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.

Protein-tannic acid multilayer films: A multifunctional material for microencapsulation of food-derived bioactives.

The benefits of various functional foods are often negated by stomach digestion and poor targeting to the lower gastrointestinal tract. Layer-by-Layer assembled protein-tannic acid (TA) films are suggested as a prospective material for microencapsulation of food-derived bioactive compounds. Bovine serum albumin (BSA)-TA and pepsin-TA films demonstrate linear growth of 2.8±0.1 and 4.2±0.1nm per bi-layer, correspondingly, as shown by ellipsometry. Both multilayer films are stable in simulated gastric fluid but degrade in simulated intestinal fluid. Their corresponding degradation constants are 0.026±0.006 and 0.347±0.005nm-1min-1. Milk proteins possessing enhanced adhesion to human intestinal surface, Immunoglobulin G (IgG) and ß-Lactoglobulin (BLG), are explored to tailor targeting function to BSA-TA multilayer film. BLG does not adsorb onto the multilayer while IgG is successfully incorporated. Microcapsules prepared from the multilayer demonstrate 2.7 and 6.3 times higher adhesion to Caco-2 cells when IgG is introduced as an intermediate and the terminal layer, correspondingly. This developed material has a great potential for oral delivery of numerous active food-derived ingredients.

Formulation for Oral Delivery of Lactoferrin Based on Bovine Serum Albumin and Tannic Acid Multilayer Microcapsules.

Lactoferrin (Lf) has considerable potential as a functional ingredient in food, cosmetic and pharmaceutical applications. However, the bioavailability of Lf is limited as it is susceptible to digestive enzymes in gastrointestinal tract. The shells comprising alternate layers of bovine serum albumin (BSA) and tannic acid (TA) were tested as Lf encapsulation system for oral administration. Lf absorption by freshly prepared porous 3 µm CaCO3 particles followed by Layer-by-Layer assembly of the BSA-TA shells and dissolution of the CaCO3 cores was suggested as the most efficient and harmless Lf loading method. The microcapsules showed high stability in gastric conditions and effectively protected encapsulated proteins from digestion. Protective efficiency was found to be 76 ± 6% and 85 ± 2%, for (BSA-TA)4 and (BSA-TA)8 shells, respectively. The transit of Lf along the gastrointestinal tract (GIT) of mice was followed in vivo and ex vivo using NIR luminescence. We have demonstrated that microcapsules released Lf in small intestine allowing 6.5 times higher concentration than in control group dosed with the same amount of free Lf. Significant amounts of Lf released from microcapsules were then absorbed into bloodstream and accumulated in liver. Suggested encapsulation system has a great potential for functional foods providing lactoferrin.

Differential Ability of Bovine Antimicrobial Cathelicidins to Mediate Nucleic Acid Sensing by Epithelial Cells.

Cathelicidins encompass a family of cationic peptides characterized by antimicrobial activity and other functions, such as the ability to enhance the sensing of nucleic acids by the innate immune system. The present study aimed to investigate the ability of the bovine cathelicidins indolicidin, bactenecin (Bac)1, Bac5, bovine myeloid antimicrobial peptide (BMAP)-27, BMAP-28, and BMAP-34 to inhibit the growth of bacteria and to enhance the sensing of nucleic acid by the host's immune system. BMAP-27 was the most effective at killing Staphylococcus aureus, Streptococcus uberis, and Escherichia coli, and this was dependent on its amphipathic structure and cationic charge. Although most cathelicidins possessed DNA complexing activity, only the alpha-helical BMAP cathelicidins and the cysteine-rich disulfide-bridged Bac1 were able to enhance the sensing of nucleic acids by primary epithelial cells. We also compared these responses with those mediated by neutrophils. Activation of neutrophils with phorbol myristate acetate resulted in degranulation and release of cathelicidins as well as bactericidal activity in the supernatants. However, only supernatants from unstimulated neutrophils were able to promote nucleic acid sensing in epithelial cells. Collectively, the present data support a role for certain bovine cathelicidins in helping the innate immune system to sense nucleic acids. The latter effect is observed at concentrations clearly below those required for direct antimicrobial functions. These findings are relevant in development of future strategies to promote protection at mucosal surfaces against pathogen invasion.

