Impact of FADS genotype on polyunsaturated fatty acid content in human milk extracellular vesicles: A genetic association study.

BACKGROUND: Extracellular vesicles in human milk are critical in supporting newborn growth and development. Bioavailability of dietary extracellular vesicles may depend on the composition of membrane lipids. Single-nucleotide polymorphisms (SNPs) in the fatty acid desaturase gene cluster impact the content of long-chain polyunsaturated fatty acids in human milk phospholipids. This study investigated the relation between variation in FADS1 and FADS2 with the content of polyunsaturated fatty acids in extracellular vesicles from human milk. METHODS: Milk was obtained from a cohort of mothers (N = 70) at 2-4 weeks of lactation. SNPs in the FADS gene locus were determined using pyrosequencing for rs174546 in FADS1 and rs174575 in FADS2. Quantitative lipidomic analysis of polyunsaturated fatty acids in human milk and extracellular vesicles from human milk was completed by gas chromatography-mass spectrometry. RESULTS: The rs174546 and rs174575 genotypes were independent predictors of the arachidonic acid content in extracellular vesicles. The rs174546 genotype also predicted eicosapentaenoic acid and docosahexaenoic acid in extracellular vesicles. The reduced content of long-chain polyunsaturated fatty acids in extracellular vesicles in human milk may be due to lower fatty acid desaturase activity in mothers who are carriers of the A allele in rs174546 or the G allele in rs174575. CONCLUSION: The polyunsaturated fatty acid composition of milk extracellular vesicles is predicted by the FADS genotype. These findings yield novel insights regarding extracellular vesicle content and composition that can inform the design of future research to explore how lipid metabolites impact the bioavailability of human milk extracellular vesicles.

Homogenization and thermal processing reduce the concentration of extracellular vesicles in bovine milk.

Extracellular vesicles (EVs) in bovine milk confer beneficial physiologic effects to consumers. Industrial processing treatments may affect the amount or bioactivity of EVs intrinsic to bovine milk. We investigated how the content and concentration of EVs were affected by homogenization and thermal processing of raw bovine milk. Raw milk was processed by homogenization, low-temperature (LT) heat, or pasteurization [high-temperature short-time (HTST) and ultra-high-temperature (UHT)] in a pilot processing facility. EVs were isolated from the raw and processed bovine milk using differential ultracentrifugation and quantified using a nanoparticle tracking analyzer. Bovine milk EVs were assessed for total miRNA and protein concentrations standardized to particle count using a fluorometric assay. There were 1.01 × 1010 (±3.30 × 109) EV particles per ml of bovine milk. All industrial processing treatments caused >60% decrease in EV concentration compared to the raw bovine milk. Homogenization and heat treatments independently and additively reduced the content of EVs in bovine milk. The averages of total miRNA/particle and total protein/particle concentrations were elevated threefold by low-temperature heat-processing treatment relative to HTST and UHT pasteurizations. The average diameter of EVs was reduced by 11%-16% by low temperature compared to raw milk (127 ± 13 nm). Homogenization and pasteurization indiscriminately reduce the EV concentration of bovine milk. Smaller EVs with higher protein content resist degradation when processing bovine milk at sub-pasteurization temperature. This new foundational knowledge may contribute to food product development on the preservation of EVs in processed dairy products, including bovine milk-based infant formulas that some newborns are dependent on for adequate growth and development.

Ganglioside Alters Phospholipase Trafficking, Inhibits NF-κB Assembly, and Protects Tight Junction Integrity.

