Synbiotics Combined with Glutamine Stimulate Brain Development and the Immune System in Preterm Pigs.

Background: Preterm infants are born with an immature gut, brain, and immune system, predisposing them to short- and long-term complications. Objective: We hypothesized that a milk diet supplemented with pre- and probiotics (i.e. synbiotics) and glutamine would improve gut, brain, and immune maturation in preterm neonates, using preterm pigs as a model. Methods: Preterm pigs (Landrace x Yorkshire x Duroc, n = 40, delivered by c-section at 90% of gestation) were reared individually until day 23 after birth under highly standardized conditions. Piglets in the intervention group (PPG, n = 20) were fed increasing volumes of bovine milk supplemented with prebiotics (short-chain galacto- and long chain fructo-oligosaccharides 9:1, 4-12 g/L), probiotics (Bifidobacterium breve M16-V, 3 × 109 CFU/d) and l-glutamine [0.15-0.30 g/(kg · d)], and compared with pigs fed bovine milk with added placebo compounds as control (CON, n = 20). Clinical, gastrointestinal, immunological, cognitive, and neurological endpoints were measured. Results: The PPG pigs showed more diarrhea but weight gain, body composition, and gut parameters were similar between the groups. Cognitive performance, assessed in a T-maze, was significantly higher in PPG pigs (P < 0.01), whereas motor function and exploratory interest were similar between the groups. Using ex vivo diffusion imaging, the orientation dispersion index in brain cortical gray matter was 50% higher (P = 0.04), and fractional anisotropy value was 7% lower (P = 0.05) in PPG pigs compared with CON pigs, consistent with increased dendritic branching in PPG. In associative fibers, radial diffusivity was lower and fractional anisotropy was higher in PPG pigs compared with CON pigs (all P < 0.05), while measures in the internal capsule showed a tendency towards reduced radial diffusivity and mean diffusivity (both P = 0.09). On day 23 pigs in the PPG group showed higher blood leukocyte numbers (+43%), neutrophil counts (+100%), and phagocytic rates (+24%), relative to CON, all P < 0.05. Conclusion: Preterm pigs supplemented with Bifidobacterium breve, galacto- and fructo-oligosaccharides, and l-glutamine showed enhanced neuronal and immunological development. The findings indicate the potential for targeted nutritional interventions after preterm birth, to support development of important systems such as immunity and brain.

Diet-Derived Short Chain Fatty Acids Stimulate Intestinal Epithelial Cells To Induce Mucosal Tolerogenic Dendritic Cells.

The gastrointestinal tract is continuously exposed to many environmental factors that influence intestinal epithelial cells and the underlying mucosal immune system. In this article, we demonstrate that dietary fiber and short chain fatty acids (SCFAs) induced the expression of the vitamin A-converting enzyme RALDH1 in intestinal epithelial cells in vivo and in vitro, respectively. Furthermore, our data showed that the expression levels of RALDH1 in small intestinal epithelial cells correlated with the activity of vitamin A-converting enzymes in mesenteric lymph node dendritic cells, along with increased numbers of intestinal regulatory T cells and a higher production of luminal IgA. Moreover, we show that the consumption of dietary fiber can alter the composition of SCFA-producing microbiota and SCFA production in the small intestines. In conclusion, our data illustrate that dietary adjustments affect small intestinal epithelial cells and can be used to modulate the mucosal immune system.

Lipoproteins attenuate TLR2 and TLR4 activation by bacteria and bacterial ligands with differences in affinity and kinetics.

