In Vitro Assessment of Anti-Adipogenic and Anti-Inflammatory Properties of Black Cumin (Nigella sativa L.) Seeds Extract on 3T3-L1 Adipocytes and Raw264.7 Macrophages.

Background and Objectives: This study evaluated the in vitro anti-adipogenic and anti-inflammatory properties of black cumin (Nigella sativa L.) seed extract (BCS extract) as a potential candidate for developing herbal formulations targeting metabolic disorders. Materials and Methods: We evaluated the BCS extract by assessing its 2,2-diphenyl-1-picrohydrazyl (DPPH) radical scavenging activity, levels of prostaglandin E2 (PGE2) and nitric oxide (NO), and mRNA expression levels of key pro-inflammatory mediators. We also quantified the phosphorylation of nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and mitogen-activated protein kinases (MAPK) signaling molecules. To assess anti-adipogenic effects, we used differentiated 3T3-L1 cells and BCS extract in doses from 10 to 100 µg/mL. We also determined mRNA levels of key adipogenic genes, including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/BEPα), adipocyte protein 2 (aP2), lipoprotein lipase (LPL), fatty acid synthase (FAS), and sterol-regulated element-binding protein 1c (SREBP-1c) using real-time quantitative polymerase chain reaction (qPCR). Results: This study showed a concentration-dependent DPPH radical scavenging activity and no toxicity at concentrations up to 30 µg/mL in Raw264.7 cells. BCS extract showed an IC50 of 328.77 ± 20.52 µg/mL. Notably, pre-treatment with BCS extract (30 µg/mL) significantly enhanced cell viability in lipopolysaccharide (LPS)-treated Raw264.7 cells. BCS extract treatment effectively inhibited LPS-induced production of PGE2 and NO, as well as the expression of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), inducible NO synthase (iNOS), interleukin (IL)-1ß and IL-6, possibly by limiting the phosphorylation of p38, p65, inhibitory κBα (I-κBα), and c-Jun N-terminal kinase (JNK). It also significantly attenuated lipid accumulation and key adipogenic genes in 3T3-L1 cells. Conclusions: This study highlights the in vitro anti-adipogenic and anti-inflammatory potential of BCS extract, underscoring its potential as a promising candidate for managing metabolic disorders.

Assessment of Nonalcoholic Fatty Liver Disease Symptoms and Gut-Liver Axis Status in Zebrafish after Exposure to Polystyrene Microplastics and Oxytetracycline, Alone and in Combination.

BACKGROUND: Environmental pollution may give rise to the incidence and progression of nonalcoholic fatty liver disease (NAFLD), the most common cause for chronic severe liver lesions. Although knowledge of NAFLD pathogenesis is particularly important for the development of effective prevention, the relationship between NAFLD occurrence and exposure to emerging pollutants, such as microplastics (MPs) and antibiotic residues, awaits assessment. OBJECTIVES: This study aimed to evaluate the toxicity of MPs and antibiotic residues related to NAFLD occurrence using the zebrafish model species. METHODS: Taking common polystyrene MPs and oxytetracycline (OTC) as representatives, typical NAFLD symptoms, including lipid accumulation, liver inflammation, and hepatic oxidative stress, were screened after 28-d exposure to environmentally realistic concentrations of MPs (0.69mg/L) and antibiotic residue (3.00µg/L). The impacts of MPs and OTC on gut health, the gut-liver axis, and hepatic lipid metabolism were also investigated to reveal potential affecting mechanisms underpinning the NAFLD symptoms observed. RESULTS: Compared with the control fish, zebrafish exposed to MPs and OTC exhibited significantly higher levels of lipid accumulation, triglycerides, and cholesterol contents, as well as inflammation, in conjunction with oxidative stress in their livers. In addition, a markedly smaller proportion of Proteobacteria and higher ratios of Firmicutes/Bacteroidetes were detected by microbiome analysis of gut contents in treated samples. After the exposures, the zebrafish also experienced intestinal oxidative injury and yielded significantly fewer numbers of goblet cells. Markedly higher levels of the intestinal bacteria-sourced endotoxin lipopolysaccharide (LPS) were also detected in serum. Animals treated with MPs and OTC exhibited higher expression levels of LPS binding receptor (LBP) and downstream inflammation-related genes while also exhibiting lower activity and gene expression of lipase. Furthermore, MP-OTC coexposure generally exerted more severe effects compared with single MP or OTC exposure. DISCUSSION: Our results suggested that exposure to MPs and OTC may disrupt the gut-liver axis and be associated with NAFLD occurrence. https://doi.org/10.1289/EHP11600.