Background and Aims: Dietary gangliosides are present in human milk and consumed in low amounts from organ meats. Clinical and animal studies indicate that dietary gangliosides attenuate signaling processes that are a hallmark of inflammatory bowel disease (IBD). Gangliosides decrease pro-inflammatory markers, improve intestinal permeability, and reduce symptoms characteristic in patients with IBD. The objective of this study was to examine mechanisms by which dietary gangliosides exert beneficial effects on intestinal health. Methods: Studies were conducted in vitro using CaCo-2 intestinal epithelial cells. Gangliosides were extracted from milk powder and incubated with differentiated CaCo-2 cells after exposure to pro-inflammatory stimuli. Gut barrier integrity was assessed by electron microscopy, epithelial barrier function was examined by measuring transepithelial electric resistance, and content of HBD-2, IL-23, NF-κB, and sPLA2 was assessed by ELISA. Results: Ganglioside attenuated the decrease in integrity of tight junctions induced by pro-inflammatory stimuli and improved epithelial barrier function (P < 0.05). Ganglioside decreased the basolateral secretion of sPLA2 (P ≤ 0.05), lowered HBD-2 and IL-23 levels (P ≤ 0.05), and inhibited NF-κB activation (P ≤ 0.05). Conclusions: In summary, the present study indicates that ganglioside GD3 improves intestinal integrity by altering sPLA2 trafficking, and the production of pro-inflammatory mediators is mitigated by decreasing assembly of the NF-κB complex. Dietary gangliosides may have promising potential beneficial effects in IBD as decreased inflammatory signaling, improved intestinal integrity, and maintenance of epithelial barrier function have been demonstrated in vitro.

Effectiveness of a community program for older adults with type 2 diabetes and multimorbidity: a pragmatic randomized controlled trial.

BACKGROUND: Type II diabetes mellitus (T2DM) affects upwards of 25% of Canadian older adults and is associated with high comorbidity and burden. Studies show that lifestyle factors and self-management are associated with improved health outcomes, but many studies lack rigour or exclude older adults, particularly those with multimorbidity. More evidence is needed on the effectiveness of community-based self-management programs in older adults with T2DM and multimorbidity. The study purpose is to evaluate the effect of a community-based intervention versus usual care on physical functioning, mental health, depressive symptoms, anxiety, self-efficacy, self-management, and healthcare costs in older adults with T2DM and 2 or more comorbidities. METHODS: Community-living older adults with T2DM and two or more chronic conditions were recruited from three Primary Care Networks (PCNs) in Alberta, Canada. Participants were randomly allocated to the intervention or control group in this pragmatic randomized controlled trial comparing the intervention to usual care. The intervention involved up to three in-home visits, a monthly group wellness program, monthly case conferencing, and care coordination. The primary outcome was physical functioning. Secondary outcomes included mental functioning, anxiety, depressive symptoms, self-efficacy, self-management, and the cost of healthcare service use. Intention-to-treat analysis was performed using ANCOVA modeling. RESULTS: Of 132 enrolled participants (70-Intervention, 62-Control), 42% were 75 years or older, 55% were female, and over 75% had at least six chronic conditions (in addition to T2DM). No significant group differences were seen for the baseline to six-month change in physical functioning (mean difference: -0.74; 95% CI: - 3.22, 1.74; p-value: 0.56), mental functioning (mean difference: 1.24; 95% CI: - 1.12, 3.60; p-value: 0.30), or other secondary outcomes.. CONCLUSION: No significant group differences were seen for the primary outcome, physical functioning (PCS). Program implementation, baseline differences between study arms and chronic disease management services that are part of usual care may have contributed to the modest study results. Fruitful areas for future research include capturing clinical outcome measures and exploring the impact of varying the type and intensity of key intervention components such as exercise and diet. TRIAL REGISTRATION: NCT02158741 Date of registration: June 9, 2014.

Characterization of Extracellular Vesicles Isolated From Human Milk Using a Precipitation-Based Method.

Extracellular vesicles (EV) function in intercellular communication, and those in human milk may confer immunologic benefits to infants. Methods of EV isolation such as ultracentrifugation (UC) may not be feasible for the study of EVs in human milk due to the need for large sample volume. A technique to isolate EVs from a small volume of human milk using a precipitation reagent is described herein. Electron microscopy, nanoparticle tracking analysis, and semi-quantitative antibody array were conducted to confirm isolation of human milk EVs. Count, size, protein content, and fatty acid quantification of EVs were determined. This isolation technique yielded 8.9 x 109 (± 1.1 × 109) EV particles/mL of human milk. The present method meets the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines. An established EV isolation method suitable for a low volume of human milk will facilitate further research in this growing area.

