Effects of tamoxifen on the immune response phenotype in equine peripheral blood mononuclear cells.

Tamoxifen (TAM) is widely utilized in the prevention and treatment of human breast cancer and has demonstrated the potential to modulate the immune response. It has been proposed as a therapeutic tool for immune-mediated diseases. TAM has been investigated as a possible treatment for asthma-like conditions in horses, revealing specific impacts on the innate immune system. While the effects of TAM on equine neutrophils are well-documented, its influence on lymphocytes and the modulation of the immune response polarization remains unclear. This in vitro study employed peripheral blood mononuclear cells (PBMC) from healthy horses, exposing them to varying concentrations of the TAM and assessing the expression of genes involved in the polarization of the immune response (TBX21, IFNG, GATA3, IL4, IL10, FOXP3, and CTLA4) in PBMC stimulated or not with PMA/ionomycin. Additionally, the effect of TAM over the proportion of regulatory T cells (Treg) was also assessed. TAM did not significantly affect the expression of these genes and Treg at low concentrations. However, at the highest concentration, there was an impact on the expression of GATA3, IL4, IL10, and CTLA4 genes. These alterations in genes associated with a Th2 and regulatory response coincided with a noteworthy increase in drug-associated cytotoxicity but only at concentrations far beyond those achieved in pharmacological therapy. These findings suggest that the effects of TAM, as described in preclinical studies on asthmatic horses, may not be attributed to the modification of the adaptive response.

Andrographolide Ameliorates Inflammatory Changes Induced by D-Lactate in Bovine Fibroblast-like Synoviocytes.

During acute ruminal acidosis, the manifestation of aseptic polysynovitis and lameness in cattle has been observed. Evidence suggests that joint inflammation can be attributed to the metabolic alterations induced by D-lactate in fibroblast-like synoviocytes (FLSs). We aimed to investigate whether andrographolide could mitigate the inflammation and metabolic alterations induced by D-lactate in bovine fibroblast-like synoviocytes (bFLSs). To assess this, bFLSs were cultured in the presence or absence of andrographolide. We evaluated its potential interference with the expression of proinflammatory cytokines, COX-2, HIF-1α, and LDHA using RT-qPCR. Furthermore, we investigated its potential interference with PI3K/Akt signaling and IκBα degradation through immunoblotting and flow cytometry, respectively. Our observations revealed that andrographolide reduced the elevation of IL-6, IL-8, COX-2, HIF-1α, and LDHA induced by D-lactate. Additionally, andrographolide demonstrated interference with the PI3K/Akt and NF-κB pathways in bFLSs. In conclusion, our findings suggest that andrographolide can potentially reverse the inflammatory effects and metabolic changes induced by D-lactate in bFLSs, showing promise as a therapeutic intervention for managing these conditions associated with lameness.

Characterization of extracellular trap production and release by equine neutrophils in response to different stimuli.

This study explores Neutrophil Extracellular Trap (NET) formation in equine neutrophils, which is crucial for eliminating infections and is implicated in various equine inflammatory diseases. We investigated the molecular pathways involved in NET release by equine neutrophils in response to stimuli. We use PMA, A23187, LPS, PAF, OZ, and cytokines, observing NET release in response to PMA, PAF, and A23187. In contrast, LPS, OZ, and the cytokines tested did not induce DNA release or did not consistently induce citrullination of histone 4. Peptidyl-arginine deiminase inhibition completely halted NET release, while NADPH oxidase and mitochondrial reactive oxygen species only played a role in PMA-induced NETs. Neutrophil elastase inhibition modestly affected PAF-induced NET liberation but not in PMA or A23187-induced NET, while myeloperoxidase did not contribute to NET release. We expect to provide a foundation for future investigations into the role of NETs in equine health and disease and the search for potential therapeutic targets.

d-lactate-induced ETosis in cattle polymorphonuclear leucocytes is dependent on the release of mitochondrial reactive oxygen species and the PI3K/Akt/HIF-1 and GSK-3ß pathways.

