Effects of Curcuma longa L., Eucommia ulmoides Oliv. and Gynostemma pentaphyllum (Thunb.) Makino on Cytokine Production in Stimulated Peripheral Blood Mononuclear Cells in Patients with Tuberculosis.

BACKGROUND: Tuberculosis (TB) infection triggers the innate and adaptive immune responses. Eucommia ulmoides Oliv., Gynostemma pentaphyllum (Thunb.) Makino, and Curcuma longa L. extracts exhibit various immunomodulatory effects. OBJECTIVE: This study aimed to determine the effects of 3 extracts used in Traditional Chinese Medicine (TCM) on cytokine production in peripheral blood mononuclear cells (PBMCs) obtained from patients with TB. DESIGN: The research team performed an in vitro study with self controls. SETTING: The study took place at the Department of Pulmonary and Critical Care Medicine, Chang Gung Memorial Hospital, Taiwan. PARTICIPANTS: 18 patients diagnosed with pulmonary TB were enrolled in the study. INTERVENTION: Purified protein derivative (PPD)-stimulated PBMCs were cultured for 48 h in the presence and absence of 0.05 or 0.1 mg/mL of herbal extracts. OUTCOME MEASURES: Cytokine levels of interferon (IFN)-γ, interleukin (IL)-10, IL-12, tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-ß1 in the culture supernatant were measured. RESULTS: C longa L., E ulmoides Oliv. and G pentaphyllum (Thunb.) Makino extracts decreased IFN-γ production in PPD-stimulated PBMCs. C longa L. extract did not exhibit a marked and consistent effect on the production of IL-10, IL-12, TNF-α and TGF-ß1. E ulmoides Oliv. extract increased the production of IL-10, TNF-α and TGF-ß1. G pentaphyllum (Thunb.) Makino extract increased the production of IL-10, IL-12, TNF-α and TGF-ß1. CONCLUSION: These results show that G pentaphyllum (Thunb.) Makino might enhance cell immunity since it increased the production of IL-12 and TNF-α with dose effect.

Percutaneous absorption of resveratrol and its oligomers to relieve psoriasiform lesions: In silico, in vitro and in vivo evaluations.

Resveratrol was shown to exert anti-inflammatory effects in experimental models of psoriasis. Several natural oligomers of resveratrol have been extracted from plants. We investigated the antipsoriatic activity of topical administration of resveratrol oligomers and explored the effect of the number of resveratrol subunits on skin absorption to establish the structure-permeation relationship (SPR). Three oligomers, ε-viniferin (dimer), ampelopsin C (trimer) and vitisin A (tetramer), extracted from Vitis thunbergii root were compared to the resveratrol glycoside polydatin. Delivery to porcine skin was assessed in vitro using the Franz cell. Keratinocytes activated with imiquimod (IMQ) were utilized to evaluate cytokine/chemokine inhibition. Topical application of resveratrol and oligomers was characterized in vivo by assessing cutaneous absorption, skin physiology, proinflammatory mediator expression, and histopathology in IMQ-treated mice. Skin deposition decreased as the molecular size and lipophilicity of the permeants increased. Resveratrol exhibited highest absorption, followed by ε-viniferin. The monomers resveratrol and polydatin exhibited higher flux across skin than the larger oligomers. In silico modeling revealed the permeants that strongly interacted with stratum corneum (SC) lipids exhibited lower transport to viable skin and the receptor compartment. In vitro, resveratrol and its derivatives had comparable ability to inhibit IMQ-induced IL-1ß, IL-6, and CXCL8 secretion in activated keratinocytes. In vivo, topically applied ε-viniferin accumulated at higher levels than resveratrol (0.067 versus 0.029 nmol/mg) in psoriasis-like mouse skin with impaired barrier capacity. Topical ε-viniferin alleviated psoriasiform symptoms and reduced IL-23 secretion (by 58% vs. 37%) more effectively than resveratrol. ε-Viniferin has potential as an anti-inflammatory agent to prevent or treat psoriasis.

Efficacy and safety of indigo naturalis ointment in Treating Atopic Dermatitis: A randomized clinical trial.

