Maternal effect genes produce mRNA or proteins that accumulate in the egg during oogenesis and control the developmental program until embryonic genome activation takes place. NLRP5 (NLR family, ...Pyrin domain containing 5), also called MATER (Maternal Antigen That Embryos Require) is one of the genes required for normal early embryonic development, although its precise function remains to be elucidated. The aim of the present study was to analyze the NLRP5 gene expression pattern and protein distribution in somatic tissues and germ cells in the pig. Reverse transcription was performed on mRNA from germinal vescicle (GV) oocytes and total RNA from spermatozoa and tissues from different organs. The transcript for NLRP5 gene was identified only in ovaries and oocytes. The presence of NLRP5 protein was detected only in ovaries by western blot analysis and immunohistochemistry.
Filarial nematodes harbour intracellular endosymbiotic bacteria, which have been assigned to the genus Wolbachia. These bacteria appear to play an important role in the pathogenesis of filarial ...diseases through their lipopolysaccharides. In view of the presence of Wolbachia endosymbionts in the body of filarial nematodes, one might also expect that proteins from these bacteria play an antigenic role in humans and animals affected by filariases. To test this hypothesis, we produced in recombinant form the surface protein WSP and a portion of the cell-cycle protein FTSZ from the Wolbachia of Dirofilaria immitis. Western immunoblot assays were then performed using cat sera to test the immunogenicity of these proteins. Sera were collected from owner's cats, which were either sero-negative or sero-positive for D.immitis and from cats before and after experimental infection with D.immitis. FTSZ was recognized in Western blots by sera from both positive and negative cats and from both uninfected and experimentally infected cats.WSP was recognized only by sera from positive cats and from cats experimentally infected with D.immitis; this protein was not recognized by sera from negative cats and from cats before experimental infection with D.immitis. The results of Western blot assays on WSP thus support the hypothesis that infection with filarial nematodes induces the production of antibodies against Wolbachia proteins.
Coagulase-negative staphylococci (CNS) are the most prevalent microorganisms isolated from cow milk and are associated with subclinical mastitis and persistent increases in the bulk milk somatic cell ...count (BMSCC) of low BMSCC herds. By combining peptide enrichment, LC-ESI-MS/MS, and statistical analysis, we investigated the influence of subclinical mastitis and CNS infection on the milk peptidome. Quarter milk samples from clinically healthy Holstein cows were subjected to bacteriological culture (BC) and somatic cell counting (SCC) for two consecutive samplings and 28 (including 11 negatives and 17 positives) were selected for peptidomic analysis.
The study identified 1363 different endogenous peptides and highlighted a significant increase of peptides in CNS-positive milk, mainly represented by casein fragments. Milk peptidome changes increased with the SCC, as also demonstrated by protein electrophoresis and densitometry. Peptides significantly different in CNS or CONTROL samples were identified and characterized. Our results indicate that subclinical mastitis by CNS can induce significant changes in the milk peptidome, opening the way to future studies for the identification of a biomarker panel as well as for the understanding of their consequences for the technological and sensorial characteristics of cow milk and dairy products.
This is the first investigation on the impact of subclinical CNS mastitis on the bovine milk peptidome. The peptide enrichment strategy combined with a highly sensitive MS/MS analysis enabled the compilation of a very large peptide dataset for healthy and mastitic milk. The comparison of CNS and Control samples, also considering SCC classes, highlighted several peptides with potential for understanding milk protein and peptide dynamics in subclinical mastitis, with possible implications for its detection.
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•Detailed characterization of the milk peptidome in subclinical CNS mastitis•Very large milk peptide dataset available for healthy and mastitic milk.•Demonstration that subclinical CNS mastitis impacts significantly on the milk peptidome.•Demonstration of the direct relationship with the SCC
The effect of doxorubicin and Congo Red on prion protein (PrP) infectivity in experimental scrapie was studied to better understand
the effect of these compounds in prion diseases and to establish ...whether a dose-response correlation exists for Congo Red.
