A Novel NOD2-associated Mutation and Variant Blau Syndrome: Phenotype and Molecular Analysis

Purpose: To describe the clinical and molecular implications of a novel mutation in the NOD2/CARD15 gene on a family and its seven affected members.

Methods: We reviewed the clinical presentations of family members who came to our center for refractory uveitis. Genetic testing and molecular testing was performed.

Results: All affected members had adult onset recurrent non-granulomatous panuveitis. The inheritance pattern suggested an autosomal dominant disease and genetic analysis identified a novel mutation in the NOD2 gene that converted amino acid 600 from glutamate to alanine (E600A). Transfection of the E600A NOD2 into human embryonic kidney-293 (HEK293) cells revealed constitutive activation and a reduced ability to respond to the NOD2 ligand, muramyl dipeptide (MDP) as compared with wild-type NOD2.

Conclusions: The E600A mutation in the NOD2 gene may confer a higher penetrance of uveitis but a later onset of milder forms of non-ocular involvement.     

睫状体和小梁网上相关离子通道的研究进展

房水由睫状上皮细胞产生,约80%通过小梁网进入Schlemm管,睫状上皮细胞和小梁网细胞上存在丰富的离子通道(Cl-通道、Ca~(2+)通道、K+通道和水通道蛋白等),这些通道对房水的产生和滤过有重要影响.本文对近年来睫状体和小梁网上的离子通道研究进行综述,希望能对青光眼研究和治疗有所帮助.     

Histoautoradiographic and biochemical studies on human and monkey trabecular meshwork and ciliary body in short-term explant culture

Explants of two types of chamber angle tissue derived from five human autopsy eyes and five freshly enucleated monkey eyes were kept under tissue culture conditions for 1–3 days and then incubated with labelled¹⁴C-glucosamine for 40–48 h. In all specimens an intense labelling was seen histoautoradiographically within the area of the trabecular meshwork, especially in the cribriform layer and the outer wall of Schlemm's canal. Large quantities of silver grains were also found at the tips of the ciliary processes where the nonpigmented epithelium showed the most intense labelling. Measuring the total radioactivity of the two types of specimens in the tissue-localized and the medium-released glycosaminoglycans (GAGs), we found that the type 1 specimens containing only trabecular meshwork and corneosclera excrete relatively more GAGs into the medium than the type 2 specimens consisting of corneosclera, trabecular meshwork and ciliary body. Biochemical analysis of these GAGs revealed that 57%–70% of the tissue GAGs were hyaluronic acid, 16%–33% heparan sulphate and 12%–22% various types of chondroitin sulphate.The findings show that both the ciliary epithelium and the trabecular meshwork possesses the ability to synthesize large amounts of GAGs, probably in the form of proteoglycans. The differences between the in vitro behaviour of type 1 and type 2 specimens indicate a functional interrelationship between the ciliary body and the trabecular meshwork, at least with regard to the GAG metabolism.Supported by the Deutsche Forschungsgemeinschaft (Ro 81/18-2), D-5300 Bonn-Bad Godesberg     

Age-related changes in the trabecular meshwork of the normal human eye

Specimens from 17 human eyes, ranging in age from 3 to 80 years, were subjected to morphometric studies using light microscope, transmission electron microscope and scanning electron microscope, in order to clarify the age-related changes in the normal trabecular tissue. Statistical analyses showed that the cellularity in the various regions of the trabecular meshwork significantly declined with age. The spaces corresponding to the aqueous outflow pathway in each region of the meshwork also significantly decreased with age. On the other hand, extracellular materials significantly increased in amount with age in all regions of the trabecular meshwork. However, the decrease in the cellularity and the outflow pathway spaces did not show any statistically significant regional difference. These results suggest that general narrowing of the outflow pathway spaces due to the accumulation of extracellular materials with age is the cause of the increase in aqueous outflow resistance with age, and that each region of the trabecular meshwork is equally responsible for the increased resistance.     

