The multiple immune-evasion genes of murine cytomegalovirus are not redundant: m4 and m152 inhibit antigen presentation in a complementary and cooperative fashion.

Both human cytomegaloviruses (HCMVs) and murine cytomegaloviruses (MCMVs) encode multiple genes that interfere with antigen presentation by major histocompatibility complex (MHC) class I, and thus protect infected targets from lysis by virus-specific cytotoxic T lymphocytes (CTLs). HCMV has been shown to encode four such genes and MCMV to encode two. MCMV m152 blocks the export of class I from a pre-Golgi compartment, and MCMV m6 directs class I to the lysosome for degradation. A third MCMV gene, m4, encodes a glycoprotein which is expressed at the cell surface in association with class I. Here we here show that m4 is a CTL-evasion gene which, unlike previously described immune-evasion genes, inhibited CTLs without blocking class I surface expression. m152 was necessary to block antigen presentation to both K(b)- and D(b)-restricted CTL clones, while m4 was necessary to block presentation only to K(b)-restricted clones. m152 caused complete retention of D(b), but only partial retention of K(b), in a pre-Golgi compartment. Thus, while m152 effectively inhibited D(b)-restricted CTLs, m4 was required to completely inhibit K(b)-restricted CTLs. We propose that cytomegaloviruses encode multiple immune-evasion genes in order to cope with the diversity of class I molecules in outbred host populations.

Class I major histocompatibility complex-restricted cytotoxic T lymphocytes specific for Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines against which they were raised.

We have raised CD8+ cytotoxic T lymphocytes (CTL) from three Epstein-Barr virus-seropositive donors by incubating peripheral blood lymphocytes with irradiated autologous B95.8-strain EBV-transformed B lymphoblastoid cells (LCL). However, to detect lysis in a standard 51Cr release assay of the LCL against which these CTL were raised, superinfection with recombinant vaccinia expressing the appropriate EBV protein or incubation with the peptide epitope was necessary. The untreated LCL were not lysed, even though Western blotting demonstrated that they expressed the EBV antigens containing the CTL epitopes. We have found CTL of this phenotype that are restricted by human leukocyte antigen-A2, -A3, -B7, or -B39, and which recognize the EBV latent proteins, EBV nuclear antigen (EBNA)-3A, EBNA-3C, or terminal protein. During these experiments, we identified a new human leukocyte antigen-A3-restricted EBNA-3A epitope, residues 603-611, RLRAEAGVK. We raised a spontaneous LCL, transformed by endogenous EBV, from one donor, but this was also not lysed. For at least one of the epitopes, CTL from another donor lysed the LCL without superinfection or addition of peptides. We conclude that the CTL were unable to achieve a high enough avidity of interaction with untreated LCL to trigger effector function, although the LCL were able to stimulate them to grow in vitro for up to 4 mo. To assess whether a small percentage of the LCL might possess a higher antigen density, we used an assay of tumor necrosis factor release from a CTL clone, which was able to detect antigen-bearing cells representing only 1% of a stimulating LCL population. Nevertheless, the untreated autologous LCL line failed to stimulate tumor necrosis factor release.

Transient inhibition of DNA synthesis results in increased dihydrofolate reductase synthesis and subsequent increased DNA content per cell.

We examined the role that blockage of cells in the cell cycle may play in the stimulation of gene amplification and enhancement of drug resistance. We found that several different inhibitors of DNA synthesis, which were each able to block cells at the G1-S-phase boundary, induced an enhanced cycloheximide-sensitive synthesis of an early S-phase cell cycle-regulated enzyme, dihydrofolate reductase, and of other proteins as well. This response was specific, in that blockage at the G2 phase did not result in overproduction of the enzyme. When the cells were released from drug inhibition, DNA synthesis resumed, resulting in a cycloheximide-sensitive elevation in DNA content per cell. We speculate that the excess DNA synthesis (which could contribute to events detectable later as gene amplification) is a consequence of the accumulation of S-phase-specific proteins in the affected cells, which may then secondarily influence the pattern of DNA replication.

Increased gene amplification in L5178Y mouse lymphoma cells with hydroxyurea-induced chromosomal aberrations.

