Brendan J. Haigh

Immune components of bovine colostrum and milk

Colostrum and milk provide a complete diet for the neonate. In ruminants, colostrum is also the sole source of initial acquired immunity for the offspring. Milk therefore plays an important role in mammalian host defense. In colostrum, the concentration of immunoglobulins is particularly high, with IgG being the major immunoglobulin class present in ruminant milk, in contrast to IgA being the major immunoglobulin present in human milk. Immunoglobulins are transported into mammary secretions via specialized receptors. In addition to immunoglobulins, both colostrum and milk contain viable cells, including neutrophils and macrophages, which secrete a range of immune-related components into milk. These include cytokines and antimicrobial proteins and peptides, such as lactoferrin, defensins, and cathelicidins. Mammary epithelial cells themselves also contribute to the host defense by secreting a range of innate immune effector molecules. A detailed understanding of these proteins and peptides offers great potential to add value to the dairy industry. This is demonstrated by the wide-ranging commercial applications of lactoferrin derived from bovine milk. Knowledge of the immune function of milk, in particular, how the gland responds to pathogens, can be used to boost the concentrations of immune factors in milk through farm management practices and vaccination protocols. The latter approach is currently being used to maximize yields of bovine milk-derived IgA directed at specific antigens for therapeutic and prophylactic use. Increasingly sophisticated proteomics technologies are being applied to identify and characterize the functions of the minor components of milk. An overview is presented of the immune factors in colostrum and milk as well as the results of research aimed at realizing this untapped value in milk.

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.

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 Ebners 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.

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.

Host-defence-related proteins in cows' milk

Milk is a source of bioactive molecules with wide-ranging functions. Among these, the immune properties have been the best characterised. In recent years, it has become apparent that besides the immunoglobulins, milk also contains a range of minor immune-related proteins that collectively form a significant first line of defence against pathogens, acting both within the mammary gland itself as well as in the digestive tract of the suckling neonate. We have used proteomics technologies to characterise the repertoire of host-defence-related milk proteins in detail, revealing more than 100 distinct gene products in milk, of which at least 15 are known host-defence-related proteins. Those having intrinsic antimicrobial activity likely function as effector proteins of the local mucosal immune defence (e.g. defensins, cathelicidins and the calgranulins). Here, we focus on the activities and biological roles of the cathelicidins and mammary serum amyloid A. The function of the immune-related milk proteins that do not have intrinsic antimicrobial activity is also discussed, notably lipopolysaccharide-binding protein, RNase4, RNase5/angiogenin and cartilage-glycoprotein 39 kDa. Evidence is shown that at least some of these facilitate recognition of microbes, resulting in the activation of innate immune signalling pathways in cells associated with the mammary and/or gut mucosal surface. Finally, the contribution of the bacteria in milk to its functionality is discussed. These investigations are elucidating how an effective first line of defence is achieved in the bovine mammary gland and how milk contributes to optimal digestive function in the suckling calf. This study will contribute to a better understanding of the health benefits of milk, as well as to the development of high-value ingredients from milk.

The abundance of milk cathelicidin proteins during bovine mastitis

Current on-farm methods for detecting mastitis in dairy cows have limitations with their specificity and sensitivity, particularly at an early stage of infection. There is therefore a need to explore new approaches for detecting early and subclinical mastitis. This study examined the expression of a group of neutrophil-specific proteins, the cathelicidins, in milk samples from naturally occurring as well as experimentally induced mastitis infections. Immunoblot analysis indicated that cathelicidin proteins are only observed in infected quarters and demonstrate a high correlation with somatic cell count (SCC) during the onset of infection. In most of the infections examined, cathelicidin was detected prior to the observation of clinical symptoms and at SCC counts as low as 6.2 × 10(3)cells/mL. In naturally occurring mastitis the correlation between cathelicidin and infection status is not as strong, with 25% of pathogen-positive milk samples containing no detectable cathelicidin. This may reflect the varying levels of neutrophil concentration and activity at different stages or severities of infection. Our results indicate that milk cathelicidin levels increase following intramammary infection and cathelicidin-based biomarkers may assist in the detection of preclinical mastitis or determining the stage of infection.

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

BackgroundMilk 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.ResultsWe 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.ConclusionsThese 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.

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.

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 β-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.

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.