Probiotics or biotherapeutic agents can influence physiology through direct or indirect effects occurring in the gastrointestinal tract. Knowledge on their pharmacokinetics is needed 1) to answer the questions how much probiotic should be consumed?, how often?, how long?; 2) to correlate the effects with the concentration of the probiotic at the target site; 3) to validate hypotheses such as “a probiotic should be of human origin, … have a high survival capacity, … adhere to the intestinal epithelium” …; 4) to anticipate the effects of other probiotics; 5) to establish what concentrations should be present in the commercial preparations; 6) for safety. Some in vitro models have been developed to predict the survival of probiotics in vivo or their adherence to the intestinal epithelium, however, the best way to establish the pharmacokinetics of a probiotic is to measure it in vivo. Three techniques can be used: collection of feces, pixigraphy, and intestinal intubation. The use of transit markers such as spores of Bacillus stearothermophilus is necessary to study the potential for probiotics to colonize (i.e. to persist for longer period than the inert marker). Knowledge on the pharmacokinetics of probiotics is reviewed. Some strains can adhere to cell lines such as CaCo₂ or HT29. The survival of ingested probiotics differs greatly between genus and strains. Some strains are rapidly destroyed in the stomach while others such as some Bifidobacterium sp. or Lactobacillus sp. survive till feces.

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To avoid the problems involved in culturing, the intestinal microflora has been studied using digital image analysed microscopy. Several such applications have been developed in Groningen, for morphometry, fluorometry, and combined use of both. The response of the intestinal flora to antibiotics and probiotics could be determined readily with these methods, along with measurements of the systemic immune response to the intestinal microflora. More recently 16S-rRNA targeted fluorescence in situ hybridization (FISH) has been used in combination with image analysis, to allow identification of individual cells. Assessment of physiological activity of bacteria is now also possible through mRNA targeted FISH.

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The role of PrP^c (cellular isoform of prion protein) will control the survival or death of neuronal cells in vivo. Since many neurons that are activated for a long time survive, they may undergo cell death in vivo. As this is just a possibility, further and more detailed examinations are necessary.

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The development of a prophylactic and therapeutic vaccine against human herpesvirus infection represents the best hope for controlling the continuing and devastating worldwide herpes endemic. A successful preventative herpesvirus vaccine need to induce both systemic and mucosal protective immunity. We previously described the possibility of oral immunization with live HSV-1 as a vaccine and presented evidence suggesting that the best immunization method for preventing this viral infection is the oral route rather than other routes such as cutaneous or intravenous ones. Oral administration induces the and-HSV-1 antibody on the mucosal surfaces where most viruses enter the body and provides systemic immunity. Further work is required to establish optimal immunization conditions and to explore the use of safer immunogens. We outline here the future plans for developing the oral engineering of recombinant live vectors such as vaccinia expressing the combination of two or more epitopes that induce excellent cellular and humoral immunity against acute and latent herpesvirus infection.

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A modified continuous flow system was used to test the regulative effect of Enterococcus faecium SF68 on single strain cultures of Lactobacillus reuteri and Lactobacillus acidophilus representative of the autochthonous intestinal microflora. Experiments were conducted in duplicate using serially arranged fermentation cells. The second (A) and third (B) cells were filled with agar blocks containing an immobilized culture of either L. reuteri or L. acidophilus. The data obtained confirmed observations from previous clinical tests on the influence of E. faecium SF68 in the treatment of intestinal disorders. In our in vitro investigations with the continuous flow system E. faecium SF68 accelerated the restoration of the L. reuteri and L. acidophilus populations when applied after antibiotic treatment. The application of E. faecium SF68 together with the antibiotic had a protective effect on the L. reuteri and L. acidophilus populations, as cell numbers decreased at a significantly lower rate compared to the control unit.

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Klebsiella pneumoniae IFO 13541 is a bacterium which produces vitamin B₁₂. The effect of vitamin B₁₂ on the growth of bifidobacteria was investigated. The addition of vitamin B₁₂ and /or PQQ to the culture medium showed a significant stimulatory effect on the growth of bifidobacteria. The interactions between K. pneumoniae IFO 13541 and Bifidobacterium bifidum A234-4 were investigated in a mixed culture. Under conditions in which both bacteria grew well in pure cultures, K. pneumoniae promoted the growth of B. bifidum.

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The implication of an abnormal spectrum of intestinal microflora in the pathogenesis of diseases with immune disorders was examined in patients with sternocostoclavicular hyperostosis, rheumatoid arthritis and atopic dermatitis. In these patients, frequent episodes of severe constipation or diarrhea were observed before the onset of the diseases. Serum biotin levels in the patients were low and remained almost unchanged even after oral administration of biotin. However, supplementary administration of a probiotic agent with biotin significantly increased serum biotin levels and maintained the vitamin levels high enough to improve clinical manifestations. Biotin is mainly synthesized by members of the microflora in the intestine and absorbed from the intestine into the circulation. Therefore, low serum biotin levels may be attributable to the proliferation of‘Harmful’intestinal microflora to degrade or ingest biotin. The results presented here suggest that biotin-limited conditions produced by ‘Harmful’ intestinal microflora in the patients with immune disorders play a causal role in the pathogenesis of the diseases. Biotin administration with a probiotic agent may provide a therapeutic effect on the diseases.

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More than 90% of 75 clinical isolates of Helicobacter pylori strains had the vacuolating cytotoxin gene vacA as demonstrated by polymerase chain reaction (PCR). A PCR restriction fragment-length polymorphism (RFLP) analysis revealed various gene subtypes, and some of the RFLPs correlated significantly with gastric cancer or duodenal ulcer. These findings suggest that specific types of H. pylori have factors that cause gastric cancer or duodenal ulcer.

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