Frances Lloyd

The spread of transgene expression at the site of gene construct injection

Seven 2‐day‐old golden retriever pups were given focal intramuscular injections of a first generation adenovirus–dystrophin minigene construct and adenovirus–β‐galactosidase construct as a 2:1 mixture into the left anterior tibial muscle. The spread of transgene expression within the anterior tibial muscle was compared with the spread of methylene blue dye after identical injection into the contralateral muscle. Transgene expression 5–7 days after intramuscular injection was shown to extend between 5.8 and 11.6 mm along the biopsied muscle length (range of biopsy lengths 11.1–12.2 mm). The level of transgene expression at 2–2.5‐mm intervals from the site of injection was significantly related to the distance from the site of injection (dystrophin, P = 0.009; β‐galactosidase, P = 0.015). The spread of methylene blue dye within the anterior tibial muscle ≤24 h after identical intramuscular injection demonstrated a similar pattern to the transgene expression, with dye staining measured between 5.5 and 8.5 mm along the muscle sample length (range of biopsy lengths 5.6–15.6 mm). The greatest transgene expression and dye staining was measured 2–2.5 mm proximal to the site of injection with a maximum of 23% of muscle fibers expressing the dystrophin transgene, 95.2% expressing the β‐galactosidase transgene, and 98% of the tissue section stained with methylene blue dye. These results suggest transgene expression after focal intramuscular injection is relatively localized around the site of injection. Further research is required to develop techniques that will provide transgene expression throughout the length and breadth of a muscle.

Secreted Protein Acidic and Rich in Cysteine (SPARC) Exacerbates Colonic Inflammatory Symptoms in Dextran Sodium Sulphate-Induced Murine Colitis

Background Secreted 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. Methods Wild-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. Results KO 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. Conclusions Compared 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.

Direct dystrophin and reporter gene transfer into dog muscle in vivo

Bacterial β‐galactosidase cDNA was injected without lipofectin into 41 sites in dog muscle and expression was seen in 22 of them. The cDNA and lipofectin was injected into 35 similar sites and expression was seen in 21. Expression was seen in a maximum of 2.5% of muscle fibers and 23.21% of nonmuscle cells. A total of 106 muscle sites were injected with the minigene with and without lipofectin. In 4 of the 45 sites injected with the minigene without lipofectin human dystrophin was expressed around the periphery of 0.3% of the fibers. Bacterial β‐galactosidase cDNA was injected into the peritoneal cavity of 4 pups, 2 of which also received lipofectin. In all 4, expression was seen in liver, spleen, and mesenteric lymph node. In the 2 pups that received lipofectin, expression was also seen in the diaphragm, intercostal, and abdominal muscles of 1 and in the diagphragm and intercostal muscles of the other. These experiments show that human dystrophin transgene expression can be obtained in dog muscle. However, other methods will be required to increase the degree of expression before gene therapy trials can be undertaken.

High Dose Vitamin D supplementation alters faecal microbiome and predisposes mice to more severe colitis

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.

Ultraviolet Irradiation of Skin Alters the Faecal Microbiome Independently of Vitamin D in Mice

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.

Tu1934 SPARC Modifies Colonic Tissue Healing and Inflammation by Regulating Collagen and MMP Expression

OP07 Use of fecal calprotectin as marker of disease activity in patients under maintenance treatment with infliximab for ulcerative colitis M. De Vos1 *, J. Jahnsen2, J. Vandervoort3, G. D’Haens4, O. Dewit5, E. Louis6, D. Franchimont7, F. Baert8, R. Torp9, P. Potvin10, P. Van Hootegem11, M. Henriksen12, B. Vander Cruyssen1, S. Vermeire13, on behalf of BIRD14. 1Ghent University Hospital, Department of Gastroenterology, Gent, Belgium, 2Oslo University Hospital, Department of Gastroenterology, Aker, Norway, 3OLV Hospital, Department of Gastroenterology, Aalst, Belgium, 4Imelda Hospital, Department of Gastroenterology, Bonheiden, Belgium, 5Clinique Universitaire St Luc, Department of Gastroenterology, Brussel, Belgium, 6University of Liege and CHU Liege, Department of Gastroenterology, Liege, Belgium, 7Erasme Hospital, Department of Gastroenterology, Brussel, Belgium, 8H. Hart Hospital, Department of Gastroenterology, Roeselare, Belgium, 9Innlandet Hospital, Department of Gastroenterology, Hammar, Norway, 10St-Jozephs Hospital, Department of Gastroenterology, Bornem, Belgium, 11AZ St Lucas, Department of Gastroenterology, Brugge, Belgium, 12Ostfold Fredrikstad Hospital, Department of Gastroenterology, Fredrikstad, Norway, 13University Hospital Gasthuisberg, Department of Gastroenterology, Leuven, Belgium, 14, Belgium