ETHNOPHARMACOLOGICAL RELEVANCE: Indigo naturalis, a herbal medicine with a history of use dating back to ancient times, may be a good alternative topical treatment for atopic dermatitis (AD). AIM OF THE STUDY: To provide empirical evidence of the efficacy and safety of Indigo naturalis ointment in treating AD. MATERIALS AND METHODS: In this randomized double-blind clinical trial, participants aged 6 to 65 years with AD affecting less than 40% of their body surface area (BSA) and an Investigator's Global Assessment (IGA) score of 2 to 4 were randomized (2:1) to receive either Lindioil ointment or a vehicle ointment twice daily for 6 weeks. The primary endpoint was the percentage change in the Eczema Area Severity Index (EASI) from baseline to week 6. Secondary endpoints were as follows: EASI improvement ≥50%, 75%, and 90%; IGA score; BSA affected by AD; pruritus severity; and Dermatology Life Quality Index. The safety assessment included adverse events (AEs), laboratory tests, and physical examinations. RESULTS: The Lindioil group (32 participants) and vehicle group (16 participants) achieved mean percentage EASI reductions of 49.9% ± 36.5% (95% CI 36.8%-63.1%) and 19.6% ± 52.2% (95% CI -8.2%-47.4%), respectively (P = 0.0235). The Lindioil group also showed greater improvement in every secondary assessment category. No significant AEs occurred. CONCLUSION: Indigo naturalis ointment is effective for treating mild to severe AD topically, and appears to be safe. This is the first clinical trial to provide evidence supporting topical indigo-based AD treatment. ClinicalTrials.gov identifier: NCT02669888.

Effect of Eucommia ulmoides Oliv., Gynostemma pentaphyllum (Thunb.) Makino, and Curcuma longa L. on Th1- and Th2-cytokine responses and human leukocyte antigen-DR expression in peripheral blood mononuclear cells of septic patients.

ETHNOPHARMACOLOGICAL RELEVANCE: Many traditional Chinese medicines (TCM), such as Eucommia ulmoides Oliv., Gynostemma pentaphyllum (Thunb.) Makino, and Curcuma longa L., have been reported to have various immune-modulatory effects. AIM OF THE STUDY: To determine the effects of extracts from these three TCM on type 1 T help (Th1)- and Th2-cytokine responses and human leukocyte antigen (HLA)-DR expression in peripheral blood mononuclear cells (PBMCs) obtained from septic patients. MATERIALS AND METHODS: Lipopolysaccharide (LPS)-stimulated PBMCs of healthy controls and septic patients were cultured for 48 hs with or without 0.05/0.1 mg/ml of TCM extract. HLA-DR expression in monocytes was detected using flow cytofluorimetry. The interferon [IFN]-γ, tumor necrosis factor [TNF]-α, interleukin (IL)- 2, IL-5, IL-10, and IL-13 levels in supernatants were measured with a human enzyme-linked immunosorbent assay. RESULTS: Treatment with either 0.05 or 0.1 mg/ml of C. longa L. extract significantly restored the percentage of HLA-DR-positive monocytes, which was decreased by LPS in control and patient groups. Treatment with 0.05 or 0.1 mg/ml E. ulmoides Oliv. and C.longa L. extract decreased IL-10 production from LPS-stimulated PBMCs of controls and patients. In patients with sepsis, C. longa L. extract decreased IL-10 production to a greater degree than did E. ulmoides Oliv extract. Although IFN-γ, TNF-α, or IL-13 productions from LPS-stimulated PBMCs were influenced by E. ulmoides Oliv., G. pentaphyllum (Thunb.) Makino, or C. longa L. in control or sepsis groups in this study, only the influence of IL-10 was consistent in both control and sepsis groups. CONCLUSIONS: By enhancing monocyte HLA-DR expression and decreasing IL-10 production, C. longa L. might help restore inflammatory responses in septic patients to eradicate pathogens.

Wild Bitter Melon Leaf Extract Inhibits Porphyromonas gingivalis-Induced Inflammation: Identification of Active Compounds through Bioassay-Guided Isolation.

