Everolimus improves experimental autoimmune uveoretinitis.

The efficacy and action mechanism of everolimus in the treatment of experimental autoimmune uveoretinitis (EAU) was analyzed. Disease was induced in B10.RIII mice by immunization with human interphotoreceptor-retinoid-binding protein peptide 161-180 (hIRBPp161-180). Everolimus was administered by oral gavage (5 mg/kg/d) beginning either two days before or 14 days after immunization. Everolimus significantly reduced the histopathological uveitis score compared to sham-treated mice. To examine the effect on the antigen-specific immune response, proliferation ([(3)H]-thymidine test) and delayed-type hypersensitivity (DTH) response were measured. Furthermore, content of T-helper-1, -2, and -17 cytokines were analyzed intraocularly (Bead Array) and in cell culture supernatants from splenocytes (sandwich ELISA). To study the effect on the humoral immune response the presence of antigen-specific serum antibodies was tested (indirect ELISA). The DTH, the humoral immune response, the proliferation of splenocytes and the intraocular Th1, Th2, Th17 cytokine content and in vitro production of Th1 and Th17 cytokines were impaired after everolimus treatment. The study of CD4+CD25+FoxP3+ regulatory T cells (T(reg)) in peripheral blood, draining lymph nodes, and spleen by flow cytometry showed an increased number of splenic T(reg) in mice of the everolimus therapy group. Furthermore the T(reg) of these mice had a higher suppressive capacity than cells from sham-treated mice. These results indicate that the immunosuppressive effect of everolimus on EAU was associated with the suppression of pathogenic effector responses and induction of regulatory T cells.

Renal insufficiency is independently associated with a distal distribution pattern of symptomatic lower-limb atherosclerosis.

OBJECTIVES: The purpose of this study was to assess the impact of renal insufficiency (RI) on the distribution pattern of peripheral arterial disease (PAD). We hypothesised that RI is associated with a distally accentuated involvement of the peripheral arterial tree. DESIGN: This is a retrospective analysis. MATERIALS AND METHODS: Analysis was based on a consecutive series of 2709 patients with chronic PAD of atherosclerotic origin undergoing primary endovascular treatment of lower-extremity arteries. Atherosclerotic pattern was grouped into femoropopliteal (n=2085) and infragenicular (n=892) disease according to target lesions treated while using iliac disease (n=1133) as reference. Univariable and multivariable multinomial regression analyses were performed to assess relation with RI. Results are shown as relative risk ratio (RRRs) with 95% confidence intervals (95% CIs). A p<0.05 was considered statistically significant. RI was defined as glomerular filtration rate (GFR)<60 ml min(-1) 1.73 m(-2). RESULTS: Presence of RI was an independent risk factor for a centrifugal lesion pattern (RRR 1.48, 95% CI: 1.17-1.86, p=0.001). Moreover, a decrease in GFR by 10 ml min(-1) 1.73 m(-2) was associated with an RRR of 1.08 for below-the-knee arterial disease (95% CI: 1.03-1.13, p=0.003). CONCLUSION: Presence and severity of RI are independent predictors of a distal obstructive pattern in patients with symptomatic PAD.

[Do antisense oligonucleotides improve viral immunopathology?]. / Verbessern Antisense-Oligonukleotide die virale Immunpathologie? Lokale Gabe gegen TNF-alpha-mRNA bei experimenteller Herpeskeratitis.

BACKGROUND: Tumor necrosis factor (TNF)-alpha as a proinflammatory cytokine is of great importance during the development of herpes simplex virus-1 keratitis (HSK). In this study the local administration of antisense oligonucleotides (ASON) targeting TNF-alpha was examined for its usefulness in ameliorating this disease. METHODS: Uptake and efficacy of the oligonucleotides were studied in vitro by fluorescence microscopy and flow cytometry. Substance- and sequence-specific influences on the development of HSK were scrutinized in an animal model. RESULTS: Quick and stable uptake of FITC-labeled ASON by isolated spleen and lymph node cells was proved. The production of TNF-alpha by these cells after stimulation with HSV antigen or concanavalin A (ConA) was clearly downregulated after addition of ASON. In vivo, incidence and development of HSK were ameliorated after subepithelial corneal injection of ASON targeting TNF-alpha. When buffer and control oligonucleotides were given, no significant influence on the disease was found. CONCLUSION: The ASON effectively reduced TNF-alpha secretion in vitro and suppressed the development of experimental HSK in vivo.

[Amniotic membrane transplantation improves herpetic keratitis by local and not by systemic effects]. / Amnionmembrantransplantation. Verbesserung der experimentellen herpetischen Keratitis durch lokale und nichtsystemische Effekte.

