Evaluation of a xenogeneic vascular endothelial growth factor-2 vaccine in two preclinical metastatic tumor models in mice.

In this study, a xenogeneic DNA vaccine encoding for human vascular endothelial growth factor receptor-2 (hVEGFR-2) was evaluated in two murine tumor models, the B16-F10 melanoma and the EO771 breast carcinoma model. The vaccine was administered by intradermal injection followed by electroporation. The immunogenicity and the biological efficacy of the vaccine were tested in (1) a prophylactic setting, (2) a therapeutic setting, and (3) a therapeutic setting combined with surgical removal of the primary tumor. The tumor growth, survival, and development of an immune response were followed. The cellular immune response was measured by a bioluminescence-based cytotoxicity assay with vascular endothelial growth factor-2 (VEGFR-2)-expressing target cells. Humoral immune responses were quantified by enzyme-linked immunosorbent assay (ELISA). Ex vivo bioluminescence imaging and immunohistological observation of organs were used to detect (micro)metastases. A cellular and humoral immune response was present in prophylactically and therapeutically vaccinated mice, in both tumor models. Nevertheless, survival in prophylactically vaccinated mice was only moderately increased, and no beneficial effect on survival in therapeutically vaccinated mice could be demonstrated. An influx of CD3+ cells and a slight decrease in VEGFR-2 were noticed in the tumors of vaccinated mice. Unexpectedly, the vaccine caused an increased quantity of early micrometastases in the liver. Lung metastases were not increased by the vaccine. These early liver micrometastases did however not grow into macroscopic metastases in either control or vaccinated mice when allowed to develop further after surgical removal of the primary tumor.

Safety assessment of the butyrate-producing Butyricicoccus pullicaecorum strain 25-3(T), a potential probiotic for patients with inflammatory bowel disease, based on oral toxicity tests and whole genome sequencing.

Inflammatory bowel disease (IBD) is a chronic inflammation of the digestive tract, characterized by dysbiosis of the intestinal microbiota. Probiotics have been suggested as a strategy to reduce active disease or extend remission. We isolated and characterized the butyrate-producing strain Butyricicoccus pullicaecorum 25-3(T) and identified it as a potential probiotic for patients with IBD. To evaluate the safety of 25-3(T) for use in humans, we conducted a standard acute oral toxicity test and a 28-day repeated oral dose toxicity test. The complete genome of B. pullicaecorum 25-3(T) was sequenced to search for virulence factors and antibiotic resistance determinants. The minimum inhibitory concentration (MIC) of 21 antimicrobials was determined. Results showed no adverse effects in the oral toxicity tests. B. pullicaecorum 25-3(T) is resistant against aminoglycosides and trimethoprim. The genome of 25-3(T) contains no virulence factors, one gene related to harmful metabolites and 52 sequences with high similarity to antimicrobial and toxic compound resistance genes, that did not correspond with a resistant phenotype. This first report of a safety assessment of a butyrate-producing strain from Clostridium cluster IV shows that B. pullicaecorum 25-3(T) is a non-pathogenic strain, but carries antibiotic resistance genes with the risk of transfer, that need further investigation.

Butyricicoccus pullicaecorum in inflammatory bowel disease.

OBJECTIVE: Many species within the phylum Firmicutes are thought to exert anti-inflammatory effects. We quantified bacteria belonging to the genus Butyricicoccus in stools of patients with ulcerative colitis (UC) and Crohn's disease (CD). We evaluated the effect of Butyricicoccus pullicaecorum in a rat colitis model and analysed the ability to prevent cytokine-induced increases in epithelial permeability. DESIGN: A genus-specific quantitative PCR was used for quantification of Butyricicoccus in stools from patients with UC or CD and healthy subjects. The effect of B pullicaecorum on trinitrobenzenesulfonic (TNBS)-induced colitis was assessed and the effect of B pullicaecorum culture supernatant on epithelial barrier function was investigated in vitro. RESULTS: The average number of Butyricicoccus in stools from patients with UC and CD in active (UC: 8.61 log10/g stool; CD: 6.58 log10/g stool) and remission phase (UC: 8.69 log10/g stool; CD: 8.38 log10/g stool) was significantly lower compared with healthy subjects (9.32 log10/g stool) and correlated with disease activity in CD. Oral administration of B pullicaecorum resulted in a significant protective effect based on macroscopic and histological criteria and decreased intestinal myeloperoxidase (MPO), tumour necrosis factor α (TNFα) and interleukin (IL)-12 levels. Supernatant of B pullicaecorum prevented the loss of transepithelial resistance (TER) and the increase in IL-8 secretion induced by TNFα and interferon γ (IFN gamma) in a Caco-2 cell model. CONCLUSIONS: Patients with inflammatory bowel disease have lower numbers of Butyricicoccus bacteria in their stools. Administration of B pullicaecorum attenuates TNBS-induced colitis in rats and supernatant of B pullicaecorum cultures strengthens the epithelial barrier function by increasing the TER.