The mycobacterial 65 kD heat-shock protein and autoimmune arthritis

Summary Arthritis — induced experimentally in rats by immunization with mycobacteria has been shown to depend on specific T cell recognition of an epitope present on the mycobacterial 65-kD heat-shock protein. This particular epitope has been observed to have a structural mimicry with a cartilage-associated molecule present in the joints. Since the bacterial heat-shock proteins and the cartilage-associated molecules are of a conserved nature, one might infer from the experimental model that in humans similar mimicry could play a role in the initiation of autoimmune arthritis. Recent findings from the analysis of immunological reactivity to the 65-kD in rheumatoid arthritis patients seem to support such a role for the mycobacterial 65-kD heat-shock protein in human disease.     

Hyaline cartilage changes in diastrophic dwarfism

Summary Hyaline cartilage of the talus of a diastrophic dwarf was studied by light and transmission electron microscopy before and after proteoglycan extraction or digestion, glycogen digestion, and enzyme marking.The nuclei of the chondrocytes were as a rule large and round and the cytoplasm contained large vacuoles. Best's carmine stained the cytoplasm of most cells red; after diastase digestion the cytoplasm remained unstained. This suggested that the cells contained glycogen. This observation was complimented by the ultrastructural demonstration of large amounts of glycogen. Cell scars were frequent. The shape and state of activity of the cells as well as the development of intracytoplasmic organelles showed great variability. The matrix showed many areas of degeneration and a general dearth of sulphated acid mucopolysaccharides. A lacey pattern of unmasked collagen fibers was frequently seen. Collagen fibers showed a great variability in diameter and often appeared frayed when examined by electron microscopy.These observations suggest an enzymatic deficiency in chondrocyte mucopolysaccharide and glucose metabolism. The techniques we used when added to biochemical studies should prove useful in the investigation of human dwarfism.This work was supported in part by the James A. and Jane C. Folger Foundation     

共同性外斜视患者内直肌纤维细胞外基质的变化

目的 探讨共同性外斜视患者内直肌纤维细胞外基质的变化及其与共同性外斜视中间歇性外斜视和恒定性外斜视的关系。方法对31例共同性外斜视患者（间歇性外斜视17例,恒定性外斜视14例;具有阳性家族史7例）行内直肌缩短术,术中切除前段内直肌作为患者组;21例正常人对应前段内直肌作为对照组。采用定量酶联免疫吸附法分别测定两组内直肌中蛋白聚糖和纤维连接蛋白的含量,比较患者组与对照组、间歇性外斜视患者与恒定性外斜视患者、不同性别患者间、具有不同家族史患者间的差异,并分析不同年龄患者间蛋白聚糖和纤维连接蛋白含量的变化。结果患者组内直肌纤维连接蛋白含量（23．56μg/g）明显低于对照组（444．59μg／g）,差异具有统计学意义（P〈0．01）,而蛋白聚糖的含量差异无统计学意义（P〉0．05）。间歇性外斜视患者的纤维连接蛋白含量（103．88μg/g）明显高于恒定性外斜视患者（11．2μg／g）,差异具有统计学意义（P〈0．01）,而蛋白聚糖含量差异无统计学意义（P〉0．05）。斜视患者内直肌蛋白聚糖含量随年龄增长而减少（r=-0．8712,P〈0．01）,而纤维连接蛋白含量与年龄增长无关（r=-0．1718,P〉0．05）。蛋白聚糖和纤维连接蛋白含量与患者的性别、家族史均无关（P〉0．05）。结论共同性外斜视患者内直肌纤维连接蛋白含量的改变可能与共同性外斜视、间歇性外斜视发展为恒定性外斜视有关,在今后的斜视研究中应对纤维连接蛋白给予重视。（中华腰科杂志,2007,43：618-621）     

Polyhexanide and hydrogen peroxide inhibit proteoglycan synthesis of human chondrocytes

Abstract

The use of local antiseptics is a common method in septic joint surgery. We tested polyhexanide and hydrogen peroxide, two of the most frequently used antiseptics with high efficacy and low toxicity. The purpose of this study was to evaluate the effects of both antiseptics on the extracellular cartilaginous matrix synthesis of human chondrocytes. Chondrocytes were isolated from donated human knee joints, embedded in alginate beads, and incubated for 10 and 30 minutes with polyhexanide (0·04%), hydrogen peroxide (3%), or phosphate-buffered saline (PBS) for control. Cartilaginous matrix production was quantified through light microscopic analysis of Alcian blue staining. Cell number and morphology were detected by histological analysis. Chondrocytes showed a decreased intensity of blue colouring after antiseptic treatment versus PBS. In contrast to that, neither the cell number per view field nor the cell morphology differed between the groups. Polyhexanide has more toxic potential than hydrogen peroxide. Based on the fact that the cell number and morphology was not altered by the substances at the examined concentrations, the lower intensity of Alcian blue staining of treated chondrocytes indicates a decreased cartilage-specific matrix synthesis by polyhexanide more than by hydrogen peroxide and control.     

