Luc Missotten

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.

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.

Chemokines in the limbal form of vernal keratoconjunctivitis

BACKGROUND/AIMS Chemokines are a family of low molecular weight cytokines that attract and activate leucocytes. The CC chemokines act on eosinophils, basophils, monocytes, and lymphocytes, suggesting that they play an important part in allergic diseases. The aims of this study were to investigate the expression of the CC chemokines, RANTES, eotaxin, monocyte chemotactic protein (MCP) 1, MCP-2, and MCP-3 in the conjunctiva of patients with vernal keratoconjunctivitis (VKC) and to determine the cellular source of these chemokines. METHODS Conjunctival biopsy specimens from nine subjects with active VKC, and six control subjects were studied by immunohistochemical techniques using a panel of monoclonal and polyclonal antibodies directed against RANTES, eotaxin, MCP-1, MCP-2, and MCP-3. The phenotype of inflammatory cells expressing chemokines was examined by sequential double immunohistochemistry. RESULTS In the normal conjunctiva, superficial epithelial cells showed a constitutive, weak cytoplasmic expression of eotaxin. Few inflammatory cells in the perivascular areas expressed RANTES, MCP-1, MCP-2, and MCP-3. In VKC specimens, the epithelium showed intense cytoplasmic eotaxin staining in all cells, and cytoplasmic RANTES staining mainly in the superficial layers. Furthermore, RANTES and eotaxin were expressed on the vascular endothelium mainly in the upper substantia propria. Compared with normal controls, VKC specimens showed significantly more inflammatory cells expressing RANTES, eotaxin, MCP-1, and MCP-3 (p<0.001, 0.0028, 0.0092, and <0.001, respectively). In VKC specimens, the numbers of inflammatory cells expressing RANTES were significantly higher than the numbers of inflammatory cells expressing eotaxin, MCP-1, and MCP-2 (all p values <0.001). Colocalisation studies revealed that the majority of inflammatory cells expressing chemokines were CD68 positive monocytes/macrophages. CONCLUSIONS These results demonstrate an increase in the expression of RANTES, eotaxin, MCP-1, and MCP-3 in the conjunctiva of patients with VKC compared with control subjects. These data suggest a potential role for these chemokines in the pathogenesis of VKC. Antagonists of chemokine receptors may provide new therapeutic modalities in VKC.

Expression of hypoxia-inducible factor-1α and the protein products of its target genes in diabetic fibrovascular epiretinal membranes

Aims: To investigate the expression of the hypoxia-inducible factor-1α (HIF-1α) and the protein products of its target genes vascular endothelial growth factor (VEGF), erythropoietin (Epo) and angiopoietins (Angs), and the antiangiogenic pigment epithelium-derived factor (PEDF) in proliferative diabetic retinopathy (PDR) epiretinal membranes. Methods: Sixteen membranes were studied by immunohistochemical techniques. Results: Vascular endothelial cells expressed HIF-1α, Ang-2 and VEGF in 15 (93.75%), 6 (37.5%) and 9 (56.25%) membranes, respectively. There was no immunoreactivity for Epo, Ang-1 and PEDF. There were significant correlations between the number of blood vessels expressing the panendothelial marker CD34 and the numbers of blood vessels expressing HIF-1α (r = 0.554; p = 0.026), Ang-2 (r = 0.830; p<0.001) and VEGF (r = 0.743; p = 0.001). The numbers of blood vessels expressing Ang-2 and VEGF in active membranes were higher than that in inactive membranes (p = 0.015 and 0.028, respectively). Conclusions: HIF-1α, Ang-2 and VEGF may play an important role in the pathogenesis of PDR. The findings suggest an adverse angiogenic milieu in PDR epiretinal membranes favouring aberrant neovascularisation and endothelial abnormalities.