Porphyromonas gingivalis has been identified as one of the major periodontal pathogens. Activity-directed fractionation and purification processes were employed to identify the anti-inflammatory active compounds using heat-killed P. gingivalis-stimulated human monocytic THP-1 cells in vitro. Five major fractions were collected from the ethanol/ethyl acetate extract of wild bitter melon (Momordica charantia Linn. var. abbreviata Ser.) leaves and evaluated for their anti-inflammatory activity against P. gingivalis. Among the test fractions, Fraction 5 effectively decreased heat-killed P. gingivalis-induced interleukin (IL)-8 and was subjected to separation and purification by using chromatographic techniques. Two cucurbitane triterpenoids were isolated from the active fraction and identified as 5ß,19-epoxycucurbita-6,23-diene-3ß,19,25-triol (1) and 3ß,7ß,25-trihydroxycucurbita-5,23-dien-19-al (2) by comparing spectral data. Treatments of both compounds in vitro potently suppressed P. gingivalis-induced IL-8, IL-6, and IL-1ß levels and the activation of mitogen-activated protein kinase (MAPK) in THP-1 cells. Both compounds effectively inhibited the mRNA levels of IL-6, tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2 in P. gingivalis-stimulated gingival tissue of mice. These findings imply that 5ß,19-epoxycucurbita-6,23-diene-3ß,19,25-triol and 3ß,7ß,25-trihydroxycucurbita-5,23-dien-19-al could be used for the development of novel therapeutic approaches against P. gingivalis infections.

Efficacy and safety of Indigo naturalis extract in oil (Lindioil) in treating nail psoriasis: a randomized, observer-blind, vehicle-controlled trial.

Treating nail psoriasis is notoriously difficult and lacks standardized therapeutic regimens. Indigo naturalis has been demonstrated to be safe and effective in treating skin psoriasis. This trial was conducted to evaluate the efficacy and safety of refined indigo naturalis extract in oil (Lindioil) in treating nail psoriasis. Thirty-one outpatients with symmetrically comparable psoriatic nails were enrolled. Lindioil (experimental group) or olive oil (control group) was applied topically to the same subjects' two bilaterally symmetrical psoriatic nails twice daily for the first 12 weeks and then subjects applied Lindioil to both hands for 12 additional weeks. Outcomes were measured using Nail Psoriasis Severity Index (NAPSI) for five nails on one hand and for the single most severely affected nail from either hand. The results show a reduction of NAPSI scores for the 12-week treatment for the Lindioil group (49.8% for one hand and 59.3% for single nail) was superior to the reduction in the scores for the control group (22.9%, 16.3%, respectively). There were no adverse events during the 24 weeks of treatment. This trial demonstrates that Lindioil is a novel, safe and effective therapy for treating nail psoriasis.

Successful treatment of pediatric nail psoriasis with periodic pustular eruption using topical indigo naturalis oil extract.

Psoriasis of the nail greatly affects quality of life because of the difficulty in achieving long-lasting remission. Pustular psoriasis of the nail apparatus is characterized by the formation of sterile pustules, starting on one or two fingers or less often on the toes, and spontaneous improvement has rarely been observed. This case presents a girl with refractory nail psoriasis accompanied by periodic pustular eruption that responded well to topical treatment with indigo naturalis oil extract drops, achieving a remission of longer than 1 year.

Indigo naturalis upregulates claudin-1 expression in human keratinocytes and psoriatic lesions.

ETHNOPHARMACOLOGICAL RELEVANCE: Indigo naturalis is used in traditional Chinese medicine to treat various dermatoses. Our previous clinical studies showed that indigo naturalis is an effective treatment for psoriasis. Herein, the capabilities of indigo naturalis extract and its derivatives to increase claudin-1 expression and tight junction (TJ) function in human keratinocytes and psoriatic lesions were further studied. MATERIALS AND METHODS: Claudin-1 expression in psoriatic plaques with or without indigo naturalis treatment was analyzed by immunohistochemical methods. In primary human keratinocytes, the expression of claudin-1 was analyzed by fluorescent immunostaining, a real-time RT-PCR, and Western blot analysis. The effect of indigo naturalis on TJs was evaluated by measuring the transepithelial electrical resistance (TEER) and paracellular tracer flux. RESULTS: The indigo naturalis extract upregulated mRNA and protein expressions of claudin-1 and function of TJs in primary human keratinocytes in concentration-dependent manners. Its main components, indirubin, indigo, and tryptanthrin, exerted synergistic effects on upregulating TJ functions in primary human keratinocytes. In addition, indigo naturalis increased the activity of protein kinase C (PKC), and a known potent PKC inhibitor, Ro318220, attenuated the indigo naturalis-induced claudin-1 expression. Significantly, restoration of claudin-1 was observed in healed psoriatic lesions after indigo naturalis treatment. CONCLUSIONS: Indigo naturalis upregulates claudin-1 expression and restores TJ function in keratinocytes. Our data also suggest that indirubin, indigo, and tryptanthrin have a synergistic effect on TJ function.

