In vitro screening of 51 birdsfoot trefoil (Lotus corniculatus L.; Fabaceae) strains for anti-parasitic effects against Haemonchus contortus.

Secondary plant compounds have shown bioactivity against multi-drug resistant Haemonchus contortus in small ruminants. This study screened 51 strains of birdsfoot trefoil (BFT, Lotus corniculatus) crude aqueous extracts (BFT-AqE) for anti-parasitic activity in vitro against egg hatching, and of those 51 strains, 13 were selected for further testing of motility of first (L1) and third stage (L3) larvae, and exsheathment of L3. Proanthocyanidin content ranged between 1.4 and 63.8 mg PAC g-1 powder across the 51 BFT strains. When tested against egg hatching, 21 of the 51 aqueous extracts had an EC50 of 1-2 mg powder mL-1, 70% of the strains were >90% efficacious at 6 mg powder mL-1 and 11 of the strains were 100% efficacious at 3 mg powder mL-1 BFT-AqE. Across the 13 strains tested against L3, efficacy ranged from 0 to 75% exsheathment inhibition, and 17 to 92% L3 motility inhibition at a concentration of 25 mg powder mL-1 BFT-AqE. There was no correlation between the PAC content of BFT powders and the anti-parasitic activity of aqueous extracts, therefore other secondary compounds may have contributed to the observed anti-parasitic effects. Further testing of BFT using bioactivity-driven fractionation and screening of BFT populations for the identified anti-parasitic compounds is needed.

Co-operative suppression of inflammatory responses in human dendritic cells by plant proanthocyanidins and products from the parasitic nematode Trichuris suis.

Interactions between dendritic cells (DCs) and environmental, dietary and pathogen antigens play a key role in immune homeostasis and regulation of inflammation. Dietary polyphenols such as proanthocyanidins (PAC) may reduce inflammation, and we therefore hypothesized that PAC may suppress lipopolysaccharide (LPS) -induced responses in human DCs and subsequent T helper type 1 (Th1) -type responses in naive T cells. Moreover, we proposed that, because DCs are likely to be exposed to multiple stimuli, the activity of PAC may synergise with other bioactive molecules that have anti-inflammatory activity, e.g. soluble products from the helminth parasite Trichuris suis (TsSP). We show that PAC are endocytosed by monocyte-derived DCs and selectively induce CD86 expression. Subsequently, PAC suppress the LPS-induced secretion of interleukin-6 (IL-6) and IL-12p70, while enhancing secretion of IL-10. Incubation of DCs with PAC did not affect lymphocyte proliferation; however, subsequent interferon-γ production was markedly suppressed, while IL-4 production was unaffected. The activity of PAC was confined to oligomers (degree of polymerization ≥ 4). Co-pulsing DCs with TsSP and PAC synergistically reduced secretion of tumour necrosis factor-α, IL-6 and IL-12p70 while increasing IL-10 secretion. Moreover, both TsSP and PAC alone induced Th2-associated OX40L expression in DCs, and together synergized to up-regulate OX40L. These data suggest that PAC induce an anti-inflammatory phenotype in human DCs that selectively down-regulates Th1 response in naive T cells, and that they also act cooperatively with TsSP. Our results indicate a novel interaction between dietary compounds and parasite products to influence immune function, and may suggest that combinations of PAC and TsSP can have therapeutic potential for inflammatory disorders.

Non-covalent pomegranate (Punica granatum) hydrolyzable tannin-protein complexes modulate antigen uptake, processing and presentation by a T-cell hybridoma line co-cultured with murine peritoneal macrophages.

In this work we characterize the interaction of pomegranate hydrolyzable tannins (HT) with hen egg-white lysozyme (HEL) and determine the effects of non-covalent tannin-protein complexes on macrophage endocytosis, processing and presentation of antigen. We isolated HT from pomegranate and complex to HEL, the resulting non-covalent tannin-protein complex was characterized by gel electrophoresis and MALDI-TOF MS. Finally, cell culture studies and confocal microscopy imaging were conducted on the non-covalent pomegranate HT-HEL protein complexes to evaluate its effect on macrophage antigen uptake, processing and presentation to T-cell hybridomas. Our results indicate that non-covalent pomegranate HT-HEL protein complexes modulate uptake, processing and antigen presentation by mouse peritoneal macrophages. After 4 h of pre-incubation, only trace amounts of IL-2 were detected in the co-cultures treated with HEL alone, whereas a non-covalent pomegranate HT-HEL complex had already reached maximum IL-2 expression. Pomegranate HT may increase rate of endocytose of HEL and subsequent expression of IL-2 by the T-cell hybridomas.

