Search

PurposeTo develop diagnostic criteria for nonparaneoplastic autoimmune retinopathy (AIR) through expert panel consensus and to examine treatment patterns among clinical experts.DesignModified Delphi process.MethodsA survey of uveitis specialists in the American Uveitis Society, a face-to-face meeting (AIR Workshop) held at the National Eye Institute, and 2 iterations of expert panel surveys were used in a modified Delphi process. The expert panel consisted of 17 experts, including uveitis specialists and researchers with expertise in antiretinal antibody detection. Supermajority consensus was used and defined as 75% of experts in agreement.ResultsThere was unanimous agreement among experts regarding the categorization of autoimmune retinopathies as nonparaneoplastic and paraneoplastic, including cancer-associated retinopathy and melanoma-associated retinopathy. Diagnostic criteria and tests essential to the diagnosis of nonparaneoplastic AIR and multiple supportive criteria reached consensus. For treatment, experts agreed that corticosteroids and conventional immunosuppressives should be used (prescribed) as first- or second-line treatments, though a consensus agreed that biologics and intravenous immunoglobulin were considered appropriate in the treatment of nonparaneoplastic AIR patients regardless of the stage of disease. Experts agreed that more evidence is needed to treat nonparaneoplastic AIR patients with long-term immunomodulatory therapy and that there is enough equipoise to justify randomized, placebo-controlled trials to determine if nonparaneoplastic AIR patients should be treated with long-term immunomodulatory therapy. Regarding antiretinal antibody detection, consensus agreed that a standardized assay system is needed to detect serum antiretinal antibodies. Consensus agreed that an ideal assay should have a 2-tier design and that Western blot and immunohistochemistry should be the methods used to identify antiretinal antibodies.ConclusionsConsensus was achieved using a modified Delphi process to develop diagnostic criteria for nonparaneoplastic AIR. There is enough equipoise to justify randomized, placebo-controlled trials to determine whether patients with nonparaneoplastic AIR should be treated with long-term immunomodulatory therapy. Efforts to develop a standardized 2-tier assay system for the detection of antiretinal antibodies have been initiated as a result of this study.

Background: Cytokines are known to be a key a factor in numerous malignancies and to exert an important regulatory role in the tumor microenvironment. Interest has grown in understanding how cytokines modulate the tumor microenvironment and which cytokines may serve as markers of the tumor process; however, a complete picture of the cytokine landscape in bladder cancer remains unclear. Methods: Fresh urine specimens with sufficient volume were collected at random intervals. The urine concentrations of IL‐8 (CXCL8), CCL18, and CXCL9 were determined using the standard commercially available enzyme immunoassay. The urine concentrations of IL‐6 were determined using the high sensitivity enzyme immunoassay kit. Urinary cytokine concentrations were normalized with urinary creatinine concentrations. Results: Significantly elevated concentrations of IL‐6 and IL‐8 were detected in the urine from patients with urothelial carcinoma on follow‐up compared to patients with benign follow‐up. The presence of both IL‐6 and IL‐8 in the urine samples from the high grade urothelial carcinoma (HGUC) cohort revealed a clear discrimination when compared to samples from patients with benign follow‐up. The presence of the combination of both IL‐6 and IL‐8 had a sensitivity of 90.0% and a specificity of 81.25%. Similar data were obtained when receiver operating characteristic analysis was performed on both IL‐6 and IL‐8 concentrations in the urine from patients with HGUC vs. the hematuria cohort. Conclusions: The presence of IL‐6 and IL‐8 in urine specimens may have predictive value for urothelial carcinoma. However, a large longitudinal study is required to statistically eliminate confounding factors and support this theory. Cytokines are known to be a key a factor in numerous malignancies and to exert an important regulatory role in the tumor microenvironment. The authors show that a combination of two regulatory molecules, IL‐6 and IL‐8, are present in 100% of patients with high‐grade bladder cancer, whereas this dual combination signature was observed in only 13.6% of patients with hematuria and without any diagnosis of urothelial carcinoma on follow‐up. [ABSTRACT FROM AUTHOR]

