Differential Gene Expression in Porcine Lung Compartments after Experimental Infection with Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (Mhyo) is the causative agent of porcine enzootic pneumonia (EP), as well as one of the main pathogens involved in the porcine respiratory disease complex. The host-pathogen interaction between Mhyo and infected pigs is complex and not completely understood; however, improving the understanding of these intricacies is essential for the development of effective control strategies of EP. In order to improve our knowledge about this interaction, laser-capture microdissection was used to collect bronchi, bronchi-associated lymphoid tissue, and lung parenchyma from animals infected with different strains of Mhyo, and mRNA expression levels of different molecules involved in Mhyo infection (ICAM1, IL-8, IL-10, IL-23, IFN-α, IFN-γ, TGF-ß, and TNF-α) were analyzed by qPCR. In addition, the quantification of Mhyo load in the different lung compartments and the scoring of macroscopic and microscopic lung lesions were also performed. Strain-associated differences in virulence were observed, as well as the presence of significant differences in expression levels of cytokines among lung compartments. IL-8 and IL-10 presented the highest upregulation, with limited differences between strains and lung compartments. IFN-α was strongly downregulated in BALT, implying a relevant role for this cytokine in the immunomodulation associated with Mhyo infections. IL-23 was also upregulated in all lung compartments, suggesting the potential involvement of a Th17-mediated immune response in Mhyo infections. Our findings highlight the relevance of Th1 and Th2 immune response in cases of EP, shedding light on the gene expression levels of key cytokines in the lung of pigs at a microscopic level.

Impact of supplementation with dihydroxylated vitamin D3 on performance parameters and gut health in weaned Iberian piglets under indoor/outdoor conditions.

BACKGROUND: Vitamin D may improve innate antimicrobial response and the integrity of the intestinal mucosal barrier representing an alternative to antibiotics for improving pig health. Therefore, benefits of dietary supplementation with a product based on vitamin D3 metabolite-rich plant extracts were assessed in 252 purebred Iberian piglets for a period of 60 days. The study group received 1,25 dihydroxyvitamin D (1,25(OH)2D) (100 ppm) in the conventional feed, which already included vitamin D (2000 IU in the starter and 1000 IU in the adaptation diets, respectively). Average daily gain (ADG), feed conversion ratio (FCR) and coefficient of variation of body weight (CV-BW) were assessed along the study. Blood samples, from 18 animals of the study group and 14 animals of the control group, were collected at selected time points to determine white blood cell count, concentration of vitamin D3 and its metabolites, and IgA and IgG in serum. Histopathology, morphometry, and immunohistochemistry (IgA and FoxP3) from small intestine samples were performed on days 30 and 60 of the study from 3 animals per group and time point. RESULTS: The ADG (493 vs 444 g/day) and FCR (2.3 vs 3.02) showed an improved performance in the supplemented animals. Moreover, the lower CV-BW indicated a greater homogeneity in the treated batches (13.17 vs 26.23%). Furthermore, a mild increase of IgA and in the number of regulatory T cells in the small intestine were observed in treated pigs. CONCLUSIONS: These results highlight the benefits of this supplementation and encourage to develop further studies along other production stages.

Fermented rye with Agaricus subrufescens and mannan-rich hydrolysate based feed additive to modulate post-weaning piglet immune response.

