Membranes were blocked with 5% skim milk and labelled with ImmunoPure anti-mouse IgG HRP (Pierce), anti-mouse IgA HRP (SouthernBiotech, Birmingham, AL, USA) or anti-β-actin then anti-rabbit HRP (both from Cell Signaling Technology, Beverly, MA, USA). Membranes were visualized using ECL chemiluminescence EGFR inhibitor reagent

(GE Healthcare) and an ImageQuant LAS 4000 (GE Healthcare), with densitometry performed using ImageJ software. RNA from one submandibular and one sublingual salivary gland was extracted using Tri Reagent (Ambion), then converted to cDNA using the Quantitect Reverse Transcription Kit (Qiagen, Hilden, Germany) which was diluted out to 150 μL in Tris-EDTA buffer. For qPCR, duplicate reactions of 25 μL containing 12.5 μL QuantiTect SYBR Green PCR Master Mix (Qiagen), 0.2 μmol/L primers and 3 μL of cDNA were performed in an Mx3000P cycler (Stratagene, La Jolla, CA, USA). Primer efficiencies within each run were determined with LinRegPCR [22] and gene expression calculated relative to

Actb. For statistical analyses, data were log-transformed then compared by analysis of variance (ANOVA), with Dunnett’s post hoc analysis using SPSS software, version 20.0 (IBM, Armonk, NY, USA). To examine whether changes in salivary Idelalisib datasheet cytokine or mucin expression correlated with vaccine-mediated protection, mice were immunized orally with H. pylori lysate and CT 上海皓元医药股份有限公司 adjuvant. Vaccination was confirmed to induce a significant reduction in H. pylori colonization upon subsequent challenge with live bacteria, when compared with unimmunized controls (Fig. 1). To determine whether this protective response correlated with an increase in immune activity in the salivary glands, cytokine levels were compared in these glands from infected and immunized/challenged mice, as well as from negative controls (uninfected/unimmunized). Not only was there no evidence of an increase, but surprisingly the total levels of many cytokines (IL-1ß, TNFα, IL-10, IL-6 and IL-17A) were

significantly reduced in the salivary glands of immunized, infected mice (Fig. 2). Further analysis revealed that salivary glands from the immunized/challenged mice in this experiment contained significantly more total protein than non-immunized mice (Fig. 2). Salivary gland weights were not recorded, so it was not possible to determine whether this was due to an increase in salivary gland size (although no obvious increase was noted at extraction), or increased protein concentrations within the glands. Given salivary glands are a major source of mucosal secretory antibody, in particular IgA, we theorized the increase in protein concentration in immunized infected mice was most likely to be due to increased levels of IgA production, and this was confirmed by Western blot (Fig. 3). The key aim of this study was to evaluate the effect of vaccination on salivary mucin production.