Our body, especially the intestine, is colonized by a huge number of microbes – ten times more than the body’s own eukaryotic cells. This overcrowding, which seems rather alarming at first glance, fulfills important functions. Although bacteria are commonly associated with diseases, many of these microbes are harmless or even beneficial for humans. Indeed, the interaction between the healthy intestinal microbiota and the host is crucial for e. g. an effective digestion, degradation of toxins, defense of pathogens and the development of the host immune system – in regard of this last aspect, the relationship is of particular importance.
Colonization of the gastrointestinal tract starts at the time point of birth and shapes host immunity to tolerate “good” (commensal) bacteria and fight the “bad” (pathogenic) microbes. In genetically predisposed humans, however, an imbalance between the microbiota and the mucosal immunity may lead to a breakdown of tolerance and a constant activation of the immune system by commensal bacteria. This manifests in tissue inflammation and the production of antibodies against autoantigens and the microbiota. The most prevalent forms of such chronic inflammatory bowel diseases (CIBD) are Crohn’s disease (CD) and ulcerosa colitis (UC).
CD may involve inflammation of the complete gastrointestinal tract, most frequently the colon and the small intestine. Characteristic symptoms are abdominal pain, diarrhea, vomitus, weight loss and fever. In nearly half of the patients, extra intestinal signs, primarily cutaneous and joint manifestations, occur. Antibodies against the baker’s yeast Saccharomyces cerevisiae reveal a prevalence of 70% in CD patients and are accepted as important indicators for this disease. Furthermore, approximately 40% of patients exhibit autoantibodies against the acinus cells of the exocrine part of the pancreas (pancreatic antibodies, PAbs). They are also highly indicative for CD and have been commonly detected by indirect immunofluorescence on tissue sections of human pancreas. In 2012 finally, the specific target antigens of the PAbs could be identified (Komorowski et al. J Crohn’s Colitis 2012). These are two glycosylated membrane proteins called GP2 and CUZD1 which are located in the secretory vesicles of the pancreatic cells. Both belong to protein families that are involved in regulatory processes of the immune system. Thus, GP2 and CUZD1 could as well play a role in the maintenance of homeostasis between tolerance and defense of intestinal microbes. The characterization of the antigens provided the opportunity to develop new immunofluorescence assays based on recombinant cell substrates. The transfected cells express GP2 and CUZD1, respectively, and allow precise determination of PAbs specificities. This may contribute to a better stratification of CD patients, since the different autoantibodies seem to be associated with distinct clinical phenotypes, according to Papp et al. (J Crohn’s Colitis, 9(8): 659-68, 2015). Anti-GP2 antibodies, for instance, were more prevalent in patients with paediatric onset of CD and were associated with a more extensive disease with particular ileal involvement. Anti-CUZD1 antibodies, instead, were frequently detected in patients with inflammation of the colon.
In UC, inflammation is restricted to the colon, leading to diarrhea, intestinal bleeding and colics. Extraintestinal symptoms also manifest in the skin, the joints and also the liver. Autoantibodies against intestinal goblet cells and cytoplasmic components of neutrophil granulocytes (ANCA) are important markers of UC. Anti-intestinal goblet cell antibodies are exclusively found in UC patients, the prevalence is around 30%. Detection of the autoantibodies is performed by indirect immunofluorescence on primate intestinal tissue sections or cultivated goblet cells. ANCA, in particular perinuclear ANCA (pANCA) are present in 70% of UC patients. Major target is DNA-bound lactoferrin (Teegen et al, Ann NY Acad Sci 1173: 161–165, 2009). Determination of the antibodies relies on interaction of the protein with DNA – a requirement which is not given in many anti-lactoferrin specific ELISA. Therefore, a more sensitive lactoferrin-specific substrate has been developed for indirect immunofluorescence analysis: Initially, granulocytes are pretreated with high-salt buffer to remove pANCA-specific target antigens while DNA stays intact. Afterwards, lactoferrin is reconstituted by incubation of the cells with a lactoferrin solution. This preparation of the substrate prevents disturbing antibody reactivities with other antigens which is why the sensitivity of the immunofluorescence test is significantly increased compared to anti-lactoferrin ELISA – from 5% to more than 70% as shown in the study of Teegen et al (2009).
The BIOCHIP mosaic CIBD profil 3 combines all relevant substrates within on test: the pancreatic antigens GP2 and CUZD1 in form of transfected cell lines, Saccharomyces cerevisiae, tissue section of the intestine and the pancreas, intestinal goblet cells, granulocytes and lactoferrin-specific granulocytes. The multiparametric testing allows systematic diagnostics of CIBD and supports the clinician to differentiate between the clinically related diseases MC and UC.