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Persistent and symptomatic reflux of gastric and duodenal contents, known as gastroesophageal reflux disease (GERD), is the strongest risk factor for esophageal adenocarcinoma (EAC). Despite similar rates of GERD and other risk factors across racial groups, EAC progression disproportionately impacts Caucasians. We recently reported that elevated tissue levels of the detoxification enzyme GSTT2 in the esophagi of Blacks compared to Caucasians may contribute protection. Herein, we extend our research to investigate whether cranberry proanthocyanidins (C-PAC) mitigate bile acid-induced damage and GSTT2 levels utilizing a racially diverse panel of patient-derived primary esophageal cultures. We have shown that C-PACs mitigate reflux-induced DNA damage through GSTT2 upregulation in a rat esophageal reflux model, but whether effects are recapitulated in humans or differentially based on race remains unknown. We isolated normal primary esophageal cells from Black and Caucasian patients and assessed GSTT2 protein levels and cellular viability following exposure to a bile acid cocktail with and without C-PAC treatment. Constitutive GSTT2 levels were significantly elevated in Black (2.9-fold) compared to Caucasian patients, as were GSTT2 levels in Black patients with GERD. C-PAC treatment induced GSTT2 levels 1.6-fold in primary normal esophageal cells. GSTT2 induction by C-PAC was greatest in cells with constitutively low GSTT2 expression. Overall, C-PAC mitigated bile-induced reductions of GSTT2 and subsequent loss of cell viability regardless of basal GSTT2 expression or race. These data support that C-PAC may be a safe efficacious agent to promote epithelial fitness through GSTT2 induction and in turn protect against bile acid-induced esophageal injury.

Esophageal adenocarcinoma (EAC) is the major subtype of esophageal cancer in Westernized countries, characterized by rising incidence and poor prognosis with a five-year survival rate less than 20%. Only 15% of EAC patients have a complete histopathological response to standard chemotherapy, indicating the urgent need to identify mechanisms associated with therapeutic resistance for improved therapeutic efficacy and patient outcomes. Herein, we performed transcriptional profiling utilizing tissues collected from EAC patients prior to treatment initiation. First, EAC patients (n=67) were stratified into 5 therapeutic response groups based on the changes in TNM pre- to post-treatment; second, bioinformatic approaches were utilized to identify molecular changes associated with response to treatment and patient survival. Third, we employed OE19 and OE33 human EAC cell lines to investigate the efficacy of cranberry proanthocyanidins (C-PAC), a promising anti-inflammatory and anti-cancer agent which shows synergistic effects with carboplatin in other cancer types, at mitigating molecular pathways which drive therapeutic resistance. Our therapeutic response data set revealed epithelial-mesenchymal transition (EMT) as a top pathway associated with differential response to therapy and patient survival. Subsequent gene-set enrichment analysis identified that EMT is significantly associated with NFκB and STAT3 signaling pathways, two nodes with documented proinflammatory roles in EAC progression. Deconvolution analysis was conducted further supporting EMT enrichment creates an immune-suppressive tumor microenvironment in EAC patients. Next, EAC cell viability, migration and interrogation of EMT signaling proteins were conducted following treatment with paclitaxel and carboplatin (chemo drugs) alone or in combination with C-PAC. Combination treatment of C-PAC and chemo drugs significantly inhibited EAC cell viability and migration capability, compared to cells treated with chemo drugs alone, with synergistic effects observed in OE33 cells. Moreover, combination treatment also significantly decreased NFκB1 expression and expressions of EMT markers and transcription factors (Snail, MMP-2, MMP-9, and Vimentin) whereas cells treated only with chemo drugs showed minimal modulation. Knockdown experiments of NFκB1 using RNA interference are underway to investigate the relationship between canonical NFκB1 signaling and EMT. Primary cell lines and organoids developed from isolated EAC patient tissues will also be used in the future to validate experimental findings. The results suggest C-PAC may be used as a safe and novel therapeutic strategy to improve chemotherapy efficacy targeting EAC. Citation Format: Yun Zhang, Katherine M. Weh, Connor L. Howard, Jean-Jack Riethoven, Jennifer L. Clarke, Michelle P. Lee, Kiran H. Lagisetty, Jules Lin, Rishindra M. Reddy, Andrew C. Chang, Amy B. Howell, Laura A. Kresty. Targeting therapeutic resistance of esophageal adenocarcinoma through modulating the epithelial-to-mesenchymal transition via NFκB-mediated signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1745.

Blueberry polyphenols are known for their high antioxidant and antimicrobial potential. Aichi virus (AiV) is an emerging human enteric virus that causes gastroenteritis outbreaks worldwide. This study aimed to (1) determine the time- and dose-dependent effects of blueberry proanthocyanidins (B-PAC) against AiV over 24 h at 37 °C; (2) gain insights on their mode of action using pre- and post-treatment of host cells and Transmission Electron Microscopy; and (3) determine their anti-AiV effects in model foods and under simulated gastric conditions. AiV at ∼5 log PFU/ml was incubated with equal volumes of commercial blueberry juice (BJ, pH 2.8), neutralized BJ (pH 7.0), B-PAC (2, 4, and 10 mg/ml) prepared either in 10% ethanol, apple juice (AJ), 2% milk, simulated gastric fluid (SGF, pH 1.5) or simulated intestinal fluid (SIF, pH 7.5), and controls (malic acid (pH 3.0), phosphate buffered saline (pH 7.2), apple juice (pH 3.6) and 2% milk) over 24 h at 37 °C, followed by standard plaque assays. Each experiment was replicated thrice and data were statistically analyzed. Differences in AiV titers with 1 mg/ml B-PAC were 2.13 ± 0.06 log PFU/ml lower after 24 h and ≥3 log PFU/ml (undetectable levels) lower with 2 and 5 mg/ml B-PAC compared to AiV titers in PBS after 24 h and 3 h, respectively. BJ at 37 °C resulted in titer differences (lower titers compared to PBS) of 0.17 ± 0.06, 1.27 ± 0.01, and 1.73 ± 0.23 log PFU/ml after 1, 3, and 6 h and ≥3 log PFU/ml after 24 h. Pre- and post-treatment of host cells with 0.5 mg/ml B-PAC caused titer decreases of 0.62 ± 0.33 and 0.30 ± 0.06 log PFU/ml, respectively suggesting a moderate effect on viral-host cell binding. B-PAC at 2 mg/ml in AJ caused titer differences of ≥3 log PFU/ml after 0.5 h, while differences of 0.84 ± 0.03 log PFU/ml with 5 mg/ml B-PAC in milk, and ≥3 log PFU/ml with B-PAC at 5 mg/ml in SIF after 30 min were obtained. This study shows the ability of BJ and B-PAC to decrease AiV titers to potentially prevent AiV-related illness and outbreaks.