Heme oxygenase-1 ameliorates endotoxin-induced acute lung injury by modulating macrophage polarization via inhibiting TXNIP/NLRP3 inflammasome activation.

Acute lung injury (ALI) remains a global public health issue without specific and effective treatment options available in the clinic. Alveolar macrophage polarization is involved in the initiation, development and progression of ALI; however, the underlying mechanism remains poorly understood. Heme oxygenase-1 (HO-1) acts as an antioxidant in pulmonary inflammation and has been demonstrated to be linked with the severity and prognosis of ALI. In this study, the therapeutic effects of HO-1 were examined, along with the mechanisms involved, mainly focusing on alveolar macrophage polarization. HO-1 depletion induced higher iNOS and CD86 (M1 phenotype) expression but was significantly decreased in Arg-1 and CD206 (M2 phenotype) expression in BALF alveolar macrophages after equivalent LPS stimulation. We also found that HO-1 deletion distinctly accelerated the expression of inflammasome-associated components NLRP3, ASC and caspase-1 in vivo and in vivo and in vitro. Moreover, on the basis of LPS for MH-S cells, levels of TXNIP, NLRP3, ASC and caspase-1 were increased and HO-1 depletion exacerbated these changes, whereas double depletion of HO-1 and TXNIP partially mitigated these elevations. Also, HO-1 knockdown induced more M1 phenotype and less M2 phenotype compared with LPS alone, whereas double silence of HO-1 and TXNIP partially changed the polarization state. Taken together, we demonstrated that HO-1 could modulate macrophage polarization via TXNIP/NLRP3 signaling pathway, which could be a potential therapeutic target for ALI treatment.

AdMSC-derived exosomes alleviate acute lung injury via transferring mitochondrial component to improve homeostasis of alveolar macrophages.

Rationale: Aberrant activation of macrophages with mitochondria dismiss was proved to be associated with pathogenesis of ALI (acute lung injury). Exosomes from adipose-derived mesenchymal stem cells (AdMSC-Exos) have been distinguished by their low immunogenicity, lack of tumorigenicity, and high clinical safety, but their role in treating ALI and the mechanism involved need to be defined. In this study, we sought to investigate whether the mitochondrial donation from AdMSC-Exos provides profound protection against LPS-induced ALI in mice, accompanied by improvement of macrophage mitochondrial function. Methods: C57BL/6 mice were orotracheally instilled with LPS (1 mg/kg). AdMSC-Exos were administered via the tail vein 4 h after LPS inhalation. Flow cytometry, H&E, Quantitative Real-Time PCR, immunofluorescence (IF), confocal microscopy imaging was conducted to investigate lung tissue inflammation and macrophage mitochondrial function. And further observe the transfer of exosomes and the effect on mitochondrial function of MH-S cells through in vitro experiments. Results: AdMSC-Exos can transfer the stem cell-derived mitochondria components to alveolar macrophages in a dose-dependent manner. Likely through complementing the damaged mitochondria, AdMSC-Exos exhibited the ability to elevate the level of mtDNA, mitochondrial membrane potential (MMP), OXPHOS activity and ATP generation, while reliving mROS stress in LPS-challenged macrophages. Restoring mitochondrial integrity via AdMSC-Exos treatment enabled macrophages shifting to anti-inflammatory phenotype, as featured with the down-regulation of IL-1ß, TNF-α and iNOS secretion and increase in production of anti-inflammatory cytokines IL-10 and Arg-1. As we depleted alveolar macrophages using clodronate liposomes, the protective role for AdMSC-Exos was largely abrogated. Conclusions: AdMSC-Exos can effectively donate mitochondria component improved macrophages mitochondrial integrity and oxidative phosphorylation level, leading to the resumption of metabolic and immune homeostasis of airway macrophages and mitigating lung inflammatory pathology.