Adhesion molecules in vernal keratoconjunctivitis

AIMS/BACKGROUND Adhesion molecules play a key role in the selective recruitment of different leucocyte population to inflammatory sites. The purpose of the present study was to investigate the presence and distribution of adhesion molecules in the conjunctiva of patients with vernal keratoconjunctivitis (VKC). METHODS The presence and distribution of adhesion molecules were studied in 14 conjunctival biopsy specimens from seven patients with active VKC and in four normal conjunctival biopsy specimens. We used a panel of specific monoclonal antibodies (mAbs) directed against intercellular adhesion molecule-1 (ICAM-1), intercellular adhesion molecule-3 (ICAM-3), lymphocyte function associated antigen-1 (LFA-1), very late activation antigen-4 (VLA-4), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leucocyte adhesion molecule-1 (ELAM-1). In addition, a panel of mAbs were used to characterise the composition of the inflammatory infiltrate. RESULTS In the normal conjunctiva, ICAM-1 was expressed on the vascular endothelium only, LFA-1 and ICAM-3 on epithelial and stromal mononuclear cells , and VLA-4 on stromal mononuclear cells. The expression of VCAM-1 and ELAM-1 was absent. The number of cells expressing adhesion molecules was found to be markedly increased in all VKC specimens. This was concurrent with a heavy inflammatory infiltrate. Strong ICAM-1 expression was induced on the basal epithelial cells, and vascular endothelial cells. Furthermore, ICAM-1 was expressed on stromal mononuclear cells. LFA-1 and ICAM-3 were expressed on the majority of epithelial and stromal infiltrating mononuclear cells. VLA-4 expression was noted on stromal mononuclear cells. Compared with controls, VKC specimens showed significantly more ICAM-3+, LFA-1+, and VLA-4+ cells. VCAM-1 and ELAM-1 were induced on the vascular endothelial cells. CONCLUSIONS Increased expression of adhesion molecules may play an important role in the pathogenesis of VKC.

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.

Immunopathogenesis of conjunctival scarring in trachoma

Purpose Trachoma, a chronic follicular conjunctivitis caused by infection with Chlamydia trachomatis, is the leading cause of preventable blindness. The blinding complications are associated with progressive conjunctival scarring that may result from immunologically mediated responses. We studied the processes involved in the regulation of inflammation and fibrosis in trachoma by investigating the expression of fibrogenic cytokines in the conjunctiva.Methods We studied conjunctival biopsy specimens obtained from nine subjects with active trachoma and from four control subjects. We used immunohistochemical techniques and a panel of monoclonal and polyclonal antibodies directed against interleukin-1α (IL-1α), interleukin-lβ (IL-1β), tumour necrosis factor-α (TNF-α) and platelet-derived growth factor (PDGF). In addition, we characterised the composition of the inflammatory infiltrate by the use of a panel of monoclonal antibodies. Sirius red and Van Gieson stains were used to characterise the extent of fibrous tissue in the substantia propria.Results Trachoma specimens showed greater numbers of inflammatory cells than control specimens. The expression of cytokines was absent in the normal conjunctiva. Cytoplasmic IL-1α and IL-1β expression was noted in the conjunctival epithelium in all trachoma specimens. IL-1α, IL-1β, TNF-α and PDGF were detected in macrophages infiltrating the substantia propria. B lymphocytes predominated over T lymphocytes in six trachoma biopsies with fibrosis confined to the deep substantia propria, whereas T lymphocytes predominated over B lymphocytes in three biopsies with more extensive fibrosis. In all trachoma biopsies helper/inducer T lymphocytes outnumbered suppressor/cytotoxic T lymphocytes.Conclusions The upregulated local production of IL-1α, IL-β, TNF-α and PDGF might contribute to conjunctival damage and scarring in trachoma.

