Dov Sulitzeanu

Immunosuppressive factors in human cancer.

Publisher Summary This chapter reviews the factors on immunosuppressive molecules that are elaborated by tumor cells or found in sera and effusions. Immunosuppressive factors can be found in normal sera, but their level is increased in cancer sera. These factors may contribute to the defective cellular responses of patients. Some well-characterized immunosuppressive molecules are transforming growth factor β (TGF-β), lymphocyte blastogenesis inhibitory factor (LBIF), p15E, and suppressive E-receptor (SER). TGF-β is produced by most cells and has an extraordinarily wide range of biological activities. p15E protein possesses a remarkable range of immunosuppressive activities. The SER is isolated from malignant effusions derived from patients with several types of cancer and inhibited several cellular immune responses. The exact mechanism of the immunosuppression is still uncertain, but a multiplicity of factors contributes to its occurrence: suppressor T cells and suppressor macrophages, immune complexes, acute phase proteins, tumor-derived suppressor molecules, suppressor substances in sera and effusion fluids of patients, and chemotherapy and irradiation. The colony-stimulating factors, acute-phase proteins, and miscellaneous molecules are some of the immunosuppressive factors in human cancer.

Human cancer-associated antigens: present status and implications for immunodiagnosis.

Publisher Summary The successful demonstration of specific antigens in animal tumors essentially provided a major impetus to the search for comparable antigens in human neoplasms. This chapter examines the considerable amount of data that have accumulated in recent years on tumor-associated antigens (TAAs) of human solid tumors to assess their specificity and their possible usefulness for the early diagnosis of cancer. The selection of material included is arbitrary, as there is an absence of a clear-cut and universally accepted definition for TAA. TAAs are an extremely heterogeneous group of molecules and their overlap with normal antigens is so great that it is practically impossible to define TAAs in such a way so as to distinguish them clearly from ordinary cellular components. The chapter considers a molecule as a TAA if its presence renders the cancer cell quantitatively or qualitatively different from its normal counterpart. TAAs appear to be oncofetal or digerentiation antigens and their apparent specificity is due to their generally increased concentration in the tumor cells.

Simplified technique for preparative disc electrophoresis: II. Further improvements in apparatus and some details of performance

Abstract Several improvements have been incorporated in the instrument and technique described here as compared to the technique and apparatus described originally. The modified instrument7 can fractionate 200–400 mg protein (contained in a volume of up to 12 ml) in one run, requiring only 3–4 hr. Once the position in the polyacrylamide gel of the desired fraction is known, that fraction can be obtained in solution in another 3–4 hr in a concentrated form (3–3.5 ml). If necessary, the portion of the gel containing the desired fraction can be refractionated on a fresh column in order to obtain the material in a higher degree of purity. The effectiveness of the instrument has been confirmed by working with colored proteins. The proteins became concentrated in very narrow discs in the spacer gel and separated well from each other as fairly narrow discs (3–5 mm) in the separating gel.

Cross-reactivity between the EBNA-1 p107 peptide, collagen, and keratin: Implications for the pathogenesis of rheumatoid arthritis

An unusually heavy load of Epstein-Barr virus (EBV) infection and autoimmunity to collagen are believed to be contributing factors to the pathogenesis of rheumatoid arthritis (RA). The present report presents data showing that p107, the major epitope of the EBV-encoded EBNA-1 antigen, cross-reacts with denatured collagen (DC) and keratin (K), suggesting a new likely link among RA, EBV-1, and these autoantigens. A radioimmunoassay using antigen-coated microtiter plates was used to demonstrate antibodies in sera of patients with RA and sera of healthy donors against p107, DC, and K. Specificity of the antibodies was ascertained by inhibition tests with the homologous antigens. Cross-reactivity among anti-p107, anti-DC, and anti-K antibodies was assayed by the ability of a given antigen to block the binding of nonpurified or affinity-purified antibodies to plates coated with another antigen. Most of the sera contained antibodies to all three antigens, but only anti-DC antibodies were present in higher titers in RA sera. Preincubation of sera with p107 appreciably reduced their binding to plates coated with DC or K. On the other hand, preincubation with DC (in solution or bound to Sepharose) did not result in consistent reduction of anti-p107 titers. Tests with affinity-purified antibodies revealed the existence of two antibodies populations, one of which reacted preferentially with p107, the other with DC. The cross-reactivity of the anti-p107 antibodies with DC and K suggests that such antibodies, produced by RA patients following persistent stimulation with EBV, might react in vivo with collagen (and keratin) exposed in previously damaged areas and thus reinforce the disease process.

A highly sensitive solid-phase radioimmunoassay for the assay of Plasmodium falciparum antigens and antibodies.

A highly sensitive radioimmunoassay for detection of P. falciparum antibodies and antigens is described. A partially purified P. falciparum antigen preparation is obtained from in vitro cultured parasites enriched after gelatin sedimentation by sonicating the infected red blood cells and precipitating the proteins with 50% saturated ammonium sulfate. The precipitate is dissolved in buffer, ultracentrifuged and used to coat wells of microtiter plates. Anti-P. falciparum antibodies are detected by incubating antiserum dilutions in the coated wells and detecting the bound IgG with radioiodinated staphylococcal protein A. P. falciparum antigens are detected by their ability to inhibit binding of antibodies to the coated wells. Sera of individuals with a history of P. falciparum infection contain antibodies detectable at a dilution of 1:75,000. P. falciparum RBC infected in vitro can be detected at levels of parasitemia of the order of 1 parasite or less per 10(6) RBC.

