La présente étude vise à étudier le potentiel de bifidobactéries à protéger les cellules contre l’infection par le Coxsackie B4 (CV-B4). Le criblage de bifidobactéries a identifié deux des cinq souches qui protégeaient les cellules HEp-2 lorsque les bifidobactéries sont pré-incubées avec les particules virales avant l'inoculation sur les cellules Hep-2. En revanche, aucun effet protecteur n'a été observé en incubant les cellules Hep-2 avec des bifidobactéries avant l'inoculation de CV-B4. Les lipoprotéines des parois cellulaires (LpAs) sécrétées par les souches sélectionnées sont testées pour leur activité antivirale. Les deux LpAs présentaient une activité antivirale quand ils sont incubés avec les particules virales avant d’être inoculées aux cellules HEp-2. Aucun effet protecteur n'a été induit par incubation des LpAs avec les cellules HEp-2 avant l'inoculation de CV-B4. La protéine recombinante présente une activité antivirale identique. Pour identifier les séquences peptidiques interagissant avec les particules virales, les protéines de LpAs sont alignées avec les séquences peptidiques du nord bord du canyon et avec l’empreinte de la région puff sur le Coxsackievirus et sur le récepteur de l’adénovirus (CAR). L'étude d'amarrage moléculaire in silico (Docking) utilisant le CV-B3 en tant que modèle a montré une faible énergie de liaison indiquant un système stable pour les peptides sélectionnés et par conséquent une interaction probable avec le CV-B. Les peptides de B.longum et de B.breve qui sont homologues à l’empreinte du rebord nord viral sur la séquence CAR, forment des liaisons d’hydrogène avec plusieurs résidus viraux dans la région du rebord nord du canyon, qui sont déjà décrits pour leur interaction avec le CAR.En conclusion, les protéines de LPAS bifidobactéries peuvent inhiber l'infection par le CV-B4 probablement par liaison aux acides aminés de la capside qui interagissent avec le CAR. / The present study aims at investigating the potential of bifidobacteria in protecting cells from Coxsackievirus B4 (CV-B4) infection. The bifidobacterial screening identified two out of five strains that protected HEp-2 cell viability when bifidobacteria were incubated with the viral particles prior inoculation. In contrast, no effect was shown by incubating HEp-2 cells with bifidobacteria prior CV-B4 inoculation. Cell-wall lipoproteins secreted by the selected strains (LpAs) were assayed for their anti-viral activity. The two LpAs exhibited anti-viral activity when they were incubated with the viral particles prior inoculation to HEp-2 cells. No effect was induced by incubating LpAs with HEp-2 cells prior CV-B4 inoculation. The recombinant LpAs derived-protein exhibited identical anti-viral activity. To identify the peptide sequences interacting with the virus particles, LpAs proteins were aligned with the peptide sequences of north canyon rim and puff footprint onto coxsackievirus and adenovirus receptor (CAR). The in silico molecular docking study using CV-B3 as template showed a low energy binding indicating a stable system for the selected peptides and consequently a likely binding interaction with CV-B. B.longum and B.breve peptides homologous to the viral north rim footprint onto CAR sequence formed hydrogen bonds with several viral residues in the north rim of the canyon, which were already predicted as interacting with CAR. In conclusion, proteins from bifidobacterial LpAs can inhibit the infection with CV-B4 likely through binding to the capsid aminoacids that interact with CAR.

