Barry D. Kahan

Human colonic adenocarcinoma cells: I. Establishment and description of a new line

SummaryA series of human colonic epithelial cell lines have been cultured from a single patient: LS-180 the original adenocarcinoma, LS-174T a trypsinized variant, and normal colonic tissue. The malignant cells, 20 to 40 µm in diameter and oval to polygonal, exhibited characteristics of normal colonic mucosal cells, namely, abundant microvilli prominent in secretory cells, and the presence of intracytoplasmic mucin vacuoles. The cultured adenocarcinoma cells, but not normal, demonstrated neoplastic properties by producing high levels of carcinoembryonic antigen (CEA) and by the ability to be propagated in hamster cheek pouches and in immunodeprived mice. The CEA production by the newly established line LS-180 released 900 times more CEA per cell into the culture medium and bore 30 times more cell-associated material than the established line, HT-29. These cell lines may permit detection of distinctive chemical, physiological, pharmacologic, and immunologic characteristics of neoplastic colonic cells.

Specific tumor immunity induced with soluble materials: Purification of antigens inducing tumor resistance

Abstract The tumor-specific transplantation antigen (TSTA) in crude three molar potassium chloride (3M KCl) extracts of a chemically-induced, murine fibrosarcoma was purified by ammonium sulfate salt fraction at 20% saturation (S20S) and by polyacrylamide gel electrophoresis (PAGE). Mice, which had been pretreated with the S20S precipitate, displayed retarded outgrowth of a 100-fold supralethal dose of the corresponding, but not of a non-cross-reactive syngeneic tumor. Analysis of PAGE gels by Coomassie Blue staining revealed at least 30 bands in the crude 3M KCl extract, and only two components (Rf 0.34 and 0.43) in the ammonium sulfate fraction. That these two components bore TSTA activity was demonstrated by the observation that the immunoprotective activity of crude 3M KCl extracts was localized to the Rf 0.25–0.50 region. The two components present in the S20S fraction had isoelectric points of 5.05 and 6.9, and estimated molecular weights of 40,000 and 75,000, thus demonstrating the soluble nature of the active principle. These findings offer the prospect of a chemical dissection of the polymorphic TSTA surface markers on MCA-induced murine tumors.

Specific immunoprotection with 3 M KCI solubilized tumor antigen

Abstract Tumor-specific transplantation antigens were extracted from two methylcholanthrene-induced murine sarcomas. Administration of the soluble material to syngeneic hosts yielded immunoprotection against a challenge of supralethal numbers of tumor cells. The protective effect was only induced over a narrow dosage range; higher doses were ineffective. The engendered immune state could be overcome with larger numbers of cells in the tumor challenge. The restricted range of immunogenicity of soluble extracts in this experimental system cautions against premature clinical application of these materials as immunotherapeutic tools.

Purification of tumor-specific antigens. An overview of the relevance to human colon carcinoma.

Methods which dissociate intramolecular noncovalent bonds have been used to prepare soluble derivatives of cell‐surface antigens. Applications of these techniques to human colon carcinoma are underway. Continuous tissue‐culture strains derived from primary lesions were developed and shown to be composed of malignant epithelial elements. Parallel data on the preparation and activity of soluble materials in a murine model methylcholanthrene system reveal that although cultured cells are a satisfactory source for antigen extraction, they are poor targets of the immune response. The development of methods to quantitate the biologic activity of colon‐specific, soluble materials may provide indicator systems to define the antigenic determinants, to permit purification, and to serve as assays of the efficacy of immunotherapy.

A role for cAMP in the preparation of human platelets for the extraction of histocompatibility antigens.

Abstract Conditions were developed for the preparation of platelets to obtain optimal yields of HL-A antigens by hypertonic salt (3 M KCl) extraction. Although these antigens were readily demonstrable on the surface of platelets prepared in 10 −2 M EDTA, they could not be solubilized by 3 M KCl. However, antigens could readily be solubilized from platelets prepared in 10 −2 M caffeine, 10 −2 M theophylline or a combination of 10 −6 M prostaglandin E 1 , and 10 −3 M theophylline. Using these conditions, three fractions of HL-A antigenic activity, as determined by specific inhibition of cytotoxicity, were obtained; (1) a fraction extractable by 3 M KCl; (2) a fraction not extractable by 3 M KCl but extractable by isotonic saline; and (3) residual activity not extractable by either solvent. Significantly higher intracellular cAMP levels were observed in those platelet preparations from which HL-A antigens were solubilized. Quantitative inhibition studies of whole platelets, performed using monospecific HL-A antisera, indicated that increased cAMP levels did not alter the immunologic potency of cell-bound HL-A antigens. In addition, extractions of cell surface-radioiodinated platelets demonstrated that a generalized increase in the extractability of cell membrane proteins, by hypertonic salt, resulted from increased intracellular cAMP levels. These results indicate that the levels of platelet cAMP, prior to 3 M KCl extraction, are important in determining whether HL-A antigens can be effectively solubilized.

Leukocyte Aggregation Test for Evaluating Cell-Mediated Immunity

Publisher Summary Cell-mediated immunity as demonstrated in vitro consists of at least three steps: recognition, proliferation, and performance. The leukocyte aggregation test (LAT) was developed to evaluate the cellular immunity of patients undergoing renal transplantation. The LAT is based upon the adhesion of host leukocytes to target cells secondary to an immunospecific recognition of donor transplantation antigens by sensitized T lymphocytes. After this event, predominantly nonimmune leukocytes are captured at the reaction foci and appear as aggregates. The reaction requires the expenditure of metabolic energy; it is dependent upon physiological temperatures and is susceptible to inhibitors of protein and RNA synthesis. The LAT has several advantages that recommend it as a routine tool for assessing cell-mediated immunity in transplant patients: (1) completion within 5 hours, (2) requirement for only 10 ml of whole blood, permitting daily testing, (3) relative resistance to immunosuppressive agents, (4) immunological specificity, and (5) sensitivity to the modulating effects of serum factors on cellular immunity.