Sarah S. Comstock

Human milk oligosaccharides shorten rotavirus-induced diarrhea and modulate piglet mucosal immunity and colonic microbiota.

The impact of human milk oligosaccharides (HMO) on mucosal immunity, gut microbiota and response to rotavirus (RV) infection was investigated in the piglet model. Newborn piglets were fed with formula alone (FF) or formula supplemented with 4 g l−1 HMO (HMO) or a prebiotic mixture of 9:1 short-chain galactooligosaccharides (3.6 g l−1) and long-chain fructooligosaccharides (0.4 g l−1) (PRE) (n=19–21 per group) for 15 days. Piglets (n=7–8) in each dietary group were orally infected with porcine rotavirus (RV) OSU strain on d10, and stool consistency was assessed daily. Blood, small intestine and colonic contents were collected at day 15. Serum RV-specific antibody concentrations, intestinal histomorphology, RV non-structural protein-4 (NSP4) and cytokine mRNA expression were assessed. Colonic content pH, dry matter (DM) and short-chain fatty acid concentrations were measured. Ascending colonic microbiota was analyzed by 16S rRNA gene v1-3 region pyrosequencing. HMO- and PRE-fed groups had shorter duration of diarrhea than FF piglets. Infection changed intestinal histomorphology, increased serum RV-specific antibody response and intestinal RV NSP4 expression, and modulated ileal cytokine expression. HMO enhanced T helper type 1 (interferon-gamma) and anti-inflammatory (interleukin-10) cytokines in the ileum, while prebiotics promoted RV-specific immunoglobulin M response to the infection. RV infection and HMO supplementation altered intraluminal environment and gut microbiota. HMO increased pH and lowered DM of colonic contents and enhanced the abundance of unclassified Lachnospiraceae, which contains numerous butyrate-producing bacteria. In conclusion, HMO and prebiotics did not prevent the onset of RV infection but reduced the duration of RV-induced diarrhea in piglets, in part, by modulating colonic microbiota and immune response to RV infection.

Adipokines and obesity are associated with colorectal polyps in adult males: A cross-sectional study

Background Obesity increases the risk of colon cancer. It is also known that most colorectal cancers develop from adenomatous polyps. However, the effects of obesity and adipokines on colonic polyp formation are unknown. Methods To determine if BMI, waist circumference or adipokines are associated with colon polyps in males, 126 asymptomatic men (48–65 yr) were recruited at time of colonoscopy, and anthropometric measures as well as blood were collected. Odds ratios were determined using polytomous logistic regression for polyp number (0 or ≥3) and polyp type (no polyp, hyperplastic polyp, tubular adenoma). Results 41% of the men in our study were obese (BMI ≥30). The odds of an obese individual having ≥3 polyps was 6.5 (CI: 1.3–33.0) times greater than those of a lean (BMI<25) individual. Additionally, relative to lean individuals, obese individuals were 7.8 (CI: 2.0–30.8) times more likely to have a tubular adenoma than no polyp. As BMI category increased, participants were 2.9 (CI: 1.5–5.4) times more likely to have a tubular adenoma than no polyps. Serum leptin, IP-10 and TNF-α were significantly associated with tubular adenoma presence. Serum leptin and IP-10 were significantly associated with increased likelihood of ≥3 polyps, and TNF-α showed a trend (p = 0.09). Conclusions Obese men are more likely to have at least three polyps and adenomas. This cross-sectional study provides evidence that colonoscopy should be recommended for obese, white males.

Plasma phospholipids, non-esterified plasma polyunsaturated fatty acids and oxylipids are associated with BMI

The obese lipid profile is associated with increased free fatty acids and triacylglycerides. Currently, little is known about the plasma lipid species associated with obesity. In this study, we compared plasma lipid fatty acid (FA) profiles as a function of BMI. Profiling phospholipid (PL) FAs and their respective oxylipids could predict which obese individuals are more likely to suffer from diseases associated with chronic inflammation or oxidative stress. We investigated the relationship between BMI and plasma PL (PPL) FA composition in 126 men using a quantitative gas chromatography analysis. BMI was inversely associated with both PPL nervonic and linoleic acid (LA) but was positively associated with both dihomo-γ-linolenic and palmitoleic acid. Compared to lean individuals, obese participants were more likely to have ω-6 FAs, except arachidonic acid and LA, incorporated into PPLs. Obese participants were less likely to have EPA and DHA incorporated into PPLs compared to lean participants. Non-esterified plasma PUFA and oxylipid analysis showed ω-6 oxylipids were more abundant in the obese plasma pool. These ω-6 oxylipids are associated with increased angiogenesis (i.e. epoxyeicosatrienoates), reactive oxygen species (i.e. 9-hydroxyeicosatetraenoate), and inflammation resolution (i.e. Lipoxin A4). In summary, BMI is directly associated with specific PPL FA and increased ω-6 oxylipids.

