Detection and Localization of IL-8 and CXCR1 in Rainbow Trout Larvae in Response to Pseudomonas aeruginosa Lipopolysaccharide.

The salmonid industry faces challenges due to the susceptibility of fish to opportunistic pathogens, particularly in early developmental stages. Understanding the immunological capacity during these stages is crucial for developing effective disease control strategies. IL-8R, a member of the G-protein-coupled receptor family, acts as a receptor for Interleukin 8 (IL-8). The binding of IL-8 to IL-8R plays a major role in the pathophysiology of a wide spectrum of inflammatory conditions. This study focused on the immune response capacity of rainbow trout (Oncorhynchus mykiss) larvae by analyzing IL-8/CXCR1 response to lipopolysaccharide (LPS) from Pseudomonas aeruginosa. Previous research demonstrated that LPS from P. aeruginosa acts as a potent immunostimulant in teleost, enhancing pro-inflammatory cytokines. The methodology included in silico analysis and the synthesis and characterization of an omCXCR1-derived epitope peptide, which was used to produce omCXCR1-specific anti98 serum in mice. The research revealed that rainbow trout larvae 19 days post-hatching (dph) exhibited pronounced immune responses post-stimulation with 1 µg/mL of LPS. This was evidenced by the upregulated protein expression of IL-8 and omCXCR1 in trout larvae 2 and 8 h after LPS challenge, as analyzed by ELISA and immunohistochemistry. Furthermore, fluorescence microscopy successfully revealed the colocalization of IL-8 and its receptor in cells from mucosal tissues after LPS challenge in larvae 19 dph. These findings underscore the efficacy of LPS immersion as a method to activate the innate immune system in trout larvae. Furthermore, we propose IL-8 and its receptor as molecular markers for evaluating immunostimulation in the early developmental stages of salmonids.

Repeated Administration of a Full-Spectrum Cannabidiol Product, Not a Cannabidiol Isolate, Reverses the Lipopolysaccharide-Induced Depressive-Like Behavior and Hypolocomotion in a Rat Model of Low-Grade Subchronic Inflammation.

Background: Mounting evidence suggests that the phytocannabinoid cannabidiol (CBD) holds promise as an antidepressant agent in conditions underlined by inflammation. Full-spectrum CBD extracts might provide greater behavioral efficacy than CBD-only isolates and might require lower doses to achieve the same outcomes due to the presence of other cannabinoids, terpenes, and flavonoids. However, investigations in this area remain limited. Methods: We evaluated the behavioral response to the administration for 7 days of 15 and 30 mg/kg of a CBD isolate and a full-spectrum CBD product in a rat model of subchronic lipopolysaccharide (LPS, 0.5 mg/kg/day/7 days, intraperitoneal)-induced depressive-like and sickness behavior. The forced swim test was used to assess depressive-like behavior, the open field test (OFT) to assess locomotion, and the elevated plus maze to assess anxiety-like behavior. Results: The full-spectrum CBD extract at both doses, but not the CBD isolate, reversed the LPS-induced depressive-like behavior in the forced swim test. Moreover, the full-spectrum CBD extract at the higher dose but not the CBD isolate restored the subchronic LPS-induced hypolocomotion in the OFT. Repeated administration of both formulations elicited an anxiogenic-like trend in the elevated plus maze. Conclusion: Full-spectrum CBD products might have greater therapeutic efficacy in resolving inflammation-induced depressive and sickness behavior compared to a CBD-only isolate.

Pereskia sacharosa Griseb. (Cactaceae) Prevents Lipopolysaccharide-Induced Neuroinflammation in Rodents via Down-Regulating TLR4/CD14 Pathway and GABAA γ2 Activity.

Pereskia sacharosa Griseb. is a plant used in traditional herbal medicine to treat inflammation. We analyzed the phenolic content of P. sacharosa leaves (EEPs) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and investigated the anti-inflammatory properties of EEPs and its flavonoid fraction (F10) in animal models subjected to acute neuroinflammation induced by bacterial lipopolysaccharide (LPS). Coronal brain sections of C57BL/6JN male mice or Wistar male rats administered with EEPs or F10 before LPS were subjected to in situ hybridization to determine c-fos and CD14 mRNA levels in the hypothalamus or GABAA γ2 mRNA levels in the hippocampus. Theta oscillations were recorded every 6 h in the hippocampus of Wistar rats. In total, five flavonoids and eight phenolic acids were identified and quantified in P. sacharosa leaves. Either EEPs or F10 crossed the blood-brain barrier (BBB) into the brain and reduced the mRNA expression of c-fos, CD14, and GABAA γ2. A decrease in theta oscillation was observed in the hippocampus of the LPS group, while the F10 + LPS group overrode the LPS effect on theta activity. We conclude that the bioactive compounds of P. sacharosa reduce the central response to inflammation, allowing the early return of ambulatory activity and well-being of the animal.

