Viral causes of unexplained anterior uveitis in Thailand

Aims

To assess the possible role of virus infection in patients with unexplained anterior uveitis (AU).

Methods

Intraocular fluid and plasma samples of 30 HIV-negative AU patients who were unresponsive or poorly responsive to topical steroid therapy were analyzed for nucleic acid of cytomegalovirus (CMV), herpes simplex virus (HSV), and varicella zoster virus (VZV) by real-time polymerase chain reaction (PCR) and for intraocular antibodies against these viruses by Goldmann–Witmer coefficient (GWC) analysis. Of these 30 cases, 21 were tested for rubella virus by GWC analysis, 16 of which also had PCR assessment of aqueous for rubella virus.

Results

Viral uveitis determined by either real-time PCR and/or GWC was documented in 20 out of 30 patients (67%). Of 30 paired samples tested by both methods for HSV, CMV, and VZV, 15 showed positive results (CMV (10), HSV (4), and VZV (1)). Real-time PCR was positive in 8/15 (53%), whereas GWC was positive in 10/15 (67%). Out of 10 CMV-positive patients, four had endotheliitis, two had Posner–Schlossman syndrome, and one Fuchs heterochromic uveitis syndrome (FHUS). Five out of 21 (24%) samples tested by GWC for Rubella virus were positive, three of which exhibited clinical features of FHUS.

Conclusions

Our results indicate that CMV is a major cause of AU in Thailand and show that FHUS can be caused by both CMV and Rubella virus.     

BIGH3基因的Arg555Trp突变引起颗粒状角膜营养不良

目的　阐明颗粒状角膜营养不良(CDGGⅠ )的发病机制。 方法　采用聚合酶链式反应 -单链构象多态性(PCR SSCP)分析,对 1个CDGGⅠ病例进行BIGH3基因第 12号外显子的突变筛查,并对发现的异常泳动带进行DNA测序,以确定突变位点。 结果　CDGGⅠ患者在 12号外显子有Arg555Trp突变。 结论　BIGH3基因突变Arg555Trp是导致CDGG1的原因。     

RANTES与SPARC基因在大鼠角膜移植排斥反应中的表达

目的：动态检测激活正常T细胞表达和分泌的调节物（regulated on activation nomal T expressed and secreted,RANTES)和酸性富含胱氨酸分泌型蛋白（secreted protein,acidic and rich in cysteine,SPARC)在异体角膜移植排斥反应中的表达，为角膜移植排斥反应的早期诊断提供依据。方法：首先建立大鼠异体角膜移植动物模型，并设同体角膜移植，角膜碱烧伤模型为对照组，在不同时间段提取各组角膜上皮细胞总RNA。分别进行逆转录－聚合酶链反应（RT－PCR）扩增，电泳，半定量检测RANTES和SPARCmRNA的表达水平，然后酶切验证表达结果。结果：（1）正常角膜上皮细胞未见RANTES基因表达，可见SPARC基因的低表达；（2）异体角膜移植3天后各时间点均见排斥眼RANTES基因的高表达，同体移植及碱烧伤后未见表达。（3）SPARC在角膜移植及碱烧伤后均见较高水平表达，且与正常细胞表达有显著差异；（4）用RT－PCR方法检测基因变化诊断排斥反应早于裂隙灯检查结果。结论：大鼠角膜上皮RANTESmRNA表达上调与角膜移植排斥反应特异性相关。同时，SPARC在角膜移植排斥反应，碱烧伤和创伤过程中具有高水平表达，依时间顺序，RANTES和SPARC早期高表达早于临床观察的排斥反应至少一周，由此，选择性对细胞因子mRNA扩增，是早期诊断角膜移植排斥反应的有效方法。     

大鼠视网膜中转化生长因子β1和β2基因表达的定量检测(英文)

