Soluble Fas ligand expression in the ocular fluids of uveitis patients

PURPOSE. The expression of Fas ligand (FasL) in ocular tissues is thought to play a critical role in maintaining immune privilege in the eye. In this study, to clarify the involvement of the Fas-FasL system in inflammatory processes of the eye,we examined soluble FasL (sFasL) in ocular inflammation. METHODS. Using ELISA systems recently developed, sFasL concentrations in aqueous humor (AH) and/or vitreous fluid (VF) were measured. AH was obtained from 17 eyes of 17 uveitis patients and from 12 eyes of 12 non-uveitis (cataract) patients. VF was obtained from 22 eyes of 22 uveitis patients and 7 eyes of 7 non-uveitis (macular hole) patients. Serum levels of sFasL were also determined. RESULTS. sFasL in AH and VF was below the detection limit of the ELISA systems in all non-uveitis eyes. On the other hand, sFasL was detected in AH from uveitis patients where it measured 367.0 +/- 154.7 pg/ml (mean +/- SEM). sFasL was also detected in VF from uveitis patients where it measured 1132.2 +/- 281.7 pg/ml. None of the sera from patients with or without uveitis contained a detectable level of sFas L. CONCLUSIONS. sFasL levels in AH and VF are elevated in the eye during ocular inflammation. Fas-FasL mediated apoptosis may play an important role in the regulation of inflammation during uveitis.     

Soluble Fas and Fas ligand in human tear fluid after photorefractive keratectomy

BACKGROUND/AIMS: The Fas-Fas ligand system is thought to be involved in stromal cell apoptosis after corneal wounding. The aim was to measure changes in human tear fluid levels of soluble Fas (sFas) and Fas ligand (sFasL) following myopic photorefractive keratectomy (PRK). METHODS: Tear samples of 59 patients were collected preoperatively, and 1 or 2 days after PRK. Tear fluid sFas or sFasL concentrations were determined using sandwich ELISAs. Subsequently, tear flow corrected concentrations (releases) were calculated to compensate for the postoperative tear hypersecretion. RESULTS: The preoperative tear fluid flows (TFF) were 6.4 (1.7) microl/min (mean (SEM)) in sFas group (n = 18), and 7.5 (1.5) microl/min in sFasL group (n = 39). Postoperatively TFFs increased to 37.9 (10.9) microl/min (p = 0.003) and 58.3 (7.0) microl/min (p = 0.000), respectively. The mean preoperative sFas concentration (24.4 (11.6) U/ml) decreased to 9.7 (4.1) U/ml (p = 0.001) postoperatively, and the mean sFasL concentration (299.1 (28.8) ng/l) to 118.7 (15.9) ng/l (p = 0.000). However, the release of both substances increased significantly: sFas from 87.3 (29.4) mU/min to 229.4 (82.9) mU/min (p = 0.002) and sFasL from 1620.6 (226.4) fg/min to 4777.1 (596.1) fg/min (p = 0.000). CONCLUSIONS: Both sFas and sFasL are normal constituents of human tears. Despite a decrease in concentrations related to reflex tears, the release of sFas and sFasL increases significantly after excimer laser photorefractive keratectomy, which suggests that they are involved in corneal healing after PRK in humans.     

Elevated soluble Fas in aqueous humor of patients with Behcet's uveitis: correlation with uveitis severity