Peripheral versus central venous blood sampling does not influence the assessment of platelet activation in cirrhosis.

Cirrhotic patients have an increased risk of bleeding and thromboembolic events, with platelets being involved as key players in both situations. The impact of peripheral versus central blood sampling on platelet activation remains unclear. In 33 cirrhotic patients, we thus analyzed platelet function in peripheral (P) and central (C) blood samples. Platelet surface expression of P-selectin, activated glycoprotein (GP) IIb/IIIa, and leukocyte-platelet aggregate formation were measured by flow cytometry in response to different agonists: thrombin receptor-activating peptide-6, adenosine diphosphate, collagen-related peptide (CrP), epinephrine, AYPGKF, Pam3CSK4, and lipopolysaccharide. Unstimulated platelet surface expression of P-selectin (p = .850) and activated GPIIb/IIIa (p = .625) were similar in peripheral and central blood samples. Stimulation with various agonists yielded similar results of platelet surface expression of P-selectin and activated GPIIb/IIIa in peripheral and central samples, except for CrP-inducible expression of activated GPIIb/IIIa (median fluorescence intensity, MFI in P: 7.61 [0.00-24.66] vs. C: 4.12 [0.00-19.04], p < .001). The formation of leukocyte-platelet aggregate was similar in central and peripheral blood samples, both unstimulated and after stimulation with all above-mentioned agonists. In conclusion, peripheral vs. central venous blood sampling does not influence the assessment of platelet activation by flow cytometry in cirrhosis.Abbreviations: ACLD: advanced chronic liver disease; ADP: adenosine diphosphate; ALD: alcoholic liver disease; AYPGKF: PAR-4 agonist AYPGKF; CrP: collagen related protein; EPI: epinephrine; FACS: fluorescence-activated cell sorting; GP: glycoprotein; HVPG: hepatic venous pressure gradient; IQR: interquartile range; LPS: lipopolysaccharide; LSM: liver stiffness measurement; MFI: median fluorescence intensity; NAFLD: nonalcoholic fatty liver disease; PAM: lipopeptide Pam3CSK4; PAR: protease-activated receptor; PBS: phosphate-buffered saline; PH: portal hypertension; TIPS: transjugular intrahepatic portosystemic stent shunt; TLR: toll-like receptor; TRAP-6: thrombin receptor-activator peptide-6; vWF: von Willebrand factor.

Racial differences across pregnancy in maternal pro-inflammatory immune responsivity and its regulation by glucocorticoids.

BACKGROUND: The distribution of adverse pregnancy, birth and subsequent child developmental and health outcomes in the U.S. is characterized by pronounced racial (particularly Black-white) disparities. In this context, chronic stress exposure represents a variable of considerable importance, and the immune/inflammatory system represents a leading candidate biological pathway of interest. Previous pregnancy studies examining racial disparities in immune processes have largely utilized circulating cytokine levels, and have yielded null or mixed results. Circulating cytokines primarily represent basal secretion and do not necessarily represent functional features of immune responsivity and regulation. Thus, in order to conduct a more in-depth characterization of racial differences in functional immune properties during pregnancy, we utilized an ex vivo stimulation assay, a dynamic measure of immune function at the cellular level, to investigate Black-white racial differences in in mid- and late-gestation in i) pro-inflammatory (IL-6) responsivity of leukocytes to antigen [lipopolysaccharide (LPS)] challenge, and ii) regulation (dampening) of this pro-inflammatory response by glucocorticoids. METHOD: 177 women (N = 42 Black (24%), n = 135 white (76%)) with a singleton, intrauterine pregnancy provided 20 mL venous blood in mid- (16.6 ± 2.4 wks) and late (33.3 ± 1.1 wks) pregnancy. Maternal pro-inflammatory responsivity of leukocytes was quantified by assessing the release of the pro-inflammatory cytokine IL-6 in response to LPS stimulation, and regulation of the pro-inflammatory response was quantified by assessing the suppression of the stimulated IL-6 response after co-incubation with progressively increasing levels of dexamethasone [10-7, 10-6, 10-5 M] (i.e., glucocorticoid receptor resistance (GRR)). A priori model covariates included maternal age, parity, SES (socioeconomic status), and pre-pregnancy BMI. RESULTS: Maternal pro-inflammatory responsivity (LPS-stimulated IL-6) and GRR increased significantly across mid- and late gestation (adjusted ß = 0.157, p = 0.007; ß = 0.627, p < 0.001, respectively). Across both time points in pregnancy Black women exhibited significantly higher LPS-stimulated IL-6 release and reduced glucocorticoid regulation of the IL-6 response (i.e., higher GRR) relative to white women, before and after adjusting for covariates (ß = 0.381, p = 0.0030; ß = 0.391, p = 0.0075, respectively). There was no racial difference in the concentrations of circulating IL-6 (p = 0.9199). CONCLUSION: Our findings support the hypothesis postulating significant racial (Black-white) differences in key functional properties of the maternal immune system in pregnancy, which were not apparent using circulating cytokine measures. These data elucidate a potentially important physiological mechanism underlying the transduction of environmental conditions into racial disparities in reproductive and subsequent child health outcomes, and the use of these ex vivo measures should be considered in future studies.