目的:定量检测转化生长因子-β1(Transforming growth factor β1,TGF-β1)和转化生长因子-β2(TGF-β2)在大鼠正常视网膜中的表达水平,探讨TGF-β1和TGF-β2在视网膜中表达的差异及其意义。方法:分离取出大鼠正常视网膜,抽提RNA并逆转录,实时荧光定量PCR技术分析TGF-β1和TGF-β2的mRNA含量。结果:大鼠视网膜RNA保持完好未被降解,能够用于表达水平分析。大鼠视网膜中TGF-β2相对于β-actin的基因表达水平为0.0378±0.009,TGF-β1为0.0008±0.0003,前者明显高于后者,统计学上差异有显著性(t=12.37,P<0.001),说明在视网膜中TGF-β的表达以TGF-β2为主,TGF-β2和TGF-β1的比值为55.00±26.61。结论:实时荧光定量PCR技术能够针对性地精确分析极少量组织细胞的基因表达。TGF-β在视网膜中以TGF-β2表达为主,提示可能是TGF-β2在视网膜病变中起主导作用。     

接合黏附分子-1在大鼠角膜中的表达研究

背景 接合黏附分子-1(JAM-1)是新发现的跨膜蛋白,参与细胞紧密连接的结构组成和功能发挥.在眼组织方面,紧密连接对维持角膜的透明性十分重要,但是目前就JAM-1在角膜紧密连接结构和功能方面的研究较少. 目的 确定JAM-1在大鼠角膜上皮层、基质层和内皮层的构成.方法 选取4只SPF级Wistar大鼠,2只用于JAM-1基因在角膜组织中表达的逆转录聚合酶链反应(RT-PCR)检测,另2只用于免疫组织化学检测.动物过量麻醉处死后获得角膜组织并制备角膜上皮、基质和内皮标本,RT-PCR法检测角膜标本中JAM-1、occludin和claudin-1 mRNA的表达.反应产物行质量分数1.5％琼脂糖凝胶电泳并用凝胶成像系统进行分析.用兔抗鼠JAM-1单克隆抗体对角膜石蜡切片、上皮及内皮铺片行免疫组织化学检测,评估JAM-1蛋白在大鼠角膜组织各层的表达部位和表达强度. 结果 在大鼠角膜各层均可检测到JAM-1、occludin和claudin-1 mRNA的表达,PCR熔解曲线为清晰的单峰.角膜组织各层中JAM-1 mRNA表达水平与occludin mRNA相似,均高于claudin-1 mRNA.3种黏附分子均在上皮层表达最强,角膜基质层表达较弱.免疫组织化学检测显示,JAM-1蛋白在角膜各层均有明确的阳性染色,角膜上皮基底层的表达强于基质层和内皮层.角膜上皮、内皮铺片检测显示,JAM-1蛋白主要表达于上皮细胞和内皮细胞的连接部位,而角膜内皮中JAM-1蛋白的阳性染色广泛而弥散.结论 JAM-1作为细胞连接的构成成分,在角膜上皮层、内皮层和基质层均有表达,但其表达的形态和水平因组织层次的不同而不同.     

单纯疱疹病毒性角膜炎角膜组织病毒潜伏感染的PCR检测分析

 目的  探讨通过穿透性角膜移植获取的单纯疱疹病毒性角膜炎病变角膜组织中1型单纯疱疹病毒（HSV-1）DNA的表达情况及意义。设计 实验研究。研究对象  2010年5-12月北京同仁医院因病毒性角膜炎角膜白斑行穿透性角膜移植术后角膜标本20例,圆锥角膜、大泡性角膜病变和角膜营养不良等非感染性角膜病变的角膜标本20例。方法  对角膜组织标本中HSV-1 DNA进行聚合酶链反应（PCR）检测。 主要指标  HSV-1 DNA的阳性率。结果  单纯疱疹病毒性角膜炎静止期患者角膜组织中12/20例（60%）检出HSV-1 DNA,非感染性角膜组织中6/20例（30%）检出HSV-1 DNA（χ2=3.64,P=0.057）。结论  单纯疱疹病毒性角膜炎静止期角膜组织多数表达HSV-1 DNA,角膜内潜伏病毒是引起单纯疱疹病毒性角膜炎的可能原因,正常人角膜也可能有HSV-1的DNA存在。（眼科,2013,22：45-48）     

Current approach in the diagnosis and management of posterior uveitis

Posterior uveitic entities are varied entities that are infective or non-infective in etiology. They can affect the adjacent structures such as the retina, vitreous, optic nerve head and retinal blood vessels. Thorough clinical evaluation gives a clue to the diagnosis while ancillary investigations and laboratory tests assist in confirming the diagnosis. Newer evolving techniques in the investigations and management have increased the diagnostic yield. In case of diagnostic dilemma, intraocular fluid evaluation for polymerase chain testing for the genome and antibody testing against the causative agent provide greater diagnostic ability.     