内毒素耐受对内毒素诱导的葡萄膜炎炎症程度及虹膜睫状体中PI3K/AKT 信号通路的影响

目的探讨低剂量脂多糖(lipopolysaccharide,LPS)预处理对内毒素诱导葡萄膜炎(endotoxin induced uveitis,EIU)动物模型炎症程度的影响,并分析虹膜睫状体组织中PI3K/AKT表达水平的改变。设计实验研究。研究对象8-10周龄健康雄性Wistar大鼠90只。方法随机分为正常对照(normal control,NC)组、内毒素耐受(endotoxin tolerance,ET)组和内毒素诱导的葡萄膜炎(EIU)组,每组30只。通过皮下注射1 mg/kg LPS建立内毒素诱导的葡萄膜炎动物模型。ET组:EIU模型诱导之前连续5日腹腔注射0.1 mg/kg LPS诱导内毒素耐受。EIU组及NC组连续5日注射等体积生理盐水。第6天相同时间ET组及EIU组接受200μg LPS皮下注射,NC组接受等体积生理盐水注射。注射后24小时,在裂隙灯显微镜下进行临床评分。眼球摘除行HE染色组织病理学观察。ELISA测量房水TNF-α浓度,蛋白印迹法检测虹膜睫状体PI3K和AKT蛋白的表达。主要指标眼前节炎症反应评分,虹膜切片HE染色观察,房水TNF-α浓度及虹膜睫状体PI3K/AKT蛋白表达。结果临床评分ET组、EIU组、NC组分别为(1.65±0.49)、(6.65±0.59)、(0.20±0.41)分(χ~2=53.261,P=0.001);组织病理评分ET组、EIU组、NC组分别为(0.6±0.5)、(3.8±0.4)、(0.2±0.4)分(χ~2=46.137,P=0.001)。房水TNF-α浓度ET组、EIU组、NC组分别为(10.58±0.67)、(30.96±1.55)、(10.32±0.61)pg/ml(F=260.08,P=0.03)。蛋白印迹法显示虹膜睫状体组织中PI3K和AKT的蛋白水平ET组、EIU组、NC组分别为(0.197±0.019)、(0.539±0.017)、(0.453±0.014)(F=210.66,P=0.002)和(0.234±0.019)、(0.553±0.013)、(0.428±0.002)(F=275.35,P=0.001)。结论内毒素耐受可以降低大鼠内毒素诱导的葡萄膜炎的炎症反应程度,下调虹膜睫状体组织中PI3K和AKT的表达,揭示内毒素耐受对EIU的保护机制可能与PI3K/AKT信号通路有关。     

水通道蛋白-1在小梁切除术之切除组织中的表达

目的观察青光跟患者小梁和虹膜组织与正常跟组织水通道蛋白-1（AQP-1）的表达差异。方法收集开角型和闭角型青光跟小梁切除术时切除的小梁和虹膜组织，免疫组织化学法检测AQP-1的表达，并与正常跟相应组织对照。结果正常眼小梁网组织、Schlemm’s管内皮细胞、周边虹膜组织中上皮和基质组织可见AQP-1呈强阳性着色，开角型青光眼和闭角型青光眼组织标本小梁网AQP-1阳性染色较正常弱；部分急性闭角型青光眼患者周边虹膜组织标本上皮层较基质组织染色明显弱。结论开角型青光眼小梁网AQP-1的表达减少可能与小梁网的发育有关，闭角型青光眼虹膜上皮和小梁网AQP-1的表达减少可能与虹膜萎缩或高眼压有关。     

Expression and distribution of matrix metalloproteinases and their inhibitors in the human iris and ciliary body

AIM: To determine the expression and distribution of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in the normal human iris and ciliary body. METHODS: Seven postmortem human eyes were fixed with formalin. The iris and ciliary body were dissected out and embedded in paraffin. The expression of MMPs -1, 2, 3, and 9, and TIMPs 1-4 in the iris and ciliary body was determined by a novel immunofluorescence technique and the results graded by masked observers. RESULTS: Positive staining for MMPs and TIMPs was observed in all regions of the anterior uvea, and was more intense in the ciliary body than in the iris. Most MMPs and TIMPs showed similar patterns in their distribution. In the ciliary body, staining was strongest in the epithelium, and was localised to the epithelial cell cytoplasm, except for TIMP-3 which was strongly expressed in the basement membranes. In the iris, staining was most noticeable in the anterior border and anterior epithelial layer. Blood vessels in the stroma of the iris and ciliary body also stained moderately for MMPs and TIMPs. CONCLUSION: Both MMPs and TIMPs are widely expressed in the anterior uvea, with a positive correlation between their expressions. Their differential localisation in the ciliary body suggests they may have a role in maintaining homeostasis in the uveal tract.     