3-O-trans-caffeoyloleanolic acid improves acute lung injury via anti-inflammation and antioxidative stress-involved PI3K/AKT pathway.

3-O-trans-caffeoyloleanolic acid (COA) is a pentacyclic triterpenoid compound, with significant anti-inflammatory effects. In this study, we report the protective effects of COA on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and explored its mechanism of action. LPS was used to construct in vivo mouse ALI models to observe the effects of COA pretreatment on lung pathology, inflammation, and oxidative stress. In vitro, mouse alveolar macrophages MH-S cells were cultured and stimulated with LPS to investigate the effects of COA pretreatment on inflammation and oxidative stress. Western blotting was used to investigate the expression of iNOS, TLR4, p-p65, p-AKT, and p-PI3K from in vivo and in vitro samples. The results showed that COA significantly improved lung injury, inhibited neutrophil infiltration, prevented macrophage infiltration, inhibited the release of inflammatory factors, reduced oxidative stress, and down-regulated the expression of iNOS, TLR4, p-p65, p-AKT, and p-PI3K in ALI mice caused by LPS. In vitro, COA inhibited the release of inflammatory factors, reduced oxidative stress, and down-regulated the expression of iNOS, TLR4, p-p65, p-AKT, and p-PI3K in MH-S cells stimulated with LPS. Of interest, the protective effects of COA were significantly attenuated in MH-S cells pretreated with the PI3K phosphopeptide activator 740Y-P with no effect on TLR4 expression observed. Taken together, these findings confirm the protective effects of COA on ALI. We further demonstrate that the anti-inflammation and antioxidant effects of COA are mediated through its effects on PI3K/AKT and potentially TLR4.

Gut Dysbiosis Dysregulates Central and Systemic Homeostasis via Suboptimal Mitochondrial Function: Assessment, Treatment and Classification Implications.

The gut and mitochondria have emerged as two important hubs at the cutting edge of research across a diverse array of medical conditions, including most psychiatric conditions. This article highlights the interaction of the gut and mitochondria over the course of development, with an emphasis on the consequences for transdiagnostic processes across psychiatry, but with relevance to wider medical conditions. As well as raised levels of circulating lipopolysaccharide (LPS) arising from increased gut permeability, the loss of the short-chain fatty acid, butyrate, is an important mediator of how gut dysbiosis modulates mitochondrial function. Reactive cells, central glia and systemic immune cells are also modulated by the gut, in part via impacts on mitochondrial function in these cells. Gut-driven alterations in the activity of reactive cells over the course of development are proposed to be an important determinant of the transdiagnostic influence of glia and the immune system. Stress, including prenatal stress, also acts via the gut. The suppression of butyrate, coupled to raised LPS, drives oxidative and nitrosative stress signalling that culminates in the activation of acidic sphingomyelinase-induced ceramide. Raised ceramide levels negatively regulate mitochondrial function, both directly and via its negative impact on daytime, arousal-promoting orexin and night-time sleep-promoting pineal gland-derived melatonin. Both orexin and melatonin positively regulate mitochondria oxidative phosphorylation. Consequently, gut-mediated increases in ceramide have impacts on the circadian rhythm and the circadian regulation of mitochondrial function. Butyrate, orexin and melatonin can positively regulate mitochondria via the disinhibition of the pyruvate dehydrogenase complex, leading to increased conversion of pyruvate to acetyl- CoA. Acetyl-CoA is a necessary co-substrate for the initiation of the melatonergic pathway in mitochondria and therefore the beneficial effects of mitochondria melatonin synthesis on mitochondrial function. This has a number of treatment implications across psychiatric and wider medical conditions, including the utilization of sodium butyrate and melatonin. Overall, gut dysbiosis and increased gut permeability have significant impacts on central and systemic homeostasis via the regulation of mitochondrial function, especially in central glia and systemic immune cells.