d-lactate is a metabolite originating from bacterial metabolism that accumulates as a result of dietary disturbances in cattle, leading to ruminal acidosis. d-lactate exerts functions as a metabolic signal inducing metabolic reprogramming and extracellular trap (ET) release in polymorphonuclear leucocytes (PMNs). We previously demonstrated that d-lactate induces metabolic reprogramming via hypoxia-induced factor 1 alpha (HIF-1α) stabilization in bovine fibroblast-like synoviocytes (FLSs). In the present study, the role of HIF-1 in ET formation induced by d-lactate was assessed. HIF-1α stabilization in PMNs was controlled by mitochondrial reactive oxygen species (mtROS) release. Furthermore, inhibition of mitochondrial complex I and scavenging of mtROS decreased d-lactate-triggered ETosis. d-lactate-enhanced HIF-1α accumulation was dependent on the PI3K/Akt pathway but independent of GSK-3ß activity. Pharmacological blockade of the PI3K/Akt/HIF-1 and GSK-3ß axes inhibited d-lactate-triggered ETosis and downregulated PDK1 and LDHA expression. However, only GSK-3ß inhibition decreased the expression of glycogen metabolism enzymes and prevented the decline in glycogen stores induced by d-lactate exposure. The results of this study suggest that mtROS, PI3K/Akt/HIF-1 and GSK-3ß axes regulate carbohydrate metabolism adaptations that support d-lactate-induced ET formation in cattle.

Bovine tumor necrosis factor-alpha Increases IL-6, IL-8, and PGE2 in bovine fibroblast-like synoviocytes by metabolic reprogramming.

Lameness is a common condition in dairy cattle caused by infectious or noninfectious agents. Joint lesions are the second most common cause of lameness and can be diagnosed in association with the presentation of digit injuries. Fibroblast-like synoviocyte (FLS) are predominant cells of synovia and play a key role in the pathophysiology of joint diseases, thus increasing the expression of proinflammatory mediators. Tumor necrosis factor-alpha (TNF-α) is a potent proinflammatory cytokine involved in cyclooxygenase 2 (COX-2) and proinflammatory cytokine expression in FLS. Previously, TNF-α was demonstrated to increase hypoxia-inducible Factor 1 (HIF-1), a transcription factor that rewires cellular metabolism and increases the expression of interleukin (IL)-6 in bovine FLS (bFLS). Despite this, the proinflammatory effects of TNF-α in bFLS on metabolic reprogramming have been poorly studied. We hypothesized that TNF-α increases glycolysis and in this way controls the expression of IL-6, IL-8, and COX-2 in bFLS. Results first, gas chromatography/mass spectrometry (GC/MS)-based untargeted metabolomics revealed that bTNF-α altered the metabolism of bFLS, increasing glucose, isoleucine, leucine, methionine, valine, tyrosine, and lysine and decreasing malate, fumarate, α-ketoglutarate, stearate, palmitate, laurate, aspartate, and alanine. In addition, metabolic flux analysis using D-glucose-13C6 demonstrated an increase of pyruvate and a reduction in malate and citrate levels, suggesting a decreased flux toward the tricarboxylic acid cycle after bTNF-α stimulation. However, bTNF-α increased lactate dehydrogenase subunit A (LDHA), IL-6, IL-8, IL-1ß and COX-2 expression, which was dependent on glycolysis and the PI3K/Akt pathway. The use of FX11 and dichloroacetate (DCA), an inhibitor of LDHA and pyruvate dehydrogenase kinase (PDK) respectively, partially reduced the expression of IL-6. Our results suggest that bTNF-α induces metabolic reprogramming that favors glycolysis in bFLS and increases IL-6, IL-8, IL-1ß and COX-2/PGE2.

Hydroxycarboxylic acid receptor 2 (HCA2) agonists induce NET formation and MMP-9 release from bovine polymorphonuclear leukocytes.

