炎症因子在大鼠慢性牙周炎合并动脉粥样硬化模型中的表达

目的:检测和分析大鼠慢性牙周炎(chronic periodontitis,CP)合并动脉粥样硬化(atherosclerosis,As)模型中牙周组织白细胞介素-6(Interleukin-6,IL-6)和血清C反应蛋白(C-reactive protein,CRP)的表达.方法:60只成年Wistar雄鼠随机分为4组,每组15只,A为正常对照组,B为CP组,C为As组,D为CP+As组;各组接受相应的建模处理.观察牙周组织和动脉血管病理改变,免疫组织化学方法半定量分析牙厨组织IL-6表达,酶联免疫吸附法(ELISA)检测血清CRP水平.结果:牙周病理学观察,B、D组实验牙牙周炎症表现明显,附着丧失(AL)水平较A、C组有明显增加(P＜0.05).动脉病理学观察,C、D组主动脉和胸、腹主动脉形成粥样硬化病变.B、D组牙周组织IL-6表达和血清CRP水平均高于其他组(P＜0.05).相关性分析显示,B、D组血清CRP与牙周组织IL-6均呈正相关.结论:CP与As可能相互影响、促进,两疾病的发生发展与炎症因子表达升高有关.     

牙周干预措施对动脉粥样硬化影响的实验动物研究

目的模拟牙周炎患者日常生活中的牙周干预措施,研究各种牙周干预措施对SD大鼠动脉粥样硬化(As)发生、发展的影响。方法 SD大鼠随机分为4组:正常对照组(A组)、As组(B组)、As合并牙周炎组(C组)、牙周炎组(D组),将C组根据牙周干预措施不同再分为不治疗组(C1组)、刮治组(C2组)、药物治疗组(C3组)和拔除患牙组(C4组)。对各组进行相应的建模处理,苏木精-伊红染色,光学显微镜下观察牙周组织、颈动脉血管壁组织的病理变化,酶联免疫吸附(ELISA)法检测血清超敏C反应蛋白(hsCRP)的含量。结果病理切片发现B组、C3组和D组颈动脉血管壁均可见大量泡沫细胞形成、聚集;C1组和C4组可见内膜下有钙盐沉积,中膜弹力纤维紊乱、破坏;C2组可见纤维帽的形成及斑块破裂。牙周干预处理后,所有建模组和干预处理组的血清hsCRP含量均较A组明显升高(P<0.05);C1组、C2组、C3组的hsCRP含量较B组明显升高(P<0.05);且C2组hsCRP的含量高于C1组,差异具有统计学意义(P<0.05)。结论对于SD大鼠,无论有无高脂状态,牙周炎均可引起或加重As的发生发展;而在高脂状态下,直接牙周干预都可能加重As病变,其中牙周直接刮治处理的影响在短期内可能会更严重,且hsCRP可能参与了As加重的病变过程。     

白细胞介素-6在大鼠慢性牙周炎合并动脉粥样硬化模型中的表达

目的探讨大鼠慢性牙周炎（CP）合并动脉粥样硬化（As）模型中牙周组织、As斑块中血清白细胞介素-6（IL-6）的表达。方法60只成年Wistar雄鼠随机分为4组：正常对照组（A组）、CP组（B组）、As组（C组）、CP加As组（D组）,每组15只,各组接受相应的建模处理。观察牙周组织及动脉血管病理改变,免疫组织化学方法半定量分析牙周组织及As斑块中IL-6表达,酶联免疫吸附（ELISA）法检测血清IL-6水平。结果牙周病理学观察：B、D组实验牙牙周炎症表现明显,附着丧失（AL）水平较A、C组有明显增加（P〈0.05）。动脉病理学观察：C、D组主动脉形成粥样硬化病变。免疫组化染色：B、D组IL-6在牙周组织及血清中的表达均高于其他组（P〈0.05）,D组As斑块中IL-6表达高于其他组（P〈0.05）。相关性分析显示,D组IL-6在牙周组织与As斑块中的表达呈正相关。结论CP与As的发生发展可能与IL-6表达升高有关。     