Chemokine reeptor-3 (CCR-3) was shown to be associated with choroidal neovascularization (CNV) in age-related macular degeneration (AMD). AMD is a vision threatening retinal disease that affects the aging population world-wide. Retinal pigment epithelium and choroid in the posterior part of the retina are the key tissues targeted in the pathogenesis of CNV in AMD. We used human retinal pigment epithelial (HRPE) and choroidal fibroblast (HCHF) cells, prepared from aged adult human donor eyes, to evaluate the expression of major CCR-3 ligands, CCL-5, CCL -7, CCL-11,CCL-24 and CCL-26. Microarray analysis of gene expression in HRPE cells treated with inflammatory cytokine mix (ICM= IFN-γ+TNF-α+IL-1β) revealed 75 and 23-fold increase in CCL-5 and CCL-7 respectively, but not CCL-11, CCL-24 and CCL-26. Chemokine secretion studies of the production of CCL5 and CCL7 by HRPE corroborated with the gene expression analysis data. When the HRPE cells were treated with either individual cytokines or the ICM, both CCL-5 and CCL-7 were produced in a dose dependent manner. Similar to the gene expression data, the ICM did not enhance HRPE production of CCL-11, CCL-24 and CCL-26. CCL-11 and CCL-26 were increased with IL-4 treatment and this HRPE production was augmented in the presence of TNF-α and IL1α. When HCHF cells were treated with either individual cytokines or the ICM, both CCL-5 and CCL-7 were produced in a dose dependent fashion. IL-4 induced low levels of CCL-11 and CCL-26 in HCHF and this production was significantly enhanced by TNF-α. Under these conditions, neither HRPE nor HCHF were demonstrated to produce CCL-24. These data demonstrate that chronic inflammation triggers CCL-5 and CCL-7 release by HRPE and HCHF and the subsequent interactions with CCR3 may participate in pathologic processes in AMD. [ABSTRACT FROM AUTHOR]

Age-related macular degeneration (AMD) is a sight threating retinal eye disease that affects millions of aging individuals world-wide. Choroid-retinal pigment epithelium (RPE)-neuroretina axis in the posterior compartment of the eye is the primary site of AMD pathology. There are compelling evidence to indicate association of vascular endothelial growth factors (VEGF) to AMD. Here, we report the inhiβitory actions of resveratrol (RSV) on inflammatory cytokine, TGF-β and hypoxia induced VEGF secretion βy human retinal pigment epithelial cells (HRPE). HRPE cultures prepared from aged human donor eyes were used for the studies in this report. HRPE secreted βoth VEGF-A and VEGF-C in small quantities constitutively. Stimulation with a mixture of inflammatory cytokines (IFN-γ, TNF-α, IL-1β), significantly increased the secretion of βoth VEGF-A and VEGF-C. RSV, in a dose dependent (10-50 uM) manner, suppressed VEGF-A and VEGF-C secretion induced βy inflammatory cytokines significantly. RT-PCR analysis indicated that effects of RSV on VEGF secretion were possiβly due to decreased mRNA levels. TGF-β and coβalt chloride (hypoxia mimic) also upregulated HRPE cell production of VEGF-A, and this was inhiβited βy RSV. In contrast, RSV had no effect on anti-angiogenic molecules, endostatin and pigment epithelial derived factor secretion. Studies using an in vitro scratch assay revealed that wound closure was also inhiβited βy RSV. These results demonstrate that RSV can suppress VEGF secretion induced βy inflammatory cytokines, TGF-β and hypoxia. Under pathological conditions, over expression of VEGF is known to worsen AMD. Therefore, RSV may βe useful as nutraceutical in controlling pathological choroidal neovascularization processes in AMD. [ABSTRACT FROM AUTHOR]

