In vitro regulation of reporter gene transcription by the androgen receptor AF1 domain.

The androgen receptor (AR) is a ligand-activated transcription factor that regulates gene expression in response to the steroids testosterone and dihydrotestosterone. AR-dependent gene expression is likely to play an important role in a number of receptor-associated disorders, such as prostate cancer, spinal bulbar muscular atrophy, male type baldness and hirsutism. The AR contains two transactivation domains, termed AF1 (activation function 1) located in the N-terminus and AF2 (activation function 2) in the C-terminal ligand-binding domain. AF2 exhibits weak transcriptional activity, whereas AF1 is a strong regulator of transcription. Transcriptional regulation by AF1 is thought to be modulated by a number of proteins that interact with this region, and by post-translational modifications. Our focus is on the N-terminal-interacting proteins and their regulation of transcription via interaction with the receptor. To better understand the mechanism of AR-AF1 action, we have reconstituted AR activity in HeLa nuclear extracts using a unique dual reporter gene assay. Multiple LexA-binding sites in the promoter allow transcription to be driven by a recombinant AR-AF1-Lex fusion protein. The findings from initial experiments suggest an increase in transcription initiation and elongation rates by AR-AF1-Lex. The role of protein-protein interactions involving co-activators and basal transcription factors and AR-AF1 activity are discussed.

PAF receptor antagonist modulates neutrophil responses with thermal injury in vivo.

The role of platelet-activating factor (PAF) in Ca(2+) signaling and Ca(2+)-related enhancement of reactive oxygen intermediate (ROI) generation in neutrophils of burn-injured rats was ascertained by evaluating the effect of treatment of the rats with a PAF receptor antagonist. The treatment of rats with the antagonist also allowed us to evaluate the role of PAF in the priming of neutrophil ROI response with burn in vivo. A full skin thickness burn injury was produced in anesthetized rats by exposing 30% of total body surface area to 98 degrees C water for 10 s. Sham and burn rats were killed 1 day later, and their blood was collected to obtain neutrophils. Fluorescence-activated cell sorter analysis was used to quantify ROI production by the neutrophils. Cytosolic-free Ca(2+) concentration ([Ca(2+)](i)) imaging technique was employed to measure neutrophil [Ca(2+)](i) in individual cells and microfluorometry for the assessment of [Ca(2+)](i) responses in suspensions of neutrophils. There was an overt enhancement of ROI generation by burn rat neutrophils. ROI release was accompanied by a marked elevation of [Ca(2+)](i) signaling. The treatment of rats with PAF receptor antagonist before burn prevented the upregulation of both [Ca(2+)](i) and ROI generation in neutrophils. These studies indicate that enhanced ROI production in neutrophils in the early stages after burn injury results from a PAF-mediated priming of the [Ca(2+)](i) signaling pathways in vivo.

PGE2 suppresses intestinal T cell function in thermal injury: a cause of enhanced bacterial translocation.

Increased gut bacterial translocation in burn and trauma patients has been demonstrated in a number of previous studies, however, the mechanism for such an increased gut bacterial translocation in injured patients remains poorly understood. Utilizing a rat model of burn injury, in the present study we examined the role of intestinal immune defense by analyzing the T cell functions. We investigated if intestinal T cells dysfunction contributes to bacterial translocation after burn injury. Also our study determined if burn-mediated alterations in intestinal T cell functions are related to enhanced release of PGE2. Finally, we examined whether or not burn-related alterations in intestinal T cell function are due to inappropriate activation of signaling molecule P59fyn, which is required for T cell activation and proliferation. The results presented here showed an increase in gut bacterial accumulation in mesenteric lymph nodes after thermal injury. This was accompanied by a decrease in the intestinal T cell proliferative responses. Furthermore, the treatments of burn-injured animals with PGE2 synthesis blocker (indomethacin or NS398) prevented both the decrease in intestinal T cell proliferation and enhanced bacterial translocation. Finally, our data suggested that the inhibition of intestinal T cell proliferation could result via PGE2-mediated down-regulation of the T cell activation-signaling molecule P59fyn. These findings support a role of T cell-mediated immune defense against bacterial translocation in burn injury.

Up-regulation of steroid sulphatase activity in HL60 promyelocytic cells by retinoids and 1alpha,25-dihydroxyvitamin D3.

