Smad7 positively regulates keratinocyte proliferation in psoriasis.

BACKGROUND: Transforming growth factor (TGF)-ß1 exerts inhibitory effects on keratinocyte proliferation. OBJECTIVES: To examine whether Smad7, a known inhibitor of TGF-ß1 signalling, is involved in psoriasis-associated keratinocyte hyperproliferation. METHODS: Smad7 was evaluated in skin sections of patients with psoriasis and healthy controls and in mice with Aldara-induced skin pathology by real-time polymerase chain reaction and immunohistochemistry. To assess whether Smad7 positively regulates in vivo keratinocyte growth, mice treated with Aldara received daily cutaneous administration of Smad7 antisense oligonucleotide (AS). Keratin (K)6 and K16, cell-cycle-associated factors, cell-cycle and cell proliferation were evaluated in HaCaT cells either treated with Smad7 AS or transfected with Smad7 plasmid and in mice given Smad7 AS. RESULTS: Smad7 was highly expressed in keratinocytes of patients with psoriasis and of mice treated with Aldara. In HaCaT cells, Smad7 knockdown inhibited cell growth, reduced K6 and K16 expression and promoted accumulation of cells in the S-phase of the cell cycle. Smad7-deficient keratinocytes exhibited reduced levels of CDC25A protein, a phosphatase that facilitates progression of cells through the S-phase, and hyperphosphorylation of eukaryotic initiation factor 2 (eIF2)α, a negative regulator of CDC25 protein translation. Consistently, Smad7 overexpression in HaCaT cells was followed by induction of K6 and K16 and increased cell proliferation. Topical application of Smad7 AS to Aldara-treated mice reduced epidermal thickness. CONCLUSIONS: Our data show that Smad7 is overexpressed in human and murine psoriasis and suggest a key role of this molecule in the control of keratinocyte proliferation.

Serum regenerating islet-derived 3-alpha is a biomarker of mucosal enteropathies.

BACKGROUND: The clinical presentation of organic and functional intestinal disorders can overlap and clinicians often rely on invasive and time-consuming procedures to make a final diagnosis. Regenerating islet-derived 3-alpha (Reg3α) is detectable in the circulation of patients with intestinal graft-versus host disease and patients with inflammatory bowel disease (IBD). AIM: To determine whether serum Reg3α testing is useful for discriminating mucosal enteropathies from functional intestinal disorders. METHODS: We prospectively included 47 patients with active coeliac disease (ACD), 13 patients with refractory coeliac disease (RCD), seven patients with common variable immunodeficiency (CVID), 72 patients with active Crohn's disease, 22 patients with active ulcerative colitis (UC) and 28 patients with irritable bowel syndrome (IBS)-related diarrhoea. Sera were also taken from 10 CD patients before and after 6-12 months of a gluten-free diet (GFD) and from 14 patients with IBD before and after induction therapy with Infliximab (IFX). Sera of 119 healthy volunteers were used to determine the cut-off value. Reg3α levels were measured by a commercial ELISA kit. RESULTS: Levels of Reg3α exceeded the cut-off value of the assay in 43/47(91%) ACD patients, 13/13(100%) RCD patients, 7/7(100%) CVID patients, 65/72(90%) Crohn's disease patients, 17/22(77%) UC patients and one patient with IBS(4%). Reg3α levels distinguished mucosal enteropathies from IBS with a sensitivity of 90% and a specificity of 96%. Reg3α levels significantly decreased in CD patients following a GFD and in IBD patients after treatment with IFX. CONCLUSION: Reg3α is a serum biomarker of intestinal damage that, combined with clinical data, identifies patients who should undergo invasive tests for diagnosing enteropathies.

Defective expression of SIRT1 contributes to sustain inflammatory pathways in the gut.