BACKGROUND: The small intestine is a specialized compartment were close interactions take place between host, microbes, food antigens and dietary fatty acids. Dietary fats get absorbed by epithelial cells and processed into a range of lipoprotein particles after which they are basolaterally secreted and collected in the lymphatics. In contrast to the colon, the small intestine is covered only by a thin mucus coat that allows for intimate interactions between host-cells and microbes. Lipoproteins have long been recognized as protective factors in infectious diseases via the neutralization of bacterial toxins like lipopolysaccharides. Much less attention has been given to the potential role of lipoproteins as factors contributing to the maintenance of small intestinal immune homeostasis via modulating bacteria-induced immune responses. RESULTS: Lipoproteins VLDL, LDL and HDL were found to neutralize TLR responses towards specific TLR-ligands or a selection of gram-negative and gram-positive bacteria. Attenuation of TLR2 activity was acute and only slightly improved by longer pre-incubation times of ligands and lipoproteins with no differences between bacterial-lipopeptides or bacteria. In contrast, attenuation of TLR4 responses was only observed after extensive preincubation of lipoproteins and LPS. Preincubation of bacteria and lipoproteins led only to a modest attenuation of TLR4 activity. Moreover, compared to TLR2, TLR4 activity could only be attenuated by lipoproteins over a small ligand dose range. CONCLUSIONS: These results demonstrate the ability of lipoproteins VLDL, LDL and HDL to inhibit TLR responses towards bacterial-ligands and bacteria. Presence of lipoproteins was found to modulate the MAMP-induced cytokine release by primary human monocytes measured as changes in the release of IL-6, TNFα, GM-CSF and IFNγ. Using TLR2 and TLR4-reporter cells, lipoproteins were found to inhibit TLR responses with differences in affinity and kinetics. These data establish a role for lipoproteins as immunoregulatory molecules, attenuating TLR-responses and thereby positively contributing to mucosal homeostasis.

Study protocol for cholera vaccination as a model to measure the inflammatory response in the gut: A case of modulation with a Lactobacillus plantarum K8 lysate.

It is crucial for human health that the immune system of the gastrointestinal tract works effectively. Dietary modulation is one of the factors that regulate the immune response in the gut. This study aims to develop a safe human challenge model to study gastrointestinal inflammation and immune function. This study focuses on evaluating gut stimulation induced by the oral cholera vaccine in healthy people. In addition, this paper describes the study design for assessing the efficacy and safety of a probiotic lysate, identifying whether functional ingredients in food can modulate inflammatory response induced by oral cholera vaccine. Forty-six males aged 20 to 50 with healthy bowel habits will be randomly allocated to the placebo or intervention group. Participants will consume 1 capsule of probiotic lysate or placebo twice daily for 6 weeks, take oral cholera vaccines on visit 2 (day 15) and visit 5 (day 29). The level of fecal calprotectin, a marker of gut inflammation, will be the primary outcome. The changes of cholera toxin-specific antibody levels and local/systemic inflammatory responses will be evaluated in blood. The purpose of this study is to evaluate gut stimulation of the oral cholera vaccine and investigate the effect of a probiotic lysate on improving the mild inflammatory response induced by the vaccine or supporting the immune response in healthy subjects. Trial registration: * This trial is registered in the International Clinical Trials Registry Platform of WHO (ICTRP, registration number: KCT0002589).

Low doses of diarrhoeagenic E. coli induce enhanced monocyte and mDC responses and prevent development of symptoms after homologous rechallenge.

The experimental challenge with attenuated enterotoxigenic E. coli strain E1392/75-2A prevents diarrhea upon a secondary challenge with the same bacteria. A dose-response pilot study was performed to investigate which immunological factors are associated with this protection. Healthy subjects were inoculated with increasing E. coli doses of 1E6-1E10 CFU, and three weeks later, all participants were rechallenged with the highest dose (1E10 CFU). Gastrointestinal discomfort symptoms were recorded, and stool and blood samples were analyzed. After the primary challenge, stool frequency, diarrhea symptom scores, and E. coli-specific serum IgG (IgG-CFA/II) titer increased in a dose-dependent manner. Fecal calprotectin and serum IgG-CFA/II response after primary challenge were delayed in the lower dose groups. Even though stool frequency after the secondary challenge was inversely related to the primary inoculation dose, all E. coli doses protected against clinical symptoms upon rechallenge. Ex vivo stimulation of PBMCs with E. coli just before the second challenge resulted in increased numbers of IL-6+/TNF-α+ monocytes and mDCs than before the primary challenge, without dose-dependency. These data demonstrate that primary E. coli infection with as few as 1E6 CFU protects against a high-dose secondary challenge with a homologous attenuated strain. Increased serum IgG-CFA/II levels and E. coli-induced mDC and monocyte responses after primary challenge suggest that protection against secondary E. coli challenges is associated with adaptive as well as innate immune responses.