Chronic inflammation is implicated in the pathogenesis of age-related macular degeneration (AMD). Choroidal neovascularization (CNV) observed in exudative form of AMD results in vision loss. Human retinal pigment epithelial cell (HRPE) layer and choroidal tissue are the primary pathological sites in AMD. Pathological and therapeutic evidences have strongly indicated the vascular endothelial growth factor (VEGF) molecules as critical components in CNV pathogenesis. In these studies, we used human primary HRPE and choroidal fibroblast cells (HCHF) prepared from adult donor eyes. The effects of inflammatory cytokine (IFN-γ + TNF-α + IL-1β) mix (ICM) on global gene expression profiles in HRPE cells, revealed 10- and 9-fold increase in VEGF-A and VEGF-C expression, respectively. The microarray results were validated by quantitative RT-PCR and secretion of VEGFs proteins. IL-1β is the most potent in inducing VEGFs secretion followed by IFN-γ and TNF-α, and the secretion was more effective in the presence of 2 and 3 cytokines. NF-κB and JAK-STAT pathway, but not HIF-1α, Sp-1, Sp-3, and STAT-3, transcription factors were upregulated and translocated to nucleus by ICM treatment. The mRNA levels of VEGF-A and VEGF-C and secretion of these proteins were also significantly enhanced by ICM in HCHF cells. The secretion of other angiogenic molecules, PEDF, SDF-1α, endostatin, and angiopoietins was not affected by ICM. Our results show that the inflammatory cytokines enhance secretion of VEGF-A and VEGF-C by HRPE and HCHF cells. These studies indicate that VEGFs secreted by these cells initiate and promote pathological choroidal and retinal noevascularization processes in AMD. J. Cell. Physiol. 227: 116-126, 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

In the herpetic stromal keratitis (HSK), HSV DNA fragments and HSV-IgG immune complexes (HSV-IC) are present in most of the corneas long after infective virus has disappeared. These viral components are highly immunogenic and potentiate production of proinflammatory cytokines and chemokines via Toll-like receptors (TLRs) expressed on the corneal cells and macrophages. In addition angiogenic factors, such as vascular endothelium growth factor (VEGF) and the tissue damaging enzyme matrix metalloproteinase 9 (MMP-9) deeply involved in the pathogenesis of HSK, are also induced by corneal cells and macrophages through the recognition of these viral components. These processes elicited by residual viral DNA and HSV-IC are likely one of the sustained driving force in the development of HSK. Hence, strategies developed to alter these pathways should lead to new preventative and therapeutic measures. [ABSTRACT FROM AUTHOR]

Retinal pigment epithelium (RPE) and transforming growth factor-β (TGF-β) have been shown to be involved in various retinal diseases. We have studied the role of inflammatory cytokines on the expression and secretion of TGF-β in human RPE cells (HRPE). Confluent cultures of HRPE derived from donor eyes were used. RT-PCR analyses showed that TNF-α and IL-1β increased the mRNA levels of both TGF-β1 and TGF-β2. IFN-γ enhanced constitutively expressed, as well as, TNF-α-and IL-1β-induced TGF-β1 mRNA levels but decreased TGF-β2 mRNA. The effects of these cytokines on TGF-β1 and TGF-β2 secretion correlated with the mRNA levels. TGF-β1 was always produced as the latent form while 21–31% of TGF-β2 was in the active form. IFN-γ reduced the production of active form of TGF-β2 to 4–9%. TGF-β3 secretion was not detectable under any of the conditions. The Real-Time PCR analysis of TGF-β mRNAs confirmed the observed results. The TGF-β1 and TGF-β2 secretion was induced by TGF-β2 and TGF-β1, respectively. Under these conditions, the contrasting effects of IFN-γ on TGF-β1 and TGF-β2 secretion were also observed. JAK inhibitor selectively inhibited IFN-γ induced TGF-β1 secretion and mRNA levels while reversing the inhibitory effects of IFN-γ on TGF-β2. Analyses of transcription factor activity strongly indicated the role of STAT-1 but not NFκB, C-Myc, C-Jun, SP-1, MEF-2. Our data demonstrate that IFN-γ differentially regulates constitutively expressed, as well as, cytokine-induced TGF-β1 and TGF-β2 mRNA levels and secretion of TGF-βs by HRPE. J. Cell. Physiol. 210: 192–200, 2007. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Abstract: Cytomegalovirus (CMV) retinitis is characterized by alterations in retinal cell function and host responses to virus replication. The goal of this study was to evaluate the induction of cyclooxygenase-2 (COX-2) and prostaglandin (PGE) in CMV infected human retinal pigment epithelial (RPE) cells and to determine their effect on virus replication. CMV immediate early (IE) protein and COX-2 proteins were identified in RPE cells in retinal tissue sections from patients with CMV retinitis. COX-2 mRNA and protein were induced after CMV infection of human RPE cell cultures. CMV infection of RPE cells induced translocation of NF-κB from the cytoplasm to the nucleus. PGE1 and PGE2 were significantly (p <0.001) increased in human RPE cell cultures infected with CMV. Inhibition of CMV IE gene by antisense oligonucleotides abrogated induction of mRNA for COX-2 and protein synthesis of COX-2 and PGE2. PGE enhanced CMV plaque formation and real time PCR analysis revealed that PGE treatment significantly increased CMV DNA copy numbers. These studies demonstrate that when CMV replicates within human RPE cells, COX-2 induction augments virus replication via the PGE pathway. The induction of COX-2 and PGE during retinal CMV infection may augment virus replication and alter a variety of retinal physiological responses. [Copyright &y& Elsevier]

