Search

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.

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.

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., (© 2022 Wiley Periodicals LLC.)

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.

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., (© AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cranberry proanthocyanidin-chitosan nanoparticles (PAC-CHT NPs) loaded with antibiotic gentamicin (GEN) (PAC-CHT-GEN NPs) were formulated and characterized according to size, polydispersity (PDI), surface charge, morphology, and encapsulation efficiency (EE). PAC-CHT-GEN NPs were evaluated for their ability to agglutinate E. coli, S. aureus, and P. aeruginosa and their bacteriostatic and bactericidal activity. Results indicate that the PAC-CHT-GEN NPs at 0.5:1.0, 1.0:1.0, and 2.0:1.0 weight ratios formed stable nanoparticles with sizes from 242.9 to 277.4 nm, a PDI from 0.344 to 0.391, and a zeta potential from 34.5 to 38.5 mV, and up to 94% EE. Results indicate that PAC-CHT-GEN NPs have the ability to agglutinate E. coli, S. aureus, and P. aeruginosa. Furthermore, PAC-CHT-GEN NPs exhibited greater bactericidal activity than GEN alone. Results suggested PAC-CHT-GEN NPs form stable, round-shaped, and bioactive nanoparticles with the potential to be use in the treatment of bacterial infections., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Proanthocyanidin (PAC, condensed tannin) containing forages have well-documented anti-parasitic effects against gastrointestinal nematodes (GIN) of small ruminants. Although extensive research has been conducted on the inhibition of exsheathment of the L 3 stage of Haemonchus contortus by in vitro exposure to the extracts of PAC containing plants, only one study has previously attempted to replicate this process in vivo and it was found that consumption of fresh sainfoin slowed the exsheathment rate. No similar studies have explored the effect of feeding condensed tannin forages in the form of hay on in vivo exsheathment of GIN. Another PAC containing forage, birdsfoot trefoil (Lotus corniculatus, BFT), has a large area of adaptation globally and feeding BFT has been shown to reduce fecal egg counts and total worm burdens. However, its effect on the in vivo exsheathment of H. contortus in the rumen is unknown. Recent work from this laboratory showed that BFT populations differ in the ability of aqueous extracts of freeze-dried plants to reduce exsheathment of H. contortus in vitro, and that the reduced exsheathment caused by BFT populations did not directly correlate with PAC content. Therefore, the objective of this study was twofold: 1) to evaluate the ability of birdsfoot trefoil hay to impair ruminal exsheathment of H. contortus in vivo and 2) to measure the difference in exsheathment between three commercially available cultivars of birdsfoot trefoil representing a broad range of in vitro efficacy against H. contortus. Four rumen fistulated ewes were fed three cultivars of birdsfoot trefoil (cv. Bruce, Empire, and Pardee) hay or a control hay (alfalfa/grass hay) in a Latin 4 × 4 design. The effect of consumption of birdsfoot trefoil on the exsheathment of H. contortus larvae in vivo was evaluated. For each exsheathment test, two capsules with 2000 ensheathed third-stage larvae per capsule were placed in the rumen of each ewe for eight hours. Larval containment capsules were made by capping each end of a short piece of Tygon® tubing (ID 9.5 mm, OD 14.3 mm) with an 8 μm Nunc TM Cell Culture Insert. Larval exsheathment and motility were examined pre and post rumen exposure. Three exsheathment tests were run per diet cycle. Consumption of BFT hay did not significantly alter larval exsheathment. These results highlight the importance of further in vivo testing on the role of condensed tannins and other plant secondary compounds on larval exsheathment in the rumen., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Chitosan (CHT) interacts with proanthocyanidins (PAC) by a mechanism involving hydrogen bonding and ion-dipole interactions, allowing the spontaneous formation of PAC-CHT composite nanoparticles (PAC-CHT NPs). The interaction between PAC and CHT was characterized by ellipsometry, infrared spectroscopy, and surface plasmon resonance (SPR) to determine the effect of CHT molecular weight (MW), PAC to CHT ratios, and pH on the formulation of PAC-CHT NPs. These parameters also affect the size and morphology of PAC-CHT NPs. Results indicate that CHT MW and pH of the solution impact the interactions of PAC-CHT in two ways: (1) greater CHT MW increases the amount of PAC molecules that attach to the CHT chain, and (2) lower pH of the CHT solutions increases the amount PAC molecules that attach to the CHT chain. Results also show that higher CHT MW, CHT concentration, and pH of the CHT solutions increase the size of PAC-CHT NPs. In contrast, greater PAC concentrations decreases the size of PAC-CHT NPs. This study demonstrates that SPR is a useful technique for measuring the effect of changes in the interaction between PAC and CHT, which in turn affects the size and morphology of PAC-CHT NPs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

Total polyphenol content (TPC), total flavonoid content (TFC), total anthocyanin content (TAC), and proanthocyanidin (PAC) content were determined in fruit from three tropical Vaccinium species ( Vaccinium consanguineum , Vaccinium floribundum , and Vaccinium poasanum ) from Costa Rica sampled at three stages of fruit development. Results show that TAC increased as the fruit developed, while TPC, TFC, and PAC content decreased. Anthocyanin profiles were evaluated using electrospray ionization tandem mass spectrometry. Cyanidin and delphinidin glycosides were the predominant anthocyanins for the three tropical Vaccinium species. Proanthocyanidins were characterized using attenuated total reflection Fourier transform infrared spectroscopy, nuclear magnetic resonance, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The presence of procyanidin structures with B-type interflavan bonds were observed, but deconvolution of mass spectrometry isotope patterns indicated that PACs with one or more A-type interflavan bonds accounted for more than 74% of the oligomers at each degree of polymerization.