Prebiotic Galacto-Oligosaccharides Impact Stool Frequency and Fecal Microbiota in Self-Reported Constipated Adults: A Randomized Clinical Trial.

Constipation is a major issue for 10-20% of the global population. In a double-blind randomized placebo-controlled clinical trial, we aimed to determine a dose-response effect of galacto-oligosaccharides (GOS) on stool characteristics and fecal microbiota in 132 adults with self-reported constipation according to Rome IV criteria (including less than three bowel movements per week). Subjects (94% females, aged: 18-59 years) received either 11 g or 5.5 g of BiotisTM GOS, or a control product, once daily for three weeks. Validated questionnaires were conducted weekly to study primarily stool frequency and secondary stool consistency. At base- and endline, stool samples were taken to study fecal microbiota. A trend towards an increased stool frequency was observed after the intervention with 11 g of GOS compared to control. While during screening everybody was considered constipated, not all subjects (n = 78) had less than three bowel movements per week at baseline. In total, 11 g of GOS increased stool frequency compared to control in subjects with a low stool frequency at baseline (≤3 bowel movements per week) and in self-reported constipated adults 35 years of age or older. A clear dose-response of GOS was seen on fecal Bifidobacterium, and 11 g of GOS significantly increased Anaerostipes hadrus. In conclusion, GOS seems to be a solution to benefit adults with a low stool frequency and middle-aged adults with self-reported constipation.

Butyrate and hexanoate-enriched triglycerides increase postprandrial systemic butyrate and hexanoate in men with overweight/obesity: A double-blind placebo-controlled randomized crossover trial.

Background: Short chain fatty acids (SCFA) are increasingly recognized for their potential ability to alleviate obesity-associated chronic low-grade inflammation and disturbed energy homeostasis. Evidence suggests that an increase in circulating SCFA might be necessary to induce beneficial alterations in energy metabolism. Objective: To compare the bioaccessibility of two different SCFA-enriched triglycerides: Akovita SCT (butyrate and hexanoate esterified with long chain fatty acids) and tributyrin/caproin (solely butyrate and hexanoate) and investigate whether the SCFA from orally administrated Akovita SCT reach the circulation and affect postprandial metabolism in men with overweight/obesity. Methods: The site, speed, and amount of SCFA release from Akovita SCT and tributyrin/caproin were assessed in a validated In vitro Model of the stomach and small intestine (TIM-1). Subsequently, a double-blind placebo-controlled randomized crossover study was conducted at Maastricht University with fourteen men with overweight/obesity (BMI 25-35 kg/m2) of which twelve men finished all testdays and were included for analysis. The participants received a liquid high fat mixed meal test containing either a low (650 mg), medium (1,325 mg), or high dose (2,000 mg) of Akovita SCT or a placebo (sunflower oil) in randomized order. Blood was sampled at baseline and after ingestion for 6 h for the primary outcome plasma butyrate and hexanoate concentration. Secondary outcomes included hydrogen breath, appetite, gastrointestinal complaints, circulating glucagon-like peptide 1, free fatty acids, glucose, triglycerides, insulin, and cytokines concentrations. Results: In TIM-1, tributyrin/caproin was rapidly cleaved in the gastric compartment whereas the release of SCFA from Akovita SCT occurred predominantly in the small intestine. In vivo, all doses were well-tolerated. The medium dose increased (P < 0.05) and the high dose tended to increase (P < 0.10) postprandial circulating butyrate and both doses increased circulating hexanoate (P < 0.05) compared to placebo. Nevertheless, Akovita SCT supplementation did not affect any secondary outcomes compared to placebo. Conclusion: Esterifying SCFA-enriched triglycerides with long chain fatty acids delayed SCFA release from the glycerol backbone. Akovita SCT increased postprandial circulating butyrate and hexanoate without changing metabolic parameters in men with overweight/obesity. Future randomized clinical trials should investigate whether long-term Akovita SCT supplementation can aid in the treatment or prevention of metabolic disorders. Clinical trial registration: www.ClinicalTrials.gov, identifier: NCT04662411.

A free amino acid-based diet partially prevents symptoms of cow's milk allergy in mice after oral sensitization with whey.

