Intravenous Injection Of Lipopolysaccharide Research Articles

Abstract 5246: Therapeutic efficacy of glypican-3 peptide-linked chimeric antigen receptor-macrophages in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the third most lethal cancer and sixth most diagnosed cancer worldwide. Recently, chimeric antigen receptor-T cell (CAR-T) therapy has been demonstrated to be a promising immunotherapy for the treatment of hematologic malignancies such as leukemia. However, its application in solid tumors has proven to be challenging. To overcome this limitation, chimeric antigen receptor-macrophages (CAR-Ms) have emerged as a new immunotherapy against solid cancers, capitalizing on the unique attributes of macrophages in penetrating the tumor microenvironment, promoting antigen presentation, and priming T cells. Traditionally, CAR-M therapy involves the transfer of an edited CAR gene into macrophages via viral infection. However, this method is expensive, involves tedious procedures, and has long-term safety concerns. In the present study, we employed peptides to mimic the genetic approach for producing CAR-Ms to reduce the above uncertainties. To achieve this, we developed a novel bioconjugation method called the phthalaldehydeamine capture (PAC) reaction to produce peptidic CAR-Ms (pCAR-Ms) targeting Glypican-3 (GPC3) in HCC cells. This was achieved by linking GPC3 specific peptides to Raw264.7 murine macrophages. Using an in vitro co-culture system, we demonstrated the specificity and efficacy of pCAR-Ms against GPC3, as evidenced by a significant increase in the number of phagocytosed cells with high GPC3 expression when compared to their low-expression counterparts, by flow cytometry and confocal microscopy. The specificity of pCAR-Ms was further proven in GPC3-knockdown HCC cells, with a significant decrease in the phagocytic rate. Using an HCC xenograft model derived from MHCC97L cells, we confirmed the specific homing of pCAR-Ms to tumor sites via intravenous injection of lipopolysaccharide (LPS)-differentiated pCAR-Ms. Strikingly, pCAR-Ms suppressed HCC tumor growth when compared with untagged macrophages and PBS control. In conclusion, we provided evidence of the potency and specificity of non-viral-based pCAR-Ms in targeting HCC with simple and short production time and increased flexibility. Citation Format: Wing Hei Leung, Oi Ning Leung, Tsun Ting Wong, Kin Wah Lee. Therapeutic efficacy of glypican-3 peptide-linked chimeric antigen receptor-macrophages in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5246.

Plasma S1P Orchestrates the Reverse Transendothelial Migration of Aortic Intimal Myeloid Cells in Mice.

Myeloid cells (MCs) reside in the aortic intima at regions predisposed to atherosclerosis. Systemic inflammation triggers reverse transendothelial migration (RTM) of intimal MCs into the arterial blood, which orchestrates a protective immune response that clears intracellular pathogens from the arterial intima. Molecular pathways that regulate RTM remain poorly understood. S1P (sphingosine-1-phosphate) is a lipid mediator that regulates immune cell trafficking by signaling via 5 G-protein-coupled receptors (S1PRs [S1P receptors]). We investigated the role of S1P in the RTM of aortic intimal MCs. Intravenous injection of lipopolysaccharide was used to model a systemic inflammatory stimulus that triggers RTM. CD11c+ intimal MCs in the lesser curvature of the ascending aortic arch were enumerated by en face confocal microscopy. Local gene expression was evaluated by transcriptomic analysis of microdissected intimal cells. In wild-type C57BL/6 mice, lipopolysaccharide induced intimal cell expression of S1pr1, S1pr3, and Sphk1 (a kinase responsible for S1P production). Pharmacological modulation of multiple S1PRs blocked lipopolysaccharide-induced RTM and modulation of S1PR1 and S1PR3 reduced RTM in an additive manner. Cre-mediated deletion of S1pr1 in MCs blocked lipopolysaccharide-induced RTM, confirming a role for myeloid-specific S1PR1 signaling. Global or hematopoietic deficiency of Sphk1 reduced plasma S1P levels, the abundance of CD11c+ MCs in the aortic intima, and blunted lipopolysaccharide-induced RTM. In contrast, plasma S1P levels, the abundance of intimal MCs, and lipopolysaccharide-induced RTM were rescued in Sphk1-/- mice transplanted with Sphk1+/+ or mixed Sphk1+/+ and Sphk1-/- bone marrow. Stimulation with lipopolysaccharide increased endothelial permeability and intimal MC exposure to circulating factors such as S1P. Functional and expression studies support a novel role for S1P signaling in the regulation of lipopolysaccharide-induced RTM and the homeostatic maintenance of aortic intimal MCs. Our data provide insight into how circulating plasma mediators help orchestrate intimal MC dynamics.