Silybin restores glucose uptake after tumour necrosis factor-alpha and lipopolysaccharide stimulation in 3T3-L1 adipocytes.

Obesity is associated with a low-grade chronic inflammatory process characterized by higher circulating TNFα levels, thus contributing to insulin resistance. This study evaluated the effect of silybin, the main bioactive component of silymarin, which has anti-inflammatory properties, on TNFα levels and its impact on glucose uptake in the adipocyte cell line 3T3-L1 challenged with two different inflammatory stimuli, TNFα or lipopolysaccharide (LPS). Silybin's pre-treatment effect was evaluated in adipocytes pre-incubated with silybin (30 or 80 µM) before challenging with the inflammatory stimuli (TNFα or LPS). For the post-treatment effect, the adipocytes were first challenged with the inflammatory stimuli and then post-treated with silybin. After treatments, TNFα production, glucose uptake, and GLUT4 protein expression were determined. Both inflammatory stimuli increased TNFα secretion, diminished GLUT4 expression, and significantly decreased glucose uptake. Silybin 30 µM only reduced TNFα secretion after the LPS challenge. Silybin 80 µM as post-treatment or pre-treatment decreased TNFα levels, improving glucose uptake. However, glucose uptake enhancement induced by silybin did not depend on GLUT4 protein expression. These results show that silybin importantly reduced TNFα levels and upregulates glucose uptake, independently of GLUT4 protein expression.

Omega-3 Attenuates Disrupted Neurotransmission and Partially Protects Metabolic Dysfunction Caused by Obesity in Wistar Rats.

Omega-3 (n3) is a polyunsaturated fatty acid well known for its anti-inflammatory and neuroprotective properties. Obesity is linked to chronic inflammation that disrupts metabolism, the intestine physiology and the central nervous system functioning. This study aims to determine if n3 supplementation can interfere with the effects of obesity on the mitochondrial activity, intestinal barrier, and neurotransmitter levels in the brain of Wistar rats that received cafeteria diet (CAF). We examined adipose tissue, skeletal muscle, plasma, intestine, and the cerebral cortex of four groups: CT (control diet), CTn3 (control diet with n3 supplementation), CAF, and CAFn3 (CAF and n3). Diets were offered for 13 weeks, with n3 supplementation in the final 5 weeks. Adipose tissue Electron Transport Chain complexes I, II, and III showed higher activity in CAF groups, as did complexes III and IV in skeletal muscle. Acetate levels in plasma were reduced in CAF groups, and Lipopolysaccharide (LPS) was higher in the CAF group but reduced in CAFn3 group. Claudin-5 in the intestine was lower in CAF groups, with no n3 supplementation effect. In the cerebral cortex, dopamine levels were decreased with CAF, which was reversed by n3. DOPAC, a dopamine metabolite, also showed a supplementation effect, and HVA, a diet effect. Serotonin levels increased in the CAF group that received supplementation. Therefore, we demonstrate disturbances in mitochondria, plasma, intestine and brain of rats submitted to CAF and the potential benefit of n3 supplementation in endotoxemia and neurotransmitter levels.

Effects of high temperature and LPS injections on the hemocytes of the crab Neohelice granulata.

Temperature fluctuations, particularly elevated temperatures, can significantly affect immune responses. These fluctuations can influence the immune system and alter its response to infection signals, such as lipopolysaccharide (LPS). Therefore, this study was designed to investigate how high temperatures and LPS injections collectively influence the immune system of the crab Neohelice granulata. Two groups were exposed to 20 °C (control) or 33 °C for four days. Subsequently, half were injected with 10 µL of physiological crustacean (PS), while the rest received 10 µL of LPS [0.1 mg.kg-1]. After 30 min, the hemolymph samples were collected. Hemocytes were then isolated and assessed for various parameters using flow cytometry, including cell integrity, DNA fragmentation, total hemocyte count (THC), differential hemocyte count (DHC), reactive oxygen species (ROS) level, lipid peroxidation (LPO), and phagocytosis. Results showed lower cell viability at 20 °C, with more DNA damage in the same LPS-injected animals. There was no significant difference in THC, but DHC indicated a decrease in hyaline cells (HC) at 20 °C following LPS administration. In granular cells (GC), an increase was observed after both PS and LPS were injected at the same temperature. In semi-granular cells (SGC), there was a decrease at 20 °C with the injection of LPS, while at a temperature of 33 °C, the SGC there was a decrease only in SGC injected with LPS. Crabs injected with PS and LPS at 20 °C exhibited higher levels of ROS in GC and SGC, while at 33 °C, the increase was observed only in GC and SGC cells injected with LPS. A significant increase in LPO was observed only in SGC cells injected with PS and LPS at 20 °C and 33 °C. Phagocytosis decreased in animals at 20 °C with both injections and exposed to 33 °C only in those injected with LPS. These results suggest that elevated temperatures induce changes in immune system parameters and attenuate the immune responses triggered by LPS.

