Estrogen interconversions in the induced cycle in female rhesus monkeys

To determine the extractions and interconversions of estrone and estradiol across and within the uterus, [3H]estradiol and [14C]estrone were infused at a constant rate in six ovariectomized female rhesus (Macaca mulatta) monkeys. Studies were done on Days 9, 14, and 23 of artificial menstrual cycles induced by the timed insertion and removal of Silastic capsules of estradiol and progesterone. Measurements of estrogen radioactivity were made from peripheral arterial blood and uterine venous blood as well as from endometrial biopsy samples. A significant increase occurred in the conversion of estradiol to estrone measured within the uterus on Day 23 compared to Days 9 and 14. The conversion of estrone to estradiol, measured within the uterus, fell progressively from Day 9 to Day 23, but this decrease was not significant. The extractions and interconversions across the uterus, and the overall interconversions of estrone and estradiol were not significantly different on Days 9, 14, or 23 of the cycle. Thus, we have been able to confirm in vivo the increase in the activity of the 17 beta-hydroxysteroid dehydrogenase, the enzyme responsible for estradiol to estrone interconversions, shown earlier by studies done in vitro. However, the increase in 17 beta-hydroxysteroid activity in the uterus is not reflected in the overall interconversions of estrone and estradiol as reflected by measurements in peripheral arterial blood.     

Enhanced susceptibility to cytotoxic T lymphocytes of target cells isolated from virus-infected or interferon-treated mice

Bone marrow cells and thymocytes isolated from virus-infected or interferon (IFN)-treated mice had marked increases in sensitivity to lysis by allospecific cytotoxic T lymphocytes (CTL) and in expression of class I histocompatibility antigens. Cultured fibroblasts treated with IFN in vitro yielded similar findings in addition to having increased sensitivity to lysis by virus-specific CTL. This indicates that virus-induced IFN may condition target cells in vivo for surveillance by CTL.     

Pathogenesis of murine cytomegalovirus infection in natural killer cell-depleted mice.

The effect of natural killer (NK) cells on the course of acute and persistent murine cytomegalovirus (MCMV) infection was examined by selectively depleting NK cell activity by inoculation of mice with antibody to asialo GM1, a neutral glycosphingolipid present at high concentrations on NK cells. The dose of MCMV required to cause 50% mortality or morbidity in control C57BL/6 mice dropped 4- and greater than 11-fold, respectively, in mice first treated with anti-asialo GM1. NK cell-depleted mice had higher (up to 1,000-fold) virus titers in their lungs, spleens, and livers at days 3, 5, 7, and 9 postinfection. Spleens and livers of control mice were virus-free by day 7 postinfection, and their lungs showed no signs of active infection at any time. In contrast, MCMV had disseminated to the lungs of NK cell-depleted mice by day 5, and these mice still had moderate levels of virus in their lungs, spleens, and livers at day 9. Markedly severe pathological changes were noted in the livers and spleens of NK cell-depleted, MCMV-infected mice. These included ballooning degeneration of hepatocytes and spleen necrosis. MCMV-infected, NK cell-depleted mice had severe spleen leukopenia, and their spleen leukocytes exhibited a significantly lower (up to 13-fold) response to the T cell mitogen concanavalin A when compared with those of uninfected and MCMV-infected controls. It appeared that NK cells exerted their most potent antiviral effect early in the infection, in a pattern correlating with interferon production and NK cell activation; treatment with anti-asialo GM1 later in infection had no effect on virus titers. The relative effect of NK cell depletion on MCMV pathogenesis depended on the injection route of the virus. NK cell depletion greatly augmented MCMV synthesis and pathogenesis in mice inoculated either intravenously or intraperitoneally but had no effect on the course of disease after intranasal inoculation, at any time point examined. One month after intraperitoneal inoculation of virus, NK cell depletion resulted in a six- to eightfold increase in salivary gland virus titers in persistently infected mice, suggesting that NK cells may be important in controlling virus synthesis in the salivary gland during persistent infection. This treatment did not, however, induce dissemination of virus to other organs. These data support the hypothesis that NK cells limit the severity, extent, and duration of acute MCMV infection and that they may also be involved in regulating the persistent infection.     

Dengue virus-specific cross-reactive CD8+ human cytotoxic T lymphocytes

Stimulation with live dengue virus of peripheral blood mononuclear cells from a dengue virus type 4-immune donor generated virus-specific, serotype-cross-reactive, CD8+, class I-restricted cytotoxic T lymphocytes (CTL) capable of lysing dengue virus-infected cells and cells pulsed with dengue virus antigens of all four serotypes. These CTL lysed autologous fibroblasts infected with vaccinia virus-dengue virus recombinant viruses containing the E gene or several nonstructural dengue virus type 4 genes. These results demonstrate that both dengue virus structural and nonstructural proteins are targets for the cytotoxic T-cell-mediated immune response to dengue virus and suggest that serotype-cross-reactive CD8+ CTL may be important mediators of viral clearance and of virus-induced immunopathology during secondary dengue virus infections.     

Isolation of multiple classes of mutants of CHO cells resistant to cyclic AMP

From mutagenized Chinese hamster ovary (CHO) cells we have isolated, in a single step, 11 independent mutants resistant to the growth-inhibitory effects of 8-Br-cyclic AMP, cholera toxin, and methylisobutylxanthine. Two major classes and several subclasses of mutants were obtained. Mutants from all classes have a normal doubling time. None of the mutants respond to cyclic AMP treatment with increased flattening and elongation as do the parental cells. Members of the first class have an altered protein kinase activity which has either an increased Ka for cyclic AMP or an absent response to cyclic AMP. Most of those mutations which result in a protein kinase with increased Ka for cyclic AMP (6/11) are dominant in somatic cell hybrids. Those mutations which result in a protein kinase with little or no response to cyclic AMP (3/11) are recessive. Members of the second major class (2/11) have normal levels of basal and cyclic AMP-dependent protein kinase activity. One is recessive and one is dominant by genetic tests. The basis for the defect in this second class of mutants has not been determined.