A technique for the purification of immune complexes using rheumatoid factor

A method for the purification of immune complexes (IC) from human serum is described, using as a model complexes of tetanus toxoid with human antitoxoid antibodies (TAT). The technique is based on the ability of IC to bind to tubes coated with rheumatoid factor (RF). Small amounts of TAT IC were added to human serum and the mixtures were rotated overnight in tubes coated with RF. The tubes were then washed and the bound material was eluted with sodium dodecyl sulphate and iodinated with 125I. Analysis of the labeled preparation by electrophoresis in polyacrylamide gels revealed the presence of the toxoid component. The technique should be useful in isolating small amounts of IC for analytical purposes.

Culture supernatants of lymphokine-activated killer (LAK) cells contain a high-molecular-weight cytotoxic lymphokine

Supernatants of human lymphokine-activated killer (LAK) cells grown in vitro were tested for cytotoxic activity against several mouse and human neoplastic cell lines. All LAK preparations tested (14/14) exhibited cytotoxic activity (40-90% killing of the target cells). Sephacryl S-300 Gel filtration experiments indicated that the biological activity of the LAK supernatant is associated with molecular moieties ranging from 800 kDa or more, to less than 10 kDa. The finding of strong cytotoxic activity in LAK supernatants against several tumor lines points to the possibility of employing soluble products of these cells, rather than the living cells themselves, for therapeutic purposes.

A technique for the identification of glycoprotein antigens in immune complexes and its application to the detection of a common glycomprotein in sera of patients with burkitt's lymphoma and nasopharyngeal carcinoma

A new technique for the detection of glycoprotein antigens in immune complexes (IC) isolated from serum is described. The technique was developed with a model IC system consisting of ovalbumin (OVA)-rabbit anti-ovalbumin antibodies (aOVA), at 3 times antigen excess. OVA-aOVA IC added to normal human serum (NHS) were purified by absorption onto and elution from tubes coated with rheumatoid factor (RF) and were subjected to electrophoresis in polyacrylamide gels. Concanavalin A (Con A)-binding proteins were detected by treating the gels with radioiodinated Con A (125Con A), followed by autoradiography. IC isolated from sera of patients with Burkitts lymphoma (BL) and Nasopharyngeal Carcinoma (NPC) were analyzed before and after reduction with dithiothreitol. Two closely spaced proteins of about 40 kdalton were identified in the reduced samples in 26 of 30 BL sera (86%) and in 24 of 30 NPC sera (80%) but were not seen in 30 sera of African patients with a variety of unrelated tumors nor in 12 sera of European blood bank donors.

Isolation and electrophoretic analysis of immune complexes from patients with breast cancer

Abstract Immune complexes (IC) were isolated from sera of patients with breast cancer (BC), from effusions from patients with breast and ovarian cancer (OC) and from effusions from patients with non-malignant diseases. Isolation was carried out with the aid of tubes coated with rheumatoid factor (RF) as immunoadsorbents. The effusions were first filtered through Sephacryl S-300 columns and the large molecular weight material eluted in front of the IgG peak was used as the starting material, while most of the sera were processed without prior filtration. The IC eluted from the RF-coated tubes were radioiodinated and analyzed by electrophoresis on polyacrylamide gels, followed by autoradiography. Four putative IC-derived proteins were identified in the RF eluates and designated according to their molecular weights (in Kdalton): p94, p46, p 17 and p 13. p94 was found in 1/8 BC effusions, but in none of the other samples tested; “p46”, which was actually a complex of several closely spaced proteins, was identified in at least 3/16 BC sera, 6/8 BC effusions, 3/4 OC effusions and 1/5 non-malignant effusions, but in none of 17 normal sera. The upper band of the complex seemed to be present mainly, if not exclusively, in effusions from patients with neoplasia. p17 and p13 were found more often and in higher concentrations in the malignant effusions. All of these proteins could be precipitated by treating the RF eluates with anti human Ig serum, suggesting that they were associated with Ig, presumably as immune complexes.

A radioactive antibody binding-inhibition assay, for the detection of cell-membrane related antigens in body fluids.

Antisera raised in rabbits against glutaraldehyde-fixed human breast cancer cells contain antibodies to human cell membrane components, as determined by immunofluorescence. Adsorption of such antisera onto polymerized human serum, followed by acid elution, yields purified antibodies reacting with human cell surface antigens, indicating that membrane related antigens are present in the serum. The purified antibodies were radioiodinated and shown to bind to an immunoadsorbent prepared by entrapping in a polyacrylamide gel pleural exudate of breast cancer patients. The specificity of the binding was confirmed by inhibition experiments. Data are presented demonstrating that at least some of the antibodies reacting in this radioimmunoassay are directed against antigens related to cell surface components.