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Strain-2 inbred guinea pigs were infected intradermally with 10(5)-10(7) viable BCG (Pasteur) organisms by means of multiple scarifications of shaven midflank skin. The spread of the BCG to the draining lymph nodes and on to the spleen was followed quantitatively for 28 days. The population of bacilli at the inoculation site increased as much as tenfold the first 14 days. The number of viable BCG organisms recovered from the primary draining superficial dorsal axillary and inguinal lymph nodes varied from 0.1 to 1.0% of the inoculum, with a further tenfold to 100-fold drop in counts for the secondary subclavian and lumbar lymph nodes. The bacterial counts for the various nodes increased substantially the first 14 days. By 28 days, as many as 1,000 viable bacilli were recovered from the spleen. Increasing the inoculum size or the number of inoculation sites increased the primary node counts and promoted a more extensive and rapid spread by the BCG population to the secondary lymph nodes and spleen. Prior vaccination of the host with living BCG decreased the spread of the BCG inoculum from the scarification site to the various draining lymph nodes. Multiple injections of cortisone tended to reverse this effect.

The inoculation of a mixture of drugs and guinea pig hepatoma cells (line-10) induced tumor-specific immunity in about 20% of guinea pigs. When guinea pigs with established intradermal tumors were given various drugs ip, no cures were observed; in contrast, multiple intralesional injections of actinomycin D, 1,3-bis(2-chlorethyl)-1-nitrosourea, adriamycin, mitomycin C, and melphalan were effective in curing animals of their intradermal tumors at a time when there were tumor cells in the draining lymph nodes; dimethyl-triazenoimidazole carboxamide, methotrexate, 5-fluorouracil, and 6-mercaptopurine were not effective. More than 80% of the cured animals were immune to rechallenge with 10(6) line-10 tumor cells.

Sera of strain-2 guinea pigs (cured of line-10 tumor by BCG therapy) were tested for complement-dependent, cytotoxic antibody. About 30% of the sera tested contained significant cytotoxic acitivity with the addition of human, but not syngeneic, complement. Using papain pretreated line-10 cells, we detected antibody in about 50% of the sera with syngeneic sera as the source of complement. Antibody to line-10 was also demonstrated in selected sera by indirect fluorescence and the C1 fixation and transfer test.

The inadvertent extravasation of anticancer drugs in clinical use may result in the development of indolent lesions at the site of injection. Investigation of this phenomenon in Hartley guinea pigs showed that the pathogenesis was based on direct tissue toxicity and not on sensitization. The risk of a serious reaction appeared to be related to the localization of the injurious agent within the dermis rather than at the subcutaneous site.

Subtoxic doses of azaserine induced atypical acinar cell nodules (AACN) in the pancreases of outbred Wistar rats, inbred W/LEW and F344 rats, and outbred Charles River CD-1 albino mice 4-6 months after initiation of treatment in the growing animal. These AACN apparently represented preneoplastic lesions, some of which have the potential to develop into adenomas or adenocarcinomas. Wistar and W/LEW rats were highly responsive to nodule induction; AACN developed in about 90% of the outbred Wistar rats and in all of the W/LEW rats tested. F344 rats were less susceptible and developed about 10% as many AACN as the Wistar rats. Female rats developed approximately half as many AACN as males. The mouse was intermediate in response between the F344 and the two Wistar rats. Syrian golden hamsters and strain 13 guinea pigs were relatively unresponsive. These studies of azaserine-induced AACN provided a basis for selection of carcinogenic azaserine regimens and suggested that the young male W/LEW rat was the most sensitive of the animals studied.

Resistance to line-10 hepatocarcinoma was studied in the newborn offspring of inbred strain-2 Sewall Wright guinea pigs that had recovered from this tumor after prior administration of subcellular components of Mycobacterium bovis strain BCG. These mothers gave birth to 15 offspring, 53% of which resisted repeated challenges with this tumor. Offspring of mothers who had been given only BCG components were similarly tested. Complete protection was observed in 23.5% of 17 babies and attenuation of disease in many others. All control offspring from normal, untreated mothers succumbed to this tumor. Resistance was associated with delayed cutaneous hypersensitivity reactions to tumor and mycobacterial antigens.

