Isolation, partial characterization, and concentration in experimental sepsis of baboon lipopolysaccharide-binding protein.

Lipopolysaccharide-binding protein (LBP) is important for mediating host responses to lipopolysaccharide (LPS). The structure and properties of human, rabbit, and murine LBP have been previously described. In this study we partially characterized baboon LBP and investigated its appearance in experimental sepsis. Recurrent bacteremia was induced in baboons by infusion of live Escherichia coli organisms over a 2-hour period at 0, 24, and 48 hours. To assay baboon plasma LBP levels, an enzyme-linked immunosorbent assay with cross-reactive antibodies to human LBP was developed. Control baboon plasma LBP concentrations were 2 to 5 microg/mL. During experimental sepsis, baboon plasma LBP levels increased to between 200 and 350 microg/mL and in parallel with the increase in C-reactive protein levels. Baboon LBP was isolated from acute phase serum by ion-exchange chromatography followed by immuno-affinity chromatography. Its NH2-terminal sequence (XNPGLVARTTNKGLEYSAQE) and its molecular weight (approximately 60 kd) were determined and were proved to be highly homologous to human LBP.

Immune responses in human colon cancer. I. Microcytotoxicity assay for measuring killing of adherent human colon cancer cell lines.

A short term microcytotoxicity assay system using radioisotope release as an index of target cell damage has been developed to evaluate immune killing of cultured adherent human colon cancer cell lines. Using this assay system, complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and spontaneous cell-mediated cytoxicity of adherent human colon cancer cell lines can be assessed in 4 hr or less. An essential step in the development of this system was the successful 51Cr-labeling of human colon cancer target cells with subsequent low spontaneous release. This was achieved through careful attention to cellular growth phase and medium pH during the labeling and assay period. In this microsystem, labeled colon cancer cells spontaneously released 51Cr at a mean rate of 2% per hr during the assay, a level low enough not to obscure specific cytotoxic responses. Complement-dependent cytoxicity was measured most conveniently over a 2-hr period, whereas antibody-dependent cell-mediated cytotoxicity and spontaneous cell-mediated cytotoxicity were optimal when measured over a 4-hr period.

Induction and properties of cytotoxic T cells specific for hapten-coupled tumor cells.

Techniques are reported for the induction and assay of cytotoxic effector cells capable of specifically lysing hapten-coupled EL4 leukemia targets. It is shown that EL4 cells survive coupling with TNP-sulfonic acid and retain the hapten on their cell surface for a prolonged period of time. Although TNP-EL4 cells are readily lysed by anti-TNP serum in a complement-mediated reaction, they are inefficiently killed in an antibody-dependent cell-mediated reaction. Cytotoxic effector cells, able to lyse TNP-EL4 targets, are induced when C57BL/6 spleen H-2-b cells are cultured with the following cell types which have been coupled with TNP: 1) ALLOGENEIC P815 tumor cells (H-2-d), 2) syngeneic EL4 tumor cells, 3) allogeneic BALB/c spleen cells (H-2-d), 4) syngeneic C57BL/6 spleen cells. Further experiments show that TNP-coupled xenogeneic chicken erythrocytes, which by themselves are unable to induce cytotoxic effectors, are capable of doing so if uncoupled P815 cells are present simultaneously. On the basis of these findings, it can be hypothesized that two stimuli are required for induction of these cytotoxic effector cells--one provided by the hapten, and the other by the P815 cell. Treatment of cytotoxic spleen cells induced by hapten-coupled allogeneic tumor cells with anti-Thy-1 serum and complement abrogates their cytotoxicity, indicating that T cells play a central role in the cytotoxic reaction. TNP-coupled erythrocytes do not serve as targets for these cytotoxic T cells, but do cause competitive inhibition of TNP-EL4 when added to the reaction mixture at high ratios. However, because the inhibition is relatively low, and because no such inhibition can be demonstrated with TNP-lysine, it is concluded that the receptor on the cytotoxic effector cell has a low affinity for hapten. This low affinity could be due to the receptor recognizing an antigen comprising mouse cell surface antigen in addition to the TNP moiety. Supporting this interpretation is the finding that TNP-EL4 cells competitively inhibit cytotoxicity much more efficiently than TNP-CRBC and that even uncoupled EL4 cells inhibit to some extent.

Effects of two bisdioxopiperazines on mouse B-and T-cell function.

The immunosuppresive effects of the bisdioxopiperazines ICRF 154 and ICRF 159 on the function of bone marrow-derived lymphocytes (B cells) and thymusderived lymphocytes (T cells) were assessed and compared with those of cyclophosphamide (CPA). Mice given foreign erythrocytes with any of these drugs for 3 or 5 days showed suppressed antibody responses due to the inhibition of thymus-derived cooperating lymphocyte (T-helper cell) priming and inactivation of B cells in the spleen. In contrast, 3 days of drug treatment after the injection of allogeneic tumor cells only partially inhibited T cell-dependent cytotoxicity. Moreover, when irradiated tumor cells were given for 3 days with CPA or ICRF 154, enhancement rather than inhibition of cytotoxicity was the usual response. However, with both types of immunization, no thymus-derived cytotoxic lymphocytes (T killer cells) could be generated after prolonged treatment (6 daily injections) with any of the 3 drugs. Administration of drugs before antigen also resulted in selective drug action, i.e., a relative increase in the proportion of thy-1-positive cells in the spleen. After challenge in vivo or in vitro with erythrocyte antigens, the plaque-forming cell responses of these spleens were raised up to twofold, probably because they had higher T-helper cell activity than untreated controls. Similar pretreatment before immunization with allogeneic tumor cells also led to enhanced T-killer cell activity, but only in ICRF 154- and CPA-treated mice. These observations suggest that under certain conditions, the bisdioxopiperazines and CPA have selective effects on B-cell rather than T-cell function.