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Inflammasomes are multiprotein complexes that trigger processes through caspase-1 activation, leading to the maturation of proinflammatory cytokines, such as IL-1ß and IL-18. The gene encoding the inflammasome stimulatory protein NLRP3 is conserved in canines. Caspase-1/4 homologues have been identified in multiple carnivores, including canines, and caspase-1 activity has been shown in humans. The NLRP3 inflammasome has also been described in some canine inflammatory diseases. Andrographolide, a labdane diterpene, is the principal active ingredient in the herb Andrographis paniculate. The objective of this study was to determine the effect of andrographolide on the gene expression of the components of the NLRP3 inflammasome, proinflammatory cytokines, and IL-1ß secretion in canine peripheral blood mononuclear cells. For this, MTT assays and real-time PCR were employed to assess the cytotoxicity and gene expression. Further, an ELISA test was performed to measure the IL-1ß concentration. The findings reveal that andrographolide significantly reduces the expression of NLRP3, caspase-1/4, IL-1ß, and IL-18. Additionally, it decreases the secretion of IL-1ß and other proinflammatory cytokines, including IL-6, IL-8, and TNF-α. The results show that andrographolide decreases the expression of NLRP3, caspase-1/4, IL-1ß, and IL-18. Andrographolide also reduces proinflammatory cytokines expression, and decreases IL-1ß secretion. This indicates that andrographolide can interfere with the activation and function of the inflammasome, resulting in a decrease in the inflammatory response in canines. Research in this area is still budding, and more studies are necessary to fully understand andrographolide's mechanisms of action and its therapeutic potential in relation to the NLRP3 inflammasome in dogs.

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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.

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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.

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Leptospirosis is an infectious, zoonotic disease of worldwide distribution, the cause of which is infection by pathogenic Leptospira. In Chile, dairy cattle are recognized a significant source in the maintenance and transmission of this infection, which causes economic losses and represents an infection threat to workers in the dairy industry. The infection is underestimated in cattle, due to the lack of clinical, pathognomonic signs, as well as the low efficiency of current diagnostic techniques. In this study, we developed antigen ELISA and dot blot assays, based on polyclonal antibodies, to detect pathogenic Leptospira in the urine samples of dairy cattle. The proposed tests showed an acceptable diagnostic accuracy, based on an analytical sensitivity of 1·104 Leptospira per mL for ELISA, and 3.2·103 for dot blot. These results corresponded with those obtained by qPCR, and the use of urine samples allowed us to propose new diagnostic alternatives for pathogenic Leptospira infection at a low cost, which can provide information on active infection status, which is a key element in control programs both at individual and herd level.

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Our study compared the behavior of prepartum dairy cows that either remained in an outdoor paddock until calving (OP) during winter or were moved to an indoor maternity pen either early (EM) or late (LM) relative to calving. Forty-two multiparous Holstein cows were divided into three treatments (OP, EM, or LM) and monitored from 3 weeks before to 1.5 h after calving. Cows in EM and LM were moved to a maternity pen starting at week three and week one before the expected calving date, respectively. We assessed the cleanliness of the cows at calving, immunoglobulin G concentration in colostrum, and the behavior and vitality of calves across treatments. Cows spent more time lying in EM compared to OP and LM during the weeks -3 and -2 relative to calving, but lying time was increased in LM cows compared with OP cows during the week -1 relative to calving. Prepartum rumination time was lowest in OP cows but not different between EM or LM. Calves from OP cows spent more time lying and had lower vitality after calving than those from LM and EM cows, respectively; calves from EM and LM cows were intermediate for lying and vitality, respectively, but did not differ from either group. The cleanliness was greatest in cows that calved indoors (EM or LM); nevertheless, precalving management did not affect the IgG concentration in colostrum. Our study demonstrates that, in comparison with OP, EM and LM have positive implications for the welfare of the dam and its newborn calf during winter.