ATP-binding cassette (ABC) transporters play a pivotal role in physiology and pathology. We identified and cloned two novel mRNA isoforms (ABCB 5α andABCB 5β) of the ABC transporterABCB 5 in human melanoma cells. The deducedABCB 5α protein appears to be an altered splice variant containing only a putative ABC, whereas theABCB 5β isoform shares approximately 70% similarity withABCB1 (MDR1) and has a deduced topological arrangement similar to that of the whole carboxyl terminal half of theABCB1 gene product, P-glycoprotein, including an intact ABC. Northern blot, real-time PCR, and conventional RT-PCR were used to verify the expression profiles ofABCB 5α/β. We found that the melanomas included among the NCI-60 panel of cell lines preferentially expressed bothABCB 5α andABCB 5β. However,ABCB 5α/β expression was undetectable in two amelanotic melanomas (M14 and LOX-IMVI). The expression profile ofABCB 5α/β in all of the other melanomas of the panel was confirmed both by RT-PCR and by sequencing. NeitherABCB 5α norABCB 5β expression was found in normal tissues such as liver, spleen, thymus, kidney, lung, colon, small intestines or placenta.ABCB 5α/β mRNAs were also expressed in normal melanocytes and in retinal pigment epithelial cells, suggesting thatABCB 5α/β expression is pigment cell-specific and might be involved in melanogenesis. Our findings indicate that expression ofABCB 5α/β might possibly provide two novel molecular markers for differential diagnosis of melanomas and constitute potential molecular targets for therapy of melanomas. [ABSTRACT FROM AUTHOR]

The clinical signs, laboratory data, and histological features of recurrent aphthous ulcers (RAU) and Behçet's syndrome suggest a viral etiology. In fact, there are reports of adenovirus isolations in herpetiform oral ulcers and on the isolation of a filterable agent in sporadic cases of Behçet's syndrome. However, isolation studies on the major and minor aphthous ulcers and more recent studies on Behçet's syndrome have been negative. A review of the literature on the role of viruses and autoimmunity in RAU and Behçet's syndrome is presented. Biopsy specimens of ulcerative lesions were grown in vitro for up to 300 days. Those cultures, along with leukocytes and body fluids, were examined by a variety of techniques for the presence of virus or viral antigens. Although a persistent or latent virus was not detected, these negative studies cannot elude a viral etiology. In fact, the hypothesis of an infectious and viral etiology is still reasonable. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factors (VEGF) are the primary causative agents involved in retinal and choroidal neovascularization that may lead to blinding retinal diseases. There is increasing evidence suggesting a role for inflammatory processes in retinal diseases of angiogenic etiology. We used primary cultures of human retinal pigment epithelial cells (HRPE) to evaluate the effect of inflammatory mediators on VEGF expression. HRPE cultures were incubated in serum-free medium for 24 hr in the presence of inflammatory mediators (IM), IFN-γ,TNF-α or IL-1 and various combinations of these cytokines. The secretion of VEGF-A and VEGF-C by HRPE were significantly increased in response to IFN-γ TNF-α and IL-1. The secretion of VEGF-B, VEGF-D, and angiopoitin-1 and -2 were not observed in control and IM treated HRPE. VEGF-A secretion by control and IM treated HRPE were 57 and 823 pg/ml (n=4) respectively. VEGF-C secretion by control and IM treated HRPE were 221 and 3574 pg/ml (n=4) respectively. The secretion of VEGF-A and VEGF-C was enhanced by IFN-γ, TNF-α and IL-1 individually and cumulatively. RT-PCR analysis showed significant elevation of VEGF-A and VEGF-C mRNA levels in HRPE treated with IM. Augumented secretion of VEGF-A and VEGF-C by HRPE under the influence of IM may affect retinal and choroidal vasculature leading to retinal and choroidal neovascularization. [ABSTRACT FROM AUTHOR]