PURPOSE: To examine the role of soluble Fas (sFas) in patients with Behcet's uveitis. METHODS: We measured the sFas levels in both sera and aqueous humor (AH) of patients (n = 40) with uveitis and of non-uveitis controls (n = 27) using an enzyme-linked immunosorbent assay. The patients with uveitis comprised 24 with Behcet's disease, 6 pan-uveitis, 5 anterior uveitis, 2 lens-induced uveitis, 1 Vogt-Koyanagi-Harada disease, 1 sarcoidosis, and 1 retinal vasculitis. The severity of uveitis was determined by the Hogan grading method (0--4 grade) at the time of sampling. RESULTS: The concentration of aqueous sFas in uveitis patients was significantly higher than that in non-uveitis controls, while there was no difference in the serum concentration of sFas between the two groups. In the paired samples of serum and AH, obtained simultaneously, the aqueous sFas levels were higher than serum Fas levels in patients with uveitis, whereas the non-uveitis controls displayed significantly lower sFas levels in AH than in the serum. The sFas levels in AH or serum were not different between Behcet's uveitis patients and non-Behcet's uveitis patients. However, in patients with Behcet's uveitis, circulating sFas strongly correlated with aqueous sFas, which was not so in those with non-Behcet's uveitis. Patients (n = 29) with more active (grade greater-than-or-equal 2) uveitis had significantly higher levels of aqueous sFas than those (n = 11) with less active (grade < 2) uveitis. After treatment with steroids and/or immunosuppressive agents, aqueous sFas levels decreased in parallel with a reduction in the number of inflammatory cells. CONCLUSIONS: The levels of sFas were elevated in patients with Behcet's uveitis and correlated well with the uveitis severity in these patients.     

Increased presence of Epstein-Barr virus DNA in ocular fluid samples from HIV negative immunocompromised patients with uveitis

AIMS—To investigate whether routine testing for Epstein-Barr virus (EBV) is necessary in the examination of a patient with uveitis.
METHODS—Intraocular EBV DNA was determined in 183 ocular fluid samples taken from patients with AIDS and uveitis, HIV negative immunocompromised uveitis, acute retinal necrosis, toxoplasma chorioretinitis, intraocular lymphoma, anterior uveitis, and miscellaneous uveitis of unknown cause. In 82 samples from this group of patients paired serum/ocular fluid analysis was performed to detect local antibody production against EBV. Controls (n=46) included ocular fluid samples taken during surgery for diabetic retinopathy, macular pucker, or cataract.
RESULTS—Serum antibody titres to EBV capsid antigen proved to be significantly increased in HIV negative immunocompromised patients with uveitis (p<0.01) compared with controls. Local antibody production revealed only three positive cases out of 82 patients tested, two results were borderline positive and one patient had uveitis caused by VZV. EBV DNA was detected in three out of 46 control ocular fluid samples. In the different uveitis groups EBV DNA was noted, but was not significantly higher than in the controls, except in six out of 11 HIV negative immunocompromised patients (p=0.0008). In four out of these six cases another infectious agent (VZV, HSV, CMV, or Toxoplasma gondii) had previously been identified as the cause of the uveitis.
CONCLUSIONS—When comparing various groups of uveitis patients, EBV DNA was found more often in HIV negative immunocompromised patients with uveitis. Testing for EBV does not have to be included in the routine management of patients with uveitis, since indications for an important role of this virus were not found in the pathogenesis of intraocular inflammation.

Keywords: Epstein-Barr virus; intraocular fluid; polymerase chain reaction; uveitis     

Soluble ICAM-1 serum levels in patients with intermediate uveitis

AIM: To investigate whether serum levels of soluble intercellular adhesion molecule 1 (sICAM-1) can serve as a marker of the presence of systemic disease in intermediate uveitis. METHODS: In a multicentre study sICAM-1 serum levels were measured in 61 patients with idiopathic intermediate uveitis, controls included 56 uveitis patients with a systemic disease (26 sarcoid associated uveitis and 30 HLA-B27 positive acute anterior uveitis), 58 uveitis patients without systemic disease (30 toxoplasma chorioretinitis and 28 Fuchs' hetrochromic cyclitis), and 21 normal controls. The clinical records of the patients with intermediate uveitis were analysed for disease characteristics at the time of blood sampling and for a relation with the development of a systemic disease after a mean follow up of 4.5 years. RESULTS: Increased serum levels of sICAM-1 were found in 34 out of 61 patients with intermediate uveitis and were significantly different when compared with toxoplasmosis, Fuchs' cyclitis, and healthy controls (p<0.001). Elevated sICAM-1 levels were also found in 18 out of 26 patients with sarcoid uveitis and in 11 out of 30 patients with HLA-B27 associated anterior uveitis. Raised sICAM-1 levels in the intermediate uveitis group were significantly associated with active ocular disease (p<0.01) and the presence of vitreous exudates (p<0.05). Increased levels of sICAM-1 correlated with interleukin 8 levels (IL-8) (tested in a previous study in the same group of intermediate uveitis patients) in patients with active systemic involvement. Follow up of the patients showed that an established or suspected systemic disease was found more often in the 21 intermediate uveitis patients with increased sICAM-1 and IL-8 levels compared with the other 40 patients with intermediate uveitis (p<0.01). CONCLUSIONS: The measurement of both sICAM-1 and IL-8 can be used as a marker for ocular disease activity and for a predisposition of developing an associated systemic disease in intermediate uveitis patients.     

