侵袭性牙周炎龈沟液中白介素-8 的检测

目的：检测侵袭性牙周炎（AgP）龈沟液中白细胞介素（IL-8）的总量和浓度并探讨其与牙周临床指标的关系。方法：采用常规滤纸条法收集侵袭性牙周炎实验组患牙（T1）和健康牙（T2）各位点及正常对照组（C）各位点的龈沟液（GCF）样本,用ELISA法检测各样本中IL-8的总量和浓度。结果：3组受检牙龈沟液中IL-8的总量和浓度不同。侵袭性牙周炎患牙组GCF中IL-8总量高于健康牙位组（P〈0．05）及正常对照组;而3组中IL-8浓度差异也有显著性,侵袭性牙周炎患牙组GCF中IL-8的浓度高于健康牙位组（P〈0．05）和正常对照组;虽然GCF中IL-8浓度与牙周探诊深度（PD）、牙龈出血指数（BOP）、附着丧失（AL）无相关关系;但IL-8总量与以上牙周临床指标相关。结论：在侵袭性牙周炎患者龈沟液中IL-8是参与牙周炎症反应的重要调节因子。     

侵袭性牙周炎龈沟液中硫化物的检测

目的:检测侵袭性牙周炎(AgP)龈沟液中硫化物浓度水平变化并探讨其与牙周临床指标的关系。方法:采用金刚牙周诊断仪进行龈沟液硫化物和牙周临床指标测定。选定实验组(T)16例侵袭性牙周炎病人,56个牙,共336个位点。其中健康牙(T1)16个,位点96个;炎症牙(T2)40个,位点240。对照组(C):全身、牙周健康者10例,20个牙,共120个位点。测定所选位点龈沟液(gingival crevicu lar flu id,GCF)中硫化物水平(su lcus su lph ide level,SUL),牙周袋探诊深度(pocket prob ing depth,PPD),牙周临床附着丧失水平(c lin i-cal attachm ent level,CAL),龈沟出血指数(su lcus b leed ing index,SB I)。结果:①3组受检牙龈沟液中硫化物浓度不同。侵袭性牙周炎炎症牙位组GCF中硫化物浓度(0.2210±0.0415)×10-6mol/L高于健康牙位组(P<0.05)、正常对照组;健康牙位组GCF中硫化物浓度(0.1025±0.0198)×10-6mol/L高于正常对照组(0.0523±0.0044)×10-6mol/L。②经相关性分析,侵袭性牙周炎炎症牙位组(T1)GCF中硫化物的浓度均值与PD、SB I和CAL均呈正相关关系,健康牙位组(T1)、正常对照组(C)GCF中硫化物浓度均值与PD、SB I及CAL无相关性。结论:侵袭性牙周炎病人龈沟液中的硫化物是参与牙周炎症反应的重要调节因子,其水平变化可反映牙周组织的炎症状态。     

慢性牙周炎龈沟液中硫离子水平与临床相关性研究

目的:分析慢性牙周炎(CP)患者龈沟液中硫离子(su lfides)水平的变化与临床牙周指数的相关关系及其对诊断预后的意义。方法:采用金刚牙周诊断仪进行龈沟液硫离子和牙周临床指标测定。选定实验组(T):36例慢性牙周炎患者,57颗牙位,共342个位点。其中健康牙位(T1)21颗,位点126个;炎症牙位(T2)36颗,位点216。对照组(C):全身及牙周健康者8例,16颗牙位,共96个位点。测定所选位点龈沟液(GCF)中硫化物水平(su lcussu lph ide level,SUL),牙周袋探诊深度(prob ing depth,PD),牙周临床附着丧失水平(c lin ical attachm ent level,CAL),龈沟出血指数(su lcus b leed ing index,SB I)。所有统计结果均采用SPSS11.0进行统计学分析。结果:1)牙周健康对照组(C)GCF中硫离子SUL的浓度均值为(0.0648±0.0169)pg/mL,明显低于慢性牙周炎炎症牙位组(T2)(0.3249±0.0489)pg/mL及慢性牙周炎健康牙位组(T1)(0.1160±0.0271)pg/mL;慢性牙周炎炎症牙位组(T2)GCF中硫离子(SUL)的浓度均值均高于正常对照组及慢性牙周炎健康牙位组(T1)。2)经相关性分析,慢性牙周炎炎症牙位组(T1)GCF中SUL的浓度均值与PD、SB I和CAL均呈正相关关系。而慢性牙周炎健康牙位组(T1)及正常对照组(C)GCF中SUL的浓度均值与PD、SB I及CAL间无相关性。结论:慢性牙周炎(CP)炎症牙位组龈沟液中硫离子(SUL)的浓度均值与牙周临床指标之间具有相关关系,其水平的高低变化可客观反映牙周组织的炎症状态。     