Indirubin, an acting component of indigo naturalis, inhibits EGFR activation and EGF-induced CDC25B gene expression in epidermal keratinocytes.

BACKGROUND: Topical indigo naturalis ointment is clinically proved to be an effective therapy for plaque-type psoriasis. Indirubin, as the active component of indigo naturalis, inhibits cell proliferation of epidermal keratinocytes. However, the detailed underlying mechanism is not fully understood. OBJECTIVE: To further investigate the anti-proliferating effects of indigo naturalis and indirubin on epidermal keratinocytes. METHODS: The decreased expression of CDC25B in indigo naturalis- or indirubin-treated epidermal keratinocytes, as revealed by cDNA microarray analysis, was studied. The CDC25B expression was examined under different serum concentrations and compared between primary and immortalized keratinocytes. The activation of EGFR and the effect of EGF on the cell proliferation and CDC25B expression were also investigated in epidermal keratinocytes. RT/real-time PCR and western blot method were used to analyze the CDC25B expression at the mRNA and protein levels, respectively. RESULTS: Indigo naturalis and indirubin were confirmed to down-regulate CDC25B expression significantly at both the mRNA and protein levels. The growth-dependent expression of CDC25B was demonstrated by the increased expression in serum-stimulated and immortalized keratinocytes. The activation of EGF receptor, known to be highly expressed in psoriatic lesions, was inhibited by indigo naturalis or indirubin. The cell proliferation and CDC25B expression of epidermal keratinocytes were induced by EGF alone and confirmed to be inhibited by indigo naturalis or indirubin. CONCLUSION: Except being a common therapeutic target in various cancers, CDC25B also plays an important role in the hyper-proliferation of epidermal keratinocytes which can be suppressed by anti-psoriatic drug indigo naturalis and its component, indirubin.

Protective effect of indigo naturalis extract against oxidative stress in cultured human keratinocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Indigo naturalis is used in traditional Chinese medicine to treat various skin disorders. AIM OF THE STUDY: The aims were to explore the effect of indigo naturalis on suppressing oxidative stress and protein modifications by hydrogen peroxide (H(2)O(2)) and 4-hydroxy-2-nonenal (HNE), a lipid peroxidation product, in cultured primary human keratinocytes. MATERIALS AND METHODS: Indigo naturalis extract at a dose that did not cause cytotoxicity was added to cultured keratinocytes in the absence or the presence of H(2)O(2) or HNE. The degree of cytotoxicity, levels of reactive oxygen species (ROS), and amount of protein carbonyl groups were evaluated. RESULTS: Indigo naturalis extract at the concentration of 10µg/ml had no protective effect against H(2)O(2) or HNE-induced cytotoxicity, but decreased intracellular levels of ROS after H(2)O(2) treatment and suppressed the increase of protein carbonyl groups induced by HNE. CONCLUSION: Indigo naturalis possesses an inhibitory effect on formation of intracellular ROS induced by exogenous ROS and protein modification induced by HNE in human keratinocytes, which is relevant to the alleviation of inflammatory skin diseases.

Treatment of psoriatic nails with indigo naturalis oil extract: a non-controlled pilot study.

BACKGROUND: In the treatment of nail psoriasis, standardized therapeutic regimens are currently lacking. OBJECTIVE: To evaluate the therapeutic efficacy of indigo naturalis oil extract in patients with nail psoriasis. METHODS: Patients with nail psoriasis applied indigo naturalis oil extract on affected nails twice daily for 24 weeks. Efficacy was evaluated using the Nail Psoriasis Severity Index (NAPSI) and modified target NAPSI for the single most severely affected nail. RESULTS: Twenty-eight out of 32 patients completed the study. The mean NAPSI was 36.1 ± 14.7 at baseline and decreased to 14.9 ± 11.1 at week 24 while the mean modified target NAPSI was 11.7 ± 3.9 at baseline and decreased to 3.6 ± 3.2 at week 24. CONCLUSIONS: Indigo naturalis oil extract appeared to improve nail psoriasis. Although preliminary, these results indicate that it could provide a novel therapeutic option for nail psoriasis, a disease notoriously difficult to treat.