Metabolomic study on urine of chronic inflammation rats treated with Buyang Huanwu Decoction based on UPLC-Q-TOF-MS

The study investigated the effect of Buyang Huanwu Decoction(BYHWD) on endogenous biomarkers in the urine of rats with chronic inflammation induced by lipopolysaccharide(LPS) using ultra-high performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry(UPLC-Q-TOF-MS), aiming to elucidate the molecular mechanism underlying the therapeutic effect of BYHWD on chronic inflammation from a metabolomics perspective. Male SD rats were randomly divided into a normal group, a model group, and low-, medium-, and high-dose BYHWD groups(7.5, 15, and 30 g·kg~(-1)). The model group and BYHWD groups received tail intravenous injection of LPS(200 μg·kg~(-1)) on the first day of each week, followed by oral administration of BYHWD once a day for four consecutive weeks. Urine samples were collected at the end of the administration period, and UPLC-Q-TOF-MS was used to analyze the metabolic profiles of the rat urine in each group. Multivariate statistical analysis methods such as principal component analysis(PCA), partial least squares-discriminant analysis(PLS-DA), and orthogonal partial least squares-discriminant analysis(OPLS-DA) were used to analyze the effect of BYHWD on endogenous metabolites. One-way ANOVA and variable importance for the projection(VIP) were used to screen for potential biomarkers related to chronic inflammation. The identified biomarkers were subjected to pathway and enrichment analysis using MetaboAnalyst 5.0. A total of 25 potential biomarkers were screened and identified in the rat urine in this experiment. Compared with the normal group, the model group showed significant increases in the levels of 14 substances(P<0.05) and significant decreases in the levels of 11 substances(P<0.05). BYHWD was able to effectively reverse the trend of most endogenous biomarkers. Compared with the model group, BYHWD significantly down-regulated 13 biomarkers(P<0.05) and up-regulated 10 biomarkers(P<0.05). The metabolic products were mainly related to the biosynthesis of pantothenic acid and coenzyme A, tryptophan metabolism, retinol metabolism, and propionate metabolism. BYHWD has therapeutic effect on chronic inflammation induced by LPS, which may be related to its ability to improve the levels of endogenous metabolites, enhance the body's anti-inflammatory and antioxidant capabilities, and restore normal metabolic activity.

Defibrotide modulates pulmonary endothelial cell activation and protects against lung inflammation in pre-clinical models of LPS-induced lung injury and idiopathic pneumonia syndrome.

A multiple organ dysfunction syndrome (MODS) workshop convened by the National Institute of Child Health and Human Development in 2015 identified acute respiratory distress syndrome (ARDS) and complications of allogeneic blood and marrow transplantation (allo-BMT) as contributors to MODS in pediatric patients. Pulmonary dysfunction also remains a significant complication of allo-BMT. Idiopathic pneumonia syndrome (IPS) defines non-infectious, acute, lung injury that occurs post-transplant. Injury and activation to endothelial cells (ECs) contribute to each form of lung inflammation. Two murine models were employed. In an ARDS model, naïve B6 mice receive an intravenous (i.v.) injection of lipopolysaccharide (LPS). In the established model of IPS, naïve B6D2F1 mice receive lethal total body irradiation followed by BMT from either allogeneic (B6) or syngeneic (B6D2F1) donors. Lung inflammation was subsequently assessed in each scenario. Intravenous injection of LPS to B6 mice resulted in enhanced mRNA expression of TNFα, IL-6, Ang-2, E-, and P-selectin in whole lung homogenates. The expression of Ang-2 in this context is regulated in part by TNFα. Additionally, EC activation was associated with increased total protein and cellularity in broncho-alveolar lavage fluid (BALF). Similar findings were noted during the development of experimental IPS. We hypothesized that interventions maintaining EC integrity would reduce the severity of ARDS and IPS. Defibrotide (DF) is FDA approved for the treatment of BMT patients with sinusoidal obstruction syndrome and renal or pulmonary dysfunction. DF stabilizes activated ECs and protect them from further injury. Intravenous administration of DF before and after LPS injection significantly reduced mRNA expression of TNFα, IL6, Ang-2, E-, and P-selectin compared to controls. BALF showed decreased cellularity, reflecting less EC damage and leak. Allogeneic BMT mice were treated from day -1 through day 14 with DF intraperitoneally, and lungs were harvested at 3 weeks. Compared to controls, DF treatment reduced mRNA expression of TNFα, IL6, Ang-2, E-, and P- selectin, BALF cellularity, and lung histopathology. The administration of DF modulates EC injury in models of ARDS and IPS. Cytokine inhibition in combination with agents that stabilize EC integrity may be an attractive strategy for patients in each setting.