BACKGROUND: The process of weaning in piglets is often associated with an increased inflammation response in the intestine and compromised intestinal integrity and morphology, favoring a delay in intestinal maturation and a predisposal to diseases. Research has shown the potential of different nutritional strategies to reduce the production of pro-inflammatory cytokines, with the main goal to manipulate health and performance of pigs. Promising examples of nutritional strategies are fungal fermented products and their derivatives which are described to contain several compounds that may play a role in gastrointestinal health and pathogenic bacteria control. Products from Agaricus subrufescens mushroom are reported to contain prophylactic and therapeutic properties including antimicrobial and immunomodulatory properties. RESULTS: This study analysed the post-weaning immune status in intestinal tissue and blood of piglets, with the objective to evaluate the gastrointestinal health and immune modulation response induced by a blend of mannan-rich hydrolyzed copra meal and fermented rye with A. subrufescens. Intestinal histomorphology demonstrated a villus height reduction in jejunum and increase in ileum on day 15, while increased villous height in jejunum and ileum on day 30. The results showed that in post-weaning piglets, the feed additive stimulates an immunomodulation effect most evident at 15 days post-weaning, with significant lower expression of cytokines Interferon (IFN) γ, Interleukin (IL) 1α, IL-1ß, IL-6, IL-8, IL-10 and Transforming Growth Factor (TGF) ß in jejunum, accompanied with an increase in peripheral blood mononuclear cells (PBMC) cytokine gene expression of IL-1ß, IL-6, IL-8, IL-10, IL-12p35 (IL-12α), IL-12p40 (IL-12ß), Tumor Necrosis Factor (TNF) α, IFN-α, and TGF-ß. In piglets fed the feed additive, the quantity of Immunoglobulin (Ig) A producing cells in jejunum, ileum was reduced on day 15 and 30 post-weaning, and on day 30 and 45 post-weaning in colon tissue. Natural Killer (NK) cells count in blood were increased on day 15 post-weaning in the piglets fed the feed additive. CONCLUSION: This study implies the potential of the blend including mannan-rich hydrolyzed copra meal and fermented rye with A. subrufescens on immune modulation in the intestine of post-weaning piglets.

Up-Regulation of Immune Checkpoints in the Thymus of PRRSV-1-Infected Piglets in a Virulence-Dependent Fashion.

Virulent porcine reproductive and respiratory syndrome virus (PRRSV) strains, such as the Lena strain, have demonstrated a higher thymus tropism than low virulent strains. Virulent PRRSV strains lead to severe thymus atrophy, which could be related to marked immune dysregulation. Impairment of T-cell functions through immune checkpoints has been postulated as a strategy executed by PRRSV to subvert the immune response, however, its role in the thymus, a primary lymphoid organ, has not been studied yet. Therefore, the goal of this study was to evaluate the expression of selected immune checkpoints (PD1/PDL1, CTLA4, TIM3, LAG3, CD200R1 and IDO1) in the thymus of piglets infected with two different PRRSV-1 strains. Thymus samples from piglets infected with the low virulent 3249 strain, the virulent Lena strain and mock-infected were collected at 1, 3, 6, 8 and 13 days post-infection (dpi) to analyze PRRSV viral load, relative quantification and immunohistochemical staining of immune checkpoints. PD1/PDL1, CTLA4, TIM3, LAG3 and IDO1 immune checkpoints were significantly up-regulated in the thymus of PRRSV infected piglets, especially in those infected with the virulent Lena strain from 6 dpi onwards. This up-regulation was associated with disease progression, high viral load and cell death. Co-expression of these molecules can affect T-cell development, maturation and selection, negatively regulating the host immune response against PRRSV.

Weaning causes a prolonged but transient change in immune gene expression in the intestine of piglets.

Controlling gut inflammation is important in managing gut disorders in the piglet after weaning. Establishing patterns of inflammation markers in the time subsequent to weaning is important for future research to determine whether interventions are effective in controlling gut inflammation. The objective of this study was to evaluate the intestinal inflammatory response during the postweaning period in piglets. A 45-d study included 108 piglets (weaned at 22 d, body weight 5.53 ± 1.19 kg), distributed in 12 pens with nine pigs per pen. Histomorphometry, gene expression of pro- and anti-inflammatory cytokines, and the quantity of immunoglobulin (Ig) A producing cells were measured in jejunum, ileum, and colon on days 0, 15, 30, and 45 postweaning. Cytokine gene expression in peripheral blood mononuclear cells and Ig quantities were analyzed in blood from piglets on days 0, 15, 30, and 45 postweaning. Histomorphometrical results showed a lower villus length directly after weaning. Results demonstrated a postweaning intestinal inflammation response for at least 15 d postweaning by upregulation of IgA producing cells and IFN-γ, IL-1α, IL-8, IL-10, IL-12α, and TGF-ß in jejunum, ileum, and colon. IgM and IgA were upregulated at day 30 postweaning. IgG was downregulated at day 15 postweaning. The results indicate that weaning in piglets is associated with a prolonged and transient response in gene expression of pro- and anti-inflammatory cytokines and IgA producing cells in the intestine.

Virulent Lena strain induced an earlier and stronger downregulation of CD163 in bronchoalveolar lavage cells.