In inflammatory bowel disease (IBD), tissue damage is driven by an excessive immune response, poorly controlled by counter-regulatory mechanisms. SIRT1, a class III NAD+-dependent deacetylase, regulates negatively the expression of various proteins involved in the control of immune-inflammatory pathways, such as Stat3, Smad7, and NF-κB. Here we examined the expression, regulation, and function of SIRT1 in IBD. SIRT1 RNA and protein expression was less pronounced in whole biopsies and lamina propria mononuclear cells (LPMCs) of IBD patients in comparison with normal controls. SIRT1 expression was downregulated in control LPMC by tumor necrosis factor (TNF)-α and interleukin (IL)-21, and upregulated in IBD LPMC by neutralizing TNF-α and IL-21antibodies. Consistently, SIRT1 expression was increased in mucosal samples taken from IBD patients successfully treated with Infliximab. Treatment of IBD LPMC with Cay10591, a specific SIRT1 activator, reduced NF-κB activation and inhibited inflammatory cytokine synthesis, whereas Ex527, an inhibitor of SIRT1, increased interferon (IFN)-γ in control LPMC. SIRT1 was also reduced in mice with colitis induced by 2,4,6-trinitrobenzenesulphonic acid or oxazolone. Cay10591 prevented and cured experimental colitis whereas Ex527 exacerbated disease by modulating T cell-derived cytokine response. Data indicate that SIRT1 is downregulated in IBD patients and colitic mice and suggest that SIRT1 activation can help attenuate inflammatory signals in the gut.

A functional role for Smad7 in sustaining colon cancer cell growth and survival.

Initially identified as an inhibitor of transforming growth factor (TGF)-ß mainly owing to its ability to bind TGF-ß receptor type I and abrogate TGF-ß-driven signaling, Smad7 can interact with additional intracellular proteins and regulate TGF-ß-independent pathways, thus having a key role in the control of neoplastic processes in various organs. Genome-wide association studies have shown that common alleles of Smad7 influence the risk of colorectal cancer (CRC), even though the contribution of Smad7 in colon carcinogenesis is not fully understood. In this study, we assessed the expression and role of Smad7 in human and mouse models of sporadic CRC. We document a significant increase of Smad7 in human CRC relative to the surrounding nontumor tissues and show that silencing of Smad7 inhibits the growth of CRC cell lines both in vitro and in vivo after transplantation into immunodeficient mice. Knockdown of Smad7 results in enhanced phosphorylation of the cyclin-dependent kinase (CDK)2, accumulation of CRC cells in S phase and enhanced cell death. Smad7-deficient CRC cells have lower levels of CDC25A, a phosphatase that dephosphorylates CDK2, and hyperphosphorylated eukaryotic initiation factor 2 (eIF2)α, a negative regulator of CDC25 protein translation. Consistently, knockdown of Smad7 associates with inactivation of eIF2α, lower CDC25A expression and diminished fraction of proliferating cells in human CRC explants, and reduces the number of intestinal tumors in Apc(min/+) mice. Altogether, these data support a role for Smad7 in sustaining colon tumorigenesis.

IL-15 positively regulates IL-21 production in celiac disease mucosa.

Celiac disease (CD)-associated inflammation is characterized by high interleukin- 21 (IL-21), but the mechanisms that control IL-21 production are not fully understood. Here we analyzed IL-21 cell sources and examined how IL-21 production is regulated in CD. Intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs), isolated from CD patients and non-CD controls, were analyzed for cell markers, cytokines, and transcription factors by flow cytometry. IL-21 was highly produced by CD4+ and CD4+/CD8+ IELs and LPLs in active CD. IL-21-producing cells coexpressed interferon-γ (IFN-γ) and to a lesser extent T helper type 17 (Th17) cytokines. Treatment of control LPLs with IL-15, a cytokine overproduced in CD, activated Akt and STAT3 (signal transducer and activator of transcription 3), thus enhancing IL-21 synthesis. Active CD biopsies contained elevated levels of Akt, and blockade of IL-15 in those samples reduced IL-21. Similarly, neutralization of IL-15 in biopsies of inactive CD patients inhibited peptic-tryptic digest of gliadin-induced IL-21 expression. These findings indicate that in CD, IL-15 positively regulates IL-21 production.

A phase 1 open-label trial shows that smad7 antisense oligonucleotide (GED0301) does not increase the risk of small bowel strictures in Crohn's disease.