Periparturient cows are commonly fed diets supplemented with Niacin (nicotinic acid, NA) because of its anti-lipolytic properties. NA confers its anti-lipolytic effects by activating the hydroxycarboxylic acid 2 receptor (HCA2). HCA2 is also activated by the ketone body beta-hydroxybutyrate (BHB) and circulating BHB levels are elevated in postpartum dairy cows. The HCA2 receptor is highly expressed in bovine polymorphonuclear leukocytes (PMN) and could link metabolic and innate immune responses in cattle. We investigated how HCA2 agonists affected bovine PMN function in vitro. We studied different PMN responses, such as granule release, surface expression of CD11b and CD47, generation of neutrophil extracellular traps (NETs), and apoptosis. NA, BHB, and 4,4aR,5,5aR-tetrahydro-1H-cyclopropa [4,5] cyclopenta [1,2-c] pyrazole-3-carboxylic acid (MK-1903) treatment triggered the release of matrix metalloproteinase 9 (MMP-9), a component of the tertiary granule, from neutrophils. Additionally, all HCA2 agonists induced NETs formation but did not affect surface expression of CD11b and CD47. Finally, none of the HCA2 agonists triggered apoptosis in bovine PMN. This information will give new insights into the potential role of the HCA2 receptor in the bovine innate immune response.

Novel Proteoliposome-Based Vaccine against E. coli: A Potential New Tool for the Control of Bovine Mastitis.

Escherichia coli is an important causative agent of clinical mastitis in cattle. Current available vaccines have shown limited protection. We evaluated the efficacy of a novel vaccine based on bacterial proteoliposomes derived from an E. coli field strain. Female BALB/c mice were immunized subcutaneously with two doses of the vaccine, 3 weeks apart. Between days 5 and 8 after the first inoculation, the females were mated. At 5-8 days postpartum, the mice were intramammary challenged with the same E. coli strain. Two days after bacterial infection, mice were euthanized, and the mammary glands were examined and removed to evaluate the efficacy and safety of the vaccine as well as the immune response generated by the new formulation. The vaccinated mice showed mild clinical symptoms and a lower mammary bacterial load as compared to non-vaccinated animals. The vaccination induced an increase in levels of IgG, IgG1 and IgG2a against E. coli in blood and mammary glands that showed less inflammatory infiltration and tissue damage, as compared to the control group. In summary, the vaccine based on bacterial proteoliposomes is safe, immunogenic, and effective against E. coli, constituting a new potential tool for mastitis control.

d-lactate-triggered extracellular trap formation in cattle polymorphonuclear leucocytes is glucose metabolism dependent.

D-lactic acidosis is a metabolic disease of cattle caused by the digestive overgrowth of bacteria that are highly producers of d-lactate, a metabolite that then reaches and accumulates in the bloodstream. d-lactate is a proinflammatory agent in cattle that induces the formation of extracellular traps (ETs) in polymorphonuclear leucocytes (PMN), although information on PMN metabolic requirements for this response mechanism is insufficient. In the present study, metabolic pathways involved in ET formation induced by d-lactate were studied. We show that d-lactate but not l-lactate induced ET formation in cattle PMN. We analyzed the metabolomic changes induced by d-lactate in bovine PMN using gas chromatography-mass spectrometry (GC-MS). Several metabolic pathways were altered, including glycolysis/gluconeogenesis, amino sugar and nucleotide sugar metabolism, galactose metabolism, starch and sucrose metabolism, fructose and mannose metabolism, and pentose phosphate pathway. d-lactate increased intracellular levels of glucose and glucose-6-phosphate, and increased uptake of the fluorescent glucose analog 2-NBDG, suggesting improved glycolytic activity. In addition, using an enzymatic assay and transmission electron microscopy (TEM), we observed that d-lactate was able to decrease intracellular glycogen levels and the presence of glycogen granules. Relatedly, d-lactate increased the expression of enzymes of glycolysis, gluconeogenesis and glycogen metabolism. In addition, 2DG (a hexokinase inhibitor), 3PO (a 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 inhibitor), MB05032 (inhibitor of fructose-1,6-bisphosphatase) and CP-91149 (inhibitor of glycogen phosphorylase) reduced d-lactate-triggered ETosis. Taken together, these results suggest that d-lactate induces a metabolic rewiring that increases glycolysis, gluconeogenesis and glycogenolysis, all of which are required for d-lactate-induced ET release in cattle PMN.