Human Breast-Milk Feeding Enhances the Humoral and Cell-Mediated Immune Response in Neonatal Piglets.

Background: The benefits of breastfeeding infants are well characterized, including those on the immune system. However, determining the mechanism by which human breast milk (HBM) elicits effects on immune response requires investigation in an appropriate animal model. Objective: The primary aim of this study was to develop a novel porcine model and to determine the differential effects of feeding HBM and a commercial milk formula (MF) on immune response and gastrointestinal microbial colonization in a controlled environment. Methods: Male piglets were fed HBM (n = 26) or MF (n = 26) from day 2 through day 21. Piglets were vaccinated (n = 9/diet group) with cholera toxin and cholera toxin subunit B (CTB) and tetanus toxoid at 21 d or were fed placebo (n = 6/diet group) and then weaned to a standard solid diet at the age of 21 d. Humoral and cell-mediated immune responses were assessed from blood on days 35 and 48. Immune response was further examined from tissues, including mesenteric lymph nodes (MLNs), Peyer's patches (PPs), and spleen. The colonization of gut microbiota was characterized from feces on days 16 and 49. Results: Serum antibody titers in piglets fed HBM were 4-fold higher (P < 0.05) to CTB and 3-fold higher (P < 0.05) to tetanus toxoid compared with piglets fed MF on day 48. Compared with MF, the numbers of immunoglobulin A antibody-producing cells to CTB were 13-fold higher (P < 0.05) in MLNs and 11-fold higher (P < 0.05) in PPs in the HBM diet group on day 51. In addition, significantly increased T cell proliferation was observed in the HBM group relative to the MF group. Furthermore, microbial diversity in the HBM group was lower (P < 0.05) than in the MF group. Conclusions: This porcine model appears to be valid for studying the effects of early postnatal diet on immune responses and the gastrointestinal microbiome. Our results lay the groundwork for future studies defining the role of infant diet on microbiota and immune function.

Fish oil mitigates myosteatosis and improves chemotherapy efficacy in a preclinical model of colon cancer.

BACKGROUND: This study aimed to assess whether feeding a diet containing fish oil was efficacious in reducing tumor- and subsequent chemotherapy-associated myosteatosis, and improving tumor response to treatment. METHODS: Female Fischer 344 rats were fed either a control diet for the entire study (control), or switched to a diet containing fish oil (2.0 g /100 g of diet) one week prior to tumor implantation (long term fish oil) or at the start of chemotherapy (adjuvant fish oil). Chemotherapy (irinotecan plus 5-fluorouracil) was initiated 2 weeks after tumor implantation (cycle-1) and 1 week thereafter (cycle-2). Reference animals received no tumor or treatment and only consumed the control diet. All skeletal muscle measures were conducted in the gastrocnemius. To assess myosteatosis, lipids were assessed histologically by Oil Red O staining and total triglyceride content was quantified by gas chromatography. Expression of adipogenic transcription factors were assessed at the mRNA level by real-time RT-PCR. RESULTS: Feeding a diet containing fish oil significantly reduced tumor- and subsequent chemotherapy-associated increases in skeletal muscle neutral lipid (p<0.001) and total triglyceride content (p<0.03), and expression of adipogenic transcription factors (p<0.01) compared with control diet fed animals. The adjuvant fish oil diet was as effective as the long term fish oil diet in mitigating chemotherapy-associated skeletal muscle fat content, and in reducing tumor volume during chemotherapy compared with control fed animals (p<0.01). CONCLUSION: Long term and adjuvant fish oil diets are equally efficacious in reducing chemotherapy-associated myosteatosis that may be occurring by reducing expression of transcription factors involved in adipogenesis/lipogenesis, and improving tumor-response to chemotherapy in a neoplastic model.

Data for Improvement and Clinical Excellence: a report of an interrupted time series trial of feedback in home care.