BACKGROUND: Amino acid-based formulas (AAFs) are used for the dietary management of cow's milk allergy (CMA). Whether AAFs have the potential to prevent the development and/or symptoms of CMA is not known. OBJECTIVE: The present study evaluated the preventive effects of an amino acid (AA)-based diet on allergic sensitization and symptoms of CMA in mice and aimed to provide insight into the underlying mechanism. METHODS: C3H/HeOuJ mice were sensitized with whey protein or with phosphate-buffered saline as sham-sensitized control. Starting 2 weeks before sensitization, mice were fed with either a protein-based diet or an AA-based diet with an AA composition based on that of the AAF Neocate, a commercially available AAF prescribed for the dietary management of CMA. Upon challenge, allergic symptoms, mast cell degranulation, whey-specific immunoglobulin levels, and FoxP3+ cell counts in jejunum sections were assessed. RESULTS: Compared to mice fed with the protein-based diet, AA-fed mice had significantly lower acute allergic skin responses. Moreover, the AA-based diet prevented the whey-induced symptoms of anaphylaxis and drop in body temperature. Whereas the AA-based diet had no effect on the levels of serum IgE and mucosal mast cell protease-1 (mMCP-1), AA-fed mice had significantly lower serum IgG2a levels and tended to have lower IgG1 levels (P = .076). In addition, the AA-based diet prevented the whey-induced decrease in FoxP3+ cells. In sham-sensitized mice, no differences between the two diets were observed in any of the tested parameters. CONCLUSION: This study demonstrates that an AA-based diet can at least partially prevent allergic symptoms of CMA in mice. Differences in FoxP3+ cell counts and serum levels of IgG2a and IgG1 may suggest enhanced anti-inflammatory and tolerizing capacities in AA-fed mice. This, combined with the absence of effects in sham-sensitized mice indicates that AAFs for the prevention of food allergies may be an interesting concept that warrants further research.

In vitro and in vivo modulation of cartilage degradation by a standardized Centella asiatica fraction.

Osteoarthritis (OA) is a degenerative joint disease in which focal cartilage destruction is one of the primary features. The present study aims to evaluate the effect of a Centella asiatica fraction on in vitro and in vivo cartilage degradation. Bovine cartilage explants and bovine chondrocytes cultured in alginate were stimulated with IL-1 beta in the presence or absence of different concentrations (2, 5 and 10 microg/ml) of a standardized Centella asiatica triterpenes (CAT) fraction. The CAT fraction inhibited the IL-1 beta-induced proteoglycan (PG) release and nitric oxide (NO) production by cartilage explants in a dose-dependent manner. The IL-1 beta-induced reduction in PG synthesis and proliferation of chondrocytes cultured in alginate were counteracted by the CAT fraction at a concentration of 10 microg/ml. In a zymosan-induced acute arthritis model, the CAT fraction inhibited PG depletion without modulating joint swelling and inflammatory cell infiltration. In conclusion, the present study demonstrated for the first time that the tested Centella asiatica fraction was able to inhibit the zymosan-induced cartilage degradation in vivo without affecting the zymosan-induced inflammatory cell infiltration and joint swelling. The in vitro data indicate that the cartilage protective activity might at least partially be induced by the inhibition of NO production. The overall results indicate a possible disease modifying osteoarthritic activity of the Centella asiatica fraction.

Locomotion and muscle mass measures in a murine model of collagen-induced arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is characterized by chronic poly-arthritis, synovial hyperplasia, erosive synovitis, progressive cartilage and bone destruction accompanied by a loss of body cell mass. This loss of cell mass, known as rheumatoid cachexia, predominates in the skeletal muscle and can in part be explained by a decreased physical activity. The murine collagen induced arthritis (CIA) model has been proven to be a useful model in RA research since it shares many immunological and pathological features with human RA. The present study explored the interactions between arthritis development, locomotion and muscle mass in the CIA model. METHODS: CIA was induced in male DBA/1 mice. Locomotion was registered at different time points by a camera and evaluated by a computerized tracing system. Arthritis severity was detected by the traditionally used semi-quantitative clinical scores. The muscle mass of the hind-legs was detected at the end of the study by weighing. A methotrexate (MTX) intervention group was included to study the applicability of the locomotion and muscle mass for testing effectiveness of interventions in more detail. RESULTS: There is a strong correlation between clinical arthritis and locomotion. The correlations between muscle mass and locomotion or clinical arthritis were less pronounced. MTX intervention resulted in an improvement of disease severity accompanied by an increase in locomotion and muscle mass. CONCLUSION: The present data demonstrate that registration of locomotion followed by a computerized evaluation of the movements is a simple non invasive quantitative method to define disease severity and evaluate effectiveness of therapeutic agents in the CIA model.