Cranberry proanthocyanidin-chitosan composite nanoparticles (PAC-CHT NPs) were formulated using 2:1, 5:1, 10:1, 15:1 20:1, 25:1, and 30:1 PAC to CHT weight ratio to form round shaped particles. The PAC-CHT NPs were characterized by size, polydispersity, surface charge, morphology, and PAC content. PAC-CHT NPs bioactivity was measured by agglutination of extra-intestinal pathogenic Escherichia coli (ExPEC) and inhibition of gut epithelial cell invasion by ExPEC. Results indicate that by increasing the PAC to CHT ratio 10:1 to 30:1 formed stable nanoparticles with diameters of 122.8 to 618.7 nm, a polydispersity index of approximated 0.4 to 0.5, and a zeta potential of 34.5 to 54.4 mV. PAC-CHT NPs ratio 30:1 agglutinated ExPEC and decreased the ability of ExPEC to invade epithelial cells in a dose-dependent manner. PAC-CHT NPs ratio 10:1 to 30:1 form stable, round-shaped, and bioactive nanoparticles for potential applications in the treatment of ExPEC bacterial infections., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

Chitosan interacts with proanthocyanidins through hydrogen-bonding, which allows encapsulation and development of stable nanoparticles via ionotropic gelation. Cranberry proanthocyanidins (PAC) are associated with the prevention of urinary tract infections and PAC inhibit invasion of gut epithelial cells by extra-intestinal pathogenic Escherichia coli (ExPEC). We determined the effect of cranberry proanthocyanidin-chitosan hybrid nanoparticles (PAC-CHTNp) on the ExPEC invasion of gut epithelial cells in vitro. PAC-CHTNp were characterized according to size, morphology, and bioactivity. Results showed a decrease in the size of the nanoparticles as the concentration of PAC was increased, indicating that PAC increases cross-linking by hydrogen-bonding on the surface of the chitosan nanoparticles. Nanoparticles were produced with diameters ranging from 367.3 nm to 293.2 nm. Additionally, PAC-CHTNp significantly inhibited the ability of ExPEC to invade the enterocytes by ~80% at 66 μg GAE/mL and by ~92% at 100 μg GAE/mL. Results also indicate that chitosan nanoparticles alone were not significantly different from controls in preventing ExPEC invasion of enterocytes (data not shown) and also there were not significant differences between PAC alone and PAC-CHTNp, suggesting that the new PAC-CHTNp could lead to an increase in the stability of encapsulated PAC, maintain the molecular adhesion of PAC to ExPEC., (Published by Elsevier B.V.)

The discovery that plant secondary compounds, including proanthocyanidins (PAC), suppress gastrointestinal nematode (GIN) infection has provided promise for alternative methods of GIN control in small ruminants. This investigation is the first to examine the anthelmintic potential of cranberry vine (CV) against the GIN Haemonchus contortus. The purpose of this study was to explore the anti-parasitic activity of CV in the form of a specific organic proanthocyanidin extract (CV-PAC) and an aqueous extract (CV-AqE) containing PAC and other compounds. In vitro egg hatching, first (L1) and third (L3) stage larval and adult worm motility and L3 exsheathment were evaluated after a 24-h incubation with CV products. In addition, CV treated worms were observed via scanning electron microscopy, and a preliminary investigation of the efficacy of CV powder against an experimental infection of H. contortus was conducted. The in vivo effect on an experimental infection was determined by administering 21.1 g CV powder to lambs (n = 9 per group) for three consecutive days, and collecting fecal egg count data for four weeks post-treatment. The effect of CV-PAC on egg hatching, L3 motility and exsheathment was limited. However, a substantial effect was observed on motility of post-hatch L1 (EC 50 0.3 mg PAC/mL) and adults (EC 50 0.2 mg PAC/mL). The CV-AqE showed more effect on egg hatching (EC 50 5.3 mg/mL containing 0.6 mg PAC/mL) as well as impacting motility of L1 (EC 50 1.5 mg/mL with 0.2 mg PAC/mL) and adults (EC 50 3.4 mg/mL with 0.4 mg PAC/mL), but like CV-PAC, did not substantially effect L3 motility or exsheathment. Scanning electron microscopy revealed an accumulation of aggregate on the cuticle around the buccal area of adult worms incubated in CV-AqE and CV-PAC. In the preliminary in vivo study, there was a significant effect of treatment over time (p = .04), although differences in individual weeks were not significant. In summary, both extracts inhibited motility of L1 and adult worms. The higher efficacy of CV-AqE than CV-PAC at levels that contained the same concentrations of PAC tested alone, suggest that other secondary compounds in the CV-AqE contributed to the observed effects on the parasites. This first study of the in vitro and in vivo effects of CV suggest that this readily available plant product may have utility in integrated control of H. contortus and support the need for additional testing to provide further information., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The relationship among diet, human health, and disease is an area of growing interest in biomarker research. Previous studies suggest that the consumption of cranberries (Vaccinium macrocarpon) could beneficially influence urinary and digestive health. The present study sought to determine if daily consumption of sweetened dried cranberries (SDC) changes the urinary proteome and fecal microbiome, as determined in a prospective sample of 10 healthy individuals. Baseline urine and fecal samples were collected from the subjects in the fasted (8-12 h) state. The subjects then consumed one serving (42 g) of SDC daily with lunch for 2 weeks. Urine and fecal samples were collected again the day after 2 weeks of SDC consumption. Orbitrap Q-Exactive mass spectrometry of urinary proteins showed that consumption of SDC resulted in changes to 22 urinary proteins. Multiplex sequencing of 16S ribosomal RNA genes in fecal samples indicated changes in relative abundance of several bacterial taxonomic units after consumption of SDC. There was a shift in the Firmicutes:Bacteroidetes ratio, increases in commensal bacteria, and decreases or the absence of bacteria associated with negative health effects. A decrease in uromodulin in all subjects and an increase in Akkermansia bacteria in most subjects were observed and warrant further investigation. Future larger clinical studies with multiomics and multitissue sampling designs are required to determine the effects of SDC consumption on nutrition and health.