This was performed in order to test the effectiveness of compounds that may easily be used in human prion diseases. Brain
homogenate containing membrane bound PrPSc monomers was used as inoculum and was previously incubated with doxorubicin 10 -3 M and with increasing concentrations of Congo Red ranging from 10 -7 to 10 -2 M. This study shows for the first time that doxorubicin, and confirms that Congo Red, may interact with pathological PrP
monomers modifying their infectious properties. Pre-incubation of infected brain homogenate with Congo Red resulted in prolonged
incubation time and survival, independently of Congo Red concentration (p<0.05). Doxorubicin and Congo Red effects do not
depend upon interaction with PrP amyloid material.
Abstract
The present study aimed to investigate whether acute pain associated with castration and tail docking of male piglets may modulate the expression of salivary microRNAs (miRNAs) and to ...explore their potential use as biomarkers. Thirty-six healthy 4-d-old piglets (Hermitage × Duroc) were randomly assigned to three groups: the first group (12 piglets) has been pretreated with anesthetic and anti-inflammatory drugs (ANA) and then castrated and tail docked; the second one (12 piglets) has been castrated and tail docked without any drugs (CONV); the third one (12 piglets) has been only handled (SHAM). Saliva was collected 10 min before (control group) and 30 to 45 min after the procedures. Salivary cortisol has been quantified. The expression concentrations of seven miRNAs, namely miR-19b, miR-27b-3p, miR-215, miR-22-3p, miR-155-5p, hsa-miR-365-5p, and hsa-miR-204, were measured and assessed as potential biomarkers of pain by quantitative Polimerase Chain Reaction using TaqMan probes. The area under the receiver operating curve (AUC) was used to evaluate the diagnostic performance of miRNAs. The concentration of salivary cortisol increased after treatment in CONV and ANA, while no significant variation was observed in the SHAM group. The comparative analysis demonstrated that the concentrations of salivary miR-19b (P = 0.001), miR-27b (P = 0.042), and miR-365 (P < 0.0001) were significantly greater in CONV as compared with pretreatment. The AUC of pretreatment vs. CONV and CONV vs. ANA were excellent for miR-19b and miR-365 and fair for miR-27b. Combining two miRNAs, namely miR-19b and miR-365, in a panel increased the efficiency of distinguishing between pre- and post-treatment groups. No differences have been identified between SHAM and ANA groups. mRNA potential targets of differentially expressed-miRNA were investigated, and genes related to pain and inflammation were identified: miR-19b potentially modulates TGF-beta and focal adhesion pathways, miR-365 regulates cytokines expression (i.e., IL-1, Tumor Necross Factor-alpha, and IL-8 cytokine), and miR-27b regulates macrophage inflammatory protein pathways (i.e., MIP1-beta). In conclusion, we demonstrated that the abundance of miR-19b, miR-27b, and miR-365 increases in the saliva of piglets castrated and tail docked without the administration of pain-relieving drugs. Further studies are needed to assess their potential during routine husbandry procedures and to extend their assessment in other stressful events, such as weaning or chronic pain.
MicroRNAs (miRNAs) are a class of noncoding RNA molecules playing a crucial role in tumor modulation targeting mRNA. This study aimed to validate the diagnostic potential of a panel of 3 miRNAs ...previously identified in canine mast cell tumors (MCTs), miR-21, miR-379, and miR-885, as markers of lymph node involvement in terms of histological absence (nonmetastatic: HN0; premetastatic: HN1) and presence (early-metastatic: HN2; overt-metastatic: HN3) of metastasis, in the saliva of mast cell tumor (MCT)-affected dogs by quantitative polymerase chain reaction (PCR). Forty-seven saliva samples were analyzed: 36 from MCT-affected dogs (12 subcutaneous 3 HN0-1 and 9 HN2-3 and 24 cutaneous 9 HN0-1 and 15 HN2-3—MCT) and 11 from healthy dogs. MCT-group effects were investigated using analysis of variance (ANOVA). The origin of the tumor affected the expression of salivary miR-21 (P = .011) with an increase in cases with subcutaneous MCTs compared with the healthy group (P = .0005) and those with cutaneous MCTs (P = .004). Salivary miR-21 was higher in the HN2-3 class compared with the healthy group (P = .004). Salivary miR-885 was not affected by the presence of MCT, while miR-379 was not detected in saliva. The diagnostic potential of salivary miR-21 in discriminating MCT-affected dogs from the healthy group (AUC = 0.8917), cutaneous from subcutaneous (AUC = 0.8111), and subcutaneous HN0-1 (AUC = 0.7250) and HN2-3 (AUC = 0.9750) classes from healthy samples was demonstrated by receiver operating characteristic curve analysis. Overall, salivary miR-21 was identified as a promising tool, representing a novel approach to detecting MCT-associated epigenetic alterations in a minimally invasive manner.
Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are omega-3 long-chain polyunsaturated fatty acids (n-3 PUFA) found mostly in fish oil. They have been commonly used as dietary integrators ...in human and animal nutrition, modulating the immune system, mostly by exerting anti-inflammatory activities as demonstrated by in vivo and in vitro studies. The precise mechanisms of action at the background of EPA and DHA immunomodulatory activity are still not fully elucidated. Moreover, no information on their effects on porcine monocytes immune response is available yet. To cover this gap, the study aimed to evaluate DHA and EPA's in vitro impact on porcine monocytes (CD14 +) defensive functions. Briefly, monocytes were isolated from the blood of twenty-six healthy pigs, using a magnetic-activated cell sorting technique (MACS). Monocytes were first treated with increasing concentrations of DHA and EPA (25, 50, 100 and 200 µM) and apoptosis and viability were measured to assess potential cytotoxic effects. Once determined EPA and DHA subtoxic working concentrations (25, 50 and 100 µM), their effects on chemotaxis, phagocytosis and total, intracellular and extracellular reactive oxygen species (ROS) production were evaluated. DHA and EPA only decreased porcine monocytes viability at the highest concentration (200 µM), but their apoptosis was unaffected. DHA (100 µM) decreased the cells’ chemotaxis, while EPA (25 µM) increased their intracellular ROS production after 60 min under non-inflammatory or resting conditions and at 90 min under pro-inflammatory conditions (PMA challenge). EPA (50 µM) decreased monocytes’ intracellular ROS levels only under resting conditions at 30 min. No effects were observed on porcine monocytes phagocytic capacity. In conclusion, this study demonstrates that DHA and EPA can exert differential in vitro immunomodulatory effects in pigs, by dampening monocytes chemotaxis and potentiating their oxidative burst, respectively. Thus, our results suggest these n-3 PUFA might exert both anti-inflammatory and/or immune-enhancing effects in pigs.
•n-3 PUFA have shown to have anti-inflammatory properties in livestock.•DHA and EPA decreased porcine monocytes viability at the highest concentration.•DHA inhibited the chemotactic activity of porcine monocytes.•EPA mainly increased monocytes intracellular ROS production.•DHA and EPA exert differential in vitro immunomodulatory effects in pigs.
Heat stress (HS) has a negative impact on dairy cows’ health, milk production, reproductive performance and immune defenses. Cellular and molecular responses to high temperatures in bovine ...polymorphonuclear cells and peripheral blood mononuclear cells (PBMCs) have been investigated so far. On the contrary, the effects of high temperatures on isolated monocytes remain almost undisclosed. The aim of this study was to unravel the in vitro effects of high temperatures, simulating a severe HS related body hyperthermia, on bovine lifespan and M1/M2 polarisation. The PBMCs were isolated from whole blood of 9 healthy dairy cattle. Monocytes were sorted by magnetic activated cell sorting and cultured over night at 39 °C (normothermia) or 41 °C (HS). Apoptotic rate and viability were assessed and mRNA abundance for heat shock proteins (HSPs), heat transcription factors (HSFs) and genes involved in monocyte/macrophage polarization (STAT1, STAT2, STAT3, STAT6, IL1β, TGF1β, IL-10, COX2) were quantified by qPCR. We found that apoptosis increased in monocytes exposed to 41 °C, as compared to control, while viability conversely decreased. HS increased the abundance of HSF1 and HSP70. The concomitant decrease of STAT1 and STAT2 and the increase of STAT6 genes abundance at 41 °C suggest, at transcriptional factors level, a polarization of monocytes from a classical activated M1 to a non-classically activated M2 monocytes. In conclusion, the exposure of bovine monocytes to high temperatures affects their lifespan as well as the abundance of genes involved in HS response and in monocyte/macrophages polarization phenotype, confirming that bovine immune response may be significantly affected by hyperthermia.
