SERUM AMYLOID A IN RUMINANTS: DIAGNOSTIC VALUE AND FOOD CONTAMINATION ASSESSMENT

The aims of the work presented in this thesis were to investigate the bovine acute phase protein Serum Amyloid A, focussing on its value as safety marker in farm animals. SAA can be considered as a natural anti-inflammatory and immunomodulatory agent and local expression of SAA, at the site of the initial acute phase reaction, could protect against the deleterious effects of inflammation. In this study whether SAA can be isolated from tissues of bovine with clinical amyloidosis was investigated. We also investigated if AA fibrils present in milk can be then found in cheese after caseification, i.e. if the process of ripeining can degrade the AA fibrils. In bovine, SAA was identified as potential marker of mastitis, and SAA milk concentration in milk increases before the raising of somatic cells. In this thesis two aspect of the involvement of SAA in food safety were explored: a)the acute phase reaction, strongly focused on the mammary gland. The animal model chosen was water buffalo, since no information is available so far about the acute phase reaction in this species. The acute phase proteins sequences are unknown, and also their concentration in physiological and pathological conditions are not established. b)The possibility that high concentration of SAA in milk induce the formation of amyloid fibrils, which are considered to be potentially dangerous for human safety. Results presented in this thesis advanced the knowledge of the acute phase reaction in water buffalo: the five APPs included in this investigation, namely Serum amyloid A, Haptoglobin, Ceruloplasmin, \u3b11-acid glycoprotein and Lipolysaccaride Binding Protein were sequenced for the first time, and two of them were quantified. In the second part of the thesis, we purified amyloid fibrils from amyloidosis-affected cows, and added purified fibrils at a given concentration in milk before ripening. Results demonstrated the presence of insoluble fibrils in cheese added with amyloid proteins, even if a lower amount of precipitated insoluble SAA could be detected also in negative control cheese

Proteomic insights on the metabolism in inflammatory bowel disease

Inflammatory bowel diseases (IBD) are chronic and relapsing inflammatory conditions of the gut that include Crohn's disease and ulcerative colitis. The pathogenesis of IBD is not completely unraveled, IBD are multi-factorial diseases with reported alterations in the gut microbiota, activation of different immune cell types, changes in the vascular endothelium, and alterations in the tight junctions\u2019 structure of the colonic epithelial cells. Proteomics represents a useful tool to enhance our biological understanding and to discover biomarkers in blood and intestinal specimens. It is expected to provide reproducible and quantitative data that can support clinical assessments and help clinicians in the diagnosis and treatment of IBD. Sometimes a differential diagnosis of Crohn's disease and ulcerative colitis and the prediction of treatment response can be deducted by finding meaningful biomarkers. Although some non-invasive biomarkers have been described, none can be considered as the \u201cgold standard\u201d for IBD diagnosis, disease activity and therapy outcome. For these reason new studies have proposed an \u201cIBD signature\u201d, which consists in a panel of biomarkers used to assess IBD. The above described approach characterizes \u201comics\u201d and in this review we will focus on proteomics

Horse bone marrow mesenchymal stem cells express embryo stem cell markers and show the ability for tenogenic differentiation by in vitro exposure to BMP-12

Background: Mesenchymal stem cells (MSCs) have been recently investigated for their potential use in regenerative medicine. MSCs, in particular, have great potential, as in various reports they have shown pluripotency for differentiating into many different cell types. However, the ability of MSCs to differentiate into tendon cells in vitro has not been fully investigated. Results: In this study, we show that equine bone marrow mesenchymal stem cells (BM-MSCs), defined by their expression of markers such as Oct4, Sox-2 and Nanog, have the capability to differentiate in tenocytes. These differentiated cells express tendon-related markers including tenomodulin and decorin. Moreover we show that the same BM-MSCs can differentiate in osteocytes, as confirmed by alkaline phosphatase and von Kossa staining. Conclusion: As MSCs represent an attractive tool for tendon tissue repair strategies, our data suggest that bone marrow should be considered the preferred MSC source for therapeutic approaches