PURPOSE: The immune mediated HSV-1 stromal keratitis (HSK) rapidly improves after amniotic membrane transplantation (AMT). This study investigated whether AMT modulates the T cell response and whether the anti-inflammatory action of AMT is due to local or systemic effects. METHODS: Corneas of BALB/c mice were infected with 10(5) (PFU) of HSV-1. Animals with ulcerating keratitis on day 14 post-infection were divided into 4 groups: group 1 ( n=12): right eye AMT;group 2 ( n=12): right eye tarsorrhaphy; group 3 ( n=8): right eye tarsorrhaphy, left eye AMT;group 4 ( n=8): both eyes tarsorrhaphy. The mice were examined for clinical signs of HSV keratitis after 2 days. Corneal sections were studied histologically and the inflammatory cell infiltration was studied by immunohistochemical staining. DTH response and the HSV-specific 3H-thymidin-uptake were compared between the groups. RESULTS: Compared to group 2, ulceration and stromal inflammation was profoundly improved in group 1 ( p<0.01). The corneas in the AMT mice had fewer inflammatory cells, CD3+,CD4+ and CD8+ cells than the control mice ( p<0.01). There were no significant differences between groups 1 and 2 with respect to the delayed type hypersensitivity reaction (DTH response) and the HSV-specific 3H-thymidin uptake. AMT or tarsorrhaphy on the left eyes in groups 3 and 4 had no influence on the course of keratitis or the T cell response. CONCLUSIONS: Ulcerating herpetic keratitis markedly improves after AMT. Our observations indicate that this is caused predominantly by local and not by systemic AMT-related effects.

[Amniotic membrane transplantation improves experimental herpetic keratitis. Modulation of matrix metalloproteinase-9]. / Amnionmembrantransplantation bessert experimentelle herpetische Keratitis. Modulierung von Matrixmetalloproteinase-9.

PURPOSE: Transplantation of human amniotic membrane (AMT) accelerates the healing of experimental ulcerative herpetic keratitis. Here the expression and activity of matrix metalloproteinase (MMP)-9 was studied. METHODS: BALB/c mice were corneally infected with HSV-1. Whereas the infected corneas of mice in group 1 were covered with AM, tarsorrhaphies were performed in others (group 2). After 2 days, the appearance of corneal ulcers and stromal inflammation was judged clinically, and the corneal PMN infiltration was studied histologically. The expression of MMP-9 in the corneas was localized by immunohistochemistry and analyzed by Western-blot technique. The MMP-9 activity in the corneas was determined by zymography. RESULTS: On day 14, the ulcerating corneas had a dense PMN infiltration, the ulcers and the majority of PMNs were highly positive for MMP-9, and the active forms of MMP-9 were detected. Gelatinolytic activity was found in these corneas by zymography. Compared with the mice of group 2, ulceration, stromal inflammation and neovascularization markedly improved clinically and histologically within 2 days in mice of group 1. This was associated with a reduced expression of MMP-9 in corneal tissue and in PMNs. The gelatinolytic activity of MMP-9 was reduced after AMT. CONCLUSIONS: These observations suggest that improvement of herpetic corneal ulcers and reduced corneal neovascularization after AMT may result from a reduced expression and activity of MMP-9.

Sub-lytic C5b-9 induces functional changes in retinal pigment epithelial cells consistent with age-related macular degeneration.

PURPOSE: There is evidence for complement dysfunction in age-related macular degeneration (AMD). Complement activation leads to formation of the membrane attack complex (MAC), known to assemble on retinal pigment epithelial (RPE) cells. Therefore, the effect of sub-lytic MAC on RPE cells was examined with regard to pro-inflammatory or pro-angiogenic mediators relevant in AMD. METHODS: For sub-lytic MAC induction, RPE cells were incubated with an antiserum to complement regulatory protein CD59, followed by normal human serum (NHS) to induce 5% cell death, measured by a viability assay. MAC formation was evaluated by immunofluorescence and FACS analysis. Interleukin (IL)-6, -8, monocytic chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor (VEGF) were quantified by enzyme-linked immunosorbent assay (ELISA). Intracellular MCP-1 was analysed by immunofluorescence, vitronectin by western blotting, and gelatinolytic matrix metalloproteinases (MMPs) by zymography. RESULTS: Incubation of RPE cells with the CD59 antiserum followed by 5% NHS induced sub-lytic amounts of MAC, verified by FACS and immunofluorescence. This treatment stimulated the cells to release IL-6, -8, MCP-1, and VEGF. MCP-1 staining, production of vitronectin, and gelatinolytic MMPs were also elevated in response to sub-lytic MAC. CONCLUSIONS: MAC assembly on RPE cells increases the IL-6, -8, and MCP-1 production. Therefore, sub-lytic MAC might have a significant role in generating a pro-inflammatory microenvironment, contributing to the development of AMD. Enhanced vitronectin might be a protective mechanism against MAC deposition. In addition, the increased expression of gelatinolytic MMPs and pro-angiogenic VEGF may be associated with neovascular processes and late AMD.

