Our research concerns the characterisation of immune responses to the pathogen Helicobacter pylori (Hp) which are linked to peptic ulceration and gastric cancer development ( Atherton, 2006 and Robinson et al., 2008). The challenges are broadly similar in other fields, particularly for gastrointestinal mucosal researchers: how to study immune responses using methodology that better reflects cytokine levels in the mucosa in vivo. Endoscopic mucosal biopsies are small (typically around 5–10 mg) and concentrations of many of the cytokines of interest are low,
so assay CX-4945 clinical trial sensitivity and sample volume requirements are critical. Other investigators have used semi-quantitative methods including immunohistochemistry ( Lindholm et al., 1998, Lehmann et al., 2002 and Holck et al., 2003) and western blotting ( Luzza et al., 2000 and Tomita et al., 2001), or PCR-based methods to quantify cytokine mRNA which are not always fully reflected at the protein level ( Luzza et JQ1 al., 2001, Robinson et al., 2008 and Serelli-Lee et al., 2012). Cytokines have been measured in gastric biopsy homogenates using ELISA ( Yamaoka et al., 2001, Shimizu et al., 2004, Caruso et al., 2008, Robinson et al., 2008 and Serelli-Lee et al., 2012), but additional volume is needed for each analyte assayed
which may require sample dilution. Therefore the number of cytokines, particularly those present at low concentrations, that can be assayed using this method is limited. Another common approach is to culture gastric biopsies in vitro, with or without stimulation, and measure cytokine concentrations in culture supernatants ( Crabtree et al., Tau-protein kinase 1991, Bodger et al., 1997 and Mizuno et al., 2005). However, these methods may alter the cytokine profile ( Veldhoen et al., 2009). The cytokine concentrations in homogenates of gastric biopsies should more closely reflect those found in the gastric mucosa in vivo. Luminex-based methods have been used to assess murine immune responses to Hp infection ( Taylor et al., 2008) and vaccination ( Taylor et al., 2007) in splenocyte culture supernatant and recently to quantify gastric
cytokine concentrations in Hp-infected mice ( Schumacher et al., 2012). A method to measure Hp-specific IgG in human saliva samples has also been developed, using Luminex beads conjugated with antigens including Hp whole cell sonicate and recombinant urease ( Griffin et al., 2011). However, to our knowledge, Luminex assays have not been optimised for human gastrointestinal mucosal tissue samples, though were recently used to quantify interleukin-1β, interleukin-1 receptor antagonist, interleukin-6 and tumour necrosis factor-α in gastric tissue samples ( Serelli-Lee et al., 2012). Careful kit selection and optimisation of tissue sample preparation in a limited volume of extraction buffer will theoretically facilitate cytokine detection in these samples.