Autocrine CCL2, CXCL4, CXCL9 and CXCL10 signal in retinal endothelial cells and are enhanced in diabetic retinopathy.

This study aimed at examining the presence and role of chemokines (angiogenic CCL2/MCP-1 and angiostatic CXCL4/PF-4, CXCL9/Mig, CXCL10/IP-10) in proliferative diabetic retinopathy (PDR). Regulated chemokine production in human retinal microvascular cells (HRMEC) and chemokine levels in vitreous samples from 40 PDR and 29 non-diabetic patients were analyzed. MCP-1, PF-4, Mig, IP-10 and VEGF levels in vitreous fluid from PDR patients were significantly higher than in controls. Except for IP-10, cytokine levels were significantly higher in PDR with active neovascularization and PDR without traction retinal detachment (TRD) than those in inactive PDR, PDR with TRD and control subjects. Exploratory regression analysis identified associations between higher levels of IP-10 and inactive PDR and PDR with TRD. VEGF levels correlated positively with MCP-1 and IP-10. Significant positive correlations were observed between MCP-1 and IP-10 levels. In line with these clinical findings Western blot analysis revealed increased PF-4 expression in diabetic rat retinas. HRMEC produced MCP-1, Mig and IP-10 after stimulation with IFN-γ, IL-1ß or lipopolysaccharide. IFN-γ synergistically enhanced Mig and IP-10 production in response to IL-1ß or lipopolysaccharide. MCP-1 was produced by HRMEC in response to VEGF treatment and activated HRMEC via the ERK and Akt/PKB pathway. On the other hand, phosphorylation of ERK induced by VEGF and MCP-1 was inhibited by PF-4, Mig and IP-10. In accordance with inhibition of angiogenic signal transduction pathways, PF-4 inhibited in vitro migration of HRMEC. Thus, regulatory roles for chemokines in PDR were demonstrated. In particular, IP-10 might be associated with the resolution of active PDR and the development of TRD.

Prognostic factors after primary vitrectomy and perfluorocarbon liquids for bullous rhegmatogenous retinal detachment.

PURPOSE: To identify prognostic factors for visual acuity and anatomic outcomes associated with bullous rhegmatogenous retinal detachment (RRD) management using primary pars plana vitrectomy, intraoperative perfluorocarbon liquids (PFCLs), and internal gas tamponade. METHODS: The authors studied a consecutive series of 115 eyes (115 patients) with a bullous RRD not complicated by proliferative vitreoretinopathy (PVR) associated with large, multiple, and/or posterior breaks in 58 (50.4%) eyes. All eyes underwent vitrectomy, injection of PFCL, and gas tamponade as the primary procedure. Encircling scleral bands were placed in all cases. The follow-up period ranged from 3 to 60 months (mean 16.6+/-14.1 months). RESULTS: Retinal reattachment was achieved in 92.2% of eyes (106/115) with one operation and in all eyes after a second procedure. PVR was observed in 1 (0.87%) eye and preretinal membranes in 3 (2.6%) eyes. Progression of pre-existing cataract and development of new cataract occurred in 45 (58.4%) of the 77 phakic eyes. The presence of inferior retinal breaks was significantly associated with redetachment after the first procedure (p=0.0156). On univariate analysis, better preoperative visual acuity (p<0.001), macular sparing retinal detachment (p<0.001), and fewer quadrants involved by the detachment (p=0.0015) were significant positive prognostic factors for final visual acuity. Logistic regression analysis highlighted that macular sparing retinal detachment and absence of trauma were associated with better final visual acuity. CONCLUSIONS: Redetachment was associated with the presence of inferior retinal breaks. Visual recovery was dependent on preoperative visual acuity, macular involvement, extent of retinal detachment, and trauma.

Prognostic factors associated with outcomes after giant retinal tear management using perfluorocarbon liquids.