Is Methylglyoxal a Potential Biomarker for the Warburg Effect Induced by the Lipopolysaccharide Neuroinflammation Model?

Methylglyoxal (MG) is considered a classical biomarker of diabetes mellitus and its comorbidities. However, a role for this compound in exacerbated immune responses, such as septicemia, is being increasingly observed and requires clarification, particularly in the context of neuroinflammatory responses. Herein, we used two different approaches (in vivo and acute hippocampal slice models) to investigate MG as a biomarker of neuroinflammation and the neuroimmunometabolic shift to glycolysis in lipopolysaccharide (LPS) inflammation models. Our data reinforce the hypothesis that LPS-induced neuroinflammation stimulates the cerebral innate immune response by increasing IL-1ß, a classical pro-inflammatory cytokine, and the astrocyte reactive response, via elevating S100B secretion and GFAP levels. Acute neuroinflammation promotes an early neuroimmunometabolic shift to glycolysis by elevating glucose uptake, lactate release, PFK1, and PK activities. We observed high serum and cerebral MG levels, in association with a reduction in glyoxalase 1 detoxification activity, and a close correlation between serum and hippocampus MG levels with the systemic and neuroinflammatory responses to LPS. Findings strongly suggest a role for MG in immune responses.

Detection of plasma anti-lipopolysaccharide (LPS)antibodies against enterohemorrhagic Escherichia coli(EHEC) in asymptomatic kindergarten teachers fromBuenos Aires province

Resumen En Argentina, el síndrome urémico hemolítico causado por Escherichia coli enterohemorrágica (EHEC) tiene la más alta incidencia del mundo. Las infecciones por EHEC tienen un comportamiento endemoepidémico y causan del 20 al 30% de los síndromes de diarrea sanguinolenta en niños menores de 5 años. En el período 2016-2020, se notificaron 272 nuevos casos por año al Sistema de Vigilancia de Salud Nacional. Múltiples factores son responsables de la alta incidencia de SUH en Argentina, incluyendo la transmisión persona-persona. Con el objetivo de detectar posibles portadores asintomáticos de EHEC, realizamos un estudio preliminar de la frecuencia de anticuerpos antilipopolisacáridos contra los serotipos de EHEC más prevalentes en Argentina. El estudio se realizó con muestras de plasma obtenidas de 61 maestras y maestros de jardines de infantes de 26 instituciones del distrito de José C. Paz, localizado en el área suburbana de la provincia de Buenos Aires, Argentina. El 51% de las muestras presentaron anticuerpos contra los serotipos de lipopolisacáridos O157, O145, O121 y O103; el 6,4% de las muestras positivas tuvieron el isotipo IgM (n=2), el 61,3% el isotipo IgG (n=19) y el 32,3% los isotipos IgM e IgG (n=10). Dado que los anticuerpos antilipopolisacáridos presentan usualmente una duración corta, la detección de IgM específica podría indicar una infección reciente. Además, el alto porcentaje de muestras positivas hallado podría indicar una exposición frecuente a las cepas de EHEC en la cohorte estudiada. Asimismo, la gran población de adultos portadores asintomáticos de estas cepas patógenas podría contribuir al comportamiento endémico, a través de la transmisión persona-persona. El perfeccionamiento de programas educacionales continuos en jardines de infantes podría constituir una medida importante para reducir los casos de síndrome urémico hemolítico, no solo en Argentina, sino también en el mundo.

Abstract In Argentina, hemolytic uremic syndrome (HUS) caused by EHEC has the highest incidence in the world. EHEC infection has an endemo-epidemic behavior, causing 20-30% of acute bloody diarrhea syndrome in children under 5 years old. In the period 2016-2020, 272 new cases per year were notified to the National Health Surveillance System. Multiple factors are responsible for HUS incidence in Argentina including person-to-person transmission. In order to detect possible EHEC carriers, we carried out a preliminary study of the frequency of kindergarten teachers with anti-LPS antibodies against the most prevalent EHEC serotypes in Argentina. We analyzed 61 kindergarten teachers from 26 institutions from José C. Paz district, located in the suburban area of Buenos Aires province, Argentina. Fifty-one percent of the plasma samples had antibodies against O157, O145, O121 and O103 LPS: 6.4% of the positive samples had IgM isotype (n=2), 61.3% IgG isotype (n=19) and 32.3% IgM and IgG (n=10). Given that antibodies against LPS antigens are usually short-lived specific IgM detection may indicate a recent infection. In addition, the high percentage of positive samples may indicate a frequent exposure to EHEC strains in the cohort studied, as well as the existence of a large non-symptomatic population of adults carrying pathogenic strains that could contribute to the endemic behavior through person-to-person transmission. The improvement of continuous educational programs in kindergarten institutions could be a mandatory measure to reduce HUS cases not only in Argentina but also globally.

