A bispecific antibody prolongs survival in mice bearing lung metastases of syngeneic mammary adenocarcinoma

In the present study we tested whether T cells retargeted witha bispecific antibody (bsAb) could block the growth of lungmetastases of syngeneic mammary adenocarcinoma in immunocompetentmice. BALB/c mice were injected i.v. with tumor and i.p. witha genetically engineered bispecific F(ab')₂ [bs(Fab')₂] havingspecificity for murine CD3 chain and for the gp52 mouse mammarytumor viral glycoprotein, which is expressed on the tumor cells.The bs(Fab₂) was physically stable in blood and serum, was removedfrom the body with a half-time of 12–15 h, and accumulatedin lymphoid tissue where it bound to T cells. We show that treatmentof tumor bearing mice with the bs(Fab')₂ significantly prolongedtheir survival relative to untreated controls. Two other geneticallyengineered bs(Fab'₂S having specificity for murine CD3 chainand irrelevant antigens did not inhibit tumor growth. In addition,survival was not affected by bsAb therapy using a variant tumorcell line that expressed low levels of the gp52 target antigen.Inhibition of tumor growth was even more evident by histologicanalysis. Treatment with the relevant bs(Fab^')₂ resulted ina marked reduction of tumor burden in lung sections taken ondays 7, 9 and 11. This is the first report demonstrating thata bsAb can inhibit the growth of syngeneic solid tumor metastasesin mice without addition of T cell activators.     

Age related differences in immunocompetence and incidence of mammary adenocarcinoma in murine mammary tumor virus-infected C3H/Bi mice

In breeder C3H/Bi female mice, infected neonatally by murine mammary tumor virus (MTV), the incidence of spontaneous mammary tumors is greater than 95% between 5 and 9 months of age. In young (2–3 months) female the probability for developing a tumor in the next month is negligible, higher than 80% in mice of middle age (5–6 months) but lower than 4% in aged (10–12 months) females. The age-related changes of some immune functions of spleen cells from these tumor free female mice have been evaluated. While the proliferative capacity of cells to Phytohemagglutinin (PHA) increases, the T cell-dependent antibody response against sheep red blood cells (SRBC) and the antibody-dependent cellular cytotoxicity (ADCC) are significantly decreased in 5–6-month-old mice as compared to the young (2–3 months) female mice.The antibody response against SRBC and the mitogenic response to PHA decline markedly in 10–12-month-old mice but the ADCC increases in this group of mice. In addition, assays with monoclonal anti-Lyt-1 and anti-Lyt-2 antibodies indicate that percentage of Lyt 1⁻ 2⁺ cells (suppressor and cytotoxic T cells) is lower in 10–12-month-old female as compared to 5–6-month-old animals. These results show that the immune alterations observed in 10–12-month-old C3H/Bi mice are not closely associated with an increase in incidence of spontaneous tumors and suggest that a high non-T killer cell activity could protect some of these older C3H/Bi female mice against mammary tumor development.     

Spontaneous mammary carcinoma in C57BL and C3H mice: a histochemical study.

The mucosubstances in a series of 12 mammary carcinomas arising in C57BL mice were compared with those in tumours in nine C3H mice and with normal "resting" and lactating glands. A strain difference rather than a difference according to tumour type was found. The epithelial elements in the C57BL tumours contained mainly sialomucin with a little neutral mucosubstance and sulphomucin was present in some. Sulphated mucopolysaccharide was prominent in the stroma of these tumours. The C3H mice tumours contained no sulphomucin, less sialomucin but more neutral mucosubstance. The mucosubstances were studied in serial transplants of four tumours. An acid to neutral mucosubstance change with dedifferentiation of the tumour has been observed and found to be similar to a series of changes previously observed in a group of human gastrointestinal tumours.     

Comparison of thymocyte and T lymphocyte gangliosides from C3H/HeN and C3H/HeJ mice

Gangliosides have been prepared from resting murine thymocytes and splenic T cells. Profoundly different two-dimensional thin layer chromatography (2D TLC) patterns were observed between these two cell types. Thymocytes contained 28-30 discrete gangliosides of which eight represented major gangliosides. Splenic T lymphocytes from both strains had much simpler patterns, with six to seven major gangliosides and 12-13 minor gangliosides. Computerized analysis of the thymocyte ganglioside patterns between LPS-responder C3H/HeN mice and lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice revealed no significant difference in the major gangliosides. However, with splenic T cell gangliosides, there is a striking difference in the relative proportion of three homologous gangliosides between the two strains. Consistent with previous observations on macrophage gangliosides, the ratio of N-acetylneuraminic acid-containing ganglioside to N-glycolylneuraminic acid-containing ganglioside was higher in both thymocytes and T-cells from the LPS-responder strain. These results show that sialic acid-containing glycolipids from thymocytes and T lymphocytes between endotoxin responder and hyporesponder strains manifest small but significant changes. These differences are present in unstimulated cell populations and may represent a manifestation of the Lps gene.     

