Proinflammatory actions of thromboxane receptors to enhance cellular immune responses

Metabolism of arachidonic acid by the cyclo-oxygenase (COX) pathway generates a family of prostanoid mediators. Nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibiting COX, thereby reducing prostanoid synthesis. The efficacy of these agents in reducing inflammation suggests a dominant proinflammatory role for the COX pathway. However, the actions of COX metabolites are complex, and certain prostanoids, such as PGE(2), in some circumstances actually inhibit immune and inflammatory responses. In these studies, we examine the hypothesis that anti-inflammatory actions of NSAIDs may be due, in part, to inhibition of thromboxane A(2) synthesis. To study the immunoregulatory actions of thromboxane A(2), we used mice with a targeted disruption of the gene encoding the thromboxane-prostanoid (TP) receptor. Both mitogen-induced responses and cellular responses to alloantigen were substantially reduced in TP(-/-) spleen cells. Similar attenuation was observed with pharmacological inhibition of TP signaling in wild-type splenocytes, suggesting that reduced responsiveness was not due to subtle developmental abnormalities in the TP-deficient mice. The absence of TP receptors reduced immune-mediated tissue injury following cardiac transplant rejection, an in vivo model of intense inflammation. Taken together, these findings show that thromboxane augments cellular immune responses and inflammatory tissue injury. Specific inhibition of the TP receptor may provide a more precise approach to limit inflammation without some of the untoward effects associated with NSAIDs.     

PGE(2) receptors and synthesis in human gastric mucosa: perturbation in cancer

Recent evidence suggests that prostanoids are an important participant in the pathobiology of gastric adenocarcinoma, but the location and identity of cells in tumor-adjacent gastric mucosa able to synthesize and/or bind specific prostanoids is not clear. Using probes for cyclooxygenase 1 and 2 mRNA and protein as well as for the EP family of PGE(2) receptors, we sought to define the biology of prostanoids in adjacent human gastric mucosa at the site of tumor invasion.In mucosa adjacent to an invasive gastric adenocarcinoma, expression of cyclooxygenase was prominent, with COX 1 primarily in mucosal T lymphocytes surrounding nests of tumor cells. Densitometry showed these tumor-adjacent cells had substantial levels of COX 1 immunoreactive protein (relative intensity, 3.2). Cyclooxygenase 2 was newly expressed among these cells as well, but was limited in number (<25% of cyclooxygenase-positive T lymphocytes) in tumor-adjacent mucosa. Further, CD3(+) mononuclear cells, adjacent to tumor, strongly expressed prostanoid receptor EP(4) (relative intensity, 8.0), but cells with this receptor were not evident in the tumor itself. In contrast, normal gastric mucosa showed a consistent and structured expression of cyclooxygenase and PGE(2) receptor immunoreactive protein among mucosal cells. Cyclooxygenase 1 and PGE(2) receptor EP(4) were expressed on mucosal CD3(+) T lymphocytes in the lumenal (upper) third of gastric mucosa; and prostanoid receptors EP(2), EP(3) and EP(4), on gastric epithelia lining gastric pits. In situ hybridization with COX cDNAs confirmed these findings, and neither COX 2-specific mRNA nor protein was detected in normal gastric tissue. Our studies suggest that synthetic machinery and receptors for PGE(2), prominently expressed by T lymphocytes in gastric mucosa at the boundary of normal mucosa with tumor cells, may play a central role in prostanoid-driven tumorigenesis of this tissue.     

COX-derived prostanoid pathways in gastrointestinal cancer development and progression: Novel targets for prevention and intervention

