Effect of Wnt-1 antisense RNA on the outgrowth of a mammary adenocarcinoma cell line expressing that oncogene.

Aims-To investigate the effect of Wnt-1 antisense RNA on the outgrowth of a mammary tumour cell line expressing that oncogene.Methods-A plasmid (pMT 70), containing Wnt-1 cDNA, was cut with appropriate enzymes and inserted into a eukaryotic expression vector (pMAMneo). A mammary tumour cell line (CAC-L153) was transfected with the expression vector and cells with the vector in sense and antisense orientation were selected.Results-Tumour cells with the expression vector in the antisense orientation had a notable reduction in expression of Wnt-1 protein and a considerable reduction in tumour outgrowth compared with controls.Conclusions-The results indicate that the Wnt-1 proto-oncogene may be a possible target for antisense therapy.

Protection against a syngeneic mammary adenocarcinoma by administration of idiotypic and anti-idiotypic antibodies.

A hybridoma secreting a monoclonal antibody (mAb) with specificity for tumor-associated cell surface antigens of a transplantable murine mammary adenocarcinoma (SMC-168) was prepared by fusion of syngeneic C3H/He spleen cells with SP2 myeloma cells. Mice which were pretreated with this mAb (C-73) were significantly resistant to the outgrowth of a tumorigenic dose of SMC-168 cells when compared to controls. The treated mice developed tumor-specific cell-mediated immunity, measured by leukocyte adherence inhibition (LAI), which was equal to that of mice immunized with live tumor cells. The IgG fraction from serum of mice receiving mAb C-73 contained antibodies which would bind to that mAb suggesting the presence of anti-idiotypic antibodies (anti-Id). This binding could be partially inhibited by a soluble 1-butanol cell surface extract of SMC-168. Rabbits were immunized with mAb C-73 to produce a polyclonal anti-Id. The purified and absorbed IgG fraction of this serum would bind only to mAb C-73 and not to other mAbs of the same isotype or normal C3H/HeN IgG. Binding of the rabbit anti-Id to mAb C-73 could be partially inhibited by soluble tumor-associated antigen extracted from SMC-168. Mice immunized with this polyclonal anti-Id vaccine developed tumor-specific cell-mediated immunity and were significantly resistant to the outgrowth of a tumorigenic dose of SMC-168.

Extraction of immunogenic murine mammary tumor antigens with 1-butanol.

Solubilized tumor associated antigens (TAA) were prepared by a single phase 1-butanol extraction of a transplantable murine mammary adenocarcinoma (H2712), other syngeneic non-mammary tumors, normal mammary tissue, and embryonic tissues. The crude butanol extract (CBE) of H2712 was found to have tumor-specific activity both in vitro in the leukocyte adherence inhibition (LAI) assay and in vivo in an immunoprophylaxis assay. The failure to detect any of the major mammary tumor virus (MTV) envelope glycoproteins or internal core protein in CBE-H2712 and the failure of tumor-bearing mice to give a LAI response to CBE prepared from embryonic tissues suggests that the CBE antigens which are responsible for LAI activity are not viral or oncofetal antigens. Enzymatic treatment of the CBE-H2712 indicated that a sialated glycoprotein formed the antigenic determinants recognized in CBE by the LAI assay. Gel filtration of LAI-reactive CBE-H2712 on a Sephadex G-75 column isolated both the in vitro and in vivo tumor-specific activity in a fraction pool. Polyacrylamide gel electrophoresis revealed the presence of a 67 kD subunit as the major species in the active fraction.

A radio (111In) capillary tube leukocyte adherence inhibition assay for the detection of specific tumor-associated immunity.

The specific tumor-associated immune response of C3H/HeJ mice was determined at various times after subcutaneous injection with a transplantable mammary adenocarcinoma (H2712) using a radio (111In) leukocyte adherence inhibition (LAI) assay carried out in capillary tubes. Solubilized tumor-associated antigen prepared by a single phase 1-butanol extraction of the specific tumor and other transplantable tumors of different histological origin were used in the evaluation of LAI reactivity. The assay was found to be capable of detecting a significant antitumor response before the subcutaneous tumors became detectable by palpation. The response remained significant until the tumors were greater than 20 mm in diameter.

The early detection of host immune response to spontaneous mouse mammary tumors by leukocyte adherence inhibition.