BACKGROUND: In Crohn's disease (CD), knockdown of Smad7, an inhibitor of Transforming Growth Factor (TGF)-ß1 activity, with a specific antisense oligonucleotide (GED0301) seems to be safe and tolerable and associates with TGF-ß1-mediated suppression of inflammatory pathways. AIM: Since TGF-ß1 has pro-fibrogenic effects in many organs, we evaluated whether GED0301 treatment associates with the formation of small bowel strictures. METHODS: Fifteen patients with active, inflammatory CD, receiving oral GED0301 once daily for 7 days, were monitored for the formation of small bowel strictures by Small Intestine Contrast Ultrasonography (SICUS). Serum basic fibroblast growth factor (bFGF) and human chitinase 3-like 1 (also known as YKL-40), two markers of CD-related intestinal strictures, and matrix metalloproteinases (MMP) and tissue inhibitor 1 of MMPs (TIMP1) were analysed at day 0 and day 180 by ELISA. Crohn's disease activity index (CDAI) changes were also monitored. RESULTS: Fourteen patients completed the 6-month study; the remaining underwent intestinal resection for a severe relapse not responsive to medical treatment. No patient developed small bowel stricture and none experienced obstructive symptoms during the study period. GED0301 treatment induced no significant change in the circulating levels of bFGF, YKL-40, MMPs and TIMP1. Seven of 12 patients who reached clinical remission following GED0301 treatment maintained a CDAI < 150 at day 180. CONCLUSION: Short-term treatment of patients with Crohn's disease using GED0301 is not associated with the development of small bowel stricture, thus reinforcing the concept that this drug is safe at least at early time points.

Th17-related cytokines in inflammatory bowel diseases: friends or foes?

T helper (Th)17 cells and other interleukin (IL)-17-producing cells are supposed to play critical roles in several human immune-mediated diseases, including Crohn's disease (CD) and ulcerative colitis (UC), the main forms of inflammatory bowel diseases (IBD) in man. Th17 cells infiltrate massively the inflamed intestine of IBD patients and in vitro and in vivo studies have shown that Th17-type cytokines may trigger and amplify multiple inflammatory pathways. Nonetheless, some Th17-related cytokines, such as interleukin (IL)-17A and IL-22, may target gut epithelial cells and promote the activation of counter-regulatory mechanisms. This observation together with the demonstration that Th17 cells are not stable and can be converted into either regulatory T cells or Th1 cells if stimulated by immune-suppressive (e.g. TGF-ß1) or inflammatory (e.g. IL-12, IL-23) cytokines have contributed to advance our understanding of mechanisms that regulate mucosal homeostasis and inflammation in the gut.

Silencing of SH-PTP2 defines a crucial role in the inactivation of epidermal growth factor receptor by 5-aminosalicylic acid in colon cancer cells.

Recent studies have suggested that 5-aminosalicylic acid (5-ASA) inhibits colorectal cancer (CRC) development. However, the mechanism underlying the antineoplastic effect of 5-ASA remains unknown. We here examined the effect of 5-ASA on epidermal growth factor receptor (EGFR) activation, a pathway that triggers mitogenic signals in CRC cells. We show that 5-ASA inhibits EGFR activation, through a mechanism that does not rely on CRC cell death induction. 5-ASA enhances the activity, but not expression, of phosphorylated (p)-EGFR-targeting phosphatases (PTPs), and treatment of cells with PTP inhibitors abrogates the 5-ASA-mediated EGFR dephosphorylation. Both SH-PTP1 and SH-PTP2 interact with EGFR upon 5-ASA treatment. However, knockdown of SH-PTP2 but not SH-PTP1 by small interference RNAs prevents the 5-ASA-induced EGFR dephosphorylation. Finally, we show that 5-ASA attenuates p-EGFR in ex vivo organ cultures of CRC explants. Data indicate that 5-ASA disrupts EGFR signalling by enhancing SH-PTP2 activity, and suggest a mechanism by which 5-ASA interferes with CRC growth.

Regulation of the T helper cell type 1 transcription factor T-bet in coeliac disease mucosa.