Abstract: Inflammatory response of the retinal pigment epithelium plays a critical role in the pathogenesis of retinal degenerative diseases such as age-related macular degeneration. Our previous studies have shown that human retinal pigment epithelial (HRPE) cells, established from adult donor eyes, respond to inflammatory cytokines by enhancing the expression of a number of cytokines and chemokines. To investigate the role of microRNA (miRNA) in regulating this response, we performed microarray analysis of miRNA expression in HRPE cells exposed to inflammatory cytokine mix (IFN-γ+TNF-α+IL-1β). Microarray analysis revealed ∼11-fold increase in miR-155 expression, which was validated by real-time PCR analysis. The miR-155 expression was enhanced when the cells were treated individually with IFN-γ, TNF-α or IL-1β, but combinations of the cytokines exaggerated the effect. The increase in miR-155 expression by the inflammatory cytokines was associated with an increase in STAT1 activation as well as an increase in protein binding to putative STAT1 binding elements present in the MIR155 gene promoter region. All these activities were effectively blocked by JAK inhibitor 1. Our results show that the inflammatory cytokines increase miR-155 expression in human retinal pigment epithelial cells by activating the JAK/STAT signaling pathway. [Copyright &y& Elsevier]

Abstract: Interleukin-11 (IL-11) is an anti-apoptotic, anti-inflammatory cytokine with hematopoietic potential. The expression and protective actions of IL-11 have not been explored in the eye. The expression of IL-11 in primary cultures of human retinal pigment epithelial (HRPE) and human corneal fibroblast (HCRF) cells were evaluated in these studies. Constitutive secretion of IL-11 was not observed in either HRPE or HCRF. TNF-α+IL-1 induced IL-11 secretion and this production was inhibited by NFκB pathway inhibitors. IFN-γ significantly inhibited TNF-α and IL-1 induced IL-11 secretion and inhibitors of JAK-STAT pathway reversed this inhibition. TGF-β induced IL-11 secretion that was blocked by TGF-β receptor 1 inhibitor but not by IFN-γ. RT-PCR analysis confirmed the effects of IL-1, TNF-α, IFN-γ and TGF-β on IL-11 secretion at mRNA levels. Our results demonstrate that IL-11 is dramatically up regulated in retina and cornea cells and that IFN-γ is a physiological inhibitor of IL-11 expression. [Copyright &y& Elsevier]

Abstract: Inflammatory processes within the cornea are known to be associated with corneal neovascularization (CN). We examined the effects of inflammatory mediators on the expression of angiogenic factors by corneal cells. TNF-α and IL-1 induced VEGF-A secretion by corneal fibroblasts (HCRF) and this was inhibited significantly by IFN-γ. Constitutively secreted VEGF-A by corneal epithelial cells (HCE) was not affected by these cytokines. Moreover, sVEGF-R1(sFlt-1) secretion by HCRF was stimulated significantly by IFN-γ. JAK-STAT pathway inhibitor reversed the effects of IFN-γ on VEGF-A and sFlt-1 secretion by HCRF. RT-PCR analysis showed that IFN-γ influences the expression of VEGF-A and sFlt-1 by affecting their mRNA level. IFN-γ inhibited TGF-β induced VEGF-A secretion but not sVEGF-R1secretion. This is the first report demonstrating the inhibitory and stimulatory effects of IFN-γ on VEGF-A and sFlt-1 secretion, respectively. Our results suggest that IFN-γ acts as an anti-angiogenic cytokine in the human cornea. [Copyright &y& Elsevier]