Jeffrey R. Dawson

Human natural killer cells induce morphologic changes in porcine endothelial cell monolayers

In this study, we have investigated the early in vitro effects of natural killer (NK) cells on porcine aortic endothelial cell (PAEC) monolayers. Incubation of effector cells containing about 70% CD56+ cells on PAEC monolayer led to time-dependent changes in PAEC monolayer morphology. As little as 20 min of incubation resulted in changes in PAEC shape and in the appearance of gaps between the cells. These effects have been observed for up to 6 hr, but not before 20 min or after 6 hr. When NK-depleted effector cells were used, no morphological changes were observed in comparison with the same effectors before depletion; if CD56+ cells were added back, the effects were comparable with those on nondepleted effector cells. There was no detectable NK cell-mediated cytolytic activity during the 1-6 hr of incubation of peripheral blood lymphocytes with PAEC monolayers. These data indicate that NK cells may participate in endothelial cell changes leading to xenograft rejection.

Genetic Determinants of Variable Metabolism Have Little Impact on the Clinical Use of Leading Antipsychotics in the CATIE study

Purpose: To evaluate systematically in real clinical settings whether functional genetic variations in drug metabolizing enzymes influence optimized doses, efficacy, and safety of antipsychotic medications.Methods: DNA was collected from 750 patients with chronic schizophrenia treated with five antipsychotic drugs (olanzapine, quetiapine, risperidone, ziprasidone, and perphenazine) as part of the Clinical Antipsychotic Trials of Intervention Effectiveness study. Doses for each of the medicines were optimized to 1, 2, 3, or 4× units in identically appearing capsules in a double-blind design. We analyzed 25 known functional genetic variants in the major and minor metabolizing enzymes for each medication. These variants were tested for association with optimized dose and other relevant clinical outcomes.Results: None of the tested variants showed a nominally significant main effect in association with any of the tested phenotypes in European-Americans, African-Americans, or all patients. Even after accounting for potential covariates, no genetic variant was found to be associated with dosing, efficacy, overall tolerability, or tardive dyskinesia.Conclusion: There are no strong associations between common functional genetic variants in drug metabolizing enzymes and dosing, safety, or efficacy of leading antipsychotics, strongly suggesting merely modest effects on the use of these medicines in most patients in typical clinical settings.

Natural killer cell-endothelial cell interactions in xenotransplantation

Interest in xenotransplantation derives from the documented need for more organs and tissues than can be expected from living or cadaveric donors. Although the barriers to xenotransplantation are formidable, the scientific rewards in addressing these problems have been significant. The first and most potent barrier to xenotransplantation is hyperacute rejection mediated by xenoreactive natural antibodies and serum complement. The majority of the xenoreactive antibodies appear to be directed at terminal galactose epitopes, especially gal α 1–3 gal. Significant progress has been made in surmounting hyperacute rejection, and this has led to an examination of underlying mechanisms of delayed xenograft rejection. One of these delayed mechanisms concerns the potential role of graft recipient, natural killer (NK) cells. NK cells can cause variable, low-level cytotoxicity of xenogeneic endothelial cells in vitro that may be enhanced in the presence of xenoreactive IgG. The specificity of NK cell-mediated cytotoxicity appears to overlap with a major subset of xenore active natural antibodies. These cytotoxic interactions can be regulated by “humanizing” the endothelial cells through expression of the appropriate human MHC class I genes. More important, NK cells induce endothelial cell activation, which results in changing the nature of the endothelial cell surface from an anticoagulant surface to a procoagulant surface. These findings parallel those observed in allogeneic NK cell-endothelial cell interactions and suggest these important observations may be extended to NK cell-endothelial cell interactions in general.