瓜蒌薤白半夏汤对兔动脉粥样硬化模型主动脉蛋白聚糖的作用

目的从调整动脉壁蛋白聚糖代谢角度观察化痰治法代表方剂——瓜蒌薤白半夏汤抗动脉粥样硬化的药理机制。方法3月龄新西兰兔30只,随机分为正常组、模型组、治疗组。模型组和治疗组均给予高脂饮食造成动脉粥样硬化模型,治疗组同时给予瓜蒌薤白半夏汤灌胃。6周后取材,检测血脂,观察主动脉粥样硬化病变程度,提取主动脉蛋白聚糖,酶解为糖胺多糖,再进行醋酸纤维素薄膜电泳分析,测定各种糖胺多糖组份含量。结果正常组血胆固醇和低密度脂蛋白含量分别为13.43±3.12mmol/L、8.53±1.37mmol/L,动脉壁蛋白聚糖组份硫酸软骨素、硫酸皮肤素含量分别为21.93±1.82mg/g、15.56±1.61mg/g;与正常组比较,模型组表现为典型的动脉粥样硬化病理变化,血总胆固醇和低密度脂蛋白含量明显升高,分别为23.63±4.31mmol/L、15.63±1.27mmol/L(P<0.01),动脉壁蛋白聚糖组份硫酸软骨素、硫酸皮肤素含量明显升高,分别为31.23±1.41mg/g、19.36±1.64mg/g(P<0.01或P<0.05);与模型组比较,治疗组动脉粥样硬化灶病变程度明显减轻,动脉壁蛋白聚糖组份硫酸软骨素、硫酸皮肤素含量明显降低,分别为22.33±1.58mg/g、14.36±1.71mg/g(P<0.01或P<0.05),但血胆固醇和低密度脂蛋白无明显的降低,分别为20.54±3.59mmol/L、14.53±1.32mmol/L。结论瓜蒌薤白半夏汤可降低粥样硬化病变动脉壁硫酸软骨素蛋白聚糖、硫酸皮肤素蛋白聚糖含量,从而减轻动脉粥样硬化病变,但降低血脂作用不明显。     

Structural Analysis of Glycosaminoglycans and Proteoglycans by Means of Raman Microspectrometry

Raman spectra have been determined for hyaluronan, chondroitin-4-sulfate, chondroitin-6-sulfate, aggrecan monomers and aggregates. The nature of the saccharides and the pattern of sulfation can be discerned. There were only small spectral changes with pH and ionic composition. Differences between hydroxyl vibrations, bulk water and solution conditions are shown. The spectrum of aggrecan is dominated by chondroitin sulfate contribution. The sulfation pattern and ratio of protein to glycosaminoglycan and the secondary structure of the core protein were determined.     

Proteoglycans in Predentin: The Last 15 Micrometers Before Mineralization

Small leucine-rich proteoglycans (SLRPs) regulate extracellular matrix organization. In order to investigate the distribution and potential functions of decorin, biglycan (BGN), and fibromodulin (3 SLRPs, potentially related to dentinogenesis), we performed light and electron immunochemistry on teeth from rats, and on wild-type and biglycan knockout mice (BGN KO). Immunohistochemical data demonstrate that chondroitin sulfate/dermatan sulfate (CS/DS) and keratan sulfate (KS) distributions displayed reverse gradients in predentin. The decrease of CS/DS labeling from the proximal to the distal predentin contrasted with the sharp decorin increase observed in the distal predentin near the predentin/dentin transition, an effect possibly attributable to the deglycosylation action of stromelysin-1. In contrast, BGN concentration was apparently constant throughout the whole predentin. Additional immunolabelings showed, for the first time, the presence of fibromodulin in predentin. Compared with the wild-type mouse, the mean diameter of collagen fibrils in the BGN KO was smaller in the proximal predentin but larger in the central and distal predentin, the metadentin was broader, and the dentin mineralization appeared altered and heterogeneous. Altogether, our data suggest an important role for BGN in dentin formation and mineralization.     