A number of clinical trials support the use of standardized cranberry supplement products for prevention of urinary tract infections; however, products that are not well-characterized for sufficient levels of bioactive components may contribute to negative clinical outcomes. Cranberry supplements for consumer use are not regulated and can be formulated different ways using cranberry juice, pomace or various combinations. This can lead to consumer confusion regarding effectiveness of individual products. The current study compared two commercial supplement products, one made from cranberry juice extract and the other from a blend of whole cranberry. The influence of formulation and proanthocyanidin (PAC) solubility on in vitro and ex vivo P-fimbriated Escherichia coli bacterial anti-adhesion activity (AAA) was determined. Both supplement products as well as whole, frozen cranberries were chromatographically separated into crude polyphenolic, sugar and acid fractions. In vitro AAA testing of all fractions confirmed that only those containing soluble PACs elicited activity. The cranberry juice extract product had higher soluble PAC content than the whole cranberry blended product, which contained mainly insoluble PACs. The influence of soluble and insoluble PAC levels in each product on the urinary (ex vivo) AAA was determined following ingestion. The juice extract product was associated with significantly higher urinary AAA than that of the whole berry blended product when consumed once daily over the 1-week intervention period.

Blueberry (BB) consumption is linked to improved health. The bioconversion of the polyphenolic content of BB by fermentative bacteria in the large intestine may be a necessary step for the health benefits attributed to BB consumption. The identification of specific gut microbiota taxa that respond to BB consumption and that mediate the bioconversion of consumed polyphenolic compounds into bioactive forms is required to improve our understanding of how polyphenols impact human health. We tested the ability of polyphenol-rich fractions purified from whole BB&mdash, namely, anthocyanins/flavonol glycosides (ANTH/FLAV), proanthocyanidins (PACs), the sugar/acid fraction (S/A), and total polyphenols (TPP)&mdash, to modulate the fecal microbiota composition of healthy adults in an in vitro colon system. In a parallel pilot study, we tested the effect of consuming 38 g of freeze-dried BB powder per day for 6 weeks on the fecal microbiota of 17 women in two age groups (i.e., young and older). The BB ingredients had a distinct effect on the fecal microbiota composition in the artificial colon model. The ANTH/FLAV and PAC fractions were more effective in promoting microbiome alpha diversity compared to S/A and TPP, and these effects were attributed to differentially responsive taxa. Dietary enrichment with BB resulted in a moderate increase in the diversity of the microbiota of the older subjects but not in younger subjects, and certain health-relevant taxa were significantly associated with BB consumption. Alterations in the abundance of some gut bacteria correlated not only with BB consumption but also with increased antioxidant activity in blood. Collectively, these pilot data support the notion that BB consumption is associated with gut microbiota changes and health benefits.

OBJECTIVES: Cranberry products are often used by women to prevent urinary tract infections (UTIs) but literature regarding their efficacy remains discordant. The clinical efficacy of cranberry products for prevention of UTIs in healthy women could be increased by 1) using an optimal dose of cranberry extract standardized in proanthocyanidins (PACs); 2) adequate measure of compliance. The objective of this randomized clinical trial was to assess the efficacy of an optimal dose of cranberry extract standardized in PACs for the prevention of recurrent UTI confirmed by pyuria during a 24-week intervention period. METHODS: One hundred and forty-five women with a history of recurrent UTI in the past 12 months were randomized to receive a dose of cranberry extract Urophenol™, standardized in PACs (2 × 18.5 mg PACs daily, n = 72) or a control dose (2 × 1 mg PACs daily, n = 73) for a 24-week period. Women were asked to contact the study coordinator if they presented symptoms of UTI and to provide a urine sample to evaluate the diagnosis by confirmation of pyuria. Since some women were unable to provide a urine sample in the presence of UTI symptoms, regression analyses were performed with two different approaches, 1) symptomatic UTI episodes without a urine sample were considered as no symptomatic UTIs with pyuria; 2) symptomatic UTI episodes without a urine sample were considered as symptomatic UTIs with pyuria. Analyses were adjusted for age. RESULTS: No significant differences in the number of symptomatic UTI with pyuria were found between groups during the 24-week intervention period in intention to treat analyses using either approach (approach 1: incidence rate ratio 0.98, 95% CI 0.57–1.68; approach 2: 0.86, 95% CI 0.56–1.32). Among women who experienced less than 5 UTIs in the year preceding enrolment, daily consumption of 2 × 18.5 mg PACs was associated with a decrease in the number of symptomatic UTIs compared to the control dose (approach 1: incidence rate ratio 0.65, 95% CI 0.31–1.37; approach 2: 0.54, 95% CI 0.30–0.99). Compliance to capsule intake was higher than 90% in both groups. CONCLUSIONS: In women who experience less than 5 UTIs in the last year, daily consumption of a standardized dose of 2 × 18.5 mg PACs could have a preventative impact on the incidence of symptomatic UTIs confirmed by pyuria. FUNDING SOURCES: Supported by Diana Foods and MAPAQ.

The juice of the fruit of the American cranberry (Vaccinium macrocarpon Ait.) is by far the most popular and widely used botanical preparation for the prevention and treatment of urinary tract infections. To date, most of the medical research on cranberry consists of observational clinical trials yielding mixed results or preclinical studies, the latter focusing on mechanisms of action, many of which are relevant to the putative benefits attributed to cranberry. Cranberry benefits are primarily linked to the presence of proanthocyanidin (PAC) oligomers, also referred to as condensed tannins or polyflavan-3-ols, and their capacity to prevent bacteria, particularly E. coli, from adhering to uroepithelial cells. In the following, we present a summary of the state of cranberry research in the context of urinary tract health with focus on data derived from clinical settings. The overall consensus from recent reviews and meta-analyses is that there are beneficial effects of cranberry consumption on reducing risk of UTI recurrence.

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-hour 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 = 0.04), 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 contribute to the observed effects on the parasites. This first study of the in vitro and in vivo effects of CV sugges 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.