MMX® technology and its applications in gastrointestinal diseases

The Multimatrix\uae (MMX\uae) preparation MMX\uae is a recently obtained drug formulation developed to facilitate release of high concentrations of active drugs into the colon, with a homogeneous distribution along all colonic segments, particularly the most distal ones; the distal colonic tracts, indeed, are the most difficult to reach in significant amounts when a drug is given orally. The MMX\uae formulation is characterized by a lipophilic matrix dispersed in a hydrophilic structure. Indeed, in the last few years, MMX\uae technology has been widely used in the development of various drugs for the treatment of inflammatory and infectious gastrointestinal diseases localized in the colon. In particular, MMX\uae mesalamine, budesonide and parnaparin formulations have been investigated in patients with ulcerative colitis, and the first two have reached worldwide registration for the treatment of this disease. Moreover, MMX\uae-rifamycin is being positively tested in the treatment of colonic bacterial infections, including traveler's diarrhea. MMX\uae technology is, thus, proving to be a very effective formulation for the treatment of various colonic diseases. This effectiveness has been related not only to specific colonic delivery, but also to its ability to act in a once-daily dosage, thus favouring patients' adherence to prescribed schedules of treatment. The effective delivery of the active molecule to the site of need in the colon is also associated with very low systemic absorption and very low rates of adverse events (AEs). In this paper, we have reviewed all clinical trials performed with an MMX\uc2\uae-bound drug and all possible real-life reports, in order to give an overall evaluation of MMX\uae

Effect of pre-mating nutrition on mRNA levels of developmentally-relevant genes in sheep oocytes and granulosa cells

The present study was designed to investigate the relationship between pre-mating nutrition and the relative amounts of a panel of developmentally relevant genes in ovine oocytes and granulosa cells. Cast age ewes were fed a ration providing 0.5x (0.5 M) or 1.5x (1.5 M) live weight maintenance requirements for 2 weeks before slaughter. The ewes were synchronized and superovulated with FSH and pregnant mares serum gonadotropin. At slaughter, oocytes and granulosa cells were aspirated from follicles >2 mm in diameter and the relative abundance of 8 and 17 transcripts in oocytes and granulosa cells respectively were analyzed by semi-quantitative RT-PCR. In the oocytes, no differences between groups were observed for five transcripts (GDF9, BMP15, c-kit, glucose transporter 1 (SLC2A1), and hexokinase 1), but a lower amount of glucose transporter 3 (SLC2A3), sodium/glucose cotransporter 1 (SLC5A1), and Na+/K+ ATPase mRNAs was detected in the 0.5 M group. Increased expression of PTGS2, HAS2, and the leptin receptor long form was observed in granulosa cells from the 0.5 M group. No differences between groups were observed for the other transcripts (early growth response factor-1, estrogen receptor-, LH and FSH receptors, gremlin 1, pentraxin 3, KIT ligand, glucose transporters 1, 3, and 8, IGF1, IGF1 receptor, leptin receptor, and tumor necrosis factor-stimulated gene 6). Expression of leptin and sodium/glucose cotransporter 1 was not detected in both groups. The present data indicate that pre-mating nutrition is associated with alteration in the mRNA content in oocytes and surrounding follicle cells in ewes, which may account for the reduced reproductive performance typical of ewes that are fed a restricted ration for a short period of time before mating

Dietary fatty acids on subcutaneous adipose tissue modulation in transition dairy goats

The goal of the present study was to evaluate the metabolic and immune response of peripartal dairy goats to dietary supplementation with fish oil or stearic acid. 15 multiparous alpine dairy goats were involved in the trial. Starting from the last week of gestation until 3 weeks after kidding date the experimental diets, based on alfalfa and mix hays and a concentrate mix, were added either with protected fish oil (FO) or with stearic acid (ST). Feed intake, body weight, energy balance, milk production and composition were measured weekly. Adipose tissue biopsies were performed on day -7, 7 and 21 relative to kidding date and samples were immediately fixed in formalin, paraffin embedded and Hematoxilin Eosin stained. The results discussed in the present work are relative to a subsample of 8 goats, representative of the two experimental groups. Hematological and histological data were analyzed by a Generalized Estimating Equation (GEE) in IBM SPSS 19.0 was used. Production parameters were analyzed by a MIXED repeated model in SAS 9.2. No differences were observed between FO and ST in milk production, BCS, weight, dry matter intake and milk components except for a higher milk protein percentage in the 7 to 21 d period for ST. BHB serum content was higher in ST overall the experiment, whereas NEFA and ALAT serum content were higher at day 7 in FO compared to ST (P < 0.08). ALAT was higher also at day 21 in FO. Treatment had no effect on blood cellular component except for WBC in FO group, where a significant decrease at 7 d was observed. WBC and HCM parameters were in the physiological range for dairy goats during transition period. Histologic adipose tissue analysis revealed a significant decreased adipocytes surface between -7 and 21 d in ST, whereas in FO the adipocyte surface reduction was related to the -7 to 7 d interval reaching a plateau until day 21. The EB pattern and the NEFA serum content at 7d in particular for FO are well correlated with histologic observations indicating goats were using fat depots to cope their negative energy balance. NEFA levels did not confirm the histological evidence at day 21 for ST suggesting a possible different action on subcutaneous adipose tissue during time. Results suggest a modulation in lipid storage management during peripartal negative energy balance by saturated vs. unsaturated dietary fatty acid supplementation that did not affect production levels of goats