睫状体暴露及小梁支撑术治疗青光眼的临床研究

目的 探讨睫状体暴露及小梁支撑术治疗青光眼的手术方法。方法 手术32例(32眼)，其中急闭17例，慢闭15例。术前眼压21～77mmHg，平均37．1mmHg。术前视力无光感3眼，光感～0．049眼，0．05～0．26眼，0．3及以上14眼。结果 术后观察治疗3～6月，术后1周平均眼压9．3mmHg，术后3～6月平均眼压13．3mmHg(1mmHg=0．133kPa)，术中无特殊并发症。术后并发症：①浅前房11例：I度7例。Ⅱ度4例，②前房积血5例，制动后吸收。滤过泡：术后3～6月，I级滤过泡28眼，Ⅱ级滤过泡4眼，无Ⅲ级者。结论 睫状体暴露及小梁支撑术术后超滤所致并发症明显减少，临床效果肯定。     

体外培养猴眼小梁细胞及睫状肌细胞中组织金属蛋白酶抑制剂的表达

目的：观察体外培养的正常猴眼小梁细胞及睫状肌细胞中组织金属蛋白酶抑制剂（tissue inhibitors of metalloproteinase,TIMP)的表达,探讨生理状态下基质金属蛋白酶（matrix metalloproteinase,MMP)及TIMP在小梁网房水流出及葡萄膜－巩膜房水流出的通道中的作用。方法：采用Reverse-zymography技术,检测猴眼小梁细胞及睫状肌细胞中TIMP及MMP的表达。结果;猴眼小梁细胞及睫状肌细胞培养液中均可见TMIP－1、TIMP－2及TIMP－3表达,小梁细胞中MMP／TIMP比值明显高于睫状肌细胞。结论：正常状态下小梁细胞外基质的降解能力明显高于睫状肌细胞,可能是由于传统的小梁网房水流出通道作用强于葡萄膜－巩膜房水流出通道 的作用。     

Specific receptor sites for PAF in iris and ciliary body of the rabbit eye

The existence of specific binding sites by using tritiated 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (3H-PAF) was investigated on iris and ciliary body tissue of pigmented rabbit eyes. The binding was saturable, specific, time-dependent and reversible. Scatchard's analysis indicated the presence of two types of binding sites with a Kd1 4.90 +/- 0.47 nM, a Kd2 11.60 +/- 0.33 nM, a Bmax1 3.17 +/- 0.50 pmol/mg protein and a Bmax2 12.45 +/- 2.30 pmol/mg protein for iris tissue, and a Kd1 5.71 +/- 0.09 nM, a K2d 24.40 +/- 0.91 nM, a Bmax1 3.41 +/- 1.00 pmol/mg protein and a Bmax2 16.60 +/- 0.51 pmol/mg protein for ciliary body. The binding was fully displaced by unlabelled PAF in both iris and ciliary body preparations, and partially inhibited by the PAF antagonist BN 52021 in iris tissue.     

性激素受体在牛眼小梁细胞中的表达

目的了解牛眼小梁组织是否会表达性激素受体。方法体外培养新生牛眼小梁细胞，传至3代。用逆转录聚合酶链反应的方法检测牛眼小梁组织中性激素受体蛋白mRNAs(AR，EPα和PR)的表达，用免疫组织化学技术来定位这些受体。结果体外培养细胞符合牛眼小梁细胞的特点。RT-PCR检测到这些受体的mRNAs，AR、ER、PR免疫组化染色结果呈阳性。结论牛眼小梁细胞有性激素受体表达，这些受体在房水排除及青光眼发生方面可能有重要的病理生理学意义。     