Bupleurum chinense exerts a mild antipyretic effect on LPS-induced pyrexia rats involving inhibition of peripheral TNF-α production.

Bupleuri Radix, the dried roots of Bupleurum chinense DC. (BC) or Bupleurum scorzonerifolium Willd., is one of the most frequently used traditional Chinese medicines. As the species in Xiao-Chai-Hu decoction, BC has been used as an antipyretic medicine with a long history. However, its antipyretic characteristics and underlying mechanism(s) remain unclear. To elucidate the antipyretic characteristics and mechanism(s) of BC used in its traditional way. The water extract of BC (BCE) was prepared according to the traditional decocting mode. Murine fever and endotoxemia models were induced by intravenous injection of lipopolysaccharide (LPS). In vitro complement activation assay and the levels of TNF-α, IL-6, IL-1β, and C5a were determined by ELISA. BCE exerted a confirmed but mild antipyretic effect on LPS-induced fever of rat. In vitro, it significantly lowered LPS-elevated TNF-α in the supernatant of rat complete blood cells and THP-1cells, but failed to decrease IL-6 and IL-1β. In murine endotoxemia models, BCE markedly decreased serum TNF-α, but had no impact on IL-6 and IL-1β. BCE also restricted complement activation in vitro and in vivo. Nevertheless, the mixture of saikosaponin A and D could not suppress supernatant TNF-α of monocytes and serum TNF-α of endotoxemia mice. The present study dissects the peripheral mechanism for the antipyretic effect of BC used in the traditional way. Our findings indicate that BCE directly suppresses monocyte-produced TNF-α, thus decreasing circulating TNF-α, which may be responsible for its mild but confirmed antipyretic action.

Combining accelerometers and direct visual observations to detect sickness and pain in cows of different ages submitted to systemic inflammation

Cattle suffering from inflammatory infection display sickness and pain-related behaviours. As these behaviours may be transient and last only a few hours, one may miss them. The aim of this study was to assess the benefit of combining continuous monitoring of cow behaviour via collar-attached accelerometers with direct visual observations to detect sickness and pain-related behavioural responses after a systemic inflammatory challenge (intravenous lipopolysaccharide injection) in cows of two different ages, proven by clinical, physiological and blood parameters. Twelve cloned Holstein cows (six ‘old’ cows aged 10–15 years old and six ‘young’ cows aged 6 years old) were challenged and either directly observed at five time-points from just before the lipopolysaccharide injection up to 24 h post-injection (hpi) or continuously monitored using collar-attached accelerometers in either control or challenge situations. Direct observations identified specific sickness and pain behaviours (apathy, changes in facial expression and body posture, reduced motivation to feed) expressed partially at 3 hpi and fully at 6 hpi. These signs of sickness and pain behaviours then faded, and quicker for the young cows. Accelerometers detected changes in basic activities (low ingesting, low ruminating, high inactivity) and position (high time standing up) earlier and over a longer period of time than direct observations. The combination of sensors and direct observations improved the detection of behavioural signs of sickness and pain earlier on and over the whole study period, even when direct signs were weak especially in young cows. This system could provide great benefit for better earlier animal care.

Role of Serum Inflammatory Cytokines in Sepsis Rats Following BMSCs Transplantation: Protein Microarray Analysis

Bone marrow stromal cells (BMSCs) have emerged as a potential therapy for sepsis, yet the underlying mechanisms remain unclear. In this study, we investigated the effects of BMSCs on serum inflammatory cytokines in a rat model of lipopolysaccharide (LPS)-induced sepsis. Sepsis was induced by intravenous injection of LPS, followed by transplantation of BMSCs. We monitored survival rates for 72 h and evaluated organ functions, histopathological changes, and cytokines expression. Sepsis rats showed decreased levels of white blood cells, platelets, lymphocyte ratio, and oxygen partial pressure, along with increased levels of neutrophil ratio, carbon dioxide partial pressure, lactic acid, alanine aminotransferase, and aspartate aminotransferase. Histologically, lung, intestine, and liver tissues exhibited congestion, edema, and infiltration of inflammatory cells. However, after BMSCs treatment, there was improvement in organ functions, histopathological injuries, and survival rates. Protein microarray analysis revealed significant changes in the expression of 12 out of 34 inflammatory cytokines. These findings were confirmed by enzyme-linked immunosorbent assay. Pro-inflammatory factors, such as interleukin-1β (IL-1β), IL-1α, tumor necrosis factor-α (TNF-α), tissue inhibitor of metal protease 1 (TIMP-1), matrix metalloproteinase 8 (MMP-8), Leptin, and L-selectin were upregulated in sepsis, whereas anti-inflammatory and growth factors, including IL-4, β-nerve growth factor (β-NGF), ciliary neurotrophic factor (CNTF), interferon γ (IFN-γ), and Activin A were downregulated. BMSCs transplantation led to a decrease in pro-inflammatory cytokines and an increase in anti-inflammatory and growth factors. We summarized relevant molecular signaling pathways that resulted from cytokines in BMSCs for treating sepsis. Our results illustrated that BMSCs could promote tissue repair and improve organ functions and survival rates in sepsis through modulating cytokine networks.