一氧化碳释放分子对大鼠实验性牙周炎的影响

目的 研究一氧化碳释放分子（CORM）对大鼠实验性牙周炎的影响。方法 将42只健康雄性Wistar大鼠分为正常组（ NL组）、牙周炎组（ LO组）和一氧化碳干预组（ CO组）。NL组不作任何处理;CO及LO组采用牙周丝线结扎法建立牙周炎实验模型;建模当天起 CO组经腹腔注射 CORM-2,每天 10 mg•kg-1,连续 10 d,LO组注射等体积生理盐水。分别于结扎 3、7、10 d后自心脏抽取静脉血,用酶联免疫吸附测定法检测血清中肿瘤坏死因子 -α（TNF-α）和白细胞介素 -1β（IL-1β）的质量浓度。10 d后在体视显微镜下测量牙槽骨的吸收高度,并于光镜下观察牙周组织炎症细胞的浸润情况。结果 丝线结扎10 d后,LO、CO组牙槽骨的吸收高度均明显大于NL组,而CO组明显小于 LO组（ P<0.05）; LO、CO组炎症细胞浸润指数明显高于 NL组,而 CO组明显低于 LO组（ P<0.05）。在同一观察时期内, LO组TNF-α及IL-1β质量浓度均明显高于其他两组,而 CO组虽高于 NL组,但明显低于 LO组（ P<0.05）。结论 CORM-2能有效抑制大鼠实验性牙周炎的炎症细胞浸润和牙槽骨吸收。     

IL-1家族与牙周炎研究的新进展

IL-1家族包括IL-1α,IL-1β和IL-1受体拮抗剂(IL-1ra)。本文综述了IL-1家族各成员与牙周炎发生、发展关系的研究进展。并描述了IL-1ra的临床前期动物模型研究成果。指出了IL-1ra成为一种治疗牙周炎的非激素类抗炎新药有关的问题。     

Shh蛋白与慢性牙周炎炎症程度的相关性研究

目的:探讨Sonic Hedgehog(Shh)信号通路中Shh蛋白与慢性牙周炎炎症程度的相关性。方法:选择健康对照20例(健康对照组),轻度牙周炎患者20例(轻度牙周炎组),中重度牙周炎患者20例(中重度牙周炎组),收集其龈沟液样本。采用ELISA法检测龈沟液中Shh蛋白、白细胞介素-6(IL-6)及白细胞介素-10(IL-10)的水平。结果:Shh蛋白、IL-6在慢性牙周炎组的水平均高于健康对照组(P<0.05),且中重度慢性牙周炎组高于轻度慢性牙周炎组(P<0.05);IL-10在轻度慢性牙周炎组的水平高于健康对照组及中重度慢性牙周炎组(P<0.05),且健康对照组高于中重度慢性牙周炎组(P<0.05);Shh蛋白、IL-6水平与出血指数(BI)、探诊深度(PD)均呈正相关(P<0.05)。结论:Shh信号通路可能参与了慢性牙周炎的炎症反应过程。     

IL—1家族与牙周炎研究的新进展

ＩＬ－１家族包括ＩＬ－１α，ＩＬ－１β和ＩＬ－１受体拮抗剂（ＩＬ－１ｒａ）。本文综述了ＩＬ－１家族各成员与牙周炎发生，发展关系的研究进展。并描述了ＩＬ－１ｒａ的临床前期动物模型研究成果，指出了ＩＬ－１ｒａ成为一种治疗牙周炎的非激素类抗炎新药有关的问题。     

银杏叶提取物对牙周炎正畸大鼠牙周组织中IL-6表达影响的研究

目的探讨银杏叶提取物对牙周炎正畸大鼠牙周组织的影响。方法选取60只雄性Wistar大鼠,随机选取6只为空白对照组,剩余大鼠牙周炎建模,4周后随机选取6只为炎症对照组,其余大鼠建立牙移动模型,分为炎症加力组和炎症加力给药组。炎症加力给药组以灌胃的方式给予大鼠银杏提取物溶液,炎症加力组给予等量生理盐水,每天1次,分别于加力3、7、14、21d后处死大鼠,观察牙移动距离并行HE染色、免疫组化分析。结果炎症加力组牙周组织破坏程度较炎症加力给药组严重,炎症加力给药组大鼠的牙齿移动距离均小于炎症加力组,牙周组织中白细胞介素-6(IL-6)的表达也较炎症加力组低。结论实验选取的银杏叶提取物可以降低牙周组织IL-6的表达,并且使炎性正畸大鼠在牙周组织破坏减小的情况下得到较好的牙移动。     