BACKGROUND: In coeliac disease (CD) mucosa, the histological lesion is associated with marked infiltration of T helper cell type 1 (Th1) cells. However, the molecular mechanisms which regulate Th1 cell differentiation in CD mucosa are unknown. AIMS: To analyse expression of transcription factors which control the Th1 cell commitment in CD. PATIENTS: Duodenal mucosal samples were taken from untreated CD patients and normal controls. METHODS: Interferon gamma (IFN-gamma) and interleukin (IL)-4 RNA expression was examined in T lamina propria lymphocytes by quantitative reverse transcription-polymerase chain reaction. T-bet and STAT-4, two Th1 promoting transcription factors, and STAT-6 and GATA-3, transcription factors which govern T helper cell type 2 (Th2) cell polarisation, were examined in duodenal biopsies by western blotting. The effect of gliadin and IFN-gamma on expression of T-bet was examined in an ex vivo culture of biopsies taken from normal and treated CD patients. RESULTS: As expected, IFN-gamma but not IL-4 RNA transcripts were increased in the mucosa of CD patients in comparison with controls. CD mucosal samples consistently exhibited higher levels of T-bet than controls. However, no difference in active STAT-4 expression was seen between CD patients and controls, suggesting that Th1 polarisation was not induced by local IL-12. GATA-3 and STAT-6 were also low in both CD and control mucosa. In normal duodenal biopsies, IFN-gamma stimulated T-bet through a STAT-1 dependent mechanism. Challenge of treated CD but not control biopsies with gliadin enhanced T-bet and this effect was also inhibited by STAT-1 inhibition. CONCLUSIONS: This study shows that activation of STAT-1 by IFN-gamma promotes T-bet in CD mucosa.

Cytoskeletal proteins and resident flora.

Recent observations demonstrate that enteropathogenetic and enterohaemorrhagic bacteria, as well as other non enteropathogenetic bacteria (Listeria, Coxiella Burnetii), may subvert the host cell cytoskeleton. Models from enteropathogenic bacteria demonstrate that cytoskeletal proteins are required for bacteria binding to the enterocytes and that they play a role in the immune response of the host to intestinal bacteria. The cytoskeletal protein family Tropomyosins is present in all eukaryotic cells, with multiple isoforms regulated by multiple genes. Of the different Tropomyosin isoforms, TM5 has been shown to be expressed in colonic and jejunal epithelial cells, while TM1 in colonic and jejunal smooth muscle. In vitro studies have shown the presence of serum and mucosal IgG against TM5 in almost two thirds of patients with ulcerative colitis, suggesting: a. a possible autoimmune response to Tropomyosin in these patients; b. the hypothesis that the development of pouchitis may be related to the expression of TM5 in the ileal pouch; c. the use of probiotics in the treatment of pouchitis. Overall, the new expression of cytoskeletal proteins on the cell surface appears to be possibly induced by several mechanisms, including intestinal bacteria and apoptosis. The expression of cytoskeletal proteins on the cell surface may induce tolerance or autoimmune response on target cells. Further investigations are, however needed on the possible role of cytoskeletal proteins in human diseases.

Resident bacterial flora and immune system.

The "controlled inflammation" of the normal human gut is a closely controlled phenomenon and any change in the cell type number and/or functions, including the release of soluble mediators can lead to an "uncontrolled" inflammation. The physiological inflammation in the human gut plays a crucial role in maintaining a local immune response that is appropriate, efficiently protective and which respects the gut structure and function. The intestinal mucosa represents a considerable proportion of the human immune system. Disregulation of the mucosal immune response can switch a "controlled" toward an "uncontrolled" intestinal inflammation. A key role in the maintenance of an adequate balance between antigenic stimulation and host immune response is played by the immunoregulatory molecules released by activated immunocytes in the human gut. The role of the host immune system in the maintenance of an adequate balance between luminal antigens, including the resident bacterial flora and host immune response, is strongly supported by animal models of uncontrolled intestinal inflammation. Besides the aetiology of inflammatory bowel disease, luminal antigens (including food, viral and bacterial antigens) contribute to the maintenance of the inflammatory process in inflammatory bowel disease, by stimulating the immunocompetent cells in the intestinal mucosa. Of the luminal antigens, the resident bacterial flora seems to play a major role in the development of animal models of "uncontrolled" intestinal inflammation. Recent evidence also suggest that bacterial flora can modulate the function of the intestinal mucosal cells. These observations support the role of the intestinal bacterial flora in the induction of an uncontrolled inflammation in the human gut, leading to tissue damage. Probiotics, defined as living micro-organisms which, when taken in appropriate amounts, improve the health status, have been proposed in the treatment of inflammatory bowel disease, but their mechanisms of action still remain to be fully elucidated.

Review article: monitoring the activity of Crohn's disease.

