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The L5178Y mouse lymphoma cell mutagenesis assay is used to detect the mutagenic activity of chemicals in a mammalian cell system. To evaluate this assay we compared the results of assays performed independently on 63 chemicals by laboratories at SRI International and Litton Bionetics, Inc. The two laboratories used similar protocols. The solvent and positive control mutant frequencies and cloning efficiencies obtained by the two laboratories were similar, which justified the use of the same quality-control criteria and analytical procedures for analyzing the results from both laboratories. The rate of concordance between the two laboratories was 92% for tests in the absence of S9 activation and 95% for tests in its presence. The results of the assays agreed for 57 of the 63 chemicals; three chemicals could not be compared because there were questionable calls in at least one of the laboratories; the results disagreed for the three remaining chemicals. The concordance rate for these overall assay evaluations was 95%. The interlaboratory concordance rates were similar to concordance rates for replicate experiments within the laboratories (96% at LBI, 94% at SRI). The mouse lymphoma cell mutagenicity results are concordant with the rodent chronic assay results in 78% of 50 chemicals and with the Salmonella assay results in 79% of 56 chemicals. Fifteen carcinogens were examined for genotoxic effects in mouse lymphoma, Salmonella, Chinese hamster ovary (CHO) chromosomal aberration, and CHO sister chromatid exchange assay. Eight of these were positive in all four assays. Of the seven noncarcinogens that were tested in these four assays, none was negative in all four. The main conclusion to be drawn from this study is that the mouse lymphoma cell forward mutation assay, as performed and evaluated in this study, detects chemical mutagenicity in a manner that is highly consistent with other genetic endpoints as well as rodent carcinogenicity studies. Thus the assay quality control and response criteria established in this study led not only to a high degree of reproducibility but also to an apparently reliable detection of mutagenic activity.

Published studies of the metabolism of human IgG using trace amounts of radiolabeled IgG demonstrated that the elimination of native IgG followed first-order kinetics but that the half-life of IgG was shortest in patients with the highest serum concentrations of IgG. To evaluate the effect of increasing the serum concentration of IgG on the metabolism of IgG, we determined the half-life and clearance of IgG and tetanus antibody in 16 patients with severe primary humoral immunodeficiency diseases while they received several doses of intravenous gamma-globulin (IVIG). Each patient received 100 mg/kg of IVIG each month and the half-life and clearance of IVIG were determined by following the decline in the serum IgG concentration. The dose of IVIG was adjusted to give a minimum IgG level of 200 mg/dl and the half-life was reevaluated. The dose was again adjusted to give minimum concentrations of 450 mg/dl and two additional studies were performed. Mean doses of IVIG infused increased from 100 to 346 mg/kg. The mean trough serum IgG concentration was 191 mg/dl on the standard dose and increased to 427 mg/dl at the highest dose. The serum half-lives of IgG were highly variable, ranging from 22 to 96 days. The mean decreased from 43 days in the first to 33 days in the third and fourth studies, and the clearances of IgG increased from 1.8339 to 2.4302 mg/kg/day, but the differences were not statistically significant. Patients with the highest serum IgG concentrations tended to have the longest half-lives, suggesting that intrinsic IgG production might falsely prolong the calculated half-life of IgG.(ABSTRACT TRUNCATED AT 250 WORDS)

It is often believed that the mortality intensity in the modern human population undergoes an exponential growth after 40 years, i.e. the actuarial aging rate is regarded to be constant after 40 years. To check this assumption we have calculated local aging rate values for 13 age ranges (within the interval of 30-92 years) for the male and female population of 48 states of the US (1969-1971). It was found that generally the male aging rate is not constant but lowers monotonically with time, while for females the aging rate has a pronounced approximately-shaped character with a minimum in the range of 45-60 years and a maximum within the range of 70-80 years. The results obtained are a warning to those who boldly use Gompertz or Gompertz-Makeham formulas when describing human aging on the population level.

The monoclonal antibody anti-2H4 recognizes a 220-kDa and 200-kDa glycoprotein and subdivides T4+ cells into distinct subpopulations: the T4+2H4+ inducer of suppression and the T4+2H4- inducer of help. The T4+2H4+ subset has been shown to play a crucial role in the activation of T8+ suppressor cells. In the present study, we attempted to determine whether the 2H4 molecule itself may be involved in initial triggering of suppressor inducer function. The results show that the addition of anti-2H4 antibody to a mixture of B and T cells resulted in a marked suppression of pokeweed mitogen-driven Ig synthesis. When anti-2H4 was added to B cell cultures containing T4+2H4+ or T4+2H4- cells but lacking T8 cells, no suppression was generated. In addition to the requirement for T8 cells, no suppression was generated if T4+2H4+ cells were absent. These results suggest that the anti-2H4 antibody contributes to the activation of the T4+2H4+ lymphocyte subset, which in turn induces T8 cells to suppress B cell Ig synthesis. Biochemical analysis of the T4+2H4+ subset of lymphocytes indicated that the in vitro addition of anti-2H4 antibody resulted in an increased expression of the 220-kDa and 200-kDa structure on T4 cells. We conclude that perturbation of the 2H4 molecule may potentiate the activation of the suppressor inducer subset and that the T200 molecule may be directly involved in suppressor inducer function.

