Antigen-specific synergism in the immune response of irradiated mice given marrow cells and peritoneal cavity cells or extracts.

A synergistic immune response to foreign erythrocytes may be induced in heavily irradiated mice injected with a mixture of isologous cells obtained from marrow and from the peritoneal cavity. Under appropriate conditions, homologous or heterologous peritoneal cavity cells, heat-killed cells, or cellfree extracts made from such cells are also effective. The activity of the peritoneal cavity cells or extracts is antigen-specific, in the sense that cells or extracts obtained from animals previously immunized with the test antigen produce much stronger synergistic effects than do cells from animals immunized with some other antigen; however, the peritoneal cavity cells or extracts are not immunogenic when tested in primed animals. The marrow cells, demonstrated to contain precursors of the antibody-forming cells produced during this synergistic immune response, also show a form of antigen-specificity.

Human blood group isoantigen expression on normal and malignant gastric epithelium studied with anti-A and anti-B monoclonal antibodies.

Variation in human blood group isoantigen expression on normal and malignant gastric epithelium was demonstrated with monoclonal antibodies to blood groups A and B in an indirect immunoperoxidase technique. The expected isoantigen expression was demonstrated on endoscopic biopsy specimens of normal gastric mucosa from 11 patients. Of 17 patients with gastric carcinoma (blood group A, 15; blood group AB, 2), complete loss of isoantigen expression was noted in 6 (35%). In these 6 patients, blood group isoantigen remained both in the adjacent uninvolved mucosa and at the margin of resection. The loss of isoantigen did not appear to be related to the degree of differentiation within the tumor, to the secretor status of the patient, or to the blood subgroup. Lymph node metastases reflected the isoantigen status of the primary tumor, being positive in 5 of 6 expression in all 17 patients or in an additional 15 patients studied with blood group O. These findings were discussed in the light of previously reported work on the localization of blood group isoantigens on malignant and nonmalignant gastric epithelium with the use of conventional antisera and a variety of immunohistologic techniques.

How are tumour antigens related to normal antigens?

Tumour cells share with normal cells antigens characteristic of defined states of differentiation. Is there anything else? An example of tumour antigens not expressed anywhere in normal tissue is the set of tumour-specific transplantation antigens (TSTA) of murine chemically induced sarcomas. There is evidence that at least one TSTA specificity is retrovirus-derived, is carried on the envelope protein gp70, and probably arises by the recombination events that yield the diverse gp70s of the MCF strains of murine leukaemia viruses. Whether a similar mechanism can generate human tumour antigens depends on the yet unanswered question of whether human cells have retroviruses in their genomes capable of recombination. Aside from this, the only other mechanism known for antigen expression on tumours is via their oncogenes, which seem to make normal cell products. Such products, or secondary consequences of their production, were they normally expressed only at an early stage of development, would be candidates for 'fetal antigens'. While only the two mechanisms mentioned above seem the ready sources of 'tumour-associated antigens', it would be too early--in the face of ever more startling information about gene mobility and rearrangements--to think we have exhausted possible mechanisms for generating tumour antigens.

A radioimmunoassay for alpha- and beta-gliadins.

1. A rapid, sensitive specific radioimmunoassay for alpha- and beta-gliadin has been developed using an antiserum raised in rabbits to A-gliadin, a component of alpha-gliadin. 2. The antigen used in the assay was alpha-gliadin labelled with 125I; antigen-antibody complexes were collected after adsorption to Staphylococcus aureus in suspension. 3. The sensitivity of the assay, as judged by competitive binding with unlabelled antigen, was 1 ng of alpha- or beta-gliadin, which show complete cross-reaction with this antiserum. 4. Cross-reactivity to other wheat proteins was less than 1% and no cross-reactivity to extracts of rye, barley or oats was observed. 5. This radioimmunoassay for alpha- and beta-gliadin has been used to measure their amount in different varieties of wheat flour, several foods prepared from flour, e.g. bread, biscuits and products prepared as 'gluten free'. The possibility of assaying for alpha-gliadin in prepared foods is of special value since alpha-, beta-, gamma- and omega-gliadin have been shown to exacerbate coeliac disease.

Monoclonal anti-B as a new blood-typing reagent.

Stable cloned lines of anti-B-secreting cells have been derived by fusing spleen cells of a mouse immunized by group B antigen with a mouse myeloma line. The tissue culture supernatant of one of them (NB1/19.112.28) containing secreted monoclonal anti-B antibody is an especially good blood-grouping reagent, well suited for use with manual and automated methods. The unlimited availability of a potent reagent of uniform properties, cheaply produced by a single source, makes monoclonal anti-B a serious competitor to human typing serum.