口腔干预对牙周炎大鼠动脉粥样硬化影响的研究

目的探讨牙周炎经不同口腔干预处理后对动脉粥样硬化的影响。方法 SD大鼠随机分为3大组,A组:正常对照组,B组:动脉粥样硬化模型组;C组:牙周炎模型组;牙周炎模型建立后,根据口腔干预措施的不同C组再随机2次分为4组,C1自然进程组、C2基础治疗组、C3服药组和C4拔牙组。各组接受相应口腔干预措施。用酶联免疫吸附(ELISA)法,检测干预前、中、后时段,血清hsCRP水平。处死动物后,光镜观察颈动脉血管壁组织的病理变化。结果自然状态无干预的各组在所有取样时间点,与A组相比,B组及C1组的hsCRP含量均显著升高(P0.05),其中,B组的hsCRP含量最高,且含量与C1组之间差异在第3次取样时间点,有统计学意义(P0.05)。进行口腔干预后,与C1组比,各干预组的hsCRP含量均有不同程度的升高,C4组的第2、3次血清hsCRP含量均显著高于C1组(P0.01),C2组在第2次取样时较C1组有下降趋势,但差异无统计学意义。颈动脉血管壁病理切片发现,除A组和C2组未见泡沫细胞形成外,其余各组均可见大量泡沫细胞的形成和聚集,出现动脉粥样硬化的早期病理改变。结论无高血脂存在的SD大鼠,单纯牙周炎任其自然发展或炎症期直接拔牙都可能增加动脉粥样硬化发生风险。牙周基础治疗可能降低动脉粥样硬化的发生风险。     

白细胞介素6与牙周炎的关系

白细胞介素6是一种来源广泛的多功能细胞因子，与牙周炎有密切的关系。在有害物质作用于牙周组织的初期，局部正常水平的IL-6具有一定的保护组织的作用，而增多的IL-6则对牙周组织产生破坏牙槽骨和抑制主体细胞生长等病理作用。IL-6的产生及作用受到众多因素的调控。对IL-6的检测以及抑制IL-6的分泌和破坏作用对牙周炎的诊断和治疗具有一定的临床价值。     

牙周炎和高脂血症动物模型的建立及其相关性初步分析

目的：在成功建立牙周炎和高脂血症动物模型的基础上，通过检测其临床牙周指标、血脂含量、血清中C-反应蛋白（CRP）的含量，初步探讨分析牙周炎与冠心病之问的关系。方法：选择大耳白兔作为实验动物，经结扎、创伤、感染和灌胃等手段建立起牙周炎和高脂血症模型，至第8周末将所有建立成功的动物模型按牙周炎组、高脂血症组和牙周炎＋高脂血症组分别检查牙周指标、血脂水平以及血清中免疫指标CRP的含量，进行统计学分析。结果：经过牙周检查和血脂检测，确认成功建立了实验所需的动物模型。其中牙周炎＋高脂血症组的血脂水平有明显升高、牙周炎症表现程度更重，各实验组与对照组的CRP比较均存在显著性差异（P〈0．001）。结论：牙周炎与高脂血症之间存在一定的相关联系和影响，进而可能对动脉粥样硬化和冠心病的发生发展有重要的影响。     

Effect of periodontal treatment on the C-reactive protein and proinflammatory cytokine levels in Japanese periodontitis patients