Regional modulation of toll-like receptor signaling pathway genes in acute epididymitis in mice.

BACKGROUND: Region-specific immune environments in the epididymis influence the immune responses to uropathogenic Escherichia coli (UPEC) infection, a relevant cause of epididymitis in men. Toll-like receptors (TLRs) are essential to orchestrate immune responses against bacterial infections. The epididymis displays region-specific inflammatory responses to bacterial-derived TLR agonists, such as lipopolysaccharide (LPS; TLR4 agonist) and lipoteichoic acid (LTA; TLR2/TLR6 agonist), suggesting that TLR-associated signaling pathways could influence the magnitude of inflammatory responses in epididymitis. OBJECTIVES: To investigate the expression and regulation of key genes associated with TLR4 and TLR2/TLR6 signaling pathways during epididymitis induced by UPEC, LPS, and LTA in mice. MATERIAL AND METHODS: Epididymitis was induced in mice using UPEC, ultrapure LPS, or LTA, injected into the interstitial space of the initial segment or the lumen of the vas deferens close to the cauda epididymidis. Samples were harvested after 1, 5, and 10 days for UPEC-treated animals and 6 and 24 h for LPS-/LTA-treated animals. Ex vivo epididymitis was induced by incubating epididymal regions from naive mice with LPS or LTA. RT-qPCR and Western blot assays were conducted. RESULTS: UPEC infection up-regulated Tlr2, Tlr4, and Tlr6 transcripts and their associated signaling molecules Cd14, Ticam1, and Traf6 in the cauda epididymidis but not in the initial segment. In these epididymal regions, LPS and LTA differentially modulated Tlr2, Tlr4, Tlr6, Cd14, Myd88, Ticam1, Traf3, and Traf6 expression levels. NFKB and AP1 activation was required for LPS- and LTA-induced up-regulation of TLR-associated signaling transcripts in the cauda epididymidis and initial segment, respectively. CONCLUSION: The dynamic modulation of TLR4 and TLR2/TLR6 signaling pathways gene expression during epididymitis indicates bacterial-derived antigens elicit an increased tissue sensitivity to combat microbial infection in a spatial manner in the epididymis. Differential activation of TLR-associated signaling pathways may contribute to fine-tuning inflammatory responses along the epididymis.

Calorie restriction protects against acute systemic LPS-induced inflammation.

Caloric restriction (CR) has been proposed as a nutritional strategy to combat chronic diseases, including neurodegenerative diseases, as well as to delay aging. However, despite the benefits of CR, questions remain about its underlying mechanisms and cellular and molecular targets.Objective: As inflammatory processes are the basis or accompany chronic diseases and aging, we investigated the protective role of CR in the event of an acute inflammatory stimulus.Methods: Peripheral inflammatory and metabolic parameters were evaluated in Wistar rats following CR and/or acute lipopolysaccharide (LPS) administration, as well as glial changes (microglia and astrocytes), in two regions of the brain (hippocampus and hypothalamus) involved in the inflammatory response. We used a protocol of 30% CR, for 4 or 8 weeks. Serum and brain parameters were analyzed by biochemical or immunological assays.Results: Benefits of CR were observed during the inflammatory challenge, where the partial reduction of serum interleukin-6, mediated by CR, attenuated the systemic response. In the central nervous system (CNS), specifically in the hippocampus, CR attenuated the response to the LPS, as evaluated by tumor necrosis factor alpha (TNFα) levels. Furthermore, in the hippocampus, CR increased the glutathione (GSH) levels, resulting in a better antioxidant response.Discussion: This study contributes to the understanding of the effects of CR, particularly in the CNS, and expands knowledge about glial cells, emphasizing their importance in neuroprotection strategies.

Lactobacilli Probiotics Modulate Antibacterial Response Gene Transcription of Dendritic Cells Challenged with LPS.

