急性时相血清淀粉样蛋白A致动脉粥样硬化的机制

动脉粥样硬化(Athemsclemsis,As)被认为是一种慢性炎症性疾病.急性时相血清淀粉样蛋白A(acute serum amyloid A,A-SAA)可以通过诱发炎症反应、干扰脂质转运和降低动脉粥样硬化斑块的稳定性,从而参与动脉粥样硬化的发生和发展.本文就A-SAA导致As的内在机制进行综述.     

非小细胞肺癌患者血清淀粉样蛋白A水平与放射性肺炎的相关性研究

目的:评估非小细胞肺癌患者血清淀粉样蛋白A(SAA)水平与发生放射性肺炎(RP)的相关性。方法:选取我院确诊为非小细胞肺进行肺部病灶放射治疗的患者118例,评价是否发生放射性肺炎,并在放疗前及放疗剂量达到43 GY时分别抽取患者血清样本,应用酶联免疫法(ELISA)检测患者血清淀粉样蛋白A含量。结果:43例患者发生了放射性肺炎,发生放射性肺炎组患者的放疗前血清淀粉样蛋白A水平明显高于未发生放射性肺炎组,两组对比,差异有统计学意义(P0.05)。而在放疗剂量达到43Gy时,发生放射性肺炎组患者的血清淀粉样蛋白A水平与未发生放射性肺炎组患者相比,差别无统计学意义。结论:对于非小细胞肺癌患者,通过检测放疗前血清淀粉样蛋白A水平可以预测放射性肺炎的发生。     

人血清淀粉样蛋白SAA1 蛋白的原核重组表达

目的:重组及表达人血清淀粉样蛋白SAA1融合蛋白,为进一步制备早期诊断冠心病的单克隆抗体做准备。方法:应用PCR技术,以成人肝脏的c DNA文库做模板,扩增出长度为315 bp的人血清淀粉样蛋白SAA1基因,并将其分别与带有HIS标签的PET-32a载体及GST标签的PGEX-4T-1载体连接,转化入DH5α感受态细胞中进行克隆并测序鉴定。并在大肠杆菌表达菌BL21菌中表达SAA1融合蛋白,之后分别应用SDS-PAGE及Western blot技术检验重组蛋白的纯度。结果:经SDS-PAGE凝胶电泳后,我们分别看到了分子量为32k D和分子量为38k D的两个蛋白条带,这两个条带分别为PET-32a-SAA1重组蛋白和PGEX-4T-1-SAA1重组蛋白,且这两种蛋白在超声后存在于菌液的沉淀中,证明在大肠杆菌中实现了不溶性的包涵体形式的表达,经Western blot鉴定,分别以HIS标签抗体和GST标签抗体为第一抗体,以鼠二抗为第二抗体,证明所表达的蛋白质为SAA1融合蛋白。结论:采用原核表达方法,经免疫纯化可获得高纯度的SAA1融合蛋白,为进一步制备相应的单克隆抗体和开发以SAA1增高为指标之一的冠心病诊断试剂盒打下基础,为冠心病的诊断开辟新途径。     

早老蛋白与淀粉样沉淀前体蛋白相互作用及Aβ的形成

Ａβ在脑内的沉积是Ａｌｚｈｅｉｍｅｒ病的病理现象。对Ａｌｚｈｅｉｍｅｒ病的研究揭示Ａβ的生成与早老蛋白和淀粉样沉淀前体蛋白相互作用密切相关。早老蛋白和淀粉样沉淀前体蛋白基因的突变均能改变淀粉样沉淀前体蛋白的正常切割,使得Ａβ的生成量增加。     

