Activity profiles of developmental toxicity: design considerations and pilot implementation

The available literature was searched for quantitative test results from both in vitro and in vivo assays for developmental toxicity for five model compounds: cyclophosphamide, methotrexate, hydroxyurea, caffeine, and ethylenethiourea. These compounds were chosen on the basis of their extensive utilization in a variety of assay systems for developmental toxicity as evidenced by their representation in the ETIC database (each generally has 100-500 citations encompassing multiple test systems). Nine cellular-based assays, six assays using whole embryos in culture, as well as Segment II and abbreviated exposure tests for mammalian test species are included in the database. For each assay, the critical endpoints were identified, each of which was then provided a three-letter code, and the criteria for extraction of quantitative information were established. The extracted information was placed into a computerized reference file and subsequently plotted such that the qualitative (positive/negative) and quantitative (e.g., IC50, highest ineffective dose (HID), lowest effective dose (LED] results across all test systems could be displayed. The information contained in these profiles can be used to compare qualitative and quantitative results across multiple assay systems, to identify data gaps in the literature, to evaluate the concordance of the assays, to calculate relative potencies, and to examine structure-activity relationships.     

Pituitary regulation of the male-specific steroid 6 beta-hydroxylase P-450 2a (gene product IIIA2) in adult rat liver. Suppressive influence of growth hormone and thyroxine acting at a pretranslational leve;

Oligonucleotide probes that distinguish between two closely related mRNAs encoding steroid 6 beta-hydroxylases of rat P-450 gene family CYP3A were used to individually assess their responsiveness to pituitary hormone regulation. Northern blot analysis revealed that the elevation of immunoreactive P-450 IIIA2 in livers of hypophysectomized rats reflects an elevation of the constitutive, male-specific P-450 IIIA2 (P-450 2a) and not an induction of the drug-inducible P-450 IIIA1 (P-450p). P-450 IIIA2 mRNA levels in intact adult male rats were found to be markedly reduced by GH administered as a continuous infusion at levels as low as 1 mU/h, indicating that GH acts at a pretranslational step to suppress expression of this P-450 enzyme. In hypophysectomized male rats, however, this same hormone treatment was only partially effective at suppressing P-450 IIIA2 mRNA and protein, suggesting that other pituitary-dependent factors contribute to the suppression observed in the intact rats. Further analysis revealed that T4, but not ACTH or human CG, can act in concert with GH to effect a more complete suppression of hepatic P-450 IIIA2 mRNA and protein in hypophysectomized rats. T4 also suppressed the expression of another GH-regulated, male-specific hepatic enzyme, designated P-450 IIA2 (P-450 RLM2), particularly in hypophysectomized female rats. In contrast, the GH-responsive P-450 IIA1 (P-450 3) was much less affected by T4 treatment. Thus, while T4 can modulate P-450 IIIA2 expression, it does not serve as a universal regulator for hepatic expression of GH-responsive P-450s.     

Use of a cis-acting mutation to study the role of FLP-mediated recombination in the maintenance of native yeast 2μm plasmids

The 2μm plasmid encodes a mechanism that ensures the partitioning of the plasmid at cell division. Little is known about the detailed mechanism of this partitioning system; for example, is there equal or unequal distribution of the plasmid molecules at mitosis? The plasmid also encodes a site-specific recombination system that is thought to be involved in plasmid copy-number amplification, although to date there has been no direct evidence that the recombination process itself is important for maintenance. We have identified a natural 2μm variant that has a cis-acting mutation in the FLP-mediated recombination system. We show that this plasmid is unable to amplify in vivo. Our results demonstrate that the average copy number per cell is not affected for the mutant but there is a large clonal variation. This is a direct demonstration that plasmid partitioning results in an unequal distribution of plasmids and that FLP-mediated amplification compensates for this and therefore has an important role in maintenance.     

Deletion of the membrane anchoring region of tissue factor abolishes autoactivation of factor VII but not cofactor function. Analysis of a mutant with a selective deficiency in activity.