Expression of HLA by the human trabecular meshwork and corneal endothelium

By using immunohistochemical techniques, we demonstrated that HLA class I (A, B and C) and HLA class II (DR), the major histocompatibility antigens in man, are expressed constitutively by cells of the trabecular meshwork/Schlemm's canal system and corneal endothelium, as well as by the conjunctival epithelium, Langerhans cells, vascular endothelium and uvea. Because clinical studies indicate that these antigens are involved in mediating corneal graft rejection and possibly in glaucomatous disease of the eye, the presence of both class I and II HLA in the corneal endothelium and in the trabecular cells has important implications for an understanding of immune disorders in the anterior segment of the eye. The presence of HLA on trabecular cells raises the possibility that these antigens potentiate the recognized role of Langerhans cells at the limbus and that they participate in, and/or regulate, the maintenance and defense of the aqueous outflow pathway. Our findings also open up the possibility of using HLA as a genetic marker in the determination of susceptibility to these disorders in man.     

Bradykinin activation of extracellular signal-regulated kinases in human trabecular meshwork cells

Bradykinin stimulation of B₂ kinin receptors has been shown to promote matrix metallo-proteinase (MMP) secretion from trabecular meshwork cells and to increase conventional outflow facility. Because acute secretion of MMPs can be dependent on the activity of extracellular signal-regulated MAP kinases (ERK1/2), experiments were performed to determine bradykinin effects on ERK1/2 in cultured human trabecular meshwork cells and the relationship of these effects to MMP-9 release. Treatment of cells with bradykinin produced a rapid 4-to 6-fold increase in ERK1/2 phosphorylation. Stimulation of ERK1/2 activity peaked within 2 min and then declined to control levels by 60 min. The response maximum occurred with 100 nM bradykinin and the estimated EC₅₀ was 0.7 nM. Treatment of cells with the B₂ kinin receptor agonist, Tyr⁸- bradykinin, also stimulated ERK1/2 phosphorylation while the B₁ agonist, Lys- [Des-Arg⁹]- bradykinin had no significant effect. In addition, activation of ERK1/2 by bradykinin or Tyr⁸- bradykinin was blocked by the selective B₂ receptor antagonist, Hoe-140. Inhibition of MAP kinase kinase (MEK) with U0126 also blocked bradykinin-induced ERK1/2 phosphorylation. Suppression of protein kinase C activity with the nonselective inhibitor, GF109203X, or by down-regulation with phorbol ester, diminished, but did not eliminate, bradykinin activation of ERK1/2. A similar decrease of ERK1/2 stimulation was observed when Src kinase was inhibited by 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Finally, blockade of bradykinin-induced ERK1/2 activation substantially reduced the peptide’s action to stimulate MMP-9 release into the extracellular environment. The data demonstrate that bradykinin promotes ERK1/2 activation in human trabecular meshwork cells. The effect is mediated by B₂ kinin receptors, involves two different signaling pathways, and results in increased secretion of MMP-9.     

Collagen distribution in the lamina cribrosa and the trabecular meshwork of the human eye.

Tengroth and Ammitzböll found the collagen content of the optic disc in glaucoma to differ from that of normal eyes. A theory was advanced that a primary collagen disturbance might be involved in the pathogenesis of glaucoma. The connective tissue in the body has a supportive function in almost all the organs. The tensile strength and elasticity of connective tissue is mainly due to the presence of collagen fibres and elastic fibres, which also maintain the shape of the tissues. There are many different types of collagen, three of which are discussed in this paper. Type I collagen is found in tendons, skin, and numerous other organs, for example the eye. Type III is found mainly in the blood vessels but is also present in other tissues with a mesodermal origin, and type IV is found in the basement membranes. To elucidate the precise distribution of collagen types in the ocular structures an immunhistochemical study was undertaken in normal human eyes. The amino acids proline, hydroxyproline, and hydroxylysine, which are characteristic of collagen, were also analysed. Collagen types I, III, and IV were found in the lamina cribrosa, the trabecular meshwork, and the retrolaminary optic nerve. In contrast, only type I was found in the sclera.