Future Prospects for the Treatment of Graves' Hyperthyroidism and Eye Disease.

Although there are adequate therapies for Graves' hyperthyroidism, mild to moderate Graves' orbitopathy (GO) is usually treated symptomatically whereas definitive therapy is reserved for severe, vision-threatening GO. Importantly, none of the treatment regimens for Graves' disease used today are directed at the pathogenesis of the disease. Herein, we review some aspects of what is known about the pathogenesis of these 2 major components of Graves' disease, specifically the apparent important roles of the TSH and IGF-1 receptors, and thereafter describe future therapeutic approaches directed at these receptors. We propose that targeting these receptors will yield effective and better tolerated treatments for Graves' disease, especially for GO.

Ectopic expression of thyrotropin releasing hormone (TRH) receptors in liver modulates organ function to regulate blood glucose by TRH.

Maintenance of blood glucose by the liver is normally initiated by extracellular regulatory molecules such as glucagon and vasopressin triggering specific hepatocyte receptors to activate the cAMP or phosphoinositide signal transduction pathways, respectively. We now show that the normal ligand-receptor regulators of blood glucose in the liver can be bypassed using an adenovirus vector expressing the mouse pituitary thyrotropin releasing hormone receptor (TRHR) cDNA ectopically in rat liver in vivo. The ectopically expressed TRHR links to the phosphoinositide pathway, providing a means to regulate liver function with TRH, an extracellular ligand that does not normally affect hepatic function. Administration of TRH to these animals activates the phosphoinositide pathway, resulting in a sustained rise in blood glucose. It should be possible to use this general strategy to modulate the differentiated functions of target organs in a wide variety of pathologic states.

Inhibition of constitutive signaling of Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor by protein kinases in mammalian cells in culture.

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8, which is consistently present in tissues of patients with Kaposi's sarcoma and primary effusion lymphomas, contains a gene that encodes a G protein-coupled receptor (KSHV-GPCR). We recently showed that KSHV-GPCR exhibits constitutive signaling via activation of phosphoinositide-specific phospholipase C and stimulates cell proliferation and transformation. In this study, we determined whether normal cellular mechanisms could inhibit constitutive signaling by KSHV-GPCR and thereby KSHV-GPCR-stimulated proliferation. We show that coexpression of GPCR-specific kinases (GRKs) and activation of protein kinase C inhibit constitutive signaling by KSHV-GPCR in COS-1 monkey kidney cells and in mouse NIH 3T3 cells. Moreover, GRK-5 but not GRK-2 inhibits KSHV-GPCR-stimulated proliferation of rodent fibroblasts. These data provide evidence that cell regulatory pathways of receptor desensitization may be therapeutic targets in human diseases involving constitutively active receptors.

Human interferon-gamma-inducible protein 10 (IP-10) inhibits constitutive signaling of Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor.

A G protein-coupled receptor (GPCR) is encoded within the genome of Kaposi's sarcoma- associated herpesvirus (KSHV)/human herpesvirus 8, a virus that may be involved in the pathogenesis of Kaposi's sarcoma and primary effusion lymphomas. KSHV-GPCR exhibits constitutive signaling activity that causes oncogenic transformation. We report that human interferon (IFN)-gamma-inducible protein 10 (HuIP-10), a C-X-C chemokine, specifically inhibits signaling of KSHV-GPCR. In contrast, monokine induced by IFN-gamma (HuMig), which like HuIP-10 is an agonist of C-X-C chemokine receptor 3, does not inhibit KSHV-GPCR signaling. Moreover, HuIP-10, but not HuMig, inhibits KSHV-GPCR-induced proliferation of NIH 3T3 cells. These results show that HuIP-10 is an inverse agonist that converts KSHV-GPCR from an active to an inactive state. Thus, a human chemokine inhibits constitutive signaling and cellular proliferation that is mediated by a receptor encoded by a human disease-associated herpesvirus.

Decreased TRH receptor mRNA activity precedes homologous downregulation: assay in oocytes.

