The use of human biopsy materials and other preserved tissues in in vitro
tissue binding assays allowed detection of tissue-specific adherence of H. pylori
and other pathogens and characterization of the receptors used. Early studies showed that many H. pylori
strains could bind fucosylated Lewis (or ABO histo blood group) antigens and the inflammation-associated sialylated antigens of the gastric epithelium (Aspholm-Hurtig et al., 2004
; Boren et al.
, 1993; Mahdavi et al., 2002
); that uropathogenic E. coli
bound to globoseries glycolipids, which are abundant in kidney tissue (Roberts et al., 1994
); and that Streptococcus pyogenes
–bound protein antigens in human cutaneous tissue (Okada et al., 1994
). In typical in vitro
host tissue binding assays, bacterial cells are cultured, labeled, and overlaid on histotissue sections. The binding patterns observed give insight into expression, localization, and spatial distribution of receptors in target tissues. Further characterization of receptors can emerge from studies using various inhibitors of adherence (e.g., simple glycans to titrate adhesins; glycosidases or to disrupt potential receptors) in this in vitro
tissue-binding assay. A general flowchart for initial identification of carbohydrate receptor structures is presented in the early review by Falk et al. (1994a)
Biopsy specimens from gastroscopy examinations and tissues from gastrointestinal surgery provide valuable material for adherence studies. It is often useful to choose tissues for analysis according to patient ABO and Lewis blood group status and disease condition (e.g., gastritis, peptic ulcer, gastric cancer). Also noteworthy are gender, age, medication, immune status, level of inflammation, and other associated infections, because these may also affect the types and tissue densities of various glycans that H. pylori can use as receptors and, thereby, vulnerability to infection or disease. The series of studies referred to previously also illustrates that H. pylori clinical isolates differ in types of glycans they can use as receptors and intensity of binding, reflecting diversity in their complement of adhesin genes, as noted earlier.
Tissues can be fixed by standardized formaldehyde treatment and embedded in paraffin blocks for later microtome sectioning. Most pathology laboratories use standardized protocols and robotic fixation and embedding instruments to ensure full reproducibility. Biopsy material can also be snap-frozen and embedded in cryopreservatives for later cryosectioning. There are pros and cons with both methods: Paraffin-embedded/fixed material usually cut in thinner sections than is cryopreserved material, and thus provides higher resolution images, and fixed material can be stored for years. However, cryosectioned materials retain more of the gastric mucin layer and conformational epitopes, and thereby allow better presentation of certain antigens for analyses of adherence or detection with monoclonal antibodies.
Bacterial cells used for initial in vitro
tissue adherence studies are often FITC (green fluorochrome) labeled and then overlaid on tissue sections (Falk et al., 1993
; Borén et al., 1997
; see also FITC-labeling in section 2: “Glycoprotein Array” and 8: “Identification of Bacterial Clones”). Red fluorochromes such as TRITC or Texas-Red can be used equivalently (Van de Bovenkamp et al., 2003
), although TRITC tends to leach from bacterial cells and skew adherence profiles unless tissue sections are very thoroughly washed after the bacterial overlay. It is also important to take into consideration that bacterial surface labeling involves modification of basic amino acids and thus may diminish adherence by direct inactivation of an adhesin's binding domain or indirectly through steric hindrance. When this is a concern, fluorochrome-labeled bacterial cells can be tested by RIA or ELISA assays if defined receptors are available (see the next section). If no binding activity of fluorochrome-labeled H. pylori
cells is detected, the possible influence of fluorolabeling can be tested using unlabeled cells for binding to tissue sections, followed by staining (1) with DNA intercalating agents such as acridine orange or (2) through enzymatic conversion of precursor substrate and fluorochrome-activation of bacterial cytoplasm (see Molecular Probes
), or (3) with antibodies against bacterial cells or their surface antigens.
Once H. pylori
cells are successfully fluorochrome labeled they can be aliquoted and stored frozen at −20° for 6–12 mo. Use of aliquots from the same batch of labeled cells can contribute to consistency (e.g., when many tissues or receptors are being studied) and avoids complications from any possible growth-related variation in adhesin expression and presentation on cell surfaces. This procedure depends on intactness of bacterial cells and is compatible with most H. pylori
strains. This said, there might be certain strains or species in which intrinsic fragility of bacterial cells or their adhesins, either naturally or during freezing and thawing, makes this protocol less suitable, such as Neisseria gonorrhoeae
and N. meningitidis
and various Borrelia
species. An alternative could be to use minimal levels of DMSO as solvent for the FITC staining reagent. The in vitro
tissue adherence assay is performed essentially as described by Falk et al. (1993
and Borén et al. (1997)
, with modifications in duration and frequency of washing of the histotissue section overlaid with bacterial cells to fit bacterial affinity. Slides used to test for adherence by high-affinity interactions can be washed more extensively than those used to detect low-affinity adherence or if there are relatively few adhesin proteins per cell. Identification of optimal washing conditions may sometimes need careful titration and comparison with suitable references, such as derivative strains in which the adhesin gene of interest has been switched off in expression by phase variation or has been deleted.
The biopsy specimens used for the in vitro
tissue adherence assay can alternately be cultured in vitro
for 2–3 days and used for adherence analyses (i.e., by use of the in vitro
explant culture [IVEC] technique) (Olfat et al., 2002
). This is a most useful application for studies of the impact of adherence in terms of bacterial-host crosstalk, such as cytokine release and cellular signaling in host tissue. In addition, the IVEC technique also complements the use of primary cell cultures, because the cells in biopsies are most similar to the true in vivo
conditions of the epithelium and in expression of naive
glycosylation patterns. In addition, IVEC also ensures the integrity of the spatial distribution of cell lineages in the intact biopsy materials. However, use of human gastric mucosa requires pretty fluent collaboration with the gastrointestinal surgery department, especially because the gastric mucosa has to be in good and healthy condition, which excludes the use of most cases of dysplastic and cancer tissue obtained from eradication surgery. Nevertheless, representative biopsy material can sometimes be obtained from patients undergoing vertical banded gastroplasty operation (i.e., surgical removal of stomach tissue because of morbid obesity).
The in vitro tissue binding assay is valuable for initial characterization of microbial adherence to specific host cell lineages and receptors that the microbe exploits, and of variant strains, whether generated in the laboratory or recovered during the course of natural or experimental infection.