基于裸磁珠体系检测血清淀粉样蛋白A浓度方法的建立与效果评估

传统链霉亲和素磁珠体系及酶促发光体系中通常存在生物素干扰强、发光率低、特异性差的缺陷。基于此,以裸磁珠为载体偶联血清淀粉样蛋白A（serum amyloid A, SAA）抗体,以吖啶酯作为发光标记物,依据双抗夹心原理,建立了一种快速测定SAA的方法,并对该方法的检测性能（包括：空白限、检测限、线性范围、精密度验证、干扰试验、HOOK效应、方法学比较等）进行了评估。结果表明,SAA抗体与裸磁珠成功偶联;该方法空白限为0.6 mg·mL^-1,检出限为1.0 mg·mL^-1;线性范围为1~100 mg·mL^-1 （R²＞0.990）;在精密度方面,以CV表示的重复性、室内精密度均<10%;血红蛋白、胆红素、甘油三酯对临床样本的干扰检测结果相对偏差均<10%。Bland-Altman偏倚分析表明,该方法检测值与西门子试剂的测量值差异基本在95％CI的一致性界限内波动,回归分析结果显示有较好的相关性（R²=0.987）。上述结果表明,研究所建立的裸磁珠-吖啶酯发光检测体系性能参数可满足临床检测要求,适用于血清中SAA的快速检测。     

血清淀粉样蛋白A经p38-MAPK/SR-BI途径促进巨噬细胞炎症反应北大核心CSCD

为探讨血清淀粉样蛋白A(serum amyloid A,SAA)对巨噬细胞B类I型清道夫受体(scavenger receptor class B type I,SR-BI)的表达以及炎症反应的影响及分子机制,采用SAA、p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38-MAPK)激动剂anisomycin或抑制剂SB203580处理THP-1巨噬细胞,以实时定量PCR、Western blot和ELISA分别检测细胞中SR-BI、炎症因子及磷酸化p38-MAPK的表达。结果显示,与对照组相比,SAA处理THP-1细胞后,SR-BI的表达下调,而炎症因子与磷酸化p38蛋白的表达则上调,且这种效应呈浓度和时间依赖性(P<0.05)。与SAA单独处理组比较,SAA与p38-MAPK激动剂anisomycin共孵育细胞后,细胞SR-BI表达下调,炎症因子及磷酸化p38蛋白表达增加(P<0.05);而SAA与p38-MAPK抑制剂SB203580共同处理细胞后,细胞SR-BI表达增加,炎症因子及磷酸化p38蛋白表达减少(P<0.05)。结果提示,SAA可促进THP-1巨噬细胞炎症反应,其机制与p38-MAPK的磷酸化及SR-BI表达的下调有关。     

H102对APP转基因小鼠脑内淀粉样蛋白和淀粉样蛋白前体蛋白表达的影响

目的:研究H102对APP695转基因模型小鼠脑内淀粉样蛋白和淀粉样蛋白前体蛋白表达的影响方法:9月龄转基因小鼠随机分为模型组和药物注射组,正常对照组采用月龄和性别与之相匹配的C57BL/6J小鼠。药物注射组给予侧脑室注射H102,每只每次3μl,连续10d;模型组和正常对照组给予等体积NS。应用免疫组织化学结合刚果红组织学染色,普通光学显微镜观察海马和颞叶皮层蛋白表达的变化。免疫印迹法检测小鼠大脑皮层APP蛋白的表达。结果:Aβ和APP免疫组化染色结果显示对照组海马CA1区神经元胞浆着色呈阴性或弱阳性,模型组较对照组阳性细胞增多,表达增强,胞浆着色明显加深。药物注射组同模型组相比,胞浆着色变淡,表达减弱。刚果红染色观察转基因小鼠模型组和H102注射组大脑颞叶皮层和海马的淀粉样斑块,可见H102注射组淀粉样斑块数较模型组明显减少。正常对照组未见阳性淀粉样斑块。免疫印迹检测显示模型组APP蛋白表达明显增加,给药组与模型组相比具有统计学意义。结论:APP695转基因小鼠大脑CA1区Aβ蛋白和APP蛋白表达增加,H102能够明显抑制该转基因小鼠Aβ蛋白和APP蛋白表达。     