Monoclonal antibody H9/25 reacts with functional subsets of T and B cells: killer, killer precursor and plaque-forming cells.

Monoclonal antibody (McAb) H9/25 has previously been shown to react with an alloantigen expressed on subpopulations of mouse lymphocytes. We here investigated the expression of the antigen (H9/25 Ag) on functional subsets of T and B cells. Lymphocytes were depleted of H9/25 Ag-bearing cells by complement-dependent cytolysis or by the affinity to immobilized McAb H9/25, and the residual immunological functions were tested. In these tests, killer T cells generated in mixed lymphocyte cultures and their precursors were found to express H9/25 Ag. In contrast, the activity and frequency of helper T cells specific to keyhole limpet hemocyanin carrier were not decreased by the depletion of H9/25 Ag-bearing cells. On the other hand, IgG and IgM anti-TNP plaque-forming cells were found to express H9/25 Ag, while it was not detected on unprimed B cells, as tested by using TNP-Ficoll and TNP-lipopolysaccharide. Only a small proportion of TNP memory B cells seemed to express the antigen. These results revealed significant differences between H9/25 Ag and other known alloantigens in the distribution among functional subsets of lymphocytes.

Clinical testing of bread made from nullisomic 6A wheats in coeliac patients.

The alpha-gliadins of wheat proteins exacerbate coeliac disease. Two genetic-variant wheats lacking the chromosome coding for some of these proteins were found to contain components of alpha-gliadin, and both exacerbated coeliac disease.

Study of frog sartorius muscle acetylcholine receptor using the irreversible inhibitor TDF.

The response of normal and denervated frog sartorius muscles to several agonists differing in intrinsic activity was studied using the fluid electrode technique. The response to carbamylcholine could be irreversibly blocked by exposure of the muscles top-trimethylammoniumbenzenediazonium difluoroborate (TDF), but the response could be protected from blockage by agonists and antagonists indicating that both TDF and these ligands act at the acetylcholine binding site of the receptor. It is shown that specific reversible binding of the trimethylammonium group of TDF to the receptor plays little or no role in the irreversible reaction of TDF with the receptor, which accounts for the extremely low specificity of its reaction with the receptor.

A new monoclonal anti-A. Culture supernatants with the performance of hyperimmune human reagents.

By proper selection for good growth and high avidity, we have prepared a new anti-A monoclonal antibody producing cell line that gives culture supernatants as potent as US-licensed commercial hyperimmune human reagents and which meet USA FDA standards without the need for concentration. The production and use of this reagent is cost effective and makes it a candidate to replace conventional anti-A typing reagents.

The control of transferrin receptor synthesis in mitogen-stimulated human lymphocytes.

Human lymphocytes cultured in the presence of the plant mitogenic lectin phytohemagglutin in (PHA) become activated and leave the G0 phase of the cell cycle. In the presence of PHA and lymphokines produced in situ the cells will enter S phase and undergo cell division. We have determined the time course of appearance for the receptor for transferrin as an initial attempt to understand the molecular mechanisms regulating the onset of lymphocyte differentiation and proliferation in the 48 h following PHA addition. Using three different assay methods we have shown that the increase in the number of surface receptor molecules is due to the accumulation of newly synthesized receptor and not to the redistribution of a previously existing pool of receptor molecules. The total amount of transferrin receptor increased at least four-fold. In vitro translation of RNA from activated lymphocytes indicates that the new receptor synthesis is due, at least in part, to increased availability of mRNA encoding the transferrin receptor.

A radioimmunoassay for wheat gliadin to assess the suitability of gluten free foods for patients with coeliac disease.

Coeliac disease is a clinical condition characterised by malabsorption secondary to abnormalities of the small intestine. The condition is known to be exacerbated by wheat gliadin, rye, barley and possibly oats. The only assays that are available for testing for the presence of wheat gluten in foods are double diffusion against rabbit anti-gliadin antiserum and measurement of Kjeldahl nitrogen in products derived from wheat flour. We have developed a radioimmunoassay for wheat gliadin with a detection limit of 1 ng. Nominally gluten free foods based on wheat starch have been shown to contain up to 1.9 X 10(-2)% wheat gliadin. Bread made from Nutregen wheat starch which has now been withdrawn contains 6.4 mg gliadin per standard 30 g slice. A radioimmunoassay for wheat gliadin could be used to define standards for the suitability of gluten free products based on wheat starch for patients with coeliac disease.