Ligand-induced decrease in cell-surface receptor number (homologous downregulation) is often due to rapid receptor internalization. Thyrotropin-releasing hormone (TRH), however, causes a slow downregulation of TRH receptors (TRH-Rs), with a half-time of approximately 12 hours, in GH3 rat pituitary cells. The mechanism of TRH-R downregulation was studied by monitoring TRH-evoked depolarizing currents in Xenopus oocytes injected with GH3 cell RNA as a bioassay for TRH-R messenger RNA (mRNA) activity. In GH3 cells, TRH caused a rapid decrease in TRH-R mRNA activity to 15 percent of control within 3 hours. Because the half-life of TRH-R mRNA activity in control cells was approximately 3 hours, the rapid decrease in mRNA activity was not due to inhibition of mRNA synthesis alone and may represent a post-transcriptional effect.

Thyrotropin-releasing hormone: evidence for thyroid response to intravenous injection in man.

Administration of thyrotropin-releasing hormone to normal subjects causes a prompt rise in plasma thyrotropin concentration, followed by a significant increase in circulating plasma triiodothyronine. These observations may prove to be of value in simultaneously assessing the ability of the pituitary and thyroid glands to respond to their trophic hormones.

Bihormonal regulation of the thyrotropin-releasing hormone receptor in mouse pituitary thyrotropic tumor cells in culture.

Receptors for thyrotropin-releasing hormone (TRH) are present on mouse pituitary thyrotropic tumor cells. Incubation of thyrotropes with 100 nM TRH or 4 nM L-triiodothyronine (T3) for 48 h decreased the number of TRH receptors to approximately equal to 50 and 20% of control, respectively. There was no effect on the equilibrium dissociation constant which was 3-5 nM. The depletion in the number of available TRH receptors was time- and dose-dependent. TRH, 100 nM, decreased the receptor number to 70% after 24 h, 50% after 48 h, and 45% of control after 72 h. T3, 4 nM, decreased the receptor number to 52% after 24 h, 20% after 48 h, and 17% of control after 72 h. After 48 h, half-maximal depletion occurred with 1-2 nM TRH and approximately equal to 0.15 nM T3. Incubation with 100 nM TRH and 4 nM T3 caused a significantly greater reduction in the receptor level than either hormone alone. The decrease in the receptor level was reversible within 72 h after removal of TRH, 100 nM, but was only partially reversed, from 20 to 40% of control, after removal of T3, 4 nM, after 120 h. By regulating the number of available TRH receptors on the thyrotrope. TRH and T3 interact to control thyrotropin release.

Thyrotropin-induced hyperthyroidism caused by selective pituitary resistance to thyroid hormone. A new syndrome of "inappropriate secretion of TSH".

An 18-yr-old woman with clinical and laboratory features of hyperthyroidism had persistently elevated serum levels of immunoreative thyrotropin (TSH). During 11 yr of follow-up there had been no evidence of a pituitary tumor. After thyrotropin-releasing hormone (TRH), there was a marked increase in TSH and secondarily in triiodothyronine (T3), the latter observation confirming the biologic activity of the TSH. Exogenous T3 raised serum T3 and several measurements of peripheral thyroid hormone effect, while decreasing serum TSH, thyroxine (T4), and thyroidal radioiodine uptake. After T3, the TRH-stimulated TSH response was decreased but was still inappropriate for the elevated serum T3 levels. Dexamethasone reduced serum TSH but did not inhibit TRH stimulation of TSH. Propylthiouracil reduced serum T4 and T3 and raised TSH. This patient represents a new syndrome of TSH-induced hyperthyroidism, differing from previous reports in the absence of an obvious pituitary tumor and in the responsiveness of the TSH to TRH stimulation and thyroid hormone suppression. This syndrome appears to be caused by a selective, partial resistance of the pituitary to the action of thyroid hormone. This case is also compared with previous reports in the literature of patients with elevated serum levels of immunoreactive TSH in the presence of elevated total and free thyroid hormones. A classification of these cases, termed "inappropriate secretion of TSH," is proposed.

Thyroid-stimulating hormone and insulin-like growth factor-1 synergize to elevate 1,2-diacylglycerol in rat thyroid cells. Stimulation of DNA synthesis via interaction between lipid and adenylyl cyclase signal transduction systems.

