Ethical issues in screening and testing for genetic diseases.

New analytic techniques coupled with detailed knowledge of the entire human genome soon will make possible testing of all genes in every person. Concepts of disease will change, and predisease states will be identified for genetic therapies. Patients, physicians, and laboratory personnel will be confronted with ethical and moral issues in the performance of genetic testing. The primary ethical issues will focus on who has the right to request or compel genetic testing, who has access to confidential information, and what medical or social actions may be predicated legally on genetic information.

Antibody-mediated complement activation on nucleated cells. A quantitative analysis of the individual reaction steps.

The sequential molecular events of the initiation, amplification, and membrane attack phases of classical C pathway activation on nucleated cells were investigated. As a model system, C-susceptible human melanoma cells (SK-MEL-93-2) expressing the disialoganglioside Ag GD3 were studied. Activation of the classical C pathway was initiated by the anti-GD3 mAb R24 (murine IgG3). The initiation phase is characterized by a very inefficient molar ratio of deposited C1q per Ab molecule. At an Ab density of 5.86 x 10(6) molecules/cell, only 3% of cell-bound R24 molecules form suitable pairs for C1q binding. During the amplification phase maximally 2.44 x 10(6) molecules of C4 and 0.67 x 10(6) molecules of C2/cell are being bound to form the C3 convertase. Despite the rather inefficient binding of C2, the C3 convertase is highly active in depositing high numbers of C3b molecules on the cell surface. Maximum binding of C3b occurred within 5 min of incubation with a total number of 2.1 x 10(7) molecules/cell. This indicates amplification factors at the level of C4 and C3 of 28 (C4/C1q) and 241 (C3/C1q), respectively. C3b was found to be rapidly cleaved into iC3b. As a result of this rapid C3b degradation, the membrane attack phase is initiated with a relatively inefficient C5 activation. The maximal number of 9.5 x 10(5) molecules C5b/cell corresponds to a molar ratio of C5:C3 of only 1:22. The deposition of C5b led to the subsequent maximum binding of the following numbers of molecules of terminal C components per cell: C6, 0.8 x 10(6); C7, 0.89 x 10(6); C8, 0.82 x 10(6); C9, 1.8 x 10(6). These numbers correspond to average molar ratios (calculated per C5b molecule) of C5b/C6/C7/C8/C9 of 1/0.85/0.94/0.86/1.88. In addition to the monomeric C9, dimeric and polymeric (12- to 16-mer) forms of the molecule could be demonstrated. Collectively, our data represent a first comprehensive quantitative analysis of classical pathway activation on a nucleated cell.

Cytokines and the immune response.

The recent massive growth and development of clinical immunology has been enriched by the discovery of a new family of molecules, the cytokines, which consist of various groups of polypeptide mediators involved in the communication network of the cells of the immune system. This article provides an overview of the immune system and the current status of the cytokines and their clinical application.

Molecular basis of complement resistance of human melanoma cells expressing the C3-cleaving membrane protease p65.

The molecular mechanism of complement resistance of the human SK-MEL-170 melanoma cell line was investigated. The cells have been shown to express the C3b-cleaving membrane protease p65. To delineate the molecular consequences of the C3b-cleaving activity for the complement cytotoxicity, the molecular events during the initiation (R24 monoclonal antibody, C1), amplification (C4, C3), and membrane attack (C5, C9) phases of complement were studied in comparison to a complement-susceptible human melanoma line (SK-MEL-93-2). No cleavage of C4b and C5b, 2 molecules structurally similar to C3b, was observed on the cells during classical pathway activation indicating the specificity of the p65 protease for the C3b molecule. The rapid degradation of C3b by p65 on the surface of complement-resistant SK-MEL-170 cells generates a M(r) 30,000 C3 alpha'-chain-fragment detectable as early as 1 min after complement activation, whereas no such fragment was present in detectable amounts on complement-susceptible cells. As a result of the rapid C3b proteolysis by p65 on resistant SK-MEL-170 cells, less C5 convertases are formed, which in turn results in the formation of a lower number of terminal complement components and membrane attack complexes. R24 antibody and C1q binding to the resistant cells was slightly lower as to susceptible cells. C4 binding studies, however, revealed that the observed difference in antibody and C1q binding has no influence on the complement resistance of SK-MEL-170 cells: significantly more C4b was bound to complement-resistant (1565 +/- 92 fg/cell) as compared to susceptible cells (715 +/- 31 fg/cell). On extraction of the molecular forms of C4 bound to the cell membranes, an additional high molecular weight C4 species--apparently a C4b-C4b homodimer--appeared only on the resistant SK-MEL-170 cells that may function as a residual back-up C5 convertase. Collectively, these results show that SK-MEL-170 human melanoma cells evade complement-mediated cytolysis despite sufficient activation of early components of the classical complement pathway by p65-mediated rapid degradation of surface-bound C3b, leading to a significant reduction in membrane attack complex formation. Thus, rapid cleavage of surface deposited C3b was established as a powerful mechanism of complement resistance.