[Amniotic membrane transplantation for the treatment severe acanthamoeba keratitis]. / Intérêt de la greffe de membrane amniotique dans le traitement des kératites amibiennes sévères.

BACKGROUND: It is now possible to treat ocular surface disorders by means of amniotic membrane transplantation. We performed a study to determine the efficacy of this technique in the treatment of severe Acanthamoeba keratitis. METHODS: We studied six patients with severe, painful, nonhealing Acanthamoeba keratitis who underwent one or two amniotic membrane transplantation procedures between February 2001 and January 2003. Histopathological analysis of the corneal buttons was performed in four cases. RESULTS: Eight amniotic membrane transplantation procedures were performed. The mean length of follow-up was 14 (range 3-21) months. The mean interval between institution of medical treatment and the procedure was 3.6 months. All patients had progressive stromal lesions caused by an inflammatory reaction. Complete reepithelialization occurred in four cases, and partial healing in two cases. Ocular inflammation and tissue destruction were decreased in all cases, pain was lessened in five cases, and corneal neovascularization was decreased in four cases. No postoperative complications were observed. Amniotic membrane was observed under dysplastic corneal epithelium on histologic examination. INTERPRETATION: Amniotic membrane transplantation may be a safe and effective treatment of severe Acanthamoeba keratitis, particularly during the inflammation phase. It may permit penetrating keratoplasty to be delayed.

Characterization and functionality of CXCR4 chemokine receptor and SDF-1 in human corneal fibroblasts.

PURPOSE: The aim of this study was to investigate whether cultured human corneal fibroblasts express functional chemokine CXCR4 receptors on their cell surface and to determine the presence of its specific ligand, SDF-1 (CXCL12), in human corneal fibroblasts. METHODS: Human corneal fibroblast cultures were obtained using human donor corneas. CXCR4 receptors were characterized using binding studies and autoradiography with [125I]SDF-1. The functionality of CXCR4 receptors was assessed by intracellular calcium measurement using a dynamic imaging microscopy system. CXCR4 and SDF-1 mRNA were detected in human corneal fibroblasts using reverse transcriptase polymerase chain reaction (RT-PCR). The CXCR4 protein was detected by western blot analysis. RESULTS: [125I]SDF-1 specifically bound to cultured corneal fibroblasts with a KD value of 8.3+/-1.2 nM. The presence of CXCR4 was confirmed by autoradiography of the radioligand on slices of corneal stroma. SDF-1 induced a rapid and transient intracellular calcium increase in cultured corneal fibroblasts that was blocked by the specific antagonist bicyclam. Moreover, a 48 kDa protein was detected by western blot analysis of corneal fibroblast extracts, using a specific CXCR4 polyclonal antibody. RT-PCR showed the expression of both CXCR4 and SDF-1 mRNAs in human corneal fibroblasts. CONCLUSIONS: These results indicate for the first time that cultured human corneal fibroblasts express the chemokine receptors CXCR4 and its ligand SDF-1. This latter might exert physiological effects on the cornea and could be involved in pathological conditions such as corneal angiogenesis.

Expression of neurotensin receptors in human corneal keratocytes.

PURPOSE: The purpose of the study was to investigate whether cultured human keratocytes express the neurotensin receptors (NTR1, NTR2, and NTR3), to determine the presence of neurotensin (NT) in keratocytes, and to assess the influence of NT on these cells. METHODS: Human keratocytes were cultured in medium treated with various concentrations (10(-7)-10(-9) M) of JMV449 (a weakly degradable NT agonist). Cell proliferation and viability were analyzed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay. Apoptosis was studied by nucleus labeling with a fluorescent dye and cold light fluorometry. NT, NTR1, NTR2, and NTR3 mRNA were detected in human keratocytes by means of reverse transcriptase-polymerase chain reaction (RT-PCR). NTR1 protein was detected by Western blot analysis. Functionality of NTR1 was assessed by intracellular calcium ([Ca2+]i) measurement with a dynamic imaging microscopy system. RESULTS: RT-PCR and Western blot analysis showed the expression of the NTR1 (mRNA and protein) and NTR3 mRNA in human corneal keratocytes. NT and NTR2 mRNA were undetectable. JMV449 induced a rapid and transient [Ca2+]i increase in human corneal keratocytes that was blocked by the specific antagonist SR48692. JMV449 significantly increased cell proliferation and viability after 72, 96, and 120 hours of culture, with a maximum effect at 10(-7) M (P < 0.005). Finally, JMV449 decreased keratocyte apoptosis, whatever the concentration used (P < 0.005). CONCLUSIONS: These results indicate that cultured human keratocytes express NTR1 and NTR3 and that NT may exert physiological effects on cornea such as regulation of keratocyte proliferation and apoptosis.