Date palm fruit (Phoenix dactylifera) consumption reduces serum triglyceride levels in human subjects. The objective of this study was to prepare an extract from dates and determine whether it acts as a ligand for the farnesoid x receptor (FXR), a nuclear receptor important for maintaining triglyceride and cholesterol homeostasis. Freeze-dried extracts were isolated from California-grown dates (Deglet Noor and Medjool) from the 2014 and 2015 harvests, by means of liquid extraction and solid phase separation. Each date palm extract (DPE) was characterized via HPLC and MALDI-TOF mass spectrometry, and the procyanidin content was qualitatively determined. Extracts were tested to determine their ability to modulate nuclear receptor-mediated transactivation using transient transfection. The effect of DPE on FXR-target genes regulating bile acid absorption and transport was then assessed in vitro, in Caco-2 cells. Characterization reveals that DPE is a rich source of polyphenols including hydroxycinnamic acids, proanthocyanidins, and lipohilic polyphenols, and comprises 13% proanthocyanidins. Transactivation results show that DPE acts as a co-agonist ligand for both mouse and human FXR, wherein it activates bile acid-bound FXR greater than that seen with bile acid alone. Additionally, DPE alone activated a peroxisome proliferator activated receptor alpha (PPARα) chimera in a dose-dependent manner. Consistent with DPE as a co-agonist ligand for FXR, studies in Caco-2 cells reveal that co-incubation with bile acid, dose-dependently enhances the expression of fibroblast growth factor 19 (FGF19), compared to treatment with bile acid alone. In contrast, DPE inhibited bile acid-induced expression of ileal bile acid binding protein (IBABP). Our results demonstrate that DPE acts as a potent co-agonist ligand for FXR, and that it differentially regulates FXR-target gene expression in vitro in human intestinal cells. This study provides novel insight into a potential mechanism by which dates may exert a hypotriglyceridemic effect via FXR and modulation of bile acid homeostasis.

The Rapacz familial hypercholesterolemic (FH) swine model is well-characterized and used for studies of both spontaneous and inducible atherosclerosis but has not been used for studies of metabolic dysfunction to date. We examined whether parameters of metabolic syndrome including weight and adiposity, serum cholesterol, and glucoregulatory function could be modulated by restriction of caloric intake in the FH swine. Three groups of FH swine (n = 6 per group) were fed without restriction (AL), 80% of AL caloric intake, or 60% of AL caloric intake for 8.8 ± 0.5 mo beginning 2 wk after weaning. Caloric intake influenced the rate and magnitude of body weight gain and change in adiposity, as determined by dual-emission X-ray absorptiometry. At the conclusion of the study, pigs in the AL group reached a total least-square mean body weight of 94.2 kg and fat mass of 31.1%, whereas those fed 80% AL were 71.6 kg and 24.3% fat, and swine fed 60% AL were 46.1 kg and 14.1% fat. Serum cholesterol was greater in AL than 60% AL pigs at the end of the study. At 10 mo of age, intravenous glucose tolerance testing, performed to assess glucoregulatory function, indicated significant differences in serum glucose clearance profiles and insulin sensitivity between the AL- and 60% AL-fed swine. The AL-fed animals showed almost 5-fold lower insulin sensitivity when compared with animals fed 60% AL caloric intake. These results highlight the value of the FH swine model to study metabolic dysfunction due to changes in caloric intake.

In this work we characterize the interaction of cranberry (Vaccinium macrocarpon) proanthocyanidins (PAC) with bovine serum albumin (BSA) and hen egg-white lysozyme (HEL) and determine the effects of these complexes on macrophage activation and antigen presentation. We isolated PAC from cranberry and complexed the isolated PAC with BSA and HEL. The properties of the PAC-protein complexes were studied by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), gel electrophoresis and zeta-potential. The effects of PAC-BSA complexes on macrophage activation were studied in RAW 264.7 macrophage like cells after treatment with lipopolysaccharide (LPS). Fluorescence microscopy was used to study the endocytosis of PAC-BSA complexes. The effects of the PAC complexes on macrophage antigen presentation were studied in an in vitro model of HEL antigen presentation by mouse peritoneal mononuclear cells to a T-cell hybridoma. The mass spectra of the PAC complexes with BSA and HEL differed from the spectra of the proteins alone by the presence of broad shoulders on the singly and doubly charged protein peaks. Complexation with PAC altered the electrophoretic mobility shift assay in native agarose gel and the electrophoretic mobility (ζ-potential) values. These results indicate that the PAC-protein complexes are stable and alter the protein structure without precipitating the protein. Fluorescence microscopy showed that the RAW 264.7 macrophages endocytosed BSA and PAC-BSA complexes in discrete vesicles that surrounded the nucleus. Macrophages treated with increasing amounts of PAC-BSA complexes had significantly reduced COX-2 and iNOS expression in response to treatment with lipopolysaccharide (LPS) in comparison to the controls. The PAC-HEL complexes modulated antigen uptake, processing and presentation in murine 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 the PAC-HEL complex had already reached the maximum IL-2 expression. Cranberry PAC may increase the rate of endocytosis of HEL and subsequent expression of IL-2 by the T-cell hybridomas. These results suggest that PAC-protein complexes modulate aspects of innate and acquired immune responses in 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.

