Heterogeneity of rabbit IgM antibody as detected by C'1a fixation.

The C'1a-fixing properties of purified rabbit IgM anti-benzenearsonate antibody were determined. When tested with sheep erythrocytes to which hapten had been coupled by diazo linkage, the number of C'1a molecules fixed was 21% of the number of IgM antibody molecules bound to the erythrocyte surface. This was not due to loss of C'1a-fixing capacity during the purification procedure. Preparative electrophoresis of the antibody concentrated C'1a-fixing molecules in the anodal region so that antibody fractions with greater C'1a-fixing capacity were obtained. The demonstration that C'1a fixation is a property of a subpopulation of IgM molecules provides evidence for previously unrecognized micro-chain heterogeneity.

Synthesis of the first component of human complement in vitro.

Isolated segments of human colon and to a lesser extent ileum were capable of synthesizing hemolytically active C'1. This conclusion was based on the following evidence: After elimination of C'1 from tissue with EDTA, we found that segments of the intestinal tract in short-term organ culture showed a 50-1000-fold increase in C'1 activity. The rate of production of C'1 in human intestine was highly temperature dependent; C'1 production was reversibly inhibited by puromycin and actinomycin D. Furthermore, (14)C-labeled amino acids were incorporated into molecules which behaved like C'1. No significant C'1(hu) synthesis was observed in isolated segments of jejunum, stomach, liver, kidney, lung, spleen, lymph node, and thymus.

Mouse complement: the effect of sex hormones and castration on two of the late-acting components.

The titer of late-acting complement components in sera from male mice is 8-10 times higher than the titer of sera from female mice. Using assays developed to measure the serum content of two of the late-acting components, we have shown that this difference is due to the effect of androgen and estrogen on these two late-acting complement components. These two components have been tentatively identified as C'5 and C'6. Androgen and estrogen have greater effect on C'6 than on C'5. The possibility has not been excluded that still other of the late-acting complement components are affected by androgens and estrogens. The course of homograft rejection was unchanged in mice deficient in C'5 and C'6.

Treatment by limited surgery and specific immunization of guinea pigs with stage II experimental cancer.

The malignant disease produced in guinea pigs by intradermal inoculation of line-10 was allowed to progress to stage II, at which time the dermal tumor and the first draining lymph node were grossly evident. At that stage, the external appearance of the next draining lymph node was normal, but it contained tumor cells. Limited surgery consisting of excision of the dermal tumor and first draining lymph node was not curative; palpable metastases developed in the second and other draining lymph nodes, and at autopsy, some animals were found to have gross, visible lung metastases. Immunization of guinea pigs with a mixture of irradiated syngeneic tumor cells plus mycobacterial cell walls in an oil-in-water emulsion eradicated tumor cells remaining in lymph nodes after limited surgery for stage II experimental cancer and prevented progression of the disease to stage III. Tumor intravenously implanted in the lungs of animals after limited surgery for stage II disease was also eliminated by immunization.

Efficiency of the first component of complement (C'1) in the hemolytic reaction.

With the use of the first component of guinea pig complement (C'1) labeled in vivo with (14)C-amino acids, we have obtained evidence that, under the conditions required for the assay of C'1, each molecule of C'1 capable of interaction with cell surface antigen-antibody complexes is capable of initiating the reaction sequence that leads to lysis of the cell.

Tumor immunity produced by the intradermal inoculation of living tumor cells and living Mycobacterium bovis (strain BCG).

The intradermal inoculation of mixtures containing living tumor cells and living Mycobacterium bovis (strain BCG) into unimmunized syngeneic guinea pigs results in an inflammatory reaction to the BCG, and there is no progressive tumor growth. In the absence of BCG the tumor grows progressively, metastasizes, and kills the animal. By conventional methods, it has not been possible to immunize syngeneic guinea pigs to the tumor used. Guinea pigs that receive mixtures of BCG and tumor cells, however, develop specific systemic tumor immunity as measured by delayed cutaneous hypersensitivity and by suppression of tumor growth.

Tumor immunity: tumor suppression in vivo initiated by soluble products of specifically stimulated lymphocytes.

Supernatant fluids of specifically stimulated lymphocyte cultures were purified. Fractions containing migration inhibition factor when injected intra-dermally into strain-2 guinea pigs produced a reaction similar in appearance to delayed cutaneous hypersensitivity. There was an accumulation of mononuclear cells at the injection sites and the growth of syngeneic tumor grafts at the sites was suppressed.

Mouse complement: influence of sex hormones on its activity.

Sex hormones influence the hemolytic of one or more of the late-acting components of complement measured in the presence of trisodium ethylenediaminetetraacetate. The titers of the serums of male mice, normally tenfold higher than those of females, fell after castration, becoming about the same as those of females. The titers of the serums from females rose after these mice were castrated, but castration did not affect the activities of the first, second, and fourth components of complement. Serums of normal and castrated mice of both sexes treated with testosterone showed increased late-acting component activity, whereas the estrogen caused decreased activity. Treatment in vitro of mouse serum with these hormones had no effect on the activity of late-acting components.

Neutralization of sensitized virus by the fourth component of complement.

Herpes simplex virus which had been sensitized with IgM antibody was not neutralized by the addition of the purified activated first component of complement. In the presence of an optimum concentration of the first component of complement, however, the sensitized virus was neutralized by the addition of a high concentration of the purified fourth component of complement. Under these conditions, the addition of the purified second and third components of complement failed to enhance virus neutralization. With low concentrations of the fourth component of complement, the addition of the second and third components enhanced virus neutralization.