Thyroid-stimulating hormone (TSH) and insulin-like growth factor-1 (IGF-1) synergistically stimulate DNA synthesis in thyroid cells. In this report, a novel mechanism for mediation of this synergistic interaction is described in rat thyroid (FRTL-5) cells. Because phorbol myristate acetate stimulates DNA synthesis, the effects of TSH, IGF-1 and insulin on FRTL-5 cell content of 1,2-diacylglycerol (1,2-DG), the endogenous activator of protein kinase C, were measured. After 6 d, TSH, IGF-1 and insulin caused increases in cellular 1,2-DG (mean +/- SE) to 180 +/- 10%, 540 +/- 50%, and 360 +/- 40% of control, respectively, whereas TSH plus IGF-1 and TSH plus insulin synergistically increased 1,2-DG to 1,890 +/- 310% and 1,690 +/- 230%, respectively. In the absence of insulin, the effect of TSH to elevate 1,2-DG exhibited an EC50 of approximately 2,000 microU/ml. The synergistic interaction of insulin and TSH was found to increase the potency of TSH by 300-fold (EC50 was approximately 7 microU/ml) in addition to increasing the efficacy of TSH. The effect of TSH appeared to be mediated by TSH-stimulated increases in cyclic AMP (cAMP). Forskolin and 8-bromo-cAMP, like TSH, caused modest increases in 1,2-DG and DNA synthesis, whereas forskolin plus insulin and 8-bromo-cAMP plus insulin markedly elevated 1,2-DG content and stimulated DNA synthesis. Under all conditions, increases in 1,2-DG content correlated with stimulation of DNA synthesis. These findings suggest that the synergistic stimulation of DNA synthesis in thyroid cells by TSH, via cAMP, and IGF-1 is mediated by 1,2-DG. Moreover, they implicate a novel interaction between the lipid and adenylyl cyclase signaling systems for the regulation of cell proliferation.

Thyrotropin-releasing hormone stimulation of adrenocorticotropin production by mouse pituitary tumor cells in culture: possible model for anomalous release of adrenocorticotropin by thyrotropin-releasing hormone in some patients with Cushing's disease and Nelson's syndrome.

ACTH-producing mouse pituitary tumor cells in culture (AtT-20/NYU-1 cells) were found to have binding sites for thyrotropin-releasing hormone (TRH). These putative receptors bound TRH with high affinity; the apparent equilibrium dissociation constant was 3.7 nM. The affinity of the receptors for a series of TRH analogues was similar to those previously reported for TRH-receptor interactions on thyrotropic and mammotropic cells in culture. Like some human pituitary tumors in situ, AtT-20/NYU-1 cells were found to produce the alpha subunit of the glycoprotein hormones (alpha). Alpha accumulation in the medium was constant (3.1 ng/mg cell protein per h) and was not affected by TRH. In contrast, TRH increased the amount of ACTH accumulated in the medium from AtT-20/NYU-1 cells to 190 and 420% of control at 1 and 24 h, respectively. TRH induced a dose-dependent increase in ACTH release during a 30-min incubation; half-maximal stimulation occurred at approximately 0.1 nM. TRH had no effect on ACTH release in vitro from anterior pituitary cells derived from normal rats. Because TRH stimulates release of ACTH in some untreated patients with Cushing's disease and Nelson's syndrome as well as pathological states associated with pituitary tumors (but not in normal subjects), AtT-20/NYU-1 cells may serve as an important in vitro model for human pituitary ACTH-secreting adenomas. Moreover, these findings suggest that the primary abnormality in Cushing's disease and Nelson's syndrome, allowing TRH stimulation of ACTH release, may be intrinsic to neoplastic adrenocorticotrophs rather than in neuroregulation of ACTH release.

Serum triiodothyronine: measurements in human serum by radioimmunoassay with corroboration by gas-liquid chromatography.