Probiotics are beneficial bacteria that may modulate the immune response by altering the maturation and function of antigen-presenting cells, such as dendritic cells. This study aimed to evaluate the antibacterial gene expression of dendritic cells challenged with LPS and probiotics. Immature dendritic cells were obtained from human CD14+ monocytes and challenged with E. coli LPS and probiotics Lacticaseibacillus rhamnosus (LR-32) and Lactobacillus acidophilus (LA-5) at a ratio DC:bacteria of 1:10. The analysis of gene expression was performed by RT-qPCR using the Kit RT2 human antibacterial response. In the supernatant, the cytokines secretion was determined by ELISA. Tukey post-ANOVA with p at 5% was used for statistical analysis. LPS showed the higher upregulation of 29 genes compared with the groups where probiotics were added to LPS, including genes related to an inflammatory response like BIRC3, CASP1, CCL5, CXCL1, IL12B, IL18, MYD88, NLRP3, RIPK1, and TIRAP. Similarly, LPS increased the transcription of genes enrolled with apoptosis such as CARD6, CASP1, IRF5, MAP2K1, MAP2K4, MAPK1, MYD88, NLRP3, RIPK2, TNF, TNFRSF1A, and XIAP when compared to probiotics groups (p < 0.05). Although probiotics decrease several genes upregulated by LPS, the transcription of encoded cytokines IL12A, IL12B, IL1B, IL6, CXCL8, and TNF genes was maintained upregulated by probiotics, except for IL18, which was downregulated by LA-5. LA-5 led to a higher transcription of IL1B, IL6, and CXCL-8 which was followed by the secretion of these proteins by ELISA. The results suggest that probiotics attenuate the transcription of inflammatory and immune response genes caused by LPS.

Detection of plasma anti-lipopolysaccharide (LPS) antibodies against enterohemorrhagic Escherichia coli (EHEC) in asymptomatic kindergarten teachers from Buenos Aires province.

In Argentina, hemolytic uremic syndrome (HUS) caused by EHEC has the highest incidence in the world. EHEC infection has an endemo-epidemic behavior, causing 20-30% of acute bloody diarrhea syndrome in children under 5 years old. In the period 2016-2020, 272 new cases per year were notified to the National Health Surveillance System. Multiple factors are responsible for HUS incidence in Argentina including person-to-person transmission. In order to detect possible EHEC carriers, we carried out a preliminary study of the frequency of kindergarten teachers with anti-LPS antibodies against the most prevalent EHEC serotypes in Argentina. We analyzed 61 kindergarten teachers from 26 institutions from José C. Paz district, located in the suburban area of Buenos Aires province, Argentina. Fifty-one percent of the plasma samples had antibodies against O157, O145, O121 and O103 LPS: 6.4% of the positive samples had IgM isotype (n=2), 61.3% IgG isotype (n=19) and 32.3% IgM and IgG (n=10). Given that antibodies against LPS antigens are usually short-lived specific IgM detection may indicate a recent infection. In addition, the high percentage of positive samples may indicate a frequent exposure to EHEC strains in the cohort studied, as well as the existence of a large non-symptomatic population of adults carrying pathogenic strains that could contribute to the endemic behavior through person-to-person transmission. The improvement of continuous educational programs in kindergarten institutions could be a mandatory measure to reduce HUS cases not only in Argentina but also globally.

Assessing the effects of oxytocin in changes of core body temperature during LPS-induced endotoxemia: A novel approach using Extended Poincaré Plot Analysis.

Oxytocin has shown cardioprotective effects during inflammation and may modify the core body temperature changes in LPS-induced endotoxemia. Notably, the time series analysis of core body temperature fluctuations may indicate thermoregulation alterations. This study aims to assess the effects of oxytocin on changes in the core body temperature by analyzing the fluctuations of the temperature time series of endotoxemic rats. Twelve hours of continuous core body temperature fluctuations time series were obtained from adult male Dark Agouti rats implanted with a telemetric transmitter under the following treatment: lipopolysaccharide (LPS); oxytocin (O); lipopolysaccharide + oxytocin (LPS + O), and vehicle or control (C). The temperature fluctuations time series were analyzed using the Extended Poincaré Plot Analysis (EPPA), a novel approach for measuring nonlinear features, to compute the autocorrelation by Pearson's correlation coefficient r, the standard deviation perpendicular to the line of identity (SD1), and the standard deviation parallel to the line of identity (SD2). The autocorrelation of the temperature fluctuations assessed by Pearson's coefficient was significantly higher in the LPS group compared to control rats (C). Likewise, the co-administration of oxytocin during endotoxemia (LPS + O) significantly reduced the autocorrelation and increased the short-term variability (SD1) of temperature fluctuations compared to those recorded with a single dose of LPS. Thus, we concluded that oxytocin may introduce thermoregulatory changes under LPS-induced endotoxemia. The EPPA is a simple and powerful approach to assess physiological variability that can provide valuable insights into changes in thermoregulation.