Increased CD40 ligand in patients with acute anterior uveitis

PURPOSE: The inflammatory response in acute anterior uveitis (AU) is believed to be primarily mediated by autoreactive T-cells. We wanted to evaluate whether the T-cell activation marker CD40 ligand is involved in the AU immunopathogenesis. METHODS: We evaluated the expression of the CD40 ligand on CD4+ T-cells, CD8+ T-cells and CD19+ B-cells on peripheral blood mononuclear cells using flow cytometry in six patients with unilateral AU, six patients with monosymptomatic optic neuritis (ON) as inflammatory controls, and in six healthy controls. The ex vivo induction of the CD40 ligand on T-cells in patients and controls was also studied. RESULTS: A significantly higher expression of the CD40 ligand on both CD4+ (p < 0.05) and CD8+ (p < 0.05) T-cells in patients with AU compared to ON patients and healthy controls was found. There was a significantly higher induction of the CD40 ligand on CD8+ T-cells in AU patients compared to ON patients and healthy controls (p < 0.01). No differences in the B-cell population were observed between the three groups. CONCLUSION: Patients with AU had increased expression of the CD40 ligand on T-cells in the blood and expressed higher levels of the CD40 ligand when stimulated, compared to ophthalmological inflammatory controls and healthy controls. The data suggest that the CD40 ligand is involved in the development of AU.     

Serum levels of soluble Fas in patients with Graves' ophthalmopathy

AIM: To assess levels of soluble Fas (sFas) in the sera of patients with Graves' ophthalmopathy. METHODS: The subjects in this study were 43 patients with Graves' ophthalmopathy and 11 normal subjects. Serum levels of sFas were determined by sandwich enzyme linked immunosorbent assay. In addition, serum levels of thyroid stimulating antibody (TSAB) were also measured in all the patients. RESULTS: The mean serum level of sFas was 1.35 (SD 2.03) ng/ml in patients with Graves' ophthalmopathy, and 0.93 (0.32) ng/ml in normal subjects. Serum levels of sFas in the subgroup of 24 patients with diplopia (1.98 (2.56) ng/ml) were significantly higher than those in the subgroup of 19 patients without diplopia (0.56 (0.24) ng/ml) and normal subjects (p <0.001). Serum levels of sFas in the subgroup of 27 patients with extraocular muscle hypertrophy (1.81 (2. 46) ng/ml) were significantly higher than those in the subgroup of 16 patients without extraocular muscle hypertrophy (0.58 (0.26) ng/ml) among the patients with Graves' ophthalmopathy and normal subjects (p <0.001). Serum levels of sFas were not significantly different between the subgroup of 24 patients with proptosis (1.15 (0.98) ng/ml) and the subgroup of 19 patients without proptosis (1. 61 (2.88)). In contrast, the serum levels of TSAB in the subgroup of patients with proptosis (723% (1161%)) were significantly higher than those in the subgroup of patients without proptosis (194% (122%)) (p <0.05). CONCLUSIONS: Elevated sFas levels were associated with extraocular muscle disorders but not with proptosis. On the other hand, elevated TSAB levels were associated with proptosis but not with extraocular muscle disorders, suggesting different immunological mechanisms for the extraocular muscle disorders and proptosis in Graves' ophthalmopathy. Determination of the serum levels of sFas and TSAB could provide useful markers for evaluation of the immunological processes involved in the development of Graves' ophthalmopathy.     