The RNase activity observed in the sera of leukemic guinea pigs was compared to that observed in white blood cell (WBC) lysates of the same animals. The WBC-associated RNase activity directed against polyuridylic acid decreased with the progression of neoplastic disease, though serum RNase activity remained unchanged. With certain forms of cancer, therefore, variations in cell RNase may be more sensitive markers than changes in serum RNase for the evaluation of the progression or regression of disease.

Strain 2 guinea pigs were immunized with the LE-L2C cell line and challenged with either LE-L2C or BZ-L2C, a subline of L2C leukemia deficient in la gene product and C3 receptor. The LE-L2C-immune animals were completely protected from a challenge of 5 X 10(6) cells (100 times the lethal dose) of either cell line, and the delayed skin test responses for both lines of injected cells were equivalent. Thus established in vivo immune recognition of the leukemia antigen was similar for both cell lines. After repeated immunization of strain 2 animals with BZ-L2C cells, animals were challenged with 3 X 10(5) viable cells; 9 of 13 animals survived. A subsequent 5 x 10 (6) challenge lowered survival to 8 of the 13 animals. The skin test response to BZ-L2C developed slowly during the immunization period, but survivors of the viable cell challenge exhibited good responses to skin testing with BZ-L2C or LE-L2C leukemic cells. Thus the BZ-L2C cell line possesses a leukemia-specific antigen, but the immunogenicity of this mutant line is decreased when compared to that of the LE-L2C line.

Guinea pigs with growing intradermal transplants of a syngeneic hepatoma treated by intralesional injection of living BCG at a time when lymph node metastases were detectable by palpation were not cured but survived longer than did the controls. Treatment of the animals by excision of the transplant and draining lymph nodes, instead of by BCG, resulted in a significant number of cures. The cure rate of animals receiving both treatments was not demonstrably greater than that obtained in animals receiving surgery alone.

A transplantable hepatocarcinoma of guinea pigs was used as an experimental model for immunotherapy of cancer. Earlier work showed that complete regression of 6- to 7-day-old tumors could be obtained in about 60% of cases by inoculation of the tumors with live BCG or certain fractions of BCG attached to minute oil droplets and suspended in Tween-saline. One of the most essential fractions was P3, a nonsensitizing, nonantigenic trehalose mycolate related to, but not identical with, cord factor. We now report that oil-droplet preparations containing P3 and bacterial endotoxin (ET) produced cure rates of up to 90% in the same system. In addition, regression was faster than with BCG, and older tumors could be treated successfully. The most effective ET's were from rough strains of salmonellae, known as Re mutants, which could not synthesize and attach the polysaccharide portion of endotoxin.

Line 10 hepatoma cells were incubated with antiserum specific against line 10 cells (RaL10) and then tested for growth in syngeneic Wright strain 2 guinea pigs. Palpable tumors appeared in only 11 of 23 animals inoculated id with 10(5) RaL10-treated tumor cells, compared with an incidence of 21 of 23 for nonimmune rabbit serum (NRS)-treated cells and 23 of 23 for cells treated with syngeneic guinea pig serum. Animals inoculated with RaL10-treated tumor cells did not develop systemic tumor immunity. The long-term survival of guinea pigs receiving RaL10-treated tumor cells iv was 6 of 12 with a dose of 10(5) cells and 8 of 11 with 10(4) cells. None of the animals receiving 10(4) or 10(5) control tumor cells treated with NRS survived. RaL10 antiserum was not toxic to line 10 tumor cells in vitro, but mediated tumor-specific cytolytic reactions in the presence of fresh guinea pig serum or on the addition of peritoneal exudate cells from nonimmunized syngeneic guinea pigs.

Extracts of L2C tumor cells stimulated in vitro production of macrophage migration inhibitory factor (MIF) in peritoneal exudate cells from guinea pigs immunized with L2C tumor cells. Guinea pigs immunized with extracts of L2C tumor cells that were active in vitro (in the MIF assay) were completely resistant to challenge with viable tumor cells given 2 weeks later. Furthermore, guinea pigs immunized with extracts of L2C tumor cells within 1 hour after challenge with viable L2C tumor cells survived substantially longer than did nonimmunized controls. The immunoprotective and immunotherapeutic effects seen in guinea pigs given injections of viable L2C tumor cells were obtained with extracts of L2C tumor cells but not with extracts of another guinea pig tumor (line 10 hepatoma) or with extracts of normal guinea pig lymphoid cells.