Quality of life in patients with psoriasis in northern Taiwan.

BACKGROUND: Psoriasis has a significant negative impact on quality of life. The aim of this study was to identify factors associated with the quality of life of patients with psoriasis in Taiwan. METHODS: A retrospective study analyzing data from psoriasis patients who visited the outpatient clinics in the Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital at Taipei, Taoyuan and Keelung from July 2009 to January 2010 was performed. RESULTS: A total of 480 patients who had completed the assessment of disease severity and the dermatology life quality index (DLQI) questionnaire were analyzed. Of these patients, 67.5% were men. The mean score on the DLQI was 9.16 ± 6.3 and 67% of all patients reported a moderate to extremely large impact on their quality of life (DLQI > 6). A higher psoriasis area and severity index (PASI), younger age and initial lesions on the nails significantly negatively impacted patients' quality of life. Smoking, alcohol intake and gender were also weakly correlated. CONCLUSION: The clinical severity, age and site of initial lesions are associated with negative impacts on the quality of life of patients with psoriasis. These findings provide significant new insights into factors that affect the life quality of patients with psoriasis in Taiwan.

Inhibitory effect of indigo naturalis on tumor necrosis factor-α-induced vascular cell adhesion molecule-1 expression in human umbilical vein endothelial cells.

The use of indigo naturalis to treat psoriasis has proved effective in our previous clinical studies. The present study was designed to examine the anti-inflammatory effect of indigo naturalis in primary cultured human umbilical vein endothelial cells (HUVECs). Pretreatment of cells with indigo naturalis extract attenuated TNF-α-induced increase in Jurkat T cell adhesion to HUVECs as well as decreased the protein and messenger (m)RNA expression levels of vascular cell adhesion molecule-1 (VCAM-1) on HUVECs. Indigo naturalis extract also inhibited the protein expression of activator protein-1 (AP-1)/c-Jun, a critical transcription factor for the activation of VCAM-1 gene expression. Since the reduction of lymphocyte adhesion to vascular cells by indigo naturalis extract could subsequently reduce the inflammatory reactions caused by lymphocyte infiltration in the epidermal layer and help to improve psoriasis, this study provides a potential mechanism for the anti-inflammatory therapeutic effect of indigo naturalis extract in psoriasis.

Anti-inflammatory effects of the extract of indigo naturalis in human neutrophils.

ETHNOPHARMACOLOGICAL RELEVANCE: Indigo naturalis is used by traditional Chinese medicine to treat various inflammatory diseases. AIM OF THE STUDY: Topical indigo naturalis ointment showed efficacy in treating psoriasis in our previous clinical studies. In this study, we investigated the anti-inflammatory effects of the extract of indigo naturalis (QD) and its main components indirubin, indigo, and tryptanthrin in human neutrophils. MATERIALS AND METHODS: Superoxide anion (O2(.-)) generation and elastase release were measured by spectrophotometry. Some important signals including mitogen-activated protein kinase (MAPK), cAMP, and calcium were studied by Western blot analysis, an enzyme immunoassay, and spectrofluorometry. RESULTS: QD significantly inhibited O2(.-) generation and elastase release in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils in a concentration-dependent fashion, while neither indirubin, indigo, nor tryptanthrin produced a comparable result. QD attenuated the FMLP-induced phosphorylation of extracellular regulated kinase, p38 MAPK, and c-Jun N-terminal kinase. Furthermore, QD inhibited calcium mobilization caused by FMLP. However, QD did not affect cellular cAMP levels. On the other hand, neither indirubin, indigo, nor tryptanthrin produced similar changes in human neutrophils. CONCLUSIONS: Taken collectively, these findings indicate that QD, but not indirubin, indigo, or tryptanthrin, inhibited O2(.-) generation and elastase release in FMLP-induced human neutrophils, which was at least partially mediated by the inhibition of MAPK activation and regulation of calcium mobilization.

