Metagenomic characterization of the equine endometrial microbiome during anestrus.

The equine uterus is highly interrogated during estrus prior to breeding and establishing pregnancy. Many studies in mares have been performed during estrus under the influence of high estrogen concentrations, including the equine estrual microbiome. To date, it is unknown how the uterine microbiome of the mare is influenced by cyclicity; while, the equine vaginal microbiome is stable throughout the estrous cycle. We hypothesized that differences would exist between the equine endometrial microbiome of mares in estrus and anestrus. The aim of this study was two-fold: to characterize the resident endometrial microbiome of healthy mares during anestrus and to compare this with estrus. Double-guarded endometrial swabs were taken from healthy mares during estrus (n = 16) and in the following non-breeding season during anestrus (n = 8). Microbial population was identified using 16S rRNA sequencing. Our results suggest that the equine uterine microbiome in estrus has a low diversity and low richness, while during anestrus, a higher diversity and higher richness were seen compared to estrus. Despite this difference, both the estrus and anestrus endometrial microbiome were dominated by Proteobacteria, Firmicutes, and Bacteroidota. The composition of the microbial community between anestrus and estrus was significantly different. This may be explained by the difference in the composition of the endometrial immune milieu based on the stage of the cycle. Further research investigating the function of the equine endometrial microbiome and dynamics changes within the uterine environment is required.

Limited association between stallion-like behavior and hormonal indicators of testicular remnants in geldings.

Persistent stallion-like behavior is a common sign of cryptorchidism in supposed geldings. The presence of testicular tissue can be evaluated by analyzing hormones such as testosterone and anti-Müllerian hormone (AMH). Here, we used hormonal analysis to investigate relationships between the likely presence of testicular tissue and stallion-like behavior in samples submitted from presumptive geldings (n = 1,202), retrospectively. Most geldings with stallion-like behaviors had serum concentrations of testosterone (851/1,056; 80.6 %) and AMH (682/877; 77.8 %) below the laboratory reference range for cryptorchids (< 60 pg/mL and ≤ 0.15 ng/mL for testosterone and AMH, respectively). A total of 13 samples (13/716; 1.8 %) showed AMH concentrations typical for geldings but testosterone above the cryptorchid range. Conversely, 31 samples (31/716; 4.3 %) had high AMH, suggesting cryptorchidism, but testosterone concentrations implied no testicular tissue. Among the cryptorchid stallions, the AMH and testosterone concentrations did not vary based on the season. However, age categories affected the concentration of both hormones among the presumptive true cryptorchid stallions. The results of this study demonstrate that undesirable behavior in geldings is rarely associated with the presence of testicular tissue, as assessed by these two hormonal biomarkers. This information highlights the complexity of behavior and demonstrates that persistent stallion-like behavior in geldings could be related to factors other than the presence of testicular tissue.

Effect of Sampling Method on Detection of the Equine Uterine Microbiome during Estrus.

Bacterial endometritis is among the most common causes of subfertility in mares. It has a major economic impact on the equine breeding industry. The sensitivity of detecting uterine microbes using culture-based methods, irrespective of the sample collection method, double-guarded endometrial swab, endometrial biopsy, or uterine low-volume lavage (LVL), is low. Therefore, equine bacterial endometritis often goes undiagnosed. Sixteen individual mares were enrolled, and an endometrial sample was obtained using each method from all mares. After trimming, quality control and decontamination, 3824 amplicon sequence variants were detected in the dataset. We found using 16S rRNA sequencing that the equine uterus harbors a distinct resident microbiome during estrus. All three sampling methods used yielded similar results in composition as well as relative abundance at phyla (Proteobacteria, Firmicutes, and Bacteroidota) and genus (Klebsiella, Mycoplasma, and Aeromonas) levels. A significant difference was found in alpha diversity (Chao1) between LVL and endometrial biopsy, suggesting that LVL is superior at detecting the low-abundant (rare) taxa. These new data could pave the way for innovative treatment methods for endometrial disease and subfertility in mares. This, in turn, could lead to more judicious antimicrobial use in the equine breeding industry.

Serum amyloid A, Serum Amyloid A1 and Haptoglobin in pregnant mares and their fetuses after experimental induction of placentitis.