The Gut-Immune-Brain Axis in Autism Spectrum Disorders; A Focus on Amino Acids.

Autism spectrum disorder (ASD) is a range of neurodevelopmental conditions that affect communication and social behavior. Besides social deficits, systemic inflammation, gastrointestinal immune-related problems, and changes in the gut microbiota composition are characteristic for people with ASD. Animal models showed that these characteristics can induce ASD-associated behavior, suggesting an intimate relationship between the microbiota, gut, immune system and the brain in ASD. Multiple factors can contribute to the development of ASD, but mutations leading to enhanced activation of the mammalian target of rapamycin (mTOR) are reported frequently. Hyperactivation of mTOR leads to deficits in the communication between neurons in the brain and to immune impairments. Hence, mTOR might be a critical factor linking the gut-brain-immune axis in ASD. Pharmacological inhibition of mTOR is shown to improve ASD-associated behavior and immune functions, however, the clinical use is limited due to severe side reactions. Interestingly, studies have shown that mTOR activation can also be modified by nutritional stimuli, in particular by amino acids. Moreover, specific amino acids are demonstrated to inhibit inflammation, improve gut barrier function and to modify the microbiota composition. In this review we will discuss the gut-brain-immune axis in ASD and explore the potential of amino acids as a treatment option for ASD, either via modification of mTOR activity, the immune system or the gut microbiota composition.

Supplementation of diet with non-digestible oligosaccharides alters the intestinal microbiota, but not arthritis development, in IL-1 receptor antagonist deficient mice.

The intestinal microbiome is perturbed in patients with new-onset and chronic autoimmune inflammatory arthritis. Recent studies in mouse models suggest that development and progression of autoimmune arthritis is highly affected by the intestinal microbiome. This makes modulation of the intestinal microbiota an interesting novel approach to suppress inflammatory arthritis. Prebiotics, defined as non-digestible carbohydrates that selectively stimulate the growth and activity of beneficial microorganisms, provide a relatively non-invasive approach to modulate the intestinal microbiota. The aim of this study was to assess the therapeutic potential of dietary supplementation with a prebiotic mixture of 90% short-chain galacto-oligosaccharides and 10% long-chain fructo-oligosaccharides (scGOS/lcFOS) in experimental arthritis in mice. We here show that dietary supplementation with scGOS/lcFOS has a pronounced effect on the composition of the fecal microbiota. Interestingly, the genera Enterococcus and Clostridium were markedly decreased by scGOS/lcFOS dietary supplementation. In contrast, the family Lachnospiraceae and the genus Lactobacillus, both associated with healthy microbiota, increased in mice receiving scGOS/lcFOS diet. However, the scGOS/lcFOS induced alterations of the intestinal microbiota did not induce significant effects on the intestinal and systemic T helper cell subsets and were not sufficient to reproducibly suppress arthritis in mice. As expected, we did observe a significant increase in the bone mineral density in mice upon dietary supplementation with scGOS/lcFOS for 8 weeks. Altogether, this study suggests that dietary scGOS/lcFOS supplementation is able to promote presumably healthy gut microbiota and improve bone mineral density, but not inflammation, in arthritis-prone mice.

Correction: Supplementation of diet with non-digestible oligosaccharides alters the intestinal microbiota, but not arthritis development, in IL-1 receptor antagonist deficient mice.

[This corrects the article DOI: 10.1371/journal.pone.0219366.].

Short Communication: Differences in Levels of Free Amino Acids and Total Protein in Human Foremilk and Hindmilk.