牙龈卟啉单胞菌与龈沟液中白细胞介素8关系的研究

目的:探讨龈沟液(GCF)中白细胞介素8(IL -8)在慢性牙周炎病程中的变化、IL- 8与牙周临床检测指标以及同龈下菌斑中细菌含量的相关关系。方法: 5名非牙周炎患者的10颗牙周健康牙和30名慢性牙周炎(CP)患者的70颗牙(其中10颗为健康牙, 60颗为牙周炎患牙)纳入本研究。采集观察牙的GCF样本、龈下菌斑样本,同时记录所有牙的牙龈指数(GI)、牙周袋探诊深度(PPD)、临床附着丧失水平(CAL)。用ELISA法检测样本中IL -8的水平,用厌氧菌培养技术培养菌斑标本,用PCR法检测菌斑中的牙龈卟啉单胞菌(Pg),结果:慢性牙周炎组的GCF中IL -8的总量高于临床牙周健康组,而IL -8的质量浓度在各组中无差异。GCF中IL- 8浓度与GI、PPD、CAL无相关关系,而IL -8的总量与GI、PPD、CAL呈正相关关系。龈下菌斑中Pg的检出量与GCF中IL- 8的含量间未发现有相关关系。结论:慢性牙周炎患者龈沟液中IL- -8量增加与龈沟液分泌增加有关而与牙龈卟啉单胞菌检出CFU无关。     

慢性牙周炎患者IL-8和弹性蛋白酶水平的研究

目的：探讨IL-8、弹性蛋白酶在慢性牙周炎患者龈沟液中的表达。方法：采用ELISA检测慢性牙周炎患者30名和健康对照组20名龈沟液中IL-8的总量和浓度，采用底物法检测相同人群龈沟液中细胞外弹性蛋白酶、总弹性蛋白酶的浓度和总量。采用SPSS11．0软件分析其差异性及相关性。结果：慢性牙周炎患者龈沟液中的IL-8浓度明显低于健康对照组（P〈0．01），而IL-8总量略高于健康对照组但无显著性差异（P〉0．05）；IL-8和细胞外弹性蛋白酶的浓度和总量在重度牙周炎组明显高于轻、中度牙周炎组（P〈0．01）。结论：不同浓度的IL-8和弹性蛋白酶参与了慢性牙周炎的炎症过程，二者的协同作用将加重慢性牙周炎的病情。     

维族绝经妇女龈沟液IL-6、血清雌二醇水平与牙周炎的关系

目的:探讨维吾尔族绝经早期的牙周炎患者龈沟液IL-6及血清雌二醇(E2)水平与牙周炎的关系。方法:共79例绝经年限均≤5a的妇女纳入本研究。采集30颗牙周健康牙和49颗牙周炎患牙的龈沟液(GCF),记录牙周临床观察指标。采集慢性牙周炎患者的血样本。应用放射免疫分析法检测GCF中IL-6和血清中E2的浓度。结果:牙周健康组GCF中IL-6浓度为(1088.10±102.33)pg/ml;慢性牙周炎组GCF中IL-6浓度明显高于牙周健康牙组(P<0.005);慢性牙周炎患牙GCF中IL-6浓度与GI、PPD、CAL均呈正相关(r=0.564,P<0.005;r=0.335,P<0.05;r=0.324,P<0.05)。血清E2≤30pg/ml组的妇女牙周炎患牙牙周临床指标、GCFIL-6浓度与E2>30pg/ml组无显著差异(P>0.05)。结论:健康牙GCF中含有微量IL-6。牙周炎患牙GCF中IL-6的水平反映了牙周炎症的严重程度,可以尝试作为判断维族妇女牙周病变程度的一个指标。绝经早期患慢性牙周炎的维族妇女,血清E2水平与牙周炎患牙的病变程度及GCF中IL-6的水平无关。     