[Pathogenetic concepts for pigment epithelial detachment in exudative AMD]. / Pathogenetische Konzepte zur Pigmentepithelabhebung bei exsudativer AMD.

Retinal pigment epithelial detachment (PED) as a specific manifestation in exudative age-related macular degeneration (AMD) may lead to a substantial decrease of central vision. An understanding of important events during the development of PED is necessary for a successful therapeutic intervention. At present the leading pathogenetic theory is that of reduced hydraulic conductivity of Bruch's membrane. The mechanisms underlying this process are caused by increased deposition of lipids, enhanced collagen cross-linking and alteration in the ratio of tissue-dissolving enzymes and their inhibitors. The association of newly formed vessels and an unaltered RPE pump activity can lead to the clinical picture of serous PED during exudative AMD.

Improvement of herpetic stromal keratitis with fumaric acid derivate is associated with systemic induction of T helper 2 cytokines.

Fumaric acid derivates have been shown to stimulate T helper-2-cytokines (interleukin (IL)-4, -5) without affecting the T-helper-1-cytokine (IL-2, interferon (IFN)-gamma)-response. Herein, the influence of systemic treatment with the fumaric acid derivate dimethylfumarate (DMF) on the secretion of T helper-cytokines and the development of HSV-1 stromal keratitis (HSK) was studied in mice. The corneas from BALB/c mice were infected with 10(5) PFU of HSV-1 (KOS strain). While one group of mice was treated intraperitoneally with PBS, another group of mice received DMF at 15 mg/kg of body weight. Expression of IL-2, -4, -10 and IFN-gamma was analysed in HSV-1 activated lymphocytes by ELISA. The severity of epithelial and stromal herpetic keratitis was investigated clinically. Corneas were studied for the inflammatory cell infiltration, and the CD3-, CD4- and CD8-positive cells were analysed by immunohistochemistry. The IL-2, -4, 10 and IFN-gamma content was measured in the corneas. Virus replication in the eyes was analysed by a plaque-assay. The DTH-response, the HSV-specific T cell proliferation and the serum neutralizing antibody-titres were investigated. DMF increased IL-4 and IL-10, but not IL-2 and IFN-gamma, secretion in activated lymphocytes from the spleen. Incidence and severity of stromal HSV-1 keratitis was reduced in the DMF group (P < 0.01). In the corneas from DMF-treated mice, the numbers of CD3+ and CD4+ cells were decreased and IL-4 was increased. Severity of epithelial disease and the virus-clearance from the eyes did not differ between the PBS and DMF group of mice. DTH, HSV-specific T cell proliferation and the neutralizing antibody-titres were not impaired. DMF increased the T helper-2-cytokine secretion in activated lymphocytes. After corneal HSV-1 infection, corneas from DMF treated mice had increased IL-4 content. This is associated with an improvement of herpetic stromal keratitis and reduced corneal T cell infiltration. DMF did not impair the systemic antiviral response.

Influence of dimethylfumarate on experimental HSV-1 necrotizing keratitis.

BACKGROUND: This study was performed to investigate the influence of fumaric acid esters on the course of herpes stromal keratitis (HSK). METHODS: The corneas of BALB/c mice were inoculated with 105 plaque-forming units of herpes simplex virus 1 (HSV-1, KOS strain). Groups of mice were treated intraperitoneally with phosphate buffered saline (PBS) (control mice), or with dimethylfumarate (DMF) at 15 mg/kg of body weight dissolved in PBS daily for 28 days pre-infection and for 14 days post-infection. The course of HSV-1 keratitis was studied clinically. Corneal sections were examined for inflammatory cell infiltration. The numbers of CD3, GR-1, CD11b and F4/80-expressing cells infiltrating the corneas were analyzed by immunohistochemistry. RESULTS: On day 14 after HSV infection, 72% of the mice in the control group had severe HSK. The development of HSK was reduced by DMF treatment in the DMF group (22%) (P=0.004). The total number of inflammatory cells and infiltration of polymorphonuclear-neutrophils (PMNs) were reduced in the corneas of DMF-treated mice. Compared to the PBS-treated mice, numbers of CD3, CD11b, GR-1 and F4/80-positive cells were reduced in the DMF group of mice. CONCLUSIONS: The course of experimental herpes stromal keratitis can be improved with systemic fumaric acid ester treatment. The improvement of keratitis correlates with a reduced corneal infiltration of T cells and mononuclear cells.