Components of mycobacterial cell wall(s) (CW) attached to oil droplets were evaluated for their ability 1) to inhibit the growth of line-10 tumor transplants in the skin of syngeneic guinea pigs when inoculated together with 10(6) tumor cells (suppression experiments) and 2) to regress established 7-day-old intradermal tumors and eradicate microscopic lymph node metastases upon injection into the tumors (regression experiments). CW and cell-wall skeleton (CWS) preparations from Mycobacterium phlei, a fast-growing saprophyte of group IV of the atypical mycobacteria, suppressed tumor growth in essentially all animals when 37.5-mug doses were administered; at a dose of 300 mug, they cured 50-60% of the animals in regression tests. The addition of 300 mug of a purified trehalose mycolate, isolated from M. tuberculosis strain Aoyama B, to 300 mug M. phlei CW or CWS preparations significantly increased their tumor regressive potency to provide cure rates to about 90%. Because M. phlei can be propagated more readily, it can be used advantageously in place of BCG to prepare stable, non-living immunologic adjuvants of defined composition and consistently high potency to meet the need for standards with minimal residual malignant disease.

In inbred guinea pigs, administration of Mycobacterium bovis strain BCG by scarification at a site distant from an excised skin tumor, but in the regional lymph node drainage, was evaluated for its immunotherapeutic effect on the development of lymph node metastases. Scarification was performed after surgical excision of intradermally transplanted syngeneic (line-10) hepatocarcinoma at a time when microscopic foci of tumor cells were present in regional lymph nodes. Various strains of BCG were evaluated for their immunotherapeutic potential: fresh-frozen Phipps, Pasteur, and Tice; and lyophilized Pasteur, Tice, and Connaught. Scarification commenced 3 days after surgical removal of the tumor and continued once a week for 5 weeks. Only lymph nodes from fresh-frozen Phipps- and Pasteur-scarified animals were significantly smaller than those in the control groups. Differences in lymph node weight correlated histologically with less detectable metastases. This cytostatic effect was short lived; eventually, the metastatic tumor growth was not significantly different from that of control animals. No significant differences were observed in mean survival time: All animals died as a result of metastases 3 months after tumor inoculation. These results demonstrated that limited scarification with BCG of certain strains temporarily inhibits the growth and proliferation of metastases in regional lymph nodes after removal of the primary tumor.

A transplantable fibrosarcoma induced in inbred JY-1 guinea pig strain by 3-methylcholanthrene (MCA) and designated J4, an allotransplantable subline of J4 (JH4) which was obtained by the transplantation of J4 into the inbred Hartley/F guinea pig strain and maintained by passages in this strain, and a syngeneic liposarcoma H10 induced in a Hartley/F guinea pig by MCA were tested for their immunotherapeutic response with BCG. The growth of J4 and H10 tumors was suppressed in most of the animals when tumor cells were mixed with BCG before being injected sc into BCG-immune or BCG-nonimmune recipients. The growth of the JH4 tumor was suppressed at the sites of injection with a mixture of tumor cells and BCG in BCG-immune recipients but not in nonimmune animals. All guinea pigs surviving the injection of a tumor cell-BCG mixture resisted a second tumor cell challenge. When subcutaneous sarcomas grew to about 8-15 mm in diameter, BCG was injected into the tumors. The growth of JH4 tumor was not influenced by the injection in either BCG-immune or BCG-nonimmune animals, while the regression of the established J4 transplants was produced in 2 of 3 nonimmune recipients. The growth of the H10 tumor was not inhibited with an intratumor injection into nonimmune guinea pigs, while the H10 tumor regressed in BCG-immune animals for 4-5 weeks after intratumor injection and thereafter grew progressively. Skin reactions in animals that received repeated intradermal injections of the tumor cells and BCG were tested with 10(6) viable tumor cells as eliciting antigens. Typical delayed-type hypersensitivity reactions that were specific to the homologous antigens were observed. The possible reasons for the different responses to BCG among the guinea pig tumors, including line-10 hepatocarcinoma in strain-2 guinea pigs, were discussed.