Serum triiodothyronine (T(3)) has been measured by radioimmunoassay and corroborated by analysis of the identical samples with a previously described gas-liquid chromatographic technique. Special features of the radioimmunoassay procedure which permit determinations in unextracted serum include the use of a T(3)-free serum preparation for the construction of the standard curve and of tetrachlorothyronine to inhibit binding of T(3) to thyroxine-binding globulin.T(3) values by radioimmunoassay were 138 +/-23 ng/100 ml (mean +/-SD) in 82 normal subjects, 62 +/-9 ng/100 ml in 45 hypothyroid patients, and 494 +/-265 ng/100 ml in 60 patients with toxic diffuse goiter. In the hypothyroid group, the range was similar in patients with both primary and secondary hypothyroidism. There was no overlap between the three thyroidal states. Elevated T(3) levels were seen in 40 cases that appeared clinically hyperthyroid but had normal serum thyroxine (T(3)) determinations, a syndrome we have called T(3) toxicosis. Values obtained with radioimmunoassay agreed closely with those we had previously found by gas-liquid chromatography which were 68 +/-2 ng/100 ml in hypothyroidism, 137 +/-23 ng/100 ml in normal subjects, and 510 +/-131 ng/100 ml in untreated toxic diffuse goiter. Since T(3) is very potent and its level varies in different clinical states, accurate T(3) measurements are required to assess a patient's thyroid status properly. The radioimmunoassay for T(3) appears to be sufficiently sensitive, precise, and simple to permit its routine clinical application for this purpose.

Chemokines activate Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor in mammalian cells in culture.

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8, a virus that appears to be involved in the pathogenesis of Kaposi's sarcoma and primary effusion lymphomas, encodes a G protein-coupled receptor (KSHV-GPCR) that exhibits constitutive signaling. In this report, we show that two chemokines, interleukin 8 (IL-8) and growth-related protein-alpha, activate KSHV-GPCR over constitutive levels. Moreover, as with human receptors, the integrity of the ELR motif of these chemokines is required for activation of KSHV-GPCR. Other residues that are required for IL-8 binding to human chemokine receptors CXCR1 and CXCR2 are important for KSHV-GPCR activation also. Thus, it appears that the ELR binding site and other key domains of ELR chemokine activation have been preserved in the virus KSHV-GPCR. The results suggest that KSHV-GPCR originated from CXCR1 or CXCR2 and that activation of KSHV-GPCR by endogenous chemokines may affect the pathobiology of KSHV infection in humans.

Thyrotropin-releasing hormone stimulation of prolactin release from clonal rat pituitary cells: evidence for action independent of extracellular calcium.

Thyrotropin-releasing hormone (TRH) stimulates prolactin release and (45)Ca(2+) efflux from GH(3) cells, a clonal strain of rat pituitary cells. Elevation of extracellular K(+) also induces prolactin release and increases (45)Ca(2+) efflux from these cells. In this report, we distinguish between TRH and high K(+) as secretagogues and show that TRH-induced release of prolactin and (45)Ca(2+) is independent of the extracellular Ca(2+) concentration, but the effect of high K(+) on prolactin release and (45)Ca(2+) efflux is dependent on the concentration of Ca(2+) in the medium. The increment in (45)Ca(2+) efflux induced by 50 mM K(+) during perifusion was reduced in a concentration-dependent manner by lowering extracellular Ca(2+) from 1,500 to 0.02 muM (by adding EGTA), whereas 1 muM TRH enhanced (45)Ca(2+) efflux similarly over the entire range of extracellular Ca(2+) concentrations. Although 50 mM K(+) caused release of 150 ng prolactin from 40 x 10(6) GH(3) cells exposed to 1,500 muM Ca(2+) (control), reduction of extracellular Ca(2+) to 2.8 muM decreased prolactin release caused by high K(+) to <3% of controls and no prolactin release was detected after exposure to 50 mM K(+) in medium with 0.02 muM free Ca(2+). In contrast, TRH caused release of 64 ng of prolactin from 40 x 10(6) GH(3) cells exposed to medium with 1,500 muM Ca(2+), and release caused by TRH was still 50 and 35% of control in medium with 2.8 and 0.02 muM Ca(2+), respectively. Furthermore, TRH transiently increased by 10-fold the fractional efflux of (45)Ca(2+) from GH(3) cells in static incubations with 1,500 or 3.5 muM Ca(2+), hereby confirming that the enhanced (45)Ca(2+) efflux caused by TRH in both low and high Ca(2+) medium was not an artifact of the perifusion system.Data obtained with chlortetracycline (CTC), a probe of membrane-bound Ca(2+), were concordant with those obtained by measuring (45)Ca(2+) efflux. Cellular fluorescence of CTC varied with the extracellular Ca(2+) concentration and the duration of incubation. TRH decreased the fluorescence of cell-associated CTC in a manner strongly suggesting stimulus-induced mobilization of Ca(2+), and this effect was still demonstrable in GH(3) cells incubated in 50 mM K(+). These data suggest that TRH acts to mobilize sequestered cell-associated Ca(2+) reflected as a (45)Ca(2+) efflux which is independent of the extracellular Ca(2+) concentration. Mobilization of sequestered Ca(2+) into the cytoplasm may elevate free intracellular Ca(2+) and serve to couple stimulation by TRH to secretion of prolactin.