Dietary Bovine Lactoferrin Alters Mucosal and Systemic Immune Cell Responses in Neonatal Piglets

Lactoferrin (LF) is a multifunctional immune protein found at high concentrations in human milk. Herein, the effect of dietary bovine LF (bLF) on mucosal and systemic immune development was investigated. Colostrum-deprived piglets were fed formula containing 130 [control (Ctrl)], 367 (LF1), or 1300 (LF3) mg of bLF/(kg body weight · d). To provide passive immunity, sow serum was provided orally during the first 36 h of life. Blood, spleen, mesenteric lymph node (MLN), and ascending colon (Asc) contents were collected on day 7 (n = 10-14/group) and day 14 (n = 10-12/group). Immune cell populations were quantified by flow cytometry and immunoglobulins (Igs) were measured by ELISA. Additionally, immune cells were isolated from spleen and MLNs (n = 7/group) on day 7 and stimulated ex vivo with phytohemagglutinin or lipopolysaccharide (LPS) ± LF for 72 h. Secreted cytokine concentrations were quantified by multiplex assay. Lymphocyte populations [cluster determinant (CD)4, CD8, and natural killer cells] developed normally and were unaffected by dietary bLF. LF3 piglets tended to have 1.4 to 2 times more serum IgG than Ctrl piglets (P = 0.07) or LF1 piglets (P = 0.03), but IgA in Asc contents was unaffected by bLF. Asc IgA was 4 times higher on day 14 than day 7. Spleen cells from LF3 piglets produced 2 times more interleukin (IL)-10 and tumor necrosis factor (TNF)-α ex vivo than those from Ctrl or LF1 piglets. MLN cells from LF1 and LF3 piglets produced 40% more IL-10 and tended to produce 40% more IL-6 (P = 0.05) than those from Ctrl piglets. However, ex vivo bLF did not affect the cytokine response of spleen or MLN cells to LPS. In summary, dietary bLF alters the capacity of MLN and spleen immune cells to respond to stimulation, supporting a role for LF in the initiation of protective immune responses in these immunologically challenged neonates.

Select human milk oligosaccharides directly modulate peripheral blood mononuclear cells isolated from 10-d-old pigs

Infant formulas lack the complex mixture of oligosaccharides found in human milk. These human milk oligosaccharides (HMO) may be pivotal to the development of the neonatal immune system. Few comprehensive analyses of the effects of HMO on immune cells from neonates have been undertaken. Herein, the direct effects of HMO on immune cells were analysed ex vivo. Peripheral blood mononuclear cells (PBMC) isolated from 10-d-old sow-reared (SR) or colostrum-deprived formula-fed (FF) pigs were stimulated for 72 h with single HMO, mixtures of single HMO or a complex mixture of HMO isolated from human milk (iHMO). T-cell phenotype, cytokine production and proliferation were measured by flow cytometry, immunoassay and [³H]thymidine incorporation, respectively. Stimulation with HMO had direct effects on PBMC. For instance, cells stimulated with iHMO produced more IL-10 than unstimulated cells, and cells stimulated with fucosylated HMO tended to proliferate less than unstimulated cells. Additionally, co-stimulation with HMO mixtures or single HMO altered PBMC responses to phytohaemagglutinin (PHA) or lipopolysaccharide (LPS) stimulation. Compared with PBMC stimulated with PHA alone, cells co-stimulated with iHMO and PHA proliferated more and had fewer detectable CD4⁺CD8⁺ T cells. Compared with PBMC stimulated by LPS alone, cells co-stimulated with a mixture of sialylated HMO and LPS proliferated more and tended to have fewer detectable CD4⁺ T cells. Differences in the baseline responses of PBMC isolated from the SR or FF pigs were observed. In summary, HMO directly affected PBMC populations and functions. Additionally, ex vivo measurements of PBMC phenotype, cytokine production and proliferation were influenced by the neonates diet.

