Bacterial Endotoxin Lipopolysaccharide Research Articles

First-in-class mitogen-activated protein kinase (MAPK) p38a: MAPK-activated protein kinase 2 dual signal modulator with anti-inflammatory and endothelial-stabilizing properties

We previously identified a small molecule, UM101, predicted to bind to the substrate-binding groove of p38a mitogen-activated protein kinase (MAPK) near the binding site of its proinflammatory substrate, mitogen-activated protein kinase–activated protein kinase (MK)2. UM101 exhibited anti-inflammatory, endothelial-stabilizing, and lung-protective effects. To overcome its limited aqueous solubility and p38a binding affinity, we designed an analog of UM101, GEn-1124, with improved aqueous solubility, stability, and p38a-binding affinity. Compared with UM101, GEn-1124 has 18-fold greater p38a-binding affinity as measured by surface plasmon resonance, 11-fold greater aqueous solubility, enhanced barrier-stabilizing activity in thrombin-stimulated human pulmonary artery endothelial cells in vitro, and greater lung protection in vivo. GEn-1124 improved survival from 10%–40% in murine acute lung injury induced by combined exposure to intratracheal bacterial endotoxin lipopolysaccharide instillation and febrile-range hyperthermia and from 0% to 50% in a mouse influenza pneumonia model. Gene expression analysis by RNASeq in tumor necrosis factor α–treated human pulmonary artery endothelial cells showed that the gene-modifying effects of GEn-1124 were much more restricted to tumor necrosis factor α–inducible genes than those of the catalytic site p38 inhibitor, SB203580. Gene expression pathway analysis, confocal immunofluorescence analysis of p38a and MK2 subcellular trafficking, and SPR analysis of phosphorylated p38a:MK2 binding affinity supports a novel mechanism of action. GEn-1124 destabilizes the activated p38a:MK2 complex and dissociates nuclear export of MK2 and p38a, thereby promoting intranuclear retention and enhanced intranuclear signaling by phosphorylated p38a retention and accelerated inactivation of p38-free cytosolic MK2 by unopposed phosphatases. Significance StatementWe describe a novel analog of our first-in-class small molecule modulator of p38a/MK2 signaling targeted to a pocket near the ED substrate binding domain of p38a, which destabilizes the p38a:MK2 complex without blocking p38 catalytic activity or ablating downstream signaling. The result is a rebalancing of downstream proinflammatory and anti-inflammatory signaling, yielding anti-inflammatory, endothelial-stabilizing, and lung-protective effects with therapeutic potential in acute respiratory distress syndrome.

Lipopolysaccharide from Proteus mirabilis Slows Platelet Plug Formation in Human Whole Blood

Platelets are well known for their role in hemostasis. Additionally, platelets play a crucial role in immune and inflammatory responses. Toll-like receptors (TLRs) can mediate bacterial interactions during infection, triggering platelets to initiate an inflammatory response. TLR-4 receptors enable direct interactions between platelets and the bacterial lipopolysaccharide (LPS) endotoxin. The aim of this study was to assess platelet plug formation in response to LPS from Proteus mirabilis. Human whole blood was treated with varying concentrations of LPS over a range of incubation times. Then, platelet plug formation time was measured, under high shear conditions using the platelet function analyzer PFA-100, as aperture closure time (CT). The addition of either 2 or 10 µg/mL of LPS to 80% whole blood significantly prolonged the CTs even in the absence of preincubation (p = 0.028 or p = 0.049, respectively). With added preincubation of LPS with whole blood, the measured CTs were further prolonged. If the preincubation time was set to 35 min, then even the addition of 0.2 µg/mL of LPS resulted in significant CT prolongation (p < 0.001). Taken together, the platelet plug formation in the presence of collagen/ADP is significantly prolonged by the presence of LPS in a concentration and preincubation time-dependent manner. Exposure to P. mirabilis LPS reduces the platelet aggregation response in human whole blood.

