The impacts of bovine milk, soy beverage, or almond beverage on the growing rat microbiome.

Background: Milk, the first food of mammals, helps to establish a baseline gut microbiota. In humans, milk and milk products are consumed beyond infancy, providing comprehensive nutritional value. Non-dairy beverages, produced from plant, are increasingly popular as alternatives to dairy milk. The nutritive value of some plant-based products continues to be debated, whilst investigations into impacts on the microbiome are rare. The aim of this study was to compare the impact of bovine milk, soy and almond beverages on the rat gut microbiome. We previously showed soy and milk supplemented rats had similar bone density whereas the almond supplemented group had compromised bone health. There is an established link between bone health and the microbiota, leading us to hypothesise that the microbiota of groups supplemented with soy and milk would be somewhat similar, whilst almond supplementation would be different. Methods: Three-week-old male Sprague Dawley rats were randomly assigned to five groups (n = 10/group) and fed ad libitum for four weeks. Two control groups were fed either standard diet (AIN-93G food) or AIN-93G amino acids (AA, containing amino acids equivalent to casein but with no intact protein) and with water provided ad libitum. Three treatment groups were fed AIN-93G AA and supplemented with either bovine ultra-heat treatment (UHT) milk or soy or almond UHT beverages as their sole liquid source. At trial end, DNA was extracted from caecum contents, and microbial abundance and diversity assessed using high throughput sequencing of the V3 to V4 variable regions of the 16S ribosomal RNA gene. Results: Almost all phyla (91%) differed significantly (FDR < 0.05) in relative abundance according to treatment and there were distinct differences seen in community structure between treatment groups at this level. At family level, forty taxa showed significantly different relative abundance (FDR < 0.05). Bacteroidetes (Bacteroidaceae) and Firmicutes populations (Lactobacillaceae, Clostridiaceae and Peptostreptococcaceae) increased in relative abundance in the AA almond supplemented group. Supplementation with milk resulted in increased abundance of Actinobacteria (Coriobacteriaceae and Bifidobacteriaceae) compared with other groups. Soy supplementation increased abundance of some Firmicutes (Lactobacilliaceae) but not Actinobacteria, as previously reported by others. Conclusion: Supplementation with milk or plant-based drinks has broad impacts on the intestinal microbiome of young rats. Changes induced by cow milk were generally in line with previous reports showing increased relative abundance of Bifidobacteriacea, whilst soy and almond beverage did not. Changes induced by soy and almond drink supplementation were in taxa commonly associated with carbohydrate utilisation. This research provides new insight into effects on the microbiome of three commercially available products marketed for similar uses.

Type A and B bovine milks: Heat stability is driven by different physicochemical parameters.

The value of milk hinges on its physicochemical functionality under processing conditions. We examined composition-functionality relationships with individual milks from 24 New Zealand dairy cows, sampled at 3 times over the season. Milks were classified into type A or B, according to the shape of 3-point heat coagulation time versus pH profiles. Milk type changed over the season for half of the cows in the study. Best subsets regression suggested that different factors controlled heat stability in the 2 milk types. Urea concentration was key for both types, but for type A milks, osmotic pressure and milk solids were the most important predictors of heat stability, whereas casein micelle size and ionic calcium predicted heat stability for type B milks. This study revealed that milk type is prone to change over the season, and the findings suggest that optimizing heat stability could be achieved by different means for type A versus type B milks.

Supplementation with Bovine Milk or Soy Beverages Recovers Bone Mineralization in Young Growing Rats Fed an Insufficient Diet, in Contrast to an Almond Beverage.

