正常角膜上皮细胞单纯疱疹病毒I型的检测

单纯疱疹病毒（herpes simplex virus,HSV)在眼部感染主要为HSV-Ⅰ型，其最主要的生物学科特性是能够形成潜伏感染，目前已公认HSV-Ⅰ可以潜伏在静止期单纯疱疹病毒性角膜炎（herpes simplex keratitis,HSK）角膜组织中。针对正常角膜内是否有HSV-Ⅰ的存在，近几的国内外学者采用聚合酶链反应（polymerase chain reaction,PCR）检测到正常角膜内存有HSV-Ⅰ DNA。     

单纯疱疹病毒性角膜炎复发及治疗的研究进展

单纯疱疹病毒性角膜炎(herpes simplex keratitis,HSK)是由单纯疱疹病毒(HSV)引起的一种严重的感染性角膜疾病。HSV的感染有两种形式：原发性感染和复发性感染。其中,复发性HSV感染的相关机制和干预方式是近些年来的研究焦点。本文介绍了几种常见的HSK的复发免疫机制及治疗,分析了最新研究发现的各种抗HSV复发的药物,希望能尽早开发出新型的干预方式,开发出具有较强疗效和较小毒性的化合物,使其减少潜在的副作用,提供更好的治疗效果。     

单纯疱疹病毒性角膜炎的研究进展

单纯疱疹病毒(herpes simplex virus,HSV)引起的角膜感染称为单纯疱疹病毒性角膜炎,简称单疱角膜炎(Herpes simplex keratitis,HSK)。HSK有原发和继发两种,原发者约10%有临床症状,故极为少见。临床多为继发性,继发性者只有在某种非特异性刺激如发烧、感冒、外伤等诱因及机体抵抗力低下的时候才会发病。本病多见于青壮年,多为单眼。角膜病变表现为多种形     

可溶性B、T淋巴细胞衰减因子及其配体在单纯疱疹性角膜基质炎小鼠体内的异常表达

目的 研究B、T淋巴细胞衰减因子(B and T lymphocyte attenuator,BTLA)及其配体疱疹病毒侵入介体(herpes virus entry mediator,HVEM)在单纯疱疹性角膜基质炎(herpetic stromal keratitis,HSK)小鼠角膜组织中及外周血CD4+T细胞上的表达水平,探讨共抑制信号BTLA-HVEM是否参与了CD4+T细胞介导的HSK免疫病理反应.方法 将106 PFU的单纯疱疹病毒Ⅰ型(herpes simplex virus type 1,HSV-1)KOS毒株接种于BALB/c鼠的角膜上建立HSK动物模型,分别于角膜接种病毒前(0 d),接种病毒后的第3、7、10、14、21天,用毛细管取小鼠的左眼眼眶静脉窦血1 mL,分离淋巴细胞,行荧光抗体染色,用流式细胞仪检测CD3+ CD4+ BTLA+T细胞和CD3+ CD4+ HVEM+T细胞阳性率;在裂隙灯显微镜下观察小鼠角膜变化;免疫组织化学方法检测BTLA蛋白及HVEM蛋白在角膜组织中的表达.结果 BALB/c鼠的角膜接种HSV-1后的1～5d,角膜擦拭液中均检测出HSV-1复制,表明小鼠感染了单纯疱疹病毒.裂隙灯显微镜观察显示:角膜接种HSV-1后第3天,所有小鼠均患了急性上皮性角膜炎,并于感染后1周内痊愈,自病毒接种后第8天起,小鼠出现角膜基质炎的改变,表现为角膜基质呈灰白色混浊,角膜基质混浊于病毒接种后的第10天达到高峰,持续至第14天后逐渐减轻.流式细胞仪检测显示,小鼠外周血淋巴细胞中CD3+CD4+BTLA+T细胞和CD3+ CD4+ HVEM+T细胞的阳性率,在角膜接种病毒前(0 d)分别为(3.15±0.60)％和(9.84±1.06)％,在角膜接种病毒后第10天(HSK疾病程度最严重时)分别增加到(20.47±3.15)％和(45.18±3.90)％(与0d相比差异均有显著统计学意义,均为P＜0.01).免疫组织化学方法检查HSK小鼠角膜组织中BTLA和HVEM的蛋白表达结果一致:HSK临床表现最严重时,即病毒接种后第10天时,BTLA蛋白和HVEM蛋白在角膜组织中表达最强,主要表达于角膜基质层内浸润的炎性细胞上,角膜上皮层和内皮层也有表达.结论 在HSK小鼠模型中,BTLA及其配体HVEM蛋白在角膜组织中及外周血CD4+T细胞上表达明显增强,共抑制信号BTLA-HVEM参与了CD4+T细胞介导的HSK的免疫病理过程.     