Sera from feline leukemia virus-infected cats lysed FL74 cells in the presence of an appropriate complement source; lysis was detected with the use of the 51 Cr release method. Serum complement from both rabbits and guinea pigs mediated lysis. Partial correlation of results was obtained from preliminary comparisons of lysis and immunofluorescence tests with the use of FL74 cells as targets.

In a transplantable hepatocarcinoma (L10) model of inbred Sewall Wright strain 2 guinea pigs, established micrometastatic tumor foci could be eliminated from the viscera by specific tumor immunity induced by the systemic effect of a BCG-L10 tumor cell vaccine. The conditions of vaccine preparation and regimen were rigid, and a dose dependency for both BCG and tumor cells existed. Comparison of the influence of cryobiologic preservation procedures on the tumor cells to be used in the vaccine showed that cells frozen by an optimized cryobiologic procedure, improved for maintenance of viable cells, were more effective than cells frozen by a conventional glycerol method. The glycerol method resulted in a lower percentage of viable cells, and the effectiveness of the vaccine was greatly diminished. Thus some past failures of active specific immunotherapy could be associated with acute antigenic exposures resulting from suboptimal cryobiologic preservation of tumor cells in the vaccine preparation instead of prolonged or chronic tumor cell antigenic exposure resulting from optimal cryobiologic procedures.

The effect of chronic administration of 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN) was studied in randombred guinea pigs. DHPN, dissolved in olive oil, was injected sc into 40 animals at a dose of 250 mg/kg body weight/week for 30 weeks, and the animals were observed until their death or termination of the experiment at the end of 40 weeks. Of the 32 guinea pigs that survived more than 20 weeks of DHPN treatment, 23 developed angiosarcoma of the liver between 22 and 40 weeks. Metastases to lungs, spleen, and peripancreatic lymph nodes were observed in 8 animals. Other tumors included hepatocellular carcinoma (1 animal), cholangiocarcinoma (1 animal), chronic myeloid leukemia (1 animal), acinar cell adenoma of pancreas (1 animal), and acinar cell carcinoma of pancreas (1 animal). In addition, megalocytic change of hepatic cells with intranuclear inclusions, pelliosis hepatis, and cholangiomatous lesions were also encountered frequently in the livers.

Syrian hamster embryo cells that had been transformed in vitro by guinea pig herpes-like virus (GPHLV) were found to be oncogenic when inoculated into hamster sc or ip. Of 71 animals inoculated, 30 showed tumors at the site of inoculation. Tumors appeared 4-23 weeks after inoculation of the transformed cells at passage 37 or higher. Inbred and randombred hamsters of all ages were susceptible. Upon microscopic examination the tumors were characterized as fibrosarcomas. The cultured hamster tumor cells were easily transplanted into hamsters, but produced no evidence of tumors when inoculated into guinea pigs. Infectious GPHLV was not isolated from the tumor cells, but GPHLV-specific surface antigens were detected in tumor cells by immunofluorescence of GPHLV antiserum produced in rabbits. Sera from tumor-bearing hamsters did not contain GPHLV-neutralizing antibodies, but sera from 4 of 23 hamsters bearing primary tumors and 12 of 41 bearing transplanted tumors produced nuclear fluorescence in cells infected with GPHLV, thus establishing the relationship between the guinea pig herpesvirus and the hamster tumors.

Strain-2 male guinea pigs with established intradermal (id) tumors and microscopic regional lymph node metastases were treated by systemic transfer of syngeneic peritoneal exudate (PE) cells from tumor-immune guinea pigs. This treatment produced complete regressions of established id tumor nodules (10-11 mm in diameter) and prevented the growth of lymph node metastases in 32 (80%) of the 40 treated animals. All untreated animals died with progressive id and lymphatic tumor growth. Lymph node tumor metastases that remained after id tumor excision were also suppressed by immune cell transfer. PE cells from guinea pigs immune to an antigenically distinct tumor line (line-1), BCG, or PE cells from nonimmune guinea pigs failed to produce tumor regression or prolongation of survival time. PE cells from allogeneic guinea pigs and from sheep immune to line-10 failed to transfer tumor immunity to strain-2 guinea pigs. The effectiveness of therapy was reduced by increasing the tumor burden or decreasing the number of transferred lymphoid cells. This study demonstrated that systemic transfer of cells from syngeneic immune donors could effectively eliminate tumors as well as early metastases.

