Ultrasonicated Enterococcus faecium SF68 enhances neutrophil free radical production and udder innate immunity of drying-off dairy cows.

Proper dry cow management is critical not only for subsequent milk production and fertility but also for mastitis control. A phenomenon of immunosuppression was commonly observed in transition cows, an example being the high susceptibility of the mammary gland during early the dry period to new infectious agents. Polymorphonuclear neutrophils (PMN) play important defence roles in the mammary gland of newly dried cows. One of the bactericidal mechanisms of PMN is through producing reactive oxygen species (ROS), which can be efficiently quantified by chemiluminescence (CL) assay. In the current study, the potential of intramammary application of a commercial Enterococcus faecium SF68 (SF68) product to enhance the local innate immunity of newly dried mammary glands was evaluated based on the CL assay. The preliminary experiments in vitro indicated virtual dose-responsiveness of ROS generation from three different cell preparations, bovine blood PMN, bovine blood PMN pre-conditioned with cow milk, and the post-diapedesis model somatic cells from cow milk, on their exposure to phorbol 12-myristate 13-acetate (PMA), viable SF68, and ultrasonicated SF68, but not dry-heated SF68. Because ultrasonication treatment was found to profoundly enhance the immunogenicity of SF68 in vitro, in the following animal trial, single infusion of either 5 or 10×107 original cfu of ultrasonicated SF68 was randomly applied to the front quarters and phosphate-bufferedsaline (PBS) applied to the rear quarters of each of the four experimental cows on the first day of milk stasis. The results showed that within the first post-infusion week, ultrasonicated SF68 induced a faster and greater (P<0·05) recruitment of PMN into mammary lumen with no apparent local or systemic inflammatory sign. Meanwhile, ultrasonicated SF68 also induced a greater (P<0·05) ROS production in response to PMA challenge by in situ somatic cells of mammary secretion. Taken together, ultrasonicated SF68 modulated ROS generation of bovine neutrophils, and would be a potential enhancer of udder innate immunity in drying-off dairy cows. More thorough work is warranted.

Physical, antioxidant and structural characterization of blend films based on hsian-tsao gum (HG) and casein (CAS).

The effects of hsian-tsao gum (HG) addition on the physical properties, antioxidant activities and structure of casein (CAS) film have been investigated. It has been observed that HG addition provided CAS film with better mechanical properties and resistant to moisture, stronger barrier properties against light and higher antioxidant activities than pure CAS film. Fourier transformation infrared (FTIR) data indicated that hydrogen bonding interactions and Maillard reactions occurred between CAS and HG, giving rise to a more compact structure than CAS film. The results of X-ray diffraction and differential scanning calorimetry (DSC) indicated that CAS and HG were compatible, and addition of HG destroyed the original crystalline domains of CAS film, and the blend films exhibited higher glass transition temperatures than CAS film. Moreover, nuclear magnetic resonance (NMR) analysis showed that HG addition significantly changed the mobility of water molecule in CAS film. Especially, ratio of the high mobility water of CAS/HG films significantly decreased as compared to CAS film.

Intramammary infusion of an Enterococcus faecium†SF68 preparation promoted the involution of drying off Holstein cows partly related to neutrophil-associated matrix metalloproteinase 9.

A problem for dairy cows following milk stasis is to cope with a high risk of intramammary infection and there is a need to initiate an extensive renewal of secretory modules in mammary glands so that milk production in next lactation may be optimized. We recently reported that ultrasonicated Enterococcus faecium SF68 (SF68) is compatible with cow mammary glands and an enhancer of innate immunity during the immediate post-milk stasis period. The current study further examines the concomitant effect of ultrasonicated SF68 on mammary tissue remodeling. Four Holstein cows each received intramammary infusions of regular antibiotic dry-cow formula (positive control) and two different doses of SF68 in different quarters. Analyses of individual quarter secretion samples showed faster neutrophil infiltration, earlier modifications in protein composition, including caseins and lactoferrins, as well as more prompt elevation of the specific unit of 92-kDa matrix metalloproteinase 9 (MMP9) in SF68-infused quarters compared to the positive controls. Intramammary infusion of ultrasonicated SF68 seems able to accelerate the regression of mammary synthetic capacity and potentiate the breakdown of glandular extracellular matrix, indicating a more efficient mammary gland involution. Correlation analyses imply that the ability of ultrasonicated SF68 to induce faster neutrophil chemotaxis and the associated MMP9 release is partly responsible.