Macrophage migration inhibitory factor in ocular fluids of patients with uveitis

AIMS: To investigate the levels of macrophage migration inhibitory factor (MIF) in intraocular fluids of uveitis patients, the capacity of intraocular infiltrating lymphocytes to produce MIF, and the correlation between MIF levels in the eye and intraocular inflammatory activity. METHODS: MIF levels were measured by enzyme linked immunosorbent assay (ELISA) using (1) aqueous humour (AH) of 12 uveitis patients and eight control patients with cataract, (2) vitreous fluid of 15 uveitis patients and eight control patients with idiopathic macular hole, and (3) culture supernatants of T cell clones (TCCs) established from intraocular fluids of uveitis patients. MIF expression on infiltrating cells was determined by a double staining immunofluorescence technique using a flow cytometry. RESULTS: Significant levels of MIF were detected from intraocular fluids of uveitis patients (4.0 (SD 3.0) ng/ml in AH and 16.5 (24.7) ng/ml in vitreous), whereas MIF levels in control groups were below the detectable levels. There was a significant correlation between MIF levels and vitreous inflammation (29.7 (30.0) ng/ml in active uveitis v 3.3 (2.6) ng/ml in inactive uveitis, p< 0.05). Significant levels of MIF were detected in culture supernatants of TCCs from ocular fluids of uveitis patients. MIF was expressed on infiltrating CD4+ lymphocytes from vitreous of uveitis patients. CONCLUSION: Significant levels of MIF are present in intraocular fluids of patients with uveitis. Lymphocytes infiltrating in the eye are capable of producing MIF. MIF levels in vitreous fluid are correlated with vitreous inflammation activity. These data thus indicate that MIF in the eye has a significant role in the pathophysiology of ocular inflammation.     

Non-cleavable mutant Fas ligand transfection of donor cornea abrogates ocular immune privilege

The expression of Fas ligand (FasL) in donor corneal tissue has been thought to contribute to the prolonged survival of orthotopic corneal allografts. However, in solid organ transplantation, FasL gene-transfected tissues have been reported to lead to graft destruction through neutrophil recruitment. We wished to examine whether transfection of mutant FasL cDNA, which produces only membrane-binding forms of FasL because of its cleavage site, prolonged corneal allograft survival in mice. Donor corneal tissues were transduced with two adenovirus vectors containing human mutant FasL cDNA (AxCALNmFasL) and Cre recombinase (AxCANCre). Donor corneal tissues were then transplanted orthotopically onto recipient mice, and graft clarity was examined by slit lamp microscopy. We found that donor corneal grafts transfected with mutant FasL were intensely opaque for 1-2 weeks after grafting, when grafts without transfection remained clear. Histological examination revealed polymorphonuclear cell infiltration in the transfected grafted tissue. Moreover, mutant FasL transduced to donor tissues was found to exert its effects by binding to host, rather than donor Fas molecules, since corneal grafts with mutant FasL survived as long as those without transfection only when host animals, but not donor animals, lacked Fas molecules. Our results indicate that membrane-binding FasL over expression in donor cornea does not prolong corneal allograft survival; indeed, it causes rapid graft destruction.     

Inflammatory ocular hypertension syndrome (IOHS) in patients with syphilitic uveitis

Aim

To determine the prevalence and clinical characteristics of the inflammatory ocular hypertension syndrome (IOHS) in patients with uveitis and serological evidence of syphilis.

Methods

A retrospective, observational case review of 39 consecutive patients with uveitis and serological evidence of syphilis was carried out between January 1977 and December 2001. Other causes of uveitis were excluded. The prevalence and clinical characteristics of IOHS among patients with uveitis and serological evidence of syphilis were documented. IOHS was defined as an increase in intraocular pressure (IOP) of more than 21 mm Hg that began at the onset of acute, recurrent, or chronic anterior chamber inflammation and reversed promptly with appropriate anti‐inflammatory or antimicrobial treatment.