Crohn's disease is characterized by a chronic inflammation of the intestine of unknown aetiology. One of the main problems when treating patients with Crohn's disease, is the identification of patients undergoing early clinical relapse, for timely treatment and the possible prevention of complications. No sub-clinical markers are currently available that predict relapse during remission. Several parameters have been proposed for this purpose. Although none have proven useful, growing evidence suggests a possible benefit in the clinical management of Crohn's disease. Among these, we may identify: clinical behaviour, the characteristics of the host, clinical activity, markers of intestinal inflammation and markers of immune activation. In particular, the possible relationship between cytokine pattern and the clinical behaviour of Crohn's disease has been addressed. Overall, these observations suggest that mucosal immune activation is a feature of Crohn's disease, and may persist in the form of activated immunocompetent cells during remission. On the basis of this evidence, studies are currently investigating whether the down-regulation of immune activation markers is associated with clinical remission in Crohn's disease. It has been shown that higher mucosal levels of TNF-alpha and an increased state of activation of lamina propria mononuclear cells in patients with inactive Crohn's disease, are significantly associated with an earlier clinical relapse of the disease. These observations suggest that a persistent local immune activation during remission may represent a marker of early clinical relapse of Crohn's disease.

Immunoregulation in the gut: success and failures in human disease.

In normal conditions, human gut mucosa is infiltrated with a large number of mononuclear cells. This is a reflection of the fact that human intestine is continuously subjected to a massive stimulation by luminal antigens. This state of "physiological" inflammation is a tightly controlled phenomenon, as several mucosal cells interact to generate and maintain an appropriate local immune response. Changes in cell type number and/or function, including the release of soluble mediators, have been associated with the development of chronic inflammatory diseases, such as Crohn's disease (CD) and ulcerative colitis (UC), the two major forms of inflammatory bowel disease. Evidence also indicates that the type of inflammatory response occurring in the intestine of patients with CD differs from that in UC, and this probably reflects distinct pathways of immune activation. In CD mucosa, a Th1 response with high IL-12 and IFNgamma production prevails, while in UC a humoral immunity appears to be predominant. Despite this, CD and UC share downstream inflammatory events, characterised by high levels of inflammatory cytokines, free radicals, matrix-degrading enzymes and growth factors.

Hepatitis B and C virus infection in Crohn's disease.

Patients with Crohn's disease (CD) are at higher risk of hepatitis C (HCV) and B virus (HBV) infection, because of surgical and/or endoscopic procedures. However, the prevalence of HCV and HBV infection in CD is unknown. This issue may be relevant because of the growing use of immunomodulatory drugs in CD. The purpose of this study was to assess, in a multicenter study, the prevalence and risk factors of HCV and HBV infection in CD. The effect of immunomodulatory drugs for CD on the clinical course of hepatitis virus infections and of interferon-alpha (IFN-alpha) on the course of CD was examined in a small number of patients. Sera from 332 patients with CD and 374 control subjects (C) were tested for the following: hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (HBsAb), HBcAb, HBeAg, HBeAb, anti-HCV, and HCV-RNA. An additional 162 patients with ulcerative colitis (UC) were tested as a disease control group. Risk factors were assessed by multivariate statistical analysis. Infection by either HCV or HBV was detected in 24.7% of patients with CD. In the age groups younger than 50 years, HCV prevalence was higher in CD than in C (p = 0.01). HCV infection in CD was associated with surgery (OR 1.71; 95% CI 1.00-2.93; p = 0.04), blood transfusions (OR 3.39; 95% CI 1.04-11.04; p = 0.04), and age (OR 2.3; 95% CI 1.61-3.56; p < 0.001). The event CD-related surgery appeared to be the main risk factor for HCV infection in CD. HCV prevalence was higher in CD (7.4%) than in UC (0.6%) (p = 0.001). HBcAb positivity was higher in CD (10.9%) and UC (11.5%) than in C (5.1%) (CD vs. C: p = 0.016; UC vs. C: p = 0.02), associated with age (OR 2.08; 95% CI 1.37-3.17; p = 0.001) and female gender (OR 2.68; 95% CI 1.37-3.17; p = 0.001) in CD and to UC duration (OR 1.20; 95% CI 1.06-1.36; p = 0.002). Immunomodulatory drugs did not influence the course of HBV or HCV infection in seven patients with CD, and IFN-alpha for chronic hepatitis C did not affect CD activity in six patients with CD. It is concluded that HBV prevalence is higher in CD than in C at all ages, whereas HCV prevalence is increased in young patients with CD, because of a greater need for surgery. The higher HCV (but not HBV) prevalence in CD than in UC suggests that the host immune response may influence the risk of HCV infection. Although a relatively high proportion of patients with CD showed HBV and/or HCV infections, this should not influence treatment strategies for CD.