Arachidonic acid metabolism through cyclooxygenase (COX) pathways leads to the generation of biologically active eicosanoids. Eicosanoid expression levels vary during development and progression of gastrointestinal (GI) malignancies.COX-2 is the major COX-isoform responsible for G.I. cancer development/progression. COX-2 expression increases during progression from a normal to cancerous state. Evidence from observational studies has demonstrated that chronic NSAID use reduces the risk of cancer development, while both incidence and risk of death due to G.I. cancers were significantly reduced by daily aspirin intake. A number of randomized controlled trials (APC trial, Prevention of Sporadic Adenomatous Polyps trial, APPROVe trial) have also shown a significant protective effect in patients receiving selective COX-2 inhibitors. However, chronic use of selective COX-2 inhibitors at high doses was associated with increased cardiovascular risk, while NSAIDs have also been associated with increased risk. More recently, downstream effectors of COX-signaling have been investigated in cancer development/progression. PGE₂, which binds to both EP and PPAR receptors, is the major prostanoid implicated in the carcinogenesis of G.I. cancers. The role of TXA₂ in G.I. cancers has also been examined, although further studies are required to uncover its role in carcinogenesis. Other prostanoids investigated include PGD₂ and its metabolite 15d-PGJ2, PGF_1α and PGI₂. Targeting these prostanoids in G.I. cancers has the promise of avoiding cardiovascular toxicity associated with chronic selective COX-2 inhibition, while maintaining anti-tumor reactivity.A progressive sequence from normal to pre-malignant to a malignant state has been identified in G.I. cancers. In this review, we will discuss the role of the COX-derived prostanoids in G.I. cancer development and progression. Targeting these downstream prostanoids for chemoprevention and/or treatment of G.I. cancers will also be discussed. Finally, we will highlight the latest pre-clinical technologies as well as avenues for future investigation in this highly topical research field.     

Changes in gene expression contribute to cancer prevention by COX inhibitors

Non-steroidal anti-inflammatory drugs (NSAIDs) are used primarily for the treatment of inflammatory diseases. However, certain NSAIDs also have a chemopreventive effect on the development of human colorectal and other cancers. NSAIDs inhibit cyclooxygenase-1 (COX-1) and/or cyclooxygenase-2 (COX-2) activity and considerable evidence supports a role for prostaglandins in cancer development. However, the chemopreventive effect of NSAIDs on colorectal and other cancers appears also to be partially independent of COX activity. COX inhibitors also alter the expression of a number of genes that influence cancer development. One such gene is NAG-1 (NSAID-Activated Gene), a critical gene regulated by a number of COX inhibitors and chemopreventive chemicals. Therefore, this article will discuss the evidence supporting the conclusion that the chemo-preventive activity of COX inhibitors is mediated, in part, by altered gene expression with an emphasis on NAG-1 studies. This review may also provide new insights into how chemicals and environmental factors influence cancer development. In view of the cardiovascular and gastrointestinal toxic side effects of COX-2 inhibitors and non-selective COX inhibitors, respectively, the results presented here may provide the basis for the development of a new family of anti-tumorigenic compounds acting independent of COX inhibition.     

Cyclooxygenase-dependent signalling: molecular events and consequences

Non-steroidal anti-inflammatory drugs (NSAIDs) currently attract large interest. Next to pain relief, NSAIDs have important anti-thrombotic and anti-oncogenic effects. NSAIDs exert their action by inhibition of cyclooxygenase, the enzyme responsible for the production of prostanoids. Prostanoid signal transduction is still poorly understood, but it has become clear that these inflammatory lipids influence cellular physiology at three different levels: (1) activation of a 7 x transmembrane receptor coupled to heterotrimeric G proteins, (2) the inhibition of inflammation by activating corticosteroid-like receptors, (3) participation in receptor protein tyrosine kinase signal transduction. In this review prostanoid signalling at these three different levels will be reviewed and the relevance in (patho)physiological processes will be evaluated.     

Coupling between cyclooxygenases and terminal prostanoid synthases

Biosynthesis of prostanoids is regulated by three sequential enzymatic steps, namely phospholipase A2, cyclooxygenase (COX), and terminal prostanoid synthase. Recent evidence suggests that lineage-specific terminal prostanoid synthases, including prostaglandin (PG) E2, PGD2, PGF2alpha, PGI2, and thromboxane synthases, show distinct functional coupling with upstream COX isozymes, COX-1 and COX-2. This can account, at least in part, for segregated utilization of the two COX isozymes in distinct phases of PG-biosynthetic responses. In terms of their localization and COX preference, terminal prostanoid synthases are classified into three categories: (i) the perinuclear enzymes that prefer COX-2, (ii) the cytosolic enzyme that prefers COX-1, and (iii) the translocating enzyme that utilizes both COXs depending on the stimulus. Additionally, altered supply of arachidonic acid by phospholipase A2s significantly affects the efficiency of COX-terminal prostanoid synthase coupling. In this review, we summarize our recent understanding of the coupling profiles between the two COXs and various terminal prostanoid synthases.     

