Effect of bovine milk fat-based infant formulae on microbiota, metabolites and stool parameters in healthy term infants in a randomized, crossover, placebo-controlled trial

Background: Natural enrichment of sn-2 palmitate content of infant formulae by using bovine milk fat is known to reduce formation of faecal fatty acid soaps and to improve stool consistency, but effects on gut microbiota composition are unknown. The purpose of this study was to test the influence of milk fat-based formula high in sn-2 palmitate on the infants’ gut microbiota composition and to confirm the beneficial effects of the formula on formation of faecal fatty acid soaps and stool consistency. Methods: Twenty-two healthy term, formula-fed infants were enrolled in a single-blinded randomized, crossover, placebo-controlled trial. After a 2-week run-in period, infants received either a 50% milk fat-based formula containing 39% sn-2 palmitate (MF) or a vegetable fat-based formula (VF) containing 10% sn-2 palmitate in a 2 × 2-week crossover design. Faecal microbiota composition was the primary outcome of the study. Other outcomes included faecal fatty acid soap excretion, calcium excretion, gut comfort parameters and faecal metabolites. Results: Microbiota analysis showed that bifidobacteria dominated the gut microbiota of most infants. Neither alpha- nor beta-diversity was significantly influenced by the intervention. Also, abundance of metabolic pathways was independent of the intervention. The MF formula resulted in significantly lower faecal levels of palmitic acid soap (p = 0.0002) and total fatty acid soaps (p = 0.0001) than the VF formula. Additionally, calcium excretion and palmitic acid concentration were significantly (p = 0.0335) lower in stool samples after MF intervention. Furthermore, a significant physiological effect on softer stools was observed in the MF intervention compared to the VF intervention (p = 0.02). Of the 870 measured faecal metabolites, 190 were significantly different after MF and VF intervention (FDR corrected p &lt; 0.05). Most of these were found at higher levels after MF intervention, potentially indicative of the complex structure of milk fat. Metabolites with more than twofold change between interventions were mostly lipid-derived and included several milk fat-specific fatty acids. Conclusions: Replacing part of the vegetable fat in infant formula with bovine milk fat with high sn-2 palmitate levels did not change the microbiota composition, although a reduction in faecal palmitate soaps, total fatty acid soaps and calcium excretion while improving stool consistency in the MF intervention was confirmed. In addition, 190 faecal metabolites were significantly different, many related to the fat source. Trial registration: Netherlands Trial Registry Identifier: NL7815 19/06/2019.</p

Cigarette smoke induces β2-integrin-dependent neutrophil migration across human endothelium

<p>Abstract</p> <p>Background</p> <p>Cigarette smoking induces peripheral inflammatory responses in all smokers and is the major risk factor for neutrophilic lung disease such as chronic obstructive pulmonary disease. The aim of this study was to investigate the effect of cigarette smoke on neutrophil migration and on β₂-integrin activation and function in neutrophilic transmigration through endothelium.</p> <p>Methods and results</p> <p>Utilizing freshly isolated human PMNs, the effect of cigarette smoke on migration and β₂-integrin activation and function in neutrophilic transmigration was studied. In this report, we demonstrated that cigarette smoke extract (CSE) dose dependently induced migration of neutrophils <it>in vitro</it>. Moreover, CSE promoted neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that Mac-1 (CD11b/CD18) is responsible for the cigarette smoke-induced firm adhesion of neutrophils to fibrinogen. Furthermore, neutrophils transmigrated through endothelium by cigarette smoke due to the activation of β₂-integrins, since pre-incubation of neutrophils with functional blocking antibodies against CD11b and CD18 attenuated this transmigration.</p> <p>Conclusion</p> <p>This is the first study to describe that cigarette smoke extract induces a direct migratory effect on neutrophils and that CSE is an activator of β₂-integrins on the cell surface. Blocking this activation of β₂-integrins might be an important target in cigarette smoke induced neutrophilic diseases.</p

Platelet-monocyte complexes support monocyte adhesion to endothelium by enhancing secondary tethering and cluster formation

