In-depth proteome analysis of more than 12,500 proteins in buffalo mammary epithelial cell line identifies protein signatures for active proliferation and lactation.

The mature mammary gland is made up of a network of ducts that terminates in alveoli. The innermost layer of alveoli is surrounded by the differentiated mammary epithelial cells (MECs), which are responsible for milk synthesis and secretion during lactation. However, the MECs are in a state of active proliferation during pregnancy, when they give rise to network like structures in the mammary gland. Buffalo (Bubalus bubalis) constitute a major source of milk for human consumption, and the MECs are the major precursor cells which are mainly responsible for their lactation potential. The proteome of MECs defines their functional state and suggests their role in various cellular activities such as proliferation and lactation. To date, the proteome profile of MECs from buffalo origin is not available. In the present study, we have profiled in-depth proteome of in vitro cultured buffalo MECs (BuMECs) during active proliferation using high throughput tandem mass spectrometry (MS). MS analysis identified a total of 8330, 5970, 5289, 4818 proteins in four sub-cellular fractions (SCFs) that included cytosolic (SCF-I), membranous and membranous organelle's (SCF-II), nuclear (SCF-III), and cytoskeletal (SCF-IV). However, 792 proteins were identified in the conditioned media, which represented the secretome. Altogether, combined analysis of all the five fractions (SCFs- I to IV, and secretome) revealed a total of 12,609 non-redundant proteins. The KEGG analysis suggested that these proteins were associated with 325 molecular pathways. Some of the highly enriched molecular pathways observed were metabolic, MAPK, PI3-AKT, insulin, estrogen, and cGMP-PKG signalling pathway. The newly identified proteins in this study are reported to be involved in NOTCH signalling, transport and secretion processes.

Incidence of mastitis and activity of milk neutrophils in Tharparkar cows reared under semi-arid conditions.

Rearing of indigenous Tharparkar (TP) cows (native of arid Thar deserts) under high humid conditions (>75 % humidity) has increased the incidence of mammary infections in them. A study was undertaken to see the number, activity, and expression of milk neutrophils isolated from healthy and mastitic cows. There was a significant (P < 0.05) influx in milk somatic cell counts (SCC) and neutrophils in sub-clinical and clinical mastitis cows. No change was observed in the phagocytic activity (PA) of milk neutrophils between healthy and sub-clinical mastitis (SCM) cows, but these activities decreased significantly (P < 0.05) in clinical cases. Chemotactic activity showed a significant difference between all the groups. Lactose varied significantly (P < 0.05) between healthy, sub-clinical, and clinical mastitis (CM) cows. Expression of chemokine receptor (CXCR1) was more in mastitis cows and also higher as compared to CXCR2. No change was observed in cluster of differentiation molecule (CD62L) among all the three groups of TP cows. Expression of interleukin (IL-8) and CD11b was low in healthy cows, increased significantly (P < 0.05) in both sub-clinical and mastitis cows. This study indicates that low producing TP cows are also prone to mammary infections when reared under semi-arid conditions.

DIGE based proteome analysis of mammary gland tissue in water buffalo (Bubalus bubalis): lactating vis-a-vis heifer.

Mammary gland is an exocrine and sebaceous gland made up of branching network of ducts that end in alveoli. Milk is synthesized in the alveoli and secreted into alveolar lumen. Mammary gland represents an ideal system for the study of organogenesis that undergoes successive cycles of pregnancy, lactation and involution. To gain insights on the molecular events that take place in pubertal and lactating mammary gland, we have identified 43 differentially expressed proteins in mammary tissue of heifer (non-lactating representing a virgin mammary gland), and lactating buffaloes (Bubalus bubalis) by 2D-difference gel electrophoresis (2D-DIGE) and mass spectrometry. Twenty one proteins were upregulated during lactation whereas 8 proteins were upregulated in heifer mammary gland significantly (p<0.05). Bioinformatics analyses of the identified proteins showed that a majority of the proteins are involved in metabolic processes. The differentially expressed proteins were validated by real-time PCR and Western blotting. We observed differential expressions of certain new proteins including EEF1D, HSPA5, HSPD1 and PRDX6 during lactation which have not been reported before. The differentially expressed proteins were mapped to available biological pathways and networks involved in lactation. This study signifies the importance of some proteins which are preferentially expressed during lactation and in heifer mammary gland. BIOLOGICAL SIGNIFICANCE: This work is important because we have generated information in water buffalo (B. bubalis) for the first time which is the major milk producing animal in Indian Subcontinent. Out of a present production of 133milliontons of milk produced in India, contribution of buffalo milk is around 54%. Its physiology is somewhat different from the lactating cows. Buffalo milk composition varies from cow milk in terms of higher fat and total solid content, which confers an advantage in preparation of specialized cheese, curd and other dairy products. Being a major milk producing animal in India it is highly essential to understand the lactation associated proteins in the mammary gland of buffalo. In the present investigation our attempt has been to identify new protein evidences which are expressed in lactating buffalo mammary gland and have not been reported before. The findings reported in the present study will help in understanding the lactation biology of buffalo mammary gland in particular and the mammary gland biology in general.