Chromosomal aberrations and dihydrofolate reductase gene amplification are observed in L5178Y mouse lymphoma cells after treatment with hydroxyurea. The types of aberrations include polyploidy, endoreduplication, chromosome fragmentation, and the presence of extrachromosomal DNA. Hydroxyurea-treated cells analyzed by cell sorting showed a subpopulation of cells with increased DNA and increased dihydrofolate reductase. This subpopulation shows a high incidence of chromosome aberrations and an increased frequency of dihydrofolate reductase gene amplification. Hydroxyurea-treated cells with the normal amount of DNA and dihydrofolate reductase have few aberrations and a low frequency of dihydrofolate reductase gene amplification. We propose that hydroxyurea treatment causes overreplication of DNA and that recombination of overreplicated DNA can lead to chromosome aberrations and gene amplification.

Cytogenetic and molecular characterization of tumors in nude mice derived from a multidrug-resistant human leukemia cell line.

We have evaluated whether selection of a human tumor leukemic line for resistance to vinblastine (Velban; VLB) alters its tumorigenicity. To address this question, CEM and CEM/VLB100 cells [which express the multiple drug-resistant (MDR) phenotype via amplification of the P-glycoprotein gene] were characterized by several techniques including chromosome banding, in situ hybridization, Southern blotting, RNA dot blotting, in vitro drug sensitivity, and tumorigenicity in nude mice. Analysis of the chromosome banding patterns of both drug-sensitive CEM cells and the MDR CEM/VLB100 cells revealed that the two lines differed primarily by the presence of a large metacentric marker chromosome associated with the acquisition of VLB resistance. In situ hybridization of a P-glycoprotein complementary DNA to metaphase chromosomes showed that the amplified P-glycoprotein genes in the CEM/VLB100 cell line were localized to this large marker. Tumorigenicity of both the CEM and CEM/VLB100 cell lines was measured after injection of 10(7) cells/nude mouse. The results showed that 4 of 4 drug-sensitive and 4 of 5 drug-resistant cell lines formed tumors in 5-10 wk. By comparison with the parental line, three of the four tumors arising from the CEM/VLB100 line retained their drug-resistance properties as measured by vinblastine resistance in vitro and elevated P-glycoprotein mRNA expression associated with P-glycoprotein gene amplification. In addition, tumors retaining the MDR phenotype also retained the large metacentric marker chromosome. One tumor arising from CEM/VLB100 reverted to the drug-sensitive phenotype, with a resultant decrease in P-glycoprotein mRNA expression and loss of P-glycoprotein gene amplification. This revertant was also missing the large metacentric marker present in all cells from the CEM/VLB100 parent. Our experiments show that the acquisition of the MDR phenotype resulting from overexpression of P-glycoprotein in the plasma membrane does not effect the tumorigenicity of human CEM cells.

Radiation resistance in a multidrug resistant human T-cell leukemia line.

In clinical practice, cancers refractory to chemotherapy commonly appear to be comparatively radioresistant. One mechanism by which cancer cells become resistant to chemotherapy is pleiotropic multidrug resistance, characterized by cross resistance to a number of otherwise unrelated heterocyclic antineoplastic agents, including vinca alkaloids, anthracyclines, dactinomycin, and others. We have studied a drug sensitive human leukemia cell line, CEM; a pleiotropic multidrug resistant subline of CEM, CEM/VLB100; VLB-1, a drug sensitive revertant subline arising during in vivo passage of CEM/VLB100; and a methotrexate resistant subline of CEM, CEM-MTX. Using soft-agar colony formation after graded doses of X rays as an endpoint, we found that CEM, CEM-MTX, and CEM/VLB100 had similar terminal slopes (D0 = 0.66 Gy). However, the CEM/VLB100 survival curve had a broader initial shoulder (n = 3.0, Dq = 0.75 Gy) than did CEM (n = 1.6, Dq = 0.25 Gy) or CEM/MTX (n = 1.0, Dq = 0 Gy), suggesting that CEM/VLB100 has an increased capacity to repair radiation-induced DNA damage. This was tested by comparing the cell lines' abilities to accumulate sublethal damage. In split dose recovery experiments, CEM/VLB100 demonstrated increased ability to repair sublethal radiation damage following fractionated irradiation compared with the CEM parental line. Although it no longer demonstrated multidrug resistance, VLB-1 still displayed diminished radiation sensitivity. On the basis of these and other investigators' results, we suggest that diminished radiation sensitivity is separate from, but can be closely associated with, the multidrug-resistant phenotype.