盐酸米诺四环素软膏对慢性牙周炎龈沟液中硫化物的影响

目的：分析慢性牙周炎病人局部应用盐酸米诺四环素（minocycline HCl，MINO）后，不同牙周状态下龈沟液（GCF）中硫化物（suleus sulphide level，SUL）浓度变化及其与临床指标的关系。方法：采用金刚牙周探测仪对盐酸米诺四环素治疗前后龈沟液中硫化物浓度及其临床指标进行测定。随机单盲法选择慢性牙周炎病人21例，61个患牙。用药组（T）34个患牙，204个位点，基础治疗后将盐酸米诺四环素软膏置牙周袋内；对照组（C）卯个患牙，162个位点，以单纯基础治疗为主。于基线前两周完成全口龈上洁治、口腔卫生宣教。基线时测定相应位点龈沟液中硫化物浓度，龈沟出血指数（SBI），牙周袋探诊深度（PPD），牙周临床附着丧失水平（CAL）；然后行龈下刮治术，用药组龈沟内放药，对照侧不放药。第2周各项指标检查同上。结果：用药组（T）龈沟液中硫化物浓度与其基线时和对照组相比明显下降（P〈0．05），其临床指标用药组（T）与对照组（C）相比改善明显（P〈0．05），龈沟液中硫化物的浓度与临床指标间具有相关性。结论：盐酸米诺四环素软膏（MINO）辅助治疗牙周炎，能有效降低龈沟液中硫化物浓度，改善牙周组织状况；椅旁龈沟液硫化物浓度变化可间接反映对牙周袋内细菌的杀灭、抑制作用。     

广泛型侵袭性牙周炎患者龈沟液中白介素-17的检测

目的 比较广泛型侵袭性牙周炎患者与健康人龈沟液中白介素-17(IL-17)的表达水平及与临床指标的关系。方法 选择广泛型侵袭性牙周炎患者18例和健康人16例,共68颗牙,记录临床牙周检查指标,用滤纸条法收集龈沟液。采用抗体夹心ELISA法测定广泛型侵袭性牙周炎患者治疗前、治疗后3个月及健康对照者龈沟液中IL-17总量和浓度。结果 广泛型侵袭性牙周炎患牙治疗前龈沟液中IL-17的总量和浓度高于牙周健康牙,基础治疗3个月后广泛型侵袭性牙周炎患牙龈沟液中IL-17的总量和浓度较治疗前下降。广泛型侵袭性牙周炎患牙龈沟液中IL-17浓度与探诊深度、附着丧失和出血指数呈正相关关系,而IL-17总量仅与探诊深度呈正相关关系。结论 龈沟液中IL-17浓度可能与广泛型侵袭性牙周炎牙周破坏及炎症的严重程度相关。?     

正常人、牙龈炎和牙周炎患者龈沟液内一氧化氮含量的检测

目的：检测正常人和牙周病患者龈沟液中NO含量,探讨NO在牙周病发病过程中的作用。方法：选择牙周健康组20例,牙龈炎组22例,慢性牙周炎组32例,分别采集龈沟液标本,免疫荧光法检测龈沟液内NO的含量。结果：慢性牙周炎患者和牙龈炎患者龈沟液内NO含量与牙周健康组相比均有高度显著性差异（P〈0．01）,慢性牙周炎患者龈沟液内NO含量与牙龈炎组相比有高度显著性差异（P〈0．01）。结论：牙周健康者、牙龈炎患者、慢性牙周炎患者龈沟液中能检测出NO的存在,NO参与了慢性牙周炎的发展过程,龈沟液内NO含量与慢性牙周炎炎症程度密切相关。     