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Mesenchymal stem/stromal cells (MSCs) therapy has been a cornerstone of regenerative medicine in humans and animals since their identification in 1968. MSCs can interact and modulate the activity of practically all cellular components of the immune response, either through cell-cell contact or paracrine secretion of soluble mediators, which makes them an attractive alternative to conventional therapies for the treatment of chronic inflammatory and immune-mediated diseases. Many of the mechanisms described as necessary for MSCs to modulate the immune/inflammatory response appear to be dependent on the animal species and source. Although there is evidence demonstrating an in vitro immunomodulatory effect of MSCs, there are disparate results between the beneficial effect of MSCs in preclinical models and their actual use in clinical diseases. This discordance might be due to cells' limited survival or impaired function in the inflammatory environment after transplantation. This limited efficacy may be due to several factors, including the small amount of MSCs inoculated, MSC administration late in the course of the disease, low MSC survival rates in vivo, cryopreservation and thawing effects, and impaired MSC potency/biological activity. Multiple physical and chemical pre-conditioning strategies can enhance the survival rate and potency of MSCs; this paper focuses on hypoxic conditions, with inflammatory cytokines, or with different pattern recognition receptor ligands. These different pre-conditioning strategies can modify MSCs metabolism, gene expression, proliferation, and survivability after transplantation.

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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.

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Extracellular traps (ETs) are webs of DNA, citrullinated histones, anti-microbial peptides, and proteins that were not previously reported in Atlantic salmon (Salmo salar). ETs are mainly released from polymorphonuclear neutrophils (PMN) and are considered a novel PMN-derived effector mechanism against different invasive pathogens. Here, we showed that Atlantic salmon-derived PMN released ETs-like structures in vitro in response to highly pathogenic facultative intracellular rickettsial bacteria Piscirickettsia salmonis. PMN were isolated from pre-smolt Atlantic salmon and stimulated in vitro with oleic acid and P. salmonis. Extracellular DNA was measured using the PicoGreen™ dye, while immunofluorescence image analysis was used to confirm the classical components of salmonid-extruded ETs. Future studies are required to better understand the role of Atlantic salmon-derived ETs orchestrating innate/adaptive immunity and the knowledge on regulation pathways involved in this cell death process. Thus, comprehension of salmonid-derived ETs against P. salmonis might represent novel alternative strategies to improve host innate defense mechanisms of farmed salmon against closely related rickettsial bacteria, as a complement to disease prevention and control strategies.

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Clinical manifestations of leptospirosis are diverse and very similar to other febrile diseases, hence early and accurate detection of subclinical infections is a key element in disease control. We evaluated immunomagnetic separation (IMS) capture technology coupled with a standard quantitative PCR (qPCR) system for the detection of pathogenic Leptospira in urine samples from 803 cows from dairy herds with a history of clinical cases of leptospirosis. The urine samples were first processed in a purification step, then subdivided into 2 subsamples, one that continued to DNA extraction and direct qPCR, and one that was pretreated by IMS before continuing to DNA extraction and qPCR. Overall, 133 of 803 (16.6%) samples were IMS-qPCR positive, whereas only 92 of 803 (11.5%) were positive when using direct qPCR. Statistically significant differences were observed between the mean estimated Leptospira load between the IMS-qPCR and the direct qPCR positive urine samples. The IMS-qPCR technology revealed a larger number of positive results and higher bacterial loads than direct qPCR. This difference is most likely the result of the high antigen-binding capacity and capture efficiency of the IMS system. The use of polyclonal antibodies produced by the inoculation of 3 synthetic peptides, which make up the extracellular regions of the LipL32 protein, provided a high detection capacity to the IMS-qPCR technique, resulting in performance superior to direct qPCR.

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The present work characterized the metabolomic profile of bronchoalveolar lavage fluid (BALF) in healthy horses, experimentally-induced airway inflammation by lipopolysaccharide (LPS) nebulization, and naturally-occurring asthma (n = 3 in each group). All animals underwent clinical and upper airway endoscopic examinations, and bronchoalveolar lavage. BALF supernatant samples were subjected to metabolic analysis based on gas chromatography-mass spectrometry (GC-MS). Overall, 67 peaks were obtained from BALF GC-MS analysis, corresponding to 53 metabolites which were categorized according to chemical class, such as organic acids, fatty acids, nucleosides or their derivatives, amino acids, peptides or their derivatives, carbohydrates, and other compounds. Our results showed that the airway inflammation induction model with LPS produced the same pattern of metabolite changes as in horses with naturally occurring asthma. Metabolic pathway analysis was done by means of Fisher's exact test, for detection of metabolites over-represented in asthma affected-horses and LPS-induced airway inflammation as compared with healthy horses. The most significant altered metabolic pathways were fatty acid biosynthesis, galactose metabolism and citrate cycle. These results suggest that the airway inflammation induction model with LPS is a good study model for asthma-affected horses, due to the similarity of the profile of inflammatory cells (specifically neutrophils) and similar metabolic alterations found in BALF that occur during the inflammatory process of the airways. Further research may increase understanding of metabolomics disturbances and their significance in the pathogenesis of equine asthma.