The growth in the colon wall of a diethylnitrosamine-induced hepatocarcinoma was suppressed by BCG. Injection of BCG into established colon-wall tumors caused tumor regression. Successful therapy required a limited tumor burden. Guinea pigs in which growth of the neoplasm was suppressed at the site of BCG infection developed systemic tumor transplantation immunity.

Intralesional administration of emulsified croton oil into established syngeneic transplants of murine firosarcoma no. 1023 caused complete regression of the injected tumors in C3H mice without recurrence during the period of observation. In Sewall Wright strain 2 guinea pigs, in contrast to BCG cell wall vaccine which eradicated regional lymph node metastasis as well as dermal transplants, croton oil treatment only delayed the development of metastatic disease despite the fact that the injected skin tumors did not recur. 12-O-Tetradecanoylphorbol 13-acetate (TPA), the active principle of croton oil, incorporated in mineral oil droplets in aqueous suspension, caused regression of murine tumors when injected intralesionally. Aqueous suspensions of TPA failed to eliminate the tumors. Our results suggest that tumor regression induced by croton oil of TPA emulsions was due to indiscriminate destruction of the injected tissue.

The immunogenicity of inbred strain 2/N guinea pig fibroblasts transformed to the malignant state in vitro by chemical carcinogens was evaluated with the use of a variety of in vivo and in vitro methods including delayed-type hypersensitivity skin and tumor transplantation tests and analysis of antibody production by immunofluorescence, complement fixation, and staphylococcal protein A binding tests. Neoplastic transformation was induced by direct treatment of cells in culture with benzo(a)pyrene, 3-methylcholanthrene, or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or by the host-mediated method by which fetuses were exposed to diethylnitrosamine or MNNG in vivo prior to cell culture. Rabbits and syngeneic guinea pigs were inoculated with unirradiated and X-irradiated clonally derived cells. Delayed hypersensitivity skin reactions to immunizing or other cells were equivalent in immunized or control guinea pigs, and no protection to tumor outgrowth from a challenge inoculum of immunizing cells was observed. Antibody activity induced in the sera of immunized guinea pigs was cross-reactive and removed by absorption with nontumorigenic cells. Rabbit anitsera after absorption with fetal guinea pig cells were nonreactive with the specific immunizing or other cultured cells. Chemical carcinogen-induced neoplastic transformation of guinea pig cells can, therefore, occur without formation of detectable, individually distinct cell surface tumor-specific neoantigens.

Growth of a guinea pig hepatoma was suppressed when tumor cells were mixed with viable Listeria monocytogenes (LM) before intradermal (id) injection into syngeneic recipients. Heat-killed LM were less effective than viable organisms in suppressing tumor growth. A vaccine containing oil droplets and LM cell walls lacked antitumor activity. Intratumor injection of viable LM on the 7th day after id injection of tumor cells prolonged survival of guinea pigs that did not succumb to LM infection. After intratumor injection of 0.6 times 10-8-1.0 times 10-8 LM, 5 of 22 guinea pigs died from acute infection (23 percent). In the 17 survivors, 3 tumors regressed completely (18 percent). Animals surviving injections of LM and tumor cells were immune to a second challenge with tumor cells. Immunization ofguinea pigs with an intravenous injection of LM decreased the mortality from intratumor injection of LM, but the intratumor injection of LM failed to cure a significant fraction of LM-immune animals bearing 7-day hepatoma transplants. BCG was more effective than LM in producing tumor regression. Synergism between LM and BCG was not observed, and simultaneous intratumor injection of BCG and LM was no more effective than intratumor injection of BCG alone in the treatment of 12-day tumor transplants.

Line 10 hepatocarcinoma cells derived from ascites in a strain 2 guinea pig were tumorigenic when transferred intradermally. After they had been cultured in vitro for 20 days or more in medium enriched with 10% fetal bovine serum (FBS), they became immunogenic. Injections of immunogenic cells did not cause lethal tumors, and recipients were resistant to subsequent challenges with tumorigenic line 10 cells. Resistance was specific since growth of line 1 cells, a syngeneic but antigenically distinct tumor, was not affected. Cells cultured in medium enriched with 10% calf bovine serum or 10% normal guinea pig serum or in reduced concentrations of FBS were less effective in inducing resistance. When cultured line 10 cells were injected ip into normal guinea pigs, ascites tumors developed that were tumorigenic. The growth rate of line 10 cells in culture was considerably decreased as determined by reduced [3H]thymidine incorporation and mitotic indices. The mechanism(s) responsible for enhancement of immunogenicity in cultured line 10 cells is discussed but was not determined.