Role of non-neuronal cholinergic system in breast cancer progression

We have previously reported the expression of functional muscarinic acetylcholine receptors (mAChR) in two different murine mammary adenocarcinoma cell lines LM2 and LM3. Activation of mAChR with carbachol (CARB) increased proliferation in both tumor cell lines in a concentration-dependent manner. In LM3 cells CARB promoted proliferation via M(3) receptor activation by inositol 1,4,5-triphosphate and nitric oxide (NO) production. CARB-induced LM2 cells proliferation needed both M(2) and M(1) receptor activation increasing prostaglandin E(2) liberation and arginase catabolism respectively. Our present results indicate that CARB stimulates LM2 and LM3-induced angiogenesis and tumor growth. This activation follows different patterns. In LM2 tumor, M(1) and M(2) receptors activation stimulates neovascularization by arginase II and cyclooxygenase-2 (COX-2)-derived products while M(1) and M(3) receptors mediate CARB-induced tumor growth by the same effector enzymes. In LM3 tumor, we observe that M(1) and M(2) receptors are involved in agonist-stimulated angiogenesis by COX and NOS1-derived products while tumor growth is stimulated by M(3) and M(2) receptors activation and COX-2-derived prostanoids. Taken together these data present, at least in part, a picture of the regulation that different mAChR subtypes activation exerts on angiogenesis and growth of two different murine mammary adenocarcinomas.     

Prostanoid function and cardiovascular disease

Prostanoids, including prostaglandins (PGs) and thromboxanes (TXs) are synthesized from arachidonic acid by the combined action of cyclooxygenases (COXs) and PG and TX synthases. Finally after their synthesis, prostanoids are quickly released to the extracellular medium exerting their effects upon interaction with prostanoid receptors present in the neighbouring cells. These agents exert important actions in the cardiovascular system, modulating vascular homeostasis and participating in the pathogenesis of vascular diseases as thrombosis and atherosclerosis. Among prostanoids, Tromboxane (TX)A(2), a potent platelet activator and vasoconstrictor and prostacyclin (PGI2), a platelet inhibitor and vasodilator, are the most important in controlling vascular homeostasis. Although multiple studies using pharmacological inhibitors and genetically deficient mice have demonstrated the importance of prostanoid-mediated actions on cardiovascular physiology, further analysis on the prostanoid mediated actions in the vascular system are required to better understand the benefits and risks for the use of COX inhibitors in cardiovascular diseases.     

The role of the cylooxygenase pathway in nociception and pain

Cycloxygenase (COX) pathways have long been targeted for the treatment of inflammatory pain, initially through the use of NSAIDs. With the demonstration of two major COX isoforms, COX-1 and COX-2, involved in the production of prostanoids, but with different distribution and regulation, selective COX-2 inhibitors have been developed. This review covers factors influencing COX enzyme activity, the role of their products in the development and maintenance of pain and discusses recent safety concerns of COX-2 inhibitors.     

Reduced spontaneous relaxation in immature guinea pig airway smooth muscle is associated with increased prostanoid release

Airway smooth muscle (ASM) from infant guinea pigs has less spontaneous relaxation during stimulation than ASM from adults. Inhibition of cyclooxygenase (COX), which catalyzes the production of prostanoids, increases this relaxation in infant ASM and abolishes age differences, thus suggesting that prostanoids reduce relaxation in infant ASM. In this study, we investigated whether leukotrienes are also involved in reducing spontaneous relaxation; whether the two COX isoforms, COX-1 and COX-2, differentially regulate spontaneous relaxation; and whether prostanoid release is developmentally regulated in guinea pig ASM. In different age groups, we measured relaxation during and after electrical stimulation in tracheal strips as well as prostanoid release from tracheal segments. Relaxation was studied in the absence and in the presence of a lipoxygenase inhibitor, a cysteinyl leukotriene receptor-1 antagonist, a COX-1 inhibitor, or a COX-2 inhibitor. We found that inhibition of lipoxygenase or cysteinyl leukotriene receptor-1 antagonism did not increase spontaneous relaxation at any age, thus excluding a role for leukotrienes in this phenomenon. Inhibition of COX-2, but not COX-1, promoted spontaneous relaxation. The basal release of prostanoids was more abundant in tissue from infant animals and decreased significantly with age. Thromboxane B2 was the most abundant metabolite released at all ages. Electrical stimulation and epithelium removal did not affect the age difference in prostanoid release. We conclude that increased basal prostanoid release contributes to the reduced spontaneous relaxation in immature guinea pig ASM compared with older animals. By regulating ASM relaxation, prostanoids may play a role in the airway hyperresponsiveness at a young age.     