Measurements of wall shear stress and aortic pulse wave velocity in swine with familial hypercholesterolemia.

PURPOSE: To assess measurements of pulse wave velocity (PWV) and wall shear stress (WSS) in a swine model of atherosclerosis. MATERIALS AND METHODS: Nine familial hypercholesterolemic (FH) swine with angioplasty balloon catheter-induced atherosclerotic lesions to the abdominal aorta (injured group) and 10 uninjured FH swine were evaluated with a 4D phase contrast (PC) magnetic resonance imaging (MRI) acquisition, as well as with radial and Cartesian 2D PC acquisitions, on a 3T MR scanner. PWV values were computed from the 2D and 4D PC techniques, compared between the injured and uninjured swine, and validated against reference standard pressure probe-based PWV measurements. WSS values were also computed from the 4D PC MRI technique and compared between injured and uninjured groups. RESULTS: PWV values were significantly greater in the injured than in the uninjured groups with the 4D PC MRI technique (P = 0.03) and pressure probes (P = 0.02). No significant differences were found in PWV between groups using the 2D PC techniques (P = 0.75-0.83). No significant differences were found for WSS values between the injured and uninjured groups. CONCLUSION: The 4D PC MRI technique provides a promising means of evaluating PWV and WSS in a swine model of atherosclerosis, providing a potential platform for developing the technique for the early detection of atherosclerosis.

Improved in vitro Hemagglutination Assays Utilizing P-Type and Type 1 Uropathogenic Escherichia coli to Evaluate Bacterial Anti-Adhesion Activity of Cranberry Products.

Cranberries have a long history of use in the prevention of urinary tract infections. Cranberry products vary in proanthocyanidin content, a compound implicated in preventing the adhesion of uropathogenic Escherichia coli (E. coli) to uroepithelial cells. Testing is routinely done by cranberry product formulators to evaluate in vitro bacterial anti-adhesion bioactivity, shelf-life, and potential efficacy of cranberry products for consumer use to maintain urinary tract health. Hemagglutination assays evaluate the anti-adhesion bioactivity of cranberry products by determining how effectively the products prevent agglutination of specific red blood cells with E. coli expressing P-type and Type 1 fimbriae. The current study sought to improve upon an established anti-adhesion assay method by expanding the number of E. coli strains used to broaden potential in vivo efficacy implications and presenting results using photomicrographic data to improve accuracy and build databases on products that are routinely tested. Different lots of cranberry powder ingredient and two formulated products were tested independently for anti-adhesion activity using the established method and the improved method. Positive harmonization of results on the same samples using rigorous controls was achieved and provides the substantiation needed for the cranberry industry to utilize the improved, rapid in vitro testing method to standardize cranberry products for sufficient anti-adhesion bioactivity and maintain consumer confidence.

Differences in P-Type and Type 1 Uropathogenic Escherichia coli Urinary Anti-Adhesion Activity of Cranberry Fruit Juice Dry Extract Product and D-Mannose Dietary Supplement.

BACKGROUND: Urinary tract infection (UTI) prevention benefits of cranberry intake are clinically validated, especially for women and children. To ensure the benefits of cranberry dietary supplement products, the anti-adhesion activity (AAA) against uropathogenic bacteria is routinely used in in vitro bioassays to determine the activity in whole product formulations, isolated compounds, and ex vivo bioassays to assess urinary activity following intake. D-mannose is another dietary supplement taken for UTI prevention, based on the anti-adhesion mechanism. OBJECTIVE: Compare the relative AAA of cranberry and D-mannose dietary supplements against the most important bacterial types contributing to the pathogenesis of UTI, and consider how certain components potentially induce in vivo activity. METHODS: The current study used a crossover design to determine ex vivo AAA against both P- and Type 1-fimbriated uropathogenic Escherichia coli of either D-mannose or a cranberry fruit juice dry extract product containing 36 mg of soluble proanthocyanidins (PACs), using bioassays that measure urinary activity following consumption. AAA of extracted cranberry compound fractions and D-mannose were compared in vitro and potential induction mechanisms of urinary AAA explored. RESULTS: The cranberry dietary supplement exhibited both P-type and Type 1 in vitro and ex vivo AAA, while D-mannose only prevented Type 1 adhesion. Cranberry also demonstrated more robust and consistent ex vivo urinary AAA than D-mannose over each 1-week study period at different urine collection time points. The means by which the compounds with in vitro activity in each supplement product could potentially induce the AAA in urines was discussed relative to the data. CONCLUSIONS: Results of the current study provide consumers and healthcare professionals with additional details on the compounds and mechanisms involved in the positive, broad-spectrum AAA of cranberry against both E. coli bacterial types most important in UTIs and uncovers limitations on AAA and effectiveness of D-mannose compared to cranberry.