Bovine herpesvirus-1 infection affects the peptide transport activity in bovine cells.

Infection of cattle with bovine herpesvirus-1 (BHV-1) impairs the cell-mediated immune response (CMI) of the affected host. We investigated the location of interference of BHV-1 with the major histocompatibility complex (MHC) class I antigen presentation pathway by employing an assay that allows assessment of the peptide transport activity of the Transporter associated with Antigen Presentation (TAP) from the cytoplasm into the endoplasmic reticulum (ER). We found a considerable down-regulation of the peptide transport activity in bovine epithelial cells, taking place as early as 2 h after virus infection. This down-regulation was also dose-dependent, and, at high multiplicities of infection (moi), led to an almost complete shutdown of TAP. By inhibiting peptide transport into the ER, the virus impairs loading of MHC class I molecules and their subsequent egress from the ER to the cell surface. This may lead to defective priming of cytotoxic T lymphocytes. Thus, BHV-1 is yet another member of its family Herpesviridae that selectively interferes with the host's antigen presentation machinery to evade the host's immune response in vivo.

Effects of v-src oncogene activation on radiation sensitivity in drug-sensitive and in multidrug-resistant rat fibroblasts.

Recent work has implicated the activated ras oncogene, whose gene product is a G-protein located in the plasma membrane, as well as the activated raf oncogene, whose gene product is a membrane-associated protein kinase, in contributing to radioresistance. Another transforming oncogene whose gene product is localized to the plasma membrane is v-src. We have examined a rat fibroblast line (RAT-1) infected with an avian sarcoma virus carrying a temperature-sensitive mutation in the v-src tyrosine kinase domain (LA-24). At 40 degrees C, LA-24 cells have a flat morphology and grow as a contact-inhibited monolayer, while at 35 degrees C, LA-24 cells have a transformed morphology, lose contact inhibition, grow in soft agar, and exhibit 3.5-fold higher tyrosine kinase activity. The parental RAT-1 line, not infected by the virus, grows at both temperatures as a contact-inhibited monolayer. This well-characterized system represents a good model for examining the effect of v-src transformation on radiosensitivity. RAT-1 and LA-24 cells grown at 35 and 40 degrees C were irradiated with graded doses of radiation, and clonogenic survival was assayed. For LA-24 cells grown at 35 and 40 degrees C, and for RAT-1 cells grown at 35 and 40 degrees C, calculated D0, n, alpha, and beta values did not differ significantly. To determine whether there might be differences in radiation damage repair capacity too subtle to detect by comparing radiation survival curves, sublethal damage repair capacity was assessed. There was no difference in sublethal damage repair capacity for LA-24 cells grown at 35 or 40 degrees C. Other studies have associated multidrug resistance with radioresistance. We have examined the radiation sensitivity of two colchicine-resistant LA-24 clones with four- to fivefold amplification of the P-glycoprotein gene, which are four-to fivefold more resistant to colchicine than the parental LA-24 line. In these multidrug-resistant clones, v-src activation does appear to increase radiation resistance. This did not appear to be due to alteration in cell cycle kinetics. We conclude that oncogene activation, or even protein kinase activity per se, does not necessarily lead to radiation resistance. Rather, radiation resistance following oncogene activation depends upon the oncogene and cell line studied, and perhaps upon specific protein phosphorylation.

Increased frequency of P-glycoprotein gene amplification in colchicine-resistant Rat-1 clones transformed by v-src.

A rat fibroblast cell line (Rat-1) carrying a temperature-sensitive mutation of v-src was used to determine whether inducible cellular transformation altered the ability of cells to amplify the p-glycoprotein gene in response to colchicine selection. Transformed and nontransformed Rat-1 fibroblasts selected under 4 times the LD50 generated the same number of colchicine-resistant colonies. We next examined colchicine-resistant colonies derived from transformed cells and compared them to colchicine-resistant colonies derived from nontransformed cells. When Rat-1 cells were selected at 35 degrees C (transforming temperature), 7 out of 7 clones exhibited a 3- to 5-fold p-glycoprotein gene amplification. These results contrasted to those found at the nontransforming temperature (40 degrees C); none of the 8 colchicine-resistant clones examined had amplified the p-glycoprotein gene. Thus in Rat-1 cells carrying a temperature-sensitive v-src gene, p-glycoprotein gene amplification was observed at a high frequency only in transformed clones selected at the temperature permissive for v-src activity.