Dietary Bovine Lactoferrin Increases Intestinal Cell Proliferation in Neonatal Piglets

Lactoferrin is a bioactive milk protein that stimulates cell proliferation in vitro; however, limited in vivo evidence exists to allow lactoferrin to be incorporated into infant formula. Herein, the effect of dietary bovine lactoferrin (bLF) on neonatal intestinal growth and maturation was investigated guided by the hypothesis that bLF would increase cellular proliferation leading to functional differences in neonatal piglets. Colostrum-deprived piglets were fed formula containing 0.4 [control (Ctrl)], 1.0 (LF1), or 3.6 (LF3) g bLF/L for the first 7 or 14 d of life. To provide passive immunity, sow serum was provided orally during the first 36 h of life. Intestinal cell proliferation, histomorphology, mucosal DNA concentration, enzyme activity, gene expression, and fecal bLF content were measured. Intestinal enzyme activity, DNA concentration, and villus length were unaffected by bLF. However, crypt proliferation was 60% greater in LF1- and LF3-fed piglets than in Ctrl piglets, and crypt depth and area were 20% greater in LF3-fed piglets than in Ctrl piglets. Crypt cells from LF3-fed piglets had 3-fold higher β-catenin mRNA expression than did crypt cells from Ctrl piglets. Last, feces of piglets fed bLF contained intact bLF, suggesting that some bLF was resistant to digestion and could potentially affect intestinal proliferation through direct interaction with intestinal epithelial cells. This study is the first to our knowledge to show that dietary bLF stimulates crypt cell proliferation in vivo. The increased β-catenin expression indicates that Wnt signaling may in part mediate the stimulatory effect of bLF on intestinal cell proliferation.

Human Milk Oligosaccharides Influence Neonatal Mucosal and Systemic Immunity

The immune system of the infant is functionally immature and naïve. Human milk contains bioactive proteins, lipids, and carbohydrates that protect the newborn and stimulate innate and adaptive immune development. This review will focus on the role human milk oligosaccharides (HMO) play in neonatal gastrointestinal and systemic immune development and function. For the past decade, intense research has been directed at defining the complexity of oligosaccharides in the milk of many species and is beginning to delineate their diverse functions. These studies have shown that human milk contains a higher concentration as well as a greater structural diversity and degree of fucosylation than the milk oligosaccharides in other species, particularly bovine milk from which many infant formulae are produced. The commercial availability of large quantities of certain HMO has furthered our understanding of the functions of specific HMO, which include protecting the infant from pathogenic infections, facilitating the establishment of the gut microbiota, promoting intestinal development, and stimulating immune maturation. Many of these actions are exerted through carbohydrate-carbohydrate interactions with pathogens or host cells. Two HMOs, 2′-fucosyllactose (2′FL) and lacto-N-neotetraose (LNnT), have recently been added to infant formula. Although this is a first step in narrowing the compositional gap between human milk and infant formula, it is unclear whether 1 or 2 HMO will recapitulate the complexity of actions exerted by the complex mixture of HMO ingested by breastfed infants. Thus, as more HMO become commercially available, either isolated from bovine milk or chemically or microbially synthesized, it is anticipated that more oligosaccharides will be added to infant formula either alone or in combination with other prebiotics.

Natural killer cell populations and cytotoxic activity in pigs fed mother's milk, formula, or formula supplemented with bovine lactoferrin

Background:Natural killer (NK) cells are components of the innate immune defense system, and their levels differ between breast and formula-fed (FF) infants. Lactoferrin (Lf) modulates NK cell cytotoxicity ex vivo. We hypothesized that dietary bovine Lf (bLf) would increase NK cell populations and cytotoxicity.Methods:Piglets were sow-reared (SR), FF, or 1 g/l bLf-fed (LF) for 21 d. NK cells (CD3−CD4−CD8+) in blood (peripheral blood mononuclear cells (PBMCs)), spleen, and mesenteric lymph node (MLN) were determined by flow cytometry. PBMC NK cells were tested for cytotoxic activity against target K562 cells ex vivo in the presence of media (unstimulated), interleukin-2, or bLf. NK cell mRNA expression was determined by reverse transcription-quantitative PCR.Results:SR and LF piglets had more NK cells in MLN (P = 0.0097) and spleen (P = 0.0980) than FF piglets. In PBMCs, SR piglets had more NK cells than FF piglets (P = 0.0072); LF piglets were intermediate and not different from FF or SR piglets. NK cell intelectin-2 mRNA expression was 2.5-fold higher (P = 0.0095) in LF than SR or FF piglets. NK cells in SR piglets exhibited greater (P < 0.0001) cytotoxic activity than those in LF or FF piglets, which was supported by greater perforin mRNA expression.Conclusion:Dietary bLf increased blood NK cell populations and NK Lf receptor expression but not NK cell cytotoxicity.