Free amino acids (FAAs) in human milk are indicated to have specific functional roles in infant development. Studies have shown differences between human milk that is expressed at the beginning of a feed (i.e., foremilk) and the remainder of the milk expressed (i.e., hindmilk). For example, it is well established that human hindmilk is richer in fat and energy than foremilk. Hence, exclusively feeding hindmilk is used to enhance weight gain of preterm, low birthweight infants. Whether FAAs occur differently between foremilk and hindmilk has never been reported, but given their bioactive capacities, this is relevant to consider especially in situations where hindmilk is fed exclusively. Therefore, this study analyzed and compared the FAA and total protein content in human foremilk and hindmilk samples donated by 30 healthy lactating women. The total protein content was found to be significantly higher in hindmilk (p < 0.001), whereas foremilk contained a significantly higher total content of FAAs (p = 0.015). With regards to individual FAAs, foremilk contained significantly higher levels of phenylalanine (p = 0.009), threonine (p = 0.003), valine (p = 0.018), alanine (p = 0.004), glutamine (p < 0.001), and serine (p = 0.012) than hindmilk. Although statistical significance was reached, effect size analysis of the milk fraction on FAA levels in milk revealed that the observed differences were only small. To what extent these differences are of physiological importance for infant development remains to be examined in future research.

Longitudinal Variation of Amino Acid Levels in Human Milk and Their Associations with Infant Gender.

It is discussed that specific amino acids (AAs) have functional roles in early life. Understanding the AA composition in human milk (HM) during lactation assists in specifying these roles. To this end we assessed the levels of free AAs (FAAs), total AAs (free and bound, TAAs) and protein levels in HM in the first 6 months of lactation, and evaluated possible associations with infant gender. HM samples of 25 healthy Dutch mothers participating in the PreventCD study were collected monthly during the first 6 months of lactation. Of the participating mothers, 12 gave birth to a boy and 13 gave birth to a girl. Analyses of the HM samples revealed that levels of free glutamate, glutamine, aspartate, glycine, and serine significantly increased during months 1⁻3 of lactation, both in absolute sense and relative to TAA levels. Evaluation of gender differences by mixed model analyses revealed an association between female infant gender and higher protein content (p = 0.0465) and TAA content (p = 0.0362) in HM during the first 3 months of lactation. Furthermore, there was a tendency for an association of male infant gender with higher levels of free glutamine (p = 0.0948) in HM during the first 3 months of lactation. These results show that FAA, TAA and protein levels in HM display a time-specific occurrence during lactation. Moreover, although confirmation is necessary in view of the small sample size, this study indicates that the AA composition in HM shows differential effects of the infant's sex.

Oral exposure to the free amino acid glycine inhibits the acute allergic response in a model of cow's milk allergy in mice.

The conditionally essential amino acid glycine functions as inhibitory neurotransmitter in the mammalian central nervous system. Moreover, it has been shown to act as an anti-inflammatory compound in animal models of ischemic perfusion, post-operative inflammation, periodontal disease, arthritis and obesity. Glycine acts by binding to a glycine-gated chloride channel, which has been demonstrated on neurons and immune cells, including macrophages, polymorphonuclear neutrophils and lymphocytes. The present study aims to evaluate the effect of glycine on allergy development in a cow's milk allergy model. To this end, C3H/HeOuJ female mice were supplemented with glycine by oral gavage (50 or 100 mg/mouse) 4 hours prior to sensitization with cow's milk whey protein, using cholera toxin as adjuvant. Acute allergic skin responses and anaphylaxis were assessed after intradermal allergen challenge in the ears. Mouse mast cell protease-1 (mMCP-1) and whey specific IgE levels were detected in blood collected 30 minutes after an oral allergen challenge. Jejunum was dissected and evaluated for the presence of mMCP-1-positive cells by immunohistochemistry. Intake of glycine significantly inhibited allergy development in a concentration dependent manner as indicated by a reduction in; acute allergic skin response, anaphylaxis, serum mMCP-1 and serum levels of whey specific IgE. In addition, in-vitro experiments using rat basophilic leukemia cells (RBL), showed that free glycine inhibited cytokine release but not cellular degranulation. These findings support the hypothesis that the onset of cow's milk allergy is prevented by the oral intake of the amino acid glycine. An adequate intake of glycine might be important in the improvement of tolerance against whey allergy or protection against (whey-induced) allergy development.