Esophageal adenocarcinoma (EAC) is a devastating cancer characterized by rising incidence and poor prognosis. The five-year survival rate is 20% due to late stage diagnosis and ineffective treatment. It is estimated that only 15% of EAC patients have a complete histopathological response to standard chemotherapy. Thus, new strategies are urgently needed to improve therapeutic efficacy and patient survival. Our recent analysis of prechemotherapy endoscopic biopsies of EAC patients revealed differential response to neoadjuvant therapy. Differential response was reflected by two-year mortality of 9.1% in therapy responsive patients (Complete responders, no residual tumor), 22.2% in strong responders (improved TNM), 25.0% in intermediate responders (stable TNM), 42.9% in poor responders (worse TNM) and 77.8% in non-responders (worse TNM and metastasis). Transcriptomic analysis revealed differential response to therapy is linked to immune defense, inflammation (IL-4, IFNs), cell adhesion, signal transduction and angiogenesis regulation. Similar pathways were mitigated by cranberry proanthocyanidins (C-PAC) in a rat surgical model for EAC. Herein, we investigated C-PAC pretreatment alone or in combination with Paclitaxel and Carboplatin (chemo drug) on EAC cell viability (Calcein-AM), gene expression (RNA-seq) and select protein levels (Western blot). C-PAC pre-treatment and co-treatment enhance chemotherapy-induced EAC cell death, with the greatest synergistic effects in chemo-resistant OE33 cells. C-PAC pretreatment followed by chemo drugs decreased cell viability by an additional 50% and 17% compared to chemo drugs alone in OE33 cells and OE19 cells, respectively. Greater reductions were noted with pretreatment of C-PAC, followed by co-treatment with chemo drugs (64.9% in OE33 and 49.4% in OE19 cells). C-PAC enhanced chemo drug-induced EAC cell death via increased DNA damage, apoptosis and ECM degradation as evidenced by induction of phosphorylated H2AX and cleaved caspase-3, as well as inhibition of BCLXL and MMP9. C-PAC also mitigated elevated levels of P53, a gene with a well-documented role in EAC progression and therapeutic resistance. Transcriptome analysis of cells treated with C-PAC and chemo drugs is underway and will be compared to signaling networks differentially expressed in patients stratified by treatment responsiveness. Results suggest that C-PAC pretreatment may offer a non-toxic intervention strategy for enhancing chemotherapeutic efficacy. Citation Format: Yun Zhang, Katherine Weh, Connor Howard, Kiran Lagisetty, Dyke McEwen, Jules Lin, Rishindra M. Reddy, Andrew Chang, David G. Beer, Amy Howell, Laura A. Kresty. Proanthocyanidins enhance chemotherapy-induced esophageal adenocarcinoma cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1418.

Persistent and symptomatic reflux of gastric and duodenal contents, known as gastroesophageal reflux disease (GERD), is the strongest risk factor for EAC development. GERD and esophagitis occur at similar rates among Blacks and Caucasians; yet, progression to EAC is significantly elevated among Caucasians. Unique protective factors in the epithelium of Blacks may contribute to this disparity. Our research team recently reported that the detoxification enzyme GSTT2 is higher in the esophageal squamous epithelium of Blacks compared to Caucasians with potential linkages to previously identified genomic variants in the GSTT2 locus (a 37 kb deletion and a 17 bp promoter duplication among Caucasians). Thus, the current study seeks to evaluate whether primary esophageal cell cultures isolated from Black or Caucasian cohorts can serve as discerning and relevant model systems to investigate risk factors linked to EAC progression, assess efficacy of mitigating agents and differential responses linked to race. We have shown that cranberry proanthocyanidins (C-PAC) mitigate DNA damage associated with reflux through upregulation of GSTT2 in a rat surgical model of reflux-induced EAC, but whether effects are recapitulated in humans or differentially based on race remains unknown. Herein we isolated normal primary esophageal epithelial cells from Black and Caucasian patients and assessed GSTT2 genotype, GSTT2 protein levels and cellular viability following exposure of the cultures to a bile acid cocktail (BAC) [0.2mM] with and without C-PAC [50µg/ml] treatment. Constitutive levels of GSTT2 were 1.7-fold higher in Blacks than Caucasians, with 71% of Blacks identified as high expressors compared to 33% of Caucasians. Pretreatment (48h) of primary cultures with C-PAC induced GSTT2 levels in all but one Black-derived culture which already expressed high basal levels. GSTT2 induction in normal epithelial cultures by C-PAC was greatest in cells with constitutively low GSTT2 expression; however, upon BAC challenge C-PAC effectively mitigated BAC-induced reductions in GSTT2 levels and subsequent loss of normal cell viability regardless of basal GSTT2 expression or race. C-PAC treatment pre- plus post-BAC imparted no additional benefit over pretreatment alone in preserving viability but did further increase GSTT2 levels. Next steps include expanding our panel of primary cultures and conducting nano LC-MS/MS proteomic profiling of Black and Caucasian-derived cultures treated with vehicle, BAC, C-PAC and BAC + C-PAC, with stratification based on GSTT2 basal expression. Taken together these data support that C-PAC may be used as an efficacious non-toxic agent serving to promote epithelial fitness and resiliency against the biologic and molecular sequelae linked bile acid-induced esophageal injury and progression to EAC. Citation Format: Katherine M. Weh, Danielle K. Turgeon, Amy Howell, Laura A. Kresty. Racially diverse primary esophageal cell cultures for evaluating mitigation of bile-induced injury [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2593.

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium that has been strongly associated with localized aggressive periodontitis. The capacity of A. actinomycetemcomitans to produce a leukotoxin (LtxA) that activates pyroptosis in macrophages and induces the release of endogenous danger signals is thought to play a key role in the disease process. The aim of the present study was to investigate the effects of cranberry proanthocyanidins (PACs) on gene expression and cytotoxic activities of LtxA. We showed that cranberry PACs dose-dependently attenuate the expression of genes making up the leukotoxin operon, including ltxB and ltxC, in the two strains of A. actinomycetemcomitans tested. Cranberry PACs (&ge, 62.5 &micro, g/mL) protected macrophages against the cytotoxic effect of purified LtxA. Moreover, cranberry PACs reduced caspase-1 activation in LtxA-treated macrophages and consequently decreased the release of both IL-1&beta, and IL-18, which are known as damage-associated molecular patterns (DAMPs) and contribute to the progression of periodontitis by increasing cell migration and osteoclastogenesis. In addition, cranberry PACs reduced the expression of genes encoding the P2X7 receptor and NALP3 (NACHT, LRR and PYD domains-containing protein 3), which play key roles in pore formation and cell death. Lastly, cranberry PACs blocked the binding of LtxA to macrophages and consequently reduced the LtxA-mediated cytotoxicity. In summary, the present study showed that cranberry PACs reduced LtxA gene expression in A. actinomycetemcomitans and neutralized the cytolytic and pro-inflammatory responses of human macrophages treated with LtxA. Given these properties, cranberry PACs may represent promising molecules for prevention and treatment of the aggressive form of periodontitis caused by A. actinomycetemcomitans.

Urinary tract infections (UTIs) are one of the common bacterial infections treated with antibiotics. The North American cranberry is recommended for prophylaxis in women with recurrent UTIs as a nutritional alternative. The ability of cranberry components and their metabolites to inhibit adhesion of uropathogenic Escherichia coli (E. coli) is an important mechanism by which cranberry mitigates UTIs. The objective of this study was to evaluate urinary anti-adhesion activity against type 1 and P-type uropathogenic E. coli after consumption of cranberry +health™ cranberry supplement (cranberry chew). In this randomized, double-blind, placebo-controlled, crossover design pilot trial (n = 20), subjects consumed two cranberry or placebo chews, one in the morning and one in the evening. Clean-catch urine samples collected at the baseline and post-intervention (0–3, 3–6, 6–9, 9–12, 12–24, 24–30, 30–36 h) were tested for anti-adhesion effects with a mannose-resistant human red blood cell hemagglutination assay specific for P-type E. coli, or a T24 cell line model for type 1 E. coli. Urinary anti-adhesion activity against P-type E. coli after consumption of the cranberry chew was significantly greater (p < 0.05) than that observed with placebo chew at all time points except 24–36 h. Ex vivo anti-adhesion effects on type 1 E. coli were greater (p < 0.05) after cranberry chew consumption than placebo chew at 3–6 and 6–9 h urine collections. In conclusion, consumption of cranberry +health™ cranberry supplement exhibited greater ex vivo urinary anti-adhesion activity compared to placebo, suggesting that it may have the potential to help promote urinary tract health.