Role of Cellular Metabolism in the Formation of Neutrophil Extracellular Traps in Airway Diseases.

Neutrophil extracellular traps (NETs) are a recently described mechanism of neutrophils that play an important role in health and disease. NETs are an innate defense mechanism that participate in clearance of pathogens, but they may also cause collateral damage in unrelated host tissues. Neutrophil dysregulation and NETosis occur in multiple lung diseases, such as pathogen-induced acute lung injury, pneumonia, chronic obstructive pulmonary disease (COPD), severe asthma, cystic fibrosis, and recently, the novel coronavirus SARS-CoV-2. More recently, research into immunometabolism has surged due to the possibility of reprogramming metabolism in order to modulate immune functions. The present review analyzes the different metabolic pathways associated with NETs formation, and how these impact on pathologies of the airways.

Tamoxifen triggers the in vitro release of neutrophil extracellular traps in healthy horses.

Neutrophils display an array of biological functions including the formation of neutrophil extracellular traps (NETs), web-like structures specialized in trapping, neutralizing, killing and preventing microbial dissemination within the host. However, NETs contribute to a number of inflammatory pathologies, including severe equine asthma. Tamoxifen (TX) is a selective estrogen receptor modulator which belongs to the triphenylethyllenes group of molecules, and which is used as a treatment in all stages of estrogen-positive human breast cancer. Our previous results suggest that tamoxifen can modulate neutrophil functionality and promote resolution of inflammation; this would partly explain the clinical beneficial effect of this drug in horses with airway inflammation. Enhanced NETs production has been reported with tamoxifen use in humans, but minimal data exists regarding the drug's effect on NETs in horses. The aim of this study is to assess the in vitro effect of TX on NETs formation from peripheral blood of healthy horses. Five clinically healthy mixed-breed adult horses were enrolled in the study. For this, cellular free DNA quantification, immunofluorescence for the visualization of NETs, assessment of different types of NETs, and detection of mitochondrial superoxide. TX induced NETs formation at a concentration of 10 uM. Our results show that only two types of NETs were induced by TX: 95% spread NETs (sprNETs) and 5% aggregated NETs (aggNETs). Furthermore, induction of these NETs could be influenced by mitochondrial ROS. Future research should involve an In vivo study of horses with severe asthma and TX treatment, to evaluate BALF neutrophil NET formation. In conclusion, this in vitro study suggests that the resolution of inflammation by TX in horses with airway inflammation is due to inhibition of other neutrophilic functions but not to NET formation.

Metabolic Reprogramming and Inflammatory Response Induced by D-Lactate in Bovine Fibroblast-Like Synoviocytes Depends on HIF-1 Activity.