Serum amyloid A (SAA) and Haptoglobin (Hp) are acute phase proteins, produced during inflammation, such as placentitis. In horses, SAA and SAA1 are protein coding genes. Objectives were to analyze SAA and Hp concentrations and relative abundance of SAA, SAA1 and Hp mRNA transcript in maternal and fetal tissues after experimental induction of placentitis or mares of a control group. Serum Amyloid A family proteins were in marked abundance in the stroma of the endometrium and chorioallantois associated with inflammatory cells. Maternal plasma SAA concentrations were greater (P = 0.01) in mares with experimentally induced placentitis compared to those of the control group. Maternal Hp from the groups were not different, but fetal Hp concentrations of mares with experimentally induced placentitis were greater (P = 0.02). Maternal plasma SAA and Hp concentrations were greater than fetal plasma concentrations in mares with experimentally induced placentitis (P < 0.05). Relative abundance of SAA mRNA transcript was greater in the maternal, fetal liver and chorioallantois of mares with experimentally induced placentitis (P < 0.05) compared to those in the control group. Interestingly, relative abundance of SAA1 mRNA transcript was greater in the chorioallantois of mares with experimentally induced placentitis (P < 0.05). The SAA and Hp concentrations, therefore, were greater in mares with induced placentitis. Furthermore, relative abundance of SAA1 mRNA transcript is specifically greater in the chorioallantois of mares with placentitis, which warrants further studies to elucidate the immunological response of SAA1 in the chorioallantois of mares with placentitis.

The imbalance of the Th17/Treg axis following equine ascending placental infection.

Ascending placentitis is a leading cause of abortion in the horse, but adaptive immune response to this disease is unknown. To evaluate this, sub-acute placentitis was experimentally-induced via trans-cervical inoculation of S. zooepidemicus, and endometrium and chorioallantois was collected 8 days later (n = 6 inoculated/n = 6 control). The expression of transcripts relating to Th1, Th2, Th17, and Treg maturation was assessed via RNASeq. IHC of transcription factors relating to each subtype in the same tissues (Th1: TBX21, Th2: GATA3, Th17: IRF4, Treg: FOXp3). An immunoassay was utilized to assess circulating cytokines (Th1: IFNg, IL-2; Th2: IL-4, IL-5; Th17: IL-17, IL-6; Treg: IL-10, GM-CSF). An increase in Th1 and Th17-related transcripts were noted in the chorioallantois, although no alterations were seen in the endometrium. Th2 and Treg-related transcripts altered in a dysregulated manner, as some transcripts increased in expression while others decreased. Immunolocalization of Th1, Th2, and Th17 cells was increased in diseased chorioallantois, while no Treg cells were noted in the diseased tissue. Secreted cytokines relating to Th1 (IFNg, IL-2), Th17 (IL-6), Th2 (IL-5), and Treg (IL-10) populations increased in maternal circulation eight days after inoculation. In conclusion, the Th1/Th17 response to ascending placentitis occurs primarily in the chorioallantois, indicating the adaptive immune response to occur in fetal derived placental tissue. Additionally, ascending placentitis leads to an increase in the helper T cell populations (Th1/Th17/Th2) while decreasing the Treg response. This increase in Th17-related responses alongside a diminishing Treg-related response may precede or contribute to fetal demise, abortion, or preterm labor.

New insights in equine steroidogenesis: an in-depth look at steroid signaling in the placenta.

Steroid production varies widely among species, with these differences becoming more pronounced during pregnancy. As a result, each species has its own distinct pattern of steroids, steroidogenic enzymes, receptors, and transporters to support its individual physiological requirements. Although the circulating steroid profile is well characterized during equine pregnancy, there is much yet to be explored regarding the factors that support steroidogenesis and steroid signaling. To obtain a holistic view of steroid-related transcripts, we sequenced chorioallantois (45 days, 4 months, 6 months, 10 months, 11 months, and post-partum) and endometrium (4 months, 6 months, 10 months, 11 months, and diestrus) throughout gestation, then looked in-depth at transcripts related to steroid synthesis, conjugation, transportation, and signaling. Key findings include: 1) differential expression of HSD17B isoforms among tissues (HSD17B1 high in the chorioallantois, while HSD17B2 is the dominant form in the endometrium) 2) a novel isoform with homology to SULT1A1 is the predominant sulfotransferase transcript in the chorioallantois; and 3) nuclear estrogen (ESR1, ESR2) and progesterone (PGR) expression is minimal to nonexistant in the chorioallantois and pregnant endometrium. Additionally, several hypotheses have been formed, including the possibility that the 45-day chorioallantois is able to synthesize steroids de novo from acetate and that horses utilize glucuronidation to clear estrogens from the endometrium during estrous, but not during pregnancy. In summary, these findings represent an in-depth look at equine steroid-related transcripts through gestation, providing novel hypotheses and future directions for equine endocrine research.