Altered saturated and monounsaturated plasma phospholipid fatty acid profiles in adult males with colon adenomas

Background: Altered lipid metabolism and plasma fatty acid (FA) levels are associated with colorectal cancer. Obesity and elevated waist circumference (WC) increase the likelihood of developing precancerous colon adenomas. Methods: Venous blood was collected from 126 males, ages 48 to 65 years, who received routine colonoscopies. Plasma phospholipid (PPL) FAs were isolated, derivatized, and then analyzed using gas chromatography. ORs and 95% confidence intervals were determined using polytomous logistic regression after adjusting for confounding factors [i.e., age, smoking, WC, and body mass index (BMI)]. Results: PPL palmitic acid (PA) was inversely correlated with the presence of colon adenomas (P = 0.01). For each unit increase in palmitoleic acid (OR, 3.75; P = 0.04) or elaidic acid (OR, 2.92; P = 0.04), an individual was more likely to have adenomas relative to no colon polyps. Higher enzyme activity estimates (EAE) of stearoyl-CoA desaturase-1 (SCD-1; P = 0.02) and elongation of very long chain fatty acids protein-6 (ELOVL-6; P = 0.03) were associated with an individual being approximately 1.5 times more likely to have an adenoma compared with no polyps. Conclusions: PPL FAs and EAEs, which have previously been associated with colorectal cancer, are significantly different in those with adenomas when compared with those without polyps. PPL PA, elaidic acid, and SCD-1 and ELOVL-6 EAEs are associated with adenomas independent of BMI and WC. Impact: PPL PA, elaidic acid, and SCD-1 and ELOVL-6 EAEs are associated with adenomas even after adjusting for obesity-related risk factors and may function as novel biomarkers of early colorectal cancer risk. Cancer Epidemiol Biomarkers Prev; 25(3); 498–506. ©2015 AACR.

Association of Insulin-Related Serum Factors with Colorectal Polyp Number and Type in Adult Males

Background: Dysregulated insulin signaling is thought to contribute to cancer risk. Methods: To determine if insulin-related serum factors are associated with colon polyps, 126 asymptomatic men (48–65 years) were recruited at colonoscopy. Blood was collected. Odds ratios were determined using polytomous logistic regression for polyp number and type. Results: Males with serum C-peptide concentration >3.3 ng/mL were 3.8 times more likely to have an adenoma relative to no polyp than those with C-peptide ≤1.8 ng/mL. As C-peptide tertile increased, an individual was 2 times more likely to have an adenoma (P = 0.01) than no polyp. There were no associations between insulin-like growth factor or its binding proteins with polyp number or type. Males with soluble receptor for advanced glycation end products (sRAGE) concentration >120.4 pg/mL were 0.25 times less likely to have ≥3 polyps relative to no polyps compared with males with sRAGE ≤94.5 pg/mL. For each increase in sRAGE tertile, a man was 0.5 times less likely to have ≥3 polyps than no polyps (P = 0.03). Compared with males with a serum vascular endothelial growth factor (VEGF) concentration ≤104.7 pg/mL, males with a serum VEGF concentration >184.2 pg/mL were 3.4 times more likely to have ≥3 polyps relative to no polyps. As the VEGF tertile increased, a man was 1.9 times more likely to have ≥3 polyps than no polyps (P = 0.049). Conclusions: Serum concentrations of C-peptide, sRAGE, and VEGF may indicate which men could benefit most from colonoscopy. Impact: Identification of biomarkers could reduce medical costs through the elimination of colonoscopies on low-risk individuals. Cancer Epidemiol Biomarkers Prev; 23(9); 1843–51. ©2014 AACR.