Eradication of microscopic hepatic metastases by active specific immunization.

Strain-2 guinea pigs, each with microscopic deposits of line 10 hepatocarcinoma in the liver, were treated by ID immunization with a mixture of irradiated tumor cells and an oil-in-water emulsion containing cell walls of Mycobacterium bovis strain Bacillus Calmette-Guérin (BCG CWE). Injection of line 10 hepatoma cells into the hepatic portal vein led to the development of tumor foci in the liver, metastasis in the hepatic lymph node, malignant ascites, and death. Active immunization using irradiated line 10 cells and BCG CWE was effective therapy when administered 1, 7, and 14 days after intraportal injection of line 10 cells. Effective immunization required both irradiated line 10 tumor cells and the BCG cell wall emulsion. Immunization with BCG CWE admixed with irradiated line 1 tumor cells, a hepatoma antigenically distinct from line 10, did not prevent outgrowth of line 10 deposits in the liver. Animals rendered free of disease could reject a challenge of line 10 tumor cells but not of line 1 tumor cells.

Mechanisms of immunological eradication of a syngeneic guinea pig tumor. II. Effect of methotrexate treatment and T cell depletion of the recipient on adoptive immunity.

The influence of methotrexate on the development of immunity to the line 10 hepatoma was studied in guinea pigs. Chronic methotrexate treatment had no apparent effect on the ability of immune guinea pigs to suppress the growth of inoculated tumor cells. In contrast, the same methotrexate regimen inhibited the development of tumor immunity if started before the 8th day after immunization with a vaccine containing viable line 10 cells admixed with Bacillus Calmette-Guérin (BCG) cell walls. Thus, methotrexate selectively inhibited the afferent limb of the immune response. In adoptive transfer experiments, methotrexate-treated recipient guinea pigs were capable of being passively sensitized with immune spleen cells, indicating that the primary cell-mediated immune response of the recipient was not required for adoptive immunity. The contribution of recipient T cells in adoptive immunity was further investigated in guinea pigs deleted of T cells by thymectomy, irradiation, and bone marrow reconstitution. Despite demonstrable deficiency in T lymphocyte reactions, "B" animals were fully capable of rejecting tumors after transfer of immune cells. These results suggest that the expression of adoptive immunity was independent of recipient T cell participation. In addition, sublethal irradiation of immune spleen cells prior to adoptive transfer abolished their efficacy. Proliferation of transferred immune cells in the recipient may be essential for expression of adoptive immunity.

Mechanisms of immunologic eradication of a syngeneic guinea pig tumor. I. Quantitative analysis of adoptive immunity.

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.

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

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

Adoptive immunity to the guinea pig line 10 hepatoma and the nature of in vitro lymphoid-tumor cell interactions.

Adoptive transfer of spleen cells from specifically immunized donors to nonimmunized recipients was used to study tumor immunity in vivo to the syngeneic line 10 guinea pig hepatoma. Hepatoma cells cultured as monolayers on fibronectin-coated surfaces served as targets for immune splenocytes in a 3H release cytotoxicity assay in vitro. An antigenically distinct syngeneic guinea pig hepatoma (line 1) was used to study the specificity of adoptive systemic immunity and of the cytotoxicity in vitro. The protection afforded by adoptive immunization against challenge with hepatoma cells was tumor line specific, while in most cases cytotoxicity in vitro was not. The in vitro cytotoxic effect was abolished after absorption of the immune spleen cells with monolayers of either line 10 or line 1. In contrast, the in vivo tumor-specific rejection activity of line 10 immune spleen cells was depleted after absorption with line 10 but not with line 1 or other control monolayers. These studies revealed that the immune cells mediating cytotoxicity in vitro were functionally distinct from those conveying adoptive protection in vivo. Immune cells possessed receptors for tumor-specific rejection antigens on hepatoma cells, and their interaction did not lead to destruction of the neoplastic cells in vitro.

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.

Plasma concentrations of progesterone, oestrogens and prostaglandin F in maternal blood and corticosteroids and oestrogens in foetal blood of cows during dexamethasone-induced deliveries.