淀粉样前体蛋白生理功能研究进展

阿尔茨海默病(alzheimer's diasese,AD)是一种神经退行性疾病,以β淀粉样肽(βamyloid,Aβ)为主要成分的老年斑是脑内的主要病理改变,因此Aβ的过量产生和沉积是AD主要发病机制。Aβ主要由淀粉样前体蛋白(amyloid precursor protein,APP)经β和γ分泌酶异常剪切产生,APP表达增加被认为是AD的风险因素之一,目前关于APP的研究主要集中在作为Aβ的前体如何剪切及在AD发病中的作用,而对APP的生理功能关注较少。近年研究发现APP具有广泛的生理功能,APP缺失可以影响学习记忆,这与其剪切及Aβ的产生均无关。APP广泛表达于多种器官和组织,生理功能具有多样性,因此了解APP的生理功能将为AD发病机制的研究与治疗干预提供重要的理论依据。本文对此进行综述。     

阿尔茨海默病患者血清淀粉样蛋白A、胆碱酯酶、载脂蛋白A1水平与认知功能的关系研究

目的：探讨阿尔茨海默病（AD）患者血清淀粉样蛋白A(SAA)、胆碱酯酶（CHE）、载脂蛋白A1(ApoA1)水平与认知功能的关系。方法：选取2020年12月～2023年5月在我院就诊的103例AD患者作为病例组,另外选取同期在本院体检健康的志愿者80例作为对照组。采用酶联免疫吸附法检测两组研究对象血清SAA、CHE、ApoA1水平;采用蒙特利尔认知评估量表（MoCA）、简明精神状态检查量表（MMSE）评估两组研究对象的认知功能。对比两组研究对象血清SAA、CHE、ApoA1水平及MMSE、MoCA评分。根据MMSE评分将AD患者分为轻度组、中度组和重度组,观察并比较轻度组、中度组和重度组血清SAA、CHE、ApoA1水平。采用Pearson检验分析血清SAA、CHE、ApoA1水平与MMSE评分的相关性。结果：病例组的血清SAA高于对照组,血清CHE、ApoA1水平低于对照组（P<0.05）。病例组的MoCA、MMSE评分低于对照组（P<0.05）。重度组、中度组血清SAA水平高于轻度组,且重度组高于中度组（P<0.05）。重度组、中度组血清CHE、ApoA1水平低于...     

Isolation of serum amyloid A protein by small-scale hydrophobic interaction chromatography and two-dimensional electrophoresis

A recently introduced technique to isolate serum amyloid A protein is hydrophobic interaction chromatography combined with two-dimensional electrophoresis with immobilized pH gradients. A modification of the original version of this technique is presented. Mouse serum was subjected to hydrophobic interaction chromatography on a small scale, and the eluate was applied directly to two-dimensional electrophoresis. Simple electropherogramss with optimal resolution of serum amyloid A protein were obtained. The presented technique facilitates isolation of serum amyloid A protein from small blood volumes, and might also be adapted to alternative applications.     

Immunotherapy and combined assay of serum levels of carcinoembryonic antigen and acute-phase reactants