Purification of transglutaminase and its effects on myosin heavy chain and actin of spent hens.

The purpose of this study was to purify pig plasma transglutaminase (TGase) and examine its effects on the myosin heavy chain and actin of the breast muscles from spent hens at different temperatures. TGase (0.3 units/mg) was added to myofibrillar proteins solution (0.5 ml) at 4°C for 0, 4, 8, 12, 24 and 48 h; at 25°C for 0, 1, 2, 4, 8, 12, 24 and 48 h; at 37°C for 0, 5, 10, 30 and 60 min. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that plasma TGase was composed of units of molecular weights approximately 75,000 and 80,000. TGase added to the myofibrillar proteins solution indicated that the concentration of the myosin heavy chain and actin decreased when incubated at 4°C for 48 h and when incubated at 25°C for 2 h. Moreover, the relative intensity determined by scanning densitometry of the SDS-PAGE gel indicated that the myosin heavy chain and actin concentration decreased to 45 and 64%, respectively. In addition, the relative intensity of the myosin heavy chain and actin declined to 7 and 63%, respectively, when incubated at 37°C for 5 min. The relative intensity of both the myosin heavy chain and actin decreased with time when incubated at 25 and 37°C.

Prognostic factors for cure, recurrence and long-term survival after surgical resection of thymoma.

INTRODUCTION: To determine long-term outcome and risk factors for recurrence after thymectomy. METHODS: Patients who underwent thymectomy (n = 262) for a thymic tumor (1986-2010) were identified from a prospective database. Patients were classified according to World Helath Organization (WHO) histologic classification, Masaoka staging system, and completeness of resection. Risk factors for recurrence: WHO histology, tumor size, Masaoka stage and completeness of resection were analyzed. RESULTS: Of 262 patients, 51% were female, median age was 55 years, and 39% had myasthenia gravis. Median follow-up was 7.5 years, median tumor size was 5.4 cm, and Masaoka stage distribution was: I (25%), II (47%), III (17%), IV (4%), and (7%) not classified. Of 200 patients classified under the WHO system, there were (7%) type A, (22%) type AB, and (71%) type B; 83% had complete resection. One-hundred and sixty-nine patients received adjuvant radiotherapy, eight adjuvant chemoradiotherapy and 14 neoadjuvant chemoradiotherapy. Overall survival was 95% at 5 years, 91% at 10 years and 91% at 15 years. Recurrence occurred in 12 patients and disease-related death in four patients. Five patients underwent re-resection for recurrence with survival of 2-15 years. Only Masaoka stage and tumor size were associated with statistically significant risk of recurrence on multivariate analysis. CONCLUSION: Resectable thymoma is associated with excellent prognosis. Aggressive resection of recurrent disease yielded excellent long-term results. Higher Masaoka stage is associated with a greater chance of incomplete resection. Higher Masaoka stage and increasing tumor size are independent factors associated with recurrence.

Effects of pH and temperature on microstructure and morphology of hydroxyapatite/collagen composites synthesized in vitro.

Hydroxyapatite/collagen composites (HAp/Col) were synthesized, in vitro, using the self-organization mechanism, by the co-precipitation of collagen, extracted from pork skin, and aqueous H3PO4 and Ca(OH)2. The effects of pH and temperature, on the microstructure and morphology of HAp/Col composites, were extensively studied. It was shown that the yield of the composite is closely related to the pH value, during preparation. At high pH, the white precipitate of the composites was formed in a large quantity. In contrast, the yield was quite low, at low pH, owing to the dissolution of the composite precipitates. The electrophoretogram of the as-prepared composite shows that the α1(I) chain was dominant and that there was no protein residue in the supernatant of the reaction solution. Accordingly, it is reasonable to assume that the collagen molecules had completely reacted with hydroxyapatite. A closer look at the structural evolution of HAp/Col composites revealed that, at pH=5, there was no sign of interfacial interaction between hydroxyapatite and collagen, during the first 36h. In contrast, under alkaline conditions, the amount of α(I) chains was indeterminate, at the start of precipitation, while the concentration of ß-chains decreased gradually. The chemical structure of HAp/Col, as determined by FTIR spectra, revealed that the bending vibration of phosphate contours can be attributed to the dissolution of HAp, at low pH. The crystalline of HAp was readily discerned, for all samples, as revealed by the XRD patterns. The morphology of the HAp/Col, prepared under neutral to alkaline conditions (7≤pH≤9), exhibited a compact, coral reef-like structure. In summary, HAp/Col composites can be synthesized, in vitro, under conditions native to animal physiology. The desirable conditions for synthesis are a higher pH (8 to 9) and a temperature of 40°C.