Loss of tumorigenicity in a methotrexate-resistant human leukemia cell line.

We have studied the tumorigenicity of a CCRF-CEM-derived cell line (CEM/MTX-3) resistant to MTX. Eight of nine mice inoculated with drug-sensitive CEM cells developed tumors within 5 weeks, but 16 weeks after inoculation with CEM/MTX-3 cells, none of nine mice developed tumors. We were unable to detect dihydrofolate reductase gene overexpression, amplification, or rearrangement in CEM/MTX-3 cells. Instead, the resistance in CEM/MTX-3 cells appeared to be due largely to decreased methotrexate accumulation. Because tumorigenicity could have been related to intracellular folate levels, we cultured CEM and CEM/MTX-3 cells in folate-rich and folate-deprived media. When inoculated in mice, CEM cells cultured in either medium rapidly formed tumors. As before, CEM/MTX-3 cells grown in either medium did not, suggesting that factors other than low folate levels contributed to the inability of CEM/MTX-3 cells to form tumors. Cytogenetic analysis revealed that the CEM/MTX-3 karyotype contained a unique and complex translocation marker chromosome that was not observed in the CEM cell line and which involved chromosomal breakpoints at bands 11p14, 22p11, and 22p13. Although biochemical mechanisms are not yet delineated, this remodeled chromosome could be related to the loss of tumorigenicity in CEM/MTX-3 cells.

Dialkylquinoneimine metabolites of chloroacetanilide herbicides induce sister chromatid exchanges in cultured human lymphocytes.

Some of the most widely-used herbicides are the chloroacetanilides exemplified by alachlor and butachlor (derived from 2,6-diethylaniline) and metolachlor and acetochlor (synthesized from 2-ethyl-6-methylaniline). This investigation tests the hypothesis that the previously-observed oncogenicity of these herbicides is due to genotoxic intermediates such as diethylbenzoquinoneimine, a purported alachlor metabolite. Syntheses are reported here for the corresponding 2,6-dialkylbenzoquinoneimines, selected chloroacetyldialkylbenzoquinoneimines and several other candidate or known metabolites. The possible mutagenicity of diethylbenzoquinoneimine was tested in Salmonella typhimurium strains TA98 and TA100 with a weakly-positive response in the TA100 strain indicating induction of base-pair substitution mutations. The frequency of sister chromatid exchange (SCE) in Chinese hamster ovary cells was increased by alachlor at 10 microM and diethylaniline but not ethylmethylaniline at 30 and 3 microM. Isolated and cultured peripheral lymphocytes (mostly T cells) were used from two human donors to study the effects of the chloroacetanilides and their metabolites on primary human cells. In tests at 10 microM, the SCE frequency was increased by alachlor and possibly acetochlor but not by butachlor, metolachlor, dimethachlor (a 2,6-dimethyl analog) and dimethenamid (an analog based on 2,4-dimethyl-3-thienylamine). At 0.3 microM in cultured human lymphocytes, alachlor, the corresponding chloroacetanilide (N-dealkyl-alachlor) and aniline metabolites (and their 4-hydroxy derivatives), and diethylbenzoquinone were inactive or active in only one of the two donors whereas at 0.1-0.3 microM the SCE ratio for treated cells divided by the controls was always higher for diethylbenzoquinoneimine than for ethylmethyl- and dimethylbenzoquinoneimines. All the tested compounds were toxic to lymphocytes, but the depression of the mitotic index and increased duration of the cell cycle were not directly linked with SCE induction. Previous investigations have suggested that chloroacetanilide herbicides such as alachlor derived from 2,6-dialkylanilines are metabolized to 2,6-dialkylbenzoquinoneimines and the present study provides the first direct evidence that these metabolites are genotoxic in human lymphocytes.

Radiation resistance in a doxorubicin-resistant human fibrosarcoma cell line.

