Immunoneutralisation of gonadotrophin releasing hormone: a potential treatment for oestrogen-dependent breast cancer.

The aim of this study was to assess the therapeutic potential of active immunisation with GnRH-glycys-PPD in a hormone-dependent experimental model. Mammary tumours were induced in female rats using dimethylbenzanthracene (DMBA) and the effects of GnRH immunoneutralisation on tumour development were evaluated. High titres of anti-GnRH IgG correlated with a decrease in oestrogen levels and subsequent tumour suppression. A comparison of immunised and non-immunised animals showed that when GnRH-specific IgG levels were at a maximum titre (80-100 micrograms/ml), nearly 10% of the GnRH-glycys-PPD treated animals showed mammary masses, compared with all the non-treated animals at the same stage in the study. When the antibody levels fell, tumour regrowth was observed, but to a level below that observed in the non-treated animals. Following further treatment with the analogue, the tumours regressed again, showing their retention of hormone dependency. This is consistent with other endocrine manipulations in the treatment of breast cancer; the advantages of immunisation with GnRH-glycys lies in its non-toxicity and reduction in side-effects, which were mainly adjuvant-induced.

M4HS2, a new human T-cell line for the efficient generation of human T-cell hybrids.

We have developed and characterised a variant of the human T-cell tumour, MOLT-4F, which is resistant to both 8-azaguanine and 6-thioguanine. This new cell line, M4HS2, is suitable for the efficient production of lymphokine-secreting human T-cell hybrids and is available for distribution.

Generation of human T-cell hybrids with the characteristics of human peripheral blood T-lymphocytes.

Using a selection system based on the two irreversible biochemical inhibitors, actinomycin-D and emetine hydrochloride, we have constructed human T-lymphocyte hybrids between the human T-cell line, Molt-4F, and mitogen-activated human lymphocytes. The cells were identified as true hybrids by their karyotype, differences in supernatant activities, responses to mitogen and membrane characteristics. These hybrid cells are stable in culture and express functions found in mature human T-lymphocytes.

Androgen receptor in the human thymus.

An androgen binding species has been identified in partially purified cytosol from human thymic tissue, resolved from sex steroid binding globulin by gel chromatography. This putative 5 alpha-dihydrotestosterone receptor was characterised by high affinity (Kd 6.7 X 10(-10) M) and low capacity (9 fmol/mg of cytosol protein). High affinity binding was confirmed with methyltrienolone (R1881). Competition studies were carried out with a number of androgenic compounds and 5 alpha-19 nordihydrotestosterone was shown to possess the highest affinity for the androgen receptor.

Studies of thyroglobulin autoantibody synthesis using a micro-ELISA assay.

Thyroglobulin autoantibody synthesis by Hashimoto lymphocyte cultures has been studied using an ELISA, a plaque assay and tanned red cell haemagglutination. The ELISA system was found to be the most suitable and using this method IgG-class thyroglobulin antibody synthesis was detectable in cultures of mitogen-stimulated lymphocytes from all 10 Hashimoto patients studied, but not in cultures of lymphocytes from 4 normal donors. The ELISA was also sufficiently sensitive to detect thyroglobulin antibody synthesis in mitogen-free lymphocyte cultures from 4 out of the 10 Hashimoto patients and consequently it should be possible to use this system to study the effect of various pathophysiological factors on autoantibody synthesis which would otherwise be masked by mitogenic stimulation.

Monoclonal antibody technology for mycotoxins.

Specific monoclonal antibodies (MABs) against aflatoxins, ochratoxin A, zearalenone, diacetoxyscirpenol and T-2 toxin have been prepared in various laboratories by the application of hybridoma technology to mycotoxins. These antibodies can be selected for sensitivity, reduced cross-reactivity, reliability and ease of production. When a suitable antibody is chosen it can then be used in a rapid immunological method such as an enzyme-linked or radio-immunoassay or immunoaffinity chromatography system. These assays have a lower limit of mycotoxin detection in the ng/ml range and have been applied to the determination of mycotoxins in samples such as maize, peanuts, peanut butter, milk and porcine kidneys. Using these immunoassay techniques, sample preparation has generally been simplified to a matter of solvent extraction of mycotoxins from the sample followed by dilution; under these conditions, levels of 1-5ug of mycotoxins/kg of sample can be found. The application and advantages of MABs to mycotoxins and the use of these antibodies in various assay techniques is discussed.

Proceedings: Quantitation of a new serum alpha-macroglobulin in malignant disease, steroid treatment, pregnancy and normal subjects.

PIP: Alpha-macroglobulin was quantitated in patients with malignant disease, steroid treatment, pregnancy, and in normal subjects using the rocket technique of Laurell. Women treated with combined estrogen/progestogen and with mestranol and men treated with stilbesterol showed rises in alpha-macroglobulins. Those treated with norethynodrel did not, indicating that the estrogen is the responsible agent. The level increased during pregnancy and decreased sharply in the first 2 days postpartum. 30% of normal women and 10% of normal men had detectable quantities of the protein (up to 4 mg/100 ml) in their serum. 92% of patients with malignant disease had detectable levels of protein--6 mg/100 ml or higher.^ieng

Proceedings: An investigation into the immunosuppressive properties of oestrogen.