Targeting PGE2 receptor subtypes rather than cyclooxygenases: a bridge over troubled water?

Prostaglandins (PG) are synthesized by the sequential action of phosholipases, cyclooxygenases (COX)-1 and COX-2, and specific terminal synthases, and exert their diverse biological effects through several membrane receptors. In particular, PGE2 is involved in many normal and pathological pathways that are mediated by four different E prostanoid receptors (EP1-4). Selective COX-2 inhibitors (Coxibs) have analgesic and antipyretic effects that are indistinguishable from those of nonsteroidal anti-inflammatory drugs (NSAIDs), but some possess hazardous cardiovascular side effects. Recent results indicate that EP1 and EP4 antagonists might prove useful for inhibiting the unwanted actions of COX-2. Has the time come for research to examine earnestly the selective antagonism of EP subtypes rather than further the development of direct COX-2 inhibitors?     

Indomethacin decreases EP2 prostanoid receptor expression in colon cancer cells

Nonsteroidal anti-inflammatory drugs (NSAIDs) can decrease the risk of colorectal cancer; however, it has not been established if this effect is solely through their ability to inhibit cyclooxygenase (COX). In this study the effects of indomethacin, a potent NSAID and nonselective COX inhibitor, was examined in LS174T human colon cancer cells. These cells were found to express EP2 prostanoid receptors, but not the EP1, EP3 or EP4 subtypes. Pretreatment of LS174T cells with indomethacin produced a complete inhibition of prostaglandin E(2) (PGE(2)) stimulated cyclic AMP (cAMP) formation in a dose dependent manner with an IC(50) of 21 microM. Interestingly, the inhibition of PGE(2)-stimulated cAMP formation by indomethacin was accompanied by a decrease in EP2 mRNA expression and by a decrease in the whole cell specific binding of [(3)H]PGE(2). Thus, treatment of LS174T cells with indomethacin causes a down regulation of EP2 prostanoid receptors expression that may be independent of COX inhibition.     

A novel single‐chain enzyme complex with chain reaction properties rapidly producing thromboxane A2 and exhibiting powerful anti‐bleeding functions

Uncontrollable bleeding is still a worldwide killer. In this study, we aimed to investigate a novel approach to exhibit effective haemostatic properties, which could possibly save lives in various bleeding emergencies. According to the structure‐based enzymatic design, we have engineered a novel single‐chain hybrid enzyme complex (SCHEC), COX‐1‐10aa‐TXAS. We linked the C‐terminus of cyclooxygenase‐1 (COX‐1) to the N‐terminus of the thromboxane A₂ (TXA₂) synthase (TXAS), through a 10‐amino acid residue linker. This recombinant COX‐1‐10aa‐TXAS can effectively pass COX‐1–derived intermediate prostaglandin (PG) H₂ (PGH₂) to the active site of TXAS, resulting in an effective chain reaction property to produce the haemostatic prostanoid, TXA₂, rapidly. Advantageously, COX‐1‐10aa‐TXAS constrains the production of other pro‐bleeding prostanoids, such as prostacyclin (PGI₂) and prostaglandin E₂ (PGE₂), through reducing the common substrate, PGH₂ being passed to synthases which produce aforementioned prostanoids. Therefore, based on these multiple properties, this novel COX‐1‐10aa‐TXAS indicated a powerful anti‐bleeding ability, which could be used to treat a variety of bleeding situations and could even be useful for bleeding prone situations, including nonsteroidal anti‐inflammatory drugs (NSAIDs)‐resulted TXA₂‐deficient and PGI₂‐mediated bleeding disorders. This novel SCHEC has a great potential to be developed into a biological haemostatic agent to treat severe haemorrhage emergencies, which will prevent the complications of blood loss and save lives.     

