Laura Francesca Pisani

Effects 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-alpha, 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.

In vitro modulatory effect of ω-3 polyunsaturated fatty acid (EPA and DHA) on phagocytosis and ROS production of goat neutrophils

An in vitro study was carried out to examine the influence of two fish-oil-derived long chain omega-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on goat polymorphonuclear leukocytes (PMN). Twelve Saanen healthy goats were used as blood donors. Neutrophils were isolated from blood and incubated with increasing concentration of EPA and DHA (25, 50, 100, 200muM). Control samples were incubated in the absence of omega-3 PUFAs. Phagocytosis was evaluated by fluorescein-labeled Escherichia coli incorporation, while extracellular Reactive Oxygen Species (ROS) production was determined by cytochrome c reduction assay, which was selected among the others due to its specificity for extracellular superoxide anion release. Phagocytic activity was significantly increased by EPA (P<0.05) and DHA (P<0.01). Treating PMN with EPA does not affect extracellular ROS production which is, on the contrary, down-regulated by DHA. This effect was increased in experimental conditions which mimic pro-inflammatory challenges (stimulation with PMA). This study demonstrates that EPA and DHA may have beneficial effect on neutrophil function by increasing their phagocytosis activity and, in the meanwhile, decreasing the tissue damages due to extracellular release of ROS.

Distribution of acute phase proteins in the bovine forestomachs and abomasum

Acute phase proteins (APPs) are produced mainly by the liver and their concentration is increased during the systemic inflammatory response. Expression of haptoglobin (Hp), serum amyloid A (SAA), lipopolysaccharide-binding protein (LBP) and α-1 acid glycoprotein (AGP) was determined in the mucosa of the normal bovine forestomachs and abomasum by qualitative and quantitative reverse transcriptase-PCR for mRNA and by Western blot analysis and immunohistochemistry for proteins. Although expression of SAA mRNA was evident in the forestomachs and abomasum, SAA protein was identified only in the abomasum. Expression of Hp protein was high in the forestomachs and abomasum, even though expression of Hp mRNA was negligible. The main site of expression of LBP mRNA was the omasum, whereas the highest protein expression was evident in the abomasum. AGP was expressed at low levels in the bovine forestomachs. Western blot analysis revealed a heterogeneous electrophoretic pattern for AGP, LBP and Hp, indicating that different stomach compartments produce isoforms that are different to those expressed by the liver. Expression of APPs by the bovine forestomachs and abomasum may contribute to regulation of the innate immune response against pathogens.

Escherichia coli lipopolysaccharides and Staphylococcus aureus enterotoxin B differentially modulate inflammatory microRNAs in bovine monocytes.

MicroRNAs (miRNAs) are a family of regulatory molecules involved in many physiological processes, including activation of cells of the immune system. This study investigated the effect of Escherichia coli lipopolysaccharide (LPS) and Staphylococcus aureus enterotoxin B (SEB) on the expression of five miRNAs involved in the inflammatory response, including miR-9, miR-125 b, miR-155, miR-146 a and miR-223, in bovine CD14(+) cells (monocytes). Incubation of monocytes with SEB induced down-regulation of miR-155, miR-223 and miR-125 b, but not the anti-inflammatory miRNA miR-146 a. Conversely, incubation with LPS upregulated both miR-155 and miR-146 a. In vitro incubation of isolated CD14(+) bovine monocytes with LPS and SEB elicited different and opposite expression of miRNAs reportedly involved in inflammatory reactions.

Development of 23 individual TaqMan® real-time PCR assays for identifying common foodborne pathogens using a single set of amplification conditions

Most of the acute intestinal diseases are caused by foodborne pathogens with infants and elderly people being at major risk. The aim of this study was to develop a procedure to simultaneously detect 20 foodborne pathogens in complex alimentary matrices such as milk, cheese and meat. The list of targets include, among the others, Listeria spp., Salmonella spp., Shigella spp., Escherichia coli spp., Campylobacter spp., Clostridium spp. and Staphylococcus aureus. The accuracy of detection was determined by using ATCC strains as positive and negative controls. The achieved sensitivity of each of assays was 1 pg of genomic DNA, which was equivalent to ∼1 cfu. The working ranges of the TaqMan(®) Real-time PCR assays, when used quantitatively on cheese and meat samples inoculated with serial dilution of Listeria spp., Listeria monocytogenes, S. aureus, Salmonella enterica, Shigella boydii, E. coli O157:H7, Bacillus cereus, Campylobacter coli, Yersinia enterocolitica, Enterobacter sakazakii and Pseudomonas aeruginosa was 10(8) cfu/g to 10(4) cfu/g. No matrix interferences were observed.

Microscopic Colitis: What Do We Know About Pathogenesis?

Abstract:Microscopic colitis (MC) is a common cause of chronic diarrhea. The 2 most frequent forms of MC are collagenous colitis and lymphocytic colitis. Over the past years, the incidence and prevalence of microscopic colitis are rising and this is largely attributed to a greater awareness, and concomitantly an increasing number of diagnoses. Patients with microscopic colitis report watery, nonbloody diarrhea of chronic course, abdominal pain, weight loss, and fatigue that may impair patients health-related quality of life. The underlying mechanisms involved in the pathogenesis of microscopic colitis remain unspecified but is probably multifactorial. Collagenous colitis and lymphocytic colitis may represent specific mucosal responses to different luminal agents in predisposed individuals, resulting in an uncontrolled immune response. Genetic predisposition, altered modulation of cytokines and miRNAs, and aberrant response to drugs seem to be involved in the development of MC. Despite the progress of knowledge, still many questions remain unsolved regarding the etiology, pathophysiology, and optimal management of MC. This review gives an update on the immunological aspects of collagenous colitis and lymphocytic colitis.

In vitro modulation of caprine monocyte immune functions by ω-3 polyunsaturated fatty acids.

The in vitro effects of the ω-3 polyunsaturated fatty acids (PUFAs) eicosapentanoic acid (EPA) and docosahexanoic acid (DHA) on phagocytosis and the extracellular respiratory burst in caprine monocytes were assessed. Blood monocytes incubated with increasing concentrations of EPA or DHA (25-200 μM) demonstrated increased phagocytosis compared to unexposed monocytes. Generation of reactive oxygen species (ROS) was not markedly affected in the presence of EPA and DHA, except at 200 μM, at which concentrations monocyte viability was also reduced.

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.

Biomarkers and Microscopic Colitis: An Unmet Need in Clinical Practice

One of the most common causes of chronic diarrhea is ascribed to microscopic colitis (MC). MC is classified in subtypes: collagenous colitis (CC) and lymphocytic colitis (LC). Patients with MC report watery, non-bloody diarrhea of chronic course, abdominal pain, weight loss, and fatigue that may impair patient’s health-related quality of life. A greater awareness, and concomitantly an increasing number of diagnoses over the last years, has demonstrated that the incidence and prevalence of MC are on the rise. To date, colonoscopy with histological analysis on multiple biopsies collected along the colon represents the unique accepted procedure used to assess the diagnosis of active MC and to evaluate the response to medical therapy. Therefore, the emerging need for less-invasive procedures that are also rapid, convenient, standardized, and reproducible, has encouraged scientists to turn their attention to the identification of inflammatory markers and other molecules in blood or feces and within the colonic tissue that can confirm a MC diagnosis. This review gives an update on the biomarkers that are potentially available for the identification of inflammatory activity, related to CC and LC.

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.