Quercetin Supplement to Aspirin Attenuates Lipopolysaccharide-Induced Pre-eclampsia-Like Impairments in Rats Through the NLRP3 Inflammasome.

Aspirin is a common drug for the treatment of pre-eclampsia. We aimed to explore whether quercetin as a supplement to aspirin could enhance the therapeutic outcome in pre-eclampsia rat models. We further aimed to evaluate the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome as a potential pre-eclampsia-related molecular mechanism, which can be affected by quercetin treatment. Rat pre-eclampsia models were established using an intravenous lipopolysaccharide injection after gestation. Rats were treated with aspirin and quercetin at 6-18days after pregnancy. On day 20, blood, fetus, and placenta were harvested. Blood pressure and the level of proteinuria were measured every 4days. Fetal outcomes were analyzed by pup body weight. Serum soluble Fms-like tyrosine kinase-1, PIGF, interleukin-6, and interleukin-10 levels were measured using the enzyme-linked immunosorbent assay. Caspase-1, NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain, and p-caspase-1 levels in the placenta were assessed using western blot or quantitative real-time polymerase chain reaction analyses. Pre-eclampsia rat models showed a pronounced increase in systolic blood pressure and proteinuria after 4days of pregnancy, while aspirin, quercetin, and aspirin/quercetin combinatory treatment significantly attenuated the blood pressure and proteinuria abnormalities. Notably, the aspirin/quercetin combinatory treatment showed the highest efficacy in attenuating pre-eclampsia-like symptoms. Placental caspase-1 and NLRP3 levels also showed the greatest attenuation in pre-eclampsia rats after aspirin/quercetin treatment. Our data suggested that quercetin supplementation to aspirin is more effective in attenuating symptoms of pre-eclampsia and improving pregnancy outcomes compared with quercetin or aspirin alone. Quercetin can ameliorate placental NLRP3 inflammasome activation, which might serve as an underlying mechanism for its therapeutic efficacies in pre-eclampsia.

Effects of membrane-type PBLs on the expression of TNF-α and IL-2 in pulmonary tissue of SD rats after LPS-induced acute lung injury.

As the first target organ, the lungs usually display symptoms of acute lung injury (ALI). Pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-2, are crucial in triggering the systemic inflammatory response syndrome and the subsequent cascading effects. Therefore, the inhibition of the release of inflammatory mediators has become an important strategy for the prevention and treatment of ALI. To evaluate the preventive and therapeutic effects of transmembrane peripheral blood leukocytes (PBLs) on lipopolysaccharide (LPS)-induced ALI and its mechanism. Sixty Sprague Dawley rats were randomly divided into experimental and control groups. The animal model was established through intravenous injection of LPS. Plasmid PBLs were dissolved in a saline solution and injected into the experimental group of rats in different doses (0.1 mg, 0.2 mg and 0.3 mg per rat) using the in situ injection method. After injecting the PBL solution, the rats were killed after 12 h, 24 h, 36 h, or 48 h. The expression of microRNA (miRNA)-25 and miRNA-223 was detected using the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Tumor necrosis factor alpha and IL-2 levels in bronchoalveolar lavage fluid (BALF) were detected with an enzyme-linked immunosorbent assay (ELISA). The expressions of TNF-α and IL-2 proteins in lung tissue were detected using western blotting. The expression of miRNA-25 was upregulated in tissues and BALF in a doseand time-dependent manner, while miRNA-223 was downregulated. The differences were statistically significant compared to the control group (p < 0.05). The TNF-α and IL-2 levels in the BALF of rats in the experimental group were increased in a dose-dependent manner compared to the control group (p < 0.05). In the presence of PBLs, the expressions of TNF-α, IL-2, miRNA, and proteins were inhibited. Thus, PBLs were found to alleviate pulmonary tissue damage. In summary, PBLs have a protective effect on rats with ALI through the downregulation of TNF-α and IL-2 expression.