Normal inhibition of mammary tumor metastasis in C3H/He mice

The blood of normal C3H mice contains molecular factors that can rapidly lyse mammary carcinoma cells that enter the circulation. The cytotoxic effect of blood on tumor cells during incubation was most clearly demonstrated if fibrin formation was prevented. The conversion of the mammary carcinoma cells to ascites growth increased both the resistance to lysis in plasma and the ability to form lung colonies after intravenous injection. Mice that were surgically cured of numerous 9 mm tumors and survived for an average 10 months had a lower incidence (36%) of spontaneous metastases than mice that experienced one 30 mm tumor and survived for an average 8 weeks (65%). This is interpreted to mean that mice that retained their vitality by early surgical cures, could suppress the development of metastases by the action of normal serum factors that can destroy neoplastic cells that enter the circulation.     

Decreased histamine content and metabolism in mammary cancer tissue from C3H mice

Interleukin-8 (IL-8), is a potent activator of polymorphonuclear leukocyte (PMN) functions including chemotaxis, superoxide anion production, and enzyme release and it is also chemotactic for lymphocytes. Additionally, it has recently been shown that IL-8 stimulates the formation of 5-lipoxygenase (LO) products of arachidonic acid (AA) by human PMNs. The purpose of the present study was to determine whether IL-8 also might affect the formation of 15-LO products from AA. Purified PMNs in phosphate buffered saline were preincubated with and without exogenous AA (10^–5–10^–4 M) for 10 min. Then IL-8 was added in biologically relevant concentrations ranging from 0.1 to 100 ng/ml and incubation was carried out for 5 min at 37°C. Lipids were then extracted from supernatants, and eicosanoids were determined by quantitative RP-HPLC. Compared with unstimulated cells, IL-8 resulted in a dose dependent increase in both LTB₄ and 15-HETE (up to 125% and 40% at 100 ng/ml, respectively). This increase in eicosanoid formation required the presence of exogenous AA. These results indicate that IL-8 is both a potent stimulator of 5-LO activity and of 15-LO activity. LTB₄ can induce both inflammation and contribute to hyperproliferation in the skin. 15-HETE in contrast has the ability to inhibit the effects induced by LTB₄. Because IL-8 is able to stimulate both LTB₄ and 15-HETE formation, the effect of IL-8 as a putative regulator of inflammatory processes may be dependent on the relative stimulation of 5-LO and 15-LO.Part of this work has been presented at the Annual Meeting of the European Society for Dermatological Research (E.S.D.R.), Turin, Italy, June, 1990.     

Histamine in C3H/W mice carrying spontaneous tumors of the mammary gland

Adenocarcinoma mammae, a spontaneously growing mammary cancer in C3H/W mice, contrary to many transplanted tumors does not evoke any rise in histamine level either in the tumor or in distant tissues. On the other hand, the histamine level is reduced by 90% in the tumor in comparison with the healthy gland. This seems to be a consequence of the fall of histidine decarboxylase activity to below a detectable level. There is also a significant reduction in histamine N-methyltransferase activity to onefifth of the control level. The healthy mammary gland contains a high concentration of histamine and catabolizes it exclusively through the methylation pathway.     

Immunotherapy of mammary adenocarcinoma metastases in C3H/HeN mice with chronic administration of cyclo-oxygenase inhibitors alone or in combination with IL-2

