Analysis of the immunomodulatory properties of mycobacterium cell wall fraction on the cytokine production of peripheral blood mononuclear cells of healthy dogs.

BACKGROUND: Mycobacterium cell wall fraction (MCWF) is derived from nonpathogenic Mycobacterium phlei and is used as an immunomodulatory compound in clinical practice, yet its mode-of-action requires further research. OBJECTIVE: To evaluate the host response to MCWF in canine peripheral blood mononuclear cells (PBMCs) by using enzyme-linked immunosorbent assays (ELISA) and quantitative reverse transcription (qRT)-PCR for assessment of cytokines. ANIMALS: Eight healthy Labrador retrievers. MATERIALS AND METHODS: PBMCs were isolated from whole blood using density centrifugation. The cells were cultured with different concentrations of MCWF or a potent stimulator of cytokine production, phorbol 12-myristate 13-acetate/ionomycin, or left in cell culture medium for 24, 48 and 72 h. Cytokines were measured by ELISA for interleukin (IL)-4, IL-10 and interferon-gamma (IFN-γ), and by qRT-PCR for IL-4, IL-10, IL-13, IFN-γ, tumour necrosis factor alpha (TNF-α) and transforming growth factor-beta. RESULTS: A significant increase of IL-10 messenger ribonucleic acid (mRNA) was detected at all time points for all concentrations of MCWF (p < 0.05). Protein analysis reflected this finding, with a maximum IL-10 concentration of 300.6 ± 38.3 µg/mL. Compared to the negative control, post-stimulation elevation of IFN-γ mRNA was noted at 24 h with all concentrations of MCWF (p < 0.01), and TNF-α mRNA was increased for 0.5 µg/dL MCWF only at 72 h (p < 0.05). CONCLUSIONS AND CLINICAL RELEVANCE: MCWF stimulation of PBMCs results in the elevation of both proinflammatory and regulatory cytokine mRNA. Further research into the role of MCWF as a systemically administered regulatory immunomodulator or adjuvant to allergen-specific immunotherapy should be considered.

Targeting the Wnt pathway in zebrafish as a screening method to identify novel therapeutic compounds.

Activating mutations in the Wnt signaling pathway account for the initiation of greater than 90% of all colorectal cancers and this pathway has been implicated in numerous other diseases. Therefore, identifying small molecule inhibitors of this pathway is of critical importance towards identifying clinically relevant drugs. Numerous screens have been employed to identify therapeutic reagents, but none have made it to advanced clinical trials, suggesting that traditional screening methods are ineffective at identifying clinically relevant targets. Here, we describe a novel in vivo screen to identify small molecule inhibitors of the Wnt pathway. Specifically, treatment of zebrafish embryos with LiCl inhibits GSK3 kinase function, resulting in hyperactivation of the signaling pathway and an eyeless phenotype at 1 day post fertilization. Using the small molecule XAV939, a known inhibitor of Wnt signaling, we rescued the LiCl induced eyeless phenotype, confirming efficacy of the screen. We next tested our assay with 400 known small molecule kinase inhibitors, none of which should inhibit Wnt signaling below the level of GSK3 based on their known targets. Accordingly, none of these small molecules rescued the eyeless phenotype, which demonstrates the stringency of the assay. However, several of these small molecule kinase inhibitors did generate a non-Wnt phenotype in accordance with the kinase they targeted. Therefore, combining the efficacy, sensitivity, and stringency of this preliminary screen, this model will provide an alternative to the traditional in vitro screen, generating potentially clinical relevant drugs in a rapid and cost-effective way.