Expression of antiapoptotic and proapoptotic molecules in diabetic retinas

PurposeTo investigate the expression of the antiapoptotic and proapoptotic markers in diabetic retinas.MethodsIn total, 12 donor eyes from six subjects with diabetes mellitus, and 10 eyes from five nondiabetic subjects without known ocular disease serving as control subjects were examined. Immunohistochemical techniques were used with antibodies directed against cyclooxygenase-2 (Cox-2), Akt (protein kinase B), Mcl-1, Bad, cytochrome c, apoptosis-inducing factor (AIF), tumour necrosis factor receptor-1-associated death domain protein (TRADD), and Fas-associated death domain protein (FADD).ResultsIn retinas from all subjects without diabetes, cytoplasmic immunoreactivity for the antiapoptotic molecules Cox-2, Akt, and Mcl-1 was noted in ganglion cells. Cytoplasmic immunostaining for Cox-2 was also noted in the retinal pigment epithelial cells. Weak immunoreactivity for the mitochondrial apoptogenic proteins cytochrome c, and AIF was noted in the inner segments of photoreceptors, in the inner one-third of the outer plexiform layer, in cells in the inner nuclear layer, in the inner plexiform layer, and in ganglion cells. There was no immunoreactivity for the other antibodies tested. All diabetic retinas showed de novocytoplasmic immunoreactivity for Bad in ganglion cells, and in occasional cells in the inner nuclear layer. Upregulation of cytochrome cand AIF immunoreactivity was noted. Cox-2, Akt, and Mcl-1 immunoreactivity was not altered in the diabetic retinas. There was no immunoreactivity for TRADD, and FADD.ConclusionsGanglion cells in diabetic and nondiabetic retinas express the antiapoptotic molecules Cox-2, Akt, and Mcl-1. Retinal ganglion cells express the proapoptotic molecule Bad in response to diabetes-induced neuronal injury. Diabetic retinas show upregulation of the mitochondrial proteins cytochrome c, and AIF.

Inducible nitric oxide synthase and vascular endothelial growth factor are colocalized in the retinas of human subjects with diabetes.

AbstractPurpose Nitric oxide (NO) mediates vascular endothelial growth factor (VEGF)-induced angiogenesis and vascular hyperpermeability. This study was undertaken to study the cellular distribution of inducible nitric oxide synthase (iNOS) and VEGF in the retinas from human subjects with diabetes mellitus. In addition, glial reactivity and peroxynitrite generation were detected by immunolocalization of glial fibrillary acidic protein (GFAP) and nitrotyrosine, respectively.Methods Eight post-mortem eyes from four consecutive subjects with diabetes mellitus and eight eyes from four subjects without diabetes and without known ocular disease were prospectively collected and examined. We used immunohistochemical techniques and antibodies directed against iNOS, VEGF, GFAP, and nitrotyrosine.Results In retinas from all subjects without diabetes, weak GFAP immunoreactivity was confined to nerve fibre and ganglion cell layers. There was no immunoreactivity for iNOS, nitrotyrosine, and VEGF. All diabetic retinas showed GFAP induction in Müller cells and GFAP upregulation in nerve fibre and ganglion cell layers. All diabetic retinas showed cytoplasmic immunoreactivity for iNOS, and VEGF in ganglion cells, cells in the inner nuclear layer, and glial cells. In serial sections, ganglion cells and cells in the inner nuclear layer expressing VEGF were localized in the same area of iNOS-expressing ganglion cells and cells in the inner nuclear layer. Six retinas from three subjects with diabetes showed immunoreactivity for nitrotyrosine in vascular endothelial cells in inner retinal layer.Conclusions iNOS and VEGF are colocalized in diabetic retinas. Increased GFAP immunoreactivity is a pathological event in the retina during diabetes.

Subretinal neovascular membranes associated with chronic membranoproliferative glomerulonephritis type II

Subretinal neovascular membranes were observed in three patients with chronic membranoproliferative glomerulonephritis type II (dense deposit disease). The first signs of glomerulonephritis occurred at respective ages of 13, 10 and 10 years; subretinal neovascular membranes were noted at respective ages of 25, 32 and 32 years. All patients had bilateral, widespread retinal pigment epithelial abnormalities. Our findings indicate that subretinal neovascularization is a complication of dense deposit disease. In one patient, the early recognition and laser treatment of an extrafoveal subretinal neovascular membrane prevented further loss of vision.