Detection of herpes simplex virus type 1, 2 and varicella zoster virus DNA in recipient corneal buttons

AIM: To study the value of polymerase chain reaction (PCR) analysis, to detect viral DNA in recipient corneal buttons taken at the time of penetrating keratoplasty (PKP) in patients with an initial diagnosis of herpetic stromal keratitis (HSK). Since HSK has a tendency to recur, an accurate diagnosis of previous HSK could be the reason to start antiviral treatment immediately, thereby possibly decreasing the number of graft failures due to recurrent herpetic keratitis. METHODS: Recipient corneal buttons and aqueous humour (AH) samples were obtained at the time of PKP from HSK patients (n=31) and from other patients (n=78). Eye bank corneas were also used (n=23). Herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), and varicella zoster virus (VZV) infection were assessed by PCR and antibody detection. RESULTS: The clinical diagnosis HSK could be confirmed by PCR for HSV-1 in 10/31 (32%). In these corneal buttons HSV-2 DNA was detected in 1/31 (3%) and VZV DNA in 6/31 (19%). Intraocular anti-HSV antibody production was detected in 9/28 AH samples tested (32%). In the other patient derived corneas HSV-1 DNA was detected in 13/78 (17%), including eight failed corneal grafts without clinically obvious herpetic keratitis in the medical history. In clear eye bank corneas HSV-1 was detected in 1/23 (4%). CONCLUSIONS: PCR of HSV-1 on corneal buttons can be a useful diagnostic tool in addition to detection of intraocular anti-HSV antibody production. Furthermore, the results were suggestive for the involvement of corneal HSV infection during allograft failure of corneas without previous clinical characteristic signs of herpetic keratitis.     

Improvement of HSV-1 necrotizing keratitis with amniotic membrane transplantation

PURPOSE: Stromal herpes simplex virus keratitis (HSK) is an immune-mediated disease. Previous studies have indicated that T cells, neutrophils, and macrophages contribute to the tissue damage in HSK. It has been shown that human amniotic membrane promotes epithelial wound healing and has diverse anti-inflammatory effects. In this study, the effect of amniotic membrane transplantation (AMT) on corneal wound healing and on inflammation in mice with necrotizing HSK was examined. METHODS: BALB/c mice were corneally infected with 10(5) plaque-forming units (PFU) of HSV-1 (KOS strain). In 16 mice that exhibited severe ulcerating HSK, the cornea was covered with a preserved human amniotic membrane as a patch. Corneas in 16 infected mice remained uncovered and served as a control. On days 2 and 7 after surgery, the amniotic membrane was removed (eight mice in each group), the HSV-1-infected cornea was evaluated clinically, and the eye was enucleated. Tissue sections were analyzed histologically for epithelialization and cellular infiltration and immunohistochemically with anti-CD3 mAb to T cells, anti-CD11b mAb to both macrophages and neutrophils, or anti-F4/80 mAb to macrophages. RESULTS: Profound regression of corneal inflammation and rapid closure of epithelial defects were observed clinically within 2 days in the amniotic membrane-covered eyes, whereas HSV-1 keratitis and ulceration progressed in all mice in the control group (P < 0.001). Histologically, corneal edema and inflammatory infiltration, and immunohistochemically the number of CD3(+), CD11b(+), and F4/80(+) cells in the cornea were markedly decreased at 2 and 7 days after amniotic membrane application, compared with the uncovered control corneas (P < 0.001). CONCLUSIONS: AMT promotes rapid epithelialization and reduces stromal inflammation and ulceration in HSV-1 keratitis. AMT in mice with HSV necrotizing stromal keratitis appears to be a useful model for investigating the effect and the action mechanism of human amniotic membrane.     