Thyrotropin-releasing hormone analogs.

Thyrotropin releasing hormone (TRH: pyroglutamic acid-histidine-prolineamide) regulates the activity of cells in the anterior pituitary and within the central and peripheral nervous systems. TRH, which has been the subject of much research over the past three decades, exerts its effects by acting through class A G-protein coupled receptors. The recent discovery of a second receptor subtype has generated an interest in the discovery of receptor subtype-selective TRH analogs. In this review, we describe advances in the development of TRH analogs and in the understanding of their mechanism of interaction with TRH receptors. We also describe the recent breakthrough in the identification of analogs that bind selectively at TRH-R2.

Thyroxine-binding globulin biosynthesis in isolated monkey hepatocytes.

Thyroxine-binding globulin biosynthesis was demonstrated in hepatocytes isolated from normal adult Rhesus monkeys. Dispersed cells were obtained by in situ liver perfusion with collagenase, hyaluronidase and EDTA. Conditions for optimum cell survival and incorporation of radioactive leucine into newly synthesized proteins were defined. Protein synthesis, and specifically thyroxine-binding globulin synthesis, were shown to continue throughout the incubation period, while cell survival remained high (75% excluded trypan blue after 6h). Incubation medium, cytosol and a particulate fraction (extracted with digitonin) were analyzed for thyroxine-binding globulin. After extensive dialysis and purification by affinity chromatography, newly synthesized thyroxine-binding globulin was identified by specific double-antibody immunoprecipitation and by immunodiffusion and immunoelectrophoresis with autoradiography. Newly synthesized thyroxine-binding globulin was present after 4 h of incubation. After 6 h, the total synthesized had increased to 150% of the 4 h value, while the fraction present in the medium and increased to 300%, indicating probable thyroxine-binding globulin secretion

Constitutively signaling G-protein-coupled receptors and human disease.

Dysregulation of G-protein-coupled receptor (GPCR) function has been shown to be associated with a growing number of human diseases. In some diseases, mutation of an endogenous GPCR causes the receptor to lose the ability to bind agonist or signal (;loss of function' mutation), whereas another mutation causes the receptor to be in an active state in the absence of agonist (;gain of function' mutation), leading to ;constitutive signaling activity'. A number of constitutively active GPCRs are tumorigenic in vitro and in animal models, and cause syndromes of hyperfunction and/or tumors in humans. The recent characterization of a constitutively active GPCR in the genome of a disease-associated, human herpesvirus provides a potential novel mechanism for viral tumorigenesis.

Thyrotropin-releasing hormone (TRH) and phorbol myristate acetate decrease TRH receptor messenger RNA in rat pituitary GH3 cells: evidence that protein kinase-C mediates the TRH effect.