 Our previous studies have revealed that gastric and esophageal cancer patients with abnormal sialic acid levels had a better response than those with normal levels if they received polysaccharide K (PSK), a nonspecific immunomodulator. Serum levels of carcinoembryonic antigen (CEA) and acute-phase reactants (APR) such as immunosuppressive acidic protein, acid-soluble glycoproteins, α1-antichymotrypsin, and sialic acid were analyzed in 872 gastric cancer patients who had undergone resection from March 1979 to September 1993 at the Department of Surgery of Tokai University. The patients were categorized into four groups according to the preoperative serum levels: group A had normal levels of both CEA and APR, group B had abnormal CEA and normal APR levels, group C had a normal CEA level and normal levels of one or more APR, and group D had abnormal levels of both CEA and of one or more APR. Patients in group D who received PSK showed significantly better survival than those without PSK (29.3% versus 6.9%; log-rank test, P = 0.0015; Breslow test, P = 0.0042). CEA-positive patients receiving PSK therapy exhibited a significantly better survival rate than those without PSK (38.1% versus 18.6%; log-rank test, P = 0.0136; Breslow test, P = 0.0125). Cox’s regression analysis showed that PSK therapy was significantly related to survival in group D, but not in the other groups. We conclude that the combined assay of tumor-associated factors (such as CEA) and various nonspecific reactants to the presence of cancer (such as immunosuppressive acidic protein, α1-antichymotrypsin, acid-soluble glycoproteins and sialic acid) provides a good set of preoperative indicators on which to base the selection of treatment for individual gastric cancer patients. Received: 25 July 1997 / Accepted: 5 November 1997     

Preliminary characterizations of a serum biomarker for sarcoidosis by comparative proteomic approach with tandem-mass spectrometry in ethnic Han Chinese patients

Background

The diagnosis of sarcoidosis is still a significant challenge in China because of the need to exclude other diseases including granulomatous infections and malignancies that may be clinically and radiographically similar. The specific aim of the study is to search for serum protein biomarkers of sarcoidosis and to validate their clinical usefulness in differential diagnosis.

Methods

Serum samples were collected from patients with sarcoidosis (n = 37), and compared to those from patients with tuberculosis (n = 20), other pulmonary diseases (n = 20), and healthy volunteers (n = 20) for determination of sarcoidosis-specific or -associated protein expression profiles. The first part of this study focused on proteomic analysis of serum from patients with sarcoidosis to identify a pattern of peptides capable of differentiating the studied populations using the ClinProt profiling technology based on mass spectrometry. Enzyme Linked Immunosorbent Assay (ELISA) was then used to verify corresponding elevation of the serum protein concentration of the potential biomarkers in the same patients sets. Receiver operating characteristic curve (ROC) analyses was performed to determine the optimal cutoff value for diagnosis. Immunohistochemistry was carried out to further confirm the protein expression patterns of the biomarkers in lung tissue.

Results

An unique protein peak of M/Z 3,210 Daltons (Da) was found to be differentially expressed between the sarcoidosis and control groups and was identified as the N-terminal peptide of 29 amino acids (94-122) of serum amyloid A (SAA). ELISA confirmed that the serum SAA level was significantly higher in the sarcoidosis group than that of the other 3 control groups (p < 0.05). The cutoff for serum SAA concentration determined by ROC analysis was 101.98 ng/ml, with the sensitivity and specificity of 96.3% and 52.5%, respectively. Immunohistochemical staining showed that the SAA depositions in lung tissue of the sarcoidosis patients were also significantly more intense than in non-sarcoid lung tissue (p < 0.05).

Conclusion

This is the first study to investigate serum protein markers in Chinese subjects with sarcoidosis. This study shows that the serum SAA expression profiles were different between the sarcoidosis and non-sarcoidosis groups. SAA may be a potential serum biomarker for ruling-out the diagnosis of sarcoidosis in Chinese subjects.     

Heparan sulfate dissociates serum amyloid A (SAA) from acute-phase high-density lipoprotein, promoting SAA aggregation

Inflammation-related (AA) amyloidosis is a severe clinical disorder characterized by the systemic deposition of the acute-phase reactant serum amyloid A (SAA). SAA is normally associated with the high-density lipoprotein (HDL) fraction in plasma, but under yet unclear circumstances, the apolipoprotein is converted into amyloid fibrils. AA amyloid and heparan sulfate (HS) display an intimate relationship in situ, suggesting a role for HS in the pathogenic process. This study reports that HS dissociates SAA from HDLs isolated from inflamed mouse plasma. Application of surface plasmon resonance spectroscopy and molecular modeling suggests that HS simultaneously binds to two apolipoproteins of HDL, SAA and ApoA-I, and thereby induce SAA dissociation. The activity requires a minimum chain length of 12-14 sugar units, proposing an explanation to previous findings that short HS fragments preclude AA amyloidosis. The results address the initial events in the pathogenesis of AA amyloidosis.     