Gene expression differences during the heterogeneous progression of peripheral atherosclerosis in familial hypercholesterolemic swine.

BACKGROUND: The heterogeneous progression of atherosclerotic disease in the peripheral arteries is currently not well understood. In humans, artery specific disease progression is partly attributed to the local hemodynamic environments. However, despite similar hemodynamic environments, porcine brachial arteries are protected while femoral arteries are highly susceptible to advanced lesion formation. The aim of this investigation was to determine whether artery specific gene expression patterns contribute to the uneven distribution of peripheral arterial disease (PAD) in Rapacz Familial-Hypercholesterolemic (FHC) swine. RESULTS: Histological results confirmed rapid atherosclerotic disease progression in femoral but not brachial arteries. A total of 18,922 probe sets had sufficient signal abundance. A main effect for age and artery was observed for 1784 and 1256 probe sets, respectively. A significant age x artery interaction was found for 184 probe sets. Furthermore, comparison between arteries found a decrease from 714 to 370 differentially expressed transcripts from nine months to two years of age. Gene ontology analysis of the 56 genes with a main effect for artery and an age x artery interaction identified vascular smooth muscle contraction as enhanced biological signaling pathway. CONCLUSION: This is the first investigation to report that the total number of differential genes decreases with diverging atherosclerotic disease pattern between porcine brachial and femoral arteries.

Parenteral nutrition increases susceptibility of ileum to invasion by E coli.

BACKGROUND: Parenteral nutrition (PN), with the lack of enteral feeding, compromises mucosal immune function and increases the risk of infections. We developed an ex vivo intestinal segment culture (EVISC) model to study the ex vivo effects of PN on susceptibility of the ileum to invasion by extra-intestinal pathogenic Escherichia coli (ExPEC) and on ileal secretion of antimicrobial secretory phospholipase A2 (sPLA2) in response to the pathogen. MATERIALS AND METHODS: Study 1: Using mouse (n = 7) ileal tissue, we examined the effects of ileal region (proximal versus distal) and varying ExPEC inoculum concentrations on ex vivo susceptibility to ExPEC invasion and sPLA2 secretion. Study 2: Ten mice were randomized to oral chow or intravenous PN feeding for 5 d (n = 5/group). Using the EVISC model, we compared the susceptibility of ileal tissue to invasion by ExPEC and sPLA2 secretion in response to the pathogen. RESULTS: Study 1: The proximal ileum was more susceptible to invasion (P < 0.0001) and secreted lower amounts of sPLA2 (P = 0.0002) than the distal ileum. Study 2: Ileal tissue from PN-fed animals was more susceptible (approximately 4-fold, P = 0.018) to invasion than those from chow-fed animals. Ileal tissue from PN-fed animals secreted less sPLA2 (P < 0.02) than those from chow-fed animals. CONCLUSIONS: The data illustrate EVISC as a reproducible model for studying host-pathogen interactions and the effects of diet on susceptibility to infections. Specifically, the findings support our hypothesis that PN with the lack of enteral feeding decreases mucosal responsiveness to pathogen exposure and provides a plausible mechanism by which PN is associated with increased risk of infectious complication.

Quantifying and characterizing proanthocyanidins in cranberries in relation to urinary tract health.