单纯疱疹病毒性角膜炎小鼠模型的建立及鉴定

目的：探讨建立不同感染时期HSK小鼠动物模型,为HSK的深入研究建立基础。方法：Balb/c小鼠125只麻醉后在显微镜下用刀片背面尖端于角膜＂#＂字划痕,其中100只小鼠接种HSV-Ⅰ病毒,另25只小鼠不接种病毒作为正常对照组。术后每天用10g/L荧光素钠染色后裂隙灯显微镜下观察角膜病变发生情况,并取角膜表面泪液进行HEK293T细胞检测以确定裂隙灯显微镜下有无病毒复制。对潜伏感染期小鼠模型采用紫外线B光照射以诱导HSK复发。结果：接种HSV-Ⅰ病毒的小鼠模型眼于接种后3d内全部出现急性上皮性角膜炎表现。经阿昔洛韦滴眼液治疗1wk后角膜炎症消失,但角膜和三叉神经节中PCR检测病毒仍为阳性。潜伏感染期小鼠模型经紫外线B光照射后也都在1wk内复发,并表现为以基质型角膜炎为主要临床表现的角膜病变。结论：采用角膜划痕法对Balb/c小鼠接种HSV-Ⅰ病毒和紫外线B光照射可以成功地制作出原发感染期、潜伏感染期和复发感染期等不同感染时期的HSK模型,而且操作相对简单、方便易行。     

Herpetic stromal keratitis: an immunopathologic disease mediated by CD4+ T lymphocytes.

To investigate the role of T cell subsets in the development of herpetic stromal keratitis (HSK) in a well defined model, we used an adoptive transfer approach in which thymectomized and T cell-depleted mice [T(-)] were reconstituted with different numbers of syngeneic immune T lymphocytes after topical corneal challenge with RE strain of herpes simplex virus-1. In vitro stimulated or unstimulated immune T cells obtained from cervical and retropharyngeal lymph nodes of mice with HSK were used in adoptive transfer experiments. Although T(-) mice developed an initial epithelial inflammation, stromal keratitis did not occur. Reconstitution experiments revealed that mice that received 2 x 10(7) or more unfractionated immune T cells could develop HSK lesions with severity comparable to immuno-competent control mice. In mice receiving CD8(+)-depleted populations, even fewer cells (5 x 10(6)/mouse) were able to induce significant HSK. In contrast, mice that received similar or increased numbers of cells depleted of CD4+ T lymphocytes did not develop HSK. Immune T lymphocytes transferred to mice that were mock infected on the cornea did not develop HSK, indicating that the immunopathogenic cells were virus specific and not merely reacting to autoantigens. Histopathologic examination of the diseased corneas demonstrated that the stromal inflammation in euthymic normal and T(-)-reconstituted mice was characterized by extensive polymorphonuclear leukocyte infiltration. Scattered lymphocytes, and occasional macrophages also were observed. These results provide further evidence that HSK represents an immunopathologic process mediated mainly by CD4+ T cells.     

Corneal epithelial cells and stromal keratocytes efficently produce CC chemokine-ligand 20 (CCL20) and attract cells expressing its receptor CCR6 in mouse herpetic stromal keratitis

PURPOSE: CC chemokine-ligand 20 (CCL20) is known to be selectively expressed by surface-lining mucosal epithelial cells and skin epidermal keratinocytes and to attract cells such as immature dendritic cells and effector T cells via CCR6. This study evaluated the ability of corneal epithelial cells and stromal keratocytes to produce CCL20 in vitro and in vivo. METHODS: Human corneal epithelial cells (HCE) and corneal keratocytes (HCK) were treated without or with various cytokines and expression of CCL20 mRNA and secreion of its protein were evaluated by RT-PCR and ELISA. Induction of CCL20 mRNA in HCE and HCK was also examined upon in vitro infection with HSV-1. Using a mouse model of herpetic stromal keratitis (HSK), induction of CCL20 expression and accumulation of cells expressing CCR6 were evaluated by RT-PCR and immunohistochemistry. RESULTS: Not only corneal epithelial cells but also stromal keratocytes efficiently expressed CCL20 mRNA and protein upon stimulation with IL-1beta and TNF-alpha. In vitro infection with HSV-1 also induced CCL20 mRNA in both types of cells. In a mouse herpetic stromal keratitis model, prominent accumulation of CCL20 and CCR6 mRNA was revealed in HSV-1-infected corneas. Furthermore, immunohistochemistry demonstrated production of CCL20 by corneal epithelial cells as well as stromal keratocytes and stromal infiltration of DEC205+ dendritic cells, CD4+ T cells and CD8+ T cells. Double staining revealed that CCR6-expressing cells were mostly MHC class II+ dendritic cells. CONCLUSIONS: Not only epithelial cells but also stromal keratocytes are efficient producers of CCL20 in the cornea and recruit CCR6-expressing cells such as dendritic cells into inflamed cornea.     