Increased intestinal permeability and lipopolysaccharide contribute to swainsonine-induced systemic inflammation

Long-term consumption of swainsonine could be poisonous to livestock, including facilitating apoptosis by impairing lysosomal function and inhibiting autophagic degradation, leading to liver inflammation and even death in livestock. However, the mechanism by swainsonine induced systemic inflammatory responses remained unclear, especially the effects of swainsonine on intestinal permeability, lipopolysaccharide (LPS) level and oxidative stress response were unknown. In this study, swainsonine increased intestinal permeability as evidenced by the significant down-regulation of colonic goblet cells, Akkermansia muciniphila and intestinal tight junction protein Occludin, Claudin 1 and ZO-1, and the significant up-regulation of mRNA expression level of the intestinal permeability indicator protein tyrosine phosphatase receptor type H (Ptprh) in the ileum of mice. Simultaneously, the elevated LPS biosynthetic genes in intestinal microbiota and increased intestinal permeability facilitated more bacterial endotoxin LPS to enter the blood. High concentration of free-form LPS induced high levels of proinflammatory cytokines and oxidative stress response, thereby causing the systemic inflammation. These findings provided a new perspective on swainsonine-induced systemic inflammation, suggesting that intestinal permeability and free-form LPS level may be the potential trigger factors.

Mitigating lipopolysaccharide-induced impairment in human dental pulp stem cells with tideglusib-doped nanoparticles: Enhancing osteogenic differentiation and mineralization

ObjectiveDrug-loaded non-resorbable polymeric nanoparticles (NPs) are proposed as an adjunctive treatment for pulp regenerative strategies. The present in vitro investigation aimed to evaluate the effectiveness of tideglusib-doped nanoparticles (TDg-NPs) in mitigating the adverse effects of bacterial lipopolysaccharide endotoxin (LPS) on the viability, morphology, migration, differentiation and mineralization potential of human dental pulp stem cells (hDPSCs). MethodsCell viability, proliferation, and differentiation were assessed using a MTT assay, cell migration evaluation, cell cytoskeleton staining analysis, Alizarin Red S staining and expression of the odontogenic related genes by a real-time quantitative polymerase chain reaction (RT-qPCR) were also performed. Cells were tested both with and without stimulation with LPS at various time points. One-way ANOVA and Tukey's test were employed for statistical analysis (p < 0.05). ResultsAdequate cell viability was encountered in all groups and at every tested time point (24, 48, 72 and 168 h), without differences among the groups (p > 0.05). The analysis of cell cytoskeleton showed nuclear alteration in cultures with undoped NPs after LPS stimulation. These cells exhibited an in blue diffuse and multifocal appearance. Some nuclei looked fragmented and condensed. hDPSCs after LPS stimulation but in the presence of TDg-NPs exhibited less nuclei changes. LPS induced down-regulation of Alkaline phosphatase, Osteonectin and Collagen1 gene markers, after 21d. LPS half-reduced the cells production of calcium deposits in all groups (p < 0.05), except in the group with TDg-NPs (decrease about 10 %). SignificanceLPS induced lower mineral deposition and cytoskeletal disorganization in hDPSCs. These effects were counteracted by TDg-NPs, enhancing osteogenic differentiation and mineralization.

Relationship between Endotoxin Content in Vaccine Preclinical Formulations and Animal Welfare: An Extensive Study on Historical Data to Set an Informed Threshold.

The most widely known pyrogen impurity in vaccines is the Gram-negative bacterial endotoxin lipopolysaccharide (LPS). When administered at toxic doses, endotoxin triggers inflammatory responses, which lead to endotoxic shock. The literature on endotoxic content (EC) for preclinical vaccines' formulations used in animal studies is very poor, and the recommended thresholds are solely based on commercial vaccine limits set for humans and are, therefore, not connected to the actual impact of EC on animal welfare for species used in preclinical research studies. An extensive study to evaluate the presence of a potential relationship between endotoxin content in formulations administered to mice (the most common species used in preclinical research studies) and their welfare was conducted to calculate an EC threshold for formulations of candidate vaccines. Three years of historical data, from more than 500 formulations of different antigen types (i.e., proteins, glycoconjugates, OMV/GMMA) injected into more than 5000 mice, was evaluated with two alternative statistical methodologies, both demonstrating that there is no significant relationship between actual endotoxin levels and mouse welfare. The calculation of thresholds was, therefore, performed by consistency versus formulations that demonstrated no impact on animal welfare.

Effect of dietary macronutrients and immune challenge on gut microbiota, physiology and feeding behaviour in zebra finches.

