No abstract

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease arising from defects in the dystrophin gene, typically nonsense or frameshift mutations, that preclude the synthesis of a functional protein. A milder, allelic version of the disease, Becker muscular dystrophy, generally arises from in-frame deletions that allow synthesis of a shorter but still semifunctional protein. Therapies to introduce functional dystrophin into dystrophic tissue through either cell or gene replacement have not been successful to date. We report an alternative approach where 2'-O-methyl antisense oligoribonucleotides have been used to modify processing of the dystrophin pre-mRNA in the mdx mouse model of DMD. By targeting 2'-O-methyl antisense oligoribonucleotides to block motifs involved in normal dystrophin pre-mRNA splicing, we induced excision of exon 23, and the mdx nonsense mutation, without disrupting the reading frame. Exon 23 skipping was first optimized in vitro in transfected H-2K(b)-tsA58 mdx myoblasts and then induced in vivo. Immunohistochemical staining demonstrated the synthesis and correct subsarcolemmal localization of dystrophin and gamma-sarcoglycan in the mdx mouse after intramuscular delivery of antisense oligoribonucleotide:liposome complexes. This approach should reduce the severity of DMD by allowing a dystrophic gene transcript to be modified, such that it can be translated into a Becker-dystrophin-like protein.

Vitamin D has been suggested as a possible adjunctive treatment to ameliorate disease severity in human inflammatory bowel disease. In this study, the effects of diets containing high (D++, 10,000 IU/kg), moderate (D+, 2,280 IU/kg) or no vitamin D (D−) on the severity of dextran sodium sulphate (DSS) colitis in female C57Bl/6 mice were investigated. The group on high dose vitamin D (D++) developed the most severe colitis as measured by blinded endoscopic (p < 0.001) and histologic (p < 0.05) assessment, weight loss (p < 0.001), drop in serum albumin (p = 0.05) and increased expression of colonic TNF-α (p < 0.05). Microbiota analysis of faecal DNA showed that the microbial composition of D++ control mice was more similar to that of DSS mice. Serum 25(OH)D3 levels reduced by 63% in the D++ group and 23% in the D+ group after 6 days of DSS treatment. Thus, high dose vitamin D supplementation is associated with a shift to a more inflammatory faecal microbiome and increased susceptibility to colitis, with a fall in circulating vitamin D occurring as a secondary event in response to the inflammatory process.

BackgroundSecreted Protein Acidic and Rich in Cysteine (SPARC) is expressed during tissue repair and regulates cellular proliferation, migration and cytokine expression. The aim was to determine if SPARC modifies intestinal inflammation.MethodsWild-type (WT) and SPARC-null (KO) mice received 3% dextran sodium sulphate (DSS) for 7 days. Inflammation was assessed endoscopically, clinically and histologically. IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-12/IL23p40, TNF-α, IFN-γ, RANTES, MCP-1, MIP-1α, MIP-1β, MIG and TGF-β1 levels were measured by ELISA and cytometric bead array. Inflammatory cells were characterised by CD68, Ly6G, F4/80 and CD11b immunofluorescence staining and regulatory T cells from spleen and mesenteric lymph nodes were assessed by flow cytometry.ResultsKO mice had less weight loss and diarrhoea with less endoscopic and histological inflammation than WT animals. By day 35, all (n = 13) KO animals completely resolved the inflammation compared to 7 of 14 WT mice (p<0.01). Compared to WTs, KO animals at day 7 had less IL1β (p = 0.025) and MIG (p = 0.031) with higher TGFβ1 (p = 0.017) expression and a greater percentage of FoxP3+ regulatory T cells in the spleen and draining lymph nodes of KO animals (p<0.01). KO mice also had fewer CD68+ and F4/80+ macrophages, Ly6G+ neutrophils and CD11b+ cells infiltrating the inflamed colon.ConclusionsCompared to WT, SPARC KO mice had less inflammation with fewer inflammatory cells and more regulatory T cells. Together, with increased TGF-β1 levels, this could aid in the more rapid resolution of inflammation and restoration of the intestinal mucosa suggesting that the presence of SPARC increases intestinal inflammation.

Reduced sunlight exposure has been associated with an increased incidence of Crohn’s disease and ulcerative colitis. The effect of ultraviolet radiation (UVR) on the faecal microbiome and susceptibility to colitis has not been explored. C57Bl/6 female mice were fed three different vitamin D-containing diets for 24 days before half of the mice in each group were UV-irradiated (1 kJ/m2) for each of four days, followed by twice-weekly irradiation of shaved dorsal skin for 35 days. Faecal DNA was extracted and high-throughput sequencing of the 16S RNA gene performed. UV irradiation of skin was associated with a significant change in the beta-diversity of faeces compared to nonirradiated mice, independently of vitamin D. Specifically, members of phylum Firmicutes, including Coprococcus, were enriched, whereas members of phylum Bacteroidetes, such as Bacteroidales, were depleted. Expression of colonic CYP27B1 increased by four-fold and IL1β decreased by five-fold, suggesting a UVR-induced anti-inflammatory effect. UV-irradiated mice, however, were not protected against colitis induced by dextran sodium sulfate (DSS), although distinct faecal microbiome differences were documented post-DSS between UV-irradiated and nonirradiated mice. Thus, skin exposure to UVR alters the faecal microbiome, and further investigations to explore the implications of this in health and disease are warranted.