慢性牙周炎患者唾液中miR-146a的表达及其与龈沟炎症、MMP-8/TIMP-1水平的关系

目的： 探讨慢性牙周炎患者唾液中miR-146a的表达及其与龈沟炎症、基质金属蛋白酶8（MMP-8）、基质金属蛋白酶抑制剂1（TIMP-1）水平的关系。方法： 选择2015年3月—2017年1月间收治的慢性牙周炎患者68例作为慢性牙周炎组,同期在本院进行体检的健康志愿者50例作为正常对照组。检测2组研究对象唾液中miR-146a的表达量,龈沟液中炎症因子、MMP-8/TIMP-1的水平及牙周临床症状指标。采用SPSS24.软件中的Pearson检验评估慢性牙周炎患者唾液中miR-146a的表达量与病情严重程度的相关关系。结果： 慢性牙周炎患者唾液中miR-146a的表达量显著高于正常对照组（P<0.05）,龈沟液中炎症因子（IL-1β、IL-6、IL-35、TNF-α）水平、牙周临床症状指标（PD、AL、PLI、BI）以及龈沟液中MMP-8、TIMP-1的水平显著高于正常对照组（P<0.05）。Pearson检验发现,慢性牙周炎患者唾液中miR-146a表达量与龈沟炎症程度、牙周临床症状严重程度及MMP-8/TIMP-1水平呈正相关。结论： 慢性牙周炎患者唾液中miR-146a表达量异常增高,且与龈沟炎症程度、牙周损伤程度一致。     

慢性牙周炎患者牙周治疗前后龈沟液中白介素-10水平的变化

目的探讨慢性牙周炎患者牙周治疗前后龈沟液中抗炎性细胞因子白介素(IL)-10水平的变化。方法采集12例慢性牙周炎患者的12个健康牙位和36个炎症牙位于治疗前及治疗后6、122、4周的龈沟液,用酶联免疫吸附分析法(ELISA)检测龈沟液中IL-10的浓度。另外,分别记录治疗前、后的探诊深度(PD)、临床附着丧失(CAL)、牙龈指数(GI)和菌斑指数(PlI)。结果IL-10浓度在健康牙位明显高于炎症牙位(P<0.01),且于牙周治疗后明显升高。IL-10浓度与探诊深度(PD)、临床附着丧失(CAL)呈负相关(P<0.05)。结论IL-10浓度与牙周组织破坏程度呈负相关,在牙周炎中起抗炎作用。     

种植体周龈沟液IL-17A和IL-35水平检测及意义

目的分析种植体组不同时期龈沟液(peri-implant sulcular fluid,PISF)中(interleukin,IL)-17A和IL-35水平改变,探讨其在种植体周围炎或黏膜炎发展过程中的作用和相互关系。方法收集2008—2013年在中山大学孙逸仙纪念医院口腔科行种植修复患者共40例为研究对象,分为种植体健康组20例和种植体炎症组20例,将种植体炎症组经过治疗后作为对照组。在种植体中有单冠修复、联冠修复。采集不同患者龈沟液并定量,对标本采用双抗体夹心酶联免疫吸附方法 (Enzyme Linked Immunosorbent Assay,ELISA)测定各组龈沟液中IL-17A和IL-35表达水平。并分别记录健康组、种植体炎症组、治疗后种植体炎症组探诊出血指数,改良出血指数,牙龈指数,并行统计学分析。结果种植体炎症组IL-17A,IL-35水平比健康组IL-17A,IL-35水平高(P<0.05),炎症组IL-17A水平治疗前比治疗后高(P<0.05),炎症组IL-35水平治疗后比治疗前升高(P<0.05),Pearson相关检验示,IL-35和IL-17A表达成负相关关系(P<0.05)。结论 IL-35和IL-17A的表达与种植体黏膜炎或者周围炎的发生发展密切,IL-17A和IL-35是辅助种植体周围炎诊治和评估抗炎疗效的潜在分子生物学指标。     

牙周炎患者血清、龈沟中的IL-8检测及其局部病变组织的免疫组织化学研究

目的 :探讨龈沟液中白细胞介素 8(IL - 8)的来源及IL - 8与成人牙周炎的关系。方法 :用ELISA法检测了30份血清样本 (13例牙周健康者 ,17例成人牙周炎患者血清 )和 2 7份龈沟液样本 (9例牙周健康者 ,18例成人牙周炎患者 )中IL - 8的水平。用免疫组织化学的方法检查了IL - 8分泌细胞在牙龈组织中的分布 (7例健康牙龈组织 ,18例成人牙周炎牙龈组织 )。结果 :成人牙周炎患者中血清IL - 8的检出率 (4 7% ,8 18)显著高于健康人血清中IL- 8的检出率 (7.7% ,1 13) ,P <0 .0 5 ;患者血清与患者龈沟液 ,健康者血清与健康者龈沟液之间 ,血清中IL - 8的含量均显著低于龈沟液 (P <0 .0 1)。IL - 8阳性细胞分布于牙龈的口腔龈上皮、结缔组织及沟内的上皮中 ;且结缔组织及沟内上皮中的IL - 8阳性细胞数量 ,成人牙周炎组显著高于健康对照组。结论 :龈沟液中的IL - 8主要来源于局部牙龈组织。IL - 8参与了牙周炎的病理过程。     

