Correlation between the anticellular and DNA fragmenting activities of tumor necrosis factor.

The treatment of cells sensitive to the anticellular effect of tumor necrosis factor (TNF) with TNF results in a degradation of their cellular DNA into DNA fragments that are multiples of 200 base pairs. TNF treatment of cells resistant to the anticellular effect of TNF, but bearing receptors for TNF, fails to result in any DNA fragmentation. Incubation conditions, such as temperature, the presence of metabolic inhibitors or amino acid deprivation, that modulate the effectiveness of TNF or affect the rate at which TNF exerts its anticellular effect have a similar effect on the ability of the TNF to generate DNA fragments. Thus the TNF-mediated DNA fragmentation and the rate at which it occurs correlates with the rate at which cells respond to the anticellular effect of TNF and, as such, might serve as a marker for the responsiveness of cells to TNF.

The development of antibody to the interferon-induced indoleamine 2,3-dioxygenase and the study of the regulation of its synthesis.

Interferon (IFN) treatment of cells induces the synthesis of several new proteins. Antibody to the IFN-induced 42,000 dalton protein has been prepared and used in the study of this protein. The synthesis of the 42,000 dalton protein is dependent on de novo RNA synthesis, as its induction can be blocked if actinomycin D and the IFN are added to the cells simultaneously. Several lines of evidence suggest that the IFN-induced 42,000 dalton protein is the IFN-induced indoleamine 2,3-dioxygenase (IDO). These are as follows: 1) antibody to the 42,000 dalton protein neutralizes the activity of the IDO; 2) examination of a variety of cell lines reveals a correlation between the presence of this protein and the presence of the IDO; and 3) the induction of both the IDO and the 42,000 dalton protein is blocked under conditions in which the IFN treatment is performed in the presence of cycloheximide, and actinomycin D is added to the cells prior to the removal of the cycloheximide. A study of a variety of cell lines has revealed that the induction of the IDO occurs primarily in response to IFN-gamma. Peripheral blood mononuclear cells (PBMC) were the only cell population in which IFN-alpha and IFN-gamma were observed to produce the IDO. The IDO activity induced in IFN-alpha and IFN-gamma treated PBMC is neutralized by antibody to the 42,000 dalton protein, thus demonstrating that the IDO activity induced in these cells by IFN-alpha and IFN-gamma is mediated by the same molecule or antigenically related molecules. Fractionation of the PBMC populations reveals that it is the monocyte that produces the IFN-induced IDO.

Tumor necrosis factor and IFN induce a common set of proteins.

The treatment of cells with TNF or IFN results in the development of an antiviral state and in the induction of a common set of proteins with m.w. of 80,000, 67,000, and 56,000. The induction of the 80,000- and 56,000-Da proteins after TNF treatment is dependent on the synthesis of an intermediary protein, whereas the induction of the 67,000-Da protein appears to occur as a direct result of the TNF treatment. The effects of antibodies to IFN on the TNF-mediated effects have been evaluated and reveal that the incubation of TNF-treated cells with antibody to rIFN-beta 1 greatly reduces the antiviral effectiveness of the TNF treatment and blocks the ability of TNF to induce the 80,000-Da protein. Incubation with antibodies to either IFN-alpha or IFN-gamma failed to affect the TNF-mediated responses. Thus, the induction by TNF of each of the proteins is regulated differently and is mediated through both IFN-dependent and IFN-independent mechanisms.

Production of a monoclonal antibody directed against an interferon-induced 56,000-dalton protein and its use in the study of this protein.

Interferon (IFN) treatment of cells induces the synthesis of several new proteins. A hybridoma cell line producing monoclonal antibody to the IFN-induced 56,000-dalton protein has been developed. The IFN-induced 56,000-dalton protein is synthesized by a variety of different cells and in response to IFN-alpha, IFN-beta, and IFN-gamma. The induction of this protein is dependent on de novo RNA synthesis, since its induction is inhibited if actinomycin D and the IFNs are added to the cells simultaneously. Labeling of IFN-treated cells at 4-h intervals at various times after the addition of the IFNs reveals that the synthesis of the 56,000-dalton protein in IFN-alpha-treated cells peaks within 12 h after the addition of the IFN and is no longer enhanced 20 h after exposure to the IFN. In contrast, IFN-gamma-treated cells continue to show an enhanced synthesis of this IFN-induced protein even after 20 h of exposure to the IFN. Thus, the synthesis of the IFN-induced 56,000-dalton protein is regulated differently by the different IFNs. When cells are treated with IFN-alpha or IFN-gamma in the presence of cycloheximide, and actinomycin D is added prior to the removal of the cycloheximide, the cells produce the IFN-induced 56,000-dalton protein and develop an antiviral state in response to both IFN-alpha and IFN-gamma. These results demonstrate that the synthesis of the 56,000-dalton protein is not dependent on the synthesis of an intermediary protein and that the establishment of an antiviral state occurs in the absence of multiple transcriptional events.

Measuring clinical competence of medical students.

Evidence of clinical competence for medical students entering the clinical clerkships at the University of Kansas College of Health Sciences is established by passing two different examinations: a 100 item multiple choice examination and a videotaped history and physical examination by each student of a simulated patient, being rated by that patient and two examiners. In 1976 the class of 196 medical students took an average 1.85 written examinations per student. With 70% or better constituting a passing score, 30.6% passed on the first attempt, 55.6% the second, 11.2% the third and 2.5% the fourth. Each student passed the televised practical examination and had the opportunity to review his or her videotape with a critiqued data base and the examiners' and simulated patient's evaluations in hand. Correlation coefficients for all 196 students between scores of written examinations, medicine tutors, examiners and professional patients revealed weak but significant correlations between the assessments of examiners and medical tutors and assessments of examiners and written examination scores, but not between other evaluations. This scheme of proof of competence appears to be objective and direct, and serves the convenience of both students and teaching staff.