Differential expression of natural killer cell markers: human versus baboon

The use of baboons as a model for the study of allo- and xenotransplantation has become increasingly important, but there are few studies on the basic immunological responses in baboons that might be relevant for a rejection reaction. In present study, the cell-surface phenotype, cytokine-induced activation and growth, and cytotoxicity of baboon and human natural killer (NK) and lymphokine-activated killer (LAK) cells were compared. A panel of murine monoclonal antibodies specific for human cell-surface markers expressed on lymphocytes was used to compare relevant baboon and human peripheral blood lymphocytes (PBL). Baboon PBL were 52.1+/-2.9% CD8+, 18.5+/-2.2% CD16+, 3.0+/-0.5% CD25+, and 5.5+/-1.8% CD69+. The corresponding proportions in humans were 23.8+/-7.1%, 12.8+/-3.2%, 4.5+/-1.0%, and 2.3+/-1.1%. In contrast to human PBL, less than 1% of baboon lymphocytes expressed CD56, CD57, and CD122 (interleukin [IL]-2Rbeta). Baboon lymphocytes showed NK cytotoxic activity against the human K562 and CEM cell lines, which was comparable to human NK activity. Depletion of baboon CD16+ or CD8+ cells led to dramatic decreases in NK cytotoxicity, and removal of both subsets completely abrogated NK activity. Incubation of baboon lymphocytes with human recombinant IL-2 for 1 week led to the appearance of CD56+ cells (11.3+/-2.8%). Most of the baboon CD56+ cells induced in culture were in S and G2 phases of cell cycle. Both baboon and human IL-2-activated lymphocytes were highly cytotoxic against the human LAK-sensitive cell line Daudi. Depletion of baboon CD8+ but not CD56+ cells significantly decreased LAK activity. These studies revealed differences in the NK system of humans and baboons that should be taken into consideration when analyzing immune responses to allo- and xenotransplantation in baboons.

Lymphocyte stimulation in vitro: Generation of cytotoxic effector lymphocytes using subcellular fractions of a lymphoid cell line☆

Abstract Subcellular fractions, isolated from the lymphoid cell line IM-1, are capable of stimulating a weak proliferative response in allogeneic lymphocytes. They also stimulate the generation of cytotoxic effector lymphocytes. The proliferative response to subcellular fractions, as measured by 3H-thymidine incorporation, is only one-fourth to one-sixth as great as that to intact IM-1 cells, suggesting that a component(s) synthesized during the mixed lymphocyte reaction (MLR), or a short-lived cellular constituent, may be responsible for the ability of intact cells to stimulate a lymphocyte proliferative response. This component appears to be lacking or in limiting quantity in subcellular fractions, including the soluble fractions. In contrast to the decreased proliferative response to subcellular fractions, the cytotoxic capacity of the stimulated lymphocytes is comparable to that after stimulation by intact IM-1 cells. The data demonstrate that, in this system, cytotoxic effector lymphocytes can be generated in the absence of the extensive proliferative response normally observed in the MLR. The antigenic stimulus responsible for the generation of cytotoxic effector cells appears to reside on intracellular components as well as on plasma membrane. In these reactions, specificity is shown by the failure of the cytotoxic cells to release 51Cr from autologous target cells. In fact, reactivity of lymphocytes stimulated by subcellular fractions is more specific than the reactivity of cells stimulated by intact IM-1 as judged by their lytic capacity for another target cell, RPMI 4265.

Activity of antibodies recovered from immune complexes of ovarian cancer patients

SummaryAscites fluids from ovarian cancer patients contain immune complexes (IC). Attempts were made to characterize those antigens to which the patient is reacting using antibody recovered from these complexes. Polyethylene glycol (PEG) precipitation and protein A Sepharose 4B affinity chromatography were used to isolate IC. Dissociation of the IC was achieved by G-150 gel filtration in 0.1 M acetic acid, 0.15 M NaCl. Activity of antibody preparations was measured using a binding assay with 125I-labeled staphylococcal protein A. Confluent monolayers of various cell lines (including human ovarian and cervical carcinoma lines, human lymphoblastoid cell lines, human and chick embryo fibroblasts) were used. Six of nine antibody preparations isolated from ovarian cancer patient IC contained at least some reactivity against all cell types.Specificity was further defined using cell adsorption assays and polyacrylamide gel electrophoresis. These results indicate what appears to be autoreactivity directed against a normal component(s) of many cells. No evidence for an ovarian cancer-restricted response was shown. Immunoprecipitation analyses showed several polypeptide bands on autoradiograms (49K, 46K, 33K, 25K), which deviated distinctly from the pattern obtained for whole cell extracts.