Two 111indium-labeled murine monoclonal antibodies (MoAb), D3 and 9.2.27, directed to tumor antigens of L-10 hepatocarcinoma and human melanoma, respectively, selectively localized antigen-positive target cells in guinea pigs and nude mice. The fate of MoAb differed in the two antigen-antibody systems after reacting with their corresponding tumor antigens in vivo as reflected by patterns of distribution and turnover in vivo. The 9.2.27 localized in melanoma xenograft in nude mice after intravenous administration with slow loss from tumor but more rapid loss from normal tissues and thus demonstrated optimal imaging of small tumors (approximately equal to 5 mm) between 3 and 6 days after injection of the radiolabeled antibody. In contrast, D3 demonstrated a biphasic localization in guinea pig L-10 hepatocarcinoma with a maximal activity on the 2d day after administration and showed rapid loss from both tumor and normal tissues. Nonspecific localization of antibodies in liver and in kidney was found both in syngeneic (nude mice) and xenogeneic (guinea pig) hosts but was more pronounced in the xenogeneic species. These results indicate that the nature of the antigen-antibody interaction may be of importance in selecting MoAb for both diagnosis and therapy of malignant diseases.

A correlation was found between the highly polar phospholipid (HPPL) content of serum lipids and the growth and metastasis of the line-10 hepatoma in strain 2 guinea pigs. Animals whose tumors were treated by intralesional injection of decarbazine (DTIC) or saline showed progressive tumor growth and metastasis resulting in death within 120 days of tumor implantation. The percent HPPl in the serum lipids of these animals rose to 40% by 80 days and remained elevated above 20-25% until death. Animals whose tumors were treated by intralesional injection of adriamycin were either cured of their tumors (67%) or showed little or no tumor growth. The percent HPPL in the serum lipids of these animals remained between 3 and 12% at all times. Adriamycin or DTIC injected into non-tumor-bearing animals resulted in an HPPL content of serum lipids that was not significantly different from that of control, saline-inoculated animals.

Chromosomes were examined in leukocytes from a unique transplantable granulocyte leukemia (GL-13) of strain 13 guinea pigs. The GL-13 leukemia has many similarities to human chronic myelogenous leukemia (CML). It has remained predominantly diploid after 90 transplant generations and is characterized cytogenetically by a terminal deletion of the long arm of one of the #1 pair of chromosomes. No other chromosomal abnormality was observed. These findings lend further support to the conclusion that the GL-13 leukemia may be a useful model for the study of human CML.

Intrarectal administration of 0.5 ml of a 0.25 percent solution of N-methyl-N-nitrosourea twice seekly for 42 weeks to female inbred strain-2 guinea pigs induced large-bowel adenocarcinomas in 9 of 10 animals in 38-56 weeks. Controls did not show cancer. The lesions were infiltrative or constrictive, which distinguished them from chemically induced large-bowel cancers of rats and mice.

Dunkin-Hartley and Hartley guinea pigs were fed a diet containing 1% cholesterol (C+) or a control diet (C-). The C+-fed guinea pigs showed a decrease in antitumor effector cell levels as measured by an in vitro 18-hour 51Cr release assay. Natural killer (NK) activity fell rapidly after initiation of cholesterol feeding, decreasing to 25.6% of control levels by 2 weeks. While the interferon inducer polyinosinic-polycytidylic acid increased NK activity as much as 3.6-fold in controls, the NK levels in C+-fed animals were not increased. NK activity was lower in both spleen and peripheral blood of C+-fed animals and against K562, MOLT-3, HL-60, and Raji target cells. Lectin-dependent cell-mediated cytotoxicity was increased in the C+-fed group over, the first 1-2 weeks on the diet, but it dropped to low levels by 6 weeks. Lipoprotein preparations from plasmas of both C+- and C--fed animals inhibited NK cell activity, but suppression was not due to lipoprotein cholesterol content. On the basis of lipoprotein protein, lipoproteins from C--fed animals were more suppressive. The results suggest that the decrease in cytotoxicity induced by dietary cholesterol is due to more than the high levels of plasma and lipoprotein cholesterol.