Serum angiotensin converting enzyme,Erythrocyte sedimentation rate and high sensitive-C reactive protein levels in diagnosis of cardiac sarcoidosis- where do we stand?

BackgroundCardiac sarcoidosis (CS) is increasingly being recognized in the last two decades. The diagnosis of CS depends on clustering of multiple symptoms, investigations and demonstration of a non-caseating granuloma on histopathology. Serum Angiotensin Converting Enzyme (SACE) level, one of the serological markers, is often elevated in systemic sarcoidosis. However, the yield of SACE level among patients with isolated or predominant CS is unclear. We conducted a retrospective study to assess the prevalence of elevated SACE level among patients with proven CS.Materials and methodsFrom our Granulomatous myocarditis (GM) registry, 45 biopsy proven CS patients were enrolled. Inclusion criteria: Clinical diagnosis of CS [HRS definition + Lymph Node biopsy/Endomyocardial biopsy (non-caseating granuloma)]. Exclusion criteria - Other causes of GM like cardiac tuberculosis (TB culture/AFB smear -positive) and patients taking medications affecting SACE level.ResultsAmong 143 GM cases, 45 CS were analyzed. Mean age:42 ± 11 years (Range 22–63 years, 19 females). With our laboratory reference of SACE (Normal range: 20–70 U/L), 3 out of 45 (6.7%) patients of CS had elevated SACE. In a comparative analysis we found, Erythrocyte Sedimentation Rate (ESR) and High sensitive-C Reactive Protein (Hs-CRP) are much more sensitive, although not specific for CS. Patients with pulmonary involvement more often had elevated SACE level.ConclusionSerum ACE is elevated only in approximately 6.7% of patients with biopsy proven CS. Hence, it is insensitive serological tool for diagnosis of CS even in the active phase of the disease. In contrast, ESR and Hs-CRP emerges to be more sensitive markers of active CS.     

Discovery and identification of Serum Amyloid A protein elevated in lung cancer serum

Two hundred and eighteen serum samples from 175 lung cancer patients and 43 healthy individuals were analyzed by using Surface Enhaced Laser Desorption/Ionization Time of Flight Mass Spectrome-try (SELDI-TOF-MS). The data analyzed by both Biomarker Wizard™ and Biomarker Patterns™ software showed that a protein peak with the molecular weight of 11.6 kDa significantly increased in lung cancer. Meanwhile, the level of this biomarker was progressively increased with the clinical stages of lung cancer. The candidate biomarker was then obtained from tricine one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis by matching the molecular weight with peaks on WCX2 chips and was identified as Serum Amyloid A protein (SAA) by MALDI/MS-MS and database searching. It was further validated in the same serum samples by immunoprecipitation with commercial SAA antibody. To confirm the SAA differential expression in lung cancer patients, the same set of serum samples was measured by ELISA assay. The result showed that at the cutoff point 0.446 (OD value) on the Receiver Operating Characteristic (ROC) curve, SAA could better discriminate lung cancer from healthy individuals with sensitivity of 84.1% and specificity of 80%. These findings demonstrated that SAA could be characterized as a biomarker related to pathological stages of lung cancer.     