Recent advances in cranberry research have expanded the evidence for the role of this Vaccinium berry fruit in modulating gut microbiota function and cardiometabolic risk factors. The A-type structure of cranberry proanthocyanidins seems to be responsible for much of this fruit's efficacy as a natural antimicrobial. Cranberry proanthocyanidins interfere with colonization of the gut by extraintestinal pathogenic Escherichia coli in vitro and attenuate gut barrier dysfunction caused by dietary insults in vivo. Furthermore, new studies indicate synergy between these proanthocyanidins, other cranberry components such as isoprenoids and xyloglucans, and gut microbiota. Together, cranberry constituents and their bioactive catabolites have been found to contribute to mechanisms affecting bacterial adhesion, coaggregation, and biofilm formation that may underlie potential clinical benefits on gastrointestinal and urinary tract infections, as well as on systemic anti-inflammatory actions mediated via the gut microbiome. A limited but growing body of evidence from randomized clinical trials reveals favorable effects of cranberry consumption on measures of cardiometabolic health, including serum lipid profiles, blood pressure, endothelial function, glucoregulation, and a variety of biomarkers of inflammation and oxidative stress. These results warrant further research, particularly studies dedicated to the elucidation of dose-response relations, pharmacokinetic/metabolomics profiles, and relevant biomarkers of action with the use of fully characterized cranberry products. Freeze-dried whole cranberry powder and a matched placebo were recently made available to investigators to facilitate such work, including interlaboratory comparability., (© 2016 American Society for Nutrition.)

Background: Familial hypercholesterolemia (FH) is a prevalent hereditary disease associated with increased atherosclerosis and calcific aortic valve disease (CAVD). However, in both FH and non-FH individuals, the role of hypercholesterolemia in the development of CAVD is poorly understood. This study used Rapacz FH (RFH) swine, an established model of human FH, to investigate the role of hypercholesterolemia alone in the initiation and progression of CAVD. The valves of RFH swine have not previously been examined., Methods and Results: Aortic valve leaflets were isolated from wild-type (0.25- and 1-year-old) and RFH (0.25-, 1-, 2-, and 3-year-old) swine. Adult RFH animals exhibited numerous hallmarks of early CAVD. Significant leaflet thickening was found in adult RFH swine, accompanied by extensive extracellular matrix remodeling, including proteoglycan enrichment, collagen disorganization, and elastin fragmentation. Increased lipid oxidation and infiltration of macrophages were also evident in adult RFH swine. Intracardiac echocardiography revealed mild aortic valve sclerosis in some of the adult RFH animals, but unimpaired valve function. Microarray analysis of valves from adult versus juvenile RFH animals revealed significant upregulation of inflammation-related genes, as well as several commonalities with atherosclerosis and overlap with human CAVD., Conclusions: Adult RFH swine exhibited several hallmarks of early human CAVD, suggesting potential for these animals to help elucidate CAVD etiology in both FH and non-FH individuals. The development of advanced atherosclerotic lesions, but only early-stage CAVD, in RFH swine supports the hypothesis of an initial shared disease process, with additional stimulation necessary for further progression of CAVD., (© 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Urinary tract infections (UTI) are one of the most frequent extraintestinal infections caused by Escherichia coli (ExPEC). Cranberry juice has been used for decades to alleviate symptoms and prevent recurrent UTI. The putative compounds in cranberries are proanthocyanidins (PAC), specifically PAC with "A-type" bonds. Since PAC are not absorbed, their health benefits in UTI may occur through interactions at the mucosal surface in the gastrointestinal tract. Recent research showed that higher agglutination of ExPEC and reduced bacterial invasion are correlated with higher number of "A-type" bonds and higher degree of polymerization of PAC. An understanding of PAC structure-activity relationship is becoming feasible due to advancements, not only in obtaining purified PAC fractions that allow accurate estimation, but also in high-resolution MS methodologies, specifically, MALDI-TOF MS. A recent MALDI-TOF MS deconvolution method allows quantification of the ratios of "A-type" to "B-type" bonds enabling characteristic fingerprints. Moreover, the generation of fluorescently labeled PAC allows visualization of the interaction between ExPEC and PAC with microscopy. These tools can be used to establish structure-activity relationships between PAC and UTI and give insight on the mechanism of action of these compounds in the gut without being absorbed., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2014 Wiley Periodicals, Inc.)

A novel methodology was developed to elucidate proanthocyanidins (PAC) interaction with extra-intestinal pathogenic Escherichia coli (ExPEC). PAC inhibit ExPEC invasion of epithelial cells and, therefore, may prevent transient gut colonization, conferring protection against subsequent extra-intestinal infections, such as urinary tract infections. Until now PAC have not been chemically labeled with fluorophores. In this work, cranberry PAC were labeled with 5-([4,6-dichlorotriazin-2-yl]amino) fluorescein (DTAF), detected by high-performance liquid chromatography with diode-array detection and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We report single and double fluorescent-labeled PAC with one or two chlorine atoms displaced from DTAF in alkaline pH via nucleophilic substitution. Fluorescent labeling was confirmed by fragmentation experiments using MALDI-TOF/TOF MS. Fluorescent labeled PAC were able to promote ExPEC agglutination when observed with fluorescence microscopy. DTAF tagged PAC may be used to trace the fate of PAC after they agglutinate ExPEC and follow PAC-ExPEC complexes in cell culture assays., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Rapacz familial hypercholesterolemic (RFH) swine are a well-established model of human FH, a highly prevalent hereditary disease associated with increased risk of coronary artery disease and calcific aortic valve disease (CAVD). However, while these animals have been used extensively for the study of atherosclerosis, the heart valves from RFH swine have not previously been examined. We report the analysis of valvular interstitial cell gene expression in adult (two year old) and juvenile (three months old) RFH and WT swine by microarray analysis via the Affymetrix Porcine Genome Array (GEO #: GSE53997). Principal component and hierarchical clustering analysis revealed grouping and almost no variability between the RFH juvenile and WT juvenile groups. Additionally, only 21 genes were found differentially expressed between these two experimental groups whereas over 900 genes were differentially expressed when comparing either RFH or WT juvenile swine to RFH adults.