Acute ruminal acidosis (ARA) occurs after an excessive intake of rapidly fermentable carbohydrates and is characterized by the overproduction of D-lactate in the rumen that reaches the bloodstream. Lameness presentation, one of the primary consequences of ARA in cattle, is associated with the occurrence of laminitis and aseptic polysynovitis. Fibroblast-like synoviocytes (FLS) are predominant cells of synovia and play a key role in the pathophysiology of joint diseases, thus increasing the chances of the release of pro-inflammatory cytokines. Increased D-lactate levels and disturbances in the metabolism of carbohydrates, pyruvates, and amino acids are observed in the synovial fluid of heifers with ARA-related polysynovitis prior to neutrophil infiltration, suggesting an early involvement of metabolic disturbances in joint inflammation. We hypothesized that D-lactate induces metabolic reprogramming, along with an inflammatory response, in bovine exposed FLS. Gas chromatography-mass spectrometry (GC-MS)-based metabolomics revealed that D-lactate disrupts the metabolism of bovine FLS, mainly enhancing glycolysis and gluconeogenesis, pyruvate metabolism, and galactose metabolism. The reverse-transcription quantitative PCR (RT-qPCR) analysis revealed an increased expression of metabolic-related genes, including hypoxia-inducible factor 1 (HIF-1)α, glucose transporter 1 (Glut-1), L-lactate dehydrogenase subunit A (L-LDHA), and pyruvate dehydrogenase kinase 1 (PDK-1). Along with metabolic disturbances, D-lactate also induced an overexpression and the secretion of IL-6. Furthermore, the inhibition of HIF-1, PI3K/Akt, and NF-κB reduced the expression of IL-6 and metabolic-related genes. The results of this study reveal a potential role for D-lactate in bFLS metabolic reprogramming and support a close relationship between inflammation and metabolism in cattle.

Participation of Short-Chain Fatty Acids and Their Receptors in Gut Inflammation and Colon Cancer.

Short-chain fatty acids (SCFAs) are the main metabolites produced by the bacterial fermentation of dietary fiber, and they play a critical role in the maintenance of intestinal health. SCFAs are also essential for modulating different processes, and they have anti-inflammatory properties and immunomodulatory effects. As the inflammatory process predisposes the development of cancer and promotes all stages of tumorigenesis, an antitumor effect has also been associated with SCFAs. This is strongly supported by epidemiological studies showing that a diet rich in fiber is linked to a reduced risk of colon cancer and has significant clinical benefits in patients with inflammatory bowel disease (IBD). SCFAs may signal through the metabolite-sensing G protein-coupled receptors free fatty acid receptor 3 [FFAR3 or G protein-coupled receptor 41 (GPR41)], FFAR2 (GPR43), and GPR109A (also known as hydroxycarboxylic acid receptor 2 or HCAR2) expressed in the gut epithelium and immune cells. This review summarizes the existing knowledge regarding the SCFA-mediated suppression of inflammation and carcinogenesis in IBD and colon cancer.

Role of Lactate in Inflammatory Processes: Friend or Foe.

During an inflammatory process, shift in the cellular metabolism associated with an increase in extracellular acidification are well-known features. This pH drop in the inflamed tissue is largely attributed to the presence of lactate by an increase in glycolysis. In recent years, evidence has accumulated describing the role of lactate in inflammatory processes; however, there are differences as to whether lactate can currently be considered a pro- or anti-inflammatory mediator. Herein, we review these recent advances on the pleiotropic effects of lactate on the inflammatory process. Taken together, the evidence suggests that lactate could exert differential effects depending on the metabolic status, cell type in which the effects of lactate are studied, and the pathological process analyzed. Additionally, various targets, including post-translational modifications, G-protein coupled receptor and transcription factor activation such as NF-κB and HIF-1, allow lactate to modulate signaling pathways that control the expression of cytokines, chemokines, adhesion molecules, and several enzymes associated with immune response and metabolism. Altogether, this would explain its varied effects on inflammatory processes beyond its well-known role as a waste product of metabolism.

Andrographolide, an Anti-Inflammatory Multitarget Drug: All Roads Lead to Cellular Metabolism.

Andrographolide is a labdane diterpene and the main active ingredient isolated from the herb Andrographis paniculata. Andrographolide possesses diverse biological effects including anti-inflammatory, antioxidant, and antineoplastic properties. Clinical studies have demonstrated that andrographolide could be useful in therapy for a wide range of diseases such as osteoarthritis, upper respiratory diseases, and multiple sclerosis. Several targets are described for andrographolide, including the interference of transcription factors NF-κB, AP-1, and HIF-1 and signaling pathways such as PI3K/Akt, MAPK, and JAK/STAT. In addition, an increase in the Nrf2 (nuclear factor erythroid 2-related factor 2) signaling pathway also supports its antioxidant and anti-inflammatory properties. However, this scenario could be more complex since recent evidence suggests that andrographolide targets can modulate glucose metabolism. The metabolic effect of andrographolide might be the key to explaining the diverse therapeutic effects described in preclinical and clinical studies. This review discusses some of the most recent evidence about the anti-inflammatory and metabolic effects of andrographolide.