Modifications of the defense and remodeling functionalities of bovine neutrophils inside the mammary gland of milk stasis cows received a commercial dry-cow treatment.

An appropriate length of milk stasis between two consecutive lactations of dairy cows is crucial for sustainable milk production. This dry period of cows allows extensive remodeling and sufficient cell renewal in mammary gland. Nevertheless, early dry period is one of the most risky stages in cow lactation cycle to intramammary infection. Dry-cow treatment through teats is, therefore, widely practiced at the commencement of milk stasis. Neutrophils are the most abundant cellular components in cow mammary secretion during early dry period, which in turn attribute to the meanwhile elevation of somatic cell counts and matrix metalloproteinase-9 (MMP-9) level. This study used bovine peripheral neutrophils as a cell model to examine the mode of modifications in their defense and remodeling functionalities after infiltration into mammary gland during early dry period. Results indicate a dose-dependent suppression of phorbol 12-myristate 13-acetate-stimulated free radical production and induction of MMP 9 degranulation in bovine peripheral neutrophils exposure to the d 7-dry secretion of cows received dry cow treatment at d 0 in milk stasis. Meanwhile, an enhancement of plasminogen activation and TNF-α shedding on bovine peripheral neutrophils were also observed. These two cellular events might be involved in the functional modifications on infiltrated neutrophils during early dry period. In conclusion, the opposite trend of modifications in the defense and matrix remodeling functionalities of neutrophils inside the mammary gland of cows at early dry period reflect the collaboration of infiltrated neutrophils for promoting extensive glandular remodeling at minimum compromise of local defense during the acute involution period without apparent disturbance by dry cow treatment.

Anti-peroxidation effects of vitamin E on low density lipoprotein and milk fat globule membrane of lactating goats: in vivo versus metal ion challenge in vitro.

Ruminants are animals with mild oxidation risk considering characteristics of their plasma. The purpose of the present study was to determine if surplus vitamin E supplementation further improved their peroxidation status. Four lactating goats (Capra ibex ibex) receiving a single intramuscular injection of 3000 IU d-alpha-tocopheryl acetate were monitored daily for a week. Plasma and milk levels of thiobarbituric acid reactive substances (TBARS) were measured to estimate the peroxidation status of overall body and mammary gland, respectively. Lipid hydroperoxide content of low density lipoprotein (LDL) and milk fat globule membrane (MFGM) were determined both immediately after isolation and after metal ion challenge to evaluate their spontaneous peroxidation in vivo and peroxidation susceptibility in vitro, respectively. The results showed that while plasma and milk levels of alpha-tocopherol peaked at day 2, the corresponding TBARS dropped to their lowest weekly levels. Content of preformed lipid hydroperoxides in LDL and MFGM remained unchanged (P>0.05) during the study period, while peroxidation in LDL and MFGM on extended exposure to Cu(2+) was prevented by vitamin E enrichment. Therefore, surplus vitamin E was beneficial to the peroxidation-resistant mechanisms of the overall body and those specifically within mammary gland of lactating goats. Furthermore, peroxidation resistance of LDL and MFGM to copper challenge in vitro also benefited from vitamin E enrichment despite the fact their endogenous peroxidation seemed unresponsive. The hypothesis that peroxidation of LDL and MFGM of lactating goats in situ, which proceeds with metal ion-independent mechanisms, is minor was discussed.