There is substantial interest in the role of plant secondary metabolites as protective dietary agents. In particular, the involvement of flavonoids and related compounds has become a major topic in human nutrition research. Evidence from epidemiological and human intervention studies is emerging regarding the protective effects of various (poly)phenol-rich foods against several chronic diseases, including neurodegeneration, cancer and cardiovascular diseases. In recent years, the use of HPLC-MS for the analysis of flavonoids and related compounds in foods and biological samples has significantly enhanced our understanding of (poly)phenol bioavailability. These advancements have also led to improvements in the available food composition and metabolomic databases, and consequently in the development of biomarkers of (poly)phenol intake to use in epidemiological studies. Efforts to create adequate standardised materials and well-matched controls to use in randomised controlled trials have also improved the quality of the available data. In vitro investigations using physiologically achievable concentrations of (poly)phenol metabolites and catabolites with appropriate model test systems have provided new and interesting insights on potential mechanisms of actions. This article will summarise recent findings on the bioavailability and biological activity of (poly)phenols, focusing on the epidemiological and clinical evidence of beneficial effects of flavonoids and related compounds on urinary tract infections, cognitive function and age-related cognitive decline, cancer and cardiovascular disease.

Supercritical fluid extraction (SFE) removed lipophilic compounds and low molecular weight flavonoids from cranberries. However, SFE did not extract proanthocyanidins (PAC). The SFE PAC-enriched residue was submitted to fractionation on Sephadex LH-20 using ethanol, ethanol/methanol, and 80% acetone. PAC degree of polymerization (DP) and ratios of "A-type" to "B-type" interflavan bonds were compared with those of PAC fractions without SFE. Mass spectrometry showed that when SFE was used, PAC distribution was shifted toward higher DP and contained higher amounts of two and three "A-type" bonds compared to PAC fractions without SFE. The 80% acetone fraction with SFE had significantly greater extraintestinal pathogenic Escherichia coli (ExPEC) agglutination and significantly lower ExPEC invasion of enterocytes than the fraction without SFE. Cranberry PAC with higher numbers of "A-type" interflavan bonds are more bioactive in agglutinating ExPEC and inhibiting ExPEC enterocyte invasion.

Background: Receptor for advanced glycated end product (RAGE) expression is a prominent feature of atherosclerosis. We have previously shown in apoE null mice uptake of a radiolabeled anti-RAGE antibody in atherosclerotic plaque and now evaluate RAGE-directed imaging to identify advanced plaques in a large animal model., Methods: Nine hyperlipidemic (HL) pigs were injected with 603.1 ± 129.5 MBq of (99m)Tc-anti-RAGE F(ab')2, and after 6 h (blood pool clearance), they underwent single-photon emission computed tomography/computed tomography (SPECT/CT) imaging of the neck, thorax, and hind limbs. Two HL pigs received (99m)Tc non-immune IgG F(ab')2, and three farm pigs were injected with (99m)Tc-anti-RAGE F(ab')2. After imaging, the pigs were euthanized. The aorta from the root to bifurcation was dissected, and the innominates, proximal carotids, and coronaries were dissected and counted, stained for H&E and RAGE, and AHA-classified., Results: On pathology, 24% of the arterial segments showed AHA class III or IV lesions, and these lesions were confined almost exclusively to coronaries and carotids with % stenosis from 15% to 65%. Scatter plots of %ID/g for class III/IV vs. I/II lesions showed almost complete separation. Focal vascular uptake of tracer visualized on SPECT scans corresponded to class III/IV lesions in the coronary and carotid vessels. In addition, uptake in the hind limbs was noted in the HL pigs and corresponded to RAGE staining of small arteries in the muscle sections. Correlations for the vascular lesions were r = 0.747, P = 0.001 for %ID vs. %ID/g and r = 0.83, P = 0.002 for %ID/g vs. % RAGE staining., Conclusions: Uptake of radiolabeled anti-RAGE antibody in coronary and carotid fibroatheroma and in the small arteries of the hind limbs in a relevant large animal model of atherosclerosis supports the important role of RAGE in atherosclerosis and peripheral artery disease as a target for imaging and treatment.

Gut colonization by extra-intestinal pathogenic Escherichia coli (ExPEC) increases the risk of subsequent infections, including urinary tract infection and septicemia. Previous work suggests that cranberry proanthocyanidins (PAC) interact with bacterial surface factors, altering bacterial interaction with host cells. Methods were developed to determine if ratios of "A-type" to "B-type" interflavan bonds in PAC affect ExPEC agglutination and invasion of enterocytes. In cranberries, 94.5% of PAC contain one or more "A-type" bonds, whereas in apples, 88.3% of PAC contain exclusively "B-type" bonds. Results show that cranberry "A-type" PAC have greater bioactivity than apple "B-type" PAC for increasing ExPEC agglutination and decreasing ExPEC epithelial cell invasion.

