The Topical Ocular Delivery of Rapamycin to Posterior Eye Tissues and the Suppression of Retinal Inflammatory Disease

Treatment of posterior eye diseases with intravitreal injections of drugs, while effective, is invasive and associated with side effects such as retinal detachment and endophthalmitis. In this work, we have formulated a model compound, rapamycin (RAP), in nanoparticle-based eye drops and evaluated the delivery of RAP to the posterior eye tissues in a healthy rabbit. We have also studied the formulation in experimental autoimmune uveitis (EAU) mouse model with retinal inflammation. Aqueous RAP eye drops were prepared using N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (Molecular Envelope Technology - MET) containing 0.23 ± 0.001% w/v RAP with viscosity, osmolarity, and pH within the ocular comfort range, and the formulation (MET-RAP) was stable in terms of drug content at both refrigeration and room temperature for one month. The MET-RAP eye drops delivered RAP to the choroid-retina with a Cmax of 145 ± 49 ng/g (tmax = 1 hour). The topical application of the MET-RAP eye drops to the EAU mouse model resulted in significant disease suppression compared to controls, with activity similar to dexamethasone eye drops. The MET-RAP eye drops also resulted in a reduction of RORγt and an increase in both Foxp3 expression and IL-10 secretion, indicating a mechanism involving the inhibition of Th17 cells and the up-regulation of T-reg cells. The MET-RAP formulation delivers RAP to the posterior eye segments, and the formulation is active in EAU

Post-translational regulation contributes to the loss of LKB1 expression through SIRT1 deacetylase in osteosarcomas

background: The most prevalent form of bone cancer is osteosarcoma (OS), which is associated with poor prognosis in case of metastases formation. Mice harbouring liver kinase B1 (LKB1+/−) develop osteoblastoma-like tumours. Therefore, we asked whether loss of LKB1 gene has a role in the pathogenesis of human OS. methods: Osteosarcomas (n=259) were screened for LKB1 and sirtuin 1 (SIRT1) protein expression using immunohistochemistry and western blot. Those cases were also screened for LKB1 genetic alterations by next-generation sequencing, Sanger sequencing, restriction fragment length polymorphism and fluorescence in situ hybridisation approaches. We studied LKB1 protein degradation through SIRT1 expression. MicroRNA expression investigations were also conducted to identify the microRNAs involved in the SIRT1/LKB1 pathway. results: Forty-one per cent (106 out of 259) OS had lost LKB1 protein expression with no evident genetic anomalies. We obtained evidence that SIRT1 impairs LKB1 protein stability, and that SIRT1 depletion leads to accumulation of LKB1 in OS cell lines resulting in growth arrest. Further investigations revealed the role of miR-204 in the regulation of SIRT1 expression, which impairs LKB1 stability. conclusions: We demonstrated the involvement of sequential regulation of miR-204/SIRT1/LKB1 in OS cases and showed a mechanism for the loss of expression of LKB1 tumour suppressor in this malignancy

Molecular genetics of cutaneous malignant melanoma

Cutaneous malignant melanoma is an aggressive tumor of melanocytes in the skin with rapidly increasing incidence. Patients with advanced disease have a poor prognosis since the tumor is usually resistant to current therapies. Therefore, the development of novel strategies for preventing and treating melanoma is important. To explore novel therapies we need to find appropriate targets and for that knowledge about the biology of melanoma is important. There is growing evidence suggesting that NRAS has an important role in tumorigenesis and tumor maintenance in malignant melanoma and that the RAS-RAF-ERK signaling pathway is constitutively activated through multiple mechanisms, one of which is activating mutations in NRAS gene. In an initial study, we investigated the occurrence of activating mutation in the NRAS gene in a subset of patients with hereditary melanoma carrying germ line CDKN2A alterations. From this study we found differences in the frequency of NRAS mutations between hereditary and sporadic melanomas. Activating mutations in NRAS codon 61 were found in 95% (20/21) of primary hereditary melanomas but in only 10% (1/10) of sporadic melanomas. We also detected multiple activating NRAS mutations in tumor cells from different regions of individual primary hereditary melanomas. We concluded that the high frequency of NRAS codon 61 mutations detected in these hereditary melanomas may be the result of a hypermutability phenotype associated with the hereditary predisposition for melanoma development in patients with germline CDKN2A mutations. The presence of a mutant NRAS oncogene in sporadic and familial melanomas implies that the NRAS oncogene may be an important target for prevention and treatment of melanomas. Therefore, to better define the role of this oncogene in melanoma development, we specifically targeted this mutant oncogene using RNAi techniques and studied the effect of suppression of mutant NRAS on melanoma cell lines. Suppression of oncogenic NRAS in these cell lines resulted in decreased proliferation, increased apoptosis as well as decreased phosphorylation of ERK and Akt, and also reduced expression of NF-kappaB and cyclin D1 downstream in the NRAS signaling pathway. To follow up this investigation, we studied the effect of siRNA against mutant NRAS on gene expression profiles in melanoma cell lines which carry oncogenic NRAS mutations. We could show the impact of knockdown of the NRAS oncogene on different cellular processes. For instance, we observed a disability of cells with respect to migration and invasion, which is accompanied by down-regulation of EphA2, uPAR and cytoskeleton proteins such as leupaxin, alpha-actinin, paxillin, and vinculin. These cells also showed inhibition of cell proliferation accompanied by downregulation of two cyclins, cyclin D1, cyclin E2, and up-regulation of HBP1 repressor. In summary, we conclude that the use of siRNAs against NRASQ61R is an important tool in suppressing oncogenic NRAS signaling, which might contribute to the development of more specific melanoma therapy in the subset of patients with tumor with NRAS mutations