Anti-psoriatic effects of indigo naturalis on the proliferation and differentiation of keratinocytes with indirubin as the active component.

BACKGROUND: Indigo naturalis has shown efficacy in treating psoriasis in our previous clinical studies. OBJECTIVES: To investigate the potential effect of indigo naturalis on regulating keratinocyte proliferation and differentiation. METHODS: Skin samples from six patients were analyzed for proliferating cell nuclear antigen (PCNA) and involucrin expression by immunohistochemical staining. In addition, indigo naturalis extracts from 10 to 500 microg/ml were added to cultured keratinocytes and cell viability determined. Real-time RT-PCR, Western blotting analysis and indirect immunofluorescent labeling were used to investigate the messenger (m)RNA and protein expressions of PCNA and involucrin. Finally, high-performance liquid chromatography (HPLC) was used to identify major components of indigo naturalis and their in vitro effects compared. RESULTS: Immunohistochemical results demonstrated decreased PCNA and increased involucrin in psoriatic lesions after indigo naturalis treatment. Cultured keratinocytes decreased after indigo naturalis treatment, while G(0)/G(1) arrest was observed to dose-dependently increase. Staining revealed decreased PCNA-stained nuclei and increased cytosolic involucrin in treated keratinocytes. Decreased PCNA and increased involucrin at both the mRNA and protein levels were confirmed. Both major components, indirubin and indigo, could cause G(0)/G(1) phase arrest; however, only indirubin modulated the expressions of PCNA and involucrin similar to indigo naturalis. CONCLUSIONS: Together, these findings indicate that the anti-psoriatic effects of indigo naturalis are mediated, at least in part, by modulating the proliferation and differentiation of keratinocytes, with indirubin as the major active component.

Biological and pharmacological activities of squalene and related compounds: potential uses in cosmetic dermatology.

Squalene is a triterpene that is an intermediate in the cholesterol biosynthesis pathway. It was so named because of its occurrence in shark liver oil, which contains large quantities and is considered its richest source. However, it is widely distributed in nature, with reasonable amounts found in olive oil, palm oil, wheat-germ oil, amaranth oil, and rice bran oil. Squalene, the main component of skin surface polyunsaturated lipids, shows some advantages for the skin as an emollient and antioxidant, and for hydration and its antitumor activities. It is also used as a material in topically applied vehicles such as lipid emulsions and nanostructured lipid carriers (NLCs). Substances related to squalene, including beta-carotene, coenzyme Q10 (ubiquinone) and vitamins A, E, and K, are also included in this review article to introduce their benefits to skin physiology. We summarize investigations performed in previous reports from both in vitro and in vivo models.

Clinical assessment of patients with recalcitrant psoriasis in a randomized, observer-blind, vehicle-controlled trial using indigo naturalis.

OBJECTIVE: To evaluate the efficacy and safety of treatment with indigo naturalis in patients with recalcitrant plaque-type psoriasis. DESIGN: Randomized, observer-blind, vehicle-controlled, intrapatient comparison study. SETTING: Ambulatory department of a hospital. PARTICIPANTS: Forty-two outpatients with chronic plaque psoriasis were enrolled in the study from May 1, 2004, to April 30, 2005. INTERVENTION: The patients applied either indigo naturalis ointment or vehicle ointment topically to each of 2 bilaterally symmetrical psoriatic plaque lesions for 12 weeks (depending on the date of enrollment in the study). MAIN OUTCOME MEASURES: The outcomes were assessed using the following criteria: the sum of erythema, scaling, and induration scores and the clearing percentage of the target plaque lesion assessed by 2 blinded observers. RESULTS: Significant reductions in the sum of scaling, erythema, and induration scores (P < .001) (mean score, 6.3 after indigo naturalis treatment vs 12.8 in control subjects) and plaque area percentage (P < .001) (mean percentage, 38.5% after indigo naturalis treatment vs 90% in controls) were achieved with topical application of indigo naturalis ointment. Approximately 31 of 42 patients (74%) experienced clearance or near clearance of their psoriasis in the indigo ointment-treated lesion. CONCLUSION: Topical indigo naturalis ointment was a novel, safe, and effective therapy for plaque-type psoriasis.