Background We recently reported that a cranberry proanthocyanidin rich extract (C-PAC) induces autophagic cell death in apoptotic resistant esophageal adenocarcinoma (EAC) cells and necrosis in autophagy resistant cells. EAC is characterized by high morbidity and mortality rates supporting development of improved preventive interventions. Objective The current investigation sought to investigate the role of reactive oxygen species (ROS) in the context of C-PAC induced cell death. Methods A panel of human esophageal cell lines of EAC or BE (Barrett's esophagus) origin were treated with C-PAC and assessed for ROS modulation using CellROX® Green reagent and the Amplex Red assay to specifically measure hydrogen peroxide levels. Results C-PAC significantly increased ROS levels in EAC cells, but significantly reduced ROS levels in CP-C BE cells. Increased hydrogen peroxide levels were also detected in C-PAC treated EAC cells and supernatant; however, hydrogen peroxide levels were significantly increased in medium alone, without cells, suggesting that C-PAC interferes or directly acts on the substrate. Hydrogen peroxide levels did not change in C-PAC treated CP-C BE cells. Conclusion These experiments provide additional mechanistic insight regarding C-PAC induced cancer cell death through modulation of ROS. Additional research is warranted to identify specific ROS species associated with C-PAC exposure.

Blueberry and blueberry extracts are known for their health benefits and antimicrobial properties. Natural therapeutic or preventive options to decrease the incidences of foodborne viral illnesses are becoming popular and being researched. This study aimed to determine the antiviral effects of blueberry juice (BJ) and blueberry proanthocyanidins (BB-PAC, B-type PAC structurally different from A-type PAC found in cranberries) against the infectivity of hepatitis A virus (HAV) and human norovirus surrogates (feline calicivirus (FCV-F9) and murine norovirus (MNV-1)) at 37 °C over 24 h using standard plaque assays. Viruses at ~5 log PFU/ml were mixed with equal volumes of BJ (pH 2.8), neutralized BJ (pH 7.0), BB-PAC (1, 2, 4, and 10 mg/ml), malic acid (pH 3.0), or phosphate-buffered saline (pH 7.2) and incubated over 24 h at 37 °C. Each experiment was carried out in duplicate and replicated thrice. FCV-F9 titers were found to be reduced to undetectable levels with 1 and 2 mg/ml BB-PAC after 5 min, with 0.5 mg/ml BB-PAC after 1-h, and with BJ after 3-h. MNV-1 titers were reduced to undetectable levels after 3 h with 1, 2, and 5 mg/ml BB-PAC and after 6 h with BJ. HAV titers were reduced to undetectable levels after 30 min with 2 and 5 mg/ml BB-PAC, after 3 h with 1 mg/ml BB-PAC, and by ~2 log PFU/ml with BJ after 24-h. BB-PAC shows preventive potential against infection by the tested enteric viruses in a dose- and time-dependent manner, although further in vitro studies in model food systems and in vivo studies using animal models are warranted.

Strong evidence points to Porphyromonas gingivalis, a Gram-negative anaerobic bacterium, as a keystone species in the development of the chronic form of periodontitis. The aim of the present study was to investigate the ability of highbush blueberry proanthocyanidins (PACs) to alleviate the P. gingivalis-induced deleterious effects on oral mucosal cells. We first showed that highbush blueberry PACs protect the integrity of the gingival keratinocyte barrier against P. gingivalis-mediated damage, as determined by measuring the transepithelial electrical resistance and paracellular flux of FITC-conjugated dextran. Moreover, the PACs prevented the translocation of P. gingivalis across the gingival keratinocyte barrier model. The proteinase activity of P. gingivalis was inhibited by the PACs suggesting that they may exert beneficial effects by reducing proteolytic degradation of the epithelial tight junctions. Regulation of gingival fibroblast inflammatory reactions may be one of the ways to prevent and control periodontal disease progression and severity. We showed that PACs significantly reduce IL-6 and IL-8 secretion by P. gingivalis-stimulated gingival fibroblasts. The present study showed the capacity of highbush blueberry PACs to protect the integrity of an in vitro model of gingival keratinocyte barrier against P. gingivalis, and to attenuate the secretion of pro-inflammatory cytokines by gingival fibroblasts infected with P. gingivalis. These results suggest beneficial effects of blueberry PACs thus supporting the need for future clinical trials on the potential of these bioactive molecules for periodontal disease prevention and/or treatment.

Our laboratory studies chemoprevention of esophageal adenocarcinoma (EAC) through utilization of the rat esophagogastroduodenal anastomosis (EGDA) surgical model of reflux-induced EAC. Specifically, we evaluated mechanisms by which cranberry proanthocyanidins (C-PAC) inhibit reflux-induced EAC by utilizing multi-omics integrative approaches employing multiple program platforms. Herein, we investigated whether pathway-based integration could be used to examine cross-talk between genes, metabolites and the microbial profiles. For this analysis, we utilized transcriptomic and untargeted metabolomic data from rat esophagi of animals that received water or C-PAC in the drinking water alone or combined with reflux inducing EGDA surgery. Additionally, we isolated DNA from fecal pellets and performed 16S rRNA sequencing to assess gut microbiome composition and functionality. Each omics dataset was analyzed for significant pathway enrichment and network generation using Metacore, DAVID and Metabolync. PICRUSt was utilized to predict microbiota functionality. Analysis of transcriptomic and metabolomic data suggest that EGDA upregulates inflammatory, NF-kB, DNA damage, cell cycle and immune function pathways as well as metabolic pathways related to eicosanoids, amino acids and primary and secondary bile acids, while C-PAC mitigates these alterations. Preliminary microbiome data analysis also suggests that bacteria related to inflammation, metabolite transport and DNA damage are increased in abundance in EGDA, with a decrease in abundance following C-PAC treatment. Analysis of omics datasets can provide insightful relationships into the cross-talk between different processes regulated by transcription, metabolism and the microbiome. Single dataset and integrative analysis showed similarities in altered pathways for vehicle and C-PAC treated animals in the context of reflux. Integration is limited by the infancy of multi-omics analysis programs, as well as the limited amount of research involving prediction of bacterial function and integration of microbiome data with other omics datasets. Further research and development of these omics programs is needed to determine accuracy of predicted results as well as continued investigation of identified pathways. Citation Format: Katherine M. Weh, Connor L. Howard, Bridget A. Tripp, Jennifer L. Clarke, Amy B. Howell, Laura A. Kresty. Omics integration provides insight into esophageal cancer inhibitory mechanisms of a cranberry extract [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6588.