BACKGROUND: Nondairy beverages, produced from soy, rice, oat, almond, or coconut, are increasingly being used as alternatives to dairy milk, with the perception that they are healthier and/or more sustainable products than dairy products. OBJECTIVE: The aim of this study was to compare the effects of supplementing either bovine milk, soy, or almond-based beverages to young, growing rats fed an intact-protein diet or a diet that had protein substituted with amino acids (AA-diet). METHODS: Three-week-old male Sprague-Dawley rats were randomly assigned to 5 groups (n = 10/group) and fed ad libitum for 4 wk. Two control groups were fed either standard AIN-93G food [20% casein (CN) protein] or AIN-93G with amino acids (AAs) equivalent to CN protein, and water to drink. Three treatment groups were fed AIN-93G AA and supplemented with either bovine ultra-heat treatment (UHT) milk or soy or almond UHT beverages. Rat weight gain and food intakes were recorded. During week 4, body composition was assessed using DEXA to determine lean soft tissue, fat, and bone mass. At trial end, bone biomechanical properties and blood plasma mineral concentrations were measured. RESULTS: At the end of the trial, animals supplemented with almond beverage were lightest (P > 0.05), with higher plasma calcium concentrations (P > 0.05) and lower bone mineral content (BMC) and bone density (P > 0.05) than animals supplemented with milk or soy beverage. Soy-supplemented animals had similar BMC and bone density compared with milk-supplemented animals, although the soy group gained most weight (P > 0.05) and had the highest fat:lean ratio (P > 0.05) compared with other groups. CONCLUSIONS: In the model tested, supplementing rats with bovine UHT milk and soy UHT beverage provided favorable bone health outcomes. Conversely, almond UHT beverage was not an effective supplement and could be detrimental to bone mineralization and strength outcomes.

Bovine milk derived skimmed milk powder and whey protein concentrate modulates Citrobacter rodentium shedding in the mouse intestinal tract.

Skimmed milk powder (SMP) and whey protein concentrate (WPC) were manufactured from fresh milk collected from cows producing high or low Immunoglobulin (Ig) A levels in their milk. In addition commercial products were purchased for use as diluent or control treatments. A murine enteric disease model (Citrobacter rodentium) was used to assess whether delivery of selected bioactive molecules (IgA, IgG, Lactoferrin (Lf)) or formulation delivery matrix (SMP, WPC) affected faecal shedding of bacteria in C. rodentium infected mice. In trial one, faecal pellets collected from mice fed SMP containing IgA (0.007-0.35 mg/mL), IgG (0.28-0.58 mg/mL) and Lf (0.03-0.1 mg/mL) contained fewer C. rodentium (cfu) compared to control mice fed water (day 8, p < 0.04, analysis of variance (ANOVA) followed by Fisher's unprotected least significant difference (ULSD)). In trial two, WPC containing IgA (0.35-1.66 mg/mL), IgG (0.58-2.36 mg/mL) and Lf (0.02-0.45 mg/mL) did not affect C. rodentium shedding, but SMP again reduced faecal C. rodentium levels (day 12, p < 0.04, ANOVA followed by Fisher's ULSD). No C. rodentium was detected in sham phosphate-buffered saline inoculated mice. Mice fed a commercial WPC shed significantly greater numbers of C. rodentium over 4 consecutive days (Fishers ULSD test), compared to control mice fed water. These data indicate that SMP, but not WPC, modulates faecal shedding in C. rodentium-infected mice and may impact progression of C. rodentium infection independently of selected bioactive concentration. This suggests that food matrix can impact biological effects of foods.

Comparative innate immune interactions of human and bovine secretory IgA with pathogenic and non-pathogenic bacteria.

Secretory IgA (SIgA) from milk contributes to early colonization and maintenance of commensal/symbiotic bacteria in the gut, as well as providing defence against pathogens. SIgA binds bacteria using specific antigenic sites or non-specifically via its glycans attached to α-heavy-chain and secretory component. In our study, we tested the hypothesis that human and bovine SIgA have similar innate-binding activity for bacteria. SIgAs, isolated from human and bovine milk, were incubated with a selection of commensal, pathogenic and probiotic bacteria. Using flow cytometry, we measured numbers of bacteria binding SIgA and their level of SIgA binding. The percentage of bacteria bound by human and bovine SIgA varied from 30 to 90% depending on bacterial species and strains, but was remarkably consistent between human and bovine SIgA. The level of SIgA binding per bacterial cell was lower for those bacteria that had a higher percentage of SIgA-bound bacteria, and higher for those bacteria that had lower percentage of SIgA-bound bacteria. Overall, human and bovine SIgA interacted with bacteria in a comparable way. This contributes to longer term research about the potential benefits of bovine SIgA for human consumers.