Adoptive immunity against a syngeneic hepatoma (line-10) of Sewall-Wright inbred strain 2 guinea pigs was analyzed by a two-dimensional titration of iv transferred immune lymphoid cells versus intradermal tumor challenges. Tumor resistance increased exponentially as a function of the number of immune lymphoid cells transferred. Within the tumor challenge doses analyzed, suppression of tumor growth mediated by the transferred immune lymphoid cells appeared to be independent of the primary immune response in the recipient. Quantitatively, rejection of a given number of tumor cells reflected the number of transferred immune cells and was independent of the presence of the same tumor at other skin sites. There was no evidence indicating that transferred immune cells were attracted specifically to the tumor inoculation site. The number of tumor cells that could be rejected at a skin site by adoptive immunity was greater than the estimated number of immune lymphoid cells present at the challenge site.

Aryl hydrocarbon hydroxylase (AHH) activity was detected in a variety of guinea pig lymphoid tissues and was increased by intradermal or ip administration of 3-methylcholanthrene AHH levels were generally greater in spleen than in lymph node, bone marrow, or thymus. Similar AHH activities were observed in lymphoid tissues from Sewall-Wright inbred strain 2 and strain 13 guinea pigs. Greater AHH activities were associated with peritoneal and alveolar macrophages than with lymph nodes lymphocytes in the absence of mitogens.

The carcinogenic effect of N-nitroso-2,6-dimethylmorpholine (DMNM) was studied in randombred guinea pigs after repeated administration of this compound by gavage. DMNM was administered at two dose levels (14 and 28 mg/kg body wt) weekly for 23 weeks. All animals were observed until their death or termination of the experiment at 54 and 37 weeks for the 14- and 28-mg dose levels, respectively. At 14 mg, 67% of the animals developed hemangiosarcomas of the liver and 60% developed cholangiomas between 36 and 54 weeks. In addition, a poorly differentiated malignant mesenchymal tumor was observed in 1 animal. At 28 mg, liver hemangiosarcomas were observed in 82% of the animals between 26 and 37 weeks. In addition, in 18% of the animals, bronchioalveolar adenoma (1 animal), hepatocellular carcinoma (1 animal), and malignant lymphoma (1 animal) were also induced. Metastases of hemangiosarcomas to lungs, mesenteries, and lymph nodes were observed in 3 animals of each group.

Relationships among carcinogen-induced morphlogic transformation, anchorage independence, susceptibility to growth inhibition by the immunologic hormone lymphotoxin and to natural killer cell and natural lymphoid cell cytotoxicity, natural delayed-type (tuberculin) skin reactivity, and tumorigenicity were investigated with the use of a guinea pig cell culture model of carcinogenesis characterized by sequential, extended developmental stages. Twelve cultures of passage 90 nontumorigenic fibroblast-like line 118 cells derived from a 36-day-old strain 2/N guinea pig fetus were treated with 10 micrograms benzo[a]pyrene or 1 microgram N-nitroso-N-methylnitroguanidine/ml medium for 3 days and maintained in exponential growth. One week later, all cultures exhibited morphologic alteration, and by 8 weeks two-thirds exhibited morphologic transformation. After 4 months, morphologically altered or transformed cells expressed progrssive colony growth in semisolid 0.35% agar medium. Susceptibility to lymphotoxin colony inhibitory activity developed subsequent to morphologic transformation but independently of growth in semisolid medium. Susceptibility to natural lymphoid cell destruction was also independent of growth in semisolid medium. All four phenotypic properties were expressed only after carcinogen treatment and 12 months later remained unaccompanied by either natural delayed-type skin reactivity or by the ability of the cells to grow as tumors in syngeneic guinea pigs of nu/nu mude mice. Thus while morphologic transformation, colony formation in semisolid medium, and susceptibility to lymphotoxin and to nautral lymphoid cell cytotoxicity were frequently associated with neoplasia, they could be expressed independently of one another; individually or collectively in a specific developmental sequence, they were insufficient for expression of either natural delayed-type skin reactivity of tumorigenicity.