D-Lactate Increases Cytokine Production in Bovine Fibroblast-Like Synoviocytes via MCT1 Uptake and the MAPK, PI3K/Akt, and NFκB Pathways.

Acute ruminal acidosis (ARA) is caused by the excessive intake of highly fermentable carbohydrates, followed by the massive production of D-lactate and the appearance of neutrophilic aseptic polysynovitis. Bovines with ARA develop different lesions, such as ruminitis, polioencephalomalacia (calves), liver abscess and lameness. Lameness in cattle with ARA is closely associated with the presence of laminitis and polysynovitis. However, despite decades of research in bovine lameness as consequence of ruminal acidosis, the aetiology and pathogenesis remain unclear. Fibroblast-like synoviocytes (FLSs) are components of synovial tissue, and under pathological conditions, FLSs increase cytokine production, aggravating inflammatory responses. We hypothesized that D-lactate could induce cytokine production in bovine FLSs. Analysis by qRT-PCR and ELISA revealed that D-lactate, but not L-lactate, increased the expression of IL-6 and IL-8 in a monocarboxylate transporter-1-dependent manner. In addition, we observed that the inhibition of the p38, ERK1/2, PI3K/Akt, and NF-κB pathways reduced the production of IL-8 and IL-6. In conclusion, our results suggest that D-lactate induces an inflammatory response; this study contributes to the literature by revealing a potential key role of D-lactate in the polysynovitis of cattle with ARA.

Mitochondria-derived ATP participates in the formation of neutrophil extracellular traps induced by platelet-activating factor through purinergic signaling in cows.

Neutrophil extracellular trap (NET) formation eliminates/prevents the spread of infectious agents. Platelet activating factor (PAF) is involved in infectious diseases of cattle because it recruits and activates neutrophils. However, its ability to induce NET release and the role of metabolism in this process is not known. We investigated if inhibition of glycolysis, mitochondrial-derived adenosine triphosphate (ATP) synthesis and purinergic signaling though P2X1 purinoceptors interfered with NET formation induced by PAF. We inhibited bovine neutrophils with 2-deoxy-d-glucose, rotenone, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and NF449 to evaluate PAF-mediated NET extrusion. PAF induced mitochondrial hyperpolarization and triggered extracellular ATP release via pannexin-1. Inhibition of mitochondrial metabolism prevented extracellular ATP release. Inhibition of glycolysis, complex-I activity and oxidative phosphorylation prevented NET formation induced by PAF. Inhibition of P2X1 purinergic receptors inhibited mitochondrial hyperpolarization and NET formation. We concluded that PAF-induced NET release is dependent upon glycolysis, mitochondrial ATP synthesis and purinergic signaling.

Oleic and Linoleic Acids Induce the Release of Neutrophil Extracellular Traps via Pannexin 1-Dependent ATP Release and P2X1 Receptor Activation.