BACKGROUND: Recent epidemiological studies have shown that individuals with periodontitis have a significantly increased risk of developing coronary heart disease. In addition to conventional risk factors, chronic infection and subsequent production of systemic inflammatory markers may be associated with this increased risk. OBJECTIVES: The aim of the present study was to determine whether the presence of chronic periodontitis and subsequent periodontal treatment could influence the serum levels of C-reactive protein (CRP), interleukin-6 and tumor necrosis factor-alpha (TNF-alpha) in a Japanese population. METHODS: Sera were obtained from 24 patients with moderate to advanced periodontitis at the baseline examination and at reassessment after completion of treatment. As a control, sera were also obtained from 21 subjects without periodontitis. High-sensitivity CRP (hs-CRP) was measured using nephelometry with a latex particle-enhanced immunoassay and interleukin-6 and TNF-alpha were determined by sensitive enzyme-linked immunosorbent assay. RESULTS: The levels of hs-CRP and interleukin-6 in the sera of this Japanese population seemed to be much lower than those reported in other populations. TNF-alpha on the other hand, demonstrated similar levels between this Japanese and other populations. Periodontal status demonstrated a significant improvement in all patients following treatment. There was a trend toward higher hs-CRP levels in patients at baseline compared with control subjects. Hs-CRP level tended to decrease with improvement of the periodontal condition following treatment and approached that of control subjects, although this decline was not statistically significant. interleukin-6 and TNF-alpha levels did not change following periodontal treatment. Furthermore, there was no difference in the serum levels of these inflammatory cytokines between patients either at baseline or at reassessment and control subjects. CONCLUSIONS: In this pilot study, we were unable to show that periodontal disease significantly affects the serum levels of systemic inflammatory markers. However, this does not necessarily mean that periodontitis does not contribute to the total burden of inflammation as there was a tendency for hs-CRP to decrease following successful periodontal treatment. Large-scale studies are clearly needed to determine the impact of periodontal disease on systemic inflammation.     

Elevation of C-reactive protein and interleukin-6 in plasma of patients with aggressive periodontitis

Background and Objective: Systemic levels of C‐reactive protein and interleukin‐6 have been reported to be elevated in patients with periodontitis compared with periodontally healthy individuals. Most studies included patients with chronic periodontitis and comprised predominantly Caucasians. The aim of this study was to determine the relative levels of C‐reactive protein and interleukin‐6 in plasma of patients with aggressive periodontitis in China and to examine the relationships between these two inflammatory mediators and clinical parameters, peripheral blood cells and protein variables. Material and Methods: Plasma samples were collected from 84 patients with aggressive periodontitis and from 65 control subjects. Periodontal examination consisted of taking probing depth and attachment loss measurements. The levels of plasma C‐reactive protein and interleukin‐6 were determined using enzyme‐linked immunosorbent assays. Results: The levels of plasma C‐reactive protein in patients with aggressive periodontitis were significantly higher than those in control subjects (1.87 vs. 0.52 mg/L). The level of plasma interleukin‐6 in patients with aggressive periodontitis was 1.20 pg/mL, higher than that in control subjects (0.08 pg/mL). Multiple linear regression analysis showed that log C‐reactive protein was significantly related to severe sites percentage and albumin following correction for age, gender, body mass index and smoking (p = 0.000, p = 0.008, respectively). Log interleukin‐6 was found to be significantly correlated with periodontal diagnosis, leukocyte count and level of fasting blood glucose after adjusting for the confounders (p = 0.000, p = 0.009 and p = 0.013, respectively). Conclusion: Patients with aggressive periodontitis have significantly elevated levels of plasma C‐reactive protein and interleukin‐6. These elevated inflammatory factors might potentially increase the risk for cardiovascular events and glucose dysregulation in relatively young individuals.     

辅以抗生素的牙周机械治疗对伴动脉粥样硬化牙周炎大鼠的颈动脉及血清超敏C-反应蛋白的影响

目的 研究伴动脉粥样硬化（As）的慢性牙周炎（CP）大鼠模型中血清超敏C反应蛋白（hsCRP）的表达及颈动脉血管的病理变化,探讨牙周基础治疗辅以抗生素对As的影响。方法 35只SD大鼠随机分为两大组：A组（正常对照）和B组（CP+As）,建立CP+AS模型后再将B组分为4组,自然进程组（B1）、牙周机械治疗组（B2）、机械治疗+局部药物组（B3）、机械治疗+局部药物+抗生素组（B4）,每组7只,接受相应的牙周干预治疗。通过酶联免疫吸附（ELISA）法检测血清中hsCRP的质量浓度,光镜下观察颈动脉血管组织的病理变化。结果 随着时间推移,B1组hsCRP质量浓度逐渐升高,在第2次干预后5周（第5次取样时间点）明显高于其他组（P<0.001）;B2、B3、B4组hsCRP先逐渐升高,在第2次干预后1周（第3次取样时间点）达到高峰,之后逐渐下降,低于其基线水平,但仍高于A组（P<0.05）。第2次干预后3周（第4次取样时间点）起,B3、B4组hsCRP显著低于B2组（P<0.001）。病理结果：B1组可见炎症细胞浸润及大量泡沫细胞,弹性纤维明显紊乱破坏;B2组血管壁厚薄不均,可见泡沫细胞,弹性纤维紊乱;B3组血管壁轻度增厚,弹性纤维排列较整齐;B4组血管壁厚度较均匀,弹性纤维排列整齐。结论 对于伴有As+CP的大鼠,牙周基础治疗短期内可能会增加As的发展风险,而从长期作用来看可能会改善As病变;在牙周机械治疗的基础上增加局部抗炎、全身抗生素治疗可优化其疗效。     

