Human milk oligosaccharides (HMOs) are the third most abundant component of breast milk. Our laboratory has previously revealed gene clusters specifically linked to HMO metabolism in selected bifidobacteria isolated from fecal samples of infants. Our objective was to test the hypothesis that growth of selected bifidobacteria on HMO stimulates the intestinal epithelium.

Caco-2 and HT-29 cells were incubated with lactose (LAC)- or HMO-grown Bifidobacterium longum subsp infantis (B infantis) or B bifidum. Bacterial adhesion and translocation were measured by real-time quantitative polymerase chain reaction. Expression of pro- and anti-inflammatory cytokines and tight junction proteins was analyzed by real-time reverse transcriptase. Distribution of tight junction proteins was measured using immunofluorescent microscopy.

We showed that HMO-grown B infantis had a significantly higher rate of adhesion to HT-29 cells compared with B bifidum. B infantis also induced expression of a cell membrane glycoprotein, P-selectin glycoprotein ligand-1. Both B infantis and B bifidum grown on HMO caused less occludin relocalization and higher expression of anti-inflammatory cytokine, interleukin-10 compared with LAC-grown bacteria in Caco-2 cells. B bifidum grown on HMO showed higher expression of junctional adhesion molecule and occludin in Caco-2 cells and HT-29 cells. There were no significant differences between LAC or HMO treatments in bacterial translocation.

The study provides evidence for the specific relation between HMO-grown bifidobacteria and intestinal epithelial cells. To our knowledge, this is the first study describing HMO-induced changes in the bifidobacteria-intestinal cells interaction.

α1-Antitrypsin (AAT) is the archetype member of the serine protease inhibitor (SERPIN) supergene family. The AAT deficiency is most often associated with the Z mutation, which results in abnormal Z AAT folding in the endoplasmic reticulum of hepatocytes during biogenesis. This causes intra-cellular retention of the AAT protein rather than efficient secretion with consequent deficiency of circulating AAT. The reduced serum levels of AAT contribute to the development of chronic obstructive pulmonary disease (COPD) and the accumulation of abnormally folded AAT protein increases risk for liver diseases. In this review we show that with the discovery of AAT deficiency in the early 60s as a genetically determined predisposition to the development of early-onset emphysema, intensive investigations of enzymatic mechanisms that produce lung destruction in COPD were pursued. To date, the role of AAT in other than lung and liver diseases has not been extensively examined. Current findings provide new evidence that, in addition to protease inhibition, AAT expresses anti-inflammatory, immunomodulatory and antimicrobial properties, and highlight the importance of this protein in health and diseases. In this review co-occurrence of several diseases with AAT deficiency is discussed.

Insulin-like growth factor II (IGF-II) is expressed commonly in colorectal tumors. IGF-binding protein-4 (IGFBP-4) counteracts the tumor promoting activities of IGF-II by binding this growth factor. We have shown previously that in LS1034 cells, which highly express IGF-II, overexpression of IGFBP-4 led to a strong reduction in proliferation, colony formation and invasive capacity. To investigate the effects of IGFBP-4 at the molecular level we analyzed growth parameters of LS1034 human colon cancer cells vs. cells expressing the murine IGFBP-4 (mIGFBP-4) and used a subtractive cDNA library approach in combination with cDNA array hybridization to detect changes in the mRNA expression profiles. The mRNA levels for several proteins that are known to affect important biological properties of neoplastic cells, such as proteolysis, proliferation and differentiation were altered by overexpression of IGFBP-4. Transcript levels for tumor markers, like the carcinoembryonic antigen-related cell adhesion molecule (CEACAM), were reduced by elevated mIGFBP-4. Changes at the mRNA level were confirmed by Western blotting for CST1 (proteolysis or protease inhibitor), COX-2 (cell motility) and CEACAM5 (tumor marker). Furthermore, the effect of mIGFBP-4 on apoptosis was investigated and no increase of apoptosis could be detected in the IGFBP-4 overexpressing LS1034 cells. Our data indicate that IGFBP-4 is involved in the regulation of gene products that are known or supposed to be important for the pathogenesis of colon cancer cells.

Epidemiological evidence suggests that a high intake of resistant starch and NSP protects against colo-rectal cancer. The mechanisms underlying this protection are thought to be mediated by the short-chain fatty acid butyrate, which is present in the colonic lumen in millimolar concentrations as a result of bacterial fermentation of carbohydrates that have resisted digestion in the small intestine. In vitro studies have shown that butyrate displays a host of chemo-preventative properties including increased apoptosis, reduced proliferation, down regulation of angiogenesis, enhanced immunosurveillance and anti-inflammatory effects in colo-rectal cancer cell lines. However, the molecular mechanisms underlying the apparent chemo-preventative actions of butyrate are largely unknown. The evidence supporting the role of butyrate as an anti-cancer agent is reviewed, with particular emphasis on those studies that have attempted to elucidate the mechanism of action of butyrate. Our understanding of the mechanistic action of butyrate and its role in cancer prevention is likely to advance considerably in this post-genomic era with the application of genomic and proteomic technologies. Studies are described that have used gene array and proteomic techniques to investigate the response of colo-rectal cancer cells to butyrate. These pioneering studies illustrate the potential of these technologies to help characterise the molecular responses of the cancer cell to butyrate, and to define the role of butyrate (and other nutrients) in the prevention of colo-rectal cancer.

alpha1-Proteinase inhibitor (alpha1-PI) is the main serine proteinase inhibitor in human plasma. Apart from its synthesis in the liver, this anti-inflammatory protein is also synthesized by and excreted from human intestinal epithelial cells. Antiinflammatory actions of alpha1-PI are thought to be of relevance in the pathogenesis of inflammatory bowel disease. To investigate the role of macrophage-derived cytokines on alpha1-PI secretion from intestinal epithelial cells, we cultured Caco-2 cells until differentiation (14 days in culture) on permeable filter supports. Monolayers of differentiated Caco-2 cells were then co-cultured with human peritoneal macrophages, grown on plastic in the basolateral chamber. Under these conditions, alpha1-PI secretion from Caco-2 cells was enhanced by 45%, probably by a direct action of macrophage-derived cytokines on Caco-2 cells. To extend this observation further, we treated differentiated Caco-2 cells with macrophage-derived proinflammatory cytokines (IL-1beta, IL-8, TNF-alpha), as well as with lymphocyte-derived cytokines IL-2, IL-6 and IFN-gamma. As early as after 24h treatment, IL-2 and IL-8 induced a significant and dose-dependent increase of alpha-1-PI secretion into cell culture medium; this effect was completely reversed after immunoneutralization by the antibodies against IL-2 and IL-8 alpha1-PI secretion was only slightly decreased after treatment with IFN-gamma, while IL-1beta, IL-6 and TNF-alpha had no effect. alpha1-PI secretion correlated well with the expression of this protein in differentiated Caco-2 cells after cytokine treatment, as confirmed by Western blot. Our data imply that, in vitro, alpha1-PI secretion in enterocyte-like Caco-2 cells is up-regulated by IL-2 and IL-8. Our results suggest that both lymphocyte- and macrophage-derived cytokines regulate secretion of the anti-inflammatory protein alpha1-PI in intestinal epithelial cells.