Immune-Mediated Retinal Vasculitis in Posterior Uveitis and Experimental Models: The Leukotriene (LT)B4-VEGF Axis

Retinal vascular diseases have distinct, complex and multifactorial pathogeneses yet share several key pathophysiological aspects including inflammation, vascular permeability and neovascularisation. In non-infectious posterior uveitis (NIU), retinal vasculitis involves vessel leakage leading to retinal enlargement, exudation, and macular oedema. Neovascularisation is not a common feature in NIU, however, detection of the major angiogenic factor—vascular endothelial growth factor A (VEGF-A)—in intraocular fluids in animal models of uveitis may be an indication for a role for this cytokine in a highly inflammatory condition. Suppression of VEGF-A by directly targeting the leukotriene B4 (LTB4) receptor (BLT1) pathway indicates a connection between leukotrienes (LTs), which have prominent roles in initiating and propagating inflammatory responses, and VEGF-A in retinal inflammatory diseases. Further research is needed to understand how LTs interact with intraocular cytokines in retinal inflammatory diseases to guide the development of novel therapeutic approaches targeting both inflammatory mediator pathways

Therapeutic Validation of GEF-H1 Using a De Novo Designed Inhibitor in Models of Retinal Disease

Inflammation and fibrosis are important components of diseases that contribute to the malfunction of epithelia and endothelia. The Rho guanine nucleotide exchange factor (GEF) GEF-H1/ARHGEF-2 is induced in disease and stimulates inflammatory and fibrotic processes, cell migration, and metastasis. Here, we have generated peptide inhibitors to block the function of GEF-H1. Inhibitors were designed using a structural in silico approach or by isolating an inhibitory sequence from the autoregulatory C-terminal domain. Candidate inhibitors were tested for their ability to block RhoA/GEF-H1 binding in vitro, and their potency and specificity in cell-based assays. Successful inhibitors were then evaluated in models of TGFβ-induced fibrosis, LPS-stimulated endothelial cell-cell junction disruption, and cell migration. Finally, the most potent inhibitor was successfully tested in an experimental retinal disease mouse model, in which it inhibited blood vessel leakage and ameliorated retinal inflammation when treatment was initiated after disease diagnosis. Thus, an antagonist that blocks GEF-H1 signaling effectively inhibits disease features in in vitro and in vivo disease models, demonstrating that GEF-H1 is an effective therapeutic target and establishing a new therapeutic approach

T-bet Activates Th1 Genes through Mediator and the Super Elongation Complex

SummaryThe transcription factor T-bet directs Th1 cell differentiation, but the molecular mechanisms that underlie this lineage-specific gene regulation are not completely understood. Here, we show that T-bet acts through enhancers to allow the recruitment of Mediator and P-TEFb in the form of the super elongation complex (SEC). Th1 genes are occupied by H3K4me3 and RNA polymerase II in Th2 cells, while T-bet-mediated recruitment of P-TEFb in Th1 cells activates transcriptional elongation. P-TEFb is recruited to both genes and enhancers, where it activates enhancer RNA transcription. P-TEFb inhibition and Mediator and SEC knockdown selectively block activation of T-bet target genes, and P-TEFb inhibition abrogates Th1-associated experimental autoimmune uveitis. T-bet activity is independent of changes in NF-κB RelA and Brd4 binding, with T-bet- and NF-κB-mediated pathways instead converging to allow P-TEFb recruitment. These data provide insight into the mechanism through which lineage-specifying factors promote differentiation of alternative T cell fates