HL60 promyeloid cells express both classes of oestrogen receptor (ERalpha and ERbeta). We show that hydrolysis of oestrone sulphate by steroid sulphatase is a major source of oestrone in HL60 cells, and that most of the released oestrone is not metabolized further to 17beta-oestradiol. Treatment of HL60 cells with retinoids or 1alpha,25-dihydroxyvitamin D3 increased steroid sulphatase mRNA and activity in parallel with the induction of CD11b, an early marker of myeloid differentiation that is expressed before the differentiating cells stop proliferating. Use of agonists and antagonists against retinoid receptor-alpha and retinoid receptor-X revealed that both classes of retinoid receptor can drive steroid sulphatase up-regulation. Steroid sulphatase activity fluctuates during the cell cycle, being highest around the transition from G1 to S phase. During the differentiation of HL60 cells induced by all-trans-retinoic acid or 1alpha,25-dihydroxyvitamin D3, there is increased conversion of 17beta-oestradiol into oestrone by an oxidative 17beta-hydroxysteroid dehydrogenase. Treatment of Caco-2 colon adenocarcinoma cells with all-trans-retinoic acid or 1alpha,25-dihydroxyvitamin D3 also increases 17beta-oestradiol oxidation to oestrone. An increase in local oestrone production therefore occurs in multiple cell types following treatment with retinoids and 1alpha,25-dihydroxyvitamin D3. The possible involvement of locally produced oestrogenic steroids in regulating the proliferation and differentiation of myeloid cells is discussed.

TGF-beta abrogates TCR-mediated signaling by upregulating tyrosine phosphatases in T cells.

TGF-beta is known to inhibit many of the immune cell functions including T cell proliferation and IL-2 production. The mechanism of such TGF-beta-mediated inhibition of T cell functions is poorly understood. The present study examined the effects of TGF-beta on the activation of protein tyrosine kinases (PTK) P56lck, P59fyn, and Zap-70, and protein tyrosine phosphatases (PTP) SHP-1 and SHP-2. A balance between the actions of PTK and PTP is critical for appropriate T cell activation. These studies were carried out using nylon wool-purified splenic T cells from healthy Sprague-Dawley rats. Results from these studies showed that incubation of T cells with TGF-beta inhibited the activation of P56lck, P59fyn and Zap-70. The decrease in these three protein tyrosine kinases was accompanied by an increase in the activation of the protein tyrosine phosphatase SHP-1. There was no change in the phosphorylation of SHP-2 with and without pretreatment of T cells with TGF-beta. The decrease in P56lck, P59fyn kinase activity, and Zap-70 phosphorylation was prevented when T cells were stimulated with anti-CD3 in the presence of pervanadate, an inhibitor of PTP. These results suggested that TGF-beta-mediated inhibition of P56lck, P59fyn, and Zap-70 is likely due to an up-regulation of protein tyrosine phosphatases such as SHP-1.

PGE(2)-mediated inhibition of T cell p59(fyn) is independent of cAMP.

We recently observed that prostaglandin E(2) (PGE(2))-mediated suppression of T cell functions could result from an attenuation of p59(fyn) protein tyrosine kinase activity. The present study evaluated the effects of an adenylate cyclase agonist (forskolin) and antagonist (SQ-22536), as well as those of cAMP analogues (dibutyryl cAMP and 8-bromo- cAMP), on T cell p59(fyn) kinase activity. The study allowed us to assess whether PGE(2)-mediated activation of adenylate cyclase by itself or the elevation in intracellular cAMP levels is an integral event in the modulation of anti-CD3-linked p59(fyn) activation in T cells. The experiments were carried out with splenic T cells from male Sprague-Dawley rats. A 30-50% suppression in the autophosphorylation and the kinase activity of p59(fyn) in T cells incubated with PGE(2) or forskolin was observed. Pretreatment of T cells with SQ-22536 prevented significant PGE(2)-mediated inhibition of T cell p59(fyn) kinase activity. In contrast, no change in p59(fyn) autophosphorylation and kinase activity in T cells treated with cAMP analogues was observed. These data suggest that PGE(2)-mediated suppression of p59(fyn) autophosphorylation and kinase activity in T cells is dependent on the activation of adenylate cyclase and independent of the elevation in cAMP levels.

Plasma exchange versus intravenous immunoglobulin treatment in myasthenic crisis.

We performed a retrospective multicenter chart review to compare the efficacy and tolerance of plasma exchange (PE) and intravenous immunoglobulin (i.v.Ig) in treatment of 54 episodes of myasthenic crisis. After adjustment for other variables, PE (compared with i.v.Ig) was associated with a superior ventilatory status at 2 weeks (partial F = 6.2, p = 0.02) and 1 month functional outcome (partial F = 4.5, p = 0.04). However, the complication rate was higher with PE compared with i.v.Ig (13 versus 5 episodes, p = 0.07).

Identification of a retinoic acid responsive aldoketoreductase expressed in HL60 leukaemic cells.