In this study the efficacy of treatment of two cyclo-oxygenase inhibitors, ibuprofen (Ibu) and indomethacin (Indo), are compared in the immunotherapy of metastasis designed to reverse prostaglandin E₂ (PGE₂)mediated inactivation of interleukin-2 (IL-2)-dependent host killer cell lineages. These agents were tested either alone for the prevention of metastasis or in combination with IL-2 for the eradication of established metastasis. C3H/HeN mice were placed on chronic oral Ibu (CIbT; 200 and 600 ,g/ml of water) or Indo (CIT; 10 g/ml) 5 days after s.c. transplantation of 5 × 10⁵ metastatic C3L5 mammary carcinoma for the prevention of spontaneous lung metastases. They showed intolerance to Indo at a dosage of 14 g/ml, which was well tolerated by other mouse strains in previous studies, but tolerated the Ibu dosages used. Control and treated mice were killed on day 30 to score metastatic lung colonies, to evaluate killer activity in splenocytes against natural killer (NK)-sensitive YAC-1 lymphoma or NK-resistant C3L5 adenocarcinoma and 8911 lymphoma targets, and to phenotype the surface markers of killer cells. CIbT and CIT alone at the above dosage significantly reduced the number of lung colonies, retarded local tumor growth and restored NK activity of splenic killer cells expressing AGM-1⁺, Thy-1^–, Lyt-2^– phenotype. To treat established lung metastasis, mice bearing 15-day C3L5 transplants were given CIbT or CIT alone or in combination with two 4-day rounds (days 20–23, 31–34) of IL-2 (15 000 Cetus units, i.p. every 8 h) and were killed on day 35 to score lung colonies and characterize splenic killer cells. CIbT or CIT alone reduced the number of spontaneous lung metastases and restored anti-YAC-1 killer function of splenocytes with NK-like phenotype (AGM-1⁺, Thy-1^–, Lyt-2^–); some anti-C3L5 killer function was also generated in the high dose Ibu group and the killer cell showed AGM-1⁺, Thy-1⁺ and Lyt-2⁺ phenotype. Combined therapies with CIbT or CIT plus IL-2 were more effective in reducing metastases and promoting killer cell function, the best results being achieved with high dose Ibu + IL-2. All killer cells expressed AGM-1 and Thy-1. In addition, C3L5 killer cells also expressed Lyt-2, suggesting T-cell stimulation. PGE₂ synthesis in the host was inhibited by at least 50% in mice subjected to CIbT or CIT. Thus, Ibu proved to be an excellent substitute for Indo in preventing metastasis and NK cell activation when given alone, and also in ameliorating established metastasis and activating lymphokine-activated killer cells when combined with IL-2.     

The effect of cimetidine on platelet function: a study involving gastric fluid measurements

Summary Gastric fluid samples were aspirated 30 and 60 minutes after the ingestion of two 200 mg tablets of cimetidine. The concentration of cimetidine in these samples was measured and their effect on platelet aggregation assessedin vitro. Gastric fluid samples significantly inhibited adrenaline- and ADP-induced platelet aggregationin vitro. In a further series of experiments, cimetidine solutions, at concentrations found in gastric fluid, inhibited platelet aggregation and thromboxane A₂ (TXA₂) release,in vitro. Ranitidine, another H₂-receptor antagonist, was a more potent inhibitor of platelet aggregation than cimetidine. Since ranitidine is also the more potent H₂-receptor antagonist which, unlike cimetidine, does not include an imidazole group (which is known to inhibit TXA₂ synthesis) in its structure, we conclude that H₂ blockade mediates the observed inhibition of aggregation. This platelet anti-aggregatory effect may be relevant to haemostatic mechanisms involved in bleeding peptic ulcers or gastric erosions exposed to high local concentrations of H₂-receptor antagonists.     

The kinetics of recovery of leukocyte number and lymphocyte function following an injection of a single high dose of cyclophosphamide in C3H/HeJ mice

The short-term effects of the immunosuppressive drug cyclophosphamide (Cy) are well-documented, but the long-term consequences are not as well studied. Here we report on the kinetics of leukocyte number and immune function recovery following a single intraperitoneal injection of 100 mg/kg or 300 mg/kg Cy. The leukocyte number in spleen, lymph node, bone marrow and thymus was unchanged in C3H/HeJ mice injected with the lower dose of Cy, and was severely but transiently depressed in mice injected with 300 mg/kg Cy. Recovery was complete by day 21. Humoral immunity was unchanged with the lower dose of Cy; mice injected with 300 mg/kg recovered normal antibody production after 3-5 weeks. Natural killer cell function was also transiently depressed in animals receiving the higher but not the lower concentration of Cy. Surprisingly, splenic mitogen responses were markedly inhibited in mice injected with either concentration of Cy. The T-cell proliferative response remained depressed even after 5 weeks in mice injected with 300 mg/kg Cy. Lymphocyte subpopulations in spleen were examined by flow cytometry, and, although some deviations were observed, it is unlikely that these changes are responsible for the highly depressed mitogen response. Thus, there is a loss of the proliferative response to mitogens in Cy-treated mice long after recovery of other lymphocyte functions.