Macronutrients play a vital role in host immunity and can influence host-pathogen dynamics, potentially through dietary effects on gut microbiota. To increase our understanding of how dietary macronutrients affect physiology and gut microbiota and investigate whether feeding behaviour is influenced by an immune threat, we conducted two experiments. First, we determined whether zebra finches (Taeniopygia guttata) exhibit shifts in physiology and gut microbiota when fed diets differing in macronutrient ratios. We found the type and amount of diet consumed affected gut microbiota alpha diversity, where microbial richness and Shannon diversity increased with caloric intake in birds fed a high-fat diet and decreased with caloric intake in birds fed a high protein diet. Diet macronutrient content did not affect physiological metrics, but lower caloric intake was associated with higher complement activity. In our second experiment, we simulated an infection in birds using the bacterial endotoxin lipopolysaccharide (LPS) and quantified feeding behaviour in immune challenged and control individuals, as well as birds housed near either a control pair (no immune threat), or birds housed near a pair given an immune challenge with LPS (social cue of heightened infection risk). We also examined whether social cues of infection alter physiological responses relevant to responding to an immune threat, an effect that could be mediated through shifts in feeding behaviour. LPS induced a reduction in caloric intake driven by a decrease in protein, but not fat consumption. No evidence was found for socially induced shifts in feeding behaviour, physiology or gut microbiota. Our findings carry implications for host health, as sickness-induced anorexia and diet-induced shifts in the microbiome could shape host-pathogen interactions.

Lipopolysaccharide regulation of antiinflammatory tristetraprolin family and proinflammatory gene expression in mouse macrophages

ObjectiveTristetraprolin (TTP/ZFP36) family proteins exhibit antiinflammatory effects by destabilizing proinflammatory mRNAs. Previous studies showed that bacterial endotoxin lipopolysaccharides (LPS) stimulated TTP and tumor necrosis factor (TNF) gene expression, but less was known about LPS effects on TTP homologues and other proinflammatory gene expression in macrophages. The objective was to investigate LPS regulation of TTP family gene and TTP-targeted gene expression in mouse RAW264.7 macrophages using much higher concentrations of LPS and much longer treatment time than previous studies.ResultsMTT assay showed that LPS was not toxic to the cells under LPS treatment up to 1000 ng/mL for 2–24 h. LPS mildly affected the soluble protein content in the cells. qPCR assay showed that LPS stimulated TTP mRNA rapidly but not sustainably with 40, 10, and 3 fold of the DMSO control after 2, 8 and 24 h treatment, respectively. Immunoblotting confirmed qPCR results on LPS stimulation of TTP gene expression in the mouse macrophages. LPS exhibited minimal effects on ZFP36L1, ZFP36L2 and ZFP36L3 mRNA levels. LPS increased mRNA levels of TNF, COX2, GM-CSF, INFγ and IL12b up to 311, 418, 11, 9 and 4 fold, respectively. This study demonstrated that LPS did not affect macrophage viability, dramatically increased antiinflammatory TTP gene expression as well as proinflammatory TNF and COX2 gene expression but had only mild effects on TTP homologues and other proinflammatory cytokine gene expression in the mouse macrophages.

Response of the terrestrial isopod Porcellio scaber to lipopolysaccharide challenge after microplastic and insecticide exposure

The exposure of organisms to microplastics could compromise their ability to cope with other environmental stressors, such as infections. In this context, we investigated the effects of a 14-day exposure of the terrestrial isopod Porcellio scaber to tire particles in soil (1.5 % w w−1 dry weight) on the organisms' response to a secondary exposure, i.e., injection of the bacterial endotoxin lipopolysaccharide. In addition, the insecticide chlorpyrifos (2 mg kg−1 dry weight) was tested as a positive control. The survival and immune response of P. scaber was assessed at the end of the 7- and 14-day primary exposure and two days after the secondary exposure, by analyzing selected haemolymph immune parameters (total haemocyte count, differential haemocyte count, and haemocyte viability). No change in survival was observed after primary exposure of P. scaber to tire particles or chlorpyrifos. However, primary exposure to chlorpyrifos triggered a strong activation of the immune response, which was not the case following exposure to the tire particles. Further injection of lipopolysaccharide into the body did not affect the survival of animals exposed to tire particles or chlorpyrifos, while a strong immunomodulatory change was observed, particularly with chlorpyrifos, and to some extent, tire particles. Based on these results, we conclude that exposure of P. scaber to tire particles or chlorpyrifos has no significant effect on the susceptibility of the organism to lipopolysaccharide in terms of their mortality, but primary exposure to an insecticide significantly modulates the immune response of the organisms to a second stressor. We discuss the “stress on stress” approach for testing low-toxic substances, such as microplastics, where an environmentally realistic exposure is followed by a secondary exposure.