Background: The utility of animal models for the prediction of drug-eluting stent (DES) efficacy in human clinical trials is still unclear. The familial hypercholesterolemic swine (FHS) model has been shown to induce a human-like neointimal response to bare metal stent (BMS) implantation. However, its utility to discriminate efficacy signals following DES implantation is unknown. In this study, we aimed to test the efficacy and healing response of several everolimus-eluting stent (EES) platforms in the coronary territory of the FHS., Methods: A total of 19 EES platforms (SYNERGY=6, SYNERGY½-dose=7, and PROMUS Element=6) and an identical BMS control (Element=6) were implanted into the coronary arteries of nine FHS. All implants were performed under intravascular ultrasound guidance using a 1.2 : 1 overstretch ratio. At 30 days, the vascular response to the implant was evaluated by quantitative coronary angiography, optical coherence tomography, and histology., Results: At 28 days, all EES platforms showed a significant decrease in angiographic late lumen loss (between 27 and 37%) compared with the BMS control group. This finding was confirmed both by optical coherence tomography (mean neointimal thickness=28-42% reduction) and by histology (mean neointimal thickness=44-55% reduction). All EES platforms showed similar degrees of neointimal inhibition. The presence of moderate to severe para-strut inflammation was observed in 83% of the stent sections in the BMS group compared with 28.6% in the SYNERGY½-dose group and 0% in the SYNERGY and PROMUS groups (P=0.0002). There was a 68-95% reduction in MMP9 expression in the media in all EES platforms compared with the BMS controls. The presence of mild to moderate para-strut fibrin deposits ranged from 66.7 to 83.4% in all EES platforms compared with 16.7% in the EBMS group., Conclusion: The FHS coronary injury model showed the efficacy of several EES platforms compared with an identical BMS control. Everolimus eluted from different polymeric platforms showed lower levels of inflammation and slightly higher fibrin deposits compared with BMS controls.

Polyphenol-rich Rutgers Scarlet Lettuce (RSL) (Lactuca sativa L.) was developed through somaclonal variation and selection in tissue culture. RSL may contain among the highest reported contents of polyphenols and antioxidants in the category of common fruits and vegetables (95.6 mg/g dry weight and 8.7 mg/g fresh weight gallic acid equivalents and 2721 µmol/g dry weight and 223 µmol/g fresh weight Trolox equivalents). Three main compounds accumulate at particularly high levels in RSL: chlorogenic acid, up to 27.6 mg/g dry weight, cyanidin malonyl-glucoside, up to 20.5 mg/g dry weight, and quercetin malonyl-glucoside, up to 35.7 mg/g dry weight. Major polyphenolic constituents of RSL have been associated with health promotion as well as anti-diabetic and/or anti-inflammatory activities. Daily oral administration of RSL (100 or 300 mg/kg) for up to eight days acutely reduced hyperglycemia and improved insulin sensitivity in high fat diet-induced obese hyperglycemic mice compared to vehicle (water) control. Data presented here support possible use of RSL as a functional food for the dietary management of diabetes.

Background: Elemental enteral nutrition (EEN) decreases gut-associated lymphoid tissue (GALT) function, including fewer Peyer's patch lymphocytes and lower levels of the tissue T helper 2 (Th2) cytokines and mucosal transport protein polymeric immunoglobulin receptor (pIgR), leading to lower luminal secretory immunoglobulin A (sIgA) levels. Since we recently demonstrated that cranberry proanthocyanidins (PACs) maintain the Th2 cytokine interleukin (IL)-4 when added to EEN, we hypothesized the addition of PACs to EEN would normalize other GALT parameters and maintain luminal levels of sIgA., Methods: Institute of Cancer Research mice were randomized (12/group) to receive chow, EEN, or EEN + PACs (100 mg/kg body weight) for 5 days, starting 2 days after intragastric cannulation. Ileum tissue was collected to measure IL-4 by enzyme-linked immunosorbent assay, pIgR by Western blot, and phosphorylated STAT-6 by microarray. Intestinal wash fluid was collected to measure sIgA by Western blot., Results: Compared with chow, EEN significantly decreased tissue IL-4, phosphorylated STAT-6, and pIgR. The addition of PACs to EEN prevented these alterations. Compared with chow, EEN resulted in significantly lower levels of luminal sIgA. The addition of PACs to EEN increased luminal sIgA levels compared with EEN alone., Conclusions: This study suggests the addition of PACs to EEN may support GALT function and maintain intestinal sIgA levels compared with EEN administration alone.

Cranberry pomace is a byproduct of cranberry processing and is comprised of seeds, skins and stems of the cranberry fruit. While cranberry pomace contains beneficial polyphenols, including proanthocyanidins and anthocyanins, it is not a palatable source of these compounds and is typically discarded. In this study, we have developed and optimized a method to extract polyphenols from cranberry pomace using aqueous ethanol, a food grade solvent. Biochemical characterization of the pomace extract showed the presence of a broad range of polyphenols also present in cranberry juice concentrate. By co-drying cranberry pomace extract with a protein-rich food matrix, such as soy protein isolate (SPI), we have developed a method to produce a cranberry polyphenol-SPI complex (CBP-SPI) containing 10% cranberry polyphenols. Unlike dried cranberry pomace extract alone, proanthocyanidins, anthocyanins and total polyphenols were found to be highly stable at 37 °C in the CBP-SPI powder. The extraction and stabilization of cranberry pomace polyphenols using SPI provides an innovative approach for utilizing pomace in the development of novel food ingredients., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Polyphenols extracted from Concord grape pomace were stabilized by complexation to soy protein isolate (SPI) to produce grape polyphenol-SPI complex (GP-SPI) containing 5% or 10% grape polyphenols. LC-MS and MALDI-TOF analysis showed that a broad range of phytochemicals were present in the grape pomace extract. Anthocyanins and total polyphenols in the GP-SPI complex were stable after a 16-week incubation at 37 °C but were reduced by up 60% in dried grape pomace extract. Compared to vehicle (236 ± 34 mg/dL), a single dose of 300 mg/kg GP-SPI (184 ± 32 mg/dL) or 500 mg/kg GP-SPI (177 ± 28 mg/dL) having 5% grape polyphenols significantly lowered blood glucose in obese and hyperglycemic C57BL/6 mice 6 h after administration. GP-SPI allows the capture of grape pomace polyphenols in a protein-rich food matrix and may be useful as a functional food ingredient for the management of blood glucose levels.