Neutrophil dynamics in the blood and milk of crossbred cows naturally infected with Staphylococcus aureus.

AIM: The present study was designed to evaluate the neutrophil dynamics in terms of the functional competence during subclinical mastitis (SCM) and clinical mastitis (CM). MATERIALS AND METHODS: A total of 146 Karan fries cows were screened and were divided into three groups as control (n=12), SCM, n=12 and CM, n=12 groups on the basis of California mastitis test scoring, bacteriological evaluation, gross and morphological changes in milk and by counting milk somatic cell count (SCC). Both blood and milk polymorphonuclear neutrophils (PMNs) were isolated in the study. Phagocytic activity (PA) was studied by spectrophotometrically; neutrophil extracelluar traps (NETs) were studied by scanning electron microscopy (SEM); CD44 was quantified by flow cytometry and apoptosis was studied by fluorescent microscopy. RESULTS: Significantly (p<0.05) higher SCC, PA was found in milk of CM cows as compared to SCM and control cows. Significantly lower (p<0.05) apoptosis was observed in PMNs isolated from both blood and milk of CM group of cows when compared to control and SCM group. The milk neutrophils of CM group of cows formed NETs as evidenced from the SEM images. Surface expression of CD44 revealed a significantly (p<0.05) lower expression in milk neutrophils of CM group of cows when compared to SCM and control group of cows. CONCLUSION: The study indicated a positive correlation between delayed neutrophil apoptosis, persistent staying of neutrophils at the site of infection along with formation of NETs as the strategies to fight against the pathogens in the udder during Staphylococcal mastitis. The study forms a strong base for future molecular research in terms of neutrophil recruitment and neutrophil removal from the site of infection.

A comparative study on the blood and milk cell counts of healthy, subclinical, and clinical mastitis Karan Fries cows.

AIM: The present study was aimed to study the use of cell counts as an early indicator of mammary health. MATERIALS AND METHODS: Milk and blood cell counts were estimated from 8 healthy, 8 subclinical (SCM), and 8 clinically mastitis (CM) groups of Karan Fries (KF) cows. RESULTS: Total leucocyte counts and neutrophil percent in blood and milk somatic cells and milk neutrophil percent of healthy cows increased significantly (p<0.05) in SCM cows and CM cows. Viability of blood and milk neutrophils was more in healthy cows, but decreased significantly (p<0.05) in SCM and CM cows. Significant (p<0.05) decrease were also observed in both the blood and milk lymphocytes and monocytes of SCM and CM cows. Phagocytic activity (PA) of blood neutrophils also decreased significantly (p<0.05) in SCM cows. There was no difference between the PA of SCM and CM cows. Milk neutrophil percent was more in the SCM and clinically infected milk than in the blood of these cows. About 96-97% of the neutrophils had segmented nucleus in both healthy and subclinical milk, whereas, 2-3% were having band shaped or immature nuclei. There was a significant decrease in the segmented neutrophils, whereas, band neutrophils increase significantly to about 5% in the infected milk of mastitic cows. Viability of the milk neutrophils decreased more in case of subclinical and clinical milk as compared to that of blood. PA was found to be highest in the milk of healthy group of cows, but decreased significantly (p<0.05) in subclinically infected cows. However, there was no difference between the PA of milk neutrophils of SCM and CM cows. PA of milk was also found to be significantly lower in the milk of healthy cows when compared to that of blood neutrophils. CONCLUSION: This study indicated that percent neutrophils and their type in conjunction with milk somatic cell counts can be used as a more reliable indicator of mammary health in cows.

Comparative 2D-DIGE proteomic analysis of bovine mammary epithelial cells during lactation reveals protein signatures for lactation persistency and milk yield.

Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.

Formation of NET, phagocytic activity, surface architecture, apoptosis and expression of toll like receptors 2 and 4 (TLR2 and TLR4) in neutrophils of mastitic cows.

