Effect of Udder Health Status and Lactation Phase on the Characteristics of Sardinian Ewe Milk

Mammary involution and inflammation are known to negatively affect milk quality. A trial was carried out to elucidate the mechanism by which udder health status and lactational phase determine compositional modifications in ovine milk. A total of 60 individual milk samples was collected from a group of 20 pluriparous Sardinian ewes from mid to late lactation. Each sample was assessed for its chemical characteristics, quantitative distribution of casein fractions, lactodynamographic characteristics, and enzymatic activity. Udders were classed as healthy, doubtful, or infected on the basis of repeated somatic cell counts, and samples were grouped in 3 classes of days in milk. Results indicated that both udder inflammation and mammary involution can increase plasmin (PL) activity (15.6 vs. 18.4 U/mL in healthy vs. infected udders; 14.0 vs. 20.2 U/mL in phase 1 vs. 3), which is responsible for an evident protein breakdown in milk. Significant differences between groups were observed for several characteristics. With regard to udder heath status, casein index was lower in the infected vs. healthy udders (74.8 vs. 76.6%), and beta(tot)-casein showed a similar trend (43.9 vs. 46.6%). As a consequence of protein degradation, gamma-casein (5.78 vs. 2.82%) and proteolysis index (7.60 vs. 3.82) increased in the infected group with respect to the healthy group. Udder health status also affected milk technological traits. Udder inflammation resulted in longer clotting time (20.7 vs. 16.5 min for infected vs. healthy, respectively) and in poorer curd firmness (35.6 vs. 47.6 mm for infected vs. healthy, respectively). Frequency of samples reactive to rennet was 100, 93, and 67%, respectively, for healthy, doubtful, and infected groups. With regard to lactational phase, a decrease in alpha(s1)-casein (39.13 vs. 29.36%) and beta(1)-casein (23.41 vs. 19.36%) occurred during phase 1 vs. 3, whereas kappa + alpha(s2)-casein increased (12.30 vs. 21.56%, phase 1 vs. 3). Correlation coefficients confirmed the role of PL in protein degradation. It was concluded that PL activity was strongly affected by both lactational phase and udder health status and, in turn, could be an important agent enhancing milk quality detriment

The Kraft Heinz Company global nutrition targets for the innovation and reformulation of food and beverages: Current and future directions

Reformulating packaged foods has the potential to improve the nutrient density of the global diet. The present perspective illustrates The Kraft Heinz Company’s approach to product (re)formulation to develop healthier product lines that are lower in saturated fats, total sugars, and sodium, and contain health promoting components. Here we present the rationale for The Kraft Heinz Company’s global nutrition targets used for the global innovation and renovation of foods and beverages. The global nutrition targets use a category specific approach to set maximum levels for the main nutrients of public health concern: saturated fat, total sugars and sodium, taking into account product characteristics (typical portion size, eating occasion, role in the diet, etc.) as well as regulatory, technological, sensory and safety constraints. Benchmarking examples illustrate how the nutrition targets are positioned within the United States, France, and Australia. These global nutrition targets serve as part of The Kraft Heinz Company’s environmental, social and governance nutrition commitments and demonstrates how the food industry is improving the nutritional value of packaged foods and beverages both now and into the future

An in vitro system for the comparison of excision and wet-dry swabbing for microbiological sampling of beef carcasses.

An in vitro system for the comparison of wet-dry swabbing and surface tissue excision was developed to ascertain whether the commonly accepted statement of the advantage (in terms of bacterial recovery) of the tissue excision method is also legitimate when different kinds of bacteria are used. A total of 1,770 sections (2.5 by 10 cm) of bovine skin were individually inoculated on the subcutaneous fat side by spreading various suspensions of marker organisms (nalidixic acid-resistant Escherichia coli, vancomycin-resistant Enterococcus faecalis, and methicillin-resistant Staphylococcus aureus) at different concentrations and sampled by two standard methods: cotton wet-dry swabbing and excision. Most counts from cuts sampled by excision were significantly (P < 0.05) higher than the wet-dry swabs; however, no differences were observed between the control and the sampling method when sections were inoculated with bacterial solutions at a concentration of 10(3) CFU/ml and sampled by excision. For sections inoculated with bacterial solutions at a concentration of 10(3) CFU/ml, counts given as log CFU/25 cm2 ranged from 1.97 (S. aureus sampled by wet-dry swab) to 3.06 (S. aureus sampled by excision). For sections inoculated at a concentration of 10(4), counts given as log CFU/25 cm(2) ranged from 2.15 (E. faecalis sampled by wet-dry swab) to 3.19 (S. aureus sampled by excision). For sections inoculated at 10(5), counts given as log CFU/25 cm(2) ranged from 2.94 (E. faecalis, wet-dry swab) to 3.98 (S. aureus, excision), and for sections inoculated at 106, counts given as log CFU/25 cm(2) ranged from 3.53 (E. coli, wet-dry swab) to 4.69 (S. aureus, excision). The proposed system, which enabled a considerable amount of samples to be analyzed under controlled experimental conditions and a large number of data to be generated in a short time, demonstrated among the tested microorganisms that whereas the excision method recovered the highest number of bacteria, control means were always (with the exception of an inoculum of 10(3)/ml) significantly higher than means from either of the sampling methods. Our results indicate that particular attention should be paid to the diverse microflora that can contaminate carcasses in a given slaughterhouse and that it is not appropriate to generalize by saying that the destructive method is the reference technique for the bacteriological sampling of carcasses in slaughterhouses, especially when the contamination is higher than 10(3) CFU/25 cm(2)

