Loss Of Cellular Response Research Articles

Activation of signaling lymphocytic activation molecule triggers a signaling cascade that enhances Th1 responses in human intracellular infection.

T cell production of IFN-gamma contributes to host defense against infection by intracellular pathogens, including mycobacteria. Lepromatous leprosy, the disseminated form of infection caused by Mycobacterium leprae, is characterized by loss of cellular response against the pathogen and diminished Th1 cytokine production. Relieving bacterial burden in Ag-unresponsive patients might be achieved through alternative receptors that stimulate IFN-gamma production. We have previously shown that ligation of signaling lymphocytic activation molecule (SLAM) enhances IFN-gamma in mycobacterial infection; therefore, we investigated molecular pathways leading from SLAM activation to IFN-gamma production in human leprosy. The expression of the SLAM-associated protein (an inhibitory factor for IFN-gamma induction) on M. leprae-stimulated cells from leprosy patients was inversely correlated to IFN-gamma production. However, SLAM ligation or exposure of cells from lepromatous patients to a proinflammatory microenvironment down-regulated SLAM-associated protein expression. Moreover, SLAM activation induced a sequence of signaling proteins, including activation of the NF-kappaB complex, phosphorylation of Stat1, and induction of T-bet expression, resulting in the promotion of IFN-gamma production, a pathway that remains quiescent in response to Ag in lepromatous patients. Therefore, our findings reveal a cascade of molecular events during signaling through SLAM in leprosy that cooperate to induce IFN-gamma production and strongly suggest that SLAM might be a focal point for therapeutic modulation of T cell cytokine responses in diseases characterized by dysfunctional Th2 responses.

Effect of cycloheximide and tunicamycin on the gonadotrophin-releasing hormone stimulated distal glycosylation of luteinizing hormone by rat pituitary cells

We have previously demonstrated that gonadotrophin-releasing hormone (GnRH) induces not only changes in quantity but also in quality on secreted luteinizing hormone (LH), by increasing [14C]Leu (translation) and [3H]Gal (distal glycosylation) incorporation into newly synthesized hormone. In the present report, we have further examined the GnRH-induced [3H]Gal-LH synthesis and release by treating anterior pituitary cells with polypeptide synthesis and glycosylation inhibitors (cycloheximide and tunicamycin, respectively). Pituitary cells from ovariectomized adult rats were cultured for 4 days and then incubated for different periods (0-5 h) in medium containing [14C]Leu plus [3H]Man or [14C]Leu plus [3H]Gal in the absence (basal) or presence of 10 nmol/L GnRH with or without (control) cycloheximide (1.0 and 4.0 µg/mL) or tunicamycin (0.5 and 2.0 µg/mL). At the end of each incubation period, the cells and the medium were separated and processed for DNA uptake and newly synthesized LH (labeled LH, by immunoprecipitation with a-betaLH) determinations. The velocity of synthesis and release (between 0 and 2 h, and between 2 and 5 h) was calculated by regression analysis and the statistical significance of differences was determined by the slope test. GnRH enhanced the rates of synthesis and release of [14C]Leu-, [3H]Man-, and [3H]Gal-LH to 157 and 237; 164 and 190; and 272 and 508% of basal values, respectively. Cycloheximide totally blocked synthesis and release of [14C]Leu-LH and greatly reduced those of [3H]Man-LH, resulting in the loss of cellular responsiveness to GnRH. Addition of tunicamycin to the pituitary cells inhibited the rates of synthesis and release of [3H]Man-LH which had been induced by GnRH, without altering those of [14C]Leu-LH. These findings indicate that glycosylation is not a condition for GnRH-stimulated LH translation. The GnRH-increased [3H]Gal-LH rates of synthesis and release were affected to a lesser extent by the inhibitors. Thus, GnRH stimulation of distal glycosylation can occur, albeit at a reduced rate, even though protein synthesis and glycosylation are blocked. In conclusion, the present results corroborate that GnRH stimulates the addition of galactose residues into LH molecule. This effect is not simply the consequence of stimulating LH polypeptide chain synthesis. In addition, it is shown that GnRH-increased LH translation is independent of glycosylation.Key words: gonadotrophin-releasing hormone, luteinizing hormone, glycosylation, cycloheximide, tunicamycin.

Origin of the decline in pancreatic amylase secretion with time in vitro.

The release of alpha-amylase (EC 3.2.1.1) into the medium bathing strips of rabbit pancreas decreased gradually with time in vitro so that after 3 h of incubation the secretory rate was only 20% of that seen initially. This decrease was not the result of an intrinsic loss of cellular responsiveness or viability, because a cholinergic agonist was still able to augment amylase secretion from the tissue after this incubation period to the same, or greater, degree than from "fresh" tissue. Rather, the inhibition of secretion was apparently due to the release of a nondialyzable substance from the tissue, which appears to be amylase itself. The observations that led to this conclusion are 1) replacing the bathing medium every 30 min eliminated the time-dependent decrease in enzyme secretion, 2) this replacement was ineffective if the tissue was separated from the medium by a dialysis membrane, and 3) adding an excess of the amylase substrate glycogen to the medium also prevented the falloff in amylase secretion, the effect of glycogen presumably being due to the formation of an enzyme-substrate complex in the medium. These observations are consistent with other observations that demonstrate that the rate of enzyme secretion by the pancreas depends on the rate of removal of product from the site of its secretion [Isenman and Rothman, Proc. Natl. Acad. Sci. USA 74: 4068-4072, 1977; Isenman and Rothman, Science 204: 1212-1215, 1979; Ho and Rothman, Am. J. Physiol. 242 (Gastrointest. Liver Physiol. 5): G32-G39, 1982].