Transcriptomic analysis reveals the key regulators and molecular mechanisms underlying myometrial activation during equine placentitis†.

The key event in placentitis-induced preterm labor is myometrial activation with the subsequent initiation of labor. However, the molecular mechanisms underlying myometrial activation are not fully understood in the mares. Therefore, the equine myometrial transcriptome was characterized during placentitis (290.0 ± 1.52 days of GA, n = 5) and the prepartum period (330 days of GA, n = 3) in comparison to normal pregnant mares (289.8 ± 2.18 days of GA, n = 4). Transcriptome analysis identified 596 and 290 DEGs in the myometrium during placentitis and the prepartum period, respectively, with 138 DEGs in common. The placentitis DEGs included eight genes (MMP1, MMP8, S100A9, S100A8, PI3, APOBEC3Z1B, RETN, and CXCL2) that are exclusively expressed in the inflamed myometrium. Pathway analysis elucidated that inflammatory signaling, Toll-like receptor signaling, and apoptosis pathways dominate myometrial activation during placentitis. The prepartum myometrium was associated with overexpression of inflammatory signaling, oxidative stress, and 5-hydroxytryptamine degradation. Gene ontology enrichment analysis identified several chemoattractant factors in the myometrium during placentitis and prepartum period, including CCL2, CXCL1, CXCL3, and CXCL6 in common. Upstream regulator analysis revealed 19 potential upstream regulators in placentitis dataset including transcription regulators (E2F1, FOXM1, HIF1A, JUNB, NFKB1A, and STAT1), transmembrane receptors (FAS, ICAM1, SELP, TLR2, and TYROBP), growth factors (HGF and TGFB3), enzymes (PTGS2 and PRKCP), and others (S100A8, S100A9, CD44, and C5AR1). Additionally, three upstream regulators (STAT3, EGR1, and F2R) were identified in the prepartum dataset. These findings revealed the key regulators and pathways underlying myometrial activation during placentitis, which aid in understanding the disease and facilitate the development of efficacious therapies.

Ascarids exposed: a method for in vitro drug exposure and gene expression analysis of anthelmintic naïve Parascaris spp.

Ascarid parasites infect a variety of hosts and regular anthelmintic treatment is recommended for all species. Parascaris spp. is the only ascarid species with widespread anthelmintic resistance, which allows for the study of resistance mechanisms. The purpose of this study was to establish an in vitro drug exposure protocol for adult anthelmintic-naïve Parascaris spp. and report a preliminary transcriptomic analysis in response to drug exposure. Live worms were harvested from foal necropsies and maintained in RPMI-1640 at 37 °C. Serial dilutions of oxibendazole (OBZ) and ivermectin (IVM) were prepared for in vitro drug exposure, and worm viability was monitored over time. In a second drug trial, worms were used for transcriptomic analysis. The final drug concentrations employed were OBZ at 40.1 µm (10 µg mL-1) and IVM at 1.1 µm (1 µg mL-1) for 24 and 3 h, respectively. The RNA-seq analysis revealed numerous differentially expressed genes, with some being potentially related to drug detoxification and regulatory mechanisms. This report provides a method for in vitro drug exposure and the phenotypic responses for Parascaris spp., which could be extrapolated to other ascarid parasites. Finally, it also provides preliminary transcriptomic data following drug exposure as a reference point for future studies of Parascaris spp.

Steroid synthesis and metabolism in the equine placenta during placentitis.