Candida albicans is one of the most common causes of hospital-acquired urinary tract infections (UTIs). However, azoles are poorly active against biofilms, echinocandins do not achieve clinically useful urinary concentrations, and amphotericin B exhibits severe toxicities. Thus, novel strategies are needed to prevent Candida UTIs, which are often associated with urinary catheter biofilms. We previously demonstrated that cranberry-derived proanthocyanidins (PACs) prevent C. albicans biofilm formation in an in vitro urinary model. To elucidate functional pathways unique to urinary biofilm development and PAC inhibition, we investigated the transcriptome of C. albicans in artificial urine (AU), with and without PACs. C. albicans biofilm and planktonic cells were cultivated with or without PACs. Genome-wide expression analysis was performed by RNA sequencing. Differentially expressed genes were determined using DESeq2 software; pathway analysis was performed using Cytoscape. Approximately 2,341 of 6,444 total genes were significantly expressed in biofilm relative to planktonic cells. Functional pathway analysis revealed that genes involved in filamentation, adhesion, drug response and transport were up-regulated in urinary biofilms. Genes involved in carbon and nitrogen metabolism and nutrient response were down-regulated. In PAC-treated urinary biofilms compared to untreated control biofilms, 557 of 6,444 genes had significant changes in gene expression. Genes downregulated in PAC-treated biofilms were implicated in iron starvation and adhesion pathways. Although urinary biofilms share key features with biofilms formed in other environments, many genes are uniquely expressed in urinary biofilms. Cranberry-derived PACs interfere with the expression of iron acquisition and adhesion genes within urinary biofilms.

Cranberries are rich in bioactive constituents known to improve urinary tract health and more recent evidence supports cranberries possess cancer inhibitory properties. However, mechanisms of cancer inhibition by cranberries remain to be elucidated, particularly in vivo. Properties of a purified cranberry-derived proanthocyanidin extract (C-PAC) were investigated utilizing acid-sensitive and acid-resistant human esophageal adenocarcinoma (EAC) cell lines and esophageal tumor xenografts in athymic NU/NU mice. C-PAC induced caspase-independent cell death mainly via autophagy and low levels of apoptosis in acid-sensitive JHAD1 and OE33 cells, but resulted in cellular necrosis in acid-resistant OE19 cells. Similarly, C-PAC induced necrosis in JHAD1 cells pushed to acid-resistance via repeated exposures to an acidified bile cocktail. C-PAC associated cell death involved PI3K/AKT/mTOR inactivation, pro-apoptotic protein induction (BAX, BAK1, deamidated BCL-xL, Cytochrome C, PARP), modulation of MAPKs (P-P38/P-JNK) and G2-M cell cycle arrest in vitro. Importantly, oral delivery of C-PAC significantly inhibited OE19 tumor xenograft growth via modulation of AKT/mTOR/MAPK signaling and induction of the autophagic form of LC3B supporting in vivo efficacy against EAC for the first time. C-PAC is a potent inducer of EAC cell death and is efficacious in vivo at non-toxic behaviorally achievable concentrations, holding promise for preventive or therapeutic interventions in cohorts at increased risk for EAC, a rapidly rising and extremely deadly malignancy.

In this study, we examined the ex vivo urinary anti-adhesion activity of low-calorie cranberry extract beverages in both a pilot study (n = 10) and a randomized, double-blind, placebo controlled clinical trial (n = 59). In the pilot study, subjects consumed a cranberry extract beverage (CEB) or a cranberry extract and juice beverage (CEJB), compared to placebo. Both cranberry beverages utilized a standardized cranberry extract powder at a level equivalent to low-calorie cranberry juice cocktail (LCJC) on a PAC content basis. Clean-catch urine samples collected at baseline and post intervention were tested for anti-adhesion activity utilizing a mannose-resistant human red blood cell hemagglutination assay specific for P-fimbriated E. coli. Results from the pilot study indicated that ex vivo anti-adhesion activity for both cranberry treatments were higher (p < 0.05) than placebo. In the clinical trial, we compared CEJB to LCJC and a placebo beverage. Post-consumption urine from both cranberry treatment groups showed significantly higher (p < 0.05) anti-adhesion activity compared to placebo. There were no differences observed in anti-adhesion activity between CJEB and LCJC, indicating similar bioactivity. Therefore, acute beverage consumption of cranberry extract and/or juice provides ex vivo anti-adhesion activity, which may help to improve urinary tract health.

Concise summary Given that the laryngopharynx is more susceptible to damage by gastric refluxate compared to the esophagus and the lack of intrinsic defense mechanisms present in their esophageal counterparts (such as peristalsis, saliva, and bicarbonate production), it is plausible that chronic uncontrolled laryngopharyngeal reflux (LPR) could not only cause inflammatory, but perhaps also neoplastic, pathologies in the laryngopharynx, as it does in the esophagus. Patients with LPR have pepsinmediated laryngeal mucosal injury, including dilation of intracellular spaces and mitochondrial damage. Pepsin is taken up by laryngeal epithelial cells through receptor-mediated endocytosis. Receptors and their ligands are typically sorted in late endosomes and the transreticular Golgi (TRG). Using immunoelectron microscopy, colocalization of pepsin with Rab-9 and TRG-46, markers of late endosomes, and the transreticular Golgi, respectively, was documented. These findings reveal an entirely novel mechanismforpepsin-induced cellular injury and may explain why many patients with reflux-attributed laryngeal injury and disease often have persistent symptoms despite maximal acidsuppression therapy and association with nonacid reflux. Thus, the activity/stability data and finding of pepsin uptake and intracellular pepsin reveal a role for pepsin in extraesophageal reflux, where the acidity of the refluxate may not be as clinically relevant.

Blueberry juice and blueberry polyphenols reportedly have antimicrobial properties against foodborne pathogens, without much currently known on their effects against Cronobacter sakazakii. This study evaluated the antimicrobial effects of blueberry proanthocyanidins (PAC) and commercial blueberry juice (BJ) against two strains of C. sakazakii, ATCC 29004 and 29544. BJ (pH 2.8), blueberry PAC (5 mg/ml) and controls (phosphate buffered saline (PBS), pH 7.2, and malic acid pH 3.0) were mixed with equal volumes of washed overnight cultures of C. sakazakii and incubated for 30 min, 1 h, 3 h and 6 h at 37°C. Reductions of ∼1 and 1.50 log CFU/ml were obtained for strains 29004 and 29544, respectively after 30 min with BJ or blueberry PAC. Both C. sakazakii strains 29004 and 29544 were reduced to undetectable levels from 8.25 ± 0.12 log CFU/ml and 8.48 ± 0.03 log CFU/ml, respectively with BJ (pH 2.8) or blueberry PAC after 1 h, while malic acid (pH 3.0) showed ∼1.3 log CFU/ml reduction for both strains. Scanning electron microscopy studies showed differences in cell membrane morphology with clumping and formation of blebs of the treated strains compared to untreated controls. These results warrant further in vivo studies with blueberry bioactives to determine potential for preventing and treating C. sakazakii infections.