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Mesenchymal stem/stromal cells (MSCs) are increasingly explored for the treatment of degenerative and inflammatory diseases in human and veterinary medicine. One of the key characteristics of MSCs is that they modulate inflammation mainly through the secretion of soluble mediators. However, despite widespread clinical use, knowledge regarding the effector mechanisms of equine MSCs, and consequently their effectiveness in the treatment of diseases, is still unknown. The objectives of this study were to determine the mechanisms underlying inhibition of lymphocyte proliferation by equine bone marrow-derived MSCs, and to evaluate the effect of pre-conditioning of equine MSCs with different pro-inflammatory cytokines on inhibition of lymphocyte proliferation. We determined that inhibition of lymphocyte proliferation by equine MSCs depends on activity of prostaglandin-endoperoxide synthase 2 and indoleamine 2,3-dioxygenase. Additionally, pre-conditioning of MSCs with TNF-α, IFN-γ or their combination significantly increased the expression of prostaglandin-endoperoxide synthase 2, indoleamine 2,3-dioxygenase, iNOS and IL-6. This upregulation correlated with an increased inhibitory effect of MSCs on lymphocyte proliferation. In conclusion, pre-conditioning of bone marrow-derived MSC increases their inhibitory effect on lymphocyte proliferation in horses.

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Neutrophils participate in innate immunity as the first line of host defence against microorganisms. However, persistent neutrophil activity and delayed apoptosis can be harmful to surrounding tissues; this problem occurs in diverse inflammatory diseases, including asthma-affected horses. Previous studies in horses with acute lung inflammation indicated that treatment with tamoxifen (TX), a selective oestrogen receptor modulator, produces a significant decrease in bronchoalveolar lavage fluid (BALF) neutrophil content. The aim of this study was to investigate the effect of tamoxifen and its metabolites (N-desmethyltamoxifen and endoxifen) on the mitochondrial membrane potential assay by flow cytometry, and the activation of effector caspase-3 through immunoblotting, in peripheral blood neutrophils obtained from healthy horses (n = 5). Results show that tamoxifen, N-desmethyltamoxifen and endoxifen depolarize the mitochondrial membrane and activate caspase-3 in healthy equine neutrophils in vitro. These findings suggest that tamoxifen and its metabolites may activate the intrinsic apoptotic pathway in equine neutrophils. However, more studies are necessary to further explore the signalling pathways of these drugs in the induction of apoptosis.

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BACKGROUND: Tamoxifen (TAM), a selective modulator of estrogen receptors (SERMs) has been recently explored as a therapeutic option for the oral treatment of airway inflammation in the horse. The objective of this work was to establish pharmacokinetic parameters of TAM and its main metabolites in equines, as well as to determine its clinical safety in short-term treatments. RESULTS: We determined TAM and its three main metabolites (4-OH tamoxifen, endoxifen, and N-desmethyl tamoxifen) in plasma after single administration of 0.25 mg/kg in healthy adult horses (n = 12). A maximum concentration of TAM was achieved 3 h after the oral administration (4.65 pg/mL ± 1.69); 4-OH tamoxifen was the metabolite that reached the highest concentration (78 pg/mL ± 70), followed by N-desmethyl tamoxifen (0.43 pg / mL ± 0.48), and finally endoxifen (0.17 pg/mL ± 0.17). All metabolites showed peak concentration 2 h after oral administration of the drug. Oral TAM bioavailability was 13,15% ± 4,18, with a steady state volume of distribution of 7831 ± 2922 (L/kg). Elimination half-life was 15.40 ± 5.80 h, and clearance was 5876 ± 699 (mL/kg/min). Clinical safety of TAM was determined over a 7-day course of treatment (0.25 mg/kg, orally q 24 h, n = 20). No adverse effects were observed through clinical examination, blood hematology, serum biochemistry, ophthalmological and reproductive examinations. Endometrial edema observed in some mares was attributed to normal cyclic activity. CONCLUSIONS: Tamoxifen has moderate oral bioavailability and a large volume of distribution, with three main metabolites in horses. Additionally, oral TAM administration over a 7-day treatment period demonstrated to be clinically safe, without adverse effects on clinical, hematological or serum biochemical parameters. These data could contribute to the continued research into this drug's potential for the treatment of different inflammatory conditions in equine species.