Equine placentitis is associated with alterations in maternal peripheral steroid concentrations, which could negatively affect pregnancy outcome. This study aimed to elucidate the molecular mechanisms related to steroidogenesis and steroid-receptor signaling in the equine placenta during acute placentitis. Chorioallantois (CA) and endometrial (EN) samples were collected from mares with experimentally induced placentitis (n = 4) and un-inoculated gestationally age-matched mares (control group; n = 4). The mRNA expression of genes coding for steroidogenic enzymes (3ßHSD, CYP11A1, CYP17A1, CYP19A1, SRD5A1, and AKR1C23) was evaluated using qRT-PCR. The concentration of these enzyme-dependent steroids (P5, P4, 5αDHP, 3αDHP, 20αDHP, 3ß-20αDHP, 17OH-P, DHEA, A4, and estrone) was assessed using liquid chromatography-tandem mass spectrometry in both maternal circulation and placental tissue. Both SRD5A1 and AKR1C23, which encode for the key progesterone metabolizing enzymes, were downregulated (P < 0.05) in CA from the placentitis group compared to controls, and this downregulation was associated with a decline in tissue concentrations of 5αDHP (P < 0.05), 3αDHP (P < 0.05), and 3ß-20αDHP (P = 0.052). In the EN, AKR1C23 was also downregulated in the placentitis group compared to controls, and this downregulation was associated with a decline in EN concentrations of 3αDHP (P < 0.01) and 20αDHP (P < 0.05). Moreover, CA expression of CYP19A1 tended to be lower in the placentitis group, and this reduction was associated with lower (P = 0.057) concentrations of estrone in CA. Moreover, ESR1 (steroid receptors) gene expression was downregulated (P = 0.057) in CA from placentitis mares. In conclusion, acute equine placentitis is associated with a local withdrawal of progestins in the placenta and tended to be accompanied with estrogen withdrawals in CA.

Cytotoxic and Antiproliferative Activity of Polyisoprenoids in Seventeen Mangroves Species Against WiDr Colon Cancer Cells

Background: Secondary metabolites from the group of isoprenoid compounds are widely distributed in mangrove plants. Polyisoprenoids (dolichol and polyprenol) are known to have benefits as anticancer agents. The present study was conducted to determine the cytotoxic potential of polyisoprenoids in leaves from seventeen selected mangrove species against colon cancer (WiDr) cells. Methods: Cytotoxic activity was evaluated by MTT assay in vitro using WiDr human colon cancer cells and 3T3 fibroblasts from Swiss albino mouse embryo tissue as controls. Mechanisms of action were approached by assessing apoptosis and the cell cycle using flow cytometry and fluorescence microscopy with annexin V-FITC, as well as expression of Bcl-2 and cyclin D1 by immunocytochemistry. Results: Polyisoprenoids from N. fruticans leaves demonstrated the highest anticancer activity, with an IC50 of 180.2 µg/mL, as compared to 397.7 µg/mL against 3T3 normal cells. Significant decrease in the expression of Bcl-2 and cyclin D1 was also noted, facilitating apoptosis and arrest of the cell cycle in the G0-G1 phase in WiDr cells. The present study showed for the first time that polyisoprenoids from N. fruticans exhibit concrete anticancer activity in vitro, decreasing cell proliferation and inducing apoptosis in colon cancer cells. Conclusions: Polyisoprenoids isolated from N. fruticans leaves may have promise as a source of anticancer agents.

Is less also better? A single-institution experience on treatment of early stage Malignant Pleural Mesothelioma.

OBJECTIVES: No clear evidence of which surgical procedure should be performed for early stage mesothelioma is available to date. We analyzed our 10-year experience in the treatment of early stage mesothelioma with surgery and Hyperthermic IntraTHOracic Chemotherapy. METHODS: We retrospectively analyzed all cases of histologically proven epithelioid or biphasic IMIG stage I and II mesothelioma that we operated between 2005 and 2014. We performed an open pleurectomy and partial decortication of any visible lesion on the visceral pleura in all cases and both diaphragm and pericardium were always spared; Hyperthermic IntraTHOracic Chemotherapy was ran using Cisplatin 80 mg/m2 and Doxorubicin 25 mg/m2 at a target temperature of 42.5 °C for 60 min. RESULTS: We operated on 26 patients (23 male and 3 female); epithelioid tumor was diagnosed in 23 cases. Twelve patients were in IMIG stage I and 14 in IMIG stage II; median overall survival for all patients, stage I and II were 35.6, 46 and 23 months respectively and disease free survival was 18, 18 and 16 months respectively. Our results for stage I were better than those reported in literature and were similar for stage II. We observe no 30- and 90- mortality and the rate of severe complication (all CTCAE stage 3) were 30%; the median postoperative stay was 7.5 days. CONCLUSIONS: Our lung sparing approach for the treatment of pleural mesothelioma in early stages allows promising long term outcomes with a complete sparing of pulmonary and diaphragmatic function. Larger studies are needed to confirm our good results.

Prevalence of cytological endometritis and effect on pregnancy outcomes at the time of insemination in nulliparous dairy heifers.