HSV-1 antigens and DNA in the corneal explant buttons of patients with non-herpetic or clinically atypical herpetic stromal keratitis

Background Little is known about the role of HSV-1 in keratitis not primarily attributed to herpetic origin. This study therefore aimed to prospectively evaluate the corneal explant buttons of patients with non-herpetic or clinically atypical herpetic stromal keratitis (experimental group: non-HSK) for the presence of HSV-1 antigens and DNA, and to compare the findings with those from individuals with typical herpetic stromal keratitis (positive control group: HSK) or non-inflammatory degenerative keratopathy (negative control group).Methods Corneal buttons derived from 51 patients with HSK, from 72 with non-HSK and from 30 with degenerative keratopathy were prospectively collected and subjected to immunohistochemical analysis for HSV-1 antigens and to HSV-1 DNA amplification.Results In corneal buttons derived from patients with non-HSK, viral antigens were detected immunohistochemically in 8/72 cases and DNA amplified in 16/72. Corresponding values for the HSK group were 16/51 and 11/51. Taking viral antigen and DNA findings together, HSV-1 was detected in 18/72 (25%) patients with non-HSK and in 19/51 (37%) with HSK (p=0.2), but in only 2/30 (6%) individuals with non-inflammatory degenerative keratopathy.Conclusion Since the detection frequencies for HSV-1 antigens and DNA were comparable in the HSK and non-HSK groups, Herpes may play an underestimated and as yet undefined role in non-herpetic and clinically atypical herpetic stromal keratitis, either as a primary trigger of the disease or as a secondary contributor to it. In this category of individuals, early anti-herpetic therapy should be considered if patients do not respond in the expected manner to treatment for non-herpetic stromal keratitis.Commercial interests: none     

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.     

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     

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.     

白细胞介素10对小鼠单纯疱疹性角膜基质炎作用的实验研究

研究白细胞介素10(IL-10)在小鼠单纯疱疹性角膜基质炎(herpetic stromal keratitis,HSK)中的作用。方法雌性BALB／c小鼠80只分为2组,每组40只;将单纯疱疹病毒1型接种于小鼠角膜上建立HSK动物模型;分别于鼠角膜接种病毒前的6h、当日及接种后2、4d,向实验组鼠的角膜内注射重组IL-10 20ng,腹腔内注射鼠重组IL-10 500 ng;对照组同步注射生理盐水;观察IL-10对小鼠HSK的发病率、临床特征、角膜病毒滴度、角膜细胞因子含量、角膜病理改变及迟发型超敏反应(DTH)的影响。结果IL-10降低了HSK发病率,减轻了HSK角膜混浊程度、角膜新生血管化程度及角膜内炎性细胞浸润,降低了角膜内IL-2和IL-6的含量,抑制了鼠的DTH反应。IL-10不影响病毒在角膜内的复制和清除。结论IL-10可抑制鼠的DTH反应和HSK的发病进展。     

Comparison of an immortalized human corneal epithelial cell line with Vero cells in the isolation of Herpes simplex virus-1 for the laboratory diagnosis of Herpes simplex keratitis

Background  

Herpes simplex keratitis (HSK) is a sight threatening ocular infection often requiring a specific and prompt laboratory diagnosis. Isolation of Herpes simplex virus (HSV-1) in culture provides the most reliable and specific method and is considered as the "Gold Standard" in the laboratory diagnosis of HSK in spite of its low sensitivity. Using "cell lines of corneal origin" for virus isolation may be beneficial under such circumstances, since these cells have been shown to be excellent substrates for the growth of HSV-1 isolated from the cornea. We report a comparative study of a novel human corneal epithelial cell line (HCE) and the Vero cell line in the isolation of HSV-1 from corneal scrapings employing a shell vial assay.     