Effects of Pasturella Multocida B:2 and its immunogens (LPS and OMP) on reproductive hormones in Nili-Ravi Buffaloes

Abstract Livestock is a fundamental part of the agriculture industry in Pakistan and contributes more than 11.53% to GDP. Among livestock species, the buffaloes are regarded as the black gold of Pakistan. Being the highest milk producers globally, Nili-Ravi buffaloes are the most famous ones. Buffaloes are affected by many endemic diseases, and "Hemorrhagic septicemia" (HS) is one of them. This study was designed to ascertain the effects of experimental exposure ofP. multocida B:2 (oral) and its immunogens, i.e., LPS (oral and intravenous) and OMP (oral and subcutaneous) on reproductive hormonal profiles in Nili-Ravi buffaloes. Repeated serum samples were collected from the jugular vein of experimental animals for 21 days (0, 02, 04, 08, 12, 16, 20, 24, 36, 48, 72, 120, 168, 216, 264, 360, 456 and 504 hours). Hormonal assays to determine the serum concentrations of Gonadotropin-releasing hormone (GnRH), Follicle-stimulating hormone (FSH), Luteinizing hormone (LH), Estrogen (E2) and progesterone (P4) were performed using (MyBioSource) commercial Elisa kits. The hormonal profile of all treatment groups of the buffalo heifers exhibited significant (P 0.05) variations as compared to the control group (G-1). These results indicate suppression in Nili-Ravi buffaloes' reproductive hormonal profile on exposure to P. multocida B:2 and its immunogens. This influence warrants that exposure to H.S may be a possible reason for delayed puberty and poor reproduction performance in Nili-Ravi buffaloes.

Resumo A pecuária é uma parte fundamental da indústria agrícola no Paquistão e contribui com 11,53% do PIB nacional. Entre as espécies de gado, os búfalos são considerados o ouro negro do Paquistão. Sendo os maiores produtores de leite em todo o mundo, os búfalos Nili-Ravi são os mais famosos. Os búfalos são afetados por muitas doenças endêmicas, entre as quais a septicemia hemorrágica (SH). Este estudo busca verificar os efeitos da exposição experimental de P. multocida B:2 (oral) e seus imunógenos, ou seja, LPS (oral e intravenoso) e OMP (oral e subcutâneo), nos perfis hormonais reprodutivos em búfalos Nili-Ravi. Amostras de soro repetidas foram coletadas da veia jugular de animais experimentais por 21 dias (0, 2, 4, 8, 12, 16, 20, 24, 36, 48, 72, 120, 168, 216, 264, 360, 456 e 504 horas). Os ensaios hormonais para determinar as concentrações séricas do hormônio liberador de gonadotrofina (GnRH), hormônio foliculoestimulante (FSH), hormônio luteinizante (LH), estrogênio (E2) e progesterona (P4) foram realizados usando kits comerciais Elisa (MyBioSource). O perfil hormonal de todos os grupos de tratamento das novilhas bubalinas apresentou variações significativas (P 0,05) em relação ao grupo controle (G-1). Esses resultados indicam supressão no perfil hormonal reprodutivo de búfalos Nili-Ravi na exposição a P. multocida B:2 e seus imunógenos. Essa influência garante que a exposição à SH possa ser uma possível razão para o atraso da puberdade e o baixo desempenho reprodutivo em búfalos Nili-Ravi.

Effects of Pasturella Multocida B: 2 and its immunogens (LPS and OMP) on reproductive hormones in Nili-Ravi Buffaloes / Efeitos de Pasteurella multocida B: 2 e seus imunógenos (LPS e OMP) em hormônios reprodutivos em búfalos Nili-Ravi

Abstract Livestock is a fundamental part of the agriculture industry in Pakistan and contributes more than 11.53% to GDP. Among livestock species, the buffaloes are regarded as the black gold of Pakistan. Being the highest milk producers globally, Nili-Ravi buffaloes are the most famous ones. Buffaloes are affected by many endemic diseases, and "Hemorrhagic septicemia" (HS) is one of them. This study was designed to ascertain the effects of experimental exposure ofP. multocida B:2 (oral) and its immunogens, i.e., LPS (oral and intravenous) and OMP (oral and subcutaneous) on reproductive hormonal profiles in Nili-Ravi buffaloes. Repeated serum samples were collected from the jugular vein of experimental animals for 21 days (0, 02, 04, 08, 12, 16, 20, 24, 36, 48, 72, 120, 168, 216, 264, 360, 456 and 504 hours). Hormonal assays to determine the serum concentrations of Gonadotropin-releasing hormone (GnRH), Follicle-stimulating hormone (FSH), Luteinizing hormone (LH), Estrogen (E2) and progesterone (P4) were performed using (MyBioSource) commercial Elisa kits. The hormonal profile of all treatment groups of the buffalo heifers exhibited significant (P<0.05) variations as compared to the control group (G-1). These results indicate suppression in Nili-Ravi buffaloes' reproductive hormonal profile on exposure to P. multocida B:2 and its immunogens. This influence warrants that exposure to H.S may be a possible reason for delayed puberty and poor reproduction performance in Nili-Ravi buffaloes.