Feeding bovine milks with low or high IgA levels is associated with altered re-establishment of murine intestinal microbiota after antibiotic treatment.

Antibiotics are a vital and commonly used therapeutic tool, but their use also results in profound changes in the intestinal microbiota that can, in turn, have significant health consequences. Understanding how the microbiota recovers after antibiotic treatment will help to devise strategies for mitigating the adverse effects of antibiotics. Using a mouse model, we have characterized the changes occurring in the intestinal microbiota immediately after five days exposure to ampicillin, and then at three and fourteen days thereafter. During the fourteen day period of antibiotic recovery, groups of mice were fed either water, cows' milk containing high levels of IgA, or cows' milk containing low levels of IgA as their sole source of liquid. Effects on microbiota of feeding milks for 14 days were also assessed in groups of mice that had no ampicillin exposure. Changes in microbiota were measured by high throughput sequencing of the V4 to V6 variable regions of the 16S ribosomal RNA gene. As expected, exposure to ampicillin led to profound changes to the types and abundance of bacteria present, along with a loss of diversity. At 14 days following antibiotic exposure, mice fed water had recovered microbiota compositions similar to that prior to antibiotics. However, feeding High-IgA milk to mice that has been exposed to antibiotics was associated with altered microbiota compositions, including increased relative abundance of Lactobacillus and Barnesiella compared to the start of the study. Mice exposed to antibiotics then fed Low-IgA milk also showed increased Barnesiella at day 14. Mice without antibiotic perturbation, showed no change in their microbiota after 14 days of milk feeding. Overall, these findings add to a knowledge platform for optimizing intestinal function after treatment with antibiotics in the human population.

Immunoglobulin A in Bovine Milk: A Potential Functional Food?

Immunoglobulin A (IgA) is an anti-inflammatory antibody that plays a critical role in mucosal immunity. It is found in large quantities in human milk, but there are lower amounts in bovine milk. In humans, IgA plays a significant role in providing protection from environmental pathogens at mucosal surfaces and is a key component for the establishment and maintenance of intestinal homeostasis via innate and adaptive immune mechanisms. To date, many of the dairy-based functional foods are derived from bovine colostrum, targeting the benefits of IgG. IgA has a higher pathogenic binding capacity and greater stability against proteolytic degradation when ingested compared with IgG. This provides IgA-based products greater potential in the functional food market that has yet to be realized.

Rhinovirus-16 induced release of IP-10 and IL-8 is augmented by Th2 cytokines in a pediatric bronchial epithelial cell model.

BACKGROUND: In response to viral infection, bronchial epithelial cells increase inflammatory cytokine release to activate the immune response and curtail viral replication. In atopic asthma, enhanced expression of Th2 cytokines is observed and we postulated that Th2 cytokines may augment the effects of rhinovirus-induced inflammation. METHODS: Primary bronchial epithelial cell cultures from pediatric subjects were treated with Th2 cytokines for 24 h before infection with RV16. Release of IL-8, IP-10 and GM-CSF was measured by ELISA. Infection was quantified using RTqPCR and TCID50. Phosphatidyl inositol 3-kinase (PI3K) and P38 mitogen activated protein kinase (MAPK) inhibitors and dexamethasone were used to investigate differences in signaling pathways. RESULTS: The presence of Th2 cytokines did not affect RV replication or viral titre, yet there was a synergistic increase in IP-10 release from virally infected cells in the presence of Th2 cytokines. Release of IL-8 and GM-CSF was also augmented. IP-10 release was blocked by a PI3K inhibitor and IL-8 by dexamethasone. CONCLUSION: Th2 cytokines increase release of inflammatory cytokines in the presence of rhinovirus infection. This increase is independent of effects of virus replication. Inhibition of the PI3K pathway inhibits IP-10 expression.