Reduced dietary intake of micronutrients with antioxidant properties negatively impacts muscle health in aged mice.

BACKGROUND: Inadequate intake of micronutrients with antioxidant properties is common among older adults and has been associated with higher risk of frailty, adverse functional outcome, and impaired muscle health. However, a causal relationship is less well known. The aim was to determine in old mice the impact of reduced dietary intake of vitamins A/E/B6/B12/folate, selenium, and zinc on muscle mass, oxidative capacity, strength, and physical activity (PA) over time. METHODS: Twenty-one-month-old male mice were fed either AIN-93-M (control) or a diet low in micronutrients with antioxidant properties (=LOWOX-B: 50% of mouse recommended daily intake of vitamins A, E, B6, and B12, folate, selenium, and zinc) for 4 months. Muscle mass, grip strength, physical activity (PA), and general oxidative status were assessed. Moreover, muscle fatigue was measured of m. extensor digitorum longus (EDL) during an ex vivo moderate exercise protocol. Effects on oxidative capacity [succinate dehydrogenase (SDH) activity], muscle fibre type, number, and fibre cross-sectional area (fCSA) were assessed on m. plantaris (PL) using histochemistry. RESULTS: After 2 months on the diet, bodyweight of LOWOX-B mice was lower compared with control (P < 0.0001), mainly due to lower fat mass (P < 0.0001), without significant differences in food intake. After 4 months, oxidative status of LOWOX-B mice was lower, demonstrated by decreased vitamin E plasma levels (P < 0.05) and increased liver malondialdehyde levels (P = 0.018). PA was lower in LOWOX-B mice (P < 0.001 vs. control). Muscle mass was not affected, although PL-fCSA was decreased (~16%; P = 0.028 vs. control). SDH activity and muscle fibre type distribution remained unaffected. In LOWOX-B mice, EDL force production was decreased by 49.7% at lower stimulation frequencies (P = 0.038), and fatigue resistance was diminished (P = 0.023) compared with control. CONCLUSIONS: Reduced dietary intake of vitamins A, E, B6, and B12, folate, selenium, and zinc resulted in a lower oxidative capacity and has major impact on muscle health as shown by decreased force production and PA, without effects on muscle mass. The reduced fCSA in combination with similar SDH activity per fibre might explain the reduced oxidative capacity resulting in the increased fatigue after exercise in LOWOX-B mice.

Anti-inflammatory effects of orally ingested lactoferrin and glycine in different zymosan-induced inflammation models: evidence for synergistic activity.

There is a growing awareness of the interaction of food constituents with the immune system. The present study aims to evaluate the anti-inflammatory effects of two of these nutritional components (glycine and bovine-lactoferrin (b-LF)) using two different mouse models. In a zymosan-induced ear-skin inflammation model both components decreased the inflammatory response locally (ear swelling and inflammatory cytokine concentration in the ears) and systemically (number of TNF-alpha producing spleen cells). Glycine effects (20, 50 or 100 mg/mouse/day) were concentration dependent. B-LF (0.1 or 1 mg/mouse/day) inhibited the inflammatory response although higher doses (5 and 25 mg/mouse/day) were not effective. A combination of b-LF 0.1 mg/mouse/day and glycine 20 or 50 mg/mouse/day counteracted the zymosan-induced ear swelling synergistically and enhanced the decrease in the number of TNF-alpha producing spleen cells of the individual components. In a zymosan-induced acute arthritis model glycine (50 mg/mouse/day) inhibited joint swelling, inflammatory cell infiltration and cartilage proteoglycan depletion. A b-LF dose of 5 mg/mouse/day reduced the zymosan-induced joint swelling without modulating inflammatory cell infiltration and cartilage proteoglycan depletion. The present study indicates that the anti-inflammatory effects of glycine are independent of the used models. B-LF displays a reversed concentration dependency and the activity is model dependent. A combination of glycine and lactoferrin demonstrated a synergistic anti-inflammatory effect on zymosan-induced skin inflammation and an enhanced decrease in the number of TNF-alpha producing spleen cells compared to the effect of the single components. Therefore, this nutritional concept might be a new option for the treatment of chronic inflammatory diseases.