In a previous report we showed that TRH-induced down-regulation of the density of its receptors (TRH-Rs) on rat pituitary tumor (GH3) cells was preceded by a decrease in the activity of the mRNA for the TRH-R, as assayed in Xenopus oocytes. Here we report the effects of TRH, elevation of cytoplasmic free Ca2+ concentration, phorbol myristate acetate (PMA), and H-7 [1-(5-isoquinolinesulfonyl)2-methylpiperazine dihydrochloride], an inhibitor of protein kinases, on the levels of TRH-R mRNA, which were measured by Northern analysis and in nuclease protection assays using probes made from mouse pituitary TRH-R cDNA, in GH3 cells. These agents were studied to gain insight into the mechanism of the TRH effect, because signal transduction by TRH involves generation of inositol 1,4,5-trisphosphate and elevation of cytoplasmic free Ca2+ concentration, which leads to activation of Ca2+/calmodulin-dependent protein kinase, and of 1,2-diacylglycerol, which leads to activation of protein kinase-C. TRH (1 microM TRH, a maximally effective dose) caused a marked transient decrease in TRH-R mRNA that attained a nadir of 20-45% of control by 3-6 h, increased after 9 h, but was still below control levels after 24 h. Elevation of the cytoplasmic free Ca2+ concentration had no effect on TRH-R mRNA. A maximally effective dose of PMA (1 microM) caused decreases in TRH-R mRNA that were similar in magnitude and time course to those induced by 1 microM TRH. H-7 (20 microM) blocked the effects of TRH and PMA to lower TRH-R mRNA to similar extents.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphatidylinositol depletion in GH3 rat pituitary cells inhibits sustained responses to thyrotropin-releasing hormone. Reversal with myo-inositol.

TRH stimulation of rat pituitary (GH3) cells causes biphasic changes in cytoplasmic free Ca2+ concentration [( Ca2+]i) and PRL secretion. It has been proposed, based primarily on indirect evidence, that the first phase effects are mediated by inositol 1,4,5-trisphosphate, which releases Ca2+ from cellular stores, and the sustained effects are mediated by 1,2-diacylglycerol, which activates protein kinase C. To determine more directly if inositol lipid hydrolysis leading to protein kinase C activation is involved in the sustained effects of TRH, GH3 cells were depleted of phosphatidylinositol (PtdIns) by prestimulation and incubation in myo-inositol-free, Li(+)-containing medium. Cells depleted of PtdIns (to 53 +/- 3.2% of control) had unchanged PtdIns 4,5-bisphosphate content, and responded to TRH with a rapid elevation of inositol trisphosphate, and a first phase (or burst) elevation of [Ca2+]i and PRL secretion that was not different from that found in control cells. In contrast, in PtdIns-depleted cells, the prolonged generation of inositol phosphates, which are produced in equimolar amounts with 1,2-diacylglycerol, caused by TRH was virtually abolished, and the second phase (or sustained) elevation of [Ca2+]i and PRL secretion were inhibited by 50% and 40%, respectively. The inhibition of both sustained effects was reversed by adding 100 mM myo-inositol to the medium, which allowed for synthesis of PtdIns. Last, in cells in which protein kinase C was down-regulated by pretreatment with a phorbol ester, the sustained effects of TRH were inhibited also.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased levels of internalized thyrotropin-releasing hormone receptors after uncoupling from guanine nucleotide-binding protein and phospholipase-C.

Internalization of TRH receptor (TRH-R) is dependent on sequences/structures in the receptor carboxyl-terminal tail. Here, we studied whether coupling to guanine nucleotide-binding protein (G-protein) and phospholipase-C (PLC) is involved in internalization. We constructed two mutant TRH-Rs: delta 218-263 TRH-R, in which most of the residues that form the putative third intracellular loop were deleted, and D71A TRH-R, in which an Asp in the putative second transmembrane helix was mutated to Ala; these TRH-Rs did not activate PLC when expressed transiently in COS-1 cells. In contrast to wild-type (WT) TRH-Rs, approximately 60% of which were internalized at steady state after binding methyl-HisTRH, only approximately 15% of delta 218-263 and D71A TRH-Rs were internalized. Thus, mutant TRH-Rs that do not activate PLC, most likely because they are uncoupled from G-proteins, are internalized to lesser extents than WT TRH-Rs. We also studied the effects of U73122 (1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione), an amino steroid that inhibits receptor-mediated activation of PLC. In COS-1 and AtT-20 cells transfected with WT TRH-Rs and in GH3 cells, U73122 virtually abolished TRH activation of PLC and partially reduced the fraction of WT TRH-Rs internalized. Thus, uncoupling WT TRH-Rs from PLC decreases internalization. We conclude that TRH-R coupling to G-protein and PLC increases the number of TRH-Rs internalized at steady state even though the primary signals for agonist-induced internalization are present in the receptor. These data support the idea that a quaternary complex of TRH/TRH-R/G protein/PLC is normally internalized.