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.

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

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.

Background: Lamina propria Th2 cytokines, interleukin (IL)-4 and IL-13, stimulate goblet cell (GC) proliferation and MUC2 production, which protect the intestinal mucosa. Elemental enteral nutrition (EEN) reduces tissue IL-4 and impairs barrier function. Proanthocyanidins (PACs) stimulate oral mucin levels. We hypothesized that adding PAC to EEN would maintain Th2-without stimulating Th1-cytokines and preserve luminal MUC2 vs EEN alone., Materials and Methods: Seventy mice were randomized to 5 diet groups-standard chow, intragastric EEN, or EEN with lowPAC, midPAC (50 mg), or highPAC (100 mg PAC/kg BW)-for 5 days, starting 2 days after gastric cannulation. Ileal tissue was analyzed for histomorphology and the cytokines IL-4, IL-13, IL-1β, IL-6, and TNF-α by enzyme-linked immunosorbent assay. MUC2 was measured in intestinal washes., Results: EEN lowered IL-13 (P < .05) compared with standard chow, whereas IL-4 was not significant (P < .07). LowPAC and midPAC increased IL-13 (P < .05), whereas highPAC increased both IL-4 and IL-13 (P < .05) compared with EEN. All EEN diets reduced (P < .05) crypt depth compared with the chow group. Compared with standard chow, GC numbers and luminal MUC2 were reduced with EEN (P < .05). These effects were attenuated (P < .05) with midPAC and highPAC. No changes were observed in tissue Th1 cytokines., Conclusions: Adding PACs to EEN reverses impaired intestinal barrier function following EEN by improving the gut mucous layer and function through increased GC size and number as well as levels of MUC2 and ileal IL-4 and IL-13.

A method to deconvolute overlapping isotope patterns in positive mode matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was developed to determine ratios of A- to B-type interflavan bonds in proanthocyanidins that were isolated from cranberry (Vaccinium macrocarpon, Ait.) press cake (c-PAC). Precision and accuracy was validated for binary mixtures of procyanidins A2 and B2. Deconvolution of c-PAC spectra indicated that oligomers with one or more A-type interflavan bonds occur in a higher proportion than oligomers with all B-type interflavan bonds. c-PAC with at least one A-type bond accounted for more than 91% of the oligomers between trimers and undecamers. The c-PAC isotope patterns are highly repeatable, suggesting that the method can be applied to authentication, standardization and efficacy of cranberry products in relationship to urinary tract health. This is the first time MALDI-TOF MS has been used for estimating ratios of A- to B-type bonds in PAC., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objectives: The authors aimed to validate a novel iliofemoral in-stent restenosis (ISR) model for the efficacy evaluation of paclitaxel-coated balloons (PCB) using the familial hypercholesterolemic swine (FHS)., Background: Most of the validation work regarding PCB technologies has been performed in the coronary territory of juvenile domestic swine. Although invaluable for safety evaluation, this model is not suited for the evaluation of the efficacy of peripheral PCB technologies., Methods: Twenty-four iliofemoral segments in 12 FHS underwent balloon injury and self-expanding stent placement. After 21 days, the resulting ISR lesions were treated with either 1 μg/mm(2) dose (n = 8), or 3 μg/mm(2) dose (n = 8) PCB (Cotavance, Bayer Pharma AG/MEDRAD, Indianola, Pennsylvania), or with an identical uncoated control balloon (n = 8)., Results: At termination (28 days after treatment), the percent diameter stenosis by quantitative vascular analysis in the control group was higher (31.2 ± 13.7%) compared with the 1 μg/mm(2) (19.3 ± 14.0%, 38% reduction) and 3 μg/mm(2) (8.6 ± 10.7%, 72% reduction) PCB groups. Intravascular ultrasound analysis showed 36% (1 μg/mm(2) dose, p = 0.04) and 55% (3 μg/mm(2) dose, p < 0.01) reductions in neointimal volume stenosis. In the histological analysis, the control group showed the highest degree of percent area stenosis (65 ± 14.3%). The reductions in percent area stenosis was 13.2% (p = 0.5) and 26% (p = 0.04) in the 1 μg/mm(2) and 3 μg/mm(2) dose groups, respectively., Conclusions: The FHS model of iliofemoral ISR demonstrated a dose-dependent effect on the inhibition of neointimal proliferation of a clinically validated PCB technology. This model represents a positive step toward the efficacy evaluation of PCB in the peripheral vascular territory., (Copyright © 2012 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Purpose: To track variations in the deformation of the arterial wall noninvasively by estimating the accumulated displacement and strain over a cardiac cycle may provide useful indicators of vascular health., Methods: In this paper, we propose an approach to track a region of interest (ROI) locally and estimate arterial stiffness variation in a familial hypercholesterolemic swine model of spontaneous atherosclerosis that allows for systematic and reproducible study of progression of the disease mechanism., Results: Strain and displacement indices may be derived from the variations of the accumulated displacement and accumulated strain (obtained from the gradient of the accumulated displacement) over a cardiac cycle to predict not only the likelihood of developing vascular diseases, but also the sites where they may occur. Currently, an ROI thickness value of less than one mm within the arterial wall is necessary for the axial accumulated displacement and strain to obtain reproducible estimates., Conclusions: Accumulated axial displacement and strain estimation on the artery wall shown in this paper indicate the repeatability of these measurements over several cardiac cycles and over five familial hypercholesterolemic swine. Our results also demonstrate the need for a small region of interest within the arterial walls for accurate and robust estimates of arterial function.