Infusing dexamethasone at rates of 0.1, 1.0 or 10 mg/day into 6 foetal calves at day 240 of gestation (2 calves per infusion rate), induced premature calving in a mean 12, 9 and 3 days respectively. Maternal plasma concentrations of progesterone, oestrone, oestradiol-17 beta and prostaglandin F or 13,14-dihydro-15-keto-prostaglandin F, and foetal plasma concentrations of oestrone, oestradiol-17 beta and corticosteroids were monitored until calving. The maternal hormone changes observed for all cows were a decline in progesterone, and a rise in both oestrogen and prostaglandin F concentrations before calving. Dexamethasone infused at a rate of 1.0 and 10.0 mg/day suppressed plasma corticosteroids in the foetus; however at 0.1 mg/day, foetal corticosteroids increased tenfold during the last 3 days before calving. At the lowest rate of infusion, dexamethasone had no effect on foetal oestrogen levels, but at rates of 1.0 and 10.0 mg/day there was a marked rise in foetal plasma levels of oestrogen.

Dexamethasone concentrations in plasma and milk of cows following the injection of long- and short-acting dexamethasone esters.

A radioimmunoassay has been developed for the measurement of dexamethasone in plasma and milk of cows injected with long- and short-acting dexamethasone esters. Dexamethasone antiserum was produced by injecting cows with a dexamethasone-21-hemisuccinate-human serum albumin complex. The antisera was highly specific for dexamethasone, cross-reacting less than 0.7% for all endogenous steroids tested. Plasma concentrations of dexamethasone in cows injected intramuscularly with either 20 mg dexamethasone-21 trimethyl acetate (n = 2) or the tributyl derivative (n = 2) reached a peak level of 0.6-1.1 ng/ml in 2-6 days then declined to undetectable levels (less than 0.15 ng/ml) and 14 days after injection. In general, dexamethasone concentrations in milk were 0.3-0.5 times the plasma concentrations but showed the same pattern of values. Plasma dexamethasone concentrations were also determined in three lactating dairy cows injected intramuscularly with tritiated dexamethasone-21 trimethyl acetate. In these cows plasma dexamethasone concentrations, as determined by isotopic dilution, reached maximal levels of 1.1-1.6 ng/ml in 1-3 days then declined to levels of around 0.05 ng/ml within 30 days. The concentrations of dexamethasone in milk of two of these cows were, in general, similar to those found in plasma. In three cows injected intramuscularly with 20 mg dexamethasone sodium phosphate the concentrations of dexamethasone in plasma rose sharply to maximum levels of 24-70 ng/ml within 2-20 min after injection and fell to undetectable levels (less than 0.15 ng/ml) after 72 hr.

Adjuvant-antigen requirements for active specific immunotherapy of microscopic metastases remaining after surgery.

We studied the conditions required for eradication by immunization of occult lymph node metastases which remained after surgical removal of an intradermally transplanted cavian hepatoma. Guinea pigs that received no postsurgical treatment all died with progressively growing lymph node metastases. The growth of these metastases could be prevented in a significant proportion of the animals by postsurgical treatment with vaccines containing oil-in-water emulsions of Mycobacterium bovis strain Bacillus Calmette-Guérin (BCG) cell walls admixed with live or irradiated tumor cells. Vaccines containing living tumor cells cured most of the guinea pigs but produced tumors at the vaccine sites in a few animals. Irradiated tumor cell vaccines were not tumorigenic but required more tumor cells for successful therapy. Therapy was dependent both on the dose of tumor cells and on that of BCG cell walls. Microgram doses of BCG cell walls were required for a therapeutic effect; milligram doses of BCG cell walls inhibited the therapeutic response. Animals rendered tumor free by postsurgical vaccine therapy rejected an intradermal challenge with living tumor cells.

Immunotherapy of guinea pigs with dermal and visceral tumor implants: comparison of living and nonliving BCG.

Emulsified cell walls of Mycobacterium bovis (BCG) were immunotherapeutically at least as active as living BCG in prolonging survival of guinea pigs with established dermal tumors and microscopic lymph node and visceral metastases.

Immunotherapy of bilateral lymph node metastases in guinea pigs by intralesional or paralesional injection of Mycobacterium bovis (BCG).