The activation of human blood coagulation factor VII can occur by the feedback activity of either factor VIIa (autoactivation) or factor Xa. Both of these reactions are known to be enhanced by the presence of tissue factor, an integral membrane protein and the cofactor for factor VIIa. We examine here the activation of 125I-factor VII by both factor VIIa and factor Xa employing a mutant soluble form of tissue factor which has had its transmembrane and cytoplasmic domains deleted (sTF1-219). This mutant soluble tissue factor retains cofactor activity toward factor VIIa in a single-stage clotting assay but shows a strong dependence on initial plasma levels of factor VIIa (from 1 to 10,000 ng/ml) when compared to wild-type tissue factor. We show that this dependence is due to a deficiency of sTF1-219 in ability to both promote autoactivation and enhance the factor Xa-catalyzed activation of 125I-factor VII. sTF1-219 does not, however, inhibit the tissue factor-independent activation of 125I-factor VII by factor Xa. The results strongly suggest that the phospholipid anchoring region of tissue factor is essential for autoactivation and beneficial for factor Xa-catalyzed activation of 125I-factor VII. In addition, when taken together with the dependence of clotting times on initial factor VIIa levels observed with sTF1-219, these results indicate that factor VII autoactivation may be of greater importance in the initiation of blood coagulation via tissue factor than has been previously realized.     

Functional tissue factor is entirely cell surface expressed on lipopolysaccharide-stimulated human blood monocytes and a constitutively tissue factor-producing neoplastic cell line

Tissue factor (TF) is an integral membrane glycoprotein which, as the receptor and essential cofactor for coagulation factors VII and VIIa (FVII and FVIIa, respectively), is the primary cellular activator of the coagulation protease cascade. Previous studies on the procoagulant activity of a variety of cell types (either lysed or in the intact state) have variously been interpreted as showing that TF is either stored intracellularly or is present in a cryptic form in the surface membrane. Using mAbs to TF, we have directly investigated the subcellular localization and functional activity of TF in lipopolysaccharide-stimulated blood monocytes and J82 bladder carcinoma cells. Blocking of surface TF of viable cells with inhibitory anti-TF mAbs abolished greater than 90% of TF activity of the intact cells as well as of lysed cells. Furthermore, quantitative analysis of the binding of FVII and anti-TF mAb to J82 cells demonstrated that all surface-expressed TF molecules were capable of binding the ligand, FVII. By immunoelectron microscopy, TF was present only in the surface membrane of monocytes and J82 cells, although the latter also contained apparently inactive TF antigen in multivesicular bodies. On the intact cell surface the catalytic activity of the TF-FVIIa complex was investigated and found to be markedly less relative to cell lysates. Membrane alterations that affect the cofactor activity of TF may be a means of regulating the extent of initiation of the coagulation protease cascade in various cellular settings.     

Effect of IL-7 on the growth of fetal thymocytes in culture

The effects of IL-7 on the in vitro growth and differentiation of day 12 to 14 murine fetal thymocytes were examined in three culture systems. In single cell suspension cultures, IL-7 and IL-2 induced a DNA synthetic response in a short term (1 day) assay, but neither cytokine supported continued cell growth. In conventional fetal thymus organ cultures, the addition of exogenous IL-7 resulted in a twofold increase in cell number over that which normally develops in unsupplemented fetal thymus organ cultures during a 7-day period. The most striking effects of IL-7 were noted in lobe submersion cultures (LSC), a system in which thymocyte growth was totally dependent on the addition of exogenous cytokine. Cells proliferated for a period of approximately 2 wk in IL-7, and cell viability could be maintained even longer. A high percentage of cells recovered after 7 to 14 days from IL-7-supplemented LSC resembled the earliest detectable fetal thymocytes with regard to cell surface markers: they expressed Pgp-1, lacked CD4, CD8, and CD3 and many expressed the IL-2R. These results suggest that IL-7 promotes the growth of cells that occur early in the T cell lineage. Cell populations recovered from LSC supplemented with IL-7 and IL-2 exhibited differential expression of some surface markers, particularly CD3 and NK1.1. In addition, cells from LSC supplemented with IL-7 were found to proliferate upon subsequent exposure to IL-2, but cells from LSC containing exogenous IL-2 were no longer responsive to IL-7. These results imply that IL-7 and IL-2 may act at different stages of thymocyte differentiation. Together with previous observations of IL-7-specific mRNA expression in the thymus, this study provides evidence highly suggestive of a pivotal role for IL-7 in T cell development.     