The "A-type" proanthocyanidins in cranberry fruit (Vaccinium macrocarpon Ait.) are bioactive components associated with prevention of urinary tract infections (UTI). Cranberry juice, fruit (fresh and dried), functional foods, and cranberry dietary supplements are promoted for prevention of UTI and for maintenance of urinary tract health (UTH), on the basis of their content of cranberry proanthocyanidins (c-PAC) with "A-type" interflavan bonds. With increasing consumer use of cranberries for maintenance of UTH and an expanding number of commercial cranberry products of different types, the availability of unified methods for measuring levels of c-PAC is important. This review discusses quantitative and qualitative analysis of c-PAC with "A-type" interflavan bonds in relation to their biological activity for UTI prevention. The integrity (including authenticity, standardization, efficacy, and safety) of cranberry fruit, juices, and dietary supplements may now be measured by using recent advances in mass spectrometry, liquid chromatography, production of c-PAC standards, and improved simple quantitative techniques.

Consumption of sweetened, dried cranberries may reduce urinary tract infection incidence in susceptible women--a modified observational study.

BACKGROUND: Urinary tract infections (UTIs) are one of the most common bacterial infections, and over 50% of women will have a UTI during their lifetimes. Antibiotics are used for prophylaxis of recurrent UTIs but can lead to emergence of drug-resistant bacteria. Therefore, it is reasonable to investigate nutritional strategies for prevention of UTIs. Cranberry juices and supplements have been used for UTI prophylaxis, but with variable efficacy. Because dried cranberries may contain a different spectrum of polyphenolics than juice, consuming berries may or may not be more beneficial than juice in decreasing the incidence of UTIs in susceptible women. The primary objectives of this study were to determine if consumption of sweetened, dried cranberries (SDC) decreases recurrent UTIs and whether this intervention would alter the heterogeneity, virulence factor (VF) profiles, or numbers of intestinal E. coli. METHODS: Twenty women with recurrent UTIs were enrolled in the trial and consumed one serving of SDC daily for two weeks. Clinical efficacy was determined by two criteria, a decrease in the six-month UTI rates pre- and post-consumption and increased time until the first UTI since beginning the study. Strain heterogeneity and virulence factor profiles of intestinal E. coli isolated from rectal swabs were determined by DNA fingerprinting and muliplex PCR, respectively. The numbers of intestinal E. coli eluted from rectal swabs pre- and post-consumption were also quantified. RESULTS: Over one-half of the patients did not experience a UTI within six months of SDC consumption, and the mean UTI rate per six months decreased significantly. Kaplan-Meier analysis of infection incidence in women consuming SDC compared to patients in a previous control group showed a significant reduction in time until first UTI within six months. The heterogeneity, VF profiles, and prevalence of intestinal E. coli strains were not significantly different after cranberry consumption. CONCLUSIONS: Results of this study indicate a beneficial effect from consuming SDC to reduce the number of UTIs in susceptible women. Because there were no changes in the heterogeneity or VF profiles of E. coli, additional studies are needed to determine the mechanism of action of SDC for reduction of UTIs.

Cranberry proanthocyanidins composite electrospun nanofibers as a potential alternative for bacterial entrapment applications.

The interaction between A-type interflavan bonds from cranberry proanthocyanidins (PAC) and surface virulence factors of extra-intestinal pathogenic Escherichia coli (ExPEC) was studied. Electrospun nanofibers (ESNF) were fabricated using PAC and polycaprolactone (PCL) solutions and their physical and chemical properties were characterized. The ability of PAC:PCL composite ESNF to interact with and entrap ExPEC strain 5011 (ExPEC-5011) was evaluated in vitro by plate culturing and when formulated as a biofilter and nanocoating. As a biofilter, the PAC:PCL ESNF exhibited a dose-dependent ability to entrap ExPEC-5011. Images from scanning electron and fluorescent microscopies revealed that ESNF sections with higher amounts of PAC led to higher bacterial entrapment. The effectiveness PAC:PCL ESNF to bind ExPEC when applied as a nanocoating was studied using ESNF-coated polyvinyl chloride intermittent catheter. Results indicate that ExPEC-5011 was entrapped well into the PAC:PCL ESNF coating on the catheter. Overall, our results suggest that incorporating the biomolecule PAC in ESNF is a potential means for applications requiring bacterial entrapment, such as biofunctionalization, biofiltration, and surface coating, among others.

Gene expression differences in healthy brachial and femoral arteries of Rapacz familial hypercholesterolemic swine.