PURPOSE: To identify prognostic factors for visual acuity and anatomic outcomes associated with giant retinal tear management using intraoperative perfluorocarbon liquids. METHODS: All patients with giant retinal tears without proliferative vitreoretinopathy (PVR) who underwent management with intraoperative perfluorocarbon liquids between 1994 and 2005 were reviewed. RESULTS: The study included 115 patients (117 eyes), 93 (80.9%) males and 22 (19.1%) females, with a mean age of 30.3+/-15.2 years. Mean follow-up period was 29.7+/-26.7 months. Success rate with primary procedure was 78.6%, which increased to 94% with multiple surgeries. On univariate analysis, factors significantly associated with final visual acuity better than 20/200 included phakic/clear lens at presentation (p=0.0113), partial retinal detachment (p=0.0233), absence of all postoperative complications (p=0.0122), absence of recurrent retinal detachment (p=0.0406), and absence of postoperative PVR (p=0.0062). Logistic regression analysis highlighted that phakic/clear lens at presentation, unfolded flap of the giant tear, absence of postoperative cataract, and absence of postoperative PVR were associated with final visual acuity better than 20/200. On univariate analysis, use of gas tamponade was significantly associated with recurrent retinal detachment (p=0.0190). Logistic regression analysis highlighted that placement of an encircling scleral buckle and use of silicone oil tamponade were associated with anatomic reattachment with primary procedure. CONCLUSIONS: Encircling scleral buckling and silicone oil tamponade decrease the risk of recurrent retinal detachment.

Full preoperative panretinal photocoagulation improves the outcome of trabeculectomy with mitomycin C for neovascular glaucoma.

PURPOSE: To investigate the efficacy of full panretinal photocoagulation (PRP) followed by trabeculectomy with mitomycin C (MMC) in the management of eyes with neovascular glaucoma (NVG). METHODS: This study is based on 30 consecutive eyes of 27 patients with NVG who underwent full PRP followed by trabeculectomy with MMC. NVG was secondary to proliferative diabetic retinopathy (23 eyes) and central retinal vein occlusion (7 eyes). Kaplan-Meier survival analysis of the surgical outcome was performed. Operative success was defined as an intraocular pressure (IOP) of < or = 21 mmHg without medical therapy. RESULTS: Kaplan-Meier cumulative success rates at the 6-, 12-, and 24-month intervals were 86.5%, 74.7%, and 57.6%, respectively. Pseudophakia was the only identified significant risk factor for failure (p=0.0138; Fisher exact test). Additional surgical procedures were performed in 8 (26.6%) eyes. The mean IOP decreased from 41.0+/-10.2 mmHg to 18.2+/-9.2 mmHg (p<0.001; Wilcoxon signed rank test). The number of anti-glaucoma medications was reduced from 3.1+/-0.5 preoperatively to 0.3+/-0.7 postoperatively (p<0.001; Wilcoxon signedrank test). Twenty-four (80%) eyes were classified as surgical success after a mean followup period of 17.3+/-22.1 months. Twenty-two (73.3%) eyes had improved vision or retained preoperative vision. CONCLUSIONS: Full PRP followed by trabeculectomy with MMC can effectively reduce the elevatedIOP associated with NVG. Presence of pseudophakia is a significant negative predictor of surgical outcome.

Sympathetic ophthalmia after successful retinal reattachment surgery with vitrectomy.

PURPOSE: To report a case of sympathetic ophthalmia (SO) following one successful pars plana vitrectomy (PPV) for rhegmatogenous retinal detachment. METHODS: Case report. RESULTS: A 50-year-old man developed SO 5 weeks after successful repair of rhegmatogenous retinal detachment with PPV and intraocular gas tamponade. The patient presented with bilateral multifocal exudative retinal detachments and inflamed optic nerve with characteristic changes of SO detected by fluorescein angiography, indocyanine green angiography, and optical coherence tomography. Prompt use of systemic steroids and cyclosporin A resulted in control of the uveitis with significant visual improvement. CONCLUSIONS: PPV should be viewed as a major risk factor for development of SO.

Cyclooxygenase-2 in human and experimental ischemic proliferative retinopathy.