Exogenous IFN-ß has antiviral and anti-inflammatory properties in primary bronchial epithelial cells from asthmatic subjects exposed to rhinovirus.

BACKGROUND: Rhinoviruses are the major cause of asthma exacerbations. Previous studies suggest that primary bronchial epithelial cells (PBECs) from asthmatic subjects are more susceptible to rhinovirus infection because of deficient IFN-ß production. Although augmenting the innate immune response might provide a novel approach for treatment of virus-induced asthma exacerbations, the potential of IFN-ß to modulate antiviral and proinflammatory responses in asthmatic epithelium is poorly characterized. OBJECTIVES: We sought to compare responses of PBECs from nonasthmatic and asthmatic subjects to exogenous IFN-ß and test the inflammatory effects of IFN-ß in response to rhinovirus infection. METHODS: PBECs were treated with IFN-ß and infected with a low inoculum of human rhinovirus serotype 1B to simulate a natural viral infection. Expression of interferon-responsive genes and inflammatory responses were analyzed by using reverse transcription-quantitative real-time PCR, cytometric bead arrays, or both; viral titers were assessed by using the 50% tissue culture infection dose. RESULTS: Expression of IFN-ß-stimulated antiviral genes was comparable in PBECs from nonasthmatic or asthmatic donors. Exogenous IFN-ß significantly protected PBECs from asthmatic donors against rhinovirus infection by suppressing viral replication. Interferon-inducible protein 10 (IP-10), RANTES, and IL-6 release in response to rhinovirus infection was triggered only in PBECs from asthmatic donors. Although exogenous IFN-ß alone stimulated some release of IP-10 (but not IL-6 or RANTES), it significantly reduced rhinovirus-induced IP-10, RANTES, and IL-6 expression when tested in combination with rhinovirus. CONCLUSIONS: PBECs from asthmatic donors have a normal antiviral response to exogenous IFN-ß. The ability of IFN-ß to suppress viral replication suggests that it might limit virus-induced exacerbations by shortening the duration of the inflammatory response.

Epigenetic mechanisms silence a disintegrin and metalloprotease 33 expression in bronchial epithelial cells.

BACKGROUND: A disintegrin and metalloprotease 33 (ADAM33) polymorphism is strongly associated with asthma and bronchial hyperresponsiveness. Although considered to be a mesenchymal cell-specific gene, recent reports have suggested epithelial expression of ADAM33 in patients with severe asthma. OBJECTIVES: Because dysregulated expression of ADAM33 can contribute to disease pathogenesis, we characterized the mechanism or mechanisms that control its transcription and investigated ADAM33 expression in bronchial biopsy specimens and brushings from healthy and asthmatic subjects. METHODS: The ADAM33 promoter and CpG island methylation were analyzed by using bioinformatics, luciferase reporters, and bisulfite sequencing of genomic DNA. Epithelial-mesenchymal transition was induced by using TGF-beta1. ADAM33 mRNA was scrutinized in bronchial biopsy specimens and brushings by using reverse transcriptase-quantitative polymerase chain reaction, melt-curve analysis, and direct sequencing. RESULTS: The predicted ADAM33 promoter (-550 to +87) had promoter transcriptional activity. Bisulfite sequencing showed that the predicted promoter CpG island (-362 to +80) was hypermethylated in epithelial cells but hypomethylated in ADAM33-expressing fibroblasts. Treatment of epithelial cells with 5-aza-deoxycytidine caused demethylation of the CpG island and induced ADAM33 expression. In contrast, phenotypic transformation of epithelial cells through a TGF-beta-induced epithelial-mesenchymal transition was insufficient to induce ADAM33 expression. ADAM33 mRNA was confirmed in bronchial biopsy specimens, but no validated signal was detected in bronchial brushings from healthy or asthmatic subjects. CONCLUSION: The ADAM33 gene contains a regulatory CpG island within its promoter, the methylation status of which tightly controls its expression in a cell type-specific manner. ADAM33 repression is a stable feature of airway epithelial cells, irrespective of disease.