The mechanisms underlying the unequal distribution of atherosclerotic disease in the peripheral arteries are currently unclear. Gene expression differences in healthy arteries may influence the heterogeneous distribution of atherosclerosis. Therefore, this investigation compares gene expression in healthy atheroprotected brachial and atherosusceptible femoral arteries of young and disease free Rapacz familial hypercholesterolemic (FHC) swine. We hypothesized that transcripts related to atherosusceptibility would be differentially expressed between these arteries prior to the onset of disease. Femoral and brachial arteries were harvested from four 13-day-old Rapacz FHC swine. No atherosclerotic disease was detected using Sudan IV, Verhoeff-van Gieson, and hematoxylin-eosin staining. Gene expression was quantified using Affymetrix GeneChip Porcine Genome Arrays. An average of 15,552 probe sets had detectable transcripts, while 430 probe sets showed a significant differential expression between arteries (false discovery rate < 0.05). The human orthologs of 63 probe sets with differential expression and a 1.5-fold or greater transcript abundance between arteries are associated with Wnt/ß-catenin, lysophospholipid, and Ca-signaling, as well as apoptosis. This is the first investigation reporting that differences in relative abundance of gene expression exist between brachial and femoral arteries in young Rapacz FHC swine prior to the development of atherosclerotic lesions.

Classification of proanthocyanidin profiles using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectra data combined with multivariate analysis.

Proanthocyanidin (PAC) profiles of apples (a-PAC), cranberries (c-PAC), and peanut skins (p-PAC) were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Deconvolution of overlapping isotopic patterns indicated that in apples, only 5% of the PAC oligomers contain one or more A-type bonds, whereas in cranberries and peanut skins, 96% of the PAC oligomers contain one or more A-type bonds. MALDI-TOF MS data combined with multivariate analysis, such as principal component analysis (PCA) and linear discriminant analysis (LDA), were used to differentiate and discriminate a-PAC, c-PAC, and p-PAC from one another. Mixtures of c-PAC with either a-PAC or p-PAC at different w/w ratios were evaluated by LDA modeling. The LDA model classified the training, testing, and validation sets with 99.4%, 100%, and 94.2% accuracy. Results suggest that MALDI-TOF MS and multivariate analysis are useful in determining authenticity of PAC from different sources and mixtures of PAC sources.

Identification of A-Type Proanthocyanidins in Cranberry-Based Foods and Dietary Supplements by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry, First Action Method: 2019.05.

BACKGROUND: Cranberry proanthocyanidins (c-PAC) are oligomeric structures of flavan-3-ol units, which possess A-type interflavan bonds. c-PAC differs from other botanical sources because other PAC mostly have B-type interflavan bonds. Cranberry products used to alleviate and prevent urinary tract infections may suffer from adulteration, where c-PAC are replaced with less expensive botanical sources of PAC that contain B-type interflavan bonds. OBJECTIVE: Identifying the presence of A-type interflavan bonds in cranberry fruit and dietary supplements. METHODS: Thirty-five samples reported to contain A-type PAC (cranberry fruit and cranberry products) and 36 samples reported to contain B-type PAC (other botanical sources) were identified and differentiated using MALDI-TOF MS, deconvolution of overlapping isotope patterns, and principal component analysis (PCA). RESULTS: Our results show that both MALDI-TOF MS and deconvolution of overlapping isotope patterns were able to identify the presence of A-type interflavan bonds with a probability greater than 90% and a confidence of 95%. Deconvolution of MALDI-TOF MS spectra also determined the ratio of A-type to B-type interflavan bonds at each degree of polymerization in cranberry fruit and cranberry products, which is a distinguishing feature of c-PAC in comparison to other botanical sources of PAC. PCA shows clear differences based on the nature of the interflavan bonds. CONCLUSIONS: MALDI-TOF MS, deconvolution of overlapping isotope patterns of MALDI-TOF MS spectra, and PCA allow the identification, estimation, and differentiation of A-type interflavan bonds in cranberry-based foods and dietary supplements among other botanical sources containing mostly B-type interflavan bonds.

Inter-Laboratory Validation of 4-(Dimethylamino) Cinnamaldehyde (DMAC) Assay Using Cranberry Proanthocyanidin Standard for Quantification of Soluble Proanthocyanidins in Cranberry Foods and Dietary Supplements, First Action Official MethodSM: 2019.06.