Resumo A pecuária é uma parte fundamental da indústria agrícola no Paquistão e contribui com 11,53% do PIB nacional. Entre as espécies de gado, os búfalos são considerados o ouro negro do Paquistão. Sendo os maiores produtores de leite em todo o mundo, os búfalos Nili-Ravi são os mais famosos. Os búfalos são afetados por muitas doenças endêmicas, entre as quais a "septicemia hemorrágica" (SH). Este estudo busca verificar os efeitos da exposição experimental de P. multocida B:2 (oral) e seus imunógenos, ou seja, LPS (oral e intravenoso) e OMP (oral e subcutâneo), nos perfis hormonais reprodutivos em búfalos Nili-Ravi. Amostras de soro repetidas foram coletadas da veia jugular de animais experimentais por 21 dias (0, 2, 4, 8, 12, 16, 20, 24, 36, 48, 72, 120, 168, 216, 264, 360, 456 e 504 horas). Os ensaios hormonais para determinar as concentrações séricas do hormônio liberador de gonadotrofina (GnRH), hormônio foliculoestimulante (FSH), hormônio luteinizante (LH), estrogênio (E2) e progesterona (P4) foram realizados usando kits comerciais Elisa (MyBioSource). O perfil hormonal de todos os grupos de tratamento das novilhas bubalinas apresentou variações significativas (P < 0,05) em relação ao grupo controle (G-1). Esses resultados indicam supressão no perfil hormonal reprodutivo de búfalos Nili-Ravi na exposição a P. multocida B:2 e seus imunógenos. Essa influência garante que a exposição à SH possa ser uma possível razão para o atraso da puberdade e o baixo desempenho reprodutivo em búfalos Nili-Ravi.

Chromosome-Mediated Colistin Resistance in Clinical Isolates of Klebsiella pneumoniae and Escherichia coli: Mutation Analysis in the Light of Genetic Background.

Purpose: Colistin resistance mechanisms involving mutations in chromosomal genes associated with LPS modification are not completely understood. Mutations in genes coding for the MgrB regulator frequently account for colistin resistance in Klebsiella pneumoniae, whereas mutations in genes coding for PhoPQ and PmrAB are frequent in E. coli. Our aim was to perform a genetic analysis of chromosomal mutations in colistin-resistant (MIC ≥4 µg/mL) clinical isolates of K. pneumoniae (n = 8) and E. coli (n = 7) of different STs. Methods: Isolates were obtained in a 3-year period in a university hospital in Santiago, Chile. Susceptibility to colistin, aminoglycosides, cephalosporins, carbapenems and ciprofloxacin was determined through broth microdilution. Whole genome sequencing was performed for all isolates and chromosomal gene sequences were compared with sequences of colistin-susceptible isolates of the same sequence types. Results: None of the isolates carried mcr genes. Most of the isolates were susceptible to all the antibiotics analyzed. E. coli isolates were ST69, ST127, ST59, ST131 and ST14, and K. pneumoniae isolates were ST454, ST45, ST6293, ST380 and ST25. All the isolates had mutations in chromosomal genes analyzed. K. pneumoniae had mutations mainly in mgrB gene, whereas E. coli had mutations in pmrA, pmrB and pmrE genes. Most of the amino acid changes in LPS-modifying enzymes of colistin-resistant isolates were found in colistin-susceptible isolates of the same and/or different ST. Eleven of them were found only in colistin-resistant isolates. Conclusion: Colistin resistance mechanisms depend on genetic background, and are due to chromosomal mutations, which implies a lower risk of transmission than plasmid-mediated genes. Colistin resistance is not associated with multidrug-resistance, nor to high-risk sequence types.

Curcumin modulates astrocyte function under basal and inflammatory conditions.

Curcumin is a pleiotropic molecule with well-known anti-inflammatory effects. This molecule has attracted attention due to its capacity to pass the blood-brain-barrier and modulate central nervous system (CNS) cells, such as astrocytes. Astrocytes are the most numerous CNS cells, and play a pivotal role in inflammatory damage, a common feature in neurodegenerative diseases such as Alzheimer's Disease. Although the actions of curcumin have been studied extensively in peripheral cells, few studies have investigated the effect of curcumin on astrocytes under basal and inflammatory conditions. The aim of this study was to characterize the effect of curcumin on astrocytic function (glutamatergic metabolism, GFAP and S100B), and investigate a possible synergic effect with another molecule, piperine. For this purpose, we used primary cultured astrocytes; our results showed that curcumin increases GSH and GFAP content, but decreases S100B secretion under basal conditions. Under inflammatory conditions, provoked by lipopolysaccharide (LPS), curcumin and piperine reversed the LPS-induced secretion of TNF-α, and piperine reverted the LPS-induced upregulation of GFAP content. Interestingly, curcumin decreases S100B secretion even more than LPS. These results highlight important context-dependent effects of curcumin and piperine on astrocytes. Although we did not observe synergic effects of co-treatment with curcumin and piperine, their effects were complementary, as piperine modulated GFAP content under inflammatory conditions, and curcumin modulated S100B secretion. Both curcumin and piperine had important anti-inflammatory actions in astrocytes. We herein provide new insights into the actions of curcumin in the CNS that may aid in the search for new molecular targets and possible treatments for neurological diseases.

