Consumption of synbiotic twice daily over four weeks significantly reduced mucosal inflammatory markers in active UC. This was concurrent with a reduction in colitis at the macroscopic and microscopic level. However, although lowered SS were observed in the synbiotic group, they were not as marked as the reductions in HS and inflammatory markers. This may indicate that changes in inflammatory mediators at the molecular level could precede gross clinical changes scored through sigmoidoscopy by several weeks. This contention is supported by the relationship seen in HS and not SS after synbiotic feeding, with both hBD2 and 4 showing that although local inflammation was resolving in these patients, more general changes in colonic appearance, such as vessel pattern, take longer to return to normal. Alternatively, this therapy may be resolving the local inflammation through direct contact with the epithelium but is insufficient in all cases of UC (three of eight patients receiving synbiotic therapy had no improvement in SS) in reducing the systemic chronic inflammatory condition. It is possible that these patients may improve their clinical scores if this treatment is given in conjunction with other therapies or through selection of a different probiotic organism. Unfortunately, no significant difference in SS or CAI between the synbiotic and placebo groups was found. This may be due to the small number of placebos who completed the study. Two placebos requested withdrawal because of exacerbation of symptoms, and refused to undergo sigmoidoscopy or provide a final biopsy due to discomfort. There were no withdrawals from the synbiotic group after commencement of the study and no reports of adverse reactions.
Markers of active disease in biopsies from UC patients include proinflammatory cytokines such as TNF-α, IL-8, IL-1α, and IL-1β.29
These molecules are upregulated in active UC but they are not specific for the epithelium as the large numbers of infiltrating leucocytes in the mucosa contribute greatly to their formation.28–30
When using a probiotic or synbiotic, it is important to be able to assess directly its effects on the epithelial barrier as well as the underlying immune system as the epithelium is the first point of host contact for the organisms. Human beta defensins are good indicators of mucosal immunity because they are uniquely expressed by epithelial cells.31,32
Two types of expression occur: hBD1 is a non-inducible constitutively expressed molecule while hBD2–4 are upregulated by bacterial challenge and proinflammatory cytokines.33,34,41
These molecules are produced by the inflammatory infiltrate and by the epithelium itself30
directly affecting expression levels of inducible hBD in epithelial cells. IL-1 has been shown to induce upregulation of hBD2 in gastric and colonic epithelial cell lines.34,41
In this study, a direct relation between mRNA synthesis for inducible hBD and severity of UC (SS) has been shown for the first time, together with the relationship between inducible hBD and histology scores in UC patients. We were unable to demonstrate a significant difference between the inducible hBD in the post-synbiotic group versus the post-placebo group but the proinflammatory cytokines TNF-α and IL-1α were significantly reduced in the synbiotic group compared with the placebo group. This can be explained by the relationship between SS and levels of inducible hBD. As our placebo group had lower clinical scores than the synbiotic group, resulting from the small sample size and random assignment to each group, we have inadvertently biased against finding a significant difference in inducible hBD between the two groups due to the direct linear relationship between SS and hBD. This is not true for the inflammatory cytokines that have a more all or nothing expression pattern and can be produced by any infiltrating cell in the biopsy. Furthermore, they are compared against the usual housekeeping genes (GAPD/β-actin) for biopsy size standardisation, unlike hBD which are only produced by colonic epithelial cells and epithelial cell numbers can be accurately standardised by hBD1 levels.
The functions of these induced hBDs are unknown in UC disease progression. However, hBD are antimicrobial peptides that are particularly good at destroying Gram negative bacteria33
so their production may be an attempt by the epithelium to modify the composition of the mucosal biofilm. The bifidobacterial probiotic strain used in this study was found to be insensitive to hBD mediated killing by recombinant hBD1, 2, and 3 (results not shown), and therefore, as part of a therapy for UC this organism would exhibit better survival characteristics on the inflamed mucosa and interact directly with the epithelial surface and modify the immune response. We detected higher numbers of total bifidobacteria on the mucosal surface in patients fed the synbiotic compared with those taking placebo. We cannot separately identify the probiotic organism from the indigenous bifidobacteria that may have been stimulated by inclusion of a prebiotic in the feeding regimen. Bifidobacterium longum
is a very complicated organism with at least five different subgroups contained within the species when 16S rDNA sequences are compared (data not shown). With the present technology it is impossible to distinguish the probiotic strain from other Bifidobacterium longum
in a small pinch biopsy. It is unknown what direct effects probiotic bacteria have on the inflamed epithelium. They could simply be out competing microorganisms involved in disease aetiology for microniches on the gut wall, thereby removing inflammatory stimuli. However, they may also be directly influencing the expression of inflammatory cytokines by the epithelium through ligation of particular pattern recognition receptors on the luminal cell surface, as supported by studies using HT29 cells in preliminary screening experiments with the probiotic. Alternatively, the probiotic could be directly influencing dendritic cells that are present in high numbers in the lamina propria42
with access to the epithelial surface,43
by downregulating proinflammatory cytokine pathways and stimulating a more immunomodulatory and tolerant immune response.
In conclusion, this short term pilot study has provided the first evidence that synbiotics have the potential to be developed into acceptable therapies for patients suffering from acute UC. A large scale clinical trial is now needed to investigate the long term effects of synbiotic use in inducing and maintaining remission in patients with active disease.