Results

Of the 39 patients with uveitis and serological evidence of syphilis, eight eyes from seven patients (18%) presented with IOHS, a significantly higher prevalence than in the uveitis population at large (2.3%; p<0.001). Best‐corrected visual acuity varied from 20/20 to 20/200, with a median of 20/40, and three of the eight eyes (37.5%) had granulomatous features, including large keratic precipitates and, in two eyes, Koeppe nodules. Intraocular pressure varied from 23 to 51 mm Hg, with a mean of 36 and a median of 34. One patient had bilateral IOHS.

Conclusions

Syphilitic uveitis should be included in the differential diagnosis of IOHS along with more commonly recognised causes.     

视网膜母细胞瘤中Fas/Fas配体表达及 与凋亡的关系

目的观察视网膜母细胞瘤(retinoblastoma, RB)　Fas/Fas 配 体 (Fas ligand ,FasL)表达及与凋亡的相关性。方法应用免疫组织化学染色法观察32例RB标本中 Fas/FasL的表达及分布,并对32例用光镜、其中4例用电镜及12例用末端脱氧核苷酸转移酶介导的生物素化脱氧尿苷三磷酸末端标记 (t erminal deoxynucleotidyl transferase mediated biotin dUTP nick end labeling, T UNEL)染色观察细胞凋亡,分析与Fas/FasL表达的关系。结果光镜下凋亡细胞主要位于RB退化区,电镜下可见染色质边集和凋亡小体等凋亡特征 ;TUNEL显示阳性标识主要位于RB退化区及死亡区。Fas及 FasL在所有RB中均呈阳性表达 。Fas和 FasL表达与凋亡指数 (apoptosis index, AI)呈正相关 (P<0．01 ,P<0．001)。结论凋亡在RB中普遍存在,可能是触发RB细胞死亡的 主要方式;Fas系统在RB的发生发展中起重要作用。Fas系统上调可能会诱导RB细胞凋亡。(中华眼底病杂志,2001,17:21-23)     

Testing ocular fluids in uveitis

Infectious uveitis is a serious condition requiring rapid diagnosis and therapeutic management. Molecular tools, such as PCR and its variants, have significantly changed the diagnostic approach during the last 10 years. Presumed and empirical diagnosis should be excluded in the face of atypical clinical presentations, but also inadequate response to anti-inflammatory drugs. It is of particular importance to note that most of these tests need to be processed in specialized central laboratories to ensure the best sensitivity and specificity. Molecular techniques have their own limits, sometimes caused by inadequate primers or PCR-inhibitors and contaminants. PCR positivity means detection of pathogen DNA, but does not confirm a productive infection. Cultures from ocular fluids should be improved to obtain complementary data about pathogen life cycles and resistance to antibiotics or antivirals. Furthermore, diagnostic yield is significantly increased when PCR and local antibody production are associated, especially in viral infections. This step is of particular importance to identify new infectious entities in cases that are presently considered to be idiopathic or autoimmune disorders.     

Soluble P-selectin glycoprotein ligand 1 inhibits ocular inflammation in a murine model of allergy.

PURPOSE: To assess the anti-inflammatory modality of a soluble extracellular form of P-selectin glycoprotein ligand 1 (sPSGL-1) in a mouse model of ocular allergic response. METHODS: Potential anti-inflammatory effects of sPSGL-1 were investigated in SWR/J mice sensitized by topical application of short ragweed pollen to the nasal mucosa followed by a challenge of the ocular mucosa with the same allergen. Five experimental groups were included in these studies: A, mice neither sensitized nor challenged with pollen (control group 1); B, animals sensitized but not challenged (control group 2); C, animals not sensitized but challenged (control group 3); D, animals sensitized and challenged; and E, sensitized animals treated with sPSGL-1 before pollen challenge. All experimental groups were evaluated for gross morphologic ocular changes, and histologic assessments were made to determine the onset/progression of inflammatory reactions and to look for evidence of eosinophil infiltration. RESULTS: Mice sensitized and challenged with pollen developed clinical signs consistent with human allergic conjunctivitis. These signs correlate with histologic changes in the conjunctival epithelium and stroma (e.g., edema and extensive eosinophil infiltration). Moreover, the ocular changes also correlated with evidence of eosinophil degranulation. However, sensitized and challenged mice concurrently treated with sPSGL-1 displayed no inflammatory ocular changes associated with a ragweed-induced type-1 hypersensitivity reaction. The lack of ocular changes included the absence of histologic late-phase inflammatory changes of the conjunctiva and a 97% reduction in the induced eosinophil infiltrate. CONCLUSIONS: The antagonistic intervention of cell- cell interactions through the blockade of selectin-dependent leukocyte adhesion may offer novel therapeutic strategies to modulate inflammatory responses. The potent inhibitory effects on eosinophil recruitment and late-phase inflammation suggest a role for sPSGL-1 in the treatment of ocular allergic diseases.     