The genetics of asthma: ADAM33 as an example of a susceptibility gene.

The ability to identify novel disease genes by positional cloning led to the identification of a disintegrin and metalloprotease (ADAM)33 gene on chromosome 20p13 as a susceptibility gene for asthma. Case-control and family-based association studies have mostly confirmed a link between ADAM33 and asthma. Its restricted expression to mesenchymal cells as well as its association with bronchial hyperresponsiveness and accelerated decline in lung function over time point strongly to its involvement in the structural airway components of asthma, such as remodeling. Extensive alternative splicing, expression during branching morphogensis in the developing fetus, impaired lung function in childhood, the production of a soluble form linked to chronic asthma, and tight epigenetic regulation indicate a level of complexity in the way ADAM33 influences disease phenotype. Its recent association with chronic obstructive pulmonary disease as well as with asthma and lung development points to functions relating to airway wall modeling and remodeling as a general morphogenetic repair gene rather than being restricted to asthma.

Association of asthma with a functional promoter polymorphism in the IL16 gene.

BACKGROUND: IL-16, a multifunctional cytokine with increased expression in the airways of asthmatic subjects, inhibits allergic airway inflammation in animal models. A T-->C single nucleotide polymorphism (SNP) at the -295 position in the promoter region of the IL16 gene has been described. OBJECTIVE: We sought to examine the functional significance of this promoter SNP and its relationship to asthma. METHODS: We examined the effect of the -295 SNP on promoter activity in cell-line (HBE4-E6/E7) transfection experiments. We investigated the association of the IL16 -295 genotype with asthma among 341 affected sib-pair white families and 184 unrelated nonasthmatic control subjects. We analyzed the association between the IL16 genotype and asthma using family-based association test and case-control analyses. RESULTS: In in vitro transfection experiments the T allele in the -295 position was associated with substantially reduced promoter activity compared with the C allele. In the family study the more common T allele at the -295 position was significantly associated with all asthma phenotypes (P = .002 to P = .015). In the case-control analysis asthmatic subjects were more likely than unrelated nonasthmatic control subjects to have the -295 TT genotype, but this did not reach statistical significance (odds ratio, 1.36; 95% CI, 0.92-2.02). CONCLUSIONS: The T allele at the -295 position in the IL16 promoter region is associated with reduced promoter activity relative to the C allele and with asthma in this white population. Further investigation is needed to delineate the mechanisms underlying these findings and the relationship of the IL16 -295 genotype to asthma in other populations.

Polymorphisms in a disintegrin and metalloprotease 33 (ADAM33) predict impaired early-life lung function.

RATIONALE: Asthma commonly originates in early life in association with impaired lung function, which tracks to adulthood. OBJECTIVES: Within the context of a prospective birth cohort study, we investigated the association between single nucleotide polymorphisms (SNPs) in a disintegrin and metalloprotease 33 (ADAM33) gene and early-life lung function. METHODS: Children were genotyped for 17 SNPs in ADAM33. Lung function at age 3 (n = 285) and 5 years (n = 470) was assessed using plethysmographic measurement of specific airway resistance (sRaw). At age 5, we also measured FEV(1). SNPs were analyzed individually using logistic regression, followed by linkage disequilibrium mapping to identify the causal locus. MAIN RESULTS: Carriers of the rare allele of F+1 SNP had reduced lung function at age 3 years (p = 0.003). When the recessive model was considered, four SNPs (F+1, S1, ST+5, V4) showed association with sRaw at age 5 years (p < 0.04). Using linkage disequilibrium mapping, we found evidence of a significant causal location between BC+1 and F1 SNPs, at the 5' end of the gene. Four SNPs were associated with lower FEV(1) (F+1, M+1, T1, and T2; p < or = 0.04). The risk of transient early wheezing more than doubled among children homozygous for the A allele of F+1 (odds ratio, 2.39; 95% confidence intervals, 1.18-4.86; p = 0.02), but there was no association between any SNP and allergic sensitization or physician-diagnosed asthma. CONCLUSIONS: Polymorphisms in ADAM33 predict impaired early-life lung function. The functionally relevant polymorphism is likely to be at the 5' end of the gene.

