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Upregulation of neurotrophic factor-related gene expression in retina with experimental autoimmune uveoretinitis by intravitreal injection of tacrolimus (FK506).

ABSTRACT: AIM: The current study was designed to determine whether intravitreal injection of tacrolimus (FK506) modulates the gene expression of neurotrophic factor-related molecules in the retina from eyes with induced experimental autoimmune uveoretinitis (EAU) in rats. METHODS: Rats were immunised with interphotoreceptor retinoid binding protein peptide (R14) and given intravitreal injection of tacrolimus on day 12 after immunisation. As control, immunised rats received intravitreal injection of vehicle. On day 15 after immunisation, changes in the genetic programme associated with neuroprotection and inflammatory responses in the retinas from both groups were determined by DNA microarray analyses and confirmed by real-time PCR analyses. RESULTS: The gene expression of inflammatory responses was markedly reduced in tacrolimus-treated eyes. Genes for molecules associated with neuroprotection (oestrogen receptor, erythropoietin receptor, gamma-aminobutyric acid receptor, protein kinase C, glial cell line-derived neurotrophic factor receptor, fibroblast growth factor and neuropeptide Y receptor) were upregulated in the retinas from tacrolimus-treated eyes. CONCLUSIONS: Intravitreal injection of tacrolimus modulated the genes related to neuroprotection in the retina during the ongoing process of EAU. This treatment may be useful for the neuroprotection of retina with severe uveitis as well as for immunosuppression in the uveitic eyes.

SUBMITTER: Oh-i K

PROVIDER: S-EPMC2095409 | biostudies-literature | 2007 Nov

REPOSITORIES: biostudies-literature

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Upregulation of neurotrophic factor-related gene expression in retina with experimental autoimmune uveoretinitis by intravitreal injection of tacrolimus (FK506).

Oh-i Keiko K Keino Hiroshi H Goto Hiroshi H Yamakawa Naoyuki N Takeuchi Masaru M Usui Masahiko M Iwasaki Takuya T

The British journal of ophthalmology 20071016 11

<h4>Aim</h4>The current study was designed to determine whether intravitreal injection of tacrolimus (FK506) modulates the gene expression of neurotrophic factor-related molecules in the retina from eyes with induced experimental autoimmune uveoretinitis (EAU) in rats.<h4>Methods</h4>Rats were immunised with interphotoreceptor retinoid binding protein peptide (R14) and given intravitreal injection of tacrolimus on day 12 after immunisation. As control, immunised rats received intravitreal inject ...[more]

PMID: 17940132

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Project description:BACKGROUND: Advances in gene transfer techniques have provided long-term, safe and stable transduction of retinal cells following subretinal injection with adeno-associated viral (AAV) vectors. In this study we investigated whether subretinal injection of AAV2-murine IL-27p28 vector was effective in inhibiting experimental autoimmune uveoretinitis (EAU) induced in B10RIII mice. METHODOLOGY/PRINCIPAL FINDINGS: An AAV2 vector encoding the murine IL-27p28 gene (rAAV2-CMV-mIL-27p28) was prepared and subretinally injected into B10RIII mice (4.35×10(8) vector genome (v.g.)). AAV2 vector mediating green fluorescent protein (rAAV2-CMV-GFP) served as a control (5×10(8) v.g.). The concentration of mIL-27p28 in homogenized eyes and serum was assayed by enzyme linked immunosorbent assay (ELISA) after subretinal injection. Human IRBP(161-180) peptide and Complete Freund's Adjuvant were injected into mice receiving either the rAAV2-CMV-mIL-27p28 or rAAV2-CMV-GFP vector. EAU was evaluated clinically and pathologically. The level of IL-17 and IL-10 in homogenized eyes was measured on day 12 and day 21 following immunization. Delayed type hypersensitivity (DTH) and IRBP(161-180)-specific proliferation of lymphocytes from the spleen and lymph nodes were assayed to examine the influence of the subretinal delivery of rAAV2-CMV-mIL-27p28 on the systemic immune response. IL-27p28 was detectable by ELISA within the eyes from two weeks following subretinal injection of the rAAV2-CMV-mIL-27p28 vector and showed a sustained high expression from day 14 to 9 months with a highest expression at 5 months. Subretinal injection of the vector significantly attenuated the severity of EAU disease clinically and pathologically in association with a significantly decreased IL-17 expression and an increased IL-10 expression. The IL-27p28 vector did not affect the systemic immune response, as determined by DTH and IRBP(161-180)-specific lymphocyte proliferation. CONCLUSIONS/SIGNIFICANCE: A high and stable expression of IL-27p28 was observed for at least 9 months following subretinal delivery of rAAV2-CMV-mIL-27p28. The amelioration of EAU disease severity was associated with a decreased IL-17 expression and an increased IL-10 expression.