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Neuroinflammation constitutes a fundamental process involved in Parkinson's disease (PD). Microglial cells play a central role in the outcome of neuroinflammation and consequent neurodegeneration of dopaminergic neurons in the substantia nigra. Current evidence indicates that CD4+ T-cells infiltrate the brain in PD, where they play a critical role determining the functional phenotype of microglia, thus regulating the progression of the disease. We previously demonstrated that mice bearing dopamine receptor D3 (DRD3)-deficient CD4+ T-cells are completely refractory to neuroinflammation and consequent neurodegeneration induced by the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In this study we aimed to determine whether DRD3-signalling is altered in peripheral blood CD4+ T-cells obtained from PD patients in comparison to healthy controls (HC). Furthermore, we evaluated the therapeutic potential of targeting DRD3 confined to CD4+ T-cells by inducing the pharmacologic antagonism or the transcriptional inhibition of DRD3-signalling in a mouse model of PD induced by the chronic administration of MPTP and probenecid (MPTPp). In vitro analyses performed in human cells showed that the frequency of peripheral blood Th1 and Th17 cells, two phenotypes favoured by DRD3-signalling, were significantly increased in PD patients. Moreover, naïve CD4+ T-cells obtained from PD patients displayed a significant higher Th1-biased differentiation in comparison with those naïve CD4+ T-cells obtained from HC. Nevertheless, DRD3 expression was selectively reduced in CD4+ T-cells obtained from PD patients. The results obtained from in vivo experiments performed in mice show that the transference of CD4+ T-cells treated ex vivo with the DRD3-selective antagonist PG01037 into MPTPp-mice resulted in a significant reduction of motor impairment, although without significant effect in neurodegeneration. Conversely, the transference of CD4+ T-cells transduced ex vivo with retroviral particles codifying for an shRNA for DRD3 into MPTPp-mice had no effects neither in motor impairment nor in neurodegeneration. Notably, the systemic antagonism of DRD3 significantly reduced both motor impairment and neurodegeneration in MPTPp mice. Our findings show a selective alteration of DRD3-signalling in CD4+ T-cells from PD patients and indicate that the selective DRD3-antagonism in this subset of lymphocytes exerts a therapeutic effect in parkinsonian animals dampening motor impairment.

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Neutrophils play an important role in the exacerbation and maintenance of severe equine asthma; persistent neutrophil activity and delayed apoptosis can be harmful to surrounding tissues. Tamoxifen (TX) is a nonsteroidal estrogen receptor modulator with immunomodulatory effects and induces early apoptosis of blood and bronchoalveolar lavage neutrophils from horses with acute lung inflammation. This study investigated if the in vitro effects of tamoxifen are produced by its action on nuclear (α and ß) and membrane (GPR30) estrogen receptors in healthy equine neutrophils. Results showed that TX inhibits neutrophil respiratory burst induced by opsonized zymosan in a dose-dependent manner. Nuclear (17-ß-Estradiol) and GPR30 cell membrane (G1) estrogen receptor agonists and their antagonists (ICI 182,780 and G15, respectively) do not block or reproduce the effect of TX. Therefore, TX does not inhibit respiratory burst through estrogen receptors. TX (8.5 µM) also increased phosphatidylserine translocation, a marker of early apoptosis, which did not occur with any of the estrogen receptor agonists or antagonists. Thus, tamoxifen generates dose-dependent inhibition of respiratory burst and increased early apoptosis in healthy equine neutrophils, independently of nuclear or membrane estrogen receptors. Further studies are necessary to explore the signaling pathways of tamoxifen-induced ROS inhibition and phosphatidylserine translocation.