In clinical practice, cancers refractory to chemotherapy can appear relatively radioresistant. Recent work in multidrug-resistant cell lines has yielded conflicting results concerning the relationship between drug resistance and radiation resistance. The current study examines the radiation response of a human fibrosarcoma (HT1080) and a doxorubicin-resistant subline (HT1080/DR4). Using soft-agar colony formation after graded doses of x-rays as an endpoint, HT1080/DR4 had an increased D0 (D0 = 2.1 Gy) and a broader initial shoulder (n = 2.7, Dq = 2.1 Gy) than the parental HT1080 line (D0 = 0.7, Gy, n = 1.2, Dq = 0.3 Gy), suggesting that HT1080/DR4 has an increased capacity to repair radiation-induced DNA damage. This possibility was tested by comparing the cell lines' ability to accumulate sublethal damage. In split-dose recovery experiments, HT1080/DR4 demonstrated increased ability to repair sublethal radiation damage following fractionated irradiation, compared with the HT1080 parental line. The mechanism for this radiation resistance is not clear, but a variety of cellular alterations seen in drug-resistant cell lines are discussed with reference to areas of further study.

Peritonitis.

Intraperitoneal infections or inflammatory disease in elderly patients are a diagnostic challenge that will be encountered with increasing frequency in the aging population of the United States. Diagnostic clues are not overt and frequently appear late in the course of the disease. Confusion and subtle changes in clinical status are important signals. A high index of suspicion is required to initiate diagnostic procedures. Therapy will frequently be modified not only to the disease process but also to the patient to incorporate concepts of minimally invasive procedures. The advent of laparoscopic surgery may be a great advance in the diagnosis and treatment of intra-abdominal crisis in geriatric patients. It also may make elective surgery conform more to the goals of the geriatric patient and thus prevent the high morbidity and mortality associated with emergency surgery in this age group.

Dynamic cardiomyoplasty for treatment of heart failure.

Dynamic cardiomyoplasty is a new surgical procedure proposed for treatment of the failing heart. Clinically, the latissimus dorsi muscle is raised as a pedicled flap and wrapped around the heart. The skeletal muscle is transformed to produce a myocardium-like fatigue-resistant muscle. It is stimulated to contract in synchrony with the heart in hope of assisting the myocardial contraction. An R-wave synchronous pacemaker provides a pulse-train form of stimulation to simulate, for the skeletal muscle, the contractile characteristics of the myocardial syncytium. We have undertaken a critical review of the clinical results of dynamic cardiomyoplasty reported to date. Objective evidence of clinical improvement after dynamic cardiomyoplasty resulting from the contractile assistance of the myoplasty has been modest. Many of the beneficial results reported could be explained by concomitant procedures done, such as aneurysmectomy or coronary artery bypass grafting. Hemodynamic studies have failed to demonstrate consistent and convincing improvement as a result of the cardiomyoplasty stimulation. We have, however, identified an interesting subgroup of patients, in whom a striking hemodynamic response to cardiomyoplasty stimulation has been reported. These patients all possess large resting heart volumes characteristic of dilated cardiomyopathy. Thus, case selection may ultimately be one of the most important factors in determining the success of dynamic cardiomyoplasty for the treatment of heart failure.

Selective processing of shape-related words in women with eating disorders, and those who have recovered.

OBJECTIVES: This study sought to investigate whether women with anorexia or bulimia nervosa and women who had recovered showed cognitive bias towards shape, food and adolescent issues. DESIGN: A five-group analysis of variance design was used, in which the different client groups were the independent variables. The dependent variable was performance on an emotional Stroop task. METHODS: Current anorexia sufferers (N = 31), current bulimia sufferers (N = 24), recovered anorexics (N = 23), recovered bulimics (N = 11) and women who had never suffered from eating disorders (N = 33) were recruited through health-care professionals, support groups and newspapers. Colour-naming times for target and comparison Stroop cards were measured. RESULTS: Women currently suffering from bulimia, and women who had recovered from anorexia, were found to be more distracted by shape concerns than women who had never suffered eating disorders and women who had recovered from bulimia. CONCLUSIONS: The results suggest that there may be an enduring cognitive bias among women who have recovered from anorexia. This is the first study in which impairment on an emotional Stroop task has been found to persist after recovery from a clinical condition.