Adhesion of monocytes to endothelium can be supported by monocyte-monocyte interactions resulting in the formation of cell aggregates at the vessel wall (clusters). Since platelets that are bound to the injured vessel wall support monocyte adhesion and platelet activation in the circulation leads to formation of platelet-monocyte complexes (PMCs), we examined whether adhesion of PMCs to the vessel wall enhances monocyte clustering. The effect of PMC formation in monocyte adhesion and clustering on human umbilical vein endothelial cells (HUVECs) was studied in vitro with a perfusion system. In the presence of 10% to 20% PMCs, monocyte adhesion and cluster formation to stimulated HUVECs increased 2-fold above levels obtained with pure monocytes. While the observed effects increased with higher PMC levels, blocking-monoclonal antibodies directed against platelet-associated P-selectin or monocyte P-selectin glycoprotein ligand-1 (PSGL-1) reversed adhesion and clustering to control values. In the presence of PMCs, blocking L-selectin decreased adhesion by 25%. When PMCs were present, clustering was only supported by L-selectin at higher shear. These data indicate that monocyte adhesion to the vessel wall is enhanced by PMC-mediated monocyte secondary tethering. These interactions are mainly mediated by P-selectin and PSGL-1. PMCs in the circulation might be proatherogenic, and prevention of their formation is a possible therapeutic goa

Fibrin and activated platelets cooperatively guide stem cells to a vascular injury and promote differentiation towards an endothelial cell phenotype

Bone marrow-derived progenitor cells play a role in vascular regeneration. However, their homing to areas of vascular injury is poorly understood. One of the earliest responses to an injury is the activation of coagulation and platelets. In this study we assessed the role of hemostatic components in the recruitment of CD34+ cells to sites of injury. Using an ex vivo injury model, representing endothelial cell (EC) injury or vessel denudation, we studied homing of CD34+ under flow. Platelet aggregates facilitated initial tethering and rolling of CD34+ cells through interaction of P-selectin expressed by platelets and P-selectin glycoprotein ligand-1 (PSGL-1), expressed by CD34+ cells. Ligation of PSGL-1 activated adhesion molecules on CD34+ cells, ultimately leading to firm adhesion of CD34+ cells to tissue factor-expressing ECs or to fibrin-containing thrombi formed on subendothelium. We also demonstrate that fibrin-containing thrombi can support migration of CD34+ cells to the site of injury and subsequent differentiation toward a mature EC phenotype. Additionally, intravenously injected CD34+ cells homed in vivo to denuded arteries in the presence of endogenous leukocytes. We provide evidence that hemostatic factors, associated with vascular injury, provide a regulatory microenvironment for re-endothelialization mediated by circulating progenitor cell

Bovine respiratory syncytial virus infection influences the impact of α(4)- and β(2)-integrin-mediated adhesion of peripheral blood neutrophils

Neutrophil migration into the airways and pulmonary tissue is a common finding in bovine respiratory syncytial virus (BRSV) infections. Although neutrophil trans-endothelial migration in general depends on β(2)-integrins, alternative integrins such as the α(4)-integrins have been implicated. In this study, rolling and firm adhesion of peripheral blood neutrophils isolated from healthy and BRSV-infected calves to tumour necrosis factor (TNF)-α activated pulmonary endothelium was investigated under flow conditions in vitro. For neutrophils obtained from healthy animals, inhibition of the β(2)-integrin reduced firm adhesion to 63% and inhibition of α(4)-integrin to 73% compared with untreated controls. Inhibition of both integrins reduced firm adhesion to 25%. Rolling velocity, which is used as a parameter for integrin involvement in neutrophil rolling, increased 1·7-fold by blocking β(2)-integrin and was significantly augmented to 2·5-fold by blocking both α(4)- and β(2)-integrins. For neutrophils obtained from BRSV-infected animals, however, rolling velocities at 10 days after infection (p.i.) were not influenced by blocking adhesion of α(4)- and β(2)-integrins, indicating that these integrins did not support neutrophil rolling. In addition, the inhibition of firm adhesion by blocking both α(4)- and β(2)-integrins was reduced significantly 9 days post-infection, resulting in a residual 68% neutrophil binding at 9 days p.i. Non-blocked firm adherence was not reduced, indicating that binding was achieved by other mechanisms than through α(4)- and β(2)-integrins. These results demonstrate an important function for α(4)- and β(2)-integrins in rolling and firm adherence of bovine neutrophils, to TNF-α-activated endothelium and show the dynamic use of these integrins for adhesion and migration by neutrophils in the course of BRSV infection

Dietary Intervention with Whey Protein Concentrate Does Not Affect Toll-like Receptor Responses and Gene Expression Patterns in Peripheral Blood Mononuclear Cells of Healthy Volunteers