单纯疱疹病毒性角膜炎的临床特点及诊疗思维

目前临床上眼部病毒感染性疾病以单纯疱疹病毒(herpes simplex virus,HSV)、巨细胞病毒(cy-tomegalovirus,CMV)和腺病毒为主要感染源。其中以HSV感染引起的单纯疱疹病毒角膜炎为最常见,其患病率近年来有明显上升趋势,在角膜病中其致盲率占第一位。由于反复发作,重症病例增多,严重威胁视功能。特别是其临床分类多样、表现差异性     

单纯疱疹病毒性角膜炎的免疫学

单纯疱疹病毒性角膜炎 (herpes simplex keratitis,HSK)是世界范围内角膜盲的最常见原因。单纯疱疹病毒 (herpes simplexvirus,HSV)不仅可直接导致角膜的感染性破坏 ,而且还可通过免疫病理机制诱导某些难以处理的疾病 ,如基质型角膜炎。1　免疫学机制1 .1　动物模型　目前 ,已有数种 HSK的动物模型 ,比较常用的是兔模型和小鼠模型。兔模型比较理想 ,因为兔眼球大小与人类相似易于观察 ;另外 ,存在有自发性病毒释放和疾病复发现象。而小鼠模型则缺乏病毒的自发性释放和疾病的复发 ,但在感染后经过一段时间才发生角膜的炎症反应 ,该特征类…     

单纯疱疹病毒性角膜炎动物模型制备

单纯疱疹病毒性角膜炎(herpes simplex keratitis,HSK)是由单纯疱疹病毒Ⅰ型(herpessimplex virus type Ⅰ,HSV-1)感染引起的具有高复发率、高致盲率及难根治性特点的疾病,可导致角膜溶解、新生血管形成及溃疡穿孔,是目前角膜病中最常见的致盲眼病之一.本文参考目前国内外实验研究时使用的各种HSK动物模型建模方法来进行综述,如建立原发感染模型的划痕法、环钻法、外植体培养法以及潜伏感染模型和诱导复发模型的建立方法.力求探索如何建立一种可靠的HSK动物模型,在为研究人类HSK发病机制、药物治疗和对比以及角膜移植等方面均有重要意义.     

细胞毒性T淋巴细胞相关抗原-4融合蛋白对鼠单纯疱疹性角膜基质炎抑制作用的研究

研究细胞毒性T淋巴细胞相关抗原-4融合蛋白(cytotoxic Tlymphocyte-associated antigen-4 immunoglobulin,CTIA-4Ig)对鼠单纯疱疹性角膜基质炎(herpetic stromal kerafifis,HSK)的抑制作用。方法用单纯疱疹病毒1型(herpes simplex virus type 1,HSV-1)接种于BALB／c鼠角膜上建立HSK动物模型,采用CTIA-4Ig阻断B7：CD28／CTIA-4协同刺激途径,抑制T淋巴细胞增殖、分化为效应细胞;观察HSK的发病率、临床特征、角膜组织学改变、角膜病毒滴度、迟发型超敏反应及抗原刺激脾细胞分泌细胞因子的情况。结果CTIA-4Ig可减少小鼠外周血中CD4^ T淋巴细胞(81．6％)及CD8^ T淋巴细胞(67．9％),阻止鼠发生HSK、减轻角膜混浊程度及角膜内炎性细胞的浸润、损伤鼠的迟发型超敏反应能力,抑制鼠脾细胞分泌辅助性T淋巴细胞1型(T-helper 1,Th1)细胞因子;但不影响角膜病毒滴度及小鼠死亡率。结论用CTIA-4Ig阻断B7：CD28／CTIA-4协同刺激途径,能够抑制T淋巴细胞增殖并抑制CIM^ Th1细胞的功能,阻止HSK发病,减轻HSK的严重程度。     

T淋巴细胞因子与单纯疱疹病毒性角膜炎的研究进展

单纯疱疹病毒性角膜炎(herpes simplex keratitis,HSK)是由病毒抗原引起的T淋巴细胞介导的迟发型变态反应。T淋巴细胞如何介导HSK免疫反应的详细机制目前仍不清楚,但从目前研究可总结:T淋巴细胞的细胞因子如IL-2,IL-10,IL-17,IFN-γ等促炎性因子在HSK的复发过程中起重要作用。本文将对T淋巴细胞及其细胞因子与HSK的研究进展做简要综述。     