Longitudinal testing of C3H/HeJ female mice was carried out over a 52-week period with the leukocyte adherence inhibition (LAI) assay using solubilized tumor antigen prepared by 3 M KCl extraction. During this period, 75% of the mice developed histologically confirmed mammary adenocarcinomas. Of those mice 93% had a statistically significant (P less than 0.01) LAI response to soluble mammary tumor antigen prior to first detection of tumors by palpation. Five percent of the test group developed statistically significant LAI responses without development of detectable mammary tumors within a 34-week observation period. The first significant LAI reactivity of individual mice preceded the first detection of tumors by an average of more than 17 weeks.

Lymphoid source and target of murine leukocyte adherence inhibition factor (LAIF).

The cellular source of murine LAIF detected by an indirect leukocyte adherence inhibition (LAI) assay using capillary tubes was investigated using non-inducing peritoneal cells (PC) from normal and spindle-cell sarcoma-bearing A/J mice. Cell populations containing greater than 95% T-cells, B-cells or macrophages were prepared from PC using a series of elimination and enrichment procedures. The in vitro incubation of enriched T-cell populations (less than 0.05% macrophages) from tumor-bearing mice with the specific soluble tumor antigen did not result in the release of detectable levels of LAIF; however, the addition of 10% normal isologous macrophages to the T-cells resulted in a significant release of LAIF. Enriched B-cell populations did not release LAIF either by themselves of when 10% normal isologous macrophages were added. Cell populations containing both T-cells and B-cells produced significant levels of LAIF, but only when suitable numbers of macrophages were present. Treatment with anti-Thy-1.2 alloantiserum and complement resulted in the abrogation of LAIF production by mixed cell populations. Using Lyt-1.2 and Lyt-2.2 alloantisera and complement to prepare either Lyt-1.2 or Lyt-2.2 depleted T-cell populations, it was found that the Lyt-1.2 subpopulation was responsible for the release of LAIF in this test system. LAIF was found to be effective in reducing the glass adherence of macrophages but not of T-cells or B-cells.

Use of capillary tube leukocyte adherence inhibition for the detection of tumor-specific immune responses.

The tumor-specific immune response of A/J mice was determined at various times after subcutaneous injection with a transplantable spindle-cell sarcoma (SaI) using a leukocyte adherence inhibition (LAI) assay carried out in capillary tubes. Solubilized antigens prepared by 3M KC1 extraction of the specific tumor, other transplantable tumors and normal A/J liver were used in the evaluation of LAI reactivity. The assay was found to be capable of detecting a significant antitumor response before the subcutaneous tumors became detectable by palpation. The response remained significant until the tumors were greater than 25 mm in diameter. Responsive cell populations did produce an active supernatant, indicating the possible presence of a lymphokine and/or specific cytophilic antibody.

Clinical resistance to vasopressin. Detection of antibody by hemagglutination.

We describe a patient with hypothalamic diabetes insipidus who after 20 years became refractory to the effect of commercial vasopressin injection. Vasopressin antibodies were measured using a sensitive hemagglutination technique. Resistance was associated with a high titer of antibodies that disappeared once vasopressin therapy was withdrawn and the diabetes insipidus was controlled with chlorpropamide. Antibodies were also measured in four additional patients with diabetes insipidus while they were or were not receiving vasopressin. A patient who had received the drug for only two years already had a substantial titer of antibodies to vasopressin, but in this case the response to the hormone was not impaired.

Thymectomy in the adult mouse: a new approach.

Adult mice were thymectomized using a retrosternal approach. The sternothyroid muscle was divided, the sternum was elevated, and the lobes of the thymus were removed by aspiration. The technique was used to thymectomize about 300 mice of several strains. The incidence of thymic remnants was less than 1% and losses from surgery were less than 10%.

Detection of Moraxella bovis antibodies in infectious bovine keratoconjunctivitis by a passive hemagglutination test.

A passive hemagglutination test was developed to detect antibody response to Moraxella bovis in tears. Tannic acid-treated sheep erythrocytes were sensitized with sonicated antigen prepared from M bovis cultures. The test was found to be a relatively simple, specific, and reliable procedure for titrating antibodies in lacrimal secretions. The hemagglutination test could be a valuable method for seroepizootiologic investigation of infectious bovine keratoconjunctivitis.