Neutrophils employ both oxidative and non oxidative mechanisms to destroy pathogens. Function of neutrophils coming in milk during mammary invasion is not clearly understood in dairy animals. Therefore, the present study was designed in 36 Sahiwal cows to see the changes occurring in the neutrophil activity of cows suffering from subclinical (SCM) and clinical mastitis (CM). Cows were divided into three groups as healthy (n = 12), SCM (n = 12) and CM (n = 12) groups on the basis of CMT scoring, gross morphological changes in milk, bacteriological examination of milk and by counting milk SCC. Significantly (P < 0.05) higher milk SCC, neutrophil percent and significantly (P < 0.05) lower viability of both blood and milk neutrophils were observed in CM group of cows as compared to SCM and control group of cows. Phagocytic activity (PA) was significantly (P < 0.05) higher in milk neutrophils of SCM and CM cows as compared to control cows. Toll like receptors 2 and 4 in blood and milk neutrophils were found to be significantly (P < 0.05) higher, whereas, apoptosis in terms of altered mitochondrial transmembrane potential, Caspase 3 and 7 activities were found to be significantly (P < 0.05) lower in CM cows. Alterations in surface architecture of neutrophils in terms of formation of pseudopods was observed by scanning electron microscope (SEM) and found to be higher in CM cows. Blood neutrophils were found to be spherical as compared to milk neutrophils. Formation of neutrophil extracellular trap (NETs) were found milk neutrophils of CM cows, whereas, SCM and healthy cows did not exhibit NET formation. The study indicated a positive correlation between lower neutrophil apoptosis and higher expression of TLR2 and TLR4 with the formation of NETs and change in surface architecture. Formation of NET like structures seemed to be an effective mode of defense employed by neutrophils of cows suffering from clinical mastitis.

Proteome analysis of functionally differentiated bovine (Bos indicus) mammary epithelial cells isolated from milk.

Mammary gland is made up of a branching network of ducts that end in alveoli. Terminally differentiated mammary epithelial cells (MECs) constitute the innermost layer of aveoli. They are milk-secreting cuboidal cells that secrete milk proteins during lactation. Little is known about the expression profile of proteins in the metabolically active MECs during lactation or their functional role in the lactation process. In the present investigation, we have reported the proteome map of MECs in lactating cows using 2DE MALDI-TOF/TOF MS and 1D-Gel-LC-MS/MS. MECs were isolated from milk using immunomagnetic beads and confirmed by RT-PCR and Western blotting. The 1D-Gel-LC-MS/MS and 2DE-MS/MS based approaches led to identification of 431 and 134 proteins, respectively, with a total of 497 unique proteins. Proteins identified in this study were clustered into functional groups using bioinformatics tools. Pathway analysis of the identified proteins revealed 28 pathways (p < 0.05) providing evidence for involvement of various proteins in lactation function. This study further provides experimental evidence for the presence of many proteins that have been predicted in annotated bovine genome. The data generated further provide a set of bovine MEC-specific proteins that will help the researchers to understand the molecular events taking place during lactation.

Comparison of innate immune activation after prolonged feeding of milk fermented with three species of Lactobacilli.

The present investigation aimed at identifying the abilities of three different species of probiotic lactobacilli to modulate cellular immune responses in mouse neutrophils and macrophages in vivo over a study period of 60 days. Neutrophil respiratory burst enzymes (cytochrome c reductase and MPO) showed remarkable increased activity (P ≤ 0.01) after consumption of milks fermented by different species of probiotics over 30 and 60 days of feeding trials. Enzyme activities (ß-galactosidase and ß-glucuronidase) and nitric oxide production also increased considerably (P ≤ 0.01) in macrophages, both in peritoneal fluid and in enriched cell cultures. The effects of enhanced enzyme activities were corroborated by simultaneous increases in the phagocytic activities of neutrophils and macrophages. The increases in cellular functions were invariably maximal during the first 30 days of study and were maintained, but did not increase, over the next 30 days. Further, Lactobacillus helveticus-fed groups were most effective at modulating neutrophil functions whereas Lactobacillus paracasei-fed groups were more potent at enhancing macrophage functions. Together, our results indicate that probiotics have strain specific effects on stimulating cellular functions while not causing excessive stimulation of the immune system over longer feeding periods, thereby resulting in maximum and stable health benefits.

Establishment and characterization of a buffalo (Bubalus bubalis) mammary epithelial cell line.

BACKGROUND: The objective of this study was to establish the buffalo mammary epithelial cell line (BuMEC) and characterize its mammary specific functions. METHODOLOGY: Buffalo mammary tissue collected from the slaughter house was processed enzymatically to obtain a heterogenous population of cells containing both epithelial and fibroblasts cells. Epithelial cells were purified by selective trypsinization and were grown in a plastic substratum. The purified mammary epithelial cells (MECs) after several passages were characterized for mammary specific functions by immunocytochemistry, RT-PCR and western blot. PRINCIPAL FINDINGS: The established buffalo mammary epithelial cell line (BuMEC) exhibited epithelial cell characteristics by immunostaining positively with cytokeratin 18 and negatively with vimentin. The BuMEC maintained the characteristics of its functional differentiation by expression of ß-casein, κ-casein, butyrophilin and lactoferrin. BuMEC had normal growth properties and maintained diploid chromosome number (2n = 50) before and after cryopreservation. A spontaneously immortalized buffalo mammary epithelial cell line was established after 20 passages and was continuously subcultured for more than 60 passages without senescence. CONCLUSIONS: We have established a buffalo mammary epithelial cell line that can be used as a model system for studying mammary gland functions.