The utility of selective screening for carotid stenosis in cardiac surgery patients.

PURPOSE: To determine if any of 8 categorical clinical variables can be used to select patients and improve the yield of a screening program for severe carotid stenosis (> or = 80%) in elective cardiac surgery patients. METHODS: A prospective cohort analysis of 200 consecutive patients prior to elective cardiac surgery for the following variables: age, gender, smoking carotid bruit, peripheral vascular disease, hyperlipidaemia, previous neurologic symptoms and diabetes mellitus. All patients were subsequently screened with carotid duplex scanning for the presence of severe carotid stenosis. Positive scans were confirmed by angiography. RESULTS: Sixteen patients (8%) were identified with severe carotid stenosis. Univariate analysis identified three variables that increase risk for carotid stenosis: carotid bruit (relative risk (RR)=16.4, 5.4-57.6 95% confidence interval, p<0.001), neurological history (RR=10.3, 3.9-23.2, p<0.001) and peripheral vascular disease (RR=5.3, 1.9-14.9, p<0.001). Stepwise logistic regression analysis identified previous neurologic history and carotid bruit as independent predictors of stenosis. If screening for carotid stenosis was limited to patients with these two variables, then 37 (18.5% of total) patients would have been screened. Fourteen of these 37 (37.8%) had a severe carotid stenosis. Two patients with stenosis (12.5% of those with carotid stenosis, 1% of total patient population) would not have been screened. CONCLUSIONS: Clinical variables can be used to improve the yield of a preoperative screening program for carotid stenosis.

Extraversion and variety-seeking in a monotonous task.

It is hypothesized on the basis of Eysenck's theory of extraversion that extraverts should build more variety into their performance at a monotonous task than introverts. The performance of a group of extravets (n = 16) and a group of introverts (n = 16) on a simple repetitive task was compared. Comparisons were made on two measures of response variety: firstly, a simple measure of number of alternations among possible responses and, secondly, a measure of variety taken from information theory-the average entropy of the set of responses made. The hypothesis was confirmed on both measures. The results are interpreted as adding further support to Eysenck's work linking differences in extraversion to differences in arousal.

Cognitive and affective aspects of boredom.

It is argued that previous research may have been mistaken in assuming that monotony in sensory stimulation is a necessary and sufficient cause of boredom. Five investigations are reported which used personal construct theory and repertory grid techniques to investigate three hypotheses: that boredom is associated with subjective monotony, that boredom is associated with a high degree of frustration and that boredom arises when stimulation lacks meaning for the individual. Results supported hypotheses (1) and (2) but no evidence was found to support hypothesis (3).

Towards a model of boredom.

On the basis of the authors' own research it is suggested boredom may be viewed as having cognitive and affective components. The cognitive component is subjective monotony and the affective component is a high level of frustration. An attempt is made to integrate the evidence supporting this view of boredom with other evidence from the literature to present an integrated model of boredom in which the roles of personality, situational and task characteristics influencing boredom are outlined. Psychophysiological changes occurring during the performance of boring tasks are examined. It is argued that the nature and extent of changes in heart rate and heart-rate variability are not a consequence of boredom but of a task feature (mental load). It is concluded that no clear psychophysiological component of boredom can be detected at present.

Anxiety responses to subliminal experience of mild stress.

Two groups of undergraduates (n = 14 in each) matched for level of trait anxiety participated in the experiment. One group (E) was presented with 20 'emotional' words 10 per cent below detection threshold while the other group (N) was presented with 20 emotionally neutral words under the same conditions. Ratings of seven psychological variables were taken before and after stimulation and two psychophysiological measures, heart and respiration rate, were also taken. MANOVA and subsequent ANOVA analyses showed ratings of sweating and anxiety increased in the E group and decreased in the N group following stimulation. Ratings of shaking and muscular tension increased significantly in both groups but the increase was significantly greater in the E group. Ratings of palpitations and difficulty in breathing increased significantly in both groups as did measures of actual heart and respiration rate, but these increases appeared to be an artifact of task demands. It is concluded that manifest anxiety and some features of anxiety having somatic referents can be induced by subliminal experience of mild stress.