BACKGROUND: Proanthocyanidins (PAC) are oligomers and polymers of flavan-3-ols with putative health benefits. PAC are prevalent in a wide variety of natural products and dietary supplements. OBJECTIVE: An inter-laboratory study was conducted to validate the 4-(dimethylamino)cinnamaldehyde (DMAC) colorimetric assay using a 96-well plate spectrophotometer for the accurate quantification of PAC in cranberry products and to evaluate the comparison of the procyanidin A2 (ProA2) dimer and cranberry PAC (c-PAC) reference standards. METHODS: Four test materials analyzed in this study included cranberry fiber powder, cranberry extract powder, concentrated cranberry juice, and a solution of cranberry PAC (30%, w/v). The samples were homogenized, extracted, sonicated, centrifuged, and analyzed using a 96-well plate spectrophotometer. RESULTS: Linearity for both the ProA2 and c-PAC standards was determined from 4.053 to 50.666 µg/mL and from 13.520 to 135.95 µg/mL, respectively. The relative standard deviation of repeatability (RSDr) values for the four materials analyzed, using both ProA2 and c-PAC standards, met the Standard Method Performance Requirements (SMPR®). Inter-laboratory precision using Horwitz ratio (HorRat) values for the four materials analyzed, using both ProA2 and c-PAC standards, satisfies the acceptance range in Appendix K of the Official Methods of Analysis (2003): Guidelines for Dietary Supplements and Botanicals. The limit of quantification (LOQ) was estimated to be 3.16 µg/mL. CONCLUSIONS: The results produced from this study demonstrate the utility of the c-PAC standard over the ProA2 standard and the advantages of using a 96-well plate spectrophotometer for the accurate quantification of PAC. HIGHLIGHTS: The use of a 96-well plate reader and c-PAC reference standard in the DMAC method improves accuracy and percision for quantification of soluble proanthocyanidins in cranberry foods and dietary supplements.

Cranberry Proanthocyanidins-PANI Nanocomposite for the Detection of Bacteria Associated with Urinary Tract Infections.

Consumption of cranberries is associated with the putative effects of preventing urinary tract infections (UTIs). Cranberry proanthocyanidins (PAC) contain unusual double A-type linkages, which are associated with strong interactions with surface virulence factors found on UTI-causing bacteria such as extra-intestinal pathogenic Escherichia coli (ExPEC), depicting in bacterial agglutination processes. In this work, we demonstrated the efficacy of cranberry PAC (200 µg/mL) to agglutinate ExPEC (5.0 × 108 CFU/mL) in vitro as a selective interaction for the design of functionalized biosensors for potential detection of UTIs. We fabricated functionalized screen-printed electrodes (SPEs) by modifying with PAC-polyaniline (PANI) nanocomposites and tested the effectiveness of the PAC-PANI/SPE biosensor for detecting the presence of ExPEC in aqueous suspensions. Results indicated that the PAC-PANI/SPE was highly sensitive (limit of quantification of 1 CFU/mL of ExPEC), and its response was linear over the concentration range of 1-70,000 CFU/mL, suggesting cranberry PAC-functionalized biosensors are an innovative alternative for the detection and diagnosis of ExPEC-associated UTIs. The biosensor was also highly selective, reproducible, and stable.

Synthesis of Fluorescent Proanthocyanidin-Cinnamaldehydes Pyrylium Products for Microscopic Detection of Interactions with Extra-Intestinal Pathogenic Escherichia coli.

Synthesis of proanthocyanidin-cinnamaldehydes pyrylium products (PCPP) was achieved by the condensation reaction of proanthocyanidins (PAC) with cinnamaldehyde and four cinnamaldehyde derivatives. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectra of PCPP show masses that correspond to (epi)catechin oligomers attached to single, double, or triple moieties of cinnamaldehydes. Synthesized PCPP exhibited fluorescence at higher excitation and emission wavelengths than PAC. Results indicate that PCPP were more bioactive for agglutinating extra-intestinal pathogenic Escherichia coli (ExPEC) compared to PAC. Scanning electron microscopy indicates that PCPP interact with ExPEC surface structures and suggests that PCPP have a higher affinity with the fimbriae-like structures of ExPEC than PAC. Fluorescent microscopy performed on in vitro and in vivo agglutination assays show that PCPP were entrapping ExPEC in a web-like network, thus demonstrating agglutination of ExPEC. This study demonstrated the potential of PCPP to improve our understanding of the temporal and dynamic interactions of PAC in in vitro and in vivo studies.