Non-esterified fatty acids (NEFAs) such as oleic acid (OA) and linoleic acid (LA) are associated with a higher incidence of infectious diseases such as metritis and mastitis during the bovine peripartum. Fatty acids can induce an increase in the release of ATP, and changes in the expression levels of purinergic receptors in bovine polymorphonuclears (PMN) during peripartum have also been reported. PMN respond to inflammatory processes with production of ROS, release of proteolytic and bactericidal proteins, and formation of neutrophil extracellular traps (NETs). NETs formation is known to require ATP production through glycolysis. Studies have shown that the above-mentioned metabolic changes alter innate immune responses, particularly in PMN. We hypothesized that NEFAs induce the formation of NETs through ATP release by Pannexin 1 and activation of purinergic receptors. In this study, we found that OA and LA induce NET formation and extracellular ATP release. Carbenoxolone, a pannexin-1 (PANX1) inhibitor, reduced OA- and LA-induced ATP release. We also found that P2X1, P2X4, P2X5, P2X7, and PANX1 were expressed at the mRNA level in bovine PMN. Additionally, NEFA-induced NET formation was completely abolished with exposure to NF449, a P2X1 antagonist, and partially inhibited by treatment with etomoxir, an inhibitor of fatty acid oxidation (FAO). Our results suggest that OA and LA induce NET formation and ATP release via PANX1 and activation of P2X1. These new data contribute to explaining the effects of NEFA high concentrations during the transition period of dairy cattle and further understanding of pro-inflammatory effects and outcome of postpartum diseases.

Glycolysis and mitochondrial function regulate the radical oxygen species production induced by platelet-activating factor in bovine polymorphonuclear leukocytes.

Dairy cows undergo metabolic disturbances in the peripartum period, during which infectious inflammatory diseases and detrimental polymorphonuclear leukocytes (PMN) functions, such as radical oxygen species (ROS) production, are observed. Platelet-activating factor (PAF) is a key pro-inflammatory mediator that increases PMN ROS production. To date, the role of glycolysis and mitochondria in PAF-induced ROS production in bovine PMN has not been known. The aim of this study was to assess whether inhibition of glycolysis and disruption of mitochondrial function alter the oxidative response induced by PAF. We isolated PMN from non-pregnant Holstein Friesian heifers and pre-incubated them with 2-deoxy-d-glucose (2-DG; 2 mM, 30 min), carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 5 µM, 5 min), oligomycin (10 µM, 30 min) or rotenone (10 µM, 30 min). Respiratory burst was measured by luminol-chemiluminescence assay, while mitochondrial ROS (mtROS) were evaluated by MitoSOX probe and flow cytometry. Also, we detected the presence of mitochondria by MitoTracker Deep Red FM probe and changes in mitochondrial membrane potential (Δψm) were assessed by JC-1 probe and flow cytometry. We observed that all inhibitors separately were able to reduce PAF-induced ROS production. Presence of mitochondria was detected and PAF increased the Δψm, while CCCP reduced it. 2-DG and rotenone reduced the mtROS production induced by PAF. CCCP did not alter the mtROS and oligomycin administered independently increased mtROS production. We concluded that PAF-induced ROS production is glycolysis- and mitochondria-dependent. Bovine PMN have a functional mitochondrion and PAF induced mtROS via glycolysis and mitochondrial complex-I activity. Our results highlight an important modulation of cellular metabolism in the oxidative response induced by proinflammatory agents, which could contribute to PMN disfunction during peripartum in cattle.

Reproductive and Behavioral Evaluation of a New Immunocastration Dog Vaccine.

Canine immunocastration development has been of interest for many years as a complementary strategy to surgical castration. The purpose of this paper was to verify the effect of a recombinant vaccine for dog immunocastration. Two tests were done, one under controlled conditions and a second under field conditions. Animals were injected with 1 mL of 500 µg GnRXG/Q recombinant protein; 500 µg of low molecular weight chitosan as adjuvant; 1 mL NaCl 0.9% q.s. In the first trial, eight Beagle male dogs between the ages of 1 and 3 comprised the sample, randomly divided into two groups: vaccinated group (n = 7) and control group (n = 2). The second trial had 32 dogs with owners. In the first controlled conditions trial, the vaccine produced specific antibodies that remained until the end of the trial (day 270), inducing reduced testosterone and spermiogram changes in the immunized animals. In a second trial, on the field, specific immunity was induced, which remained high up to day 150. The vaccine also reduced sexual agonistic and marking behaviors. This new vaccine proved to be safe, immunogenic, capable of reducing gonadal functionality, and had a positive effect on inducing reduced sexual, agonistic, and marking behavior of the animals.