BACKGROUND: Intravitreal neovascular diseases, as in ischemic retinopathies, are a major cause of blindness. Because inflammatory mechanisms influence vitreal neovascularization and cyclooxygenase (COX)-2 promotes tumor angiogenesis, we investigated the role of COX-2 in ischemic proliferative retinopathy. METHODS AND RESULTS: We describe here that COX-2 is induced in retinal astrocytes in human diabetic retinopathy, in the murine and rat model of ischemic proliferative retinopathy in vivo, and in hypoxic astrocytes in vitro. Specific COX-2 but not COX-1 inhibitors prevented intravitreal neovascularization, whereas prostaglandin E2, mainly via its prostaglandin E receptor 3 (EP3), exacerbated neovascularization. COX-2 inhibition induced an upregulation of thrombospondin-1 and its CD36 receptor, consistent with the observed antiangiogenic effects of COX-2 inhibition; EP3 stimulation reversed effects of COX-2 inhibitors on thrombospondin-1 and CD36. CONCLUSIONS: These findings point to an important role for COX-2 in ischemic proliferative retinopathy, as in diabetes.

MK-801 protects retinal neurons from hypoxia and the toxicity of glutamate and aspartate.

The protective effect of the anticonvulsant MK-801 and the antitussive dextromethorphan, which are both N-methyl-D-aspartate receptor antagonists, and kynurenic acid, a broad-spectrum excitotoxin antagonist, was tested in cultured rat retinal cells in an hypoxic environment. The protective effect of these antagonists also was tested in cultured retinal cells and in intact adult rat retinas exposed to the exogenous excitotoxins L-glutamic acid and N-methyl-D-aspartic acid. MK-801 and kynurenic acid protected retinal neurons from hypoxic damage and from the toxicity of exogenous L-glutamic acid and N-methyl-D-aspartic acid. Dextromethorphan, a less potent antagonist, did not protect the retinal neurons from hypoxic damage or the toxicity of exogenous L-glutamic acid, but did attenuate N-methyl-D-aspartate toxicity. These results provide evidence that the synaptic release of excitatory transmitters, most likely glutamate and aspartate, mediate the death of hypoxic retinal neurons. Compounds related to MK-801 may have possible therapeutic applications in the management of retinal ischemia.

Gelatinase B in vernal keratoconjunctivitis.

OBJECTIVES: To investigate the expression of gelatinase B in the conjunctiva of patients with vernal keratoconjunctivitis (VKC) and the cellular source of this enzyme. METHODS: Conjunctival biopsy specimens from 12 patients with active VKC and 12 control subjects were studied using immunohistochemical techniques and a monoclonal antibody against gelatinase B. The phenotype of gelatinase B(+) inflammatory cells was examined using double immunohistochemical analysis and monoclonal antibodies against eosinophil peroxidase or macrophage CD68. Quantitative zymography was used to compare the activity of gelatinase B in conjunctival biopsy specimens from 10 patients with active VKC and 7 control subjects. RESULTS: Gelatinase B was detected in a few polymorphonuclear cells in 8 control specimens. All VKC specimens showed gelatinase B immunoreactivity in the epithelial and stromal inflammatory infiltrate. Compared with control specimens, VKC specimens showed significantly more gelatinase B-positive cells (mean +/- SD, 40.8 +/- 29.9 vs 10.3 +/- 2.4; P<.02). Most gelatinase B-positive cells were eosinophils (90.2% +/- 3.6%). Zymography revealed that gelatinase B levels in VKC specimens were significantly higher than the levels found in normal conjunctiva (3780.3 +/- 3541.0 vs 610.1 +/- 397.1 scanning units; P<.03). CONCLUSIONS: These findings suggest overexpression of gelatinase B by eosinophils in VKC specimens and participation of gelatinase B in the pathologic changes in VKC. CLINICAL RELEVANCE: Control of the release and/or activation of gelatinase B in eosinophils may provide a new therapeutic strategy for treating VKC.

Ocular levels of azithromycin.