Nitrite Attenuates the In Vitro Inflammatory Response of Immune Cells to the SARS-CoV-2 S Protein without Interfering in the Antioxidant Enzyme Activation.

SARS-CoV-2 induces a hyperinflammatory reaction due to the excessive release of cytokines during the immune response. The bacterial endotoxin lipopolysaccharide (LPS) contributes to the low-grade inflammation associated with the metabolic syndrome, enhancing the hyperinflammatory reaction induced by the SARS-CoV-2 infection. The intake of sodium nitrate, a precursor of nitrite and nitric oxide, influences the antioxidant and pro-inflammatory gene expression profile after immune stimulation with LPS in peripheral blood mononuclear cells from metabolic syndrome patients. We aimed to assess the inflammatory and antioxidant responses of immune cells from metabolic syndrome patients to exposure to the SARS-CoV-2 spike protein (S protein) together with LPS and the effect of nitrite in these responses. Whole blood samples obtained from six metabolic syndrome patients were cultured for 16 h at 37 °C with four different media: control medium, control medium plus LPS (100 ng/mL), control medium plus LPS (100 ng/mL) plus S protein (10 ng/mL), and control medium plus LPS (100 ng/mL) plus S protein (10 ng/mL) plus nitrite (5 µM). Immune stimulation with the LPS/S protein enhanced nitrate biosynthesis from nitrite oxidation and probably from additional organic precursors. In vitro incubations with the LPS/S protein enhanced the expression and/or release of pro-inflammatory TNFα, IL-6, IL-1β, and TLR4, as well as the expression of the anti-inflammatory IL-1ra and IL-10 and antioxidant enzymes. Nitrite attenuated the pro- and anti-inflammatory response induced by the S protein without interfering with the activation of TLR4 and antioxidant enzyme expression, raising the possibility that nitrite could have potential as a coadjutant in the treatment of COVID-19.

Electrochemical detection of bacterial endotoxin lipopolysaccharide (LPS) on gold electrode modified with DAL-PEG-DK5-PEG-OH - Antimicrobial peptide conjugate

This work describes fabrication of gold electrodes modified with peptide conjugate DAL-PEG-DK5-PEG-OH that enables ultra-sensitive detection of lipopolysaccharide (LPS) isolated from the reference strain of Escherichia coli O26:B6. The initial step of the established procedure implies immobilization of the fully protected DAL-PEG-DK5-PEG-OH peptide on the surface of the gold electrode previously modified by cysteamine. Then side chain- and Fmoc-deprotection was performed in situ on the electrode surface, followed by its incubation in 1 % of BSA solution to block non-specific bindings sites before LPS detection. The efficiency of the modification was confirmed by X-ray Photoelectron Spectroscopy (XPS) measurements. Additionally, the cyclic voltammetry (CV) and electrochemical impendance spectroscopy (EIS) were employed to monitor the effectiveness of each step of the modification. The obtained results confirmed that the presence of the surface-attached covalently bound peptide DAL-PEG-DK5-PEG-OH enables LPS detection by means of CV technique within the range from 5 × 10−13 to 5 × 10−4 g/mL in PBS solution. The established limit of detection (LOD) for EIS measurements was 4.93 × 10−21 g/mL with wide linear detection range from 5 × 10−21 to 5 × 10−14 g/mL in PBS solution. Furthermore, we confirmed the ability of the electrode to detect LPS in a complex biological samples, like mouse urine and human serum. The effectiveness of the electrodes in identifying LPS in both urine and serum matrices was confirmed for samples containing LPS at both 2.5 × 10−15 g/mL and 2.5 × 10−9 g/mL.

The effect of repeated lipopolysaccharide endotoxin challenge on immune response of breeding ewes and subsequent lamb performance.