Acute phase proteins (APP) are plasma proteins that can modify their expression in response to inflammation caused by tissue injury, infections, immunological disorders or stress. Although APP are ...produced mainly in liver, extrahepatic production has also been described. As a prerequisite to get insight the expression of APP in chicken during diseases, this study investigated the presence of five APP, including alpha1-acid glycoprotein (AGP), Serum Amyloid A (SAA), PIT54, C-Reactive protein (CRP) and Ovotransferrin (OVT) in twenty tissues collected from healthy chicken (Gallus gallus) by quantitative Real Time PCR and immunohistochemistry. As expected, APP gene abundance was higher in liver compared with other tissues. The mRNA coding for CRP, OVT and SAA was detected in all analyzed tissues with a higher expression in gastrointestinal tract, respiratory and lymphatic samples. SAA expression was particularly high in cecal tonsil, lung, spleen and Meckel’s diverticulum, whereas OVT in lung, bursa of Fabricius and pancreas. AGP and PIT54 mRNA expression were detected in all tissues but at negligible levels. Immunohistochemical expression of AGP and OVT was variably detected in different organs, being identified in endothelium of every tissue. Positive cells were present in the epithelium of the mucosal layer of gastrointestinal tract and kidney. Lung and central nervous system stained for both proteins. No positive staining was detected in lymphoid tissues and muscle. These results suggest that most tissues can express different amount of APP even in healthy conditions and are therefore capable to mount a local acute phase reaction.
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•EOs obtained from Mediterranean aromatic herbs are anti-inflammatory agents.•Coriander, geranium and wormwood EOs inhibit NF-κB and caspase-1 activation.•Linalool and β-citronellol ...explain the anti-inflammatory properties of the EOs.•α-Thujone together with camphor mimic the anti-inflammatory activity of wormwood EO.•β-citronellol inhibits caspase-1 activation in LPS-stimulated THP-1 macrophages.
In this study, the capacity of eight essential oils (EOs), sage (Salvia officinalis), coriander (Coriandrum sativum), rosemary (Rosmarinus officinalis), black cumin (Nigella sativa), prickly juniper (Juniperus oxycedrus), geranium (Pelargonium graveolens), oregano (Origanum vulgare) and wormwood (Artemisia herba-alba), on the inhibition of NF-κB activation was screened at concentrations up to 0.25 µL/mL using THP-1 human macrophages bearing a NF-κB reporter. This screening selected coriander, geranium, and wormwood EOs as the most active, which later evidenced the ability to decrease over 50 % IL-6, IL-1β, TNF-α and COX-2 mRNA expression in LPS-stimulated THP-1 macrophages. The chemical composition of selected EOs was performed by gas chromatography–mass spectrometry (GC–MS). The two major constituents (>50 % of each EO) were tested at the same concentrations presented in each EO. It was demonstrated that the major compound or the binary mixtures of the two major compounds could explain the anti-inflammatory effects reported for the crude EOs. Additionally, the selected EOs also inhibit>50 % caspase-1 activity. However, this effect could not be attributed to the major components (except for β-citronellol/geranium oil, 40 %/65 % caspase-1 inhibition), suggesting, in addition to potential synergistic effects, the presence of minor compounds with caspase-1 inhibitory activity.
These results demonstrated the potential use of the EOs obtained from Tunisian flora as valuable sources of anti-inflammatory agents providing beneficial health effects by reducing the levels of inflammatory mediators involved in the genesis of several diseases.
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