Fas／FasL系统及其在葡萄膜炎中的意义

Fas是一种具有重要功能的跨膜糖蛋白,为肿瘤坏死因子受体超家族的成员之一,其自然配体为FasL(Fas Ligand)。Fas与FasL或Fas单克隆抗体结合可诱导Fas^ 细胞的凋亡。Fas系统诱导的细胞凋亡可消除自身反应性淋巴细胞,诱导活化T、B细胞凋亡,参与免疫赦免形成,从而维持机体生理平衡和自身免疫耐受。Fas/FasL系统的异常在自身免疫性葡萄膜炎发生中可能起着一定的作用,自身免疫性淋巴细胞对Fas/FasL系统诱导的凋亡有较大抵抗性,这可能是葡萄膜炎慢性化和复发的重要机制之一。     

Level of glucocorticoid receptor mRNA expression in iris tissue and postoperative ocular inflammation in patients with uveitis

BACKGROUND: Glucocorticoids (GC) are widely used to treat uveitis, but their efficacy is known to vary substantially among patients. In this retrospective preliminary study, we measured the level of GC receptor (GCR) expression in ocular tissue taken from uveitis patients during cataract surgery as an indicator of their sensitivity to GC, divided the patients into comparatively high- and low-sensitivity groups on this basis, and compared the two groups with respect to the frequency of ocular inflammation attacks observed after operation. METHODS: Iris samples were obtained from 13 patients during surgery for cataract associated with uveitis. From each sample, RNA was extracted and used as template for cDNA construction. The cDNA was exposed to GCR gene-detecting and TaqMan probes. The level of GCR mRNA expression was determined by real-time PCR and compared with the frequency of postoperative ocular inflammation attacks that occurred despite daily temporal and, as required, subconjunctival glucocorticoid injection or other therapy. The level of 18SrRNA expression was measured and taken as the mRNA expression baseline, and the ratio between the number of GCR and housekeeping gene18SrRNA copies was calculated as the normalized ratio. RESULTS: The mean normalized ratio was 13.7 for the uveitis patients and 7.3 for the uveitis-free control patients. The median normalized ratio in the uveitis patient group was 1.5. The mean frequency of inflammation attacks was 0.24/month in the uveitis patients with a normalized ratio of 1.5 or higher and 0.53/month in those with a normalized ratio of less than 1.5, a significant difference. CONCLUSION: The level of GCR expression may relate to the response to steroids in the clinical course of inflammation. Further investigation is warranted.     

Eales''病患者眼内液血管内皮生长因子检测及意义

目的　检测Eales’病患者房水及玻璃体中血管内皮生长因子 (VEGF)含量 ,探讨VEGF对Eales’病预后的影响。方法　应用酶联免疫吸附测定法对 16例Eales’病患者行玻璃体切割术时取得的房水、玻璃体标本进行VEGF检测 ,并对同期 6例黄斑裂孔手术标本中的VEGF做同样检测作为对照。结果　Eales’病患者房水、玻璃体中VEGF质量浓度分别为 ( 1 15 6± 0 2 0 4)、( 1 2 10± 0 2 44 )ng/ml ,较对照组 ( 0 189± 0 0 3 8)、( 0 2 0 1± 0 0 5 5 )ng/ml明显增高 ,两组间差别有非常显著性意义 (P <0 0 1)。结论　Eales’病患者房水、玻璃体中VEGF质量浓度明显增高 ,提示VEGF在Eales’病发病过程中可能具有一定作用