Bovine milk contains bioactive proteins, carbohydrates, and phospholipids with immunomodulatory properties impacting human immunity, potentially contributing to resistance to infections and allergies through diverse mechanisms. One such mechanism is the enhancing of the innate immune response to secondary pathogen-related stimuli, termed innate immune training. Although in vitro studies demonstrate that milk immunoglobulin G (IgG) can train human monocytes, evidence for in vivo immune training is limited. To explore the potential of bovine IgG for inducing innate immune training in vivo, this human study utilized an IgG-rich whey protein concentrate (WPC). Healthy male volunteers were assigned to a high dose WPC, low dose WPC, or placebo group. Blood was collected pre- and post-two weeks of WPC consumption. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with TLR ligands, evaluating IL-6 and TNF-α production by monocytes, myeloid DCs, and plasmacytoid DCs. Additionally, RNA was isolated for differential gene expression (DGE) analysis. Results indicated that the two-week WPC intervention did not influence the ex vivo response of studied cells to TLR agonists. Furthermore, PBMC gene expression patterns showed no significant differences between the placebo and high dose WPC groups. The data suggests that oral WPC ingestion did not enhance immune responses in young, healthy male participants

Effect of bovine milk fat-based infant formulae on microbiota, metabolites and stool parameters in healthy term infants in a randomized, crossover, placebo-controlled trial

Background: Natural enrichment of sn-2 palmitate content of infant formulae by using bovine milk fat is known to reduce formation of faecal fatty acid soaps and to improve stool consistency, but effects on gut microbiota composition are unknown. The purpose of this study was to test the influence of milk fat-based formula high in sn-2 palmitate on the infants’ gut microbiota composition and to confirm the beneficial effects of the formula on formation of faecal fatty acid soaps and stool consistency. Methods: Twenty-two healthy term, formula-fed infants were enrolled in a single-blinded randomized, crossover, placebo-controlled trial. After a 2-week run-in period, infants received either a 50% milk fat-based formula containing 39% sn-2 palmitate (MF) or a vegetable fat-based formula (VF) containing 10% sn-2 palmitate in a 2 × 2-week crossover design. Faecal microbiota composition was the primary outcome of the study. Other outcomes included faecal fatty acid soap excretion, calcium excretion, gut comfort parameters and faecal metabolites. Results: Microbiota analysis showed that bifidobacteria dominated the gut microbiota of most infants. Neither alpha- nor beta-diversity was significantly influenced by the intervention. Also, abundance of metabolic pathways was independent of the intervention. The MF formula resulted in significantly lower faecal levels of palmitic acid soap (p = 0.0002) and total fatty acid soaps (p = 0.0001) than the VF formula. Additionally, calcium excretion and palmitic acid concentration were significantly (p = 0.0335) lower in stool samples after MF intervention. Furthermore, a significant physiological effect on softer stools was observed in the MF intervention compared to the VF intervention (p = 0.02). Of the 870 measured faecal metabolites, 190 were significantly different after MF and VF intervention (FDR corrected p < 0.05). Most of these were found at higher levels after MF intervention, potentially indicative of the complex structure of milk fat. Metabolites with more than twofold change between interventions were mostly lipid-derived and included several milk fat-specific fatty acids. Conclusions: Replacing part of the vegetable fat in infant formula with bovine milk fat with high sn-2 palmitate levels did not change the microbiota composition, although a reduction in faecal palmitate soaps, total fatty acid soaps and calcium excretion while improving stool consistency in the MF intervention was confirmed. In addition, 190 faecal metabolites were significantly different, many related to the fat source. Trial registration: Netherlands Trial Registry Identifier: NL7815 19/06/2019

The piglet as a model for studying dietary components in infant diets : effects of galacto-oligosaccharides on intestinal functions

Prebiotic oligosaccharides, including galacto-oligosaccharides (GOS), are used in infant formula to mimic human milk oligosaccharides, which are known to have an important role in the development of the intestinal microbiota and the immune system in neonates. The maturation of the intestines in piglets closely resembles that of human neonates and infants. Hence, a neonatal piglet model was used to study the multi-faceted effect of dietary GOS in early life. Naturally farrowed piglets were separated from the mother sow 24-48 h postpartum and received a milk replacer with or without the addition of GOS for 3 or 26 d, whereafter several indicators of intestinal colonisation and maturation were measured. Dietary GOS was readily fermented in the colon, leading to a decreased pH, an increase in butyric acid in caecum digesta and an increase in lactobacilli and bifidobacteria numbers at day 26. Histomorphological changes were observed in the intestines of piglets fed a GOS diet for 3 or 26 d. In turn, differences in the intestinal disaccharidase activity were observed between control and GOS-fed piglets. The mRNA expression of various tight junction proteins was up-regulated in the intestines of piglet fed a GOS diet and was not accompanied by an increase in protein expression. GOS also increased defensin porcine β-defensin-2 in the colon and secretory IgA levels in saliva. In conclusion, by applying a neonatal piglet model, it could be demonstrated that a GOS-supplemented milk replacer promotes the balance of the developing intestinal microbiota, improves the intestinal architecture and seems to stimulate the intestinal defence mechanism