Progression of reactive gliosis and astroglial phenotypic changes following stab wound-induced traumatic brain injury in mice.

Astrocytes are the main homeostatic cells in the central nervous system (CNS) and they have an essential role in preserving neuronal physiology. After brain injury, astrocytes become reactive, and that involves a profound change in the astroglial gene expression program as well as intense cytoskeleton remodeling that has been classically shown by the up-regulation of glial fibrillary acidic protein (GFAP), a pan-reactive gene over-expressed in reactive astrocytes, independently of the type of injury. Using the stab wound rodent model of penetrating traumatic injury in the cortex, we here studied the reactive astroglial morphology and reactive microgliosis in detail at 1, 3, 7, 14, and 28 days post-injury (dpi). By combining immunohistochemistry, morphometrical parameters, and Sholl analysis, we segmented the astroglial cell population into clusters of reactive astrocytes that were localized in the core, penumbra, and distal regions of the stab wound. Specifically, highly reactive clusters with more complex morphology, increased C3, decreased aquaporin-4 (AQP4), and glutamine synthetase (GS) expression, were enriched at 7 dpi when behavioral alterations, microgliosis, and neuronal alterations in injured mice were most significant. While pro-inflammatory gain of function with peripheral lipopolysaccharide (LPS) administration immediately after a stab wound expanded these highly reactive astroglial clusters, the treatment with the NF-κB inhibitor sulfasalazine reduced the abundance of this highly reactive cluster. Increased neuronal loss and exacerbated reactive microgliosis at 7 dpi were associated with the expansion of the highly reactive astroglial cluster. We conclude that highly reactive astrocytes found in stab wound injury, but expanded in pro-inflammatory conditions, are a population of astrocytes that become engaged in pathological remodeling with a pro-inflammatory gain of function and loss of homeostatic capacity. Controlling this astroglial population may be a tempting strategy to reduce neuronal loss and neuroinflammation in the injured brain.

Lipopolysaccharide impairs neurodevelopment and induces changes in astroglial reactivity, antioxidant defenses and bioenergetics in the cerebral cortex of neonatal rats.

Neonates have an immature immune system, which increases their vulnerability to infectious agents and inflammatory insults. The administration of the immunostimulatory agent lipopolysaccharide (LPS) has been shown to induce the expression of pro-inflammatory cytokines and cause behavior alterations in rodents at different ages. However, the effects of LPS administration during the neonatal period and its consequences during immune system maturation remain to be elucidated. We showed here that a single intraperitoneal administration of LPS in rats on postnatal day (PND) 7 caused early and variable alterations in TNF-α, S100B and GFAP levels in the cerebral cortex, CSF and serum of the animals, indicating long-term induction of neuroinflammation and astroglial reactivity. However, on PND 21, only GFAP levels were increased by LPS. Additionally, LPS induced oxidative stress and altered energy metabolism enzymes in the cerebral cortex on PND 21, and caused neurodevelopment impairment over time. These data suggest that neuroinflammation induction during the neonatal period induces glial reactivity, oxidative stress and bioenergetic disruption that may lead to neurodevelopment impairment and cognitive deficit in adult life.

Immune Transcriptional Response in Head Kidney Primary Cell Cultures Isolated from the Three Most Important Species in Chilean Salmonids Aquaculture.

Fish cell culture is a common in vitro tool for studies in different fields such as virology, toxicology, pathology and immunology of fish. Fish cell cultures are a promising help to study how to diagnose and control relevant viral and intracellular bacterial infections in aquaculture. They can also be used for developing vaccines and immunostimulants, especially with the ethical demand aiming to reduce and replace the number of fish used in research. This study aimed to isolate head kidney primary cell cultures from three Chilean salmonids: Salmo salar, Oncorhynchus kisutch, and Oncorhynchus mykiss, and characterize the response to bacterial and viral stimuli by evaluating various markers of the innate and adaptive immune response. Specifically, the primary cell cultures of the head kidney from the three salmonids studied were cultured and exposed to two substances that mimic molecular patterns of different pathogens, i.e., Lipopolysaccharide (LPS) (bacterial) and Polyinosinic: polycytidylic acid (POLY I:C). Subsequently, we determined the mRNA expression profiles of the TLR-1, TLR-8, IgM, TLR-5, and MHC II genes. Head kidney primary cell cultures from the three species grown in vitro responded differently to POLY I:C and LPS. This is the first study to demonstrate and characterize the expression of immune genes in head kidney primary cell culture isolated from three salmonid species. It also indicates their potential role in developing immune responses as defense response agents and targets of immunoregulatory factors.