Esophageal adenocarcinoma (EAC) is characterized by rising incidence rates and high mortality due to late stage diagnosis and a lack of efficacious options for prevention and treatment. While reasons for the rapid increase in EAC are being unraveled, persistent, symptomatic reflux of gastric and duodenal contents, known as gastroesophageal reflux disease is considered the strongest risk factor. Our laboratory utilizes a rat surgical model of reflux-induced EAC to evaluate mechanisms by which cranberry proanthocyanidins (C-PAC) delivered in the drinking water inhibit reflux-induced EAC. Findings show that C-PAC (690ug/rat/day) inhibits EAC formation by 83% at 40 weeks of study and mechanisms of inhibition are under investigation. We utilized nano LC-MS/MS proteomic profiling to identify proteins that were dysregulated due to reflux-induced EAC and reversed with C-PAC treatment. Proteomic profiling revealed that C-PAC treatment restored 63 proteins dysregulated in the context of reflux. EIF3F, PSMD2 and ACTR2 were the top proteins up-regulated by reflux; whereas, top markers restored by C-PAC included GSTT2, OGN and PCMT1. Metacore integrated software showed top pathways up-regulated by reflux and down-regulated C-PAC included Translation_regulation of translation initiation (EIF-linked), Immune response and Regulation of telomere length. Conversely, C-PAC treatment upregulated the glutathione metabolism pathway which is consistent with GSTT2 restoration. Disease enrichment analysis revealed multiple gastrointestinal tract diseases linked to reflux, which C-PAC down regulated supporting that proteomic profiling may inform other potential disease targets for C-PAC. Finally, beyond the 63 reflux-driven proteins C-PAC reversed, we considered proteins, pathways and processes which could not be reversed by C-PAC (n=269). As an example, Process networks upregulated by reflux (not reversed by C-PAC) included Translation initiation (RPL-linked), Elongation, mRNA processing, G2-M and S-phase of the cell cycle. Thus, proteomics profiling may inform the development of complementary preventive combinations for improved cancer inhibitory efficacy, especially for a cancer like EAC, with high mutational burden. Western blot analysis of several markers in the rat esophagus including GSTT2 and COX2 support the data obtained through proteomic profiling. Future directions include interrogating specific pathways that are highly modulated by reflux-induced EAC and restored by C-PAC including DNA damage, repair and extracellular matrix and adhesion. Last, proteomic profiling has also allowed for identification of post-translational modifications which may provide insight into regulation of key dysregulated proteins. Citation Format: Katherine M. Weh, Connor L. Howard, Amy B. Howell, Jennifer L. Clarke, Laura A. Kresty. Proteomic profiling reveals chemopreventive targets in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5077.

Defatted soy flour (DSF), soy protein isolate (SPI), hemp protein isolate (HPI), medium roast peanut flour (MPF) and pea protein isolate (PPI) stably bind and concentrate cranberry (CB) polyphenols, creating protein/polyphenol-enriched matrices. Proanthocyanidins (PAC) in the enriched matrices ranged from 20.75 mg/g (CB-HPI) to 10.68 mg/g (CB-SPI). Anthocyanins (ANC) ranged from 3.19 mg/g (CB-DSF) to 1.68 mg/g (CB-SPI), while total phenolics (TP) ranged from 37.61 mg/g (CB-HPI) to 21.29 mg/g (CB-SPI). LC-MS indicated that the enriched matrices contained all identifiable ANC, PAC and flavonols present in CB juice. Complexation with SPI stabilized and preserved the integrity of the CB polyphenolic components for at least 15 weeks at 37 °C. PAC isolated from enriched matrices demonstrated comparable anti-adhesion bioactivity to PAC isolated directly from CB juice (MIC 0.4 to 0.16 mg/mL), indicating their potential utility for maintenance of urinary tract health. Approximately 1.0 g of polyphenol-enriched matrix delivered the same amount of PAC available in one cup (300 mL) of commercial CB juice cocktail; which has been shown clinically to be the prophylactic dose for reducing recurring urinary tract infections. CB-SPI inhibited gram- positive and gram-negative bacterial growth. Nutritional and sensory analyses indicated that the targeted CB-matrix combinations have high potential for incorporation in functional food formulations.

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.

Proanthocyanidins and ellagitannins, referred to as "tannins", exist in many plant sources. These compounds interact with proteins due to their numerous hydroxyl groups, which are suitable for hydrophobic associations. It was hypothesized that tannins could bind to the digestive enzymes α-amylase and glucoamylase, thereby inhibiting starch hydrolysis. Slowed starch digestion can theoretically increase satiety by modulating glucose "spiking" and depletion that occurs after carbohydrate-rich meals. Tannins were isolated from extracts of pomegranate, cranberry, grape, and cocoa and these isolates tested for effectiveness to inhibit the activity of α-amylase and glucoamylase in vitro. The compositions of the isolates were confirmed by NMR and LC/MS analysis, and tannin-protein interactions were investigated using relevant enzyme assays and differential scanning calorimetry (DSC). The results demonstrated inhibition of each enzyme by each tannin, but with variation in magnitude. In general, larger and more complex tannins, such as those in pomegranate and cranberry, more effectively inhibited the enzymes than did less polymerized cocoa tannins. Interaction of the tannins with the enzymes was confirmed through calorimetric measurements of changes in enzyme thermal stability.

Blueberry proanthocyanidins (B-PAC) are known to decrease titers of human norovirus surrogates in vitro. The application of B-PAC as therapeutic or preventive options against foodborne viral illness needs to be determined using model foods and simulated gastric conditions in vitro. The objective of this study was to evaluate the antiviral effect of B-PAC in model foods (apple juice (AJ) and 2% reduced fat milk) and simulated gastrointestinal fluids against cultivable human norovirus surrogates (feline calicivirus; FCV-F9 and murine norovirus; MNV-1) over 24 h at 37 °C. Equal amounts of each virus (5 log PFU/ml) was mixed with B-PAC (1, 2 and 5 mg/ml) prepared either in AJ, or 2% milk, or simulated gastric fluids and incubated over 24 h at 37 °C. Controls included phosphate buffered saline, malic acid (pH 7.2), AJ, 2% milk or simulated gastric and intestinal fluids incubated with virus over 24 h at 37 °C. The tested viruses were reduced to undetectable levels within 15 min with B-PAC (1, 2 and 5 mg/ml) in AJ (pH 3.6). However, antiviral activity of B-PAC was reduced in milk. FCV-F9 was reduced by 0.4 and 1.09 log PFU/ml with 2 and 5 mg/ml B-PAC in milk, respectively and MNV-1 titers were reduced by 0.81 log PFU/ml with 5 mg/ml B-PAC in milk after 24 h. B-PAC at 5 mg/ml in simulated intestinal fluid reduced titers of the tested viruses to undetectable levels within 30 min. Overall, these results show the potential of B-PAC as preventive and therapeutic options for foodborne viral illnesses.

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.