白细胞介素-21在不同牙周状态龈沟液中的表达及相关性分析

目的检测白细胞介素(Interleukin,IL)-21在不同牙周状态下龈沟液中的表达,探讨IL-21在牙周免疫中的可能作用。方法本研究共纳入47例患者,分为健康对照组(15例),轻度牙周炎组(10例),中重度牙周炎组(22例);记录患者一般信息、用Florida牙周探针检查牙周袋探诊深度(PD)和临床附着丧失(CAL)情况;收集牙周治疗前的龈沟液样本,采用酶联免疫吸附法(ELISA)测定不同牙周条件下龈沟液中IL-21的水平;并进行Pearson秩相关检验统计IL-21的表达与相关临床指标的关系。结果 3组龈沟液中IL-21平均浓度分别为(107.20±2.54)、(218.90±5.11)、(367.80±7.27)pg/m L,轻度牙周炎组及中重度牙周炎组龈沟液中IL-21水平显著高于对照组(P<0.05),其中以中、重度牙周炎组增高最为显著;中、重度牙周炎组龈沟液中IL-21水平分别与PD、CAL呈正相关关系(P<0.001)。结论 IL-21在牙周疾病中有较高的表达,尤其是以中重度牙周炎患者最为显著;由此推测IL-21在牙周疾病的发生发展中占有重要地位,与牙周组织的破坏有密切关系。     

Gingival crevicular fluid glycosaminoglycan levels in patients with chronic adult periodontitis

Abstract This study investigated levels of hyaluronan and chondroitin-4-sulphate in the crevicular fluid of patients with chronic adult periodontitis at diseased and healthy sites before and after treatment. The relationship between clinical diagnostic parameters and levels of glycosaminoglycans in gingival crevicular fluid were also analysed. Within each patient. 4 sites either mesial or distal and on single rooted teeth were classified as diseased or healthy using a modified gingival index, pocket depth and attachment loss. Crevicular fluid was collected from each site using glass micropipettes and analysed for glycosaminoglycan content by cellulose acetate electrophoresis. Significantly higher levels of chondroitin-4-sulphate were detected at diseased sites prior to treatment correlating with increased pocket depth or attachment levels. Following a period of treatment consisting of oral hygiene instruction and root planing, the patients were reassessed for their response to treatment by measuring the modified gingival index, pocket depth, attachment loss and levels of glycosaminoglycans. Analysis of glycosaminoglycan levels at diseased sites that demonstrated a poor response to treatment also demonstrated significantly higher levels of chondroitin-4-sulphate than those sites that responded well to treatment. Hyaluronan levels were less significantly associated with clinically succesful treatment. This study confirmed the use of the sulphated glycosaminoglycan chondroitin-4–sulphate as a potential diagnostic aid of periodontal tissue destruction; however, further longitudinal studies are required to assess their performance.     

Crevicular fluid endothelin-1 levels in periodontal health and disease

Background and Objective:  Endothelin-1 is a 21-amino-acid peptide with multifunctional regulation. Initial research indicated that endothelin-1 levels in the gingival crevicular fluid from patients with chronic periodontitis were higher than those in the gingival crevicular fluid from healthy subjects. The aim of the present study was to assess the relationship between the clinical parameters and the concentrations of endothelin-1 within the gingival crevicular fluid from inflamed gingiva and periodontitis sites and, subsequently, after the treatment of periodontitis sites.
Material and Methods:  A total of 60 subjects were divided into three groups – healthy (group I), gingivitis (group II) and chronic periodontitis (group III) – based on gingival index, pocket probing depth and clinical attachment loss. A fourth group consisted of 20 subjects from group III, 6–8 wk after treatment (i.e. scaling and root planing). Gingival crevicular fluid samples collected from each patient were quantified for endothelin-1 using an enzymatic immunometric assay.
Results:  Endothelin-1 was not detected in any sample from any of the study groups.
Conclusion:  The results showed that all the gingival crevicular fluid samples were negative for the endothelin-1 molecule. Therefore, endothelin-1 cannot be considered as a potential biomarker of periodontal disease progression.     