Infectious disease caused by exposure to Gram-negative bacterial endotoxin lipopolysaccharide (LPS) is recognized to suppress female fertility. However, the effect of varying low-dose endotoxin exposure during distinct stages of follicle development on immune response, reproductive performance, and lamb performance has yet to be elucidated. Therefore, the objective of this study was to evaluate acute phase response, mRNA abundance of inflammatory markers, reproductive performance and lamb growth characteristics of ewes challenged with subclinical doses of LPS. Rambouillet ewes (n = 36; 68.2±1.1kg; age 3 to 7 yr) stratified by body weight (BW) and age were assigned to treatment groups. Ewes received subcutaneous injections of saline (CON, n = 12), 1.5 µg/kg BW LPS (LOW, n = 12), or 3.0 µg/kg BW LPS (HIGH, n = 12) on days 5, 10, and 15 of a synchronized follicular wave. Ewes were subsequently placed with a raddle-painted ram on day 16 for a 35-d breeding season. On treatment days 5 and 15, blood samples, peripheral blood leukocytes, and rectal temperature were collected before and at regular intervals for 12h after LPS challenge. Immune response to LPS was confirmed by increased temperature and serum cortisol concentrations on days 5 and 15. Endotoxin increased circulating plasma concentration of the acute phase protein, haptoglobin by greater than 15%, in both LPS-treated groups on days 5 and 15 at 12h compared with control (P≤ 0.05). Pro- and anti-inflammatory mRNA gene expression demonstrated no differences in expression for tumor necrosis factor-α or peroxisome proliferator-activated receptor gamma among treatment groups (P > 0.10). Likewise, Toll-like receptor 4 (TLR4), interleukin-8 (IL-8), and superoxide dismutase 2 (SOD2) expression was similar among treatment groups on day 5. However, ewes challenged with LPS on day 15 displayed greater mRNA expression for TLR4 from 2 to 6h (P < 0.05), a 7-fold increase for IL-8 from 1.5 to 2.5h (P < 0.05), and 8-fold induction for SOD2 from 2 to 6h (P < 0.05) as compared with controls. First service conception rates were 90% for control ewes and 75% for both treated groups (P = 0.84). Treated ewes demonstrated a reduction in lamb birth weight compared with controls (P ≤ 0.05) and a tendency for reduction of 60-d adjusted weaning weight (P = 0.09). Data suggest that subacute endotoxin exposure aligning with key follicle and oocyte maturation events results in detrimental growth performance of the subsequent lamb.

Insights into colistin-mediated fluorescence labelling of bacterial LPS.

Gram-negative bacterial infections are becoming untreatable due to their ability to mutate, and the gradual development of their resistance to the available antimicrobials. In recent times colistin, a drug of last resort, started losing its efficacy towards multidrug-resistant bacterial infections. Colistin targets bacterial endotoxin lipopolysaccharides (LPS) and destabilises the cytoplasmic membrane by disrupting the outer LPS membrane. In this study, we have tried to label the bacterial LPS, the main constituent of the cytoplasmic membrane of bacterial cells, to try to understand the interaction mechanism of LPS with colistin. The chemosensor, naphthaldehyde appended furfural (NAF) that selectively recognises colistin can label LPS, by showing its fluorescence signals. The computationally derived three-dimensional structure of LPS has been introduced to speculate on the possible binding mode of colistin with LPS, and this was also thoroughly studied with the help of quantum mechanics and molecular dynamics energy minimisation. Fluorescence microscopy and FE-SEM microscopic studies were also used to observe the change in the structural morphology of colistin-sensitive and resistant Salmonella typhi in different experimental conditions.

Sleep deprivation reduced LPS-induced IgG2b production by up-regulating BMAL1 and CLOCK expression

Sleep deprivation (SD) weakens the immune system and leads to increased susceptibility to infectious or inflammatory diseases. However, it is still unclear how SD affects humoral immunity. In the present study, sleep disturbance was conducted using an sleep deprivation instrument, and the bacterial endotoxin lipopolysaccharide (LPS) was used to activate the immune response. It was found that SD-pretreatment reduced LPS-induced IgG2b+ B cells and IgG2b isotype antibody production in lymphocytes of spleen. And, SD-pretreatment decreased the proportion of CD4+T cells, production of CD4+T cells derived TGF-β1 and its contribution in helping IgG2b production. Additionally, BMAL1 and CLOCK were selectively up-regulated in lymphocytes after SD. Importantly, BMAL1 and CLOCK deficiency contributed to TGF-β1 expression and production of IgG2b+ B cells. Thus, our results provide a novel insight to explain the involvement of BMAL1 and CLOCK under SD stress condition, and their roles in inhibiting TGF-β1 expression and contributing to reduction of LPS induced IgG2b production.