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。     

OX40-Ig融合蛋白对鼠单纯疱疹性角膜基质炎的抑制作用

目的研究OX40-Ig融合蛋白对鼠单纯疱疹性角膜基质炎(HSK)的免疫抑制作用。方法将1×106PFU的单纯疱疹病毒1型(HSV-1)Mckrae毒株接种于BALB/c鼠的角膜上建立HSK模型;分别于接种病毒的当天、接种后第2、4d将OX40-Ig融合蛋白100μg注射到鼠的腹膜下,观察OX40-Ig融合蛋白对鼠HSK的影响。结果OX40-Ig融合蛋白使鼠外周血中CD4 T细胞减少了78·2%,使鼠HSK发病率由83·3%下降到20·0%。OX40-Ig治疗组的小鼠角膜基质混浊程度较对照组明显减轻,角膜内炎性细胞浸润也明显减少,迟发型超敏反应能力显著下降。结论OX40-Ig融合蛋白能够阻断OX-40/OX-40L协同刺激途径,抑制CD4 T细胞增生,阻止HSK的发病,减轻HSK的严重程度。     

Detection of HSV nucleic acid sequences in the cornea during acute and latent ocular disease

This study evaluated the continued presence of herpes simplex virus (HSV) nucleic acid sequences after resolution of acute herpetic stromal keratitis in the rabbit ocular model. Forty-four rabbits were inoculated bilaterally with 10(5) plaque-forming units of RE strain HSV-1 by intrastromal injection. All eyes were cultured for the presence of HSV during acute disease and immediately before the animals were killed. Full-thickness corneal buttons were then removed and processed for in situ hybridization with a 3H-labelled HSV DNA probe representing the full-length HSV genome. HSV nucleic acid sequences were detected autoradiographically at all time intervals examined. HSV nucleic acid sequences were localized in the epithelium and the anterior stromal keratocytes during acute disease and in all corneal layers during latent infection. Retention of HSV nucleic acid sequences, either HSV DNA or HSV RNA, or both, in corneal tissues (epithelium, stroma, and endothelium) may be a contributing factor in the development of HSV-induced stromal keratitis.     

CD8 T cells mediate transient herpes stromal keratitis in CD4-deficient mice

PURPOSE: To evaluate the role of CD4(+) T cells in the development of murine herpes stromal keratitis (HSK). METHODS: The corneas of wild-type (WT) BALB/c mice and three types of CD4-deficient BALB/c mice (CD4(-/-), CD4-depleted, CD4 and CD8 double-depleted) were infected with different doses of HSV-1 RE, and HSK incidence and severity were monitored. Corneal infiltrates were quantitatively and functionally assayed by flow cytometric analysis of individually digested diseased corneas and documented histologically. RESULTS: At a relatively high infectious dose (1 x 10(5) pfu/cornea): (1) CD4-deficient and WT BALB/c mice had severe HSK with a similar incidence (80%-100%), whereas HSK did not develop in mice deficient in both CD4(+) and CD8(+) T cells; (2) neutrophils were the predominate leukocyte in the corneas of CD4-deficient and WT mice; (3) the corneas of WT mice had activated, HSV-1-specific CD4(+) T cells, but few if any CD8(+) T cells; (4) the corneas of CD4-deficient mice had activated, HSV-1-specific CD8(+) T cells; and (5) HSK in CD4-deficient mice was transient, showing loss of CD8(+) T cells at 2 to 3 weeks after infection (pi) followed by a loss of neutrophils. At a relatively low infectious dose of HSV-1 (10(3) pfu/cornea) severe HSK developed in 80% to 90% of WT mice, but in only 30% to 40% of CD4-deficient mice. CONCLUSIONS: CD4(+) T cells preferentially mediate HSK, but, in their absence, a high infectious dose of HSV-1 can induce histologically similar but transient HSK that is mediated by CD8(+) T cells.     