Changes of the liver metabolome following an intravenous lipopolysaccharide injection in Holstein cows supplemented with dietary carnitine

BackgroundCarnitine facilitates the flux of long-chain fatty acids for hepatic mitochondrial beta-oxidation, which acts to ameliorate the negative energy balance commonly affecting high-yielding dairy cows. Inflammation triggered by lipopolysaccharide (LPS) load can however pose a challenge to the metabolic integrity via the expression of pro-inflammatory mediators, leading to immune system activation and respective metabolic alterations. The effect of enhanced carnitine availability on hepatic metabolome profiles during an inflammatory challenge has not yet been determined in dairy cows. Herein, Holstein cows were supplemented with 25 g/d rumen-protected carnitine from 42 d prepartum until 126 d postpartum (n = 16) or assigned to the control group with no supplementation during the same period (n = 14). We biopsied the liver of the cows before (100 d postpartum) and after (112 d postpartum) an intravenous injection of 0.5 µg/kg LPS. Liver samples were subjected to a targeted metabolomics analysis using the AbsoluteIDQ p180 Kit (Biocrates Life Sciences AG, Innsbruck, Austria). ResultsMultivariate statistical analyses revealed that hepatic metabolome profiles changed in relation to both the carnitine supplementation and the LPS challenge. Comparing the metabolite profiles on 100 d, carnitine increased the concentration of short- and long-chain acyl-carnitines, which may be explained by an enhanced mitochondrial fatty acid shuttle and hence greater energy availability. The LPS injection affected hepatic metabolite profiles only in the carnitine supplemented group, particularly altering the concentration of biogenic amines.ConclusionsOur results point to interactions between an acute hepatic inflammatory response and biogenic amine metabolism, depending on energy availability.

Empirical Study of Anti-Inflammatory Effects of Kecombrang (Etlingera elatior) in Mus musculus Sepsis Model

Mice used as experimental animals in the laboratory belong to the genus Mus, subfamily Murinae, family Muridae, superfamily Muroidea, order Rodentia, and class M. musculus , these mice are also referred to as home mice. Mice have been used as standard animals in toxicology, teratology, and carcinogenesis tests, even today, mice have also been used for behavioral, neurologic, nutritional, genetic, immunological, infectious, metabolic, and degenerative disease studies. Animal models of sepsis with intraperitoneal or intravenous injection of lipopolysaccharide (LPS) have been widely used for sepsis research. LPS induces systemic inflammation that mimics the early phase of sepsis. LPS injection causes kidney injury, including a decrease in glomerular filtration rate, an increase in blood urea nitrogen, and an increase in neutrophil infiltration in the kidney. The injectable dose of LPS can be titrated to mimic early sepsis without hemodynamic compromise, which has been useful for studying the systemic and renal responses. The response during the early phase of sepsis is that doses of LPS are usually used to induce systemic hypotension and decrease glomerular perfusion, whereas low doses of LPS do not cause systemic hypotension but still decrease glomerular perfusion. There are several advantages of LPS compared to others, namely, the method used is simple and the model is very controlled and standardized. The dose of endotoxin that causes 50% mortality in mice is 1–25 mg/kg. In this study, mice were given intraperitoneal injection of LPS at a dose of 0.3 mg/kg BW. LPS injection was given to the positive control group and treatment group 1, treatment group 2, and treatment group 3 at the start of the study.

The effects of vasopressors with and without dobutamine on haemodynamics, metabolism and gut injury during endotoxic shock in rabbits. Acontrolled study.

Vasopressors increase arterial pressure but they may have deleterious effects on mesenteric blood flow. We aimed to evaluate the response of gut biomarkers and superior mesenteric blood flow to different vasopressors with and without dobutamine. Thirty New Zealand rabbits were included and randomly allocated to 5 groups: group A- sham group; group B - norepinephrine; group C - norepinephrine plus dobutamine; group D - vasopressin; and group E - vasopressin plus dobutamine. Mean arterial pressure (MAP) target was greater than 60 mmHg. Endotoxic shock was induced by intra-venous injection of lipopolysaccharide (LPS) in four of the five groups. Aortic blood flow (Qao), superior mesenteric artery flow (QSMA) and lactate were measured after LPS injection. Enterocyte damage was evaluated by measurements of serum citrulline and intestinal fatty acid-binding protein (I-FABP) after 4 h. The largest reduction in Qao occurred in group D (64 ± 17.3 to 38 ± 7.5 mL min-1; P = 0.04). QSMA also declined significantly in groups D and E and remained lower than in the other groups over 4 h (group D - baseline: 65 ± 31; 1 h: 37 ± 10; 2 h: 38 ± 10; 3 h: 46 ± 26; and 4 h: 48 ± 15 mL min-1; P < 0.005; group E - baseline: 73 ± 14; 1 h: 28 ± 4.0; 2 h: 37 ± 6.4; 3 h: 40 ± 11; and 4 h: 48 ± 11; P < 0.005; all in mL min-1). Serum citrulline was significantly lower in groups D (P = 0.014) and E (P = 0.019) in comparison to group A. The fluid administration regimen was similar in all groups. Vasopressin seems to negatively impact gut enterocyte function during endotoxic shock despite the association of an inodilator and adequate fluid replacement.