Role of microRNA, bta-miR-375 in Immune Sturdiness of Vechur: The Native Cattle Breed of Kerala, India #

In the present study, next generation sequencing was employed to identify and explore the differential expression profiles of microRNAs (miRNAs) in peripheral blood mononuclear cells (PBMCs) of crossbred (B. taurus x B. indicus) and Vechur (B. indicus) cattle in response to the bacterial endotoxin-lipopolysaccharide (LPS). The PBMCs from adult apparently healthy female crossbred cows and Vechur cattle, a native cattle breed of Kerala, India were stimulated with 10 μg/mL of LPS for 6 h. Among the differentially expressed miRNAs, the expression of 13 miRNAs showed statistically significant up regulation while, significant decrease in the expression of 15 miRNAs was noticed in LPS treated PBMCs of Vechur cattle compared to crossbred cows. The expression profiling of miRNA, bta-miR-375, expression of which was found to be significantly down regulated in LPS treated PBMCs of Vechur cattle with respect to crossbred cattle by the NGS studies, is presented in the present manuscript. The decrease in expression of bta-miR-375 noticed by NGS was in accordance with the results of quantitative real time PCR assay. Functional gene enrichment analysis and pathway analysis revealed significant enrichment of predicted targets of bta-miR-375 in many immune related and cell signalling mechanisms. In addition, over representation of targets of bta-miR-375 was also noticed in pathogenesis of many of the bovine diseases. The study could also identify differences in the expression of cytokines, viz. Tumour Necrosis Factor Alpha (TNFα), Interleukin 4 (IL-4) and Interferon-γ (IFNγ) between LPS treated and untreated PBMCs of crossbred and Vechur cattle.

240 Prolonged Maternofetal Inflammation in the Pregnant Ewe at Mid-Gestation Increases Subsequent Fetal Inflammatory Tone and Disrupts Skeletal Muscle Growth and Glucose Metabolism Near Term

Abstract Chronic maternofetal inflammation in late gestation (i.e., peak fetal growth) causes fetal intrauterine growth restriction (IUGR) in sheep, which was associated with deficits in growth, body composition, and metabolic function before and after birth. However, the impact of sustained maternofetal inflammation during mid-gestation (i.e., peak placental growth and development) has not been explored. Therefore, our objective was to determine the effects of sustained maternofetal inflammation during this critical window on subsequent fetal inflammatory tone, metabolism, and myoblast (i.e., muscle stem cell) function. Pregnant ewes were injected every 3rd day from the 50th to 65th d of gestational age (dGA; term = 150 dGA) with saline (control; n = 12) or bacterial lipopolysaccharide endotoxin to induce maternofetal inflammation and IUGR (MI-IUGR; n = 12). Fetuses were surgically catheterized on dGA 118 to collect blood samples, and a hyperinsulinemic-euglycemic clamp (HEC) was performed on dGA 123. Fetuses were necropsied on dGA 125 to collect semitendinosus muscle samples. Fetal plasma TNFα concentrations tended to be greater (P = 0.07) for MI-IUGR fetuses (440 ± 45 pg/m l) than for controls (348 ± 24 pg/mL). Fetal blood O2 partial pressures were 12% less (P &amp;lt; 0.05) and maternofetal O2 gradients tended to be 11% greater (P = 0.08) for MI-IUGR fetuses than for controls. Hindlimb-specific glucose uptake rates did not differ between fetal groups, but hindlimb-specific glucose oxidation rates were reduced (P &amp;lt; 0.05) by 15% under basal conditions and by 27% under hyperinsulinemic conditions for MI-IUGR fetuses compared with controls. Semitendinosus glycogen content and β2 adrenoceptor content did not differ between fetal groups, but semitendinosus content of the TNFα receptor, TNFR1, was 41% greater (P &amp;lt; 0.05) for MI-IUGR fetuses than for controls. Immunohistochemistry revealed 18% fewer (P &amp;lt; 0.05) proliferating myoblasts (pax7+/Ki67+) and 38% fewer (P &amp;lt; 0.05) differentiated myoblasts (myogenin+) in semitendinosus from MI-IUGR fetuses compared with controls, which coincided with 13% smaller (P &amp;lt; 0.05) muscle fibers. From these findings, we conclude that sustained maternofetal inflammation during peak placental development at mid-gestation resulted in enhanced fetal inflammatory tone near term, which in turn impaired myoblast function, muscle fiber hypertrophy, and muscle glucose metabolism.