Epidemiology and molecular analysis of herpes simplex keratitis requiring primary penetrating keratoplasty

AIMS: To determine whether herpes simplex keratitis (HSK) has declined as an indication for penetrating keratoplasty (PKP) at the University of California San Francisco (UCSF) over the past 30 years. METHODS: Records of the Hogan Eye Pathology Laboratory were reviewed to determine the incidence of PKP performed for HSK from 1972 through 2001. Archived corneal tissue with the diagnosis of HSK was evaluated for herpes simplex virus (HSV) DNA by polymerase chain reaction (PCR) based assays. RESULTS: The number of corneal buttons submitted with the clinical diagnosis of HSK decreased from 1972 to 2001, while the overall number of PKPs performed did not. The percentage of corneal buttons with a clinical diagnosis of HSK that contained detectable HSV DNA did not change over the course of the study period. CONCLUSION: HSK declined as an indication for PKP from 1972 to 2001 at UCSF. It is unlikely that this decline was the result of improved diagnostic accuracy since detection of HSV DNA in corneal buttons with a clinical diagnosis of HSK was similar at the beginning and end of the study period.     

复发性单疱病毒性角膜炎实验模型的研究

目的：建立一种可靠、实用的复发性单疱病毒性角膜炎（HSK）的实验模型。方法：用单纯病毒I型（HSV－1）Mckrae株行NIH鼠的角膜接种,用人的抗HSV－1血清行鼠的腹腔内注射,使病毒在三叉神经节或角膜内建立起潜伏感染。用紫外线B光照射鼠的角膜,诱导HSK复发。观察诱导HSK复发的成功率、复发性HSK的临床特征、组织学特点。结果：紫外线照射诱导鼠HSK复发的成功率为72．5％,复发性HSK主要表现为基质型角膜炎,组织切片见角膜基质层内有大量的淋巴细胞和一些中性粒细胞浸润。     

Bystander activation of CD4+ T cells accounts for herpetic ocular lesions

PURPOSE: Stromal keratitis is an immunopathologic consequence of herpes simplex virus (HSV) infection of the cornea. The lesion is immunopathologic, but the identities of molecules that drive the reaction remain unresolved. To exclude viral antigen recognition as a necessary step in the disease process, ocular HSV infection was followed in Tg-RAG mice (OVA-TCR transgenic mice crossed to RAG2-deficient mice) whose limited T-cell repertoire did not include immune responsiveness to HSV. METHODS: Mice with T-cell specificity to OVA peptide (Tg-RAG mice) as well as control DO11.10 and BALB/c mice were infected with HSV on the scarified cornea and subjected to clinical, histologic, and immunologic analysis. To evaluate involvement of OVA-specific CD4+ T cells in lesion development in Tg-RAG mice, monoclonal antibody to CD4+ T cells was used for in vivo CD4+ T-cell depletion. RESULTS: Tg-RAG mice were capable of eliciting ocular lesions in the absence of detectable reactivity to viral antigens. Lesion manifestation in Tg-RAG mice was CD4+ T-cell dependent and the cellular infiltrates and their inflammatory products in the HSV-infected cornea were comparable to similarly infected BALB/c and DO11.10 mice. CONCLUSIONS: The authors conclude that mechanisms other than viral antigen recognition, and hence molecular mimicry, are at play and are sufficient to cause HSV-induced stromal keratitis. The data imply a significant role for non-virus-specific CD4+ T cells that could become activated by an inflammatory milieu consisting of enhanced accessory molecules and proinflammatory cytokines in the cornea.     

用聚合酶链反应检测单疱病毒性角膜炎静止期角膜内HSV基因

目的:单疱病毒性角膜炎是主要的致盲眼病之一,近年来研究认为HSV可在角膜内潜伏,但未获结论性证据,本研究目的为明确角膜是否为HSV的另一潜伏地。方法:选择18例已确认为HSK病例,所有病例角膜病变静止3个月以上(平均3.7年),行部分穿透性角膜移植术取下角膜片,用聚合酶链反应方法(引物为P15’CATCACCGACC-CGGAGAGGGAC,P2 5’GGGCCAGGCGCTTGGTGTA)检测角膜片内HSV基因。结果:18例静止期HSK病例角膜片中,用聚合酶链反应方法在17例病例中检测出HSV基因,1例阴性。结论:单疱病毒性角膜炎静止期角膜内有HSV基因存留,角膜可能为HSV的另一潜伏地,用聚合酶链反应检测角膜内HSV DNA方法简单,敏感性高。眼科学报 1995;11:183—185。