Radiation-induced edema is dependent on cyclooxygenase 2 activity in mouse brain

Cerebrovascular dysfunction, characterized by compromise of the blood-brain barrier and formation of cerebral edema, is common during the acute period after brain irradiation and may contribute to delayed pathology (e.g. vascular collapse, white matter necrosis) that leads to functional deficits. Another response of normal brain tissue to radiation is the induction of inflammatory markers, such as cytokine expression and glial activation. In particular, radiation-induced neuroinflammation is associated with an elevation in cyclooxygenase 2 (COX2), one of two isoforms of the obligate enzyme in prostanoid synthesis and the principal target of non-steroid anti-inflammatory drugs. Since prostanoids serve as autocrine and paracrine mediators in numerous physiological and pathological processes, including vasoregulation, we investigated COX2 protein expression and COX2-mediated prostanoid production in radiation-induced cerebral edema in male C57/BL6 mice. We found that radiation induces COX2 protein that is accompanied by specific increases in prostaglandin E(2) and thromboxane A(2) within 4 and 24 h after brain irradiation. Furthermore, we showed that treatment with NS-398, a selective COX2 inhibitor, attenuated prostanoid induction and edema formation. These results suggest that radiation-induced changes in vascular permeability are dependent on COX2 activity, implicating this enzyme and its products as targets for potential therapeutic treatment/protection from the effects of radiation on normal brain tissue.     

Colon carcinoma cell growth is associated with prostaglandin E2/EP4 receptor-evoked ERK activation

Cyclooxygenase (COX) and its prostanoid metabolites have been implicated in the control of cell survival; however, their role as mitogens remains undefined. To better understand the role of prostanoids on cell growth, we used mouse colon adenocarcinoma (CT26) cells to investigate the role of prostaglandin E(2) (PGE(2)) in cell proliferation. CT26 cells express both COX1 and COX2 and metabolize arachidonic acid to PGE(2.) Treatment with indomethacin, or COX-selective inhibitors, prevents PGE(2) biosynthesis and CT26 cell proliferation. The anti-proliferative effects of COX inhibition are rescued specifically by treatment with PGE(2) or the EP4 receptor-selective agonist PGE(1)-OH via phosphatidylinositol 3-kinase/extracellular signal-regulated kinase (ERK) activation, thus providing a functional link between PGE(2)-induced cell proliferation and EP4-mediated ERK signaling. Indomethacin or COX2 inhibitors, but not COX1 inhibitors, reduced the size and number of CT26-derived tumors in vivo. These inhibitory effects are paralleled by marked declines in the levels of tumor PGE(2), suggesting that their anti-tumor effects are directly associated with the inhibition of COX2 enzymatic activity. The described anti-tumor effects of indomethacin are evident whether it is administered at the time of, or 7 days after, tumor cell injection, suggesting that it has tumor preventive and therapeutic actions. Furthermore, the observation that indomethacin increases the survival rates of tumor-bearing mice, even after withdrawal of the drug, indicates that its effects are long lasting and that it may be potentially useful for the prevention and the clinical management of human cancers.     

A dietary conjugated linoleic acid treatment that slows renal disease progression alters renal cyclooxygenase-2-derived prostanoids in the Han: SPRD-cy rat

A mixture of dietary conjugated linoleic acid (CLA) isomers reduces inflammation and mitigates disease progression in the Han:SPRD-cy rat model of chronic kidney disease. Since cyclooxygenase (COX) activities and prostanoid levels are higher in diseased kidneys in this rat, and dietary CLA can inhibit COX2 and prostanoid production in other tissues, the effects of dietary CLA were investigated. Kidney homogenates from normal and diseased Han:SPRD-cy rats were analyzed for prostanoid levels under various conditions: endogenous levels, steady-state levels (60-min incubations) and produced by COX isoforms. Thromboxane B(2) (TXB(2); TXA(2) metabolite), 6-keto-prostaglandin F(1α) (6-keto-PGF(1α); PGI(2) metabolite) and PGE(2) levels under these conditions were two- to ninefold higher in diseased kidneys. Dietary CLA resulted in ～32%-53% lower levels of prostanoids produced by total COX and COX2 activities in normal and diseased kidneys and partially mitigated alterations in COX2 protein levels associated with disease. The COX1 protein and activity were higher in renal disease, resulting in increased production of TXB(2) and 6-ketoPGF(1α), but not PGE(2). Dietary CLA had no effect on COX1, however. Disease resulted in up to twofold higher ratios of TXB(2)/6-ketoPGF(1α), TXB(2)/PGE(2) and 6-ketoPGF(1α) /PGE(2), and dietary CLA partially mitigated these increases under several conditions. Elevated levels of renal membrane associated cytosolic phospholipase A(2) in diseased kidneys also were reduced by 50% with CLA feeding. The effects of CLA feeding on COX2 protein levels and activity indicate that the beneficial effect of dietary CLA in this renal disorder is mediated in part via effects on COX2-derived prostanoids.     