Effect of functionalized titanium particles with dexamethasone-loaded nanospheres on macrophage polarization and activity

ObjectiveThe aim of this study was to determine the effect of titanium micro particles (TiP) previously functionalized with nanoparticles doped with dexamethasone (Dex) and doxycycline (Dox), on macrophage polarization and activity. Methods: Macrophages RAW264.7 were cultured in the presence TiP loaded with dexamethasone -NPs (Dex)- and doxycycline -NPs (Dox)-, and as control, TiP with or without doped NPs. Cells were tested with and without previous bacterial lipopolysaccharide endotoxin (LPS) stimulation. Their morphology, proliferation, cytotoxicity, phenotypic change, and cytokines release were assessed by LIVE/DEAD, DNA release, metabolic activity, brightfield and scanning electron microscopy. The test Kruskall-Wallis was used for comparisons, while the cytokine expression profiles were examined by hierarchical clustering (p < 0.05). Results: Upon exposure with TiP macrophages were activated and polarized to M1, but without depicting cytotoxic effects. The particles were phagocytised, and vacuolized. When exposed to functionalised TiP with NPs(Dex) and NPs(Dox), the ratio M1/M2 was up to forty times lower compared to TiP alone. When exposed to LPS, TiP reduced cell viability in half. Functionalised TiP with NPs(Dex) inhibited the cytokine release exerted by TiP on macrophages. When macrophages were exposed to functionalised TiPs with NPs(Dex) with and without LPS, the effect of TiP on cytokine secretion was inhibited. Significance: Functionalised TiPs with NPs(Dex) and NPs(Dox) may potentially have beneficial effects on modulating titanium and LPS-related inflammatory reactions.

Evaluation of Immunomodulatory Effects of Fusarium Mycotoxins Using Bacterial Endotoxin-Stimulated Bovine Epithelial Cells and Macrophages in Co-Culture.

Mycotoxins are secondary metabolites produced by a variety of fungi that contaminate animal food and feeds and are capable of inducing a wide range of toxicities. Predictive in vitro models represent valuable substitutes for animal experiments to assess the toxicity of mycotoxins. The complexities of the interactions between epithelial and innate immune cells, vital for upholding barrier integrity and averting infections, remain inadequately understood. In the current study, a co-culture model of bovine epithelial cells (MAC-T) and macrophages (BoMac) was used to investigate the impact of exposure to Fusarium mycotoxins, namely deoxynivalenol (DON), zearalenone (ZEN), enniatin B (ENB), and beauvericin (BEA), on the inflammatory response elicited by the bacterial lipopolysaccharide (LPS) endotoxin. The MAC-T cells and BoMac were seeded on the apical side of a Transwell membrane and in the lower chamber, respectively, and mycotoxin exposure on the apical side of the membrane was carried out with the different mycotoxins (LC20; concentrations that elicited 20% cytotoxicity) for 48 h followed by an LPS immunity challenge for 24 h. The culture supernatants were collected from the basolateral compartment and these samples were submitted for cytokine/chemokine multiplex analysis. RNA-Seq analysis was performed using total RNA extracted from the MAC-T cells to acquire a more detailed insight into their cellular functions. The multiplex analysis indicated that IFN-γ, IL-1α, IL-8, and MCP-1 were significantly induced post-DON treatment when compared to control cells, and levels of IL-1α and IL-8 were enhanced significantly in all mycotoxin-treated groups post-LPS challenge. Analysis of the sequencing results showed that there were 341, 357, and 318 differentially expressed MAC-T cell genes that were up-regulated in the DON, ENB, and BEA groups, respectively. Gene ontology and pathway analysis revealed that these DEGs were significantly enriched in various biological processes and pathways related to inflammation, apoptosis signaling, and Wnt signaling. These results provide a comprehensive analysis of the co-culture cytokine/chemokine production and MAC-T cells' gene expression profiles elicited by Fusarium mycotoxins, which further contributes to the understanding of early endotoxemia post-mycotoxin exposure.