Tumor-specific antigens detected by inhibition of macrophage migration.

Tumor-specific antigens of a guinea pig hepatoma induced by diethylnitrosamine were detected by the inhibition of migration of specifically sensitized macrophages from capillary tubes, and by the local passive transfer of delayed skin hypersensitivity and the suppression of growth of intradermally same injected tumor.

Immunotherapy of cancer: an experimental model in syngeneic guinea pigs.

Successful treatment of a solid tumor was accomplished by repeated intradermal injection of living tumor cells.

Local tumor regression after intralesional injection of croton oil.

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.

Local antitumor activity of a primary and an anamnestic response to a syngeneic guinea pig hepatoma.

After intradermal (id) injection, the line-10 hepatoma grew progressively in nonimmune guinea pigs, whereas the line-1 hepatoma grew for approximately 2 weeks, developed central necrosis, ulcerated, and regressed. Growth of the line-10 hepatoma was suppressed when line-10 hepatoma cells were mixed with antigenically distinct line-1 hepatoma cells before id injection into syngeneic strain-2 guinea pigs. Mixture of line-10 with irradiated line-1 or viable strain-2 embryo cells did not inhibit tumor growth. Preimmunization of recipients to line-1 cells abrogated the suppression of tumor growth from mixtures of line-1 and line-10.

Suppression and regression of a transplanted tumor in the guinea pig colon mediated by Mycobacterium bovis, strain BCG.

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.

Immunotherapy of metastatic cancer in guinea pigs: failure of intralesional BCG to influence the results of radical surgery.

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.

Aryl hydrocarbon (benzo[a]pyrene) hydroxylase in guinea pig lymphoid tissue.

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.

Forssman antigen content of guinea pig hepatomas induced by diethylnitrosamine: a quantitative approach to the search for tumor-specific antibodies.

The Forssman antigen content of diethylnitrosamine-induced guinea pig hepatomas was found to be greater than that of either autogenous or allogeneic guinea pig liver. The autogenous normal liver was obtained from guinea pigs before tumor induction, and comparison of normal and neoplastic tissues was based on the capacity of these tissues to inhibit (absorb) the hemolytic activity of rabbit antitumor serum. A method is presented for distinguishing quantitative from qualitative antigenic differences in the search for tumor-specific antigens. The results of these experiments indicate that, in the absence of strict quantitation, quantitative differences may be mistaken for qualitative differences.

Extraction of tumor-specific antigen from cells and plasma membranes of line-10 hepatoma.

Tumor-specific antigen was extracted with 3 M KCl from line-10 guinea pig hepatoma cells. The yield of antigenic activity, estimated by production of delayed cutaneous hypersensitivity reactions in line-10 immune guinea pigs, was 10-30% of the antigen present in intact cells. By ultracentrifugation criteria, the extracted antigen was soluble. Gel filtration, ion exchange chromatography, and salting-out studies showed that the antigen was heterogeneous in size and net charge. The possibility that 3 M KCl extracted a homogeneous population of molecules associating into polymers of various sizes at low ionic strength was ruled out by heterogeneity on Sephadex G-200 chromatography at high ionic strength. After osmotic lysis of sucrose-loaded line-10 cells, whole plasma membranes or large membrane fragments were obtained in a yield of about 20%. The isolation procedure did not cause detectable loss of membrane antigenic activity. The membranes had 33 skin test U/mg membrane protein, compared to the intact cell value of 1.7 skin test U/mg cell protein. Extracts of plasma membranes had 10-20% of the antigenic activity of the starting membrane material. In contrast to the wide variety of proteins liberated from intact cells, much of the protein extracted from the membranes was in the molecular weight range above 250,000.

Protective tumor immunity induced by potassium chloride extracts of guinea pig hepatomas.

Tumor-specific antigens of cells of the diethylnitrosamine-induced hepatomas in strain-2 guinea pigs were extracted with 3 M KCl. Immunization of normal animals with the extracted tumor antigens in adjuvant protected them against a subsequent challenge with viable tumor cells. Extracted tumor-specific antigens were less effective immunogens than viable tumor cells for both of two antigenically distinct lines.

Eradication of microscopic lymph nodes metastases after injection of living BCG adjacent to the primary tumor.

Guinea pigs with established intradermal tumors and microscopic axillary lymph node metastases were treated with a combination of surgery and BCG. The tumors were excised and BCG was given in attempts to eliminate residual malignant disease. Injection of BCG into established intradermal tumors 7 days before local excision successfully eradicated microscopic axillary lymph node metastases and cured significant numbers of animals. Injection of BCG into dermal tumors 20 minutes or 1 day before excision prolonged survival but did not cure a significant number of animals. Injection of BCG into the skin adjacent to the dermal tumor 7 days before local excision eradicated microscopic axillary lymph node metastases. However, such injection 1 day before local excision did not eradicate metastases. BCG administered by intravenous, intra-arterial, or intranodal injection did not eliminate reidual malignant disease. Several factors were evaluated as possible correlates of successful immunotherapy. The development of tuberculin hypersensitivity, the magnitude of regional adenopathy, and the number of BCG organisms in axillary nodes were not useful correlates. Histologically, the presence of tumor cells, multiple focal granuloma, or histiocytosis in axillary nodes faiiled to correlate with results of therapy. The development of tumor-specific transplantation immunity provided the best correlate of successful immunotherapy.