Background: Lipoprotein(a) [Lp(a)] is composed of a low density-lipoprotein (LDL) particle and a glycoprotein molecule known as apolipoprotein(a) [apo(a)]. Apo(a) exists in several differently-sized isoforms and is responsible for the unique properties of Lp(a). Although Lp(a) has been known for the past 40 years its relationship with coronary heart disease (CHD) has not been characterized in sufficient detail. Whether Lp(a) causes CHD is not clear. Furthermore, the role of apo(a) isoform variation and other sources of Lp(a) heterogeneity (e.g., level of oxidized phospholipids) in Lp(a)-disease association has not been determined. Objectives: To characterize in detail the association of circulating Lp(a) levels with the risk CHD To assess the nature of Lp(a)-CHD association using an integrative genetic study To explore the role of Lp(a) heterogeneity in its association with CHD Data sources: 1. The Emerging Risk Factors Collaboration (ERFC) database (36 studies, 127,000 participants) 2. The European Prospective Investigation of Cancer – Norfolk (EPIC-Norfolk) study (2200CHD cases, 2200 controls) 3. The Pakistani Risk of Myocardial Infarction Study (PROMIS) (1800 MI cases and 1800 controls) 4. Systematic quantitative reviews of published epidemiological studies Results: ERFC data - Analyses of cross-sectional data on up to 127,000 participants (predominantly of European descent) demonstrated that Lp(a) is generally not strongly correlated with known CHD risk factors. Weakly positive correlations were observed with LDL-cholesterol, apolipoprotein B100 and fibrinogen. Levels were over 2-fold higher in Blacks compared to Whites. Analyses of available data on repeat measurements in 6600 participants demonstrated that Lp(a) values have very high long-term within-person consistency (regression dilution ratio ~ 0.9). Outcome data involved 9300 incident CHD events, 1900 ischaemic strokes and 8100 nonvascular deaths. The risk ratio for CHD per 1SD higher Lp(a) concentration, adjusted for age, sex, lipids and other conventional vascular risk factors, was 1.13 (95% CI, 1.09-1.18). The corresponding risk ratios for ischaemic stroke and nonvascular death were 1.10 (1.02 – 1.18) and 1.01 (0.98-1.05), respectively. Data were too limited to assess association in nonwhites. PROMIS data – the adjusted odds ratio for MI in South Asians was comparable to that of Europeans. EPIC-Norfolk genetic data - The odds ratio for CHD per 1-SD higher Lp(a) concentration, after adjustment for cardiovascular risk factors, was 1.37 (1.20-1.56). Tagging SNPs rs10455872 and rs11751605 (minor allele frequency: 8% and 18%, respectively) were associated with 207% (95% CI, 188 - 227%) and 38% (31 - 46%) higher Lp(a) concentrations per copy of minor allele, respectively. These SNPs accounted for 35% and 5% of the variation in circulating Lp(a) levels, respectively, and were associated with an odds ratio for CHD of 1.34 (1.14-1.58) and 1.17 (1.04-1.33), respectively. The observed SNP-CHD associations were consistent with expected odds ratios corresponding to the Lp(a) effect of the SNPs. Systematic reviews – meta-analysis of published data from 40 studies (11,300 cases, 47,000 controls) demonstrated that people with smaller apo(a) isoforms have about a 2-fold higher risk of CHD or ischemic stroke than those with larger isoforms. Meta-analysis of published data from 10 studies (1500 cases, 10,200 controls) showed that people in the top third of baseline distribution of oxidized LDL levels have a 1.8-fold higher risk of CHD than those in bottom third. EPIC-Norfolk biomarker data – Levels of oxidized phospholipids were strongly correlated with Lp(a) concentration (r = 0.7, p-value < 0.0001). One SD higher concentration of oxidized phospholipids was associated with an adjusted odds ratio for CHD of 1.31 (1.15-1.49). The risk ratio was no longer significant after adjustment for Lp(a) concentration (1.08; 95% CI, 0.91-1.29). Conclusion: Lp(a) concentration is specifically, continuously and independently associated with the risk of ischaemic vascular outcomes. Available evidence supports the causal role of the particle in CHD. Lp(a) appears to induce vascular damage through causal mechanisms that involve apo(a) isoforms and oxidized phospholipids. A comprehensive study of markers of Lp(a) heterogeneity should help to understand the full impact of Lp(a) on cardiovascular diseases. In addition, further study is needed in nonwhites to assess the relevance of the factor to vascular disease risk in these populations.

No description available.