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.

Bovine milk RNases modulate pro-inflammatory responses induced by nucleic acids in cultured immune and epithelial cells.

Activation of innate immune receptors by exogenous substances is crucial for the detection of microbial pathogens and a subsequent inflammatory response. The inflammatory response to microbial lipopolysaccharide via Toll-like receptor 4 (TLR4) is facilitated by soluble accessory proteins, but the role of such proteins in the activation of other pathogen recognition receptors for microbial nucleic acid is not well understood. Here we demonstrate that RNase4 and RNase5 purified from bovine milk bind to Salmonella typhimurium DNA and stimulate pro-inflammatory responses induced by nucleic acid mimetics and S. typhimurium DNA in an established mouse macrophage cell culture model, RAW264.7, as well as in primary bovine mammary epithelial cells. RNase4 and 5 also modulated pro-inflammatory signalling in response to nucleic acids in bovine peripheral blood mononuclear cells, although producing a distinct response. These results support a role for RNase4 and RNase5 in mediating inflammatory signals in both immune and epithelial cells, involving mechanisms that are cell-type specific.

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.

Abundance of RNase4 and RNase5 mRNA and protein in host defence related tissues and secretions in cattle.

Members of the RNaseA family are present in various tissues and secretions but their function is not well understood. Some of the RNases are proposed to participate in host defence. RNase4 and RNase5 are present in cows' milk and have antimicrobial activity. However, their presence in many tissues and secretions has not been characterised. We hypothesised that these two RNases are present in a range of tissues and secretions where they could contribute to host defence. We therefore, determined the relative abundance of RNase4 and RNase5 mRNA as well as protein levels in a range of host defence related and other tissues as well as a range of secretions in cattle, using real time PCR and western blotting. The two RNases were found to be expressed in liver, lung, pancreas, mammary gland, placenta, endometrium, small intestine, seminal vesicle, salivary gland, kidney, spleen, lymph node, skin as well as testes. Corresponding proteins were also detected in many of the above tissues, as well as in seminal fluid, mammary secretions and saliva. This study provides evidence for the presence of RNase4 and RNase5 in a range of tissues and secretions, as well as some major organs in cattle. The data are consistent with the idea that these proteins could contribute to host defence in these locations. This work contributes to growing body of data suggesting that these proteins contribute to the physiology of the organism in a more complex way than acting merely as digestive enzymes.

Digestion proteomics: tracking lactoferrin truncation and peptide release during simulated gastric digestion.

The extent to which nutritional and functional benefit is derived from proteins in food is related to its breakdown and digestion in the body after consumption. Further, detailed information about food protein truncation during digestion is critical to understanding and optimising the availability of bioactives, in controlling and limiting allergen release, and in minimising or monitoring the effects of processing and food preparation. However, tracking the complex array of products formed during the digestion of proteins is not easily accomplished using classical proteomics. We here present and develop a novel proteomic approach using isobaric labelling to mapping and tracking protein truncation and peptide release during simulated gastric digestion, using bovine lactoferrin as a model food protein. The relative abundance of related peptides was tracked throughout a digestion time course, and the effect of pasteurisation on peptide release assessed. The new approach to food digestion proteomics developed here therefore appears to be highly suitable not only for tracking the truncation and relative abundance of released peptides during gastric digestion, but also for determining the effects of protein modification on digestibility and potential bioavailability.

Host defence related responses in bovine milk during an experimentally induced Streptococcus uberis infection.