Methotrexate Chronotherapy is Effective Against Rheumatoid Arthritis

Methotrexate (MTX) is the most important drug for treating rheumatoid arthritis (RA). It has been stated that cytokines play an important role in the pathogenesis of RA, and that cytokine levels increase and show 24-h rhythms in RA patients. Previously, we found that arthritis was relieved after the administration of MTX at specific times in synchronization with the 24-h rhythm of tumor necrosis factor (TNF)-α in collagen-induced arthritis (CIA) animals. Based on our findings in an earlier study of the dosing time–dependent effects of MTX in MRL/lpr mice, which develop autoimmune disorders that share similarities with human RA, we examined here the utility of MTX chronotherapy in Japanese RA patients. In an initial animal modeling study, we collected blood from MRL/lpr mice at different times (2, 6, 10, 14, 18, or 22 hours after the light was turned on [HALO]), and we measured TNF-α mRNA expression in leukocytes. MTX was administered to the mice at two different dosing times (6 or 18 HALO), and various blood parameters were measured to estimate arthritis activity. TNF-α mRNA levels showed a clear 24-h rhythm with a peak at 22 HALO and a trough at 18 HALO after RA had developed. In these MRL/lpr mice, inflammation and TNF-α were markedly reduced when the MTX dosing time was matched to the time (18 HALO) when the TNF-α level began to increase. We then applied these findings to Japanese RA patients by switching them from the standard MTX three times/wk (day 1: after breakfast and supper; day 2: after breakfast schedule), to chronotherapy, in which the dose and number of doses/wk were not changed but MTX was administered once-a-day at bedtime. Disease Activity Score (DAS)28, modified health assessment questionnaire (MHAQ), and adverse effects were assessed. With MTX chronotherapy, DAS28, which is commonly used to quantitatively assess RA symptoms, was significantly improved at all follow-up clinical visit times compared with the baseline (vs. 1 mo: p?=?.0197, 2 mos: p?=?.0107, 3 mos: p?=?.0087). Significant symptom recovery was observed in 41.2% of patients, and 23.5% of patients achieved clinical remission during the 3 mos of follow-up. Functional capacity of RA patients, as indicated by the MHAQ, was markedly improved by chronotherapy. There were no severe adverse effects. Thus, we demonstrated (i) inflammation and plasma TNF-α concentrations were significantly reduced in MRL/lpr mice treated with MTX at 18 HALO, the time when TNF-α mRNA level began to increase; and (ii) MTX bedtime chronotherapy was safe, markedly reduced disease activity, and improved the functional capacity of RA patients. The findings on RA patients show that bedtime MTX chronotherapy can improve RA symptoms compared to the current standard dosing methods. (Author correspondence: hide-to@umin.net)     

P2X7 receptor-mediated scavenger activity of mononuclear phagocytes toward non-opsonized particles and apoptotic cells is inhibited by serum glycoproteins but remains active in cerebrospinal fluid

Rapid phagocytosis of non-opsonized particles including apoptotic cells is an important process that involves direct recognition of the target by multiple scavenger receptors including P2X7 on the phagocyte surface. Using a real-time phagocytosis assay, we studied the effect of serum proteins on this phagocytic process. Inclusion of 1-5% serum completely abolished phagocytosis of non-opsonized YG beads by human monocytes. Inhibition was reversed by pretreatment of serum with 1-10 mM tetraethylenepentamine, a copper/zinc chelator. Inhibitory proteins from the serum were determined as negatively charged glycoproteins (pI < 6) with molecular masses between 100 and 300 kDa. A glycoprotein-rich inhibitory fraction of serum not only abolished YG bead uptake but also inhibited phagocytosis of apoptotic lymphocytes or neuronal cells by human monocyte-derived macrophages. Three copper- and/or zinc-containing serum glycoproteins, ceruloplasmin, serum amyloid P-component, and amyloid precursor protein, were identified, and the purified proteins were shown to inhibit the phagocytosis of beads by monocytes as well as phagocytosis of apoptotic neuronal cells by macrophages. Human adult cerebrospinal fluid, which contains very little glycoprotein, had no inhibitory effect on phagocytosis of either beads or apoptotic cells. These data suggest for the first time that metal-interacting glycoproteins present within serum are able to inhibit the scavenger activity of mononuclear phagocytes toward insoluble debris and apoptotic cells.