BACKGROUND: There is substantial evidence about the effectiveness of audit with feedback, but none that we know have been conducted in home care settings. The primary purpose of the Data for Improvement and Clinical Excellence - Home Care (DICE-HC) project was to evaluate the effects of an audit and feedback delivered to care providers on home care client outcomes. The objective of this paper is to report the effects of feedback on four specific quality indicators: pain, falls, delirium, and hospital visits. METHODS: A 10-month audit with feedback intervention study was conducted with care providers in seven home care offices in Alberta, Canada, which involved delivery of four quarterly feedback reports consisting of data derived from the Resident Assessment Instrument - Home Care (RAI-HC). The primary evaluation employed an interrupted time series design using segmented regression analysis to assess the effects of feedback reporting on the four quality indicators: pain, falls, delirium, and hospitalization. Changes in level and trend of the quality indicators were measured before, during, and after the implementation of feedback reports. Pressure ulcer reporting was analyzed as a comparator condition not included in the feedback report. Care providers were surveyed on responses to feedback reporting which informed a process evaluation. RESULTS: At initiation of feedback report implementation, the percentage of clients reporting pain and falls significantly increased. Though the percentage of clients reporting pain and falls tended to increase and reporting of delirium and hospital visits tended to decrease relative to the pre-intervention period, there was no significant effect of feedback reporting on quality indicators during the 10-month intervention. The percentage of clients reporting falls, delirium, and hospital visits significantly increased in the 6-month period following feedback reporting relative to the intervention period. About 50% of the care providers that read and understand the feedback reports found the reports useful to make changes to the way clients are cared for. CONCLUSIONS: Routinely collected data used over time for feedback is feasible in home care settings. A high proportion of care providers find feedback reports useful for informing how they care for clients. Since reporting on the frequency of quality indicators increased in the post-intervention period, this study suggests that ongoing use of audit with feedback to enhance health outcomes in home care may promote improved reporting on standardized instruments.

Ganglioside Intake Increases Plasma Ganglioside Content in Human Participants.

BACKGROUND: Preclinical studies reveal associations between intestinal ganglioside content and inflammatory bowel disease (IBD). Since a low level of ganglioside is associated with higher production of proinflammatory signals in the intestine, it is important to determine safety and bioavailability of dietary ganglioside for application as a potential therapeutic agent. MATERIALS AND METHODS: Healthy volunteers (HVs; n = 18) completed an 8-week supplementation study to demonstrate safety and bioavailabity of ganglioside consumption. HVs were randomized to consume a milk fat fraction containing 43 mg/d ganglioside or placebo, and patients with IBD (n = 5) consumed ganglioside supplement in a small pilot study. Plasma gangliosides were characterized using reverse-phase liquid chromatography-QQQ mass spectrometry. Intestinal permeability was assessed by oral lactulose/mannitol, and quality of life was assessed by quality of life in the IBD questionnaire. RESULTS: There were no adverse events associated with dietary ganglioside intake. Ganglioside consumption increased ( P < .05) plasma content of total GD3 by 35% over 8 weeks. HVs consuming ganglioside exhibited a 19% decrease in intestinal permeability ( P = .04). Consumption of ganglioside was associated with a 39% increase ( P < .01) in emotional health and a 36% improvement ( P < .02) in systemic symptoms in patients with IBD. CONCLUSION: Impaired intestinal integrity characteristic of IBD results in increased permeability to bacterial antigens and decreased nutrient absorption. Intestinal integrity may be improved by dietary treatment with specific species of ganglioside. Ganglioside is a safe, bioavailable dietary compound that can be consumed to potentially improve quality of life in patients with IBD and treat other disorders involving altered ganglioside metabolism. This study was registered at clinicaltrials.gov as NCT02139709.

Reduction of Arachidonate Is Associated With Increase in B-Cell Activation Marker in Infants: A Randomized Trial.