Biology of the Gonococcus: Disease and Pathogenesis.

Neisseria gonorrhoeae infection is a major public health problem worldwide. The increasing incidence of gonorrhea coupled with global spread of multidrug-resistant isolates of gonococci has ushered in an era of potentially untreatable infection. Gonococcal disease elicits limited immunity, and individuals are susceptible to repeated infections. In this chapter, we describe gonococcal disease and epidemiology and the structure and function of major surface components involved in pathogenesis. We also discuss the mechanisms that gonococci use to evade host immune responses and the immune responses following immunization with selected bacterial components that may overcome evasion. Understanding the biology of the gonococcus may aid in preventing the spread of gonorrhea and also facilitate the development of gonococcal vaccines and treatments.

Bioprinted 3D Primary Liver Tissues Allow Assessment of Organ-Level Response to Clinical Drug Induced Toxicity In Vitro.

Modeling clinically relevant tissue responses using cell models poses a significant challenge for drug development, in particular for drug induced liver injury (DILI). This is mainly because existing liver models lack longevity and tissue-level complexity which limits their utility in predictive toxicology. In this study, we established and characterized novel bioprinted human liver tissue mimetics comprised of patient-derived hepatocytes and non-parenchymal cells in a defined architecture. Scaffold-free assembly of different cell types in an in vivo-relevant architecture allowed for histologic analysis that revealed distinct intercellular hepatocyte junctions, CD31+ endothelial networks, and desmin positive, smooth muscle actin negative quiescent stellates. Unlike what was seen in 2D hepatocyte cultures, the tissues maintained levels of ATP, Albumin as well as expression and drug-induced enzyme activity of Cytochrome P450s over 4 weeks in culture. To assess the ability of the 3D liver cultures to model tissue-level DILI, dose responses of Trovafloxacin, a drug whose hepatotoxic potential could not be assessed by standard pre-clinical models, were compared to the structurally related non-toxic drug Levofloxacin. Trovafloxacin induced significant, dose-dependent toxicity at clinically relevant doses (≤ 4uM). Interestingly, Trovafloxacin toxicity was observed without lipopolysaccharide stimulation and in the absence of resident macrophages in contrast to earlier reports. Together, these results demonstrate that 3D bioprinted liver tissues can both effectively model DILI and distinguish between highly related compounds with differential profile. Thus, the combination of patient-derived primary cells with bioprinting technology here for the first time demonstrates superior performance in terms of mimicking human drug response in a known target organ at the tissue level.

Assessment of the Intestinal Barrier with Five Different Permeability Tests in Healthy C57BL/6J and BALB/cJ Mice.

BACKGROUND: Intestinal permeability is thought to be of major relevance for digestive and nutrition-related diseases, and therefore has been studied in numerous mouse models of disease. However, it is unclear which tools are the preferable ones, and how normal values should be defined. AIMS: To compare different in vivo permeability tests in healthy mice of commonly used genetic backgrounds. METHODS: We assessed the intestinal barrier in male and female C57BL/6J and BALB/cJ mice of different ages, using four orally administered permeability markers, FITC-dextran 4000 (FITC-D4000) and ovalbumin (OVA) measured in plasma, and polyethylene glycol (PEG) and lactulose/mannitol (Lac/Man) measured in urine, and by assessing lipopolysaccharide (LPS) in portal vein plasma. RESULTS: After gavage, FITC-D4000, OVA, Lac/Man, and PEG400, but not PEG4000, were detectable in plasma or urine. Female mice tended to have a higher permeability according to the FITC-D4000, OVA, and PEG400 tests, but the Lac/Man ratio was higher in males. No significant differences between the two mouse strains of young and old mice were observed except for mannitol recovery, which was higher in BALB/cJ mice compared to C57BL/6J mice (p < 0.05). Virtually no LPS was detected in healthy mice. For all markers, normal values have been defined based on 5th-95th percentile ranges of our data. CONCLUSION: Selected oral permeability tests, such as FITC-D4000, OVA, PEG400, and Lac/Man, as well as LPS measurements in portal vein plasma, could be suitable for the evaluation of the intestinal barrier in mice, if used in a standardized way.