OBJECTIVE: To assess azithromycin levels in human serum, aqueous humor, tear fluid, and conjunctival tissue specimens after administration of a single 1-g oral dose of azithromycin. METHODS: Sixty patients undergoing cataract surgery were included in this analysis. Serum, aqueous, and tear specimens were collected 3, 6, and 12 hours and 1, 2, 3, and 4 days after azithromycin administration. Conjunctival tissue biopsy specimens were collected 1, 2, 3, 4, 6, 8, 10, 12, and 14 days after azithromycin administration. All specimens were subjected to analysis by high-performance liquid chromatography-mass spectrometry. RESULTS: Azithromycin concentration ranges during the specified sampling times were as follows: serum, 21 to 974 ng/mL; tear, 82 to 2892 ng/mL; aqueous, 10 to 69 ng/mL; and conjunctival, 0.7 to 32 micrograms/g. Levels above the 90% minimal inhibitory concentration (MIC90) for Chlamydia trachomatis were detected after 4 days in all tear samples and after 14 days in all conjunctival tissue specimens following oral azithromycin administration. CONCLUSION: We demonstrated prolonged high levels of azithromycin in drug-targeted ocular tissue. Prolonged high concentrations of azithromycin in conjunctival tissue make this drug suitable for treatment of conjunctivitis caused by chlamydiae and other susceptible organisms.

Expression of the inducible isoform of nitric oxide synthase in the retinas of human subjects with diabetes mellitus.

PURPOSE: Inducible nitric oxide synthase has been implicated in the pathogenesis of cerebral ischemic damage, in the angiogenic process and in diabetic vascular damage. This study was undertaken to determine whether inducible nitric oxide synthase is present in the retinas from human subjects with diabetes mellitus. METHODS: This was an experimental immunohistochemical prospective study. Ten postmortem eyes from five subjects with diabetes mellitus, 10 eyes from five subjects without diabetes and without known ocular disease, and two eyes from one subject with unilateral ocular ischemic syndrome secondary to severe carotid artery obstruction were examined. We used immunohistochemical techniques and antibodies directed against inducible nitric oxide synthase, glial fibrillary acidic protein, and vimentin. The main outcome measure was immunoreactivity for these antibodies. RESULTS: Immunoreactivity for inducible nitric oxide synthase was not observed in retinas from all subjects without diabetes and without ocular disease. Six retinas from three subjects with diabetes and nonproliferative retinopathy, and the retina from the eye with ocular ischemic syndrome showed immunoreactivity for inducible nitric oxide synthase in cells with elongated processes. Based on morphology and on glial fibrillary acidic protein and vimentin immunoreactivity, this inducible nitric oxide synthase immunoreactivity appeared to localize to retinal Müller glial cells. CONCLUSIONS: These observations suggest that Müller cells may be involved in the microvascular remodeling of the diseased retina and that high concentrations of nitric oxide produced by inducible nitric oxide synthase could contribute to neurotoxicity and angiogenesis that occur in diabetic retinopathy.

An immunohistochemical study of topical cyclosporine in vernal keratoconjunctivitis.

PURPOSE: To determine the immunomodulating effects of topical cyclosporine on the immune cells in the conjunctival biopsy specimens obtained from patients with active vernal keratoconjunctivitis. METHODS: We studied six patients who had severe active vernal keratoconjunctivitis. Each patient was given topical cyclosporine 2% eyedrops four times daily. A 2 x 2-mm limbal conjunctival biopsy specimen was obtained from each patient before and three weeks after treatment. Using a panel of monoclonal and polyclonal antibodies and immunohistochemical techniques, we analyzed the conjunctival immune cells before and after cyclosporine treatment. RESULTS: Three weeks after topical cyclosporine treatment, there was marked clinical improvement and a statistically significant reduction in the number of epithelial and stromal class II MHC+ cells, UCHL1+ T cells, and stromal IgA+ and IgG+ plasma cells. The mean number of cells before and after therapy, respectively, were: class II MHC+ (epithelium), 31.5 +/- 13.1 and 8.3 +/- 5.6 (P = .031); class II MHC+ (stroma), 77.0 +/- 28.7 and 24.7 +/- 17.5 (P = .031); UCHL1+ T cells (epithelium), 24.5 +/- 14.1 and 4.2 +/- 2.9 (P = .031); UCHL1+ T cells (stroma), 78.7 +/- 31.1 and 44.5 +/- 27.5 (P = .031); IgA+ plasma cells, 66.7 +/- 32.1 and 22.2 +/- 7.8 (P = .031); and IgG+ plasma cells, 37.3 +/- 30.0 and 9.0 +/- 6.4 (P = .031). There was a statistically insignificant decrease in the epithelial class II MHC+ dendritic Langerhans cells, epithelial and stromal KP1+ macrophages, stromal OPD4+ helper/inducer T cells, and stromal L26+ B cells. The numbers of IgE+ plasma cells and mast cells were unaltered. CONCLUSION: The clinical improvement in vernal keratoconjunctivitis after topical cyclosporine therapy may result from its immunomodulating effect on the components of cell-mediated and humoral immune responses. In contrast, the drug has no immunomodulatory effect on mast cells and IgE-mediated allergic response.