The co-localization of plasma C-reactive protein (CRP) and atherogenic low-density lipoprotein (LDL) at the atherosclerotic lesions raises the possibility of a role of CRP in the disease process. In vitro, in its native pentameric structural form, CRP does not bind to oxidized LDL. However, in its non-native pentameric structural form, CRP is capable of binding to oxidized LDL. It has been proposed that CRP changes its structure at sites of inflammation to gain the oxidized LDL-binding activity. In vivo, native CRP is neither pro-atherosclerotic nor atheroprotective in animal models of atherosclerosis. We assumed that native CRP shows no effect because inflammatory microenvironment in the arterial wall in animal models of atherosclerosis is not appropriate and, therefore, the structure of CRP remains unchanged. Accordingly, we hypothesized that a CRP mutant, generated by site-directed mutagenesis, capable of binding to oxidized LDL without the requirement of any further structural change should show an effect on the disease. In the current study, we evaluated the impact of such a CRP mutant on the development of atherosclerosis employing LDL receptor knockout mouse model of atherosclerosis. We found that there was a two-week delay in the development of atherosclerotic lesions in the aortic root of mice treated with mutant CRP compared to mice that did not receive CRP. The development of atherosclerotic lesions in the whole aorta was also reduced; there was 30% reduction in the size of lesions in mice treated with mutant CRP. Overall, the data indicate that CRP is indeed an atheroprotective molecule.

Lipoprotein lipase (LPL) is a key enzyme in lipoprotein metabolism. The enzyme catalyzes hydrolysis of triacylglycerols (TG) of chylomicrons and of very low density lipoproteins (VLDL). However, the mechanisms involved in the regulation of this protein are not fully understood yet. Therefore, the aim of the theses is to study selected aspects of LPL activity regulation. Recently discovered apolipoprotein A-V (apo A-V) substantially affects triglyceridemia and it is presumed that it may function as LPL activator. However, its concentration in the blood is extremely low and we therefore investigated whether most of apo A-V could be bound to the heparan sulfate proteoglycan (HSPG) of vascular wall similarly to LPL. Intravenous heparin application in healthy volunteers resulted in an expected increase in LPL activity but apo A-V concentration did not change. Our results do not support the hypothesis that most of apo A-V is bound to HSPG of the capillary endothelium. An alcohol consumption plays also a role in LPL regulation - the long-term moderate alcohol consumption is known to increase enzyme activity; on the contrary, it is presumed that LPL activity is inhibited immediately after alcohol consumption. However, the direct evidence for such a premise is missing. The other aim of the theses was to...

C-reactive protein (CRP) is a pentameric molecule made up of identical monomers. CRP can be seen in three different forms: native pentameric CRP (native CRP), non-native pentameric CRP (nonnative CRP), and monomeric CRP (mCRP). Both native and nonnative CRP execute ligand-recognition functions for host defense. The fate of any pentameric CRP after binding to a ligand is dissociation into ligand-bound mCRP. If ligand-bound mCRP is proinflammatory, like free mCRP has been shown to be in vitro, then mCRP along with the bound ligand must be cleared from the site of inflammation. Once pentameric CRP is bound to atherogenic low-density lipoprotein (LDL), it reduces both formation of foam cells and proinflammatory effects of atherogenic LDL. A CRP mutant, that is non-native CRP, which readily binds to atherogenic LDL, has been found to be atheroprotective in a murine model of atherosclerosis. Thus, unlike statins, a drug that can lower only cholesterol levels but not CRP levels should be developed. Since non-native CRP has been shown to bind to all kinds of malformed proteins in general, it is possible that non-native CRP would be protective against all inflammatory states in which host proteins become pathogenic. If it is proven through experimentation employing transgenic mice that non-native CRP is beneficial for the host, then using a small-molecule compound to target CRP with the goal of changing the conformation of endogenous native CRP would be preferred over using recombinant non-native CRP as a biologic to treat diseases caused by pathogenic proteins such as oxidized LDL.