BACKGROUND: Milk contains a range of proteins of moderate or low abundance that contribute to host defence. Characterisation of these proteins, the extent to which their abundance is regulated by pathogenic stimuli, and the variability of their response between and within individual animals would facilitate a better understanding of the molecular basis for this important function of milk. RESULTS: We have characterised the host defence proteins in bovine milk and their responses to intra-mammary infection by a common Gram positive mastitis pathogen, Streptococcus uberis, using a combination of 2D gel electrophoresis and GeLC mass spectrometry. In total, 68 host defence-associated proteins were identified, 18 of which have a direct antimicrobial function, 23 of which have a pathogen-recognition function, and 27 of which have a role in modulating inflammatory or immune signalling. The responsiveness of seven proteins was quantified by western blotting; validating the proteomic analyses, quantifying the within- and between animal variability of the responses, and demonstrating the complexity and specificity of the responses to this pathogen. CONCLUSIONS: These data provide a foundation for understanding the role of milk in host-microbe interaction. Furthermore they provide candidate biomarkers for mastitis diagnosis, and will inform efforts to develop dairy products with improved health-promoting properties.

The mammalian secreted RNases: mechanisms of action in host defence.

The mammalian ribonucleaseA family comprises a large group of structurally similar proteins which are secreted by a range of tissues and immune cells. Their physiological role is unclear. It has been suggested that some of these RNases contribute to host defence, notably eosinophil-derived neurotoxin, eosinophil cationic protein, eosinophil-associated RNases, RNase4, angiogenin (RNase5), RNase7, RNase8 and bovine seminal RNase. This review summarises data supporting the involvement of these proteins in host defence, focusing on their antimicrobial, cytotoxic and immunomodulatory activities. The extent to which the data support possible mechanisms of action for these proteins is discussed. This compilation of findings and current hypotheses on the physiological role of these RNases will provide a stimulus for further research and development of ideas on the contribution of the RNases to host defence.

The BPI-like/PLUNC family proteins in cattle.

Members of the protein family having similarity to BPI (bactericidal/permeability increasing protein) (the BPI-like proteins), also known as the PLUNC (palate, lung and nasal epithelium clone) family, have been found in a range of mammals; however, those in species other than human or mouse have been relatively little characterized. Analysis of the BPI-like proteins in cattle presents unique opportunities to investigate the function of these proteins, as well as address their evolution and contribution to the distinct physiology of ruminants. The present review summarizes the current understanding of the nature of the BPI-like locus in cattle, including the duplications giving rise to the multiple BSP30 (bovine salivary protein 30 kDa) genes from an ancestral gene in common with the single PSP (parotid secretory protein) gene found in monogastric species. Current knowledge of the expression of the BPI-like proteins in cattle is also presented, including their pattern of expression among tissues, which illustrate their independent regulation at sites of high pathogen exposure, and the abundance of the BSP30 proteins in saliva and salivary tissues. Finally, investigations of the function of the BSP30 proteins are presented, including their antimicrobial, lipopolysaccharide-binding and bacterial aggregation activities. These results are discussed in relation to hypotheses regarding the physiological role of the BPI-like proteins in cattle, including the role they may play in host defence and the unique aspects of digestion in ruminants.

Alterations in the salivary proteome associated with periodontitis.

AIM: To identify changes in the salivary proteome associated with active periodontitis. MATERIALS AND METHODS: Quantitative proteomics (two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis) was used to investigate whole saliva from individuals with severe periodontitis and their proteomic profiles before and after periodontal treatment were compared. RESULTS: A comparison of 128 proteins across all saliva samples identified 15 protein spots with altered abundance. The predominant alteration observed was an increase in the abundance of the S100 proteins S100A8/A9/A6. Of the remaining proteins with altered abundance, haptoglobin, prolactin inducible protein and parotid secretory protein have previously been associated with host defence. CONCLUSION: These results highlight the predominant involvement of S100 proteins in the host response during periodontitis, identify host defence components that have not been linked previously to this disease and suggest new potential biomarkers for monitoring disease activity in periodontitis.

Cleavage at the caspase-6 site is required for neuronal dysfunction and degeneration due to mutant huntingtin.