Monocyte chemotactic protein-1 in proliferative vitreoretinal disorders.

PURPOSE: To investigate whether the chemokines monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) are involved in the pathogenesis of proliferative vitreoretinal disorders and to study their possible interaction with IL-6. METHODS: In a prospective study of 125 consecutive patients (125 eyes), vitreous and paired serum samples were obtained and were assayed for MCP-1 and IL-8. Levels of IL-6 were determined by proliferation of the IL-6-dependent hybridoma cell line 7TD1. RESULTS: Monocyte chemotactic protein-1 was detected in 13 (48%) of 27 vitreous samples from patients with retinal detachment, in five (63%) of eight samples from patients with macular pucker, in 31 (72%) of 43 samples from patients with proliferative vitreoretinopathy, and in 32 (76%) of 42 samples from patients with proliferative diabetic retinopathy, but not in samples from five patients with idiopathic epiretinal membrane. There was a significant (P = .049) correlation between the incidence of MCP-1 detection in retinal detachment, macular pucker, and proliferative vitreoretinopathy groups and the severity of proliferation. Interleukin-8 was detected in two vitreous samples from eyes with retinal detachment, in two samples from eyes with proliferative vitreoretinopathy, and in three samples from eyes with proliferative diabetic retinopathy. Monocyte chemotactic protein-1 levels in the vitreous samples were positively correlated with IL-6 levels (r = .31, P = .01). Interleukin-6 levels were significantly (P = .0097) greater in vitreous samples with than without detectable levels of MCP-1. CONCLUSION: Monocyte chemotactic protein-1 is present in a substantial percent of vitreous samples from eyes with proliferative vitreoretinal disorders and may help in stimulating the infiltration of monocytes and macrophages into eyes with these disorders.

Gelatinase B in proliferative vitreoretinal disorders.

PURPOSE: To investigate whether gelatinases A and B are involved in the pathogenesis of proliferative vitreoretinal disorders. METHODS: In a prospective study of 101 consecutive patients, vitreous and paired serum samples were obtained from 38 patients with rhegmatogenous retinal detachment complicated by proliferative vitreoretinopathy, 25 patients with rhegmatogenous retinal detachment with no proliferative vitreoretinopathy, and 38 patients with proliferative diabetic retinopathy. Gelatinase activities were determined by quantitative zymography. RESULTS: All vitreous samples contained comparable levels of the constitutive gelatinase A. Inducible gelatinase B was detected in eight (32%) of 25 vitreous samples from patients with rhegmatogenous retinal detachment with no proliferative vitreoretinopathy (mean +/- SD, 319.5 +/- 521.0 scanning units), in 17 (44.7%) of 38 vitreous samples from patients with proliferative vitreoretinopathy (560.6 +/- 718.9 scanning units), and in 34 (89.5%) of 38 vitreous samples from patients with proliferative diabetic retinopathy (1,707.2 +/- 1,220.3 scanning units). The incidence of detection of gelatinase B in proliferative diabetic retinopathy cases was significantly higher than it was in rhegmatogenous retinal detachment with no proliferative vitreoretinopathy and proliferative vitreoretinopathy cases (P < .001). Gelatinase B levels in the vitreous samples of patients with proliferative diabetic retinopathy were higher than the levels found in patients with rhegmatogenous retinal detachment with no proliferative vitreoretinopathy and in patients with proliferative vitreoretinopathy (P = .0152). Gelatinase A was detected in all the tested sera, whereas none of the tested paired serum samples contained detectable gelatinase B activity. CONCLUSIONS: Gelatinase B may play an important role in extracellular matrix degradation associated with neovascularization in proliferative diabetic retinopathy.