Influence of Escherichia coli endotoxemia on danofloxacin pharmacokinetics in broilers following single oral administration.

As a fluoroquinolone antimicrobial agent, danofloxacin is mainly used to treat avian bacterial and mycoplasma infections. The pharmacokinetic characteristics of danofloxacin are usually explored in healthy animals, while those in endotoxemic broilers are still rare. This study aimed to investigate the pharmacokinetics of danofloxacin in endotoxemic broilers induced by Escherichia coli (E. coli) lipopolysaccharide (LPS) after single oral administration. Ten healthy 5-week-old Arbor Acres (AA) broilers with similar body weight (BW) were randomly and equally divided into LPS and control groups. The LPS group was intravenously injected with an LPS of E. coli O55: B5 at 2.5mg/kg BW, and the control group was intravenously injected with the same volume of sterile saline. Danofloxacin was administered orally at a dose of 5mg/kg BW immediately 1h after the intravenous injection of LPS or sterile saline. Rectal temperature was measured at predetermined times points in all broilers, and plasma and serum samples were taken. The interleukin-6 (IL-6) levels in serum samples were detected by the enzyme-linked immunosorbent assay (ELISA) kits, and danofloxacin concentrations in plasma were detected through the high-performance liquid chromatography (HPLC) method and subjected to a compartmental analysis using Phoenix software. The LPS challenge led to biphasic adaptive changes in broiler body temperature and increased the levels of IL-6. Compared with the control group, LPS treatment significantly prolonged the time to the peak concentration (LPS: 8.75±3.88h; Control: 3.20±2.20h). However, there were no significant differences in the other pharmacokinetic parameters between both groups.

Bai-Hu-Tang regulates endothelin-1 and its signalling pathway in vascular endothelial cells

Ethnopharmacological relevanceBai-Hu-Tang (BHT) is traditionally used to treat human and animal fever syndrome with four symptoms: large and vigorous pulse, large thirst, high sweat, and high heat. Aim of the studyTo investigate the mechanism of vasodilation regulation of Bai-Hu-Tang in primary vascular endothelial cells stimulated by lipopolysaccharide (LPS). Materials and methodsA hydrophilic concentrate of BHT was prepared, and the main components of mangiferin and timosaponin BⅡ were determined by HLPC analysis. The rabbit fever model was constructed by intravenous injection of LPS (15 μg/kg body weight), and BHT was gavaged to treat febrile rabbits. After treatment for 6 h, animal peripheral blood was collected, and serum was isolated for endothelin-1 (ET-1) and nitric oxide (NO) assays. Rabbit vascular endothelial cells (RVECs) were isolated and stimulated with 1 μg/mL LPS, and then inflammatory cells were treated with 125 or 250 μg/mL BHT for 24 h. The supernatant cytokines TNF-ɑ, IL-1β, IL-6, and ET-1 were detected by ELISA kits. Gene expression levels of endothelin receptor type B (ETB receptor) were analysed by real-time polymerase chain reaction (RT-PCR), and protein expression levels of PI3K and Akt were detected by Western blot. A nitrite assay was used to measure intracellular nitric oxide (NO) production, and nitric oxide synthase (NOS) was measured by the T-NOS colorimetric method. ResultsAnimal experiments demonstrated that BHT significantly restored ET-1 and NO in animal peripheral blood, which were disordered in LPS-induced fever rabbits. Moreover, a cytotoxicity assay demonstrated that BHT ≤700 μg/mL is innoxious to RVECs. BHT significantly repressed cellular TNF-α, IL-1β, and ET-1, which were originally elevated by LPS in RVECs. Meanwhile, BHT elevated the gene expression level of the ETB receptor and promoted NOS and NO production in RVECs induced by LPS. ConclusionBHT can inhibit excessive ET-1 secretion induced by LPS in vascular endothelial cells and activate the classic ET-1 signalling pathway to promote NO production, which may facilitate vasodilation of smooth muscle cells.