CONSIDERATIONS ON MEAT INSPECTION PROCEDURES IN THE LIGHT OF CONDEMNATIONS AT AN ITALIAN EU SLAUGHTERHOUSE IN THE PERIOD 2004-2009

Post-mortem findings of 373.901 cattle slaughtered at an EC abattoir are reported. Results show high incidence of pneumonia and hepatic lesions. Total condemnation of the whole carcass has happened 91 times in Bovine species, 1073 times in swine species, 40 in sheep. In the light of the EC Regulation N.854/2004 of the European parliament and of the council of 29th April 2004 that lays down specific rules for the organization of official controls of products of animal origin destined for human consumption, authors outline the examples of simplification and partial redrafting of the existing laws on the basis of the opinions expresses by the EFSA. Comparison to other European countries and an estimate of economic damage are discussed

Celiac disease-associated Neisseria flavescens decreases mitochondrial respiration in CaCo-2 epithelial cells: Impact of Lactobacillus paracasei CBA L74 on bacterial-induced cellular imbalance

: We previously identified a Neisseria flavescens strain in the duodenum of celiac disease (CD) patients that induced immune inflammation in ex vivo duodenal mucosal explants and in CaCo-2 cells. We also found that vesicular trafficking was delayed after the CD-immunogenic P31-43 gliadin peptide-entered CaCo-2 cells and that Lactobacillus paracasei CBA L74 (L.&nbsp;paracasei-CBA) supernatant reduced peptide entry. In this study, we evaluated if metabolism and trafficking was altered in CD-N.&nbsp;flavescens-infected CaCo-2 cells and if any alteration could be mitigated by pretreating cells with L.&nbsp;paracasei-CBA supernatant, despite the presence of P31-43. We measured CaCo-2 bioenergetics by an extracellular flux analyser, N.&nbsp;flavescens and P31-43 intracellular trafficking by immunofluorescence, cellular stress by TBARS assay, and ATP by bioluminescence. We found that CD-N. flavescens colocalised more than control N.&nbsp;flavescens with early endocytic vesicles and more escaped autophagy thereby surviving longer in infected cells. P31-43 increased colocalisation of N.&nbsp;flavescens with early vesicles. Mitochondrial respiration was lower (P&nbsp;&lt;&nbsp;.05) in CD-N.&nbsp;flavescens-infected cells versus not-treated CaCo-2 cells, whereas pretreatment with L.&nbsp;paracasei-CBA reduced CD-N.&nbsp;flavescens viability and improved cell bioenergetics and trafficking. In conclusion, CD-N.&nbsp;flavescens induces metabolic imbalance in CaCo-2 cells, and the L.&nbsp;paracasei-CBA probiotic could be used to correct CD-associated dysbiosis

Process development and validation of expanded regulatory T cells for prospective applications: an example of manufacturing a personalized advanced therapy medicinal product

Background: A growing number of clinical trials have shown that regulatory T (Treg) cell transfer may have a favorable effect on the maintenance of self-tolerance and immune homeostasis in different conditions such as graft-versus-host disease (GvHD), solid organ transplantation, type 1 diabetes, and others. In this context, the availability of a robust manufacturing protocol that is able to produce a sufficient number of functional Treg cells represents a fundamental prerequisite for the success of a cell therapy clinical protocol. However, extended workflow guidelines for nonprofit manufacturers are currently lacking. Despite the fact that different successful manufacturing procedures and cell products with excellent safety profiles have been reported from early clinical trials, the selection and expansion protocols for Treg cells vary a lot. The objective of this study was to validate a Good Manufacturing Practice (GMP)-compliant protocol for the production of Treg cells that approaches the whole process with a risk-management methodology, from process design to completion of final product development. High emphasis was given to the description of the quality control (QC) methodologies used for the in-process and release tests (sterility, endotoxin test, mycoplasma, and immunophenotype). Results: The GMP-compliant protocol defined in this work allows at least 4.11 7 109 Treg cells to be obtained with an average purity of 95.75 \ub1 4.38% and can be used in different clinical settings to exploit Treg cell immunomodulatory function. Conclusions: These results could be of great use for facilities implementing GMP-compliant cell therapy protocols of these cells for different conditions aimed at restoring the Treg cell number and function, which may slow the progression of certain diseases