A total of 480 guinea pigs each received twice weekly intrarectal instillations of 1 mg N-nitroso-N-methylurea (NMU) during 14-35 weeks (total dose NMU, 28-70 mg) to induce colon neoplasms. At 24, 28, or 35 weeks after the start of NMU instillation, the distal colon was exposed by a ventral laparotomy and suspected neoplastic lesions were treated by one of four methods: A, intratumoral instillation of an emulsion of killed BCG cell walls attached to oil droplets; B, intratumoral instillation of a control emulsion of killed BCG cell walls in an aqueous phase rather than lipid phase; C, surgical excision of the colon lesion with formation of a ventral diverting colostomy; or D, sham operation (no treatment). All guinea pigs were allowed to recover from surgery and were then observed for a period of 1 year for the study of the effects of these treatments on NMU-induced colon neoplasms. Colon neoplasms were produced in 76% of all sham-operated control guinea pigs, and the frequency of such neoplasms was dependent on the total dose of NMU. Most neoplasms were adenocarcinomas with invasion of the bowel wall but only approximately 5% of them metastasized. Treatment with BCG failed to alter the course of NMU-induced colon carcinogenesis, as determined by the frequency of colon neoplasia, the number, the gross, or microscopic characteristics of colon neoplasms, or the rate of survival.

Adenocarcinoma of the gallbladder developed in 17 of 68 untreated and in 26 of 83 irradiated guinea pigs of inbred strains 2 and 13. The carcinomas spread widely by direct extension and through lymphatic and blood vessels to lymph nodes, mesenteries, omenta, abdominal wall, liver, lungs, bones, and spleen. Whole-body exposure to gamma or X-radiation increased both the number of tumors and metastases in male inbred guinea pigs but not in females. Significantly fewer (9 of 98) noninbred than inbred guinea pigs developed gallbladder carcinomas after irradiation. In 9 untreated noninbred guinea pigs gallbladder carcinomas were not found. Inasmuch as the effect of irradiation was not dose-dependent, an indirect systemic effect of irradiation was postulated. This is the first report on the occurrence of spontaneous gallbladder adenocarcinomas in guinea pigs.

Phytohemagglutinin (PHA)-stimulated peritoneal exudate (PE) cells from strain-2 guinea pigs were more cytotoxic in culture to syngeneic tumorigenic cells than to syngeneic nontumorigenic cells. Cytotoxicity was measured by the release of 3H-thymidine from prelabeled target cells. Tumor-producing guinea pig fetal cells transformed in culture by chemical carcinogen released up to 70 percent of their label in the presence of PHA-stimulated PE cells. Non-tumor-producing cells, regardless of their morphologic characteristics, were less affected by PHA-stimulated PE cells. Nontumorigenic cultures included untreated early passage and long-term cultures previously treated with a carcinogenic or noncarcinogenic chemical. Differential cytotoxicity to tumorigenic cultures were best distinguished from nontumorigenic cultures when 0.5-1 times 10-6 PE cells and 50-100 mug PHA were incubated with 1 times 10-4 3H-thymidine-labeled target cells for 48 hours.

The growth of line-10 hepatoma in male Sewall Wright strain 2 guinea pigs was totally suppressed when tumor cells were mixed with heat-killed Candida albicans. In a significant number of animals, injection of C. albicans into established tumors 10-12 mm in diameter caused complete, rapid tumor regression. Guinea pigs whose tumors regressed or were suppressed as a result of injection of C. albicans rejected subsequent challenges at distant sites with the line-10 hepatoma, which indicated the development of systemic immunity to the tumor. Untreated control guinea pigs had positive delayed hypersensitivity reactions to intradermally injected C. albicans, which suggested prior natural exposure of the animals to C. albicans antigens. Peritoneal macrophages from mice that had received ip injections of phosphate-buffered saline (PBS) or C. albicans were not cytocidal for mouse 3T12 tumor cells in vitro. However, macrophages from the mice given injections of C. albicans, unlike those from mice given PBS, were markedly tumoricidal in the presence of 1 ng or more endotoxin/ml in vitro. These results demonstrated that heat-killed C. albicans, when inoculated into the peritoneal cavity, increased the tumoricidal potential of peritoneal macrophages.