Expression of galectins-1, -3 and -4 varies with strain and type of experimental colitis in mice.

Galectins are increasingly the focus of biomedical research. Although they are involved at different stages in inflammation, data on galectins in colitis remain scarce. The aim of this study was to determine and compare the expression of galectins in acute and chronic experimental colitis in mice. Immunohistochemistry for galectins-1, -3 and -4 was performed on colon tissue from C57BL/6 and BALB/c mice with acute dextran sodium sulphate colitis and from 129 Sv/Ev IL-10 knock-out (IL-10(-/-)) mice. From these three mouse strains, we first detected major differences in galectin expression related to the genetic background in the control animals. With regard to inflammation, chronic colitis in IL-10(-/-) mice was associated with increased galectin-4 expression; in contrast with the two other models, no galectin-1 and -3 alterations were observed in IL-10(-/-) mice. Acute colitis in C57BL/6 and BALB/c mice showed increased galectin-3 expression in the lamina propria and the crypt epithelium, together with a decreased nuclear expression. These results suggest an involvement of galectins in the development and perpetuation of colonic inflammation and illustrate that the choice of the mouse strain for studying galectins might influence the outcome of the experiments.

AG013, a mouth rinse formulation of Lactococcus lactis secreting human Trefoil Factor 1, provides a safe and efficacious therapeutic tool for treating oral mucositis.

Non-clinical studies, focusing on the pharmacodynamics (PD), pharmacokinetics (PK) and safety pharmacology of genetically modified Lactococcus lactis (L. lactis) bacteria, engineered to secrete human Trefoil Factor 1 (hTFF1), were performed to provide proof-of-concept for the treatment of oral mucositis (OM) patients. L. lactis strain sAGX0085 was constructed by stably inserting an htff1 expression cassette into the bacterial genome, and clinically formulated as a mouth rinse (coded AG013). PD studies, using different oral dosing regimens, were performed in a clinically relevant hamster model for radiation-induced OM. The PK profile was assessed in healthy hamsters and in hamsters with radiation-induced OM. In addition, in vitro and in vivo safety pharmacology studies were conducted, in pooled, complement-preserved human serum, and in neutropenic hamsters and rats respectively. Topical administration of L. lactis sAGX0085/AG013 to the oral mucosa significantly reduced the severity and course of radiation-induced OM. PK studies demonstrated that both living L. lactis bacteria, as well as the hTFF1 secreted, could be recovered from the administration site for maximum 24h post-dosing, without systemic exposure. The in vitro and in vivo safety pharmacology studies confirmed that L. lactis sAGX0085 could not survive in systemic circulation, not even under neutropenic conditions. The results from the PD, PK and safety pharmacology studies reported here indicate that in situ secretion of hTFF1 by topically administered L. lactis bacteria provides a safe and efficacious therapeutic tool for the prevention and treatment of OM.

Intracellular accumulation of trehalose protects Lactococcus lactis from freeze-drying damage and bile toxicity and increases gastric acid resistance.

Interleukin-10 (IL-10) is a promising candidate for the treatment of inflammatory bowel disease. Intragastric administration of Lactococcus lactis genetically modified to secrete IL-10 in situ in the intestine was shown to be effective in healing and preventing chronic colitis in mice. However, its use in humans is hindered by the sensitivity of L. lactis to freeze-drying and its poor survival in the gastrointestinal tract. We expressed the trehalose synthesizing genes from Escherichia coli under control of the nisin-inducible promoter in L. lactis. Induced cells accumulated intracellular trehalose and retained nearly 100% viability after freeze-drying, together with a markedly prolonged shelf life. Remarkably, cells producing trehalose were resistant to bile, and their viability in human gastric juice was enhanced. None of these effects were seen with exogenously added trehalose. Trehalose accumulation did not interfere with IL-10 secretion or with therapeutic efficacy in murine colitis. The newly acquired properties should enable a larger proportion of the administered bacteria to reach the gastrointestinal tract in a bioactive form, providing a means for more effective mucosal delivery of therapeutics.

Active delivery of trefoil factors by genetically modified Lactococcus lactis prevents and heals acute colitis in mice.

BACKGROUND & AIMS: Effective therapeutics for treating acute colitis, caused by disruption of the intestinal epithelial barrier, are scarce. Trefoil factors (TFF) are cytoprotective and promote epithelial wound healing and reconstitution of the gastrointestinal tract, which makes them good candidate therapeutics for acute colitis. However, orally administered TFF stick to the mucus of the small intestine and are absorbed at the cecum. METHODS: We have engineered the food-grade bacterium Lactococcus lactis to secrete bioactive murine TFF. The protective and therapeutic potentials of these TFF-secreting L. lactis were evaluated in parallel with purified TFF in the dextran sodium sulfate (DSS)-induced murine model for acute colitis and in established chronic colitis in interleukin (IL)-10(-/-) mice. Disease was evaluated by blinded macroscopic and microscopic inflammatory scores and by myeloperoxidase activity. RESULTS: Intragastric administration of TFF-secreting L. lactis led to active delivery of TFF at the mucosa of the colon and, in contrast to administration of purified TFF, proved to be very effective in prevention and healing of acute DSS-induced colitis. The in situ secreted murine TFF significantly decreased morbidity and mortality and stimulated prostaglandin-endoperoxide synthase 2 expression, which represents a major therapeutic pathway. In addition, this approach was successful in improving established chronic colitis in IL-10(-/-) mice. CONCLUSIONS: We have positively evaluated a new therapeutic approach for acute and chronic colitis that involves in situ secretion of murine TFF by orally administered L. lactis. This novel approach may lead to effective management of acute and chronic colitis and epithelial damage in humans.