Treatment of acute demyelinating optic neuritis

Patients with typical acute monosymptomatic demyelinating optic neuritis should receive gadolinium-enhanced magnetic resonance imaging (MRI) of the brain and orbits to determine if they are at high risk for the subsequent development of clinically definite multiple sclerosis (CDMS). The presence of ≥2 white matter lesions (≥3 mm in diameter, at least 1 lesion periventricular or ovoid) indicates high risk for CDMS; the following treatment should be considered for such patients: 1. Intravenous methylprednisolone sodium succinate (1 gram IV/day for 3 days) followed by oral prednisone (1 mg/kg/day for 11 days) with 4-day taper (20 mg on day 1, 10 mg on days 2 and 4); 2. Interferon beta 1-a (Avonex 30μg intramuscularly [IM] weekly, or Rebif 22μg subcutaneously [SQ] weekly). These two drugs have been shown to reduce the short-term risk of CDMS in high risk monosymptomatic patients. In monosymptomatic patients with &lt;2 white matter lesions, and in patients for whom CDMS has been established, IV methylprednisolone treatment followed by oral prednisone should be considered on an individual basis. Treatment in these patients may hasten visual recovery, but does not affect long-term visual outcome. Oral prednisone alone, without prior treatment with IV methylprednisolone, may increase the risk for recurrent optic neuritis and should be avoided.     

睑裂狭小综合征一家系的FOXL2基因突变研究

目的确定一个常染色体显性遗传的睑裂狭小综合征（BPES）家系的致病基因及其突变位点和类型。方法应用聚合酶链反应和直接测序技术,对来自同一家系的4例BPES患者及家系中6例正常人和50例正常对照者的外周血DNA进行分子遗传学分析,筛查FOXL2基因的外显子序列。结果来自同一家系的4例BPES患者均发现有FOXL2基因892C〉T的改变,为无义突变,而家系中6例正常人及50例正常对照者的FOXL2基因中均未发现突变。结论FOXL2基因的一种已知突变可能是该BPES家系的重要致病因素。     

人乳头状瘤病毒在翼状胬肉发病机制中的作用

背景分子生物学研究表明,翼状胬肉为肿瘤型组织。国外一些地区的研究表明,翼状胬肉中均表达人乳头状瘤病毒（HPV）,但国内尚未见类似报道。目的定量检测翼状胬肉中HPV的表达,探讨HPV在无锡地区翼状胬肉患者发病机制中的作用。方法收集无锡市第二人民医院眼科行翼状胬肉切除术的手术标本33例48眼,其中包括6例7眼复发性翼状胬肉标本和27例41眼初发性翼状胬肉标本,翼状胬肉组织研磨后提取上清液中HPV16／18DNA、HPV6／11DNA,用荧光定量聚合酶链反应（FQ—PCR）法定量检测标本中HPV的含量,以荧光扩增曲线表示。2例人宫颈癌组织和2例人正常供体结膜组织分别作为阳性对照组和阴性对照组。结果翼状胬肉、正常结膜和宫颈癌组织中的HPV6／11扩增曲线均位于临界阳性质控曲线之下;但宫颈癌组织中HPV16／18扩增曲线位于临界阳性质控曲线之上,而翼状胬肉组织和正常结膜组织扩增曲线处于临界阳性质控曲线之下。2例人宫颈癌组织中HPV16／18检测结果为阳性（2／2）,HPV6／11检测结果为阴性（0／2）。翼状胬肉标本及正常结膜组织中HPV16／18、HPV6／11检测结果均为阴性（0／50）。结论HPV不是翼状胬肉发生和复发的必要条件,无锡地区翼状胬肉中的HPV感染率较低。     

A novel mutation of sgk-1 gene in central serous chorioretinopathy

AIM

To investigate the association of serum glucocorticoid kinase gene-1 (SGK-1) DNA variants with chronic central serous chorioretinopathy (CSC).