Anti-TNF-Mediated Modulation of Prohepcidin Improves Iron Availability in Inflammatory Bowel Disease, in an IL-6-Mediated Fashion

Background. Anaemia is common in inflammatory bowel disease (IBD), frequently resulting from a combination of iron deficiency and of anaemia of chronic disease (ACD). ACD is characterized by macrophage iron retention induced by proinflammatory cytokines. Hepcidin is the master inducer of iron accumulation during ACD, and its production is mainly regulated by IL-6 and the novel erythroid hormone erythroferrone (ERFE). This study evaluates whether anti-TNF monoclonal antibodies therapy modurates hepcidin production and the levels of its main regulators, leading to a restoration of iron homeostasis. Methods. Sera were collected from 21 IBD patients, before each anti-TNF administration, for the first 6 weeks of therapy. Prohepcidin, erythropoietin, erythroferrone, C reactive protein, interleukin-6, iron markers, and haemoglobin levels were measured and clinical activity indexes were evaluated. Results. Serum prohepcidin, IL-6, CRP, and ferritin were significantly reduced after 6-week treatment; an increase in serum iron and total transferrin was observed. No changes in the EPO-ERFE axis were found. Remarkably, haemoglobin was significantly increased. Conclusions. Anti-TNF therapy improves iron metabolism and, subsequently, anaemia in IBD. This effect appears to be related to the modulation of the cytokine network and specifically IL-6 leading to a relevant decrease of hepcidin, a master regulator of ACD

Procoagulatory state in inflammatory bowel diseases is promoted by impaired intestinal barrier function

Inflammatory and immune mediated disorders are risk factors for arterial and venous thromboembolism. Inflammatory bowel diseases (IBD) confer an even greater risk of thromboembolic events than other inflammatory conditions. It has been shown that IBD patients display defective intestinal barrier functions. Thus, pathogen-associated molecular patterns (PAMPs) coming from the intestinal bacterial burden might reach systemic circulation and activate innate immunity receptors on endothelial cells and platelets, promoting a procoagulative state. Aim of the study was to test this hypothesis, correlating the presence of circulating PAMPs with the activation of innate immune system and the activation of the coagulatory cascade in IBD patients. Specifically, we studied lipopolysaccharide (LPS), Toll-like receptor (TLR) 2, TLR4, and markers of activated coagulation (i.e., D-Dimer and prothrombin fragment F1 + 2) in the serum and plasma of IBD patients. We found that LPS levels are increased in IBD and correlate with TLR4 concentrations; although a mild correlation between LPS and CRP levels was detected, clinical disease activity does not appear to influence circulating LPS. Instead, serum LPS correlates with both D-Dimer and F1 + 2 measurements. Taken together, our data support the role of an impairment of intestinal barrier in triggering the activation of the coagulatory cascade in IBD

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

The LHCb upgrade I

The LHCb upgrade represents a major change of the experiment. The detectors have been almost completely renewed to allow running at an instantaneous luminosity five times larger than that of the previous running periods. Readout of all detectors into an all-software trigger is central to the new design, facilitating the reconstruction of events at the maximum LHC interaction rate, and their selection in real time. The experiment's tracking system has been completely upgraded with a new pixel vertex detector, a silicon tracker upstream of the dipole magnet and three scintillating fibre tracking stations downstream of the magnet. The whole photon detection system of the RICH detectors has been renewed and the readout electronics of the calorimeter and muon systems have been fully overhauled. The first stage of the all-software trigger is implemented on a GPU farm. The output of the trigger provides a combination of totally reconstructed physics objects, such as tracks and vertices, ready for final analysis, and of entire events which need further offline reprocessing. This scheme required a complete revision of the computing model and rewriting of the experiment's software