A Meta-analysis of the effect of non-surgical periodontal therapy on inflammatory factors in patients with chronic kidney disease and periodontitis

目的 系统评价牙周基础治疗对慢性肾病伴牙周炎患者炎症因子的影响。方法 计算机检索中国学术期刊全文数据库（CNKI）、万方数据库、中国生物医学文献数据库 （CBM）、PubMed、EMbase以及Cochrane Library等数据库,检索时限为从建库截止到2019年12月。由2名研究者收集所有关于牙周基础治疗（牙周非手术治疗）对于慢性肾病伴牙周炎患者炎症因子[C反应蛋白（CRP）、白细胞介素（IL）-6、肿瘤坏死因子（TNF）-α]影响的文献,并且根据纳入排除标准对文献进行筛选,对研究的质量进行严格评价和资料提取,用Revman 5.3软件对符合标准的随机对照试验进行Meta分析。结果 最终纳入了6项研究分析,Meta分析结果显示,与对照组相比,牙周基础治疗能显著降低慢性肾病伴牙周炎患者CRP水平[MD=-0.58,95%CI（-1.13,-0.02）,P=0.04]和IL-6水平[MD=-2.76,95%CI（-5.15,-0.37）,P=0.02],但TNF-α水平[MD=-3.87,95%CI（-8.79,1.05）,P=0.12]没有得到明显改善。结论 慢性肾病伴牙周炎的患者在规律治疗肾病的同时行牙周基础治疗,不仅能够缓解其牙周炎症状况,还可在一定程度上改善全身的部分炎症因子的状态,有利于慢性肾病和牙周炎的控制和治疗。     

Local and systemic biomarkers in gingival crevicular fluid increase odds of periodontitis

Aim: To determine the independent and combined associations of interleukin-1 β (IL-1 β ) and C-reactive protein (CRP) in gingival crevicular fluid (GCF) on periodontitis case status in the Australian population.
Materials and Methods: GCF was collected from 939 subjects selected from the 2004–2006 Australian National Survey of Adult Oral Health: 430 cases had examiner-diagnosed periodontitis, and 509 controls did not. IL-1 β and CRP in GCF were detected by enzyme-linked immunosorbent assays. Odds ratios (OR) and 95% confidence intervals (CIs) were calculated in bivariate and stratified analysis and fully adjusted ORs were estimated using multivariate logistic regression.
Results: Greater odds of having periodontitis was associated with higher amounts of IL-1 β (OR=2.4, 95% CI=1.7–3.4 for highest tertile of IL-1 β relative to lowest tertile) and CRP (OR=1.9, 95% CI=1.5–2.5 for detectable CRP relative to undetectable CRP). In stratified analysis, there was no significant interaction between biomarkers ( p =0.68). In the multivariate analyses that controlled for conventional periodontal risk factors, these relationships remained (IL-1 β OR=1.8, 95% CI=1.1–2.6; CRP OR=1.7, 95% CI=1.3–2.3).
Conclusions:  Elevated odds of clinical periodontitis was associated independently with each biomarker. This suggests that people with elevated biomarkers indicative of either local (IL-1 β ) or systemic (CRP) inflammation are more likely to suffer from periodontal disease.     