Cleavage of huntingtin (htt) has been characterized in vitro, and accumulation of caspase cleavage fragments represents an early pathological change in brains of Huntington's disease (HD) patients. However, the relationship between htt proteolysis and the pathogenesis of HD is unknown. To determine whether caspase cleavage of htt is a key event in the neuronal dysfunction and selective neurodegeneration in HD, we generated YAC mice expressing caspase-3- and caspase-6-resistant mutant htt. Mice expressing mutant htt, resistant to cleavage by caspase-6 but not caspase-3, maintain normal neuronal function and do not develop striatal neurodegeneration. Furthermore, caspase-6-resistant mutant htt mice are protected against neurotoxicity induced by multiple stressors including NMDA, quinolinic acid (QA), and staurosporine. These results are consistent with proteolysis of htt at the caspase-6 cleavage site being an important event in mediating neuronal dysfunction and neurodegeneration and highlight the significant role of htt proteolysis and excitotoxicity in HD.

Huntingtin-interacting protein HIP14 is a palmitoyl transferase involved in palmitoylation and trafficking of multiple neuronal proteins.

In neurons, posttranslational modification by palmitate regulates the trafficking and function of signaling molecules, neurotransmitter receptors, and associated synaptic scaffolding proteins. However, the enzymatic machinery involved in protein palmitoylation has remained elusive. Here, using biochemical assays, we show that huntingtin (htt) interacting protein, HIP14, is a neuronal palmitoyl transferase (PAT). HIP14 shows remarkable substrate specificity for neuronal proteins, including SNAP-25, PSD-95, GAD65, synaptotagmin I, and htt. Conversely, HIP14 is catalytically invariant toward paralemmin and synaptotagmin VII. Exogenous HIP14 enhances palmitoylation-dependent vesicular trafficking of several acylated proteins in both heterologous cells and neurons. Moreover, interference with endogenous expression of HIP14 reduces clustering of PSD-95 and GAD65 in neurons. These findings define HIP14 as a mammalian palmitoyl transferase involved in the palmitoylation and trafficking of multiple neuronal proteins.

The BSP30 salivary proteins from cattle, LUNX/PLUNC and von Ebner's minor salivary gland protein are members of the PSP/LBP superfamily of proteins.

Saliva influences rumen function in cattle, yet the biochemical role for most of the bovine salivary proteins (BSPs) has yet to be established. Two cDNAs (BSP30a and BSP30b) from bovine parotid salivary gland were cloned and sequenced, each coding for alternate forms of a prominent protein in bovine saliva. The BSP30 cDNAs share 96% sequence identity with each other at the DNA level and 83% at the amino acid level, and appear to arise from separate genes. The predicted BSP30a and BSP30b proteins share 26-36% amino acid identity with parotid secretory protein (PSP) from mouse, rat and human. BSP30 and PSP are in turn more distantly related to a wider group of proteins that includes lung-specific X protein, also known as palate, lung, and nasal epithelium clone (LUNX/PLUNC), von Ebner's minor salivary gland protein (VEMSGP), bactericidal permeability increasing protein (BPI), lipopolysaccharide binding protein (LBP), cholesteryl ester transfer protein (CETP), and the putative olfactory ligand-binding proteins RYA3 and RY2G5. Bovine cDNAs encoding homologs of LUNX/PLUNC and VEMSGP were isolated and sequenced. Northern blot analysis showed that LUNX/PLUNC, BSP30 and VEMSGP are expressed in bovine salivary tissue and airways, and that they have non-identical patterns of expression in these tissues. The expression of both BSP30a and BSP30b is restricted to salivary tissue, but within this tissue they have distinct patterns of expression. The proximity of the human genes coding for the PSP/LBP superfamily on HSA20q11.2, their similar amino acid sequence, and common exon segmentation strongly suggest that these genes evolved from a common ancestral gene. Furthermore, they imply that the BSP30a and BSP30b proteins may have a function in common with other members of this gene family.