Simplified low-cost production of O-antigen from Salmonella Typhimurium Generalized Modules for Membrane Antigens (GMMA).

The Novartis Vaccines Institute for Global Health is developing vaccines using outer membrane particles, known as Generalized Modules for Membrane Antigens (GMMA). These are blebs of outer membrane and periplasm, shed from the surface of living Gram-negative bacteria following the targeted deletion of proteins involved in maintaining the integrity of the inner and outer membranes. The current study investigates the use of GMMA as starting material for extraction of membrane components, focusing on the O-antigen polysaccharide portion of lipopolysaccharide from Salmonella Typhimurium. We show that the amount of O-antigen extracted from GMMA by acid hydrolysis is comparable to the quantity extracted from whole wild type bacteria, but with less protein and DNA contaminants. Compared to conventional purification, GMMA enabled a reduction in the number of purification steps required to obtain the O-antigen polysaccharide with the same purity. Purification processes from GMMA and bacteria were characterised by similar final yields. Use of GMMA as starting material provides the possibility to simplify the purification process of O-antigen, with a consequent decrease in manufacturing costs of O-antigen-based glyconjugate vaccines against Salmonella strains and potentially other Gram-negative bacteria.

Gram-negative marine bacteria: structural features of lipopolysaccharides and their relevance for economically important diseases.

Gram-negative marine bacteria can thrive in harsh oceanic conditions, partly because of the structural diversity of the cell wall and its components, particularly lipopolysaccharide (LPS). LPS is composed of three main parts, an O-antigen, lipid A, and a core region, all of which display immense structural variations among different bacterial species. These components not only provide cell integrity but also elicit an immune response in the host, which ranges from other marine organisms to humans. Toll-like receptor 4 and its homologs are the dedicated receptors that detect LPS and trigger the immune system to respond, often causing a wide variety of inflammatory diseases and even death. This review describes the structural organization of selected LPSes and their association with economically important diseases in marine organisms. In addition, the potential therapeutic use of LPS as an immune adjuvant in different diseases is highlighted.

A quantitative comparison of single-dye tracking analysis tools using Monte Carlo simulations.

Single-particle tracking (SPT) is widely used to study processes from membrane receptor organization to the dynamics of RNAs in living cells. While single-dye labeling strategies have the benefit of being minimally invasive, this comes at the expense of data quality; typically a data set of short trajectories is obtained and analyzed by means of the mean square displacements (MSD) or the distribution of the particles' displacements in a set time interval (jump distance, JD). To evaluate the applicability of both approaches, a quantitative comparison of both methods under typically encountered experimental conditions is necessary. Here we use Monte Carlo simulations to systematically compare the accuracy of diffusion coefficients (D-values) obtained for three cases: one population of diffusing species, two populations with different D-values, and a population switching between two D-values. For the first case we find that the MSD gives more or equally accurate results than the JD analysis (relative errors of D-values <6%). If two diffusing species are present or a particle undergoes a motion change, the JD analysis successfully distinguishes both species (relative error <5%). Finally we apply the JD analysis to investigate the motion of endogenous LPS receptors in live macrophages before and after treatment with methyl-ß-cyclodextrin and latrunculin B.

Crucial roles of charged saccharide moieties in survival of gram negative bacteria against protamine revealed by combination of grazing incidence x-ray structural characterizations and Monte Carlo simulations.