METHODS

We enrolled 32 eyes of 32 patients who were diagnosed with chronic CSC and composed 32 normal eyes as a control group. Peripheral blood was used for DNA extraction and polymerase chain reaction (PCR) amplification. SGK1 gene was sequenced by using BigDye^® Terminator v3.1 cycle sequencing KIT (Applied Biosystems, Foster City, CA, USA). The SGK1 gene and its variants were investigated in CSC patient group and control group.

RESULTS

We identified a new polymorphism M32V in two person in the patient group (Minor allele frequency (MAF)=0.009) on the region of 1-60 amino acids. The rs1057293 was located in the encoder region of the SGK 1 gene but not associated with CSC (P=0.68). An intrinsic rs1743966 is also not associated (P=0.28).

CONCLUSIONS

The new polymorphism M32V is located on the region of 1-60 amino acids which is necessary for localization to the mitochondria in CSC patient. This mutation is probably important for the energy metabolism and plays an important role in the cellular response to hyperosmotic stress and other stress stimuli. Both rs1057293 and rs1743966 are not associated with CSC.     

Study of a Polymerase Chain Reaction-based Method for Detection of Herpes Simplex Virus Type 1 DNA among Iranian Patients with Ocular Herpetic Keratitis Infection

Purpose To study the presence of the herpes simplex virus type 1 (HSV-1) glycoprotein D gene in tear films of Iranian patients with herpetic keratitis.Methods Twenty-five tear film and eye swab specimens from 25 herpetic keratitis patients and 10 specimens from 10 healthy volunteers were collected in the Farabi Eye Hospital, Tehran, Iran. HSV-1 DNA was detected by using the nested polymerase chain reaction (nPCR) method. Viral isolation was done using conventional viral techniques. A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was used for confirmation of positive cytopathic effect cell culture. The results of a diagnosis by an ophthalmologist team were compared with those of nPCR.Results HSV-1 DNA was identified in tear films of 88% (23/25) of suspected herpetic keratitis patients. All healthy controls (100%) had negative PCR results. HSV-1 was isolated in cell culture and confirmed by ELISA in 12% (3/25) of herpetic keratitis patients who had epithelial keratitis. The kappa value showed a high level of agreement between ophthalmologist team diagnosis and the PCR results (kappa = 0.86, P < 0.0001).Conclusions nPCR is a sensitive, rapid, and powerful tool for detection of HSV-1 DNA in tear films of ocular herpetic keratitis patients and can serve as a supplemental method for diagnosis of herpetic keratitis infection. Jpn J Ophthalmol 2004;48:328–332 © Japanese Ophthalmological Society 2004     

大鼠视网膜中转化生长因子β1和β2基因表达的定量检测

目的：定量检测转化生长因子-β1（Transfimning growth factorβ1，TGF-β1）和转化生长因子-β2（TGF-β2）在大鼠正常视网膜中的表达水平，探讨TGF-β1和TGF-β2在视网膜中表达的差异及其意义。方法：分离取出大鼠正常视网膜，抽提RNA并逆转录，实时荧光定量PCR技术分析TGF-β1和TGF-β2的mRNA含量。 结果：大鼠视网膜RNA保持宅好未被降解，能够用于表达水平分析。大鼠视网膜中TGF-β2相对于β-actin的基因表达水平为0．0378±0．009，TGF-β1为0．0008±0．0003．前者明显高干后者，统计学上差异有显著性（t=12．37，P〈0．001），说明在视网膜中TGF-β的表达以TGF-β2为主，TGF-β2和TGF-β1的比值由55．00±26．61。 结论：实时荧光定量PCR技术能够针对性地精确分析极少量组织细胞的基因表达。TGF-β在视网模中以TGF-β2表达为主，提示可能是TGF-β2在视网膜病变中起主导怍用。     