Association of tumor necrosis factor-alpha polymorphisms and ozone-induced change in lung function.

Ozone is a major air pollutant with adverse health effects which exhibit marked inter-individual variability. In mice, regions of genetic linkage with ozone-induced lung injury include the tumor necrosis factor-alpha (TNF), lymphotoxin-alpha (LTA), Toll-like receptor 4 (TLR4), superoxide dismutase (SOD2), and glutathione peroxidase (GPX1) genes. We genotyped polymorphisms in these genes in 51 individuals who had undergone ozone challenge. Mean change in FEV1 with ozone challenge, as a percentage of baseline, was -3% in TNF -308G/A or A/A individuals, compared with -9% in G/G individuals (p = 0.024). When considering TNF haplotypes, the smallest change in FEV1 with ozone exposure was associated with the TNF haplotype comprising LTA +252G/TNF -1031T/TNF -308A/TNF -238G. This association remained statistically significant after correction for age, sex, disease, and ozone concentration (p = 0.047). SOD2 or GPX1 genotypes were not associated with lung function, and the TLR4 polymorphism was too infrequent to analyze. The results of this study support TNF as a genetic factor for susceptibility to ozone-induced changes in lung function in humans, and has potential implications for stratifying health risks of air pollution.

ADAM 33 and its association with airway remodeling and hyperresponsiveness in asthma.

Asthma is known to be a Th2 inflammatory syndrome that leads to intermittent airway obstruction. However, the mechanisms involved in development of the clinical features remain enigmatic, although genetic elements clearly are involved. Recently, based on a large genome wide screen involving families in the United Kingdom and the United States with at least two siblings with asthma, a locus was identified that encoded for a family of proteases. This group of proteins is now known as the ADAM superfamily. In this review, we discuss the ADAM superfamily and, in particular, ADAM 33, a member of a family of genes which encode a subgroup of zinc dependent metalloproteinase (metzincin). The potential for therapeutic intervention with ADAM 33 is extremely attractive and further work will not only focus on the specific domains of ADAM 33, but also the mechanisms by which they lead to bronchial hyperreactivity.

The role of ADAM33 in the pathogenesis of asthma.

While asthma is a disorder of the conducting airways characterised by Th2-directed inflammation, a second set of mechanisms is being increasingly recognised as fundamental to disease chronicity and severity, for which the term "remodelling" has been used. The cellular and mediator responses underpinning airway remodelling involve aberrant communication between the airway epithelium and underlying mesenchyme, involving the generation of growth factors that lead to proliferation of fibroblasts and smooth muscle and the deposition of matrix proteins to cause airway wall thickening linked to bronchial hyperresponsiveness and fixed airflow obstruction. The identification of ADAM33 on chromosome 20p13 from positional cloning as a novel candidate gene involved in the pathogenesis of these structural and functional changes has opened the way to further insight into these processes that contribute to corticosteroid refractoriness. The preferential expression of ADAM33 in mesenchymal cells and its multiple molecular actions provide ample opportunity for incriminating this molecule in chronic asthma. Its association with progressive asthma and in predicting reduced lung function in young children suggest that ADAM33 has an important role in the natural history and possibly the origins of asthma, a disease unique to humans.