Quantitation of secretory protein levels by radioimmunoassay

A radioimmunoassay was designed for the detection of secretory protein, a component of secretory immunoglobulin A, in human serum. The assay uses free secretory protein isolated from human colostrum, and antisera raised in rabbits to the purified antigen. The mean level of secretory protein in the control group was 2.34 +/- 0.41 microgram/ml (mean +/- S.E.M.). The level in cord blood was slightly lower (0.74 +/- 0.26 microgram/ml), while the level in patients with ovarian carcinoma was significantly increased (12.67 +/- 1.43 microgram/ml). Pregnant women have increasing secretory protein levels with increasing length of gestation (5.86 +/- 2.02, 11.55 +/- 1.30 and 17.00 +/- 1.16 microgram/ml for the first, second and third trimesters, respectively.

The role of calnexin in NK-target cell interaction

In this study, a relationship between target cell sensitivity to natural killing and target cell expression of the molecular chaperone++ calnexin was assessed. The NK-resistant cell line NKR was originally derived from the NK-sensitive, human T-cell line CEM and does not synthesize calnexin protein or mRNA. The cell lines CEM, NKR and 1B9 (NKR transfected with a calnexin cDNA) were compared in a number of assays. All the lines but CEM were resistant to NK in conventional 4 h cytotoxicity assay, but were highly sensitive to IL-2 activated NK. Incubation of NK cells with CEM but not with the other two lines led to increased expression of the NK cell activation marker CD69. Treatment of effector cells with PGE2 and TGF-beta resulted in an inhibition of NK activity and CD69 expression. The calnexin transfected clone 1B9 clone had intermediate ability to block cytotoxicity in cold target inhibition assay compared to CEM and NKR. Expression of the adhesion molecules CD44 and LFA-1alpha was significantly higher on both calnexin positive cell lines compared to NKR. These data suggest that calnexin controls the expression of some, but not all, target structures that are necessary for binding and activation of NK cells.

Identification of tumor-associated antigens and their purification from cyst fluids of ovarian epithelial neoplasms

Abstract Based on several earlier reports demonstrating the existence of ovarian tumor-associated antigens in the cyst fluids of adenocarcinoma, we attempted to confirm these results, purify the tumor-associated antigens, and characterize the molecular species. Several antisera were produced in rabbits with clarified whole mucinous cystadenocarcinoma fluids. Unadsorbed antisera demonstrated very complex immunodiffusion patterns. Following adsorption with normal human serum and plasma and extracts of normal cervix and ovary strong cyst-associated reactivity remained. Further adsorption with A+ and B+ erythrocytes had no effect on antisera activity. A major cyst fluid antigen was purified from mucinous cysts by ammonium sulfate fractionation (0.20–0.60 saturation), DEAE-agarose chromatography, Sephadex G-200 gel filtration, and Bio-Gel A-5m agarose gel filtration. Polyacrylamide gel electrophoresis gave a single prominent band with minor contaminants. Approximately 70% of purified and radiolabeled antigen was precipitable by antisera, and 80% precipitated in either 10% trichloroacetic acid or 0.6 M perchloric acid. Tissue distribution was assessed by immunodiffusion analysis and showed strong association with mucinous tumors (benign and malignant) of the ovary. Purified antigen showed no reactivity by immunodiffusion analyses against antisera to other common tumor markers including carcinoembryonic antigen, alphafetoprotein, ferritin, and pregnancy-associated glycoproteins.

Oxygen-reactive metabolites are not detected at the effector-target interface during natural killing

Oxygen‐reactive metabolites are not detected at the natural killer (NK)‐target cell interface in quantities comparable to those seen for other effector‐target cell interactions. A novel luminol‐coated target cell chemiluminescence assay is described in which luminol is conjugated to the target cell surface with the bifunctional crosslinker 3,3′‐dithiobis(propionic acid N‐hydroxysuccinimide ester) (DSP). This modification of the basic chemiluminescence assay precludes exclusion of the detection system from the tightly occluded intercellular junction, a possible deficiency in previous investigations. Luminol conjugation does not affect NK‐mediated conjugate formation or cytolysis. As NK activity is enriched by a standard series of effector fractionation procedures, chemiluminescence generated against labeled target cells diminishes. Residual chemiluminescence in the most highly NK‐active effector fraction is ablated upon antibody and complement depletion of MO2+ cells. This indicates that monocyte contamination is the source of luminol‐detectable oxygen‐reactive metabolites.