Regulation of tissue factor gene expression in the monocyte procoagulant response to endotoxin.

Tissue factor is the cellular receptor and cofactor for plasma factor VIIa which initiates the coagulation protease cascade on cell surfaces. Although normally absent from all intravascular cell types, tissue factor can be induced to appear on circulating monocytes and vascular endothelial cells by specific inflammatory or immunological mediators. In this study, we have examined the regulation of endotoxin-induced tissue factor gene expression in peripheral blood monocytes.     

Matrix methods for estimating odds ratios with misclassified exposure data: extensions and comparisons

Misclassification of exposure variables is a common problem in epidemiologic studies. This paper compares the matrix method (Barron, 1977, Biometrics 33, 414-418; Greenland, 1988a, Statistics in Medicine 7, 745-757) and the inverse matrix method (Marshall, 1990, Journal of Clinical Epidemiology 43, 941-947) to the maximum likelihood estimator (MLE) that corrects the odds ratio for bias due to a misclassified binary covariate. Under the assumption of differential misclassification, the inverse matrix method is always more efficient than the matrix method; however, the efficiency depends strongly on the values of the sensitivity, specificity, baseline probability of exposure, the odds ratio, case-control ratio, and validation sampling fraction. In a study on sudden infant death syndrome (SIDS), an estimate of the asymptotic relative efficiency (ARE) of the inverse matrix estimate was 0.99, while the matrix method's ARE was 0.19. Under nondifferential misclassification, neither the matrix nor the inverse matrix estimator is uniformly more efficient than the other; the efficiencies again depend on the underlying parameters. In the SIDS data, the MLE was more efficient than the matrix method (ARE = 0.39). In a study investigating the effect of vitamin A intake on the incidence of breast cancer, the MLE was more efficient than the matrix method (ARE = 0.75).     

Use of technetium antigranulocyte monoclonal antibody Fab' fragments for the detection of osteomyelitis

Accurate early diagnosis of osteomyelitis is critical for optimal clinical management. Conventional radiology (X-rays, CT) and nuclear medicine scans (bone, gallium, and technetium/indium white blood cell [WBC]) have limitations and drawbacks. The monoclonal antibody (MAb) ImmuRAID^TM-MN3 (Immunomedics Inc., Morris Plains, NJ), a 99m-Tc Antigranulocyte Fab' fragment, recognizes a surface glycoprotein NCA-90/95 shared by granulocytes, carcino-embryonic antigen (CEA), and meconium antigen (MA). Intravenous injection of radiolabeled MAb enables in vivo labeling of human granulocytes and targets infected lesions in the bone and throughout the body. Technetium labeled Fab' fragments rapidly clear the blood pool and high-quality images can be obtained the same day, as early as 1 h postinjection. Results at our institution on 13 patients with clinically suspected osteomyelitis of infected long bones, prostheses, and diabetic foot ulcers were compared with the surgical/bacteriological verification of the presence or absence of infection. The MAb scan showed six true positives, six true negatives, and one false negative (very low grade infection). The procedure was safe, no clinical or laboratory adverse reactions were encountered. The MAb fragments are markedly less immunogenic than whole IgG, resulting in lower induction of human antimouse antibody (HAMA) titers. No HAMA to this MAb fragment has been detected in 24 patients (data from multiple institutions). Our preliminary results suggest that 99m-Tc ImmuRAID^TM-MN3 is highly accurate for detection of osteomyelitis. This study is part of an on-going multiinstitutional project sponsored by Immunomedics, Inc. to evaluate the efficacy and safety of this radiopharmaceutical.