Mutants, showing either constitutive (depressed) or non-inducible expression of chromosomally-mediated Type I beta-lactamase were obtained from clinical isolates of Enterobacter cloacae, Ent. aerogenes, Citrobacter freundii, Providencia stuartii, Morganella morganii, Serratia marcescens and Pseudomonas aeruginosa. The wild-type and mutant strains were compared for susceptibility to a range of beta-lactam antibiotics. Derepression of beta-lactamase synthesis generally, but not always, resulted in a marked reduction in susceptibility to the agents tested, including the '3rd generation' cephalosporins. In many cases, the observed resistance would preclude, or severely compromise, the therapeutic efficacy of the drugs. In this context, depressed mutants of Enterobacter spp., Citro. freundii and Ps. aeruginosa could be of primary concern although those of Ser. marcescens, Prov. stuartii and Morg. morganii often exhibited equally high resistance levels to older beta-lactams. Comparison of the susceptibilities of the non-inducible mutants with that of their inducible parents suggested variation in the beta-lactamase inductive potency of different compounds in different organisms. For example, cefoxitin was a powerful inducer in Ent. cloacae, Citro. freundii and one strain of Ps. aeruginosa; similarly cefazolin and cefuroxime were good beta-lactamase inducers in Ser. marcescens and Morg. morganii. Aminothiazolyl-oxime cephalosporins and ureido-penicillins were generally poor inducers. From such comparisons, the contribution of inducible Type I beta-lactamase to resistance phenotype could be ascertained.

SRI used the L5178Y mouse lymphoma cell forward mutation assay to determine the mutagenic activity of 63 coded chemicals from 16 chemical classes. Replicate experiments were performed to assess the reproducibility of the assay within the laboratory. The evaluations (positive or negative) of the first two repeat experiments with the chemicals were the same for 116 (87%) of 134 tests. Evaluational differences between the first two experiments were fewer in the presence of induced S9 (6 tests) than in the absence of S9 (12 tests). The most commonly observed variability was the magnitude of positive mutagenic responses; this may be attributed to factors such as compound solubilities, S9 activation conditions, and differential recovery of mutant cells. Some consistency was observed in the responses of compounds of various chemical classes. Generally, antibiotics (ABO) and the azo dyes, azoxy and hydrazo compounds, diazoalkanes, nitriles and azides (AZO), were mutagenic with S9; alkyl, acyl, and aryl halides, halogenated ethers, and halohydrins (HAL) were more strongly mutagenic with than without S9; and monofunctional polycyclic aromatic hydrocarbons and fluorenones (PAH) were mutagenic only with S9. Amine-1-oxides (AMO), alkyl and aryl epoxides (EPO), and nitroalkanes, nitroaromatics, nitroquinolines, nitrofurans, and nitroimidazoles (NIT) were mutagenic with and without S9; amides, sulfonamides, aromatic amines, aliphatic amines, hydroxylamines, and benzidine and its derivatives (AMI) were mutagenic without S9; and methyl carbamate (the only monofunctional carbamate) and thioureas (CBM) induced a negative response under both conditions.

Serological approaches have been relatively unsuccessful in defining the allelic products of the bovine major histocompatibility (MHC) class II loci. We demonstrate that bovine class II allelic products can be characterized by precipitation with a polyclonal antiserum and separation using one-dimensional isoelectric focusing. Polymorphic beta chains were present in immunoprecipitates from both biosynthetically and surface-labeled lectin-stimulated bovine T cells. Precipitates from biosynthetically labeled but not surface-labeled T cells contained a basic invariant chain and a non-polymorphic structure. The non-polymorphic structure appears to be a beta chain. The polymorphic class II beta chain co-segregated with bovine MHC class I allelic products in a half-sibling family, providing evidence for linkage between bovine class I and class II loci. This approach to the biochemical analysis of the bovine class II structures should facilitate the investigation of the association between the bovine products and disease susceptibility.

A data-based approach to formulating quality-control criteria for the mouse lymphoma cell forward mutation assay is described. Quality-control guidelines for solvent controls, positive controls, and compound-treated cultures were developed based on analysis of over 800 experiments. Frequency distributions of experimental parameters of control cultures, such as mutant frequencies, cloning efficiencies, and suspension growths, were examined. Cloning efficiency and relative total growth affected the variability only when the test chemical was highly toxic. This information was used to generate the quality-control criteria, which were applied to an experiment before it was evaluated for a response. The response categories for classifying the effect of test chemicals on the assay system are defined in terms of (1) the statistically significant differences in average mutant frequency between solvent control cultures and cultures exposed to a chemical and (2) the trend of the dose-related responses.