The objectives of the present study were to assess the prevalence of cytological endometritis (CYTO) at the time of artificial insemination (AI) and its effect on pregnancy outcomes in nulliparous dairy heifers. In total, 512 endometrial cytology samples were taken during AI from 351 nulliparous Holstein-Friesian heifers using cytotape (a 1.5-cm piece of paper tape rolled on the top of an AI catheter covered with a double guard sheet). After sampling, the top of the AI catheter was gently rolled onto a glass slide, air-dried, and stained using Diff-Quick (Fisher Diagnostics, Newark, DE). For each slide, 300 nucleated cells were counted, and the polymorphonuclear cell ratio (% PMN) was assessed at 400× magnification. We constructed a receiver operating characteristic curve to find the cutoff point at which sensitivity and specificity (% PMN) affected pregnancy outcomes. The receiver operating characteristic curve revealed that the threshold level for diagnosing CYTO in nulliparous dairy heifers was 1% PMN. An insemination was considered successful when pregnancy was confirmed by rectal palpation at least 45d post-AI. Heifers were considered not pregnant when they received a subsequent insemination or were diagnosed empty by rectal palpation. We built multilevel generalized mixed-effect models to test factors affecting pregnancy outcomes and the occurrence of CYTO at AI. We excluded 16 samples harvested from 12 heifers due to poor sample quality or unavailability of reproductive data. Of the 496 AI samples, the prevalence of CYTO at AI was 7.86% (n=39). The conception rate was 62.8% (n=287) in CYTO-negative samples (n=457) and 38.46% (n=15) in CYTO-positive samples. Risk factors for non-pregnancy were a previous AI (odds ratio 2.96; 95% confidence interval: 1.21-7.26) and the interaction between CYTO and previous AI. The only risk factor identified as being associated with the occurrence of CYTO was a previous AI (odds ratio 4.7; 95% confidence interval: 2.15-10.34). The performance of unsuccessful inseminations significantly affects reproductive outcomes in subsequent AI and may lead to CYTO in nulliparous dairy heifers.

Distribution of inflammation and association between active and chronic alterations within the endometrium of dairy cows.

Objectives of this study were twofold: (i) to assess the association between polymorphonuclear (PMN) counts and chronic alterations within the bovine endometrium and (ii) to determine the distribution of inflammation throughout the endometrium of clinically healthy dairy cows. Holstein-Friesian cows (n = 32) from a single dairy farm were selected for this experiment. Before slaughtering, a complete reproductive examination was performed to discard any type of clinical disease. After slaughtering, reproductive tracts were collected, and the endometrium was sampled at 8 pre-defined locations. At each location, endometrial biopsies (EBs) and cytology (CY) samples were harvested. Histopathology samples were stained with haematoxylin-eosin (EB-HE) and naphthol-AS-D-chloroacetate-esterase (EB-naphthol), while CY samples were stained with Wright-Giemsa. In the EB-HE samples, parameters assessed were epithelium height, mononuclear cells infiltration, lymphocytic aggregates, periglandular fibrosis, angiosclerosis and haemorrhage. In EB-naphthol and CY slides, PMNs counts were evaluated. Binomial logistic regression was used to assess the association between the number of PMNs present in both the EB-naphthol and CY samples and alterations identified in the EB-HE samples and to analyse the distribution of the histopathological alterations (EB-HE). A Poisson mixed-effect model was used to analyse the distribution of PMNs within the endometrium. A significant positive association was found between the PMN counts and the mononuclear cells infiltration. The presence of erythrocytes was associated with higher odds to detect PMNs in the stratum compactum. Significantly, higher infiltration of PMNs and mononuclear cells were detected in the uterine body and the right horn region. Concluding, CY is a technique that allows the evaluation of PMN counts and therefore only evaluates active inflammation. A complete assessment of endometrial health can only be obtained using EB. To optimize the sensitivity to diagnose endometrial inflammation in cows, adjacencies of the corpus uteri should be considered as the preferred region to harvest samples.

A novel cytologic sampling technique to diagnose subclinical endometritis and comparison of staining methods for endometrial cytology samples in dairy cows.

