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.

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.

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model.

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders.

Adhesion Molecule Targeted Therapy for Non-Infectious Uveitis.

Non-infectious uveitis (NIU) is an inflammatory eye disease initiated via CD4 T-cell activation and transmigration, resulting in focal retinal tissue damage and visual acuity disturbance. Cell adhesion molecules (CAMs) are activated during the inflammatory process to facilitate the leukocyte recruitment cascade. Our review focused on CAM-targeted therapies in experimental autoimmune uveitis (EAU) and NIU.

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.

Small-molecule antagonist of VLA-4 (GW559090) attenuated neuro-inflammation by targeting Th17 cell trafficking across the blood-retinal barrier in experimental autoimmune uveitis.

Background: The integrin VLA-4 (α4β1) plays an important role in leukocyte trafficking. This study investigated the efficacy of a novel topical α4β1 integrin inhibitor (GW559090, GW) in a mouse model for non-infectious posterior uveitis (experimental autoimmune uveitis; EAU) and its effect on intraocular leukocyte subsets.

Methods: Mice (female; B10.

Leukotriene B and Its Receptor in Experimental Autoimmune Uveitis and in Human Retinal Tissues: Clinical Severity and LTB Dependence of Retinal Th17 Cells.

Nomacopan, a drug originally derived from tick saliva, has dual functions of sequestering leukotriene B (LTB) and inhibiting complement component 5 (C5) activation. Nomacopan has been shown to provide therapeutic benefit in experimental autoimmune uveitis (EAU). Longer acting forms of nomacopan were more efficacious in mouse EAU models, and the long-acting variant that inhibited only LTB was at least as effective as the long-acting variant that inhibited both C5 and LTB, preventing structural damage to the retina and a significantly reducing effector T helper 17 cells and inflammatory macrophages.

Functionally distinct IFN-γ IL-17A Th cells in experimental autoimmune uveitis: T-cell heterogeneity, migration, and steroid response.

Immunopathogenic roles for both Th1 (CD4 IFN-γ ) and Th17 (CD4 IL-17A ) cells have been demonstrated in experimental autoimmune uveitis (EAU). However, the role for Th17/Th1 (CD4 T cells co-expressing IFN-γ and IL-17A) cells in EAU is not yet understood. Using interphotoreceptor retinoid-binding protein peptide-induced EAU in mice, we found increased levels of Th17/Th1 cells in EAU retinae (mean 9.

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.

Pharmacological Inhibition of Bromodomain Proteins Suppresses Retinal Inflammatory Disease and Downregulates Retinal Th17 Cells.

Experimental autoimmune uveitis (EAU), in which CD4 Th1 and/or Th17 cells are immunopathogenic, mimics various clinical features of noninfectious uveitis in humans. The impact of bromodomain extraterminal (BET) inhibitors on Th17 cell function was studied in a mouse model of EAU in vivo and in mouse and human Th17 cells in vitro. Two BET inhibitors (GSK151 and JQ1) were able to ameliorate the progression of inflammation in EAU and in mouse CD4 T cells in vitro, downregulating levels of Th17 cells.

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

The 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.

Meta-analysis of IDH-mutant cancers identifies EBF1 as an interaction partner for TET2.

Isocitrate dehydrogenase (IDH) genes 1 and 2 are frequently mutated in acute myeloid leukaemia (AML), low-grade glioma, cholangiocarcinoma (CC) and chondrosarcoma (CS). For AML, low-grade glioma and CC, mutant IDH status is associated with a DNA hypermethylation phenotype, implicating altered epigenome dynamics in the aetiology of these cancers. Here we show that the IDH variants in CS are also associated with a hypermethylation phenotype and display increased production of the oncometabolite 2-hydroxyglutarate, supporting the role of mutant IDH-produced 2-hydroxyglutarate as an inhibitor of TET-mediated DNA demethylation.

Ollier disease and Maffucci syndrome are caused by somatic mosaic mutations of IDH1 and IDH2.

Ollier disease and Maffucci syndrome are characterized by multiple central cartilaginous tumors that are accompanied by soft tissue hemangiomas in Maffucci syndrome. We show that in 37 of 40 individuals with these syndromes, at least one tumor has a mutation in isocitrate dehydrogenase 1 (IDH1) or in IDH2, 65% of which result in a R132C substitution in the protein. In 18 of 19 individuals with more than one tumor analyzed, all tumors from a given individual shared the same IDH1 mutation affecting Arg132.