The present study demonstrated that defatted soybean flour (DSF) can sorb polyphenols from blueberry and cranberry juices while separating them from sugars. Depending on DSF concentration and juice dilution, the concentration of blueberry anthocyanins and total polyphenols sorbed to DSF ranged from 2–22 mg/g and 10–95 mg/g, respectively while the concentration of anthocyanins and proanthocyanidins in cranberry polyphenol-enriched DSF ranged from 2.5–17 mg/g and 21–101 mg/g, respectively. Blueberry polyphenols present in one serving of fresh blueberries (73 g) were delivered in just 1.4 g of blueberry polyphenol-enriched DSF. Similarly, one gram of cranberry polyphenol-enriched DSF delivered the amount of proanthocyanidins available in three 240 ml servings of cranberry juice cocktail. The concentration of blueberry anthocyanins and total polyphenols eluted from DSF remained constant after 22 weeks of incubation at 37 °C, demonstrating the high stability of the polyphenol–DSF matrix. LC–MS analysis of eluates confirmed that DSF retained major cranberry and blueberry polyphenols intact. Blueberry polyphenol-enriched DSF exhibited significant hypoglycaemic activities in C57bl/6J mice, and cranberry polyphenol-enriched DSF showed antimicrobial and anti-UTI activities in vitro , confirming its efficacy. The described sorption process provides a means to create protein-rich food ingredients containing concentrated plant bioactives without excess sugars, fats and water that can be incorporated in a variety of scientifically validated functional foods and dietary supplements.

A volunteer was given cranberry juice cocktail (CJC) or water to drink, and urine was collected at 2 and 8 hours after consumption, in order to quantitatively determine whether adhesion forces were changed for the volunteer after CJC consumption. Atomic force microscopy (AFM) was used to measure adhesion forces between bacteria and a silicon nitride tip. Forces between Escherichia coli or Staphylococcus aureus and the AFM tip were lower in the urine after the volunteer consumed CJC, compared to drinking water. A steric model was applied to the AFM data, in order to quantify how the urine changed the properties of the bacterial surfaces. There was a small decrease in the equilibrium length of surface molecules on the bacteria when in the post-CJC urine, compared to the post-water urine. However, these changes were not statistically significant. We hypothesize that post-CJC urine imparts subtle changes on the molecules of the bacterial surfaces, and that these changes lead to the reduction in adhesion with the AFM probe.

Cranberries are rich in bioactive constituents purported to enhance immune function, improve urinary tract health, reduce cardiovascular disease and more recently, inhibit cancer in preclinical models. However, identification of the cranberry constituents with the strongest cancer inhibitory potential and the mechanism associated with cancer inhibition by cranberries remains to be elucidated. This study investigated the ability of a proanthocyanidin rich cranberry fraction (PAC) to alter gene expression, induce apoptosis and impact the cell cycle machinery of human NCI-H460 lung cancer cells. Lung cancer is the leading cause of cancer-related deaths in the United States and five year survival rates remain poor at 16%. Thus, assessing potential inhibitors of lung cancer-linked signaling pathways is an active area of investigation.

Esophageal adenocarcinoma (EAC) incidence rates have increased sharply (>500%) throughout the Westernized world since the 1980s, despite the widespread use of endoscopy and anti-reflux medications. Reasons for the rapid increase in EAC are still being unraveled; however, persistent, symptomatic reflux of gastric and duodenal contents, known as gastroesophageal disease (GERD), is considered the strongest risk factor. Our laboratory utilizes the rat esophagogastroduodenal anastomosis (EGDA) surgical model of reflux-induced EAC to evaluate mechanisms by which cranberry proanthocyanidins (C-PAC) delivered in the drinking water inhibit reflux-induced EAC. Findings show that C-PAC inhibits EAC formation by 83% with concomitant restoration of the gut microbial profile and significant reduction of primary and secondary bile acid metabolites in the esophagus of animals with reflux-inducing surgery, but through unknown mechanisms. Herein, we investigated whether transporters, important in bile acid transport, buffering capacity, excretion, and even resistance to anticancer agents, were restored by C-PAC in the context of reflux. Additionally, we utilized a publicly available GEO dataset (GSE37203) to assess transporter expression levels in patients with metaplastic Barrett's esophagus (BE) versus those with high grade dysplasia (HGD) or EAC for translational relevance. Methods included esophageal RNA isolation, transcriptome sequencing using the Illumina HiSeq platform, followed by validation utilizing the PrimePCR Drug Transporter array plate and database mining. Results show significant changes in solute carriers (SLC), ATP-binding cassette (ABC) and aquaporin transporters in the rat reflux-induced model for EAC with C-PAC mitigating alterations. EGDA altered one or more members in 71% of the SLC families with frequent changes observed in SLC25, SLC4, SLC35, SLC2 and SLC6. C-PAC favorably impacted >40% of the EGDA-induced SLC changes. A number of ABC transporters involved in glutathione transport and also implicated in drug resistance were altered by EGDA including Abcc1, Abcc3, Abcc4, Abcc5 and Abcg2 and C-PAC mitigated these changes. Aquaporins, Aqp3 and Aqp4, were significantly modulated in EGDA and restored with C-PAC. Significant differences in ABCC9, ABCC10, AQP6 and AQP9, in addition to several members of the SLC25, SLC4, SLC35, SLC2 and SLC6 families, were observed in patients with HGD/EAC compared to those with BE metaplasia. Therefore, altered expression of transporters following reflux-inducing surgery or GERD is likely a defensive mechanism by which cells attempt to adapt or protect from injurious bile acid exposure. Further research is warranted to investigate agents that restore normal transporter function, which may serve to concomitantly improve epithelial barrier function and mucosal integrity in the context of esophageal cancer progression. Citation Format: Katherine M. Weh, Bridget A. Tripp, Jennifer L. Clarke, Amy B. Howell, Jules Lin, David G. Beer, Andrew C. Chang, Laura A. Kresty. Cranberry proanthocyanidins mitigate reflux-induced transporter dysregulation in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 279.

BACKGROUND: The objective of this study was to validate an improved 4-dimethylaminocinnamaldehyde (DMAC) colorimetric method using a commercially available standard (procyanidin A2), for the standard method for quantification of proanthocyanidins (PACs) in cranberry powders, in order to establish dosage guidelines for the uropathogenic bacterial anti-adhesion effect of cranberry. RESULTS: Commercially available cranberry samples were obtained (five from U.S. sources and six from European sources) for PAC quantification in five different analytical laboratories. Each laboratory extracted and analyzed the samples using the improved DMAC method. Within-laboratory variation (mean ± SD) was 4.1 ± 1.7% RSD (range, 2.3–6.1% RSD) and the between laboratory variability was 16.9 ± 8.5% RSD (range, 8–32% RSD). For comparative purposes, the cranberry samples were alternatively quantified using weights of extracted PACs (gravimetric). The correlation coefficient between the two methods was 0.989. CONCLUSION: This improved DMAC method provides a simple, robust and relatively specific spectrophotometric assay for total PACs in cranberry samples using commercially available procyanidin A2 dimer as a standard. DMAC is most useful within a given type of food such as cranberries, but may not be appropriate for comparing concentrations across different food types, particularly in those cases where large differences exist among the relative amounts of each oligomer and polymer. Copyright © 2010 Society of Chemical Industry

Cranberry (Vaccinium macrocarpon Ait.) ingestion has long been associated with prevention of urinary tract infections. The beneficial mechanism was historically thought to be due to the fruit acids causing a bacteriostatic effect in the urine. However, recently, a group of proanthocyanidins (PACs) with A-type linkages were isolated from cranberry which exhibit bacterial antiadhesion activity against both antibiotic susceptible and resistant strains of uropathogenic P-fimbriated Escherichia coli bacteria. The link between cranberry ingestion and maintenance of urinary tract health as well as the structural diversity, pharmacokinetics, quantification, and bacterial antiadhesion bioactivity of the A-linked cranberry PACs are reviewed.