Are blood group isoantigens lost from malignant prostatic epithelium? Immunohistochemical support for the preservation of the H isoantigen.

Previous studies while demonstrating the presence of blood group isoantigens on normal prostatic epithelium have failed to identify such antigens on malignant prostatic tissue. Using a series of blood group specific monoclonal antibodies directed towards the A, B, H and Y antigens we have reinvestigated blood group isoantigen expression in both benign prostatic hypertrophy and prostatic adenocarcinoma. Results obtained from areas of benign prostatic hypertrophy are in broad agreement with those published however though we were unable to detect either A or B blood group isoantigens Type 2H and Y isoantigens were identified in 10 of the 12 tumours. These findings, while differing from previously reported results, lend support to the suggested connection between ontogenesis, oncogenesis and blood group isoantigen expression and also support the proposed link between Type 2 structures and malignant transformation.

A human-human monoclonal anti-D by direct fusion with a lymphoblastoid line.

The human lymphoblastoid cell line W1-L2-729-HF2 has been fused with B cells from a plasmaphoresed anti-D donor immunized with D+ cells. A stable monoclonal antibody-producing cell line has been produced which yields culture supernatant of good titre without the need for concentration. The production and use of this reagent as an alternative Rh D typing reagent for use by saline and enzyme manual tests and automated tests in a Technicon 16C machine is discussed. Du red cells are detected in enzyme enhanced tests.

The expression of ABH and Y blood group antigens in benign and malignant breast tissue: the preservation of the H and Y antigens in malignant epithelium.

The ABO(H) and Y antigen status of epithelial cells from 45 breast carcinomas, 14 benign breast lesions and 7 normal breasts have been assessed using an indirect immunoperoxidase histochemical assay and a series of blood group specific monoclonal antibodies. All 20 A, AB and B group tumours had lost the A and B isoantigens, 13 of these tumours were however found to express H and Y antigens. Of 25 group O tumours 17 expressed the expected H and Y antigens. These findings were not dependent on the histological nature or the invasive characteristics of the tumour. Similar results were obtained when 28 metastases from breast carcinomas were examined, the H and Y antigens being identified in the tumour elements in 24 lymph nodes while we failed to identify either the A or B antigens. The development of breast malignancy appeared therefore to correlate best with the deletion of A and B glycosyl transferases. Normal breast tissue consistently expressed the expected blood group isoantigens. Areas of benign breast disease showed a more varied pattern of antigen expression. Seven of 14 lesions lacked ABH antigens, the loss of blood group structures could not however be correlated with any specific histological features and was not limited to the loss of A and B substances.

Thyroid blood group isoantigen expression: a parallel with ABH isoantigen expression in the distal colon.

An interesting and not previously reported parallel has been observed between the known pattern of ABO (H) blood group isoantigen expression in normal and neoplastic colonic epithelium and that in the thyroid. Epithelial expression of blood group isoantigens was not observed in 16 specimens of normal or non-neoplastic thyroid tissue. This contrasts with the progressive re-expression of these antigens in neoplastic thyroid tissue. Blood group isoantigens were detected in two of eight papillary adenomas and 13 of 17 papillary carcinomas. Antigen expression was in part related to differentiation, and stained cells were less readily detected in follicular tumours, only one of five adenomas and two of seven carcinomas displaying blood group antigens while three medullary and two anaplastic carcinomas were antigen-deficient.

Photo-affinity labeling of specific acetylcholine-binding sites on membranes.

Acetylcholinesterase of intact red blood cell membranes and the acetylcholine receptor at the neuromuscular junction of whole-frog sartorius muscle have been irreversibly inactivated by photo-affinity labeling with two quaternary ammonium aryl azides. The inactivation requires that the azides, at the time of their photolytic conversion to highly reactive nitrenes, are reversibly bound to the specific acetylcholine-binding sites.

Distribution of saccharide residues on membrane fragments from a myeloma-cell homogenate: its implications for membrane biogenesis.

Ferritin conjugates of two plant agglutinins, concanavalin A and ricin, have been used as specific electron microscopic stains for covalently-bound saccharide residues on membrane fragments from a myeloma-cell homogenate. The results indicate that different saccharide residues are uniformly localized to a single surface of each membrane fragment. In particular, the ferritin-concanavalin A conjugate binds exclusively to the cisternal side of membrane fragments of the rough endoplasmic reticulum. If it is postulated that the biogenesis of eukaryotic plasma membranes involves an assembly-line process from precursor intracellular membranes, these observed asymmetric distributions of saccharides on cell membranes can be explained.