Peptidoglycan in atherosclerotic plaque formation and vulnerability

The studies described in this thesis suggest that peptidoglycan (PG) is involved in initiation and progression of atherosclerosis. PG is an antigen that can be found in large amounts in the Gram-positive bacterial wall and only in small amounts in the Gram-negative bacterial wall. PG is able to stimulate the Toll-like receptor 2 (TLR2), a receptor that plays an important role in the innate immune system. In the normal situation PG is present in the intestinal mucosal flora, but under certain pathological circumstances PG is able to promote chronic inflammation at non-mucosal sites in the body. IgM, IgG and IgA antibodies against PG are present in serum of healthy subjects. However, in atherosclerotic patients the IgM antibody levels against PG are lower compared to control subjects. Furthermore, IgM antibody levels against PG are negatively associated with intima-media thickness in coronary arteries and therefore with more pronounced human atherosclerotic disease. We found these associations in a cross-sectional study, but these IgM antibody levels against PG are not prognostic markers for development of adverse cardiovascular events after elective coronary angioplasty. We also showed that adventitial fibroblasts and human coronary arteries express TLR2 and that TLR2 stimulation of adventitial fibroblasts increases expression of pro-inflammatory cytokines and chemokines, like IL-1β, IL-6, IL-8 and MCP-1. In addition, in wild-type mice Pam3Cys-SK4 stimulation promotes intimal lesion formation, which is clearly TLR2-mediated since TLR2-/- mice show no significant increase in intimal hyperplasia. In ApoE-/- mice TLR2 stimulation increases development of atherosclerotic plaques. In addition to recognition by TLR2, degradation products of PG can also be recognized by intracellular NOD and Nalp proteins. We found that NOD1 and NOD2 protein expression is significantly increased in human atherosclerotic lesions and in those lesions NOD2 protein is present in macrophages within inflammatory areas and in endothelial cells lining the vessel wall. NOD1 protein expression is associated with the presence of smooth muscle cells (SMC) and collagen, but also with macrophages when SMC are absent. Furthermore, both NOD1 and NOD2 protein levels are inversely associated with matrix metalloproteinase (MMP)-2 levels and positively with MMP-8 and MMP-9 levels. Previous studies from our group showed that PG is present in human atherosclerotic plaques that reveal an inflammatory, vulnerable phenotype and that PG is present in the macrophage-rich regions of those plaques. In this thesis we extend this finding by showing that peripheral blood monocytes that are stimulated with PG increase expression and functionality of integrins on their surface, thereby promoting firm adhesion of the monocytes to endothelial cells and migratory properties. Furthermore, peritoneal macrophages stimulated with PG in vitro show increased homing into atherosclerotic plaques in vivo. These studies not only suggest that PG promotes atherosclerotic lesion development and progression; they also show that PG enhances accumulation of monocytes and/or macrophages at sites of inflammation thereby promoting the formation of vulnerable plaques. These vulnerable plaques are prone to rupture and thereby promote the formation of a thrombus. Such a thrombus eventually might cause a myocardial infarction or stroke

Synbiotics-supplemented amino acid-based formula supports adequate growth in cow's milk allergic infants

<p>Background: Children with cow's milk allergy (CMA) are at risk for inadequate nutritional intake and growth. Dietary management of CMA, therefore, requires diets that are not only hypoallergenic but also support adequate growth in this population. This study assessed growth of CMA infants when using a new amino acid-based formula (AAF) with prebiotics and probiotics (synbiotics) and evaluated its safety in the intended population. Methods: In a prospective, randomized, double-blind controlled study, full-term infants with diagnosed CMA received either an AAF (control; n = 56) or AAF with synbiotics (oligofructose, long-chain inulin, acidic oligosaccharides, Bifidobacterium breve M-16V) (test; n = 54) for 16 wk. Primary outcome was growth, measured as weight, length and head circumference. Secondary outcomes included allergic symptoms and stool characteristics. Results: Average age (±SD) of infants at inclusion was 4.5 ± 2.4 months. Both formulas equally supported growth according to WHO 2006 growth charts and resulted in similar increases of weight, length and head circumference. At week 16, differences (90% CI) in Z-scores (test-control) were as follows: weight 0.147 (-0.10; 0.39, p = 0.32), length -0.299 (-0.69; 0.09, p = 0.21) and head circumference 0.152 (-0.15; 0.45, p = 0.40). Weight-for-age and length-for-age Z-scores were not significantly different between the test and control groups. Both formulas were well tolerated and reduced allergic symptoms; the number of adverse events was not different between the groups. Conclusions: This is the first study that shows that an AAF with a specific synbiotic blend, suitable for CMA infants, supports normal growth and growth similar to the AAF without synbiotics. This clinical trial is registered as NCT00664768.</p