Antioxidant activity, urinary tract protective activity, and cardioprotective anti-platelet effects are among the bioactivities associated with dietary phenolics. These bioactivities were measured in vitro in fruit extracts from seven Vaccinium species and five non-Vaccinium species to determine their relationship to total phenolic content and to anthocyanin and proanthocyanidin content. Berries belonging to the genus Vaccinium were particularly high in antioxidant activity and urinary tract protective anti-adhesion activity, while anti-platelet activity varied among species. There was a positive relationship between antioxidant activity (using the oxygen radical absorbing capacity (ORAC) assay) and both the total phenolic (R2 = 0.76) and anthocyanin content (R2 = 0.43) of the fruit, although there was no relationship between ORAC and proanthocyanidin content. There were no relationships between anti-adhesion activity and total phenolic content, anthocyanin content, or proanthocyanidin content. Likewise, no relationships were observed between anti-platelet activity and total phenolic content, anthocyanin content, or proanthocyanidin content. These results suggest that while antioxidant properties are characteristic of all fruit phenolics, in vitro anti-adhesion and anti-platelet bioactivities may be due to less abundant phenolic subgroups. Copyright © 2007 Crown in the right of Canada and Society of Chemical Industry

The concentrations and profiles of phenolics of selected fruit crops common in the Western diet, including several Vaccinium species, were examined to better understand how these crops may be useful sources of phenolic phytochemicals. Vaccinium fruit had a high phenolic concentration compared to non-Vaccinium fruit. Some Vaccinium fruit were particularly rich in certain phenolic subgroups, especially anthocyanins and pro-anthocyanidins. Among the pro-anthocyanidin oligomers measured using fluorometric and mass spectroscopic detection, the trimers and tetramers were most abundant, while pro-anthocyanidins with a degree of polymerization greater than 8 were least abundant. As biomedical studies determine which phenolic structures are associated with particular bioactivities, information on the phenolic concentration and profile of selected species will be useful in developing specific uses for fruit crops in human health. Methods were compared to assess the usefulness of simpler versus more sophisticated means of phenolic analysis. The phenolic components of fruit extracts were purified approximately 20-fold, and not qualitatively altered, by C18 solid-phase extraction. However, fruit extracts obtained from C18 solid-phase extraction differed in their relative abundance of phenolic components. Colorimetric and HPLC-DAD measures of phenolic concentration were correlated (R2 = 0.79), as was pro-anthocyanidin concentration detected fluorometrically and by mass spectrometry (R2 = 0.44). Copyright © 2007 Crown in the right of Canada and Society of Chemical Industry

The antiatherogenic properties of pomegranate juice (PJ) were attributed to its antioxidant potency and to its capacity to decrease macrophage oxidative stress, the hallmark of early atherogeneis. PJ polyphenols and sugar-containing polyphenolic anthocyanins were shown to confer PJ its antioxidant capacity. In the present study, we questioned whether PJ simple or complex sugars contribute to the antioxidative properties of PJ in comparison to white grape juice (WGJ) sugars. Whole PJ decreased cellular peroxide levels in J774A.1 macrophage cell-line by 23% more than PJ polyphenol fraction alone. Thus, we next determined the contribution of the PJ sugar fraction to the decrease in macrophage oxidative state. Increasing concentrations of the PJ sugar fraction resulted in a dose-dependent decrement in macrophage peroxide levels, up to 72%, compared to control cells. On the contrary, incubation of the cells with WGJ sugar fraction at the same concentrations resulted in a dose-dependent increment in peroxide levels by up to 37%. The two sugar fractions from PJ and from WGJ showed opposite effects (antioxidant for PJ and pro-oxidant for WGJ) also in mouse peritoneal macrophages (MPM) from control as well as from streptozotocin-induced diabetic Balb/C mice. PJ sugar consumption by diabetic mice for 10 days resulted in a small but significant decrement in their peritoneal macrophage total peroxide levels and an increment in cellular glutathione content, compared to MPM harvested from control diabetic mice administrated with water. In contrast, WGJ sugar consumption by diabetic mice resulted in a 22% increment in macrophage total peroxide levels and a 45% decrement in cellular glutathione content. Paraoxonase 2 activity in macrophages increases under oxidative stress conditions. Indeed, macrophage paraoxonase 2 activity was decreased after PJ sugars supplementation, but increased after WGJ sugars supplementation. We conclude that PJ sugar fraction, unlike WGJ sugar fraction, decreases macrophage oxidative state under normal and under diabetic conditions. These antioxidant/antiatherogenic effects could be due to the presence of unique complex sugars and/or phenolic sugars in PJ.

The aim of this study was to determine whether consumption of sweetened dried cranberries elicits urinary anti-adherence properties against Escherichia coli as previously demonstrated with cranberry juice and/or sweetened cranberry juice cocktail, compared to unsweetened raisins.Uropathogenic E. coli isolates were obtained from five women with culture-confirmed urinary tract infections (UTIs). Four urine samples were collected from each subject. The first urine sample was collected before any study intervention. The second urine sample was collected 2-5 hours after consumption of one box (42.5 g) of raisins. The third urine sample was collected 5-7 days later. The final urine sample was collected 2-5 hours after consumption of approximately 42.5 g of dried cranberries.E. coli isolates were incubated separately in each of the four urine samples collected from the five subjects. Bacteria were harvested from the urine and tested for the ability to prevent adhesion of P-fimbriated E. coli bacteria using a mannose-resistant hemagglutination assay with human red blood cells (A1, Rh+).Of the urine samples collected after dried cranberry consumption, one demonstrated 50% antiadherence activity, two demonstrated 25% activity, and two did not show any increased activity. None of the control urine samples and none of the postraisin consumption samples demonstrated any inhibitory activity.Data from this pilot study on only five subjects suggest that consumption of a single serving of sweetened dried cranberries may elicit bacterial antiadhesion activity in human urine, whereas consumption of a single serving of raisins does not. Further studies are needed to verify the antiadhesion effect of sweetened dried cranberries. In addition, dose-response and pharmacokinetics of the active compounds in the dried cranberries need to be determined. If clinical research is positive, dried cranberries could potentially be a viable alternative to cranberry juice consumption for prevention of UTIs.