PIP: An investigation into the immunosuppressive properties of estrogen is reported. Normal and thymectomized mature male rats were given daily intradermal injections of .2 ml arachis oil containing 200 mcg estradiol-17beta/ml for 14 days. Lymphocytes from healthy men were incubated with the rat sera and lymphocyte subpopulations were identified by 3 surface markers: 1) cells forming rosettes with sheep erythrocytes (E-rosettes) as a measure of T lymphocytes, 2) lymphocytes with surface immunoglobulin identified by direct immunofluorescence (IgF), and 3) lymphocytes with receptors for C3 observed by a rosette technique employing sheep eryhrocytes treated with rabbit hemolytic serum and human complement (EAC-rosettes). Comparison of the effects of incubating cells with normal or treated rat serum revealed that E-rosettes dropped from 67.4 to 49% and that lymphocytes lacking all markers rose from 2.4 to 17.7%, respectively. The number of cells forming EAC-rosettes, bearing IgF or having both these markers did not change, however, it was apparent that a greater proportion of lymphocytes expressed both markers after incubation with estrogen-treated rat serum (25.7% compared with 12.1%). It appears that estrogen caused both decreased T lymphocyte response and enhanced B lymphocyte activity resulting in increased expression of related markers. None of these phenomena was apparent when lymphocytes were cultured with sera from estrogen-treated thymectomized rats, suggesting that estrogen affects the thymus such that a factor appears in the blood capable of causing changes in the immune response.^ieng

Production and characterisation of anti-cardiac troponin-I monoclonal antibodies.

Cardiac troponin-I (cTn-I) was isolated from bovine left ventricular tissue and used as immunogen. Sixteen murine hybridoma lines were produced with two of them, 1D12 and 5F4, showing a high specificity for cTn-I; both of these monoclonal antibodies (McAbs) were isotyped as IgG1 with kappa-light chains. The specificity of the McAbs for cTn-I was confirmed by ELISA, western blotting and by the ability of the antibodies to block actomyosin ATPase inhibition by cTn-I. The McAbs may be useful for human in vivo imaging of myocardial infarcts and other pathological conditions related to cardiac myocyte damage.

Pregnancy-associated alpha 2-macroglobulin - a new serum protein elevated in normal human pregnancy.

A new pregnancy-associated serum protein has been isolated and characterised - pregnancy-associated alpha 2-macroglobulin (alpha 2-PAM). The native macromolecule has a molecular weight of 2.1 x 10(6) and a sedimentation coefficient of 19.6 S. This is comprised of 28 000 dalton subunits and contains 11% carbohydrate. alpha 2-PAM was found in normal male and female blood at a concentration of 25 micrograms/ml but no significant variation in this value was apparent in individuals followed over a 12 week period. The serum level can rise up to 16-fold during the first trimester of pregnancy and returns to normal levels within 8 weeks after delivery. Concentrations in contraceptive-steroid treated women, cord blood and breast milk were in the same range as normal adult sera while alpha 2-PAM could readily be detected in amniotic fluid at 8 micrograms/ml.

Immunoneutralisation of GnRH-I, without cross-reactivity to GnRH-II, in the development of a highly specific anti-fertility vaccine for clinical and veterinary use.

In recent years, several forms of gonadotrophin releasing hormone (GnRH) molecules have been isolated from primate brain. These molecules are very similar in sequence and this raises the question of whether previously developed neutralisation vaccines based on GnRH (now termed GnRH-I) would remove other forms of GnRH (namely GnRH-II) as well. As the function of these other molecules has not yet been clearly defined, potential health risks could exist by their ablation. In view of the high sequence homology between the molecules, this paper describes the production of highly specific polyclonal antibodies against GnRH-I and GnRH-II, with negligible cross-reactivity. The ultimate aim of this is to develop an anti-fertility vaccine which does not present any inappropriate side-effects, caused by neutralisation of a GnRH molecule which may or may not be directly involved in reproduction. Several formulations were investigated, based on analogues of the following molecules, conjugated to tetanus toxoid: 1. GnRH-I pGlu-His-Trp-Ser-Try-Gly-Leu-Arg-Pro-Gly-NH2 and 2. GnRH-II pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2. The specificity of the antibodies produced was examined, together with effects on fertility and any inappropriate side-effects. Immunostaining of hypothalamic sections was carried out, using the generated antisera, to determine the regional distribution of GnRH-I and GnRH-II neurones, as well as to further evaluate the specificity of the antibodies.

Variations in the level of a pregnancy-associated alpha-macroglobulin in patients with cancer.

The concentration of a pregnancy-associated alpha-macroglobulin (PAM) was determined in the blood of patients with various cancers. PAM was detectable in subjects with all types of malignant disease studied, and the level of the serum protein correlated well with the course of the disease: the concentration increased before clinical recognition of metastases and decreased significantly on successful treatment. Periodic PAM determinations may allow detection of tumour recurrence while it is still at a treatable stage and could aid in the evaluation of therapy.