Highly virulent porcine reproductive and respiratory syndrome virus (PRRSV) strains have increasingly overwhelmed Asia and Europe in recent years. This study aims to compare the clinical signs, gross and microscopic findings as well as the expression of CD163 within live pulmonary alveolar macrophages (PAMs) from bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with two PRRSV strains of different virulence. Pigs were infected with either a subtype 1 PRRSV-1 3249 strain or a subtype 3 PRRSV-1 Lena strain and consecutively euthanized at 1, 3, 6, 8 and 13 days post-inoculation. Clinical signs were reported daily and BALF and lung tissue samples were collected at the different time-points and accordingly processed for their analysis. Pigs infected with Lena strain exhibited greater clinical signs as well as gross and microscopic lung scores compared to 3249-infected pigs. A decreased frequency of PAMs from BALF was observed early in pigs infected with Lena strain. Moreover, the frequency and median fluorescence intensity (MFI) of CD163 within PAMs were much lower in Lena-infected pigs than in 3249-infected pigs. This downregulation in CD163 was also observed in lung sections after the assessment of macrophages expressing CD163 by means of immunohistochemistry. This outcome may result from the effect of PRRSV replication, PRRSV-induced inflammation, the influx of immature macrophages to restore lung homeostasis and/or the evidence of CD163low cells after CD163+ cells decrease in BALF.

Unusual histopathologic features in thymic corpuscles associated with porcine periweaning failure-to-thrive syndrome.

Six, 5-6-wk-old pigs, from 3 farms of the same company, with significant loss of body condition were submitted for postmortem evaluation. Macroscopically, the main lesion observed in all of the pigs was thymic atrophy. Microscopically, all of the pigs had thymic atrophy, superficial lymphocytic fundic gastritis, atrophic enteritis, superficial colitis, and neutrophilic and lymphocytic rhinitis, leading to a diagnosis of porcine periweaning failure-to-thrive syndrome. In the pigs from 2 of the farms, many of the thymic corpuscles had infiltrates of neutrophils and degenerate cells, in some cases infiltrating the surrounding parenchyma.

Seminal Plasma Modifies the Transcriptional Pattern of the Endometrium and Advances Embryo Development in Pigs.

Background: Seminal plasma (SP) promotes sperm survival and fertilizing capacity, and potentially affects embryo development, presumably via specific signaling pathways to the internal female genital tract. Objectives: This study evaluated how heterologous SP, infused immediately before postcervical artificial insemination (AI) affected embryo development and the transcriptional pattern of the pig endometria containing embryos. Materials and Methods: Postweaning estrus sows (n = 34) received 40-mL intrauterine infusions of either heterologous pooled SP or Beltsville Thawing Solution (BTS; control) 30 min before AI of semen extended to 10% of homologous SP. Embryos (all sows) and endometrium samples (3 sows/group) were removed during laparotomy 6 days after the infusion of SP or BTS to morphologically evaluate the embryos to determine their developmental stage and to analyze the endometrial transcriptome using microarrays (PORGENE 1.0 ST GeneChip array, Affymetrix) followed by qPCR for further validation. Results: Embryo viability was equal between the groups (~93%), but embryo development was significantly (P < 0.05) more advanced in the SP-treated group compared to control. A total of 1,604 endometrium transcripts were differentially expressed in the SP group compared to the control group. An enrichment analysis showed an overrepresentation of genes and pathways associated with the immune response, cytokine signaling, cell cycle, cell adhesion, and hormone response, among others. Conclusions: SP infusions prior to AI positively impacted the preimplantation embryo development and altered the expression of the endometrial genes and pathways potentially involved in embryo development.

Importance of the knowledge of pathological processes for risk-based inspection in pig slaughterhouses (Study of 2002 to 2016).