Removal of water binding proteins from dentin increases the adhesion strength of low-hydrophilicity dental resins

ObjectivesTo investigate the role of proteoglycans (PGs) on the physical properties of the dentin matrix and the bond strength of methacrylate resins with varying hydrophilicities.MethodsDentin were obtained from crowns of human molars. Enzymatic removal of PGs followed a standard protocol using 1 mg/mL trypsin (Try) for 24 h. Controls were incubated in ammonium bicarbonate buffer. Removal of PGs was assessed by visualization of glycosaminoglycan chains (GAGs) in dentin under transmission electron microscopy (TEM). The dentin matrix swelling ratio was estimated using fully demineralized dentin. Dentin wettability was assessed on wet, dry and re-wetted dentin surfaces through water contact angle measurements. Microtensile bond strength test (TBS) was performed with experimental adhesives containing 6% HEMA (H₆) and 18% HEMA (H₁₈) and a commercial dental adhesive. Data were statistically analyzed using ANOVA and post-hoc tests (α = 0.05).ResultsThe enzymatic removal of PGs was confirmed by the absence and fragmentation of GAGs. There was statistically significant difference between the swelling ratio of Try-treated and control dentin (p < 0.001). Significantly lower contact angle was found for Try-treated on wet and dry dentin (p < 0.002). The contact angle on re-wet dentin was not recovered in Try-treated group (p = 0.9). Removal of PGs significantly improved the TBS of H₆ (109% higher, p < 0.001) and H₁₈ (29% higher, p = 0.002) when compared to control. The TBS of commercial adhesive was not affected by trypsin treatment (p = 0.9).SignificanceChanging the surface energy of dentin by PGs removal improved resin adhesion, likely due to more efficient water displacement, aiding to improved resin infiltration and polymerization.     

核心蛋白聚糖对转化生长因子β1诱导人近端肾小管上皮细胞转分化的影响

目的探讨核心蛋白聚糖对转化生长因子β1（TGF-β1）诱导的人。肾小管上皮细胞（HK-2）转分化的影响。方法将体外培养的HK-2细胞分为6组：阴性对照组（A组）;10ng／ml TGF-β1组（B组）;10ng／ml核心蛋白聚糖组（C组）;100ng／ml核心蛋白聚糖组（D组）;10ng／ml TGF-β1＋10ng／ml核心蛋白聚糖组（E组）;10ng／ml TGF-β1＋100ng／ml核心蛋白聚糖组（F组）。倒置显微镜下观察加入刺激因子48h后,各组细胞形态学变化;应用逆转录-聚合酶链反应（RT-PCR）检测核心蛋白聚糖对TGF-β1诱导的HK-2细胞内α平滑肌肌动蛋白（α-SMA）、波形蛋白、角蛋白mRNA表达的影响。结果B组与A组相比较,细胞形态发生明显变化,大部分细胞拉长呈梭形,似成纤维细胞;E组和F组比B组梭形样细胞明显减少,尤其是F组梭形样细胞减少更明显;C组和D组与A组细胞形态无明显区别,为椭圆形;B组与A组比较,波形蛋白、α-SMAmRNA表达明显提高,角蛋白mRNA表达减少。B组平均值分别为（0.828±0.297）、（0.332±0.025）、（0.075±0.040）,A组平均值分别为（0.033±0.060）、（0.003±0.000）、（0.348±0.012）,差异有统计学意义（P〈0.05）;E组和F组与B组相比,波形蛋白、α-SMA的表达有所下降,角蛋白的表达呈上升趋势（P〈0.05）,平均值分别为E组（0.234±0.313）、（0.214±0.196）、（0.098±0.056）,F组（0.155±0.053）、（0.122±0.130）、（0.215±0.122）。C组和D组与A组比较差异无统计学意义（P〉0.05）。结论TGF-β1能诱导正常人肾小管上皮细胞发生转分化;核心蛋白聚糖对TGF-β1诱导的HK-2细胞转分化具有抑制作用,且呈剂量依赖性。     

腰椎间盘蛋白多糖核心蛋白的基因表达

目的　探讨在椎间盘中核心蛋白ｍＲＮＡ 表达的增龄性变化。方法　通过ＲＴ－ ＰＣＲ制备核心蛋白特异性的ｃＤＮＡ 探针,应用原位杂交的方法,分别对胎儿和成人椎间盘标本进行核心蛋白ｍ ＲＮＡ表达的定位观察。结果　在胎儿椎间盘标本中,髓核内呈现高表达,纤维环和髓核交界处表达减弱,纤维环处未见阳性表达信号。在成人标本中,髓核和纤维环均未见明显的阳性表达。结论　在胎儿椎间盘核心蛋白主要在髓核高表达,从髓核到纤维环其表达依次降低。