Cytokines in the vitreous of patients with proliferative diabetic retinopathy.

Sixteen vitreous and paired serum samples from 13 patients with proliferative diabetic retinopathy, vitreous samples from seven cadaveric control subjects, and aqueous humor samples from 15 normal control subjects were assayed for the cytokines interleukin-1, tumor necrosis factor-alpha, interleukin-6, and interferon-gamma. Interleukin-6 was detected in 15 of 16 vitreous samples (94%) from diabetic patients, but it was not detected in any of the aqueous humor samples. Vitreous interleukin-6 levels positively correlated with ocular disease activity. Interleukin-1 was detected in seven of 16 vitreous samples (44%) and in four of ten aqueous humor samples (40%), whereas tumor necrosis factor-alpha and interferon-gamma were never detected in vitreous or aqueous fluid. Serum samples from diabetic patients and control subjects contained comparable low levels of interleukin-6. Interleukin-1, tumor necrosis factor-alpha, and interferon-gamma were not found in any of the sera. Because interleukin-6 can function as B-cell differentiation factor, this cytokine may have a role in immunoglobulin deposition in the ocular tissues and in the immunopathologic characteristics of proliferative retinopathy.

Full panretinal photocoagulation and early vitrectomy improve prognosis of retinal vasculitis associated with tuberculoprotein hypersensitivity (Eales' disease).

BACKGROUND/AIMS: Eales' disease is an uncommon vasoproliferative retinal disease affecting otherwise healthy young men that is characterised by obliterative retinal periphlebitis, with sequelae such as recurrent vitreous haemorrhage and traction retinal detachment. This study was undertaken to determine whether visual prognosis of Eales' disease could be improved by appropriate medical and surgical treatment. METHODS: The authors retrospectively studied 30 patients (46 eyes) who were treated from 1992 to 2001. Recorded data included patient age, sex, race, medical history, medications, results of the ophthalmological examination, results of diagnostic laboratory evaluation, and details of systemic and surgical treatments. The mean follow up was 10.6 months. RESULTS: 19 patients (23 eyes) who presented with active periphlebitis received systemic steroids and antituberculous therapy. Extensive full panretinal photocoagulation was performed in 21 eyes that presented with new vessel formation and peripheral capillary closure with or without vitreous haemorrhage. Vitrectomy and endolaser panretinal photocoagulation was necessary in 15 eyes, for severe non-clearing vitreous haemorrhage in 11 eyes and vitreous haemorrhage with traction retinal detachment in four eyes. Complete regression of the disease was achieved in all eyes. Vitrectomy resulted in a significant visual improvement with 14 of the 15 eyes (93.3%) achieving > or =20/200 visual acuity. Overall, the distribution of visual acuities among eyes improved from presentation to final follow up, with 36.4% of eyes having 20/40 or better acuity at presentation compared with 63.6% of eyes by final follow up. CONCLUSIONS: These results suggest that aggressive treatment of Eales' disease with systemic steroids and antituberculous therapy, full panretinal photocoagulation and early vitrectomy, when necessary, may result in improving the anatomic and visual outcome.

Interferon-gamma and tumour necrosis factor induce expression of major histocompatibility complex antigen on rat retinal astrocytes.

