This study investigates the influence of exogenous antigenic stimulation on the serum immunoglobulin levels and the levels of circulating natural antibodies against carbohydrate antigens. Thus, BALB/c mice, raised in a germ-free environment and fed a chemically defined, ultrafiltered diet (GF-CD), were employed. These mice had normal serum IgM levels, but IgG and IgA levels were approximately 5% of conventionally reared littermates. The concentrations of all four IgG isotypes were equally low. The variable part of the heavy chains of naturally occurring BALB/c antibodies against a number of carbohydrate antigens, including 3-fucosyllactosamine (3-FL), levan and dextran, are encoded by VH441, and these antibodies express cross-reactive idiotopes recognized by the monoclonal antibodies 6C4 and 6B1. Antibodies against levan and dextran were lower in GF-CD than in conventional mice, but levels of anti-3FL antibodies, and 6C4 and 6B1 idiotopes, were comparable to those in conventional animals. Peptidoglycan polysaccharide complexes (PPC) are carbohydrate antigens of bacterial origin, like levan and galactan. Naturally occurring antibodies against PPC were found in the serum of conventional mice, but were severely reduced in GF-CD mice. The results indicate that most naturally occurring antibodies against carbohydrate antigens of bacterial origin found in conventional mice are caused by exogenous stimulation.

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We studied the presence of bacterial antigens in rat tissues.We produced a monoclonal antibody (MAb 2E9) directed against intestinat flora-derived peptidoglycan-polpaharide complexes from human and rat feces. With several immunological techniques, the specificity of 2E9 for this bacterial product was demonstrated. Using 2E9 in an immunohistological assay, we were able to show the presence of bacterial products in macrophages in the red pulp of spleens of conventional Lewis rats. However, we found no correlation between the development of the intestinal flora and positive spleen staining with MAb 2E9. The results were confirmed

Interestingly, using a monoclonal antibody, peptidoglycan-polysaccharide complexes (PPC) were detected intracellularly in the mucosa and submucosa of the bowel wall of Crohn's disease (CD) patients. PPC are the main constituents of the gram-positive bacterial cell wall. These PPC were however detected in the normal bowel wall also. Therefore, in this study the hypothesis that an enhanced immune responsiveness to bacterial antigens plays a pivotal role in the induction or the chronicity of CD was tested. As antigens, the peptidoglycan structures of intestinal bacteria (Eubacterium aerofaciens or fecal PPC) or of Streptococcus pyogenes, the 65-kDa heat shock protein and muramyl dipeptide (MDP), the smallest bioactive subunit of peptidoglycan, were used. The proliferative responses of peripheral blood (PB) mononuclear cells (MNC) of healthy subjects and patients in a remissive stage of CD or an active CD stage were examined. Of this last patient group the MNC responses of the mesenterial lymph nodes that drain the inflamed gut area were measured also. The responses of PB-MNC of the healthy subjects and the patients in a remissive CD stage were not different. Compared to the responses in remissive CD, the PB-MNC responses in active CD to the eubacterial cell wall and streptococcal cell wall antigen were significantly higher. At the inflammation site in active CD, the lymph nodes, the responses to most of the bacterial antigens were significantly higher than in the PB. In summary, the results show the presence of bacterial peptidoglycan in the bowel wall and the immune responsiveness, especially at the inflammation site, to these antigens in active CD and therefore present suggestive evidence for the role of peptidoglycan in the etiology and/or pathogenesis of CD.

Soluble peptidoglycan-polysaccharide complexes (PPC) isolated from feces of a healthy subject shared antigens with 22 out of 40 anaerobic intestinal bacteria and, therefore, these could be considered as flora derived. IgG, IgA and IgM antibodies against PPC were found in sera from healthy subjects, patients with Crohn’s disease (CD) and other patients. In sera of CD patients with agglutinating antibodies to 4 anaerobic intestinal bacteria (used as an aid for diagnosis of CD) IgG and IgA levels to PPC were higher than in CD patients without these antibodies.