Sensory Nerve Retraction and Sympathetic Nerve Innervation Contribute to Immunopathology of Murine Recurrent Herpes Stromal Keratitis

PurposeHerpes stromal keratitis (HSK) represents a spectrum of pathologies which is caused by herpes simplex virus type 1 (HSV-1) infection and is considered a leading cause of infectious blindness. HSV-1 infects corneal sensory nerves and establishes latency in the trigeminal ganglion (TG). Recently, retraction of sensory nerves and replacement with “unsensing” sympathetic nerves was identified as a critical contributor of HSK in a mouse model where corneal pathology is caused by primary infection. This resulted in the loss of blink reflex, corneal desiccation, and exacerbation of inflammation leading to corneal opacity. Despite this, it was unclear whether inflammation associated with viral reactivation was sufficient to initiate this cascade of events.MethodsWe examined viral reactivation and corneal pathology in a mouse model with recurrent HSK by infecting the cornea with HSV-1 (McKrae) and transferring (intravenous [IV]) human sera to establish primary infection without discernible disease and then exposed the cornea to UV-B light to induce viral reactivation.ResultsUV-B light induced viral reactivation from latency in 100% of mice as measured by HSV-1 antigen deposition in the cornea. Further, unlike conventional HSK models, viral reactivation resulted in focal retraction of sensory nerves and corneal opacity. Dependent on CD4⁺ T cells, inflammation foci were innervated by sympathetic nerves.ConclusionsCollectively, our data reveal that sectoral corneal sensory nerve retraction and replacement of sympathetic nerves were involved in the progressive pathology that is dependent on CD4⁺ T cells after viral reactivation from HSV-1 latency in the UV-B induced recurrent HSK mouse model.     

Herpes simplex virus: molecular biology and the possibility of corneal latency.

Herpes simplex virus (HSV) is known to be latent in ganglionic neurons. Over the past eight years, a series of reports have described the isolation of HSV after organ culture of human corneas that had been removed in the course of penetrating keratoplasty. None of the corneas showed any clinical signs of active herpetic disease immediately before keratoplasty. Studies in rabbits and mice confirmed that HSV can be recovered from corneas by organ culture long after primary infection has subsided. Recently, sophisticated techniques of molecular biology, such as specific DNA or RNA probes, have been used to detect HSV nucleic acids in the cornea. The crux of the matter is whether the virus recovered from or detected in the cornea is 1) truly latent in cell populations that are nonneuronal; 2) resident in the cornea, replicating at a slow rate; or 3) newly arrived in the cornea following ganglionic reactivation. The evidence suggests that a guarded case can be made for limited HSV latency within corneal cells. HSV corneal latency would allow for reactivation, replication, and the immune response to occur in the absence of ganglionic HSV reactivation. Such a localized phenomenon has not, however, been demonstrated to occur clinically.     

Immunological Aspects of Herpetic Stromal Keratitis

Herpetic stromal keratitis (HSK) is an immune reaction related to herpes simplex virus (HSV) corneal infection, and has many important immunological aspects. CD4⁺ T lymphocytes, especially Th1 cells, are the principal mediators for HSK. In addition, neutrophils and antigen-presenting cells play vital roles in HSK. CD8⁺ T lymphocytes, B cells, and natural killer cells all participate in the pathogenesis of HSK under certain circumstances. Many molecules are involved in the pathogenesis of HSK. Th1 cytokines such as interleukin 2 (IL-2), IL-12 and interferon γ, and inflammatory cytokines such as IL-1α and IL-6 are especially important ones. Among various chemokines that take part in HSK, MIP-1α is one of the most important aggravating factors. Vaccination therapy against HSK has been developed; glycoprotein D is a particularly promising candidate. However, the possibility of HSK exacerbation due to vaccination is the final problem to be solved before vaccination can be clinically applied to HSK. Molecular mimicry theory and bystander activation theory are the two new autoimmune theories that have been advocated. Since genuine autoimmune HSK without HSV growth can hardly be the case in clinical practice, some part of these new theories remains controversial. In the future, better understanding of the pathogenesis of HSK is essential to resolve the paradox between suppressing the immune reaction to avoid corneal scarring and preventing viral proliferation.     