Antioxidant and antiplatelet properties of grape products are thought to be responsible for observed antiatherosclerotic effects. Diverse classes of phenolics are derived from the seed and skin (GSK) of grapes. The relative contributions of the classes of phenolics to observed properties of grape products are unknown. In this paper, GSK fractions were used to examine effects on platelet aggregation, low-density lipoprotein (LDL) oxidation in vitro, and relative binding of phenolics to LDL. GSK was separated into six fractions (fractions 1-6), and primary phenolics were characterized using high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Fractions 4, 5, and 6, enriched in polygalloyl polyflavan-3-ols (PGPFs) with 3-6, 4-8, and 6-15 degrees of polymerization, respectively, inhibited platelet aggregation. Fractions 1-3, containing various amounts of oligosaccharides, hydroxycinnamic acids, anthocyanins, flavanols, and low molecular weight PGPFs, significantly increased platelet aggregation. Fractions 4-6 were most effective in binding LDL and inhibiting LDL oxidation. Fractions 5 and 6 exhibited the greatest inhibition of platelet aggregation and LDL oxidation, suggesting that polymeric PGPFs are responsible for the beneficial effects of grape products. Conversely, phenolics in fractions 1-3 may reduce the net biological potency of the grape products and have undesirable effects on cardiovascular disease risk factors.

The 4-(dimethylamino)cinnamaldehyde (DMAC) assay is currently used to quantify proanthocyanidin (PAC) content in cranberry products. However, this method suffers from issues of accuracy and precision in the analysis and comparison of PAC levels across a broad range of cranberry products. Current use of procyanidin A2 as a standard leads to an underestimation of PACs content in certain cranberry products, especially those containing higher molecular weight PACs. To begin to address the issue of accuracy, a method for the production of a cranberry PAC standard, derived from an extraction of cranberry (c-PAC) press cake, was developed and evaluated. Use of the c-PAC standard to quantify PAC content in cranberry samples resulted in values that were 2.2 times higher than those determined by procyanidin A2. Increased accuracy is critical for estimating PAC content in relationship to research on authenticity, efficacy, and bioactivity, especially in designing clinical trials for determination of putative health benefits.

Background: Drug-coated balloons are rapidly emerging as a therapeutic alternative for the interventional treatment of peripheral vascular disease. The purpose of this study was to test the hypothesis that an angioplasty balloon coated with the mTOR inhibitor zotarolimus (ZCB) would inhibit neointimal hyperplasia in a novel injury-based superficial femoral artery model in the familial hypercholesterolemic swine., Methods and Results: A total of 44 familial hypercholesterolemic swine were included (12 designated to study tissue pharmacokinetics and 32 to study safety and efficacy). Fogarty balloon denudation was performed in all superficial femoral artery segments, followed by balloon angioplasty. In the pharmacokinetic study, a total of 24 ZCBs (300 μg/cm(2)) were used. Zotarolimus was detected in arterial tissue at 5 minutes (162 ng/mg of tissue), 24 hours (5.9 ng/mg of tissue), and 28 days (0.007 ng/mg of tissue) after ZCB inflation. In the safety and efficacy study, superficial femoral artery segments were randomized to either high-dose (600 μg/cm(2), n=16), low-dose (300 μg/cm(2), n=16), or paired uncoated balloons (high-dose ZCB control, n=16; low-dose ZCB control, n=16). At 28 days, the percentage of angiographic stenosis was similar among all tested groups. Histological analysis demonstrated a reduction in neointimal formation in both ZCB groups compared with controls (high-dose ZCB 44% reduction, P=0.007; low-dose ZCB 22% reduction, P=0.08). There was no evidence of delayed arterial healing or vascular toxicity in any of the ZCB groups., Conclusions: The single delivery of zotarolimus via coated balloon is feasible, and therapeutic levels are maintained up to 28 days. The ZCB technology appears to be effective in the reduction of neointimal proliferation in the superficial femoral artery of the 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/β-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.

Introduction: To date, most of all new developments in stent technologies are tested in normal animals. Although invaluable in the evaluation of device safety, the juvenile domestic swine (DS) do not follow the biological healing response occurring in humans following coronary stent implantation. By using a novel swine breed afflicted with familial hypercholesterolemia (FHS), we aimed to analyse the vascular response occurring following bare metal stent (BMS) implantation by comparing in vivo endovascular imaging and histological data., Methods: A total of 26 swine were included in this study (12 FHS and 14 DS). Sixty eight BMS (FHS=28 versus DS=40) were implanted using a 10% overstretch ratio. Imaging evaluation (IVUS and OCT) was conducted in all animals at 30 (n=14) or 90 (n=12) days following stent implantation. After imaging, the stented coronary segments were harvested for histological evaluation., Results: At 30 days, the degree of neointimal formation analysed by OCT (%AS=DS 21.9 ± 10% versus FHS 25.4 ± 12%; p=0.18) and histology (DS 24.6 ± 10% versus FHS 23.58 ± 10%; p=0.8) was similar between both animal groups. At 90 days, the degree of neointimal formation in the DS group decreased in all analysed variables (-40% in IVUS neointimal volume, -57% in OCT %AS, and -30% in %AS by histology) compared to the progression of neointimal formation observed in the FHS group (+29% in IVUS neointimal volume, +27% in OCT %AS and +43% in %AS by histology)., Conclusion: The pattern of neointimal formation following BMS implantation in the FHS follows a progressive course that does not occur in the DS. Therefore, by providing a progressive neointimal biological response to BMS implantation, the FHS could serve as an ideal efficacy model for the validation of drug eluting stent technologies., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

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.