Grazing incidence x-ray scattering techniques and Monte Carlo (MC) simulations are combined to reveal the influence of molecular structure (genetic mutation) and divalent cations on the survival of gram negative bacteria against cationic peptides such as protamine. The former yields detailed structures of bacterial lipopolysaccharide (LPS) membranes with minimized radiation damages, while the minimal computer model based on the linearized Poisson-Boltzmann theory allows for the simulation of conformational changes of macromolecules (LPSs and peptides) that occur in the time scale of ms. The complementary combination of the structural characterizations and MC simulation demonstrates that the condensations of divalent ions (Ca2+ or Mg2+) in the negatively charged core saccharides are crucial for bacterial survival.

Assessment of cholesteryl ester transfer protein inhibitors for interaction with proteins involved in the immune response to infection.

The CETP inhibitor, torcetrapib, was prematurely terminated from phase 3 clinical trials due to an increase in cardiovascular and noncardiovascular mortality. Because nearly half of the latter deaths involved patients with infection, we have tested torcetrapib and other CETPIs to see if they interfere with lipopolysaccharide binding protein (LBP) or bactericidal/permeability increasing protein (BPI). No effect of these potent CETPIs on LPS binding to either protein was detected. Purified CETP itself bound weakly to LPS with a Kd >or= 25 microM compared with 0.8 and 0.5 nM for LBP and BPI, respectively, and this binding was not blocked by torcetrapib. In whole blood, LPS induced tumor necrosis factor-alpha normally in the presence of torcetrapib. Furthermore, LPS had no effect on CETP activity. We conclude that the sepsis-related mortality of the ILLUMINATE trial was unlikely due to a direct effect of torcetrapib on LBP or BPI function, nor to inhibition of an interaction of CETP with LPS. Instead, we speculate that the negative outcome seen for patients with infections might be related to the changes in plasma lipoprotein composition and metabolism, or alternatively to the known off-target effects of torcetrapib, such as aldosterone elevation, which may have aggravated the effects of sepsis.

Fitness costs and stability of a high-level ciprofloxacin resistance phenotype in Salmonella enterica serotype enteritidis: reduced infectivity associated with decreased expression of Salmonella pathogenicity island 1 genes.

The fitness costs associated with high-level fluoroquinolone resistance were examined for phenotypically and genotypically characterized ciprofloxacin-resistant Salmonella enterica serotype Enteritidis mutants (104-cip and 5408-cip; MIC, >32 microg/ml). The stability of the fluoroquinolone resistance phenotype in both mutants was investigated to assess whether clones with better fitness could emerge in the absence of antibiotic selective pressure. Mutants 104-cip and 5408-cip displayed altered morphology on agar and by electron microscopy, reduced growth rates, motility and invasiveness in Caco-2 cells, and increased sensitivity to environmental stresses. Microarray data revealed decreased expression of virulence and motility genes in both mutants. Two clones, 104-revert and 1A-revertC2, with ciprofloxacin MICs of 3 and 2 microg/ml, respectively, were recovered from separate lineages of 104-cip after 20 and 70 passages, respectively, on antibiotic-free agar. All fitness costs, except motility, were reversed in 104-revert. Potential mechanisms associated with reversal of the resistance phenotype were examined. Compared to 104-cip, both 104-revert and 1A-revertC2 showed decreased expression of acrB and soxS but still overexpressed marA. Both acquired additional mutations in SoxR and ParC, and 1A-revertC2 acquired two mutations in MarA. The altered porin and lipopolysaccharide (LPS) profiles observed in 104-cip were reversed. In contrast, 5408-cip showed no reversal in fitness costs and maintained its high-level ciprofloxacin resistance for 200 passages on antibiotic-free agar. In conclusion, high-level ciprofloxacin resistance in S. Enteritidis is associated with fitness costs. In the absence of antibiotic selection pressure, isolates may acquire mutations enabling reversion to an intermediate-level ciprofloxacin resistance phenotype associated with less significant fitness costs.

Absolute quantitation of bacterial biofilm adhesion and viscoelasticity by microbead force spectroscopy.