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Neutrophils are terminally differentiated innate effector cells at the first line of host defense. Neutrophil migration within tissues is complex and involves several steps, during which these cells must be able to interpret a variety of chemical and physical signals. Exacerbated neutrophil activity can be harmful to surrounding tissues; this is important in a range of diseases, including equine asthma. Tamoxifen (TX) is a non-steroidal estrogen receptor modulator with effects on cell growth and survival. Previous studies showed that TX treatment in horses with induced acute pulmonary inflammation promoted early apoptosis of blood and bronchoalveolar lavage fluid (BALF) neutrophils, reduction of BALF neutrophil content, and improvement in animals' clinical status. Further, TX dampens chemotactic index and respiratory burst production in vitro. The aim of this study was to provide information on the effect of TX on chemokinesis in peripheral blood neutrophils from five healthy horses. Results showed that neutrophils increased migration and travelled distance in response to IL-8; but in the presence of TX, IL-8 did not produce neutrophil migration. This suggests that TX has an inhibitory effect on the kinesis of equine peripheral blood neutrophils stimulated with IL-8. However, further studies are required to fully understand the signaling pathways of TX on neutrophil chemokinesis.

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BACKGROUND: The usefulness of the abbreviated Mini-Mental State Examination included in the Chilean Functional assessment of elderly people (MM-SE-EFAM) to detect Dementia has not been determined. AIM: To assess the performance of the MMSE-EFAM to detect dementia. MATERIAL AND METHODS: We studied a non-probabilistic sample of subjects older than 65 years who had been assessed by the MMSE-EFAM in a Chilean primary care center during a period of 6 months. Patients underwent clinical evaluation by a neurologist blinded to MMSE-EFAM score, to establish the diagnosis of dementia using DSM-IV-TR criteria. Besides, the full Mini-Mental State Examination (MMSE) was applied. RESULTS: The clinical diagnosis of Dementia was established in 13 of the 54 peoples evaluated. MMSE-EFAM had a sensitivity of 30.8% (95% confidence intervals (CI); 9-61.4) and a specificity of 90.2% (95% CI; 76.9%-97.3%), while MMSE had a sensitivity of 84.6% (95% CI; 54.6-98.1) and a specificity of 58.5% (95% CI; 42.1-73.7). In a receiver operating characteristic (ROC) curve analysis, the areas under the curve (AUC) were 0.77 (95% CI; 0.61-0.93) and 0.82 (95% CI; 0.70-0.95) for MMSE-EFAM and MMSE, respectively. Socio-demographic variables did not influence test performance in both cases. CONCLUSIONS: MMSE-EFAM has a low sensitivity to detect patients with Dementia and it is not an effective screening tool. These results are in agreement with the evidence and international guidelines that do not support the use of cognitive screening tools to detect dementia in the older general population.

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Background: The usefulness of the abbreviated Mini-Mental State Examination included in the Chilean Functional assessment of elderly people (MM-SE-EFAM) to detect Dementia has not been determined. Aim: To assess the performance of the MMSE-EFAM to detect dementia. Material and Methods: We studied a non-probabilistic sample of subjects older than 65 years who had been assessed by the MMSE-EFAM in a Chilean primary care center during a period of 6 months. Patients underwent clinical evaluation by a neurologist blinded to MMSE-EFAM score, to establish the diagnosis of dementia using DSM-IV-TR criteria. Besides, the full Mini-Mental State Examination (MMSE) was applied. Results: The clinical diagnosis of Dementia was established in 13 of the 54 peoples evaluated. MMSE-EFAM had a sensitivity of 30.8% (95% confidence intervals (CI); 9-61.4) and a specificity of 90.2% (95% CI; 76.9%-97.3%), while MMSE had a sensitivity of 84.6% (95% CI; 54.6-98.1) and a specificity of 58.5% (95% CI; 42.1-73.7). In a receiver operating characteristic (ROC) curve analysis, the areas under the curve (AUC) were 0.77 (95% CI; 0.61-0.93) and 0.82 (95% CI; 0.70-0.95) for MMSE-EFAM and MMSE, respectively. Socio-demographic variables did not influence test performance in both cases. Conclusions: MMSE-EFAM has a low sensitivity to detect patients with Dementia and it is not an effective screening tool. These results are in agreement with the evidence and international guidelines that do not support the use of cognitive screening tools to detect dementia in the older general population.

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