Neutrophil and monocyte differentiation can be induced in HL60 leukaemia cells by all-trans-retinoic acid (ATRA) and 1alpha,25-dihydroxyvitamin D3 (D3), respectively, whose differentiating effects can be enhanced by exposure to 'anti-inflammatory agents' and steroids. We have provided evidence that this potentiation is via inhibition of the activity of an enzyme of the aldoketoreductase (AKR) family, but had failed to identify expression of known AKRs in HL60 cells. In this study, we have identified a previously unclassified aldoketoreductase family member (termed HAKR e) that is expressed in HL60 cells. HAKR e is dramatically and transiently up-regulated in HL60 cells within 24 h of exposure to ATRA, further supporting the proposition that a member(s) of this family of enzymes play(s) a role in controlling cell growth and/or differentiation.

Effect of CRF and related peptides on calcium signaling in human and rodent melanoma cells.

Corticotropin releasing factor (CRF) induces a rapid, within seconds, and dose-dependent increase in the intracellular Ca2+ in both human and hamster melanoma cells. This effect is inhibited by depletion of extracellular calcium using 3 mM EGTA and is attenuated by the CRF receptor antagonist, alpha-helical-CRF(9-41). Other peptides of the CRF superfamily, sauvagine and urocortin, also induce increases in cytoplasmic calcium concentration but at higher concentrations than CRF. We conclude that malignant melanocytes express CRF receptors, which are coupled to activation of plasma membrane calcium channels.

Prostaglandin E2 modulation of p59fyn tyrosine kinase in T lymphocytes during sepsis.

Prostaglandin E2 (PGE2) has been implicated in the suppression of T cell IL-2 production and proliferation during burn and sepsis. The present study evaluated the potential intracellular mechanism of suppressed T cell responses by assessing the activation of p59fyn kinase in T cells from septic rats as well as the T cells incubated with PGE2. p59fyn is known to regulate T cell functions. Sepsis was induced in rats by implanting fecal pellets containing Escherichia coli (150 CFU) and Bacteroides fragilis (10(4) CFU) into the abdominal cavity. For the assessment of PGE2 role in sepsis, a group of septic rats were treated with indomethacin, which inhibits endogenous PGE2 synthesis. As assessed by immunoblotting or in vitro kinase assay, a more than 40% inhibition of p59fyn phosphorylation and kinase activity was observed in septic rat T cells compared with the T cells from sterile or control rats. A similar inhibition in p59fyn phosphorylation and kinase activity was observed in PGE2-treated T cells compared with the T cells incubated in the absence of PGE2. The septic-related suppression in p59fyn phosphorylation and kinase activity in T cells was prevented in rats treated with indomethacin. We observed that the inhibition in p59fyn activation in septic or PGE2-treated T cells was due primarily to a suppression in p59fyn phosphorylation and not due to alterations in p59fyn protein expression. These findings suggest that PGE2 released during sepsis could contribute to the sepsis-related suppression in T cell proliferation by attenuating p59fyn phosphorylation and its kinase activity.

Transforming growth factor-beta negatively modulates T-cell responses in sepsis.

Sepsis is associated with depressed T-cell functions and increased circulating levels of immunosuppressive agents. TGF-beta is a potential anti-inflammatory cytokine that can modify T-cell growth and differentiation. The up-regulation of TGF-beta and the mechanism of its action on the T-cells during septic injury have not been resolved. We hypothesized that in sepsis TGF-beta produced by macrophages acts on T-cells in a paracrine manner to suppress interleukin (IL)-2 production and proliferation. In this study, we examined the circulating TGF-beta levels in a rat model of Gram-negative bacterial sepsis, and compared the abilities of adherent and non-adherent splenocytes to produce TGF-beta. Additionally, we investigated the causal relationships of hrTGF-beta to concanavalin A (ConA)-induced T-cell responses and the intracellular mechanism of the generation of these responses in normal splenic rat T-cells. Sepsis was induced in rats by intraabdominally implanting fecal pellets containing Escherichia coli (150 CFU) and Bacteroides fragilis (10000 CFU). Adherent and non-adherent splenocytes were isolated by differential adherence using Ficoll gradient centrifugation. T-cells were purified by use of Nylon wool columns. We observed a 3-6-fold increase in the circulating levels of TGF-beta in sepsis. Western blots and ELISA determinations revealed a 2.5-3-fold increase in cell-associated TGF-beta protein levels in adherent splenic cells. Northern analyses also showed a marked increase in TGF-beta mRNA expression in adherent cells during sepsis. On the other hand, a significant change was not observed in the TGF-beta protein and mRNA expression in non-adherent splenocytes. Pretreatment of control rat T-cells with hrTGF-beta decreased both ConA-induced proliferation (by 35-40%) and IL-2 mRNA expression (by > 50%). Further, whereas incubation of control rat T-cells with either ConA or TGF-beta for 24 h resulted in a 10-15-fold increase in cAMP generation, the addition of hrTGF-beta along with ConA resulted in a 50-60-fold increase in cAMP. These results suggest that in sepsis, TGF-beta produced by splenic macrophages can act in a paracrine manner on T-cells to depress their IL-2 mRNA expression, IL-2 production and proliferation after up-regulation of cAMP which can interfere with T-cell signaling for proliferation.