OBJECTIVE: The objective of this work was to determine the prevalence of the pathologies that caused the condemnation of pig carcasses in an area of intensive pig farming and Mediterranean climatology and to evaluate their influence in a risk-based inspection procedure for slaughterhouses. METHODS: A retrospective observational investigation was carried out from 2002 to 2016 into the pathological processes that caused the condemnation of pig carcasses in a slaughterhouse from South-eastern Spain. The seasonal effect on the causes of condemnation carcass was reported. Negative binomial model was used to evaluate the effect of season on the rate of antemortem rejections and post-mortem condemnations. Histopathological examinations were performed to confirm the diagnosis. RESULTS: The risk of antemortem rejections (0.0564%) was significantly greater in summer (risk ratio [RR] = 1.57). Autumn was associated with higher rate (RR = 1.69) of the total postmortem condemnations (0.1046%). Significantly higher rates of pronounced anaemia (0.0111%) were observed in summer (RR = 3.20). The main causes of anaemia were observed gastroesophageal ulcers and haemorrhagic enteropathies. Significantly highest risk of erysipelas (0.0074%) were observed in autumn (RR = 5.485). About other zoonosis, only eight cases (0.0013%) of carcasses were declared unfit due to tuberculosis lesions. Porcine muscular cysticercosis was not detected. Nevertheless, nonspecific causes such as generalized infections and emaciation represented the half of the condemned carcasses (50.90%). CONCLUSION: The pathologies leading to the condemnation of carcasses in this study can be considered representative of the pathologies that affect the pig population from a region with a high intensive production and Mediterranean climatology because this slaughterhouse receives a lot of animals from many farms of different size in a high intensive pig production zone (Mediterranean region). Increased knowledge of environmental factors that may foment the appearance of the diseases is essential for implementing inspection programs based on risk assessment in pig's slaughterhouses.

Comparative analysis of cytokine transcript profiles within mediastinal lymph node compartments of pigs after infection with porcine reproductive and respiratory syndrome genotype 1 strains differing in pathogenicity.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a weak immune response enabling it to persist in different organs of infected pigs. This has been attributed to the ability of PRRSV to influence the induction of cytokine responses. In this study, we investigated the cytokine transcriptional profiles in different compartments of the mediastinal lymph node of pigs infected with three genotype 1 PRRSV strains of differing pathogenicity: the low virulence prototype Lelystad virus (LV), and UK field strain 215-06 and the highly virulent subtype 3 SU1-Bel isolate from Belarus. We have used a combination of laser capture micro-dissection (LCM) followed by real time quantitative PCR (RT-qPCR) and immunohistochemical (IHC) detection of immune cell markers (CD3, CD79a and MAC387) and RT-qPCR quantification of PRRSV and cytokine transcripts. Compared to mock infected pigs, we found a significant downregulation of TNF-α and IFN-α in follicular and interfollicular areas of the mediastinal lymph node from 3 days post-infection (dpi) in animals infected with all three strains. This was accompanied by a transient B cell depletion and T cell and macrophage infiltration in the follicles together with T cell depletion in the interfollicular areas. A delayed upregulation of IFN-γ and IL-23p19 was observed mainly in the follicles. The PRRSV load was higher in all areas and time-points studied in the animals infected with the SU1-Bel strain. This paper describes the first application of LCM to study the cytokine transcript profiles and virus distribution in different compartments of the lymph node of pigs.

Evaluation of the single cervical skin test and interferon gamma responses to detect Mycobacterium bovis infected cattle in a herd co-infected with Mycobacterium avium subsp. paratuberculosis.

This study reports the performance of the single intradermal tuberculin (SIT) test and the interferon-gamma (IFN-γ) assay for Mycobacterium bovis in a cattle herd with high prevalence of paratuberculosis (PTB). A total of 58/350 animals were selected for necropsy based on one or more of the following criteria: positive to SIT, IFN-γ, a breeding cow that seroconverted to PTB and showed signs compatible with a wasting disease. Infection status was determined by post mortem diagnostic tests that included histopathology examination, mycobacterial cultures and PCR identification for M. bovis and Mycobacterium avium subsp. paratuberculosis (MAP). In 7/58 animals primary tuberculosis (TB) lesions, affecting only the retropharyngeal and/or mediastinal lymph nodes, were found; 3/7 animals were found SIT positive. PTB was confirmed in 35/58 animals, of which 30 had seroconverted and 14 had typical clinical signs. 45/58 animals were IFN-γ(+) using the most stringent criterion (cut-off point ≥ 0.05); however, IFN-γ test was only positive in 33 animals when using a higher threshold (cut-off point ≥ 0.1). Three animals co-infected also showed extensive TB and diffuse PTB lesions. These results show that the combined use of SIT and IFN-γ, as interpreted using official guidelines, detected all confirmed cases of TB. Individually, the sensitivity of the SIT was inadequate to diagnose TB-positive animals with an advanced stage of PTB. The large number of IFN-γ(+) animals with no visible TB lesion could be due, in part, to some protection conferred by prior infection with MAP.