Mucosal prostanoid receptors and synthesis in familial adenomatous polyposis

Chronic ingestion of non-steroidal anti-inflammatory medication is reported to delay or, in part, reverse development of polyps in the colon, but the mechanism for this effect is unknown. Using mRNA and immunoglobulin probes, specific for prostanoid receptors and for prostaglandin endoperoxide synthase (COX 1 and 2), we sought to define, by in situ and in vitro techniques, changes in PGE2 receptors and synthesis in cell populations of precancerous familial adenomatous polyposis (FAP) colonic mucosa. In FAP, expression of prostanoid receptors EP3 and EP4 among colonic lamina propria mononuclear and lateral crypt epithelial cells was robust, with 53.9+/-5.3% of mononuclear cells staining EP4+. When sections of normal colonic mucosa were examined by similar techniques, prostanoid receptor EP4 was expressed on only 21.3+/-1.2% of lamina propria mononuclear cells (including CD4+ T lymphocytes), as well as on surface and lateral crypt epithelium, and this distribution was found at the mRNA level as well. When receptor expression was quantitated by densitometry, immunoreactive EP3 protein on deep basolateral (but not other) FAP crypt epithelium was enhanced 2.8-fold over normal, and the number of prostanoid receptor EP4+ mononuclear cells by 2.5-fold. On the other hand, while COX 1 expression in mononuclear cells was prominent in normal and FAP mucosa, densitometric analysis showed immunoreactive prostaglandin endoperoxide synthase levels were further increased in FAP, due to a greater than fourfold elevation of COX 2 expression among mononuclear cells and epithelia. Our data suggest enhanced cell-specific prostanoid receptor expression and increased prostanoid synthesis in precancerous FAP mucosa.     

Synthetic pathways of gallbladder mucosal prostanoids: the role of cyclooxygenase-1 and 2.

Acute cholecystitis is associated with increased gallbladder prostanoid formation and the inflammatory changes and prostanoid increases can be inhibited by nonsteroidal anti-inflammatory agents. Recent information indicates that prostanoids are produced by two cyclooxygenase (COX) enzymes, COX-1 and COX-2. The purpose of this study was to determine the COX enzymatic pathway in gallbladder mucosal cells involved in the production of prostanoids stimulated by inflammatory agents. Human gallbladder mucosal cells were isolated from cholecystectomy specimens and maintained in cell culture and studied in comparison with cells from a well differentiated gallbladder mucosal carcinoma cell line. COX enzymes were evaluated by Western immunoblotting and prostanoids were measured by ELISA. Unstimulated and stimulated cells were exposed to specific COX-1 and COX-2 inhibitors. In both normal and transformed cells constitutive COX-1 was evident and in gallbladder cancer cells lysophosphatidyl choline (LPC) induced the formation of constitutive COX-1 enzyme. While not detected in unstimulated normal mucosal cells and cancer cells, COX-2 protein was induced by both lipopolysaccharide (LPS) and LPC. Unstimulated gallbladder mucosal cells and cancer cells produced prostaglandin E2 (PGE2) and prostacyclin (6-keto prostaglandin F1alpha, 6-keto PGF1alpha) continuously. In freshly isolated normal gallbladder mucosal cells, continuously produced 6 keto PGF1alpha was inhibited by both COX-1 and COX-2 inhibitors while PGE2 levels were not affected. Both LPS and LPC stimulated PGE2 and 6 keto PGF1alpha formation were blocked by COX-2 inhibitors in freshly isolated, normal human gallbladder mucosal cells and in the gallbladder cancer cells. The prostanoid response of gallbladder cells stimulated by proinflammatory agents is inhibited by COX-2 inhibitors suggesting that these agents may be effective in treating the pain and inflammation of gallbladder disease.