Bilateral axillary lymph node metastases occurred after intradermal (id) injection of line 10 hepatocellular carcinoma cells over the thoracic spine of inbred guinea pigs. Excision of the dermal tumor 7 days after injection of tumor cells did not prevent the development of metastases. Injection of BCG into dermal tumors without surgery led to their regression and prevented the growth of microscopic metastases in both right and left superficial distal axillary lymph nodes. Bilateral id injection of BCG between the dermal transplant and each of the regional lymph nodes followed by excision of the dermal tumor also prevented progression of metastases. Unilateral id injection of BCG before excision of dermal tumors failed to retard metastases in contralateral superficial distal axillary lymph nodes. These results suggested that elimination of microscopic lymph node metastases required delivery of adjuvant to or near each metastatic site. Systemic tumor immunity alone may be inadequate to eradicate lymph node metastases.

Glucan: attempts to demonstrate therapeutic activity against five syngeneic tumors in guinea pigs and mice.

Animals with established syngeneic tumor transplants were treated with glucan to study the therapeutic potential of this agent under well-defined experimental conditions. The tumors used were a guinea pig hepatoma, 2 murine fibrosarcomas, a murine melanoma, and a murine adenocarcinoma. All tumors were syngeneic to the host. Living BCG, administered directly into guinea pig tumors, cured all animals, whereas glucan, administered under the same conditions, had no significant antitumor activity. Neither BCG nor glucan, when administered iv, was active against the guinea pig hepatoma. An emulsion prepared with endotoxin, a fraction of mycobacteria related to cord factor, and mineral oil when administered intratumorally was also effective in treatment of line 10 tumor. A similar emulsion, in which glucan was substituted for endotoxin, was inactive, intralesional, ip, or iv administration of glucan was ineffective against the murine tumors. Previous reports of glucan-induced activity against a B16 murine melanoma were not confirmed. BCG was tested against the 2 murine fibrosarcomas and, when given either intratumorally or iv, was found to be effective against one of them.

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.

Fetal and maternal plasma electrolytes, blood gases, and pH in dairy cows during late gestation.

Sodium, potassium, and chloride were determined in fetal and maternal plasma of six cows between 208 and 269 days of gestation. Blood pH and partial pressures of oxygen and carbon dioxide also were measured in samples collected from the jugular vein of the cow and the caudal aorta and vena cava of the fetus. Concentration of chloride in fetal plasma was generally lower than the corresponding maternal value, and fetal potassium was greater than maternal. Potassium of fetal plasma increased towards term. Sodium concentrations were not significantly different between fetal and maternal plasms. Once fetal and maternal pH and blood gas tensions had stabilized after surgery, there was little change until shortly before delivery. In the last 24 h before calving maternal partial pressure of oxygen was high and fetal blood pH was low.

Fetal and maternal mineral concentrations in dairy cattle during late pregnancy.

Concentrations of ionic and total calcium, magnesium, and inorganic phosphorus in plasma were determined in bovine fetuses and their mothers within the last month before delivery. Mean fetal concentrations were higher than corresponding maternal contents during the 22 days before delivery. Ionic and total calcium in plasma fell towards term in mother and fetus. Fetal phosphorus was elevated before parturition while maternal phosphorus fell. Maternal magnesium was elevated slightly at parturition.

Foetal and maternal hormonal changes preceding normal bovine parturition.

Successful chronic cannulation of the foetal posterior vena cava and maternal utero-ovarian and jugular veins in five Jersey cows between days 240 and 260 of gestation enabled changes in plasma hormone levels preceding calving to be monitored. All cows delivered live calves within the expected range of gestation for the breed. Corticosteroids were assayed by competitive protein-binding and prostaglandin F, progesterone, oestrone and oestradiol-17beta by radioimmunoassay. Foetal corticosteroids rose slowly from 5.0 +/- 0.7 ng/ml at 20 days to 9.3 +/- 3.0 ng/ml at 10 days before term, then progressively increased to a mean of 74 ng/ml, though higher concentrations occurred following surgery. Foetal oestrone and oestradiol-17beta concentrations were both less than 50 pg/ml and showed little change toward term. The maternal utero-ovarian oestrogens increased slowly from 20 to 10 days pre-partum and then rose more rapidly reaching peak levels (2.9 +/- 0.6 ng/ml for oestrone and 1.4 +/- 0.3 ng/ml for oestradiol-17beta) 1 to 4 days before delivery. Maternal progesterone concentrations fell towards term, with a rapid decrease over the last 36-48 h before calving when they gradually increased until the last 24 h where was a dramatic rise, reaching peak levels (5.7 +/- 0.6 ng/ml) during labour.