Identification of an immunosuppressive factor in pregnancy serum.

Complement-inactivated allogeneic serum was found to contain a factor capable of suppressing BCG-induced inhibition of leukocyte migration in vitro. Although serum from late pregnancy was the most potent in this context, the ability to antagonize the leukocyte response appears to be an intrinsic property of human serum. The inhibitory factor was purified by salf fractionation and gel chromatography and the effect found to be associated with alpha-globulins of high lipid content which are probably alpha-lipoproteins. Estrogens, progesterone, or corticosteroids, associated with the lipoprotein, did not appear to be the cause of the observed suppression. The factor appears to block the immune response at the level of the lymphocyte and its nonspecific immunosuppressive properties could contribute to acceptance of the fetus by the mother.

Anti-gonadotropin releasing hormone vaccines and their potential use in the treatment of hormone-responsive cancers.

Gonadotropin-releasing hormone (GnRH) and its analogues have been used clinically to treat a range of hormone-dependent conditions. It is often necessary for large, toxic and expensive drug doses to be administered. Improvements in drug delivery have necessitated new developments in formulation, but these in turn can induce new adverse effects. Immunological neutralisation of GnRH has been examined as a less toxic and cheaper replacement therapy, and has been studied closely in different animal species. However, only a few clinical trials have been carried out with respect to hormone-dependent cancers. Based on clinical trials of the free peptide drug in cancer patients, it would appear that there is an increasing trend towards using GnRH and its analogues in adjuvant therapy and that antibody-based GnRH neutralisation will have a role in this treatment regimen.

Immunoscintigraphy of human ovarian cancer xenografts using a radiolabelled monoclonal antibody to human pregnancy serum-derived immune complexes.

The in vivo imaging of xenografted human ovarian cancer in nude mice with a specific and control radiolabelled monoclonal antibody (MoAb) is described. The specific MoAb was previously raised by immunizing mice with immune complexes derived from late human pregnancy serum. In the first group of mice the specific MoAb 131I-5E3 F(ab')2 was injected, while a second group received equivalent amounts of a control MoAb 131I-UJ13A F(ab')2. The mice were imaged at various times up to a maximum of 2 weeks using a gamma camera, and the tumour to non-tumour (T/NT) ratio was recorded for each group. The T/NT ratio rose to 2.02 in the specific group, while the corresponding ratio in the control group was 0.70. In addition, the count rate in the tumour and non-tumour regions was determined on each imaging occasion. Biological half-lives of the divalent fragments of 5E3 and UJ13A in the tumour were 7.53 days and 0.62 days, respectively. Following sacrifice, the tumours were excised and counted relative to the rest of the animal, and the T/NT ratio was calculated. In vitro results were in direct agreement with those recorded in vivo using the gamma camera. From the results it would appear that the divalent fragment of 5E3, which has been raised to immune complexes derived from late human pregnancy serum, is specific for human ovarian tumour xenografts in the nude mouse model.

Radiolabelled monoclonal antibodies (McAb): an alternate approach to the conventional methods for the assessment of cardiomyocyte damage in an experimental brain-death pig model.

The present study was carried out to determine the possible use of cTn-I in the cardiac myofibrillar architecture, as a potential target for in vivo radioimmunodetection of cardiac damage in a brain death pig model. Radioiodination of the anti-cTn-I 5F4 McAb was carried out by lactoperoxidase method. The percentage iodine incorporation achieved was 70-75%. The radioiodinated McAbs were purified on Sephadex G-25 column and characterised by Paper chromatography, Phast Gel electrophoresis and electroimmunoblotting. Radioiodinated anti-cTn-I 5F4 McAbs were employed alongside Pyrophosphate (Tc99m-PPi) and Thallium201 chloride (Tl201) in 24 landrace pigs (brain-dead = 18 & sham-operated = 6). The percentage cardiac uptake of the radiolabelled antibody injected dose was significantly higher in the brain dead animals (0.196%) as compared to that of sham-operated animals (0.11%). Specific in vivo localization of radiolabelled McAbs in the infarcted cardiac tissue was confirmed by computer-aided reconstruction of 3-D images of the isolated heart. The preliminary results of the study revealed preferential uptake of radiolabelled antibody at the site of myocyte damage resulting from artificially induced brain death.

Characterization of monoclonal antibodies with specificity for the core oligosaccharide of Shigella lipopolysaccharide.

Monoclonal antibodies (MAbs) were prepared against different strains of Shigella, following immunization of BALB/c mice with a heat-killed preparation of Shigella. Antibody-producing hybridomas were screened in an indirect enzyme-linked immunoadsorbent assay (ELISA) and epitope specificity determined using chemically defined lipopolysaccharide, lipid, and KDO fragments. Five MAbs were characterized and the following specificities identified: 2C32E6 and 4D64B9 (reactive to S. flexneri and S. boydii), 5E45D8 (reactive with S. flexneri), 4B33D10 and 1B52F10 (all species of Shigella). The properties of 1B52F10 revealed its potential importance in immunological detection of Shigella from unknown samples, as it was able to bind to all strains of Shigella.