评价VSX1突变与圆锥角膜合并角膜颗粒状营养不良在伊朗家族中的关联

目的:为了评估视觉系统同源框1(VSX1)基因的突变和圆锥角膜(KCN)以及颗粒状角膜营养不良(GCD)之间的关联.方法:对一个同时患有KCN和GCD的四代伊朗人家系进行了直接测序,鉴别出一个包含四代人同时患有GCD的伊朗KCN家系.从全血样品中提取基因组DNA.然后,为了研究KCN和GCD之间可能的连锁关系,通过PCR在每个样品中扩增VSX1基因的整个编码区和内含子-外显子边界.随后,对PCR产物进行直接测序,并在患者和对照组中进行突变分析.结果:VSX1基因突变分析未发现KCN和GCD疾病与VSX1基因相关的证据.我们的数据排除了VSX1作为该特定家系中KCN / GCD致病基因的可能性.结论:尽管患有GCD的KCN患者与VSX1基因变异无关联,但是仍需要对其它可能与KCN合并GCD发病机制相关的基因进行研究.     

白介素1β在干眼患者眼表的表达

目的 探讨白介素1β（IL-1β）在干眼患者眼表的表达,及其与干眼症状、体征的相关性,阐明IL-1β在干眼发病中的作用.方法 临床试验研究.选取2012年9月至2013年2月来山西省眼科医院就诊的干眼患者30例（60眼,干眼组）,无干眼症状体征且年龄与性别构成匹配的个体15例（30眼）作为对照组.所有受检者均做如下检测：眼表失衡指数（OSDI）干眼调查问卷、泪液分泌试验（SIT）、泪膜破裂时间（BUT）、角膜荧光素钠染色、结膜丽丝胺绿染色.同时应用印迹细胞法收集所有2组对象球结膜上皮细胞,分别进行免疫组化染色,以及实时PCR检测结膜细胞中IL-1β的基因表达.采用独立样本t检验,Spearman相关进行数据分析.结果 干眼组与对照组OSDI评分值分别为24.1±2.2、14.3±1.3;SIT分别为（4.13±1.68）mm、（10.53±0.74） mm;BUT分别为（4.17±1.10）s、（8.80±1.21）s;角膜荧光素钠染色评分值分别为4.3±1.5、0.6±0.5;结膜丽丝胺绿染色评分值分别为5.7±2.0、1.9±1.4.干眼组与对照组以上5个指标比较差异均有统计学意义（t=22.23、-19.88、-18.20、12.85、9.62,P＜0.05）.干眼组IL-1β相对基因含量为0.65±0.37,对照组为0.22±0.06,二者比较差异有统计学意义（t=6.31,P＜0.05）.干眼组IL-1β在结膜上皮细胞中的表达与OSDI评分、角膜荧光素钠染色评分、结膜丽丝胺绿染色评分呈正相关（r=0.81、0.58、0.48,P＜0.05）,与SIT、BUT结果呈负相关（r=-0.43、-0.45,P＜0.05）.通过免疫组化法染色,在干眼患者结膜细胞中可见棕褐色颗粒,而在正常人群中未见棕褐色颗粒.结论 IL-1β作为炎性介质不仅参与了干眼的发病,与干眼的严重程度相关,同时也引起了眼表的损害.     

聚合酶链反应检测角膜内皮炎房水中单纯疱疹病毒Ⅰ型DNA及带状疱疹病毒DNA

目的 评价聚合酶链反应技术（PCR）对角膜内皮炎的诊断价值，并以此解释病因，指导临床治疗。方法对临床诊断为角膜内皮炎的患者12例（12眼）及临床诊断为年龄相关性白内障的患者15例（15眼）分别用PCR方法进行房水中单纯疱疹病毒Ⅰ型（HSV-Ⅰ）DNA及带状疱疹病毒（VZV）DNA的检测，结果采用分类变量资料两样本率比较的四格表确切概率比较。结果共扩增角膜内皮炎患者12例（12眼），HSV-Ⅰ阳性者5例（5眼），阳性率为41．67％，共扩增对照组15例（15眼），HSV-Ⅰ阳性者0例，阳性率为0％，二者差异有显著性。结论PCR技术检测角膜内皮炎患者房水中的HSV-Ⅰ，可在分子水平上建立一种快速准确的诊断方法，为临床治疗提供可靠的依据。     

The immunoregulatory role of corneal epithelium-derived thrombospondin-1 in dry eye disease

Purpose

In this study, we examine the expression of corneal epithelium-derived thrombospondin-1 (TSP-1) and its immunomodulatory functions in a validated murine model of dry eye disease (DED).