Imaging diagnosis-infiltrative lipoma causing spinal cord and lumbar nerve root compression in a dog.

A 12-year-old, male, fox terrier dog presented with an abnormal gait of the left pelvic limb. Computed tomography revealed a large, homogeneous, hypoattenuating, noncontrast enhancing mass within the left epaxial muscles that invaded the L5-6 vertebral canal and caused spinal cord compression. Imaging findings were consistent with an infiltrative lipoma. The mass was removed and a left hemilaminectomy was performed in the affected area. Histopathology confirmed the mass to be an infiltrative lipoma. The dog recovered and regained neurologic function within 2 weeks. Computed tomography assisted preoperative planning by characterizing the shape, size, and location of the mass.

Interleukin-4, interleukin-5, and interleukin-13 gene expression in cultured mononuclear cells from porcine circovirus type 2-vaccinated pigs after cells were challenged with porcine circovirus type 2 open reading frame 2 antigen.

OBJECTIVE: To characterize the kinetics of interleukin (IL)-4, IL-5, and IL-13 secretion in peripheral blood and lymph node mononuclear cells isolated from porcine circovirus type 2 (PCV2)-vaccinated pigs after cells were challenged with PCV2 open reading frame 2 antigen. ANIMALS: 10 pigs. PROCEDURES: 5 pigs were vaccinated with a PCV2 vaccine and received a booster dose 3 weeks later. They were kept together with a similar group of 5 nonvaccinated pigs that served as controls. One week after the second vaccination, peripheral blood mononuclear cells (PBMCs) and excised retropharyngeal lymph node mononuclear cells (LNMCs) were isolated and cultured. Cells were then challenged by exposure to PCV2 open reading frame 2 and evaluated at 2, 12, 24, and 48 hours to determine the expression of IL-4, IL-5, and IL-13 via quantitative PCR assay. Changes in gene expression were analyzed relative to the results from analysis of the sample at 0 hours (calibrator). RESULTS: All ILs were upregulated differently in LNMCs and PBMCs from vaccinated pigs. Lymph node mononuclear cells from vaccinated animals produced significantly more IL-4 mRNA than did PBMCs at 2, 12, and 48 hours (relative change: 2.8 vs -3.6, 13.0 vs 3.6, and 9.8 vs 1.8, respectively) and more IL-5 mRNA at 2, 12, 24, and 48 hours (relative change: 1. 2 vs -4.8, 2.2 vs 0.2, 3.2 vs -1.9, and 4.0 vs -3.6, respectively). Interleukin-13 mRNA reached its highest concentration at 24 hours but was 11.9-fold higher in PBMCs than in LNMCs. CONCLUSIONS AND CLINICAL RELEVANCE: Results supported the importance of IL-4, IL-5, and IL-13 in pigs, suggesting that PBMCs and LNMCs express cytokines in a tissue-specific manner.

Immunopathogenesis of porcine reproductive and respiratory syndrome in the respiratory tract of pigs.

Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) impairs local pulmonary immune responses by damaging the mucociliary transport system, impairing the function of porcine alveolar macrophages andinducing apoptosis of immune cells. An imbalance between pro- and anti-inflammatory cytokines, including tumour necrosis factor-α and interleukin-10, in PRRS may impair the immune response of the lung. Pulmonary macrophage subpopulations have a range of susceptibilities to different PRRSV strains and different capacities to express cytokines. Infection with PRRSV decreases the bactericidal activity of macrophages, which increases susceptibility to secondary bacterial infections. PRRSV infection is associated with an increase in concentrations of haptoglobin, which may interact with the virus receptor (CD163) and induce the synthesis of anti-inflammatory mediators. The balance between pro- and anti-inflammatory cytokines modulates the expression of CD163, which may affect the pathogenicity and replication of the virus in different tissues. With the emergence of highly pathogenic PRRSV, there is a need for more information on the immunopathogenesis of different strains of PRRS, particularly to develop more effective vaccines.