Bacterial biofilms are the most prevalent mode of bacterial growth in nature. Adhesive and viscoelastic properties of bacteria play important roles at different stages of biofilm development. Following irreversible attachment of bacterial cells onto a surface, a biofilm can grow in which its matrix viscoelasticity helps to maintain structural integrity, determine stress resistance, and control ease of dispersion. In this study, a novel application of force spectroscopy was developed to characterize the surface adhesion and viscoelasticity of bacterial cells in biofilms. By performing microbead force spectroscopy with a closed-loop atomic force microscope, we accurately quantified these properties over a defined contact area. Using the model gram-negative bacterium Pseudomonas aeruginosa, we observed that the adhesive and viscoelastic properties of an isogenic lipopolysaccharide mutant wapR biofilm were significantly different from those measured for the wild-type strain PAO1 biofilm. Moreover, biofilm maturation in either strain also led to prominent changes in adhesion and viscoelasticity. To minimize variability in force measurements resulting from experimental parameter changes, we developed standardized conditions for microbead force spectroscopy to enable meaningful comparison of data obtained in different experiments. Force plots measured under standard conditions showed that the adhesive pressures of PAO1 and wapR early biofilms were 34 +/- 15 Pa and 332 +/- 47 Pa, respectively, whereas those of PAO1 and wapR mature biofilms were 19 +/- 7 Pa and 80 +/- 22 Pa, respectively. Fitting of creep data to a Voigt Standard Linear Solid viscoelasticity model revealed that the instantaneous and delayed elastic moduli in P. aeruginosa were drastically reduced by lipopolysaccharide deficiency and biofilm maturation, whereas viscosity was decreased only for biofilm maturation. In conclusion, we have introduced a direct biophysical method for simultaneously quantifying adhesion and viscoelasticity in bacterial biofilms under native conditions. This method could prove valuable for elucidating the contribution of genetic backgrounds, growth conditions, and environmental stresses to microbial community physiology.

Assessment of lipopolysaccharide-binding activity of Bifidobacterium and its relationship with cell surface hydrophobicity, autoaggregation, and inhibition of interleukin-8 production.

This study was performed to screen probiotic bifidobacteria for their ability to bind and neutralize lipopolysaccharides (LPS) from Escherichia coli and to verify the relationship between LPS-binding ability, cell surface hydrophobicity (CSH), and inhibition of LPS-induced interleukin-8 (IL-8) secretion by HT-29 cells of the various bifidobacterial strains. Ninety bifidobacteria isolates from human feces were assessed for their ability to bind fluorescein isothiocyanate (FITC)-labeled LPS from E. coli. Isolates showing 30-60% binding were designated LPS-high binding (LPS-H) and those with less than 15% binding were designated LPS-low binding (LPS-L). The CSH, autoaggregation (AA), and inhibition of LPS-induced IL-8 release from HT-29 cells of the LPS-H and LPS-L groups were evaluated. Five bifidobacteria strains showed high levels of LPS binding, CSH, AA, and inhibition of IL-8 release. However, statistically significant correlations between LPS binding, CSH, AA, and reduction of IL-8 release were not found. Although we could isolate bifidobacteria with high LPS-binding ability, CSH, AA, and inhibition of IL-8 release, each characteristic should be considered as strain dependent. Bifidobacteria with high LPS binding and inhibition of IL-8 release may be good agents for preventing inflammation by neutralizing Gram-negative endotoxins and improving intestinal health.

Cell membrane array fabrication and assay technology.

BACKGROUND: Microarray technology has been used extensively over the past 10 years for assessing gene expression, and has facilitated precise genetic profiling of everything from tumors to small molecule drugs. By contrast, arraying cell membranes in a manner which preserves their ability to mediate biochemical processes has been considerably more difficult. RESULTS: In this article, we describe a novel technology for generating cell membrane microarrays for performing high throughput biology. Our robotically-arrayed supported membranes are physiologically fluid, a critical property which differentiates this technology from other previous membrane systems and makes it useful for studying cellular processes on an industrialized scale. Membrane array elements consist of a solid substrate, above which resides a fluid supported lipid bilayer containing biologically-active molecules of interest. Incorporation of transmembrane proteins into the arrayed membranes enables the study of ligand/receptor binding, as well as interactions with live intact cells. The fluidity of these molecules in the planar lipid bilayer facilitates dimerization and other higher order interactions necessary for biological signaling events. In order to demonstrