moieties (sphyngomyelin, phosphatidyl serine, phosphatidyl choline, phosphatidyl glycerol, or cholesterol esters). The synthesis of cardiolipin and triglycerides, therefore, appears to be associated with the mechanism whereby these tumor cells resist antibody-C mediated killing.     

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.     

Regression of line-10 hepatocellular carcinomas following treatment with water-soluble,microbial extracts combined with trehalose or arabinose mycolates

Summary Intratumor injections of the aqueous phase of phenol-water extracts of Re mutant Salmonella typhimurium (Re glycolipid) in combination with trehalose dimycolate at dose levels of 150 to 15 g were consistently and highly effective (65–93%) in producing regression of line-10 tumors in strain-2 guinea pigs. We observed that the rate of regression was more rapid than that seen after treatment with cell walls from Mycobacterium bovis strain Bacillus of Calmette and Guèrin (BCG). Arabinose mycolate could be substituted for trehalose dimycolate in the Re glycolipid-mycolate mixture without appreciably compromising antitumor activity, providing that the level of arabinose mycolate was not reduced below 15 g. In addition to the Re glycolipid preparation, similarly prepared aqueous extracts from Mycobacterium bovis strain BCG and strain AN5 in combination with trehalose dimycolate also possessed tumor-regressive activity. The activity of these last extracts was reduced when the arabinose mycolate was substituted for the trehalose dimycolate. The aqueous extract of a rickettsia, Coxiella burnetii, in combination with either trehalose dimycolate or arabinose mycolate was also active (50 and 80% tumor regression rates, respectively). Intracutaneous administration of Re glycolipid or aqueous extracts from BCG in combination with trehalose or arabinose mycolates did not produce life-threatening, clinical signs of toxicity in young mice. If additional toxicity studies demonstrate that adverse side effects can be satisfactorily controlled, these watersoluble extracts may prove beneficial in the treatment of spontaneous tumors of humans and other animals.     

Specific 125I-iodination of cell surface lipids: plasma membrane alterations induced during humoral immune attack.

A method whereby lactoperoxidase-catalyzed 125I-iodination of plasma membrane lipids can be achieved is described. The reaction results in a uniform and stable labeling of neutral lipids, phospholipids, lysophosphatides, free fatty acids, and triacylglycerides. By the use of this method, the action of antibody plus complement (C) on the specific release of lipid from the plasma membrane of line-10 tumor cells was studied. Within 15 min after the addition of C to antibody-sensitized cells, the enhanced release of specific lipid classes from the cell surface was observed; these lipids included sphingomyelin, phosphatidylserine, and phosphatidylcholine. The release of phosphatidylethanolamine and, in some instances, triglycerides, was reduced after antibody-C attack. Neither the specificity of the antibody used to sensitize the cells nor the ability of the antibody plus C to be cytotoxic to the cells appeared to affect the identity or amounts of lipids released from the cells.     

Kupffer cells modulate natural killer cell activity in vitro by producing prostaglandins

The effect of Kupffer cells on natural killer (NK) cell-mediated cytotoxicity was examined. Kupffer cells prepared from rat liver suppressed NK activity against K562 cells and other tumor cell lines through a soluble factor secreted into the culture supernatant. When human peripheral blood mononuclear cells were incubated with the Kupffer cell-culture supernatant, a significant reduction of the cytotoxic activity was observed in the 6-hr chromium-release assay. This activity was dose dependent and was evident at various effector/target cell ratios. Lipopolysaccharide stimulated generation of the suppressive factor released from Kupffer cells in a dose-dependent manner. Suppression of the NK activity was observed when the Kupffer cell-culture supernatant was present in the assay system, whereas pretreatment of effector/target cells with the supernatant had minimal inhibitory effects. Autologous monocytes in human peripheral mononuclear cells were not related to this suppression. The suppressive factor in the fraction had a molecular weight below 10,000. Indomethacin, an inhibitor of prostaglandin synthesis, ameliorated the suppressive effects. These results suggest that Kupffer cells may modulate NK activity by producing PGs (E1, E2, and F2 alpha).     

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.