The aim of this study was to test the hypothesis that C-reactive protein (CRP) protects against the development of atherosclerosis and that a conformational alteration of wild-type CRP is necessary for CRP to do so. Atherosclerosis is an inflammatory cardiovascular disease and CRP is a plasma protein produced by the liver in inflammatory states. The co-localization of CRP and low-density lipoproteins (LDL) at atherosclerotic lesions suggests a possible role of CRP in atherosclerosis. CRP binds to phosphocholine-containing molecules but does not interact with LDL unless the phosphocholine groups in LDL are exposed. However, CRP can bind to LDL, without the exposure of phosphocholine groups, if the native conformation of CRP is altered. Previously, we reported a CRP mutant, F66A/T76Y/E81A, generated by site-directed mutagenesis, that did not bind to phosphocholine. Unexpectedly, this mutant CRP, without any more conformational alteration, was found to bind to atherogenic LDL. We hypothesized that this CRP mutant, unlike wild-type CRP, could be anti-atherosclerotic and, accordingly, the effects of mutant CRP on atherosclerosis in atherosclerosis-prone LDL receptor-deficient mice were evaluated. Administration of mutant CRP into mice every other day for a few weeks slowed the progression of atherosclerosis. The size of atherosclerotic lesions in the aorta of mice treated with mutant CRP for 9 weeks was ~40% smaller than the lesions in the aorta of untreated mice. Thus, mutant CRP conferred protection against atherosclerosis, providing a proof of concept that a local inflammation-induced structural change in wild-type CRP is a prerequisite for CRP to control the development of atherosclerosis.

The effects of oxidatively modified low density lipoprotein (oxLDL) on atherogenesis may be partly mediated by alterations in the production of nitric oxide (NO) by vascular cells. Lipid hydroperoxides (LOOH) and lysophosphatidylcholine (lysoPC) are the major primary products of LDL oxidation. The purpose of this study was to characterize the effects of oxLDL, LOOH and lysoPC on NO production and the expression of inducible nitric oxide synthase (iNOS) gene in lipopolysaccharide (LPS) stimulated macrophages. LDL was oxidized using an azo-initiator 2,2'-azobis (2- amidinopropane) HCl (ABAP) and octadecadienoic acid was oxidized by lipoxygenase to generate 13-hydroperoxyl octadecadienoic acid (13-HPODE). Our study showed that oxLDL markedly decreased the production of NO, the levels of iNOS protein and iNOS mRNA in LPS stimulated macrophages. The inhibition potential of oxLDL on NO production and iNOS gene expression depended on the levels of LOOH formed in oxLDL and was not due to oxLDL cytotoxicity. Furthermore, 13-HPODE markedly reduced NO production and iNOS protein levels, whereas lysoPC showed only slight reduction. The effects of 13-HPODE and lysoPC did not require an acetylated LDL carrier. Our results suggest that 13-HPODE is a much more potent inhibitor of NO production and iNOS gene expression than lysoPC in LPS stimulated RAW264.7 macrophages.

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Background: C-reactive protein (CRP) is an acute phase plasma protein. An important binding specificity of CRP is for the modified forms of low-density lipoprotein (LDL) in which the phosphocholine-binding sites of CRP participate. CRP, however, does not bind to native LDL. Methods: We investigated the interaction of CRP with native LDL using sucrose density gradient ultracentrifugation. Results: We found that the blocking of the phosphocholine-binding sites of CRP with phosphoethanolamine (PEt) converted CRP into a potent molecule for binding to native LDL. In the presence of PEt, CRP acquired the ability to bind to fluid-phase purified native LDL. Because purified native LDL may undergo subtle modifications, we also used whole human serum as the source of native LDL. In the presence of PEt, CRP bound to native LDL in serum also. The effect of PEt on CRP was selective for LDL because PEt-complexed CRP did not bind to high-density lipoprotein in the serum. Conclusions: The pharmacologic intervention of endogenous CRP by PEt-based compounds, or the use of exogenously prepared CRP-PEt complexes, may turn out to be an effective approach to capture native LDL cholesterol in vivo to prevent the development of atherosclerosis.