Stable, tandem dicentric chromosomes were discovered in two mutant cell colonies resulting from exposure of L5178Y mouse lymphoma cells to chemical mutagens. These unusual dicentrics were present in all metaphase cells examined from these colonies, even after approximately 65 cell generations in culture. Observation of cells in metaphase and anaphase suggests that the interstitial centromere in these dicentrics is non-functional, and that the terminal centromere is solely responsible for their orderly anaphase segregation.

Studies from our laboratory have shown that anti-T12, a mAb which recognizes CD6, is a macrophage-dependent mitogen for human T cells and can augment T cell autoreactivity in vitro. To obtain additional information regarding the potential biological role of CD6 we sought to further characterize its biochemical properties. The CD6 molecule on 125I-surface-labeled T cells and by Western blot analysis was a monomer of mol. wt 130,000 under reducing conditions and mol. wt 117,000 under non-reducing conditions, suggesting the presence of intrachain disulfide bonds. The polypeptide contains a protease sensitive site. In activated T cells, the protein was serine phosphorylated. Analysis of biosynthetically labeled CD6 in the presence of tunicamycin revealed a reduction in mol. wt from 130,000 to 100,000, indicating that the polypeptide is extensively N-glycosylated. The mAb, anti-2H1, had been shown to activate T cells in combination with PMA or the anti-T11(3) mAb but, unlike anti-T12, not in the presence of macrophages alone. The present studies demonstrate by sequential immunoprecipitation that these two mAbs recognize the same polypeptide. However, Western blot analysis and indirect immunofluorescence cross-blocking studies demonstrate that the two mAbs recognize different determinants on CD6. Anti-T12 recognizes an epitope that is present only under non-reducing/non-denaturing conditions, while anti-2H1 recognizes an epitope that is also preserved under reducing/denaturing conditions. A direct comparison of activation properties of the mAbs confirmed that anti-T12 was optimally mitogenic in the presence of macrophages but not PMA, while, conversely, anti-2H1 was optimally mitogenic in combination with PMA but not macrophages, suggesting that the differences in epitope specificity may account for the distinct activation properties of each mAb. Taken together, the structural and functional data strongly suggest that the CD6 membrane glycoprotein may function as a physiologically important receptor structure on human T lymphocytes.

Several T-cell structures are capable of generating intracellular signals linked to T-cell proliferation. Crosslinking of CD2, CD4 and CD45 with Ti/CD3 to several of these antigens can augment the minimal signal induced by antigen binding to the Ti/CD3 complex. Importantly, some of these regulatory structures (CD4, CD8 and CD45) are also expressed on subsets of T cells with distinct activation requirements and functional programs (helper, suppressor, suppressor-inducer and cytotoxic function). The CD4+ CD45RA+ (2H4+) subset responds well to self-Ia, poorly to soluble antigen and possesses suppressor-inducer function. A reciprocal subset CD4+ CD45RA- (4B4+) is preferentially activated by soluble recall antigens and possesses helper function. Each of these subsets can be distinguished by virtue of the differential expression of CD45 antigens. Importantly, the anti-2H4 antibody which reacts with a specific region near the N-terminus of two CD45 isoforms can effectively block its function. Crosslinking of CD4 with the Ti/CD3 complex preferentially activated the CD4+ CD45+ RA- subset, while soluble antibodies to CD2 preferentially affected the CD45 CD45RA+ subset. Thus, CD3 and CD4 more effectively synergize in the activation process on the CD4+ CD45RA- subset, a result consistent with the ability of this subpopulation to respond to recall antigens. The regulatory role of the CD4, CD8 and CD45 antigens may be mediated by an interactive network of protein-tyrosine phosphorylation and dephosphorylation. We have shown the CD4 and CD8 antigens to be associated with the T cell-specific protein-tyrosine kinase (p56lck). p56lck is a member of a family of protein-tyrosine kinases with an established ability to activate and transform mammalian cells. The CD4/CD8:p56lck complex is catalytically active as shown by its ability to phosphorylate various members of the Ti/CD3 complex. By contrast, the CD45 antigens possess protein-tyrosine phosphatase activity within their intracellular domains and are postulated to function by virtue of a regulatory interaction with CD4/CD8:p56lck and its potential substrates. Thus, the differences in the response of the CD4+ CD45RA+/- subsets to various stimuli and the expansion of T-cell subsets with distinct immunoregulatory programs may be governed by a pathway of tyrosine-mediated events.