Contrast-enhanced ultrasound for imaging vasa vasorum: comparison with histopathology in a swine model of atherosclerosis.

AIM: To evaluate the agreement between contrast-enhanced ultrasound imaging and histopathology in an animal model of atherosclerosis. METHODS AND RESULTS: Atherosclerosis was studied in both femoral arteries of four Rapacz familial hypercholesterolaemia (RFH) swine. Contrast-enhanced ultrasound imaging of the eight femoral arteries was performed at baseline and at 5, 12, 26, and 43 weeks follow-up after percutaneous transluminal stimulation of atherosclerosis to assess the progression of intima-media thickness (IMT) and the density and extent of the vasa vasorum network. Contrast-enhanced ultrasound imaging allowed an early detection of atherosclerosis and showed a significant gradual progression of atherosclerosis over time. IMT increased from 0.22 +/- 0.05 mm at baseline to 0.45 +/- 0.06 mm (P < 0.001) at follow-up. The density of the vasa vasorum network increased during follow-up and was significantly higher in advanced than in early atherosclerosis. The findings with contrast-enhanced ultrasound were confirmed by histopathological specimens of the arterial wall. CONCLUSION: Contrast-enhanced ultrasound is effective for in vivo detection of vasa vasorum in atherosclerotic plaques in the RFH swine model. After stimulation of atherosclerosis, contrast-enhanced ultrasound demonstrated a significantly increased IMT and significantly increased density of the vasa vasorum network in the developing atherosclerotic plaque, which was validated by histology.

In vitro uptake of lysozyme-loaded liposomes coated with chitosan biopolymer as model immunoadjuvants.

Chitosan binds to negatively charged soy lecithin liposomes by an electrostatic interaction driven by its cationic amino group. This interaction allows developing stable coated vesicles suitable as a targeted carrier and controlled release system for drugs and vaccines. In this work, we studied the effect of chitosan-coated liposomes on the uptake and antigen presentation of hen egg-white lysozyme (HEL) in Peyer's patches peritoneal macrophages isolated from mice. Chitosan-coated liposomes were characterized according to size, zeta potential, and antigen-loading and release properties. Results showed an increase in the positive net charge and size of the liposomes as the concentration of chitosan was increased, suggesting an electrostatic interaction and an effective coating, followed by fluorescence microscopy. About 85% of the antigen loaded remained in the chitosan-coated liposomes after release studies for 4 hours in phosphate-buffered saline. After 4 hours of preincubation with a T-cell hybridoma line cocultured with murine peritoneal macrophages, only trace amounts of interleukin-2 (IL-2) were detected in the cocultures treated with HEL alone, whereas cocultures treated with HEL-liposomes had an important production of IL-2, and the HEL chitosan-coated liposomes had already reached maximum IL-2 expression. Confocal microscopy studies showed that chitosan-coated liposomes had a higher uptake rate of the fluorescently labeled HEL than uncoated liposomal vesicles after 30 minutes of incubation with the peritoneal macrophages. Since uptake by macrophage cells is the first step in vaccination, our results suggest that the chitosan-coated liposomal system is a potential candidate as an immunoadjuvant for vaccine delivery systems.

Development of a Cranberry Standard for Quantification of Insoluble Cranberry (Vaccinium macrocarpon Ait.) Proanthocyanidins.

Cranberry proanthocyanidins (PACs) can be partitioned into soluble PACs, which are extracted with solvents, and insoluble PACs, which remain associated with fibers and proteins after extraction. Most research on cranberry products only quantifies soluble PACs because proper standards for quantifying insoluble PACs are lacking. In this study, we evaluated the ability of a cranberry PAC (c-PAC) standard, reflective of the structural heterogeneity of PACs found in cranberry fruit, to quantify insoluble PACs by the butanol-hydrochloric acid (BuOH-HCl) method. For the first time, a c-PAC standard enabled conversion of BuOH-HCl absorbance values (550 nm) to a weight (milligram) basis, allowing for quantification of insoluble PACs in cranberries. The use of the c-PAC reference standard for sequential analysis of soluble PACs by the method of 4-(dimethylamino)cinnamaldehyde and insoluble PACs by the method of BuOH-HCl provides analytical tools for the standardization of cranberry-based ingredients.