Anterior insula morphology and vulnerability to psychopathology-related symptoms in response to acute inflammation

IntroductionThe role of inflammation in common psychiatric diseases is now well acknowledged. However, the factors and mechanisms underlying inter-individual variability in the vulnerability to develop psychopathology-related symptoms in response to inflammation are not well characterized. Herein, we aimed at investigating morphological brain regions central for interoception and emotion regulation, and if these are associated with acute inflammation-induced sickness and anxiety responses. MethodsSystemic inflammation was induced using an intravenous injection of lipopolysaccharide (LPS) at a dose of 0.6 ng/kg body weight in 28 healthy individuals, while 21 individuals received an injection of saline (placebo). Individuals’ gray matter volume was investigated by automated voxel-based morphometry technique on T1-weighted anatomical images derived from magnetic resonance imaging (MRI). Plasma concentrations of TNF-α and IL-6, sickness symptoms (SicknessQ), and state anxiety (STAI-S) were measured before and after the injection. ResultsA stronger sickness response to LPS was significantly associated with a larger anterior insula gray matter volume, independently from increases in cytokine concentrations, age, sex and body mass index (R2 = 65.6%). Similarly, a greater LPS-induced state anxiety response was related to a larger anterior insula gray matter volume, and also by a stronger increase in plasma TNF-α concentrations (R2 = 40.4%). DiscussionAnterior insula morphology appears central in the sensitivity to develop symptoms of sickness and anxiety in response to inflammation, and could thus be one risk factor in inflammation-related psychopathologies. Because of the limited sample size, the current results need to be replicated.

Immunoregulatory Monocyte Subset Promotes Metastasis Associated With Therapeutic Intervention for Primary Tumor.

Systemic and local inflammation associated with therapeutic intervention of primary tumor occasionally promotes metastatic recurrence in mouse and human. However, it remains unclear what types of immune cells are involved in this process. Here, we found that the tissue-repair-promoting Ym1+Ly6Chi monocyte subset expanded as a result of systemic and local inflammation induced by intravenous injection of lipopolysaccharide or resection of primary tumor and promoted lung metastasis originating from circulating tumor cells (CTCs). Deletion of this subset suppressed metastasis induced by the inflammation. Furthermore, transfer of Ym1+Ly6Chi monocytes into naïve mice promoted lung metastasis in the mice. Ym1+Ly6Chi monocytes highly expressed matrix metalloproteinase-9 (MMP-9) and CXCR4. MMP-9 inhibitor and CXCR4 antagonist decreased Ym1+Ly6Chi-monocyte-promoted lung metastasis. These findings indicate that Ym1+Ly6Chi monocytes are therapeutic target cells for metastasis originating from CTCs associated with systemic and local inflammation. In addition, these findings provide a novel predictive cellular biomarker for metastatic recurrence after intervention for primary tumor.

Regulation of emotions during experimental endotoxemia: A pilot study

Even though dysfunctional emotion regulation is prominent in depression and a link between depression and inflammation is well established, there is little knowledge about how inflammation affects the regulation of emotions. The aim of this pilot study was to explore the effect of experimentally induced inflammation on the cognitive reappraisal of emotions, and to assess domain specificity by comparing success in regulation of emotions towards two unpleasant stimuli classes (general negative stimuli and disgust stimuli). In a between-subject design, ten healthy participants were injected with an intravenous injection of lipopolysaccharide (2 ng/kg body weight) and eleven were injected with saline. Participants performed a cognitive reappraisal task, in which they had to down-regulate or up-regulate their emotions towards general negative stimuli and disgust stimuli, 5–6 h post-injection. Contrary to our hypotheses, participants injected with lipopolysaccharide reported greater success in down-regulating emotional responses towards unpleasant stimuli as compared to the saline group. In addition, both groups were poorer at down-regulating emotions towards disgust stimuli as compared to general negative stimuli. The current pilot study indicates that cognitive reappraisal of emotions is affected during experimental endotoxemia, and suggests that disgust stimuli might be difficult to reappraise.

Dietary l-carnitine Supplementation Modifies the Lipopolysaccharide-Induced Acute Phase Reaction in Dairy Cows.

Simple Summaryl-carnitine might limit the mitochondrial energy generation from fatty acids, particularly in periods of enhanced energy requirement such as during lipopolysaccharide (LPS) mediated innate immune responses. This study examined the influence of a dietary l-carnitine supplementation on dairy cows in mid-lactation which were challenged by an injection with LPS. Results suggest that this intervention supported the energy metabolism of the cows during LPS-induced acute phase reaction (APR).l-carnitine plays an important role in energy metabolism through supporting the transport of activated fatty acids to the subcellular site of β-oxidation. An acute phase reaction (APR) is known as an energy consuming process. Lipopolysaccharides (LPS) are often used in animal models to study intervention measures during innate immune responses such as APR. Thus, the aim of the study was to investigate the effects of dietary l-carnitine supplementation during an LPS-induced APR in mid-lactating German Holstein cows. Animals were assigned to a control (CON, n = 26) or l-carnitine group (CAR, n = 27, 25 g rumen-protected l-carnitine/cow/d) and received an intravenous injection of LPS (0.5 μg/kg body weight) at day 111 post-partum. Blood samples were collected from day 1 pre-injection until day 14 post-injection (pi). From 0.5 h pi until 72 h pi blood samplings and clinical examinations were performed in short intervals. Clinical signs of the APR were not altered in group CAR except rumen motility which increased at a lower level compared to the CON group after a period of atonia. Group CAR maintained a higher insulin level compared to group CON even up to 72 h pi which might support glucose utilization following an APR.