BACKGROUND: Infants who are not breast-fed benefit from formula with both docosahexaenoic acid (C22:6n3) and arachidonic acid (ARA; C20:4n6). The amount of ARA needed to support immune function is unknown. Infants who carry specific fatty acid desaturase (FADS) polymorphisms may require more dietary ARA to maintain adequate ARA status. OBJECTIVE: The aim of the study was to determine whether ARA intake or FADS polymorphisms alter ARA levels of lymphocytes, plasma, and red blood cells in term infants fed infant formula. METHODS: Infants (N = 89) were enrolled in this prospective, double-blind controlled study. Infants were randomized to consume formula containing 17 mg docosahexaenoic acid and 0, 25, or 34 mg ARA/100 kcal for 10 weeks. Fatty acid composition of plasma phosphatidylcholine and phosphatidylethanolamine, total fatty acids of lymphocytes and red blood cells, activation markers of lymphocytes, and polymorphisms in FADS1 and FADS2 were determined. RESULTS: Lymphocyte ARA was higher in the 25-ARA formula group than in the 0- or 34-ARA groups. In plasma, 16:0/20:4 and 18:0/20:4 species of phosphatidylcholine and phosphatidylethanolamine were highest and 16:0/18:2 and 18:0/18:2 were lowest in the 34-ARA formula group. In minor allele carriers of FADS1 and FADS2, plasma ARA content was elevated only at the highest level of ARA consumed. B-cell activation marker CD54 was elevated in infants who consumed formula containing no ARA. CONCLUSIONS: ARA level in plasma is reduced by low ARA consumption and by minor alleles in FADS. Dietary ARA may exert an immunoregulatory role on B-cell activation by decreasing 16:0/18:2 and 18:0/18:2 species of phospholipids. ARA intake from 25 to 34 mg/100 kcal is sufficient to maintain cell ARA level in infants across genotypes.

Increased catabolism and decreased unsaturation of ganglioside in patients with inflammatory bowel disease.

AIM: To investigate whether accelerated catabolism of ganglioside and decreased ganglioside content contribute to the etiology of pro-inflammatory intestinal disease. METHODS: Intestinal mucosa from terminal ileum or colon was obtained from patients with ulcerative colitis or inflammatory Crohn's disease (n = 11) undergoing bowel resection and compared to control samples of normal intestine from patients with benign colon polyps (n = 6) and colorectal cancer (n = 12) in this observational case-control study. Gangliosides and phospholipids of intestinal mucosa were characterized by class and ceramide or fatty acid composition using liquid chromatography triple-quad mass spectrometry. Content and composition of ganglioside classes GM1, GM3, GD3, GD1a, GT1 and GT3 were compared among subject groups. Content and composition of phospholipid classes phosphatidylcholine (PC) and phosphatidylethanolamine were compared among subject groups. Unsaturation index of individual ganglioside and phospholipid classes was computed and compared among subject groups. Ganglioside catabolism enzymes beta-hexosaminidase A (HEXA) and sialidase-3 (NEU3) were measured in intestinal mucosa using western blot and compared among subject groups. RESULTS: Relative GM3 ganglioside content was 2-fold higher (P < 0.05) in intestine from patients with inflammatory bowel disease (IBD) compared to control intestine. The quantity of GM3 and ratio of GM3/GD3 was also higher in IBD intestine than control tissue (P < 0.05). Control intestine exhibited 3-fold higher (P < 0.01) relative GD1a ganglioside content than IBD intestine. GD3 and GD1a species of ganglioside containing three unsaturated bonds were present in control intestine, but were not detected in IBD intestine. The relative content of PC containing more than two unsaturated bonds was 30% lower in IBD intestine than control intestine (P < 0.05). The relative content of HEXA in IBD intestine was increased 1.7-fold (P < 0.05) and NEU3 was increased 8.3-fold (P < 0.01) compared to normal intestine. Intestinal mucosa in IBD is characterized by increased GM3 content, decreased GD1a, and a reduction in polyunsaturated fatty acid constituents in GD3, GD1a and PC. CONCLUSION: This study suggests a new paradigm by proposing that IBD occurs as a consequence of increased metabolism of specific gangliosides.