A dietary resveratrol-rich grape extract prevents the developing of atherosclerotic lesions in the aorta of pigs fed an atherogenic diet.

The presence of grape and wine polyphenol resveratrol (RES) in the diet is negligible. Therefore, the cardiovascular benefits of this molecule, in a dietary context, remain to be established. We aimed to investigate, through dietary intervention, the effects of a resveratrol-rich grape extract (GE-RES) on the prevention of early aortic lesions in pigs fed an atherogenic diet (AD). These effects were compared with those produced by a grape extract lacking RES (GE) or RES alone. Pigs fed the AD for 4 months showed early atherosclerotic lesions in the thoracic aorta: degeneration and fragmentation of elastic fibers, increase of intima thickness, subendothelial fibrosis, and accumulation of fatty cells and anion superoxide radicals. GE-RES was the most effective treatment and prevented the disruption of aortic elastic fibers, decreased their alteration (57%), and reduced the intima thickness (33%) and the accumulation of fatty cells (42%) and O(2)(•-) (38%) in aortic tissue. In addition, GE-RES moderately downregulated the expression of the suppressors of cytokine signaling 1 (SOCS1) and 3 (SOCS3), key regulators of vascular cell responses, in peripheral mononuclear blood cells. Our results suggest that the consumption of this GE-RES nutraceutical, in a dietary prevention context, could prevent early atherosclerotic events. The presence of RES in the grape extract strengthened these effects.

Enhanced expression of TGFß protein in lymphoid organs and lung, but not in serum, of pigs infected with a European field isolate of porcine reproductive and respiratory syndrome virus.

Transforming growth factor ß (TGFß) is an immunomodulatory cytokine which is able to modulate the host immune response eliciting an inefficient response against pathogens. In this sense, the role of this cytokine in porcine reproductive and respiratory syndrome (PRRS) has been poorly studied and the reported results are contradictory. Thus, in the present study, the expression of TGFß was analysed both at tissue (lymphoid organs and lung) and serum level to study its correlation with the expression of PRRS virus (PRRSV). To carry out this study, 32 pigs were inoculated with the European PRRSV field isolate 2982 and sequentially killed from 0 dpi to the end of the study (24 dpi). Blood and tissue samples were collected to determine the expression of PRRSV and TGFß. PRRSV was detected in inoculated animals from 3 dpi until the end of the study, however TGFß was not detected in sera from inoculated animals. Contrary, an increase of TGFß antigen was observed both in the lymphoid organs and in the lung of PRRSV-inoculated pigs when compared with control group. Since TGFß play a role as an immunomodulatory cytokine of the immune response and also in the differentiation of regulatory T cells (Tregs), the upregulation of the TGFß at tissue level may play a role in the impairment of the host immune response observed during PRRS, being observed a significant correlation between PRRSV and TGFß expression at lung level.

Effects of long-term consumption of low doses of resveratrol on diet-induced mild hypercholesterolemia in pigs: a transcriptomic approach to disease prevention.

Metabolic and cardiovascular diseases (CVDs) have risen to alarming proportions, and there is a need for therapeutic and preventive measures. The polyphenol resveratrol (RES) protects against CVDs, but in vivo molecular mechanisms responsible for protection are not yet understood. Peripheral blood mononuclear cells (PBMNCs) are involved in the development of atherosclerosis and metabolic disorders. The identification of PBMNCs genes responding to dietary compounds might help to understand the mechanisms underlying the effects of polyphenols. We determined gene expression differences between PBMNCs from pigs fed a high-fat diet manifesting a mild increase of cholesterol and pigs fed a high-fat diet containing low doses of RES. Although the consumption of RES did not modify the levels of cholesterol, microarray analyses indicated that some of the differentially expressed genes, collagens (COL1A, COL3A), lipoprotein lipase (LPL) and fatty-acid binding proteins (FABPs) involved in CVDs and lipid metabolism were up-regulated by the high-fat diet and down-regulated by RES. Reverse transcriptase polymerase chain reaction confirmed that RES and RES-containing grape extract prevented the induction of FABP4 in PBMNCs in female pigs fed a high-fat diet. Low micromolar concentrations of RES and its metabolite dihydroresveratrol exerted a minor but significant reducing effect on the induction of FABP4 expression in human macrophages treated with oxidized low-density lipoprotein. Our results show that the consumption of low doses of RES modulates the expression of genes related to lipid metabolism and metabolic disorders that are affected by a high-fat diet and suggest that some of the circulating RES metabolites may contribute to these effects.