The Secretome Deregulations in a Rat Model of Endotoxemic Shock.

Introduction Septic shock is a systemic inflammatory response syndrome associated with organ failures. Earlier clinical diagnosis would be of benefit to a decrease in the mortality rate. However, there is currently a lack of predictive biomarkers. The secretome is the set of proteins secreted by a cell, tissue, or organism at a given time and under certain conditions. The plasma secretome is easily accessible from biological fluids and represents a good opportunity to discover new biomarkers that can be studied with nontargeted “omic” strategies. Aims To identify relevant deregulated proteins (DEP) in the secretome of a rat endotoxemic shock model. Methods Endotoxemic shock was induced in rats by intravenous injection of lipopolysaccharides (LPS, S. enterica typhi, 0.5 mg/kg) and compared to controls (Ringer Lactate, iv). Under isoflurane anesthesia, carotid cannulation allowed mean arterial blood pressure (MAP) and heart rate (HR) monitoring and blood sampling at different time points (T0 and T50 or T0 and T90, with EDTA and protease inhibitor). Samples were prepared for large-scale tandem mass spectrometry (MS-MS) based on a label-free quantification to allow identification of the proteins deregulated upon endotoxemic conditions. A Gene Ontology (GO) analysis defined several clusters of biological processes (BP) in which the DEP are involved. Results Ninety minutes after shock induction, the LPS group presents a reduction in MAP (-45%, p < 0.05) and increased lactate levels (+27.5%, p < 0.05) compared to the control group. Proteomic analyses revealed 10 and 33 DEP in the LPS group, respectively, at 50 and 90 minutes after LPS injection. At these time points, GO-BP showed alterations in pathways involved in oxidative stress response and coagulation. Conclusion This study proposes an approach to identify relevant DEP in septic shock and brings new insights into the understanding of the secretome adaptations upon sepsis.

234 In utero heat stress alters the postnatal immune and metabolic response of growing pigs subjected to a lipopolysaccharide challenge

Abstract In utero heat stress (IUHS) reduces swine productivity and welfare but little is known about the long-term impact on immune function. The study objective was to determine the effects of IUHS on the immune and metabolic response of pigs subjected to an 8 h lipopolysaccharide (LPS) challenge during postnatal life. Twenty-four pregnant gilts were exposed to thermoneutral (TN; n = 12; 17.5 ± 2.1⁰C) or heat stress (HS; n = 12; cyclical 27°C-37°C) conditions from d 1 to 58 of gestation, and TN conditions from d 59 of gestation to farrowing. After farrowing, all piglets were housed under the same conditions. At 12 weeks post-farrowing, 16 IUHS and 16 in utero thermoneutral (IUTN) pigs were selected, balanced by sex and given an intravenous injection of LPS (2 µg/kg BW) or saline (SAL). Treatment combinations were: IUTN-SAL, IUTN-LPS, IUHS-SAL, IUHS-LPS. Body temperature was monitored in 30-min intervals and blood samples were obtained at 0, 1, 2, 3, 4, 6, and 8 h. Blood was analyzed for glucose, insulin, non-esterified fatty acids (NEFA), and cytokine concentrations. Body temperature increased (P &amp;lt; 0.01; 1.05°C) in LPS versus SAL pigs, regardless of in utero treatment. Glucose concentrations were reduced overall (P = 0.05; 5.9%) in IUHS versus IUTN pigs. Non-esterified fatty acid concentrations tended to be greater (P = 0.07; 143.4%) in IUHS-LPS pigs compared to all other treatments, and IUTN-LPS pigs tended to have greater circulating NEFA concentrations (127.4%) compared to IUTN-SAL and IUHS-SAL pigs. At 1 h, TNFα was increased (P = 0.01; 115.1%) in IUHS-LPS compared to IUTN-LPS pigs. Overall, IL-1β and IL-6 were greater (P &amp;lt; 0.04; 56.0 and 46.8%, respectively) in IUHS-LPS compared IUTN-LPS pigs. In summary, IUHS altered the postnatal immune and metabolic response of pigs during postnatal life, which has negative implications towards future disease susceptibility.