Interleukin-4, a T-helper 2 cell cytokine, is associated with the remission of periodontal disease

Background and Objectives: Interleukin‐4 (IL‐4), secreted mainly by T‐helper 2 cells, is a key cytokine for the growth and proliferation of B lymphocytes. Previous studies have proved that IL‐4 has an anti‐inflammatory effect owing to its efficient inhibition of the production of proinflammatory cytokines such as tumour necrosis factor‐α (TNF‐α), IL‐1α, IL‐1β, IL‐6 and IL‐8 by monocytes/macrophages. The aim of the present study was to assess the relation between clinical parameters and concentrations of IL‐4 within gingival crevicular fluid from inflamed gingiva and periodontitis sites and, subsequently, after treatment of the periodontitis sites. Material and Methods: A total of 60 subjects were divided into three groups based on gingival index (GI), pocket probing depth and clinical attachment loss (CAL): healthy (group 1), gingivitis (group 2) and chronic periodontitis (group 3). A fourth group (group 4) consisted of 20 subjects from group 3, 6–8 weeks after treatment (i.e. scaling and root planing). Gingival crevicular fluid samples collected from each patient were quantified for IL‐4 using the enzymatic immunometric assay. Results: The highest mean concentration of IL‐4 was obtained for group 1 (99.39 ± 49.33 pg/mL) and the lowest mean concentration of IL‐4 was obtained for group 3 (15.78 ± 21.92 pg/mL). The mean IL‐4 concentrations for group 2 (64.34 ± 39.56 pg/mL) and group 4 (68.92 ± 42.85 pg/mL) were intermediate between the levels in healthy subjects and periodontitis subjects. Conclusion: The mean concentration of IL‐4 decreased from periodontal health to disease. Thus, we suggest that type 2 helper T cell cytokine, as represented by IL‐4, was associated with the remission or improvement of periodontal disease.     

Relationship of changes in interleukin-8 levels and granulocyte elastase activity in gingival crevicular fluid to subgingival periodontopathogens following non-surgical periodontal therapy in subjects with chronic periodontitis

OBJECTIVES: To determine the effect of scaling and root planing (SRP) on the interrelations of subgingival periodontopathogens and both interleukin-8 (IL-8) and granulocyte elastase activity in gingival crevicular fluid (GCF), and to assess their relations to the short-term treatment response in management of chronic periodontitis. MATERIAL AND METHODS: GCF and subgingival plaque were collected from 16 subjects with untreated chronic periodontitis at baseline and 4 weeks after SRP. IL-8 levels were determined by ELISA. Granulocyte elastase activity was analyzed with a specific substrate, pGluProVal-pNA, and the maximal rate of elastase activity (MR-EA) was calculated. 5 DNA-probes were used to detect the presence of A. actinomycetemcomitans (A. a.), B. forsythus (B.f.), P. gingivalis (P.g.), P. intermedia (P.i.), and T. denticola (T.d.), with a sensitivity = 103 cells/paper point. RESULTS: IL-8 and MR-EA levels in GCF decreased significantly after SRP (p < 0.001) with a corresponding reduction of total count of the species. Of the sites with probing depth (PD) >/= 5.0 mm and co-infection by B.f., P.g., P.i. & T.d. at baseline, the sites without persistent co-infection of these species after SRP exhibited a significant reduction of IL-8 levels (p < 0.02), MR-EA levels (p < 0.02) and PD (p < 0.01). No such change was found in the sites where such a co-infection persisted. Moreover, reduction of IL-8 levels in those pocket sites was accompanied by a concomitant reduction of MR-EA (p < 0.02) and PD (p < 0.01), while no significant change in MR-EA levels and PD was noted in those pocket sites that exhibited an increase of IL-8 levels after SRP. At baseline, the former group of sites showed significantly higher IL-8 levels than the latter group of sites (p < 0.02). CONCLUSIONS: IL-8-related granulocyte elastase activity was related to the change in infection patterns of the target periodontopathogens following scaling and root planing. Varying initial IL-8 levels in GCF and a corresponding shifting change of granulocyte elastase activity in GCF may characterize the different short-term treatment responses.