Pathogenesis of herpes simplex keratitis: The host cell response and ocular surface sequelae to infection and inflammation

Herpes simplex virus type 1 (HSV) keratitis is a leading cause of infectious blindness. Clinical disease occurs variably throughout the cornea from epithelium to endothelium and recurrent HSV stromal keratitis is associated with corneal scarring and neovascularization. HSV keratitis can be associated with ocular pain and subsequent neutrophic keratopathy. Host cell interactions with HSV trigger an inflammatory cascade responsible not only for clearance of virus but also for progressive corneal opacification due to inflammatory cell infiltrate, angiogenesis, and corneal nerve loss. Current antiviral therapies target viral replication to decrease disease duration, severity and recurrence, but there are limitations to these agents. Therapies directed towards viral entry into cells, protein synthesis, inflammatory cytokines and vascular endothelial growth factor pathways in animal models represent promising new approaches to the treatment of recurrent HSV keratitis.     

Herpetic imprint on privileged areas of its target organs: local latency and reactivation in herpetic keratitis

Herpetic ocular disease is still one of the major causes of corneal blindness. Due to its unique morphological structure, the eye is one of the most studied organs--in both clinical and experimental models of herpetic infections. Herpes simplex virus (HSV) can react with human host cells to produce cytocidal infection, persistent infection, or latent infection. Whilst the establishment of viral latency in the sensory ganglia was demonstrated and extensively studied, recent evidences based on: (a) demonstration of viral particles by electron microscopy and of HSV positive antigen cells in human corneae with inactive stromal keratitis; (b) experimental animal and in vitro studies with the use of organ cultures, co-cultivation methods and molecular biology techniques; suggest the possibility of local latency in non-neural tissues as an additional source of dormant viruses that could reactivate and lead to local reinfection of the affected target organ. Reactivation of a herpetic infection may therefore require both the existence of a dormant herpetic reservoir in the ganglia, and a predilected target organ with possible independent mechanisms of local latency and reactivation. Possible mechanisms of triggers for reactivation of herpetic ocular disease are discussed.     

单纯疱疹病毒1功能性基因在角膜内潜伏感染的实验研究

目的 研究角膜中是否有单纯疱疹病毒１型功能性基因的潜伏。方法 在新西兰白兔角膜上制作典型的单纯疱疹病毒１型引发的角膜炎模型,然后将进入稳定期的病变角膜移植到健康兔眼上,术后２周取下植片。每下植片标本均分为三部分,一部分角膜片先行ＨＳＶ－１抗原检测,另一部分采用多聚酶链反应技术检测ＨＳＶ－１潜伏相关转录、胸苷激酶和ＤＮＡ聚合酶基因,第三部分经器官培养３周后,再与兔原代肾细胞一起培养１周,再检测培养后     

Herpes simplex stromal and endothelial keratitis. Granulomatous cell reactions at the level of Descemet's membrane, the stroma, and Bowman's layer

Fifty-three (25%) of 215 keratectomy specimens of patients with herpes simplex stromal keratitis displayed granulomatous reactions at the level of Descemet's membrane (50/53), midstroma (13/53), and Bowman's layer (5/53). Using an immunoperoxidase technique, herpes simplex virus (HSV) antigens were detected in keratocytes, endothelial cells, and foci of epithelioid histiocytes and multinucleated giant cells around Descemet's membrane. Both granulomatous reactions and HSV antigens were identified significantly more often in specimens with ulcerative necrotizing stromal keratitis than in those from patients with stromal scarring or nonulcerative nonnecrotizing keratitis (P less than 0.00001 and P less than 0.005, respectively). Herpes simplex virus antigens also were present in endothelial cells adjacent to foci of granulomatous reactions around Descemet's membrane in association with disciform stromal scarring. To our knowledge, this is the first demonstration of HSV antigens in human corneal endothelial cells and in the granulomatous reactions at the level of Descemet's membrane.     

Production of Key Molecules by Ocular Neutrophils Early After Herpetic Infection of the Cornea

Herpes simplex virus infection of the eye can result in a blinding inflammatory lesion that is a T cell mediated immunopathological reaction. A prominent early event following HSV infection is neutrophil invasion of the corneal stroma. These cells may be involved in viral clearance and may influence the nature of the anti HSV T cell response which subsequently occurs. This article measures the expression of some key molecules which could participate in viral clearance and immune modulation. Using RT-PCR and in-situ hybridization, both corneal and peritoneal neutrophils were shown to be sources of iNOS and TNFαmolecules which likely contribute to antiviral activity. Neutrophils also produce the cytokine IL-12, a key molecule which modulates the CD4+T cell response to a type which mediates immunopathology. The present results indicate that neutrophils play an important role in the pathogenesis of herpetic ocular lesions.