Although naturally occurring probiotics may have insufficient efficacy in Crohn's disease, the genetic modification of commensal bacteria for the site-specific delivery of therapeutic molecules represents a realistic pharmabiotic strategy. Proof of principle has already been demonstrated in animal models of enterocolitis. Genetically engineered Lactococcus lactis has been used to deliver anti-inflammatory IL-10 or the cytoprotective trefoil factor locally to the gut (Steidler et al., 2000; Vandenbroucke et al., 2004). The safety issues related to genetic modification have been addressed by replacing the thymidylate synthase (thy A) gene in L. lactis with a synthetic therapeutic transgene. When the modified bacteria are deprived of thymine or thymidine they are not viable. Neither thymine nor thymidine are readily available in the external environment, thereby limiting the viability of the excreted organism. Moreover, the transgene would be eliminated from the bacterial genome if the engineered organisms re-acquire thy A from a wild-type strain (Steidler et al., 2003). Recently, in the first human trial with genetically-engineered therapeutic bacteria, ten Crohn's disease patients were treated with modified L. lactis in which thy A was replaced with a synthetic sequence encoding human IL-10. The treatment was safe, disease activity was reduced, and the modified bacteria were biologically contained (Braat et al., 2006). This encouraging study indicates that bacterial-based topical delivery of biologically active proteins represents a highly promising and safe therapeutic strategy for combating mucosal diseases. Designer probiotics are also being engineered to express molecular mimics of host receptors on their surface, these receptors bind bacterial toxins, thereby preventing enteric infections (Paton et al., 2006). In a different setting, Lactobacillus jensenii, a commensal of the female genital tract, has been engineered to confer protection against HIV infectivity in vitro (Chang et al., 2003). The potential for these designer probiotics is limited only by one's imagination, but public health and other safety concerns must be resolved before routine clinical use in humans.
It is clear that significant differences exist not only between probiotic bacterial species, but also between certain strains. In addition to specific interactions between probiotic bacteria and host immune cells, microbe-microbe interactions also exist. This could explain some of the varying results observed within the different clinical trials. Several unresolved issues impede the clinical evaluation of probiotics (Shanahan, 2003, 2004). These include determination of optimal dose and vehicle of delivery, development of reliable predictors of in vivo survival and performance, regulation and verification of product stability, determination of which combinations of probiotics or other pharmabiotics are synergistic or antagonistic, and strain-strain comparisons of probiotic performance in different indications. Furthermore, the microbial, immunological, and functional characteristics of individual probiotic strains and their mechanisms of action in different clinical settings require clarification.
Individual variability in composition of the enteric microbiota may also be a determining factor for optimal strain selection. Another unresolved issue is whether probiotic administration should be preceded by antibiotic treatment to open the microbial niche as may have been achieved in some of the pouchitis trials. Clearly, rigorously designed, controlled, statistically-powered clinical trials are needed. There is evidence to suggest that many of the commercially available probiotics do not contain the advertised bacterial strain or the claimed concentration (Coeuret et al., 2004). To protect consumers, there is a pressing need for more stringent regulation of unsubstantiated health claims.
Engagement with host immune cells is central to pharmabiotic action. However, therapeutic manipulation of the indigenous microbiota with any form of pharmabiotic remains sub-optimal due to incomplete understanding of the commensal microbiota, their immunoregulatory properties, and host-
microbial interactions. Further studies of physiological interactions within the complex network of host cell-commensal-PRR-ligand signalling in gut health and disease should lead to the optimal exploitation of pharmabiotic approaches to alleviate mucosal inflammation in IBD and possibly other intestinal diseases. Mining the microbiota for metabolites that impact on host physiology is a promising source of new therapeutics. The increasing availability of commensal genomes should facilitate the identification of commensal effector molecules or other components with pharmabiotic potential. The possibility of using these molecules to specifically target distinct points of intracellular signalling cascades might alleviate inflammation in a target area and overcome the global immunosuppressive effects associated with current therapies. In patients with severe IBD, the use of designer probiotics may offer a new strategy for more targeted delivery of anti-inflammatory molecules to the inflamed mucosa.
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