Cultured rat retinal astrocytes were tested by indirect immunofluorescence staining for their ability to express class I and II major histocompatibility complex (MHC) antigens under basal culture conditions and after three days of stimulation with two recombinant cytokines, rat interferon-gamma (IFN-gamma) and human tumour necrosis factor alpha (TNF alpha). Under basal culture conditions low levels of class I antigens were detected on a small percentage of cells, but there was no visible class II. IFN-gamma and TNF alpha stimulation enhanced class I expression. TNF alpha had no effect on class II expression, whereas IFN-gamma induced the expression of class II in a dose dependent manner. These findings suggest that retinal astrocytes might play a part in immunological events occurring in the retina.

Laser photocoagulation control of diabetic macular oedema without fluorescein angiography.

This study included 40 eyes in 22 diabetic patients with focal macular oedema. Laser photocoagulation was directed at decompensated or leaking microvascular lesions clinically detected without using pretreatment fluorescein angiograms. Post-treatment fluorescein angiograms performed after adequate clinical control of disease showed complete resolution of the macular oedema in 25 eyes (62.5%), whereas persistent leakage from microvascular lesions closer than 500 microns from the centre of the foveola was noted in 15 eyes (37.5%). These were clinically detected during the pretreatment examination and were found not to impair or threaten the patient's vision. Our data confirm the clinical impression that fluorescein angiography is not necessary for effective treatment and should be used only if necessary.

Expression of gelatinase B in trachomatous conjunctivitis.

BACKGROUND/AIMS: Gelatinase B is a matrix metalloproteinase involved in extracellular matrix (ECM) breakdown often associated with scarring and other pathological disorders. It was investigated whether gelatinase B is involved in the pathogenesis of ECM degradation associated with trachomatous conjunctivitis. METHODS: Conjunctival biopsy specimens obtained from six patients with active trachoma, six patients with active vernal keratoconjunctivitis (VKC), and seven control subjects were studied. Immunohistochemical techniques and a specific monoclonal antibody against human gelatinase B were used, and a monoclonal antibody against macrophage CD68 to identify mononuclear cells with gelatinase B immunoreactivity. In addition, quantitative zymography was used to compare the activity of gelatinase B in conjunctival biopsy specimens from seven patients with active trachoma and seven control subjects. RESULTS: Gelatinase B was detected by immunohistochemistry only in polymorphonuclear cells located in the vascular lumens in three normal conjunctival biopsy specimens. In all trachoma specimens and in five VKC specimens, gelatinase B was localised in monocyte/macrophage cells, positive for the CD68 marker, and in polymorphonuclear cells. The majority of the latter cell type was located in intravascular spaces. Compared with VKC specimens, trachoma specimens showed significantly more immunoreactive gelatinase B monocyte/macrophage cells (52.3 (21.9) v 8.2 (6.4); p <0.001) and polymorphonuclear cells (23.2 (14.2) v 6.3 (5.4); p = 0. 013). Activated macrophages with giant cell morphology clearly stained with the gelatinase B specific monoclonal antibody were observed in trachoma specimens. Zymography revealed that gelatinase B levels in trachoma specimens were significantly higher than the levels found in normal conjunctiva (1739.6 (1078.3) v 609.3 (395.9) scanning units; p = 0.0127). CONCLUSIONS: The increased activity of gelatinase B and numbers of inflammatory cells containing gelatinase B in trachoma specimens suggest that this enzyme plays a part in the pathogenesis of conjunctival scarring in trachoma.

Expression of T lymphocyte chemoattractants and activation markers in vernal keratoconjunctivitis.