The present article describes a study of the diagnosis of subclinical endometritis in dairy cows having two principal aims: first, to validate a novel technique for taking endometrial cytology samples to diagnose subclinical endometritis in dairy cows. Second, to compare the percentage of polymorphonuclear cells (PMNs) in cytology samples stained with Diff-Quik versus a specific staining method for PMNs, naphthol-AS-D-chloroacetate-esterase (CIAE). In the first experiment, Holstein-Friesian cows (n = 204) were used to take two cytology samples at the same time using the conventional cytobrush (CB) and the new cytotape (CT). Both devices were assembled within the same catheter allowing sampling at the same time, and approximately at the same location. Cytotape consisted of a 1.5-cm piece of paper tape rolled on the top of an insemination catheter covered with a double guard sheet. Parameters used to evaluate both methods were: PMNs percentage, total cellularity, quality of the smears, and red blood cell contamination. The concordance correlation coefficient analysis was used to assess agreement between continuous and Pearson chi-square tests for categorical variables. Agreement between the percentage of PMNs in both methods was good ρ = 0.84 (0.79, 0.87) with a minor standard error of 2%. Both methods yielded similar total cellularity (P = 0.62). Cytotape yielded better quality smears with more intact cells (P < 0.01) while samples that were taken by CB were more likely to be bloody (P < 0.01). Hence, CT and CB methods yielded smears with a similar PMNs percentage and a total number of cells, but CT provided smears with higher quality and significantly less blood contamination. For the second experiment, 114 duplicate cytology slides were stained using both Diff-Quik and CIAE. Agreement between PMNs percentage in both staining techniques was good ρc = 0.84 (0.78, 0.89) with a standard error of only 2%. Hence, Diff-Quik was confirmed as an easy, fast, and high-quality staining technique, which can be routinely used to stain endometrial cytology samples satisfactorily.

Effect of uterine lavage on neutrophil counts in postpartum dairy cows.

Subclinical endometritis affects approximately 30% of lactating dairy cows, causing significant economic losses to the dairy industry. Yet, there is no efficient treatment available for this condition. The present study examines the effect of uterine lavage in clinically normal cows with sterile saline solution at 30 days in milk (DIM) on the percentage of polymorphonuclear cells (PMNs) detected with endometrial cytology as an indicator of subclinical endometritis. It was hypothesized uterine lavage would be a technique to reduce the number of PMNs in the uterus, and hence be beneficial for cows affected by subclinical endometritis. Cytology samples were taken by low-volume flushing from 50 Holstein Friesian cows on 30 and 40 DIM. On Day 30, cows were clinically examined and randomly assigned into a treatment and control group. In the treatment group, the cytology sampling on Day 30 was immediately followed by uterine lavage with 500-600 mL of sterile physiological saline (35-40°C). Cytology sampling was repeated in all cows at 40 DIM. Lactation numbers >2, peripheral progesterone concentrations >1 ng/mL and uterine lavage at 30 DIM all were significantly associated with lesser PMN percentages at 40 DIM (P=0.0041; 0.0187 and 0.0043, respectively). Uterine lavage might, therefore, be a useful and practical method to decrease the number of PMNs in the uterus of cattle. Results from the current study can be used as preliminary data for designing in depth therapeutic protocols for treatment of subclinical endometritis in cattle.

Consumption of both low and high (-)-epicatechin apple puree attenuates platelet reactivity and increases plasma concentrations of nitric oxide metabolites: a randomized controlled trial.

We hypothesised that consumption of flavanol-containing apple puree would modulate platelet activity and increase nitric oxide metabolite status, and that high flavanol apple puree would exert a greater effect than low flavanol apple puree. 25 subjects consumed 230 g of apple puree containing 25 and 100mg epicatechin (low and high flavanol apple puree, respectively) and aspirin (75 mg) in random order. Measurements were made at baseline, acutely after treatment (2, 6 and 24 h), and after 14 d of treatment. Low flavanol apple puree significantly attenuated ADP and epinephrine-induced integrin-ß3 expression 2 h and 6 h after consumption and ADP and epinephrine-induced P-selectin expression within 2h of consumption. High flavanol apple puree attenuated epinephrine and ADP-induced integrin-ß3 expression after 2 and 6h. ADP and epinephrine-induced integrin-ß3 expression was significantly attenuated 2, 6 and 24 h after consumption of aspirin, whilst 14 d aspirin consumption attenuated collagen-induced P-selectin expression only. The plasma total nitric oxide metabolite conc. was significantly increased 6h after consumption of both low and high flavanol apple purees. In conclusion, consumption of apple purees containing ⩾25 or 100 mg flavanols transiently attenuated ex vivo integrin-ß3 and P-selectin expression and increased plasma nitric oxide metabolite conc. in healthy subjects, but the effect was not enhanced for the high flavanol apple puree.