Metabolites and tissue distribution of resveratrol in the pig.

SCOPE: trans-Resveratrol (RES) and/(or) its metabolites exert many effects in vivo. Our aim was to study the metabolism and tissue distribution of RES using the pig, a mammal physiologically close to humans. METHODS AND RESULTS: Forty-seven tissues, organs and fluids were analyzed 6 h after intragastric RES administration (5.9 mg/kg body weight) using HPLC-MS/MS. Twelve RES and seven dihydroresveratrol (DH-RES) metabolites were detected. DH-RES was the main metabolite in cecum, colon and rectum, whereas RES-3-O-glucuronide was the most abundant one in fluids and organs. Approximately 74.5% of the total RES administered was recovered in the form of RES, DH-RES and derived metabolites (65.1% along the gastrointestinal tract, 7.7% in urine, 1.2% in bile and 0.5% in organs). We report here, for the first time, the occurrence of RES ribosyl-sulfate derivative, DH-RES diglucuronide, DH-RES sulfoglucuronide and DH-RES disulfate as well as the metabolic profile of RES and DH-RES in the aorta, lymph, lymph node, ovaries, uterus, cerebellum, pancreas, urinary bladder tissue, fat and muscle. CONCLUSION: This study contributes to the clarification of the metabolism and tissue distribution of RES and could help to further understand the mechanisms underlying its effects.

Immunohistochemical detection of extrinsic and intrinsic mediators of apoptosis in porcine paraffin-embedded tissues.

Apoptosis is a strictly regulated mechanism of cell death that involves a complex network of biochemical pathways. Whether a cell undergoes apoptosis or not depends on a delicate balance of anti- and pro-apoptotic stimuli. This phenomenon can be induced by two different pathways: intrinsic and extrinsic pathways. The main aim of this study was to determine the ideal fixative and antigen retrieval method in porcine paraffin embedded tissues for the immunohistochemical detection of apoptosis mediators, from both extrinsic and intrinsic pathways. Tonsil, retropharyngeal lymph node and lung tissue samples were fixed in 10% neutral buffered formalin, Bouin solution and zinc salts fixative (ZSF) and different unmasking methods were carried out. Both 10% neutral buffered formalin and ZSF resulted as the fixatives of election to study apoptosis phenomena. Tween 20 (0.01% in PBS), citrate buffer (microwave, pH 6.0) and/or protease type XIV were the antigen retrieval methods which displayed better labelling. Our results allow to deep in the knowledge of apoptosis and its role in the pathogenesis of porcine diseases.

Pharmacokinetic study of trans-resveratrol in adult pigs.

A number of pharmacokinetic studies have shown marked differences in the plasma metabolic profile of resveratrol (RES) between humans and animals and between individuals of the same species, which complicates the identification of the putative bioactive metabolites responsible for the beneficial effects of RES. On the basis of the physiological similarity between pigs and humans, the aim of this work was to characterize the metabolic profile and pharmacokinetics of RES in the plasma of pigs and to compare this to values reported in humans. RES (5.9 mg/kg of body weight) was orally administered to pigs. The following metabolites were identified in plasma using HPLC-MS/MS: RES-diglucuronide (1), two isomers of RES-sulfoglucuronide (2, 3), two isomers of RES-glucuronide (4, 5), RES-sulfate (6), and RES. The most abundant metabolites were 2, 5 (identified as resveratrol 3-O-glucuronide), and 6. The t(max) ranged from 0.9 h for compounds 2 and 5 to 2 h for compound 3. The highest C(max) value was 2223 ng/mL (5.5 µM) for metabolite 5, which was 2.6-, 3.3-, and 12-fold higher than that for metabolites 6, 2, and 3, respectively. Peak plasma levels of RES (53 ng/mL; 0.23 µM) were detected 0.5 h after RES ingestion. Apart from the low levels of RES aglycone, the RES metabolic profile in pigs differs from that found in humans. The identification of the actual active RES metabolites is a challenge that requires more complex studies which should take into account many possible influencing factors such as age, gender, and methodological approaches.