IDH1 and IDH2 mutations are frequent events in central chondrosarcoma and central and periosteal chondromas but not in other mesenchymal tumours.

Somatic mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 occur in gliomas and acute myeloid leukaemia (AML). Since patients with multiple enchondromas have occasionally been reported to have these conditions, we hypothesized that the same mutations would occur in cartilaginous neoplasms. Approximately 1200 mesenchymal tumours, including 220 cartilaginous tumours, 222 osteosarcomas and another ∼750 bone and soft tissue tumours, were screened for IDH1 R132 mutations, using Sequenom(®) mass spectrometry.

Species-specific in vivo engraftment of the human BL melanoma cell line results in an invasive dedifferentiated phenotype not present in xenografts.

For clinically relevant studies on melanoma progression and invasiveness, in vivo experimental systems with a human cellular microenvironment would be advantageous. We have compared tumor formation from a human cutaneous malignant melanoma cell line (BL), after injection as conventional xenografts in the mouse, or when injected into a predominantly species-specific environment of human embryonic stem cell-derived teratoma induced in the mouse (the hEST model). The resulting melanoma histology was generally analogous, both systems showing delimited densely packed areas with pleomorphic cells of malignant appearance.

Oncogenic NRAS has multiple effects on the malignant phenotype of human melanoma cells cultured in vitro.

Activating mutations in the NRAS gene, which occur predominantly in codon 61 (Q61R, Q61K) are among the most common genetic events in malignant melanoma. NRAS protein with oncogenic codon 61 mutations may therefore be good therapeutic targets. In the present study, we used gene expression profiling as a method for global characterization of gene expression alterations that resulted from treatment of melanoma cells with siRNA specifically targeting NRAS(Q61R).

Suppression of oncogenic NRAS by RNA interference induces apoptosis of human melanoma cells.

The majority of human melanomas harbor activating mutations in either the BRAF or NRAS gene. To date, the role of oncogenic NRAS in melanoma remains poorly defined and no current therapies are directed at specifically suppressing oncogenic NRAS in human melanoma tumors. The aim of our study, therefore, was to investigate the effects of suppressing oncogenic NRAS in human melanoma cell lines in vitro.

Induction of melanoma phenotypes in human skin by growth factors and ultraviolet B.

Exposure to UV radiation likely plays a key role in melanoma development, whereas other etiologic agents remain unknown. Here we show that in normal human skin an increased expression of a combination of three growth factors, basic fibroblast growth factor, stem cell factor, and endothelin-3, along with exposure to UVB can transform normal melanocytes into a melanoma phenotype within 4 weeks. Invasion of melanoma lesions was found in skin from newborn donors, whereas melanomas in adult skin were of a noninvasive in situ type only.

Frequency of UV-inducible NRAS mutations in melanomas of patients with germline CDKN2A mutations.

Background: Germline alterations in cyclin-dependent kinase inhibitor 2A (CDKN2A) are important genetic factors in familial predisposition to melanoma. Activating mutations of the NRAS proto-oncogene are among the most common somatic genetic alterations in cutaneous malignant melanomas. We investigated the occurrence of NRAS mutations in melanomas and dysplastic nevi in individuals with germline CDKN2A mutations.

Electroporation in combination with a plasmid vector containing SV40 enhancer elements results in increased and persistent gene expression in mouse muscle.

Gene transfer into muscle upon injection of plasmid DNA is feasible but occurs with low frequency. However, by using electroporation after injection of plasmid DNA into mouse muscle it has been demonstrated that gene expression can be increased more than 150-fold. In this communication, we have used this technique in combination with plasmids containing a tandem repeat of three 72-bp DNA elements from the SV40 enhancer to study gene expression.

Nonsurgical direct delivery of plasmid DNA into rat heart: time course, dose response, and the influence of different promoters on gene expression.

Transfer of genes encoding therapeutic proteins into the myocardium shows great potential for treatment of coronary artery disease. To quantitatively elucidate the behavior of plasmid DNA following cardiac gene transfer, time kinetics, dose-response relationship, systemic spread to the liver, and the influence of different promoters on plasmid DNA gene expression in rat hearts were examined using a novel nonsurgical direct delivery method that enables testing of large numbers of animals. Plasmids encoding either vascular endothelial growth factor A 165 or a fusion protein between enhanced green fluorescent protein (EGFP) luciferase were injected directly in rat hearts under echocardiographic guidance.