Assessing Larval Zebrafish Survival and Gene Expression Following Sodium Butyrate Exposure and Subsequent Lethal Bacterial Lipopolysaccharide (LPS) Endotoxin Challenge.

As aquaculture production continues to grow, producers are looking for more sustainable methods to promote growth and increase fish health and survival. Butyrate is a short-chain fatty acid (SCFA) with considerable benefits to gut health, and in recent years, butyrate has been commonly used as an alternative to antimicrobials in livestock production. In this study, we aimed to assess the protective effects of sodium butyrate (NaB) on larval zebrafish subjected to a lethal Pseudomonas aeruginosa lipopolysaccharide (LPS) endotoxin challenge and to elucidate potential protective mechanisms of action. Larval zebrafish were pre-treated with 0, 3000, or 6000 μM NaB for 24 h at 72 h post-fertilization (hpf), then immune challenged for 24 h with 60 μg/mL of LPS at 96 hpf. Our results demonstrate that larval zebrafish pre-treated with 6000 μM of NaB prior to lethal LPS challenge experienced significantly increased survival by 40%, and this same level of NaB significantly down-regulated the expression of pro-inflammatory Tumor Necrosis Factor α (TNF-alpha). Findings from this study are consistent with the beneficial effects of NaB on other vertebrate species and support the potential use of NaB in aquaculture.

Tolterodine ameliorates inflammatory response and ferroptosis against LPS in human bladder epithelial cells.

Bacterial endotoxin lipopolysaccharide (LPS)-induced inflammatory response and ferroptosis play an important role in urinary tract infections. Tolterodine has been used as a urinary tract antispasmodic and anticholinergic agent. However, the effects of Tolterodine against LPS-induced insults in human bladder epithelial cells (hBECs) have not been reported before. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release assays to determine the cell viability, reactive oxygen species (ROS) and malondialdehyde level detection were used to determine the level of oxidative stress, enzyme-linked immunosorbent assay and Western blot analysis were used to detect the protein level. In the current study, we found that Tolterodine ameliorated LPS-induced production of ROS and lipid oxidation in hBECs. Interestingly, Tolterodine inhibited the production of interleukin 6, interleukin-1β, and tumor necrosis factor α. Also, Tolterodine reduced the levels of Fe2+ and suppressed ferroptosis by reducing the levels of glutathione peroxidase 4, prostaglandin-endoperoxide synthase 2, and acyl-CoA synthetase long-chain family member 4 in LPS-challenged bladder epithelial cells. Mechanistically, it was shown that Tolterodine restored the nuclear factor E2-related factor 2 (Nrf2)/nuclear factor-κB signaling. Importantly, inhibition of Nrf2 with its specific inhibitor ML385 abolished the protective effects of Tolterodine in the inflammatory response and ferroptosis, suggesting that the effects of Tolterodine are mediated by Nrf2. Based on these findings, we conclude that Tolterodine might serve as a promising agent for the treatment of LPS-induced bladder inflammation.

Bacterial endotoxin lipopolysaccharides regulate gene expression in human colon cancer cells

ObjectiveLipopolysaccharide (LPS) is a major cell wall component of gram-negative bacteria. Colon bacteria contribute to LPS which promotes colon cancer metastasis. The objective of this study was to survey the effect of LPS on cell viability and gene expression of 55 molecular targets in human colon cancer cells.ResultsLPS did not affect the viability of COLO 225 cells under the culture conditions but affected the expression of a number of genes important in inflammatory responses and cancer development. LPS increased TTP family, GLUT family and DGAT1 mRNA levels but decreased DGAT2a and DGAT2b expression in the human colon cancer cells. LPS also increased COX2, CXCL1, ELK1, ICAM1, TNFSF10 and ZFAND5 but decreased BCL2L1, CYP19A1 and E2F1 mRNA levels in the colon cancer cells. These data suggest that LPS has profound effects on gene expression in human colon cancer cells.