BACKGROUND/AIMS: T lymphocytes are present in increased numbers in the conjunctiva of patients with vernal keratoconjunctivitis (VKC) and their activation has a central role in the pathogenesis of the chronic allergic inflammatory reactions seen in VKC. The aims of this study were to examine the expression of three recently described potent T lymphocyte chemoattractants, PARC (pulmonary and activation regulated chemokine), macrophage derived chemokine (MDC), and I-309, the MDC receptor CCR4, and T lymphocyte activation markers, CD25, CD26, CD62L, CD71, and CD30, and to correlate them with the counts of CD3(+) T lymphocytes in the conjunctiva of patients with VKC. METHOD: Conjunctival biopsy specimens from 11 patients with active VKC, and eight control subjects were studied by immunohistochemical techniques using a panel of monoclonal and polyclonal antibodies directed against PARC, MDC, I-309, CCR4, CD25, CD26, CD62L, CD71, and CD30. The numbers of positively stained cells were counted. The phenotype of inflammatory cells expressing chemokines was examined by double immunohistochemistry. RESULTS: In the normal conjunctiva, vascular endothelial cells in the upper substantia propria showed weak immunoreactivity for CD26. There was no immunoreactivity for the other antibodies. VKC specimens showed inflammatory cells expressing PARC, MDC, and I-309. The numbers of PARC(+) inflammatory cells were higher than the numbers of MDC(+) and I-309(+) inflammatory cells and the mean values of the three groups differed significantly (17.0 (SD 10.1); 9.5 (9.9), and 4.3 (7.9), respectively, p = 0.0117, ANOVA). The numbers of PARC(+) inflammatory cells had the strongest correlation with the numbers of CD3(+) T lymphocytes. Few CCR4(+) inflammatory cells were observed in only three specimens. Double immunohistochemistry revealed that all inflammatory cells expressing chemokines were CD68(+) monocytes/macrophages. The numbers of CD25(+) T lymphocytes were higher than the numbers of CD26(+), CD62L(+), CD71(+), and CD30(+) T lymphocytes and the mean values of the five groups differed significantly (46.2 (27.9), 30.7 (16.0), 20.1 (8.6), 7.8 (7.7), and 6.5 (4.0), respectively, p <0.001, ANOVA). The numbers of CD25(+) T lymphocytes had the strongest correlation with the numbers of CD3(+) T lymphocytes. CONCLUSION: These results suggest a potential role for PARC, MDC, and I-309 in attracting T lymphocytes into conjunctiva in VKC. T lymphocytes in VKC are activated and express several activation markers which might contribute to the pathogenesis of VKC.

Expression of chemokine receptors in vernal keratoconjunctivitis.

BACKGROUND/AIMS: Chemokines are small peptides which are potent activators and chemoattractants for leucocyte subpopulations. Their action is mediated by a family of seven transmembrane spanning G-protein coupled receptors. The aims of this study were to examine the expression of the chemokine receptors CCR1, CCR3, CCR5, CXCR3, and CXCR4 in the conjunctiva of patients with vernal keratoconjunctivitis (VKC) and to investigate the phenotype of inflammatory cells expressing these chemokine receptors. METHODS: Conjunctival biopsy specimens from 16 patients with active VKC, and eight control subjects were studied by immunohistochemical techniques using a panel of monoclonal antibodies directed against human CCR1, CCR3, CCR5, CXCR3, and CXCR4. The phenotype of inflammatory cells expressing chemokine receptors was examined by double immunohistochemistry. RESULTS: In the normal conjunctiva, few inflammatory cells expressed CXCR3 in five of eight specimens. There was no immunoreactivity for CCR1, CCR3, CCR5, and CXCR4. In VKC specimens, membranous immunoreactivity for CXCR3 was noted on inflammatory cells in all specimens. Compared with control specimens, VKC specimens showed significantly more inflammatory cells expressing CXCR3 (54.3 (SD 34.3) v 3.3 (5.0); p<0.001). Few CCR1+, CCR3+, CCR5+, and CXCR4+ inflammatory cells were observed in only three of 16 specimens. Double immunohistochemistry revealed that all CXCR3 positive inflammatory cells were CD3 positive T lymphocytes and that 61.7% (3.7%) of the infiltrating T lymphocytes were reactive for CXCR3. CONCLUSIONS: CXCR3 is the predominant chemokine receptor and is expressed abundantly on T lymphocytes in the conjunctiva of patients with active VKC. These data suggest a potential role for CXCR3 receptors in the regulation of lymphocyte recruitment within conjunctiva of VKC patients. New therapeutic strategies that block CXCR3 may inhibit T lymphocyte recruitment and suppress adverse inflammatory reactions.