Project description:Despite advances in clinical imaging and grading our understanding of retinal immune responses and their morphological correlates in experimental autoimmune uveoretinitis (EAU), has been hindered by the requirement for post-mortem histology. To date, monitoring changes occurring during EAU disease progression and evaluating the effect of therapeutic intervention in real time has not been possible. We wanted to establish whether optical coherence tomography (OCT) could detect intraretinal changes during inflammation and to determine its utility as a tool for accurate scoring of EAU. EAU was induced in C57BL/6J mice and animals evaluated after 15, 26, 36 and 60 days. At each time-point, contemporaneous Spectralis-OCT scanning, topical endoscopic fundal imaging (TEFI), fundus fluorescein angiography (FFA) and CD45-immunolabelled histology were performed. OCT features were further characterised on retinal flat-mounts using immunohistochemistry and 3D reconstruction. Optic disc swelling and vitreous opacities detected by OCT corresponded to CD45+ cell infiltration on histology. Vasculitis identified by FFA and OCT matched perivascular myeloid and T-cell infiltrates and could be differentiated from unaffected vessels. Evolution of these changes could be followed over time in the same eye. Retinal folds were visible and found to encapsulate mixed populations of activated myeloid cells, T-cells and microglia. Using these features, an OCT-based EAU scoring system was developed, with significant correlation to validated histological (Pearson r(2) = 0.6392, P<0.0001, n = 31 eyes) and TEFI based scoring systems (r(2) = 0.6784, P<0.0001). OCT distinguishes the fundamental features of murine EAU in vivo, permits dynamic assessment of intraretinal changes and can be used to score disease severity. As a result, it allows tissue synchronisation with subsequent cellular and functional assessment and greater efficiency of animal usage. By relating OCT signals with immunohistochemistry in EAU, our findings offer the opportunity to inform the interpretation of OCT changes in human uveitis.

Project description:BackgroundRecent reports show that gene therapy may provide a long-term, safe and effective intervention for human diseases. In this study, we investigated the effectiveness of adeno-associated virus 2 (AAV2) based human interferon-alpha (hIFN-α) gene therapy in experimental autoimmune uveoretinitis (EAU), a classic model for human uveitis.Methodology/principal findingsAn AAV2 vector harboring the hIFN-α gene (AAV2.hIFN-α) was subretinally injected into B10RIII mice at two doses (1.5×10(6) vg, 1.5×10(8) vg). AAV2 vector encoding green fluorescent protein (AAV2.GFP) was used as a control (5×10(8) vg). The expression of hIFN-α in homogenized eyes and serum was detected by ELISA three weeks after injection. The biodistribution of vector DNA in the injected eyes, contralateral eyes and distant organs was determined by PCR. EAU was induced by immunization with IRBP(161-180) three weeks following vector injections, and evaluated clinically and pathologically. IRBP-specific proliferation and IL-17 expression of lymphocytes from the spleen and lymph nodes were assayed to test the influence of the subretinal delivery of AAV2.hIFN-α on the systemic immune response. hIFN-α was effectively expressed in the eyes from three weeks to three months following subretinal injection of AAV2.hIFN-α vector. DNA of AAV2.GFP was observed only in the injected eyes, but not in the distant organs or contralateral eyes. Subretinal injection of both doses significantly attenuated EAU activity clinically and histologically. For the lower dose, there was no difference concerning lymphocyte proliferation and IL-17 production among the AAV2.hIFN-α, AAV2.GFP and PBS injected mice. However, the higher dose of AAV2.hIFN-α significantly suppressed lymphocyte proliferation and IL-17 production.Conclusions/significanceSubretinal delivery of AAV2.hIFN-α lead to an effective expression within the eye for at least three months and significantly attenuated EAU activity. AAV2.hIFN-α was shown to inhibit the systemic IRBP-specific immune response.

Dataset Information

Upregulation of neurotrophic factor-related gene expression in retina with experimental autoimmune uveoretinitis by intravitreal injection of tacrolimus (FK506).

Publications

Upregulation of neurotrophic factor-related gene expression in retina with experimental autoimmune uveoretinitis by intravitreal injection of tacrolimus (FK506).

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