Calcium signaling restitution prevents T-cell proliferative suppression by prostaglandin E2.

Previous studies from our laboratory have implicated a role for Ca2+ in prostaglandin E2 (PGE2)-induced suppression in T-cell proliferation during sepsis. The present study further elucidated the mechanism of PGE2-induced suppression in T-cell proliferation. We assessed whether prevention of the suppression in Ca2+ mobilization in PGE2-treated T-cells would restore proliferation. Rat splenic T-cell Ca2+ mobilization and proliferation were measured after stimulation of cells with concanavalin A (Con A) employing Fura-2 spectroscopy and cellular [3H]thymidine uptake techniques, respectively. PGE2 and other agents that directly up-regulate the PGE2-mediated cell signaling events (e.g., cholera toxin and forskolin), substantially suppressed both Con A-induced proliferation (p < .01) and Ca2+ mobilization in T-cells (p < .01). When stimulated with Con A plus ionomycin, [Ca2+]i in PGE2 treated T-cells (395 +/- 21, nM) was not significantly different (p > .05) from that observed in Con A-stimulated T-cells without the PGE2 exposure (351 +/- 8.6). The stimulation of PGE2-treated T-cells with ionomycin and Con A also significantly (p < .025), if not completely, prevented the PGE2-induced suppression in T-cell proliferation. These results suggest that the cross-talk between the TCR- and PGE2-mediated signaling in T-cells negatively modulates the TCR signals at the Ca2+ mobilization step and/or earlier to it.

Quercetin, a bioflavonoid, inhibits the DNA synthesis of human leukemia cells.

Quercetin, a flavonoid, is found in many plants, including edible fruits and vegetables. It has been proposed that flavonoids may have potential as anticancer agents. To test an aspect of this hypothesis, we examined the effects of the flavonoid, quercetin, on the DNA synthesis of the human leukemia cell, HL-60. Quercetin induced a dose-dependent inhibition of DNA synthesis in the test range of 1 microM to 1 mM. The inhibitory effect on DNA synthesis was evident as early as 24 h after the addition of quercetin. At the concentrations of 10 microM, 100 microM and 1 mM, 50, 82 and 85% of DNA synthesis, respectively, was inhibited by quercetin as compared to the control. After 48 and 72 h incubation of the cells with 100 microM and 1 mM quercetin, DNA synthesis was almost completely abolished. These results suggest that the inhibitory effects of quercetin on HL-60 cell DNA synthesis is not due to a non-specific cytotoxic effect, since following removal of quercetin, the treated cells regrew normally.

Sepsis-induced myofibrillar protein catabolism in rat skeletal muscle.

This study evaluated sepsis-induced changes in myosin heavy chain (Mhc) protein breakdown and synthesis in rat soleus muscles. Rats were anesthetized and their external jugular veins were cannulated. After 12-16 h, rats were implanted intraabdominally with a sterile fecal pellet, or a pellet containing bacteria (Escherichia coli, 150 CFU and Bacteroides fragilis 10(4) CFU). Thirty hours after implantations, rats were infused with 14C-Leu (60 x 10(3) Bq/h) through the jugular cannula for 4 h. Protein fractional synthetic rate coefficient (FSRC) was determined in muscles of different rat groups. In separate experiments, intact soleus muscles were removed from the three rat groups on days 1 and 2 after implantations, and processed for their wet weight, total protein and Mhc contents. No mortality occurred in sterile-implanted rats. Approximately 40-45% of all septic-implanted rats died on days 1-3, post-implantation. Whereas an approximately 15% (P < 0.01, days 1 or 2) decrease occurred in Mhc content in sterile-implanted rats compared to unoperated controls, septic insult resulted in a greater Mhc loss (a 27% decrease, P < 0.001). Rats' body weight, soleus wet weight and tolat muscle protein changes with sepsis relative to controls were also greater than in the sterile groups. The FSRC value in the septic-implanted rats was significantly lower (P < 0.05) than in non-septic rat muscle. TNF-alpha administration to the septic animals or their treatment with diltiazem did not have a significant effect on FSRC. Overall, these results indicate myosin as a major muscle protein subjected to net catabolism during sepsis, and that the net catabolic response was related to a more pronounced increased in Mhc degradation than the decrease in Mhc synthesis.