A major limitation of biopharmaceutical proteins is their fast clearance from circulation via kidney filtration, which strongly hampers efficacy both in animal studies and in human therapy. We have developed conformationally disordered polypeptide chains with expanded hydrodynamic volume comprising the small residues Pro, Ala and Ser (PAS). PAS sequences are hydrophilic, uncharged biological polymers with biophysical properties very similar to poly-ethylene glycol (PEG), whose chemical conjugation to drugs is an established method for plasma half-life extension. In contrast, PAS polypeptides offer fusion to a therapeutic protein on the genetic level, permitting Escherichia coli production of fully active proteins and obviating in vitro coupling or modification steps. Furthermore, they are biodegradable, thus avoiding organ accumulation, while showing stability in serum and lacking toxicity or immunogenicity in mice. We demonstrate that PASylation bestows typical biologics, such as interferon, growth hormone or Fab fragments, with considerably prolonged circulation and boosts bioactivity in vivo.

This study showed that an iron sulfate free diet in combination with systemic iron repletion prevents the development of chronic ileitis in a murine model of Crohn's disease. Luminal iron may directly affect IEC function or generate a pathological milieu in the intestine that triggers epithelial cell stress-associated apoptosis through changes in microbial homeostasis. These results suggest that oral replacement therapy with iron sulfate may trigger inflammatory processes associated with progression of Crohn's disease-like ileitis.

Interleukin 1 (IL1) is an important regulator of inflammatory responses and contributes to host immune defence against infection. Vaccinia virus encodes a viral soluble IL1b receptor (IL1bR), which modulates the acute-phase host response to infection and might influence the induction of immune responses against virus-associated antigens. Here, modified vaccinia virus Ankara (MVA) mutants defective in IL1bR production were produced by insertion of selectable marker gene sequences that precisely deleted the IL1bR coding sequences from the MVA genome (MVA-DIL1bR). Analysis of MVA mutants indicated that deletion of the IL1bR gene did not abrogate the formation of MVA progeny upon tissue culture propagation. After high-dose intranasal infection with MVA-DIL1bR, mice showed no signs of fever or other illness, suggesting that the avirulent phenotype remained preserved for MVA-DIL1bR. Following vaccination of mice, MVA-DIL1bR or non-mutated MVA induced similar acute-phase immune responses. Importantly, when monitored at the memory phase, significantly higher vaccinia virus-specific total CD8 + and HLA-A*0201-binding peptide epitope-specific T-cell responses were found after vaccination of HLA-A*0201-transgenic and non-transgenic mice with MVA-DIL1bR. Moreover, 4-6 months after vaccination, MVA-DIL1bR provided higher levels of protection against lethal respiratory challenge infection with virulent vaccinia virus strain Western Reserve compared with wild-type MVA. These data suggest that deletion of the viral IL1bR gene may be considered a relevant approach to amplify the virus-specific CD8 + memory T-cell response and duration of protective immunity obtained after MVA vaccination.

BackgroundObesity has been associated with a more severe disease course in inflammatory bowel disease (IBD) and epidemiological data identified dietary fats but not obesity as risk factors for the development of IBD. Crohn’s disease is one of the two major IBD phenotypes and mostly affects the terminal ileum. Despite recent observations that high fat diets (HFD) impair intestinal barrier functions and drive pathobiont selection relevant for chronic inflammation in the colon, mechanisms of high fat diets in the pathogenesis of Crohn’s disease are not known. The aim of this study was to characterize the effect of HFD on the development of chronic ileal inflammation in a murine model of Crohn’s disease-like ileitis.MethodsTNFΔARE/WT mice and wildtype C57BL/6 littermates were fed a HFD compared to control diet for different durations. Intestinal pathology and metabolic parameters (glucose tolerance, mesenteric tissue characteristics) were assessed. Intestinal barrier integrity was characterized at different levels including polyethylene glycol (PEG) translocation, endotoxin in portal vein plasma and cellular markers of barrier function. Inflammatory activation of epithelial cells as well as immune cell infiltration into ileal tissue were determined and related to luminal factors.ResultsHFD aggravated ileal inflammation but did not induce significant overweight or typical metabolic disorders in TNFΔARE/WT. Expression of the tight junction protein Occludin was markedly reduced in the ileal epithelium of HFD mice independently of inflammation, and translocation of endotoxin was increased. Epithelial cells showed enhanced expression of inflammation-related activation markers, along with enhanced luminal factors-driven recruitment of dendritic cells and Th17-biased lymphocyte infiltration into the lamina propria.ConclusionsHFD feeding, independently of obesity, accelerated disease onset of small intestinal inflammation in Crohn’s disease-relevant mouse model through mechanisms that involve increased intestinal permeability and altered luminal factors, leading to enhanced dendritic cell recruitment and promoted Th17 immune responses.

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