Up-regulation of the IL-12 receptor beta 2 chain in Crohn's disease.

Crohn' s disease (CD) is a chronic intestinal inflammatory disorder characterized by aberrant mucosal Th1 cell activation and production of IL-12, the major Th1-driving factor. The T cell response to IL-12 is dependent on the expression of a specific receptor composed of two subunits, termed IL-12Rbeta1 and IL-12Rbeta2. The content of IL-12Rbeta2, as measured at the mRNA level, is crucial in regulating Th1 differentiation. In this study we therefore investigated IL-12Rbeta2 RNA transcripts in CD. IL-12Rbeta2 expression was increased in active CD as well as Helicobacter pylori (HP)-associated gastritis and Salmonella colitis compared with that in inactive CD, ulcerative colitis, noninflammatory controls, and celiac disease. In contrast, IL-12Rbeta1 transcripts were expressed at comparable levels in all samples. In CD, IL-12Rbeta2 expression strictly correlated with tyrosine phosphorylation of STAT4, a key component of the IL-12-dependent Th1 polarization. This was associated with a pronounced expression of IFN-gamma. Transcripts for IL-12/p40 were detected in CD, HP-positive, and Salmonella colitis patients, but not in celiac disease, indicating that IL-12Rbeta2 up-regulation occurs only in IL-12-associated Th1 gastrointestinal diseases. Finally, we showed that stimulation of lamina propria mononuclear cells with IL-12 enhanced IL-12Rbeta2, suggesting that IL-12 regulates IL-12Rbeta2 expression in human gastrointestinal mucosa. The data show that the signaling pathway used by IL-12 to induce Th1 differentiation is increased at the site of disease in CD, further supporting the view that IL-12/IL-12R signals contribute to the inflammatory response in this condition.

Interferon-gamma (IFN-gamma) and prostaglandin E2 (PGE2) regulate differently IL-12 production in human intestinal lamina propria mononuclear cells (LPMC).

IL-12 modulates Th1 immune response during chronic colitis. Mechanisms regulating IL-12 synthesis in human intestine are poorly understood. The aim of this study was to investigate the effect of IFN-gamma and PGE2 on lipopolysaccharide (LPS)-stimulated LPMC IL-12 production. Normal LPMC cultures were run in the presence or absence of IFN-gamma and/or PGE2 before LPS stimulation. To examine the role of endogenous PGE2 on LPS-stimulated IL-12 release, LPMC cultures were added of indomethacin before LPS stimulation. IL-12, IL-10 and IL-8 were measured by ELISA. No IL-12 was detected in either unstimulated or LPS-stimulated LPMC cultures. In contrast, LPMC released IL-8 (650 +/- 125 pg/ml) and IL-10 (75 +/- 25 pg/ml) in response to LPS. Treatment of LPMC with IFN-gamma facilitated LPS-stimulated IL-12, whereas it completely abrogated IL-10 production. IL-12 release by LPMC stimulated with IFN-gamma and LPS was significantly inhibited by exogenous IL-10. The addition of PGE2 to IFN-gamma-treated LPMC cultures inhibited in a dose-dependent manner LPS-induced IL-12 secretion. Furthermore, IL-12 was detectable (85 +/- 25 pg/ml) in the supernatants of LPMC cultures treated with indomethacin and LPS. In contrast to the effect on IL-12, PGE2 significantly augmented LPS-stimulated LPMC IL-10 production. However, the inhibition of IL-12 by PGE2 was only partially reversed by anti-IL-10. In a simplified model of LPS tolerance, we finally showed that monocyte-derived macrophages exhibited reduced IL-12 production after repeat LPS stimulation. In these cell cultures, indomethacin abrogated the induction of LPS desensitization. IFN-gamma and PGE2 modulate differently the LPMC responsiveness to LPS in terms of IL-12 synthesis.