Deletion of cytosolic gating ring decreases gate and voltage sensor coupling in BK channels

Large conductance Ca(2+)-activated K(+) channels (BK channels) gate open in response to both membrane voltage and intracellular Ca(2+). The channel is formed by a central pore-gate domain (PGD), which spans the membrane, plus transmembrane voltage sensors and a cytoplasmic gating ring that acts as a Ca(2+) sensor. How these voltage and Ca(2+) sensors influence the common activation gate, and interact with each other, is unclear. A previous study showed that a BK channel core lacking the entire cytoplasmic gating ring (Core-MT) was devoid of Ca(2+) activation but retained voltage sensitivity (Budelli et al. 2013. Proc. Natl. Acad. Sci. USA. http://dx.doi.org/10.1073/pnas.1313433110). In this study, we measure voltage sensor activation and pore opening in this Core-MT channel over a wide range of voltages. We record gating currents and find that voltage sensor activation in this truncated channel is similar to WT but that the coupling between voltage sensor activation and gating of the pore is reduced. These results suggest that the gating ring, in addition to being the Ca(2+) sensor, enhances the effective coupling between voltage sensors and the PGD. We also find that removal of the gating ring alters modulation of the channels by the BK channel’s β1 and β2 subunits

Regulatory T cells from patients with end-stage organ disease can be isolated, expanded and cryopreserved according good manufacturing practice improving their function

Background Here, we isolated, expanded and functionally characterized regulatory T cells (Tregs) from patients with end stage kidney and liver disease, waiting for kidney/liver transplantation (KT/LT), with the aim to establish a suitable method to obtain large numbers of immunomodulatory cells for adoptive immunotherapy post-transplantation. Methods We first established a preclinical protocol for expansion/isolation of Tregs from peripheral blood of LT/KT patients. We then scaled up and optimized such protocol according to good manufacturing practice (GMP) to obtain high numbers of purified Tregs which were phenotypically and functionally characterized in vitro and in vivo in a xenogeneic acute graft-versus-host disease (aGVHD) mouse model. Specifically, immunodepressed mice (NOD-SCID-gamma KO mice) received human effector T cells with or without GMP-produced Tregs to prevent the onset of xenogeneic GVHD. Results Our small scale Treg isolation/expansion protocol generated functional Tregs. Interestingly, cryopreservation/thawing did not impair phenotype/function and DNA methylation pattern of FOXP3 gene of the expanded Tregs. Fully functional Tregs were also isolated/expanded from KT and LT patients according to GMP. In the mouse model, GMP Tregs from LT or KT patient proved to be safe and show a trend toward reduced lethality of acute GVHD. Conclusions These data demonstrate that expanded/thawed GMP-Tregs from patients with end-stage organ disease are fully functional in vitro. Moreover, their infusion is safe and results in a trend toward reduced lethality of acute GVHD in vivo, further supporting Tregs-based adoptive immunotherapy in solid organ transplantation

Finding a new therapeutic approach for no-option Parkinsonisms : Mesenchymal stromal cells for progressive supranuclear palsy

Background: The trophic, anti-apoptotic and regenerative effects of bone marrow mesenchymal stromal cells (MSC) may reduce neuronal cell loss in neurodegenerative disorders. Methods: We used MSC as a novel candidate therapeutic tool in a pilot phase-I study for patients affected by progressive supranuclear palsy (PSP), a rare, severe and no-option form of Parkinsonism. Five patients received the cells by infusion into the cerebral arteries. Effects were assessed using the best available motor function rating scales (UPDRS, Hoehn and Yahr, PSP rating scale), as well as neuropsychological assessments, gait analysis and brain imaging before and after cell administration. Results: One year after cell infusion, all treated patients were alive, except one, who died 9 months after the infusion for reasons not related to cell administration or to disease progression (accidental fall). In all treated patients motor function rating scales remained stable for at least six-months during the one-year follow-up. Conclusions: We have demonstrated for the first time that MSC administration is feasible in subjects with PSP. In these patients, in whom deterioration of motor function is invariably rapid, we recorded clinical stabilization for at least 6 months. These encouraging results pave the way to the next randomized, placebo-controlled phase-II study that will definitively provide information on the efficacy of this innovative approach. Trial registration ClinicalTrials.gov NCT0182412