Periodontal therapy: a novel non-drug-induced experimental model to study human inflammation

BACKGROUND: Chronic periodontitis causes a low-grade systemic inflammatory response; its standard treatment, however, induces an acute inflammatory response. The aim of this study was to describe the systemic inflammatory reactions to an intensive periodontal treatment regimen. METHODS: Fourteen otherwise healthy subjects suffering from severe chronic periodontitis were enrolled in a 1 month pilot single-blind trial. Intensive periodontal treatment, consisting of full-mouth subgingival root debridement delivered within a 6-h period, was performed. Periodontal parameters were recorded before and 1 month after completion of treatment. Blood samples were taken at baseline and 1, 3, 5, 7 and 30 days after treatment. Interleukin-1 receptor antagonist (IL-1Ra), Interleukin-6 (IL-6) and C-reactive protein (CRP) serum concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Complete blood counts were also performed. RESULTS: One day after treatment, mild neutrophilia and monocytosis (p < 0.05) and lymphopenia (p < 0.01) were accompanied by a sharp increase in inflammatory markers (IL-1Ra, IL-6, p < 0.01). A 10-fold increase in CRP (p < 0.001) was detected on day 1 and its kinetics followed a pattern of a classical acute phase response (significantly raised concentrations up to 1 week, p < 0.01). At 3-7 days after treatment, subjects presented also with a mild tendency towards a normocytic anaemic state (p < 0.01) and a degree of lympho-thrombocytosis (p < 0.05). The observed changes were similar to those expected following the well-characterized endotoxin-challenge model of inflammation. CONCLUSIONS: Intensive periodontal treatment produced an acute systemic inflammatory response of 1 week duration and might represent an alternative to classic endotoxin-challenge or drug-induced models to study acute inflammation in humans.     

���ܻ������ƶ԰�򲻰��֬Ѫ֢�����������״���������Ӳ��������Ӱ���о�

目的通过观察血清超敏C反应蛋白(high-sensitivity C-reactive protein,hsCRP)水平的变化探讨牙周基础治疗对伴或不伴高脂血症的慢性牙周炎(chronic periodontitis,CP)大鼠动脉粥样硬化(atherosclerosis,As)发生的影响。方法本研究于2011年5—9月在山西医科大学口腔医学院完成。将24只6周龄雄性SD大鼠随机分为4组,即正常对照组(A组)、高脂血症组(B组)、CP组(C组)和高脂血症+CP组(D组),每组6只。各组接受相应的建模及牙周干预处理,同时定期进行牙周临床检查,酶联免疫吸附(ELISA)法检测牙周干预前及每次干预后1周(共2次)3个取样时间点的血清hsCRP水平。结果牙周临床检查结果显示,整个实验过程中A、B组大鼠实验牙牙龈未见炎症性改变;C、D组大鼠建模后实验牙牙龈炎症明显,有深牙周袋,部分实验牙松动度达Ⅱ~Ⅲ度,基础治疗后炎症明显减轻,牙齿均无松动。同一时间点上,B、C及D组大鼠血清hsCRP水平均显著高于A组,差异有统计学意义(P<0.01)。在不同时间点上,B、C及D组大鼠治疗后血清hsCRP水平均较治疗前显著增高,差异有统计学意义(P<0.01)。其中,C组大鼠2次治疗后血清hsCRP水平趋于稳定,D组大鼠在2次治疗后1周血清hsCRP水平显著增高。结论对于慢性牙周炎大鼠,无高脂血症情况存在时,牙周基础治疗可能会在短期内增高As的发生风险;在存在高脂血症状态下,直接牙周基础治疗可能会在较长时间内增高As的发生风险。     

Immune responses to heat shock protein in Porphyromonas gingivalis-infected periodontitis and atherosclerosis patients

BACKGROUND: It has been widely thought that heat shock protein might be involved in autoimmune disease mechanisms in humans. OBJECTIVES: The present study was performed to evaluate the recognition of Porphyromonas gingivalis heat shock protein 60 (hsp60) and human hsp60 by immune sera in P. gingivalis-infected periodontitis and atherosclerosis patients. MATERIALS AND METHODS: Mononuclear cells from atheroma lesions were stimulated with P. gingivalis hsp and sera from periodontitis or atherosclerosis patients were subjected to Western immunoblotting to P. gingivalis hsp or human hsp, respectively. RESULTS: Western immunoblot analysis demonstrated the dual reactivity of anti-P. gingivalis antisera with P. gingivalis hsp and human hsp. We could also establish P. gingivalis hsp-specific T cell lines from the atheroma lesions, a mixture of CD4+ and CD8+ cells producing the cytokines characteristic of both Th1 and Th2 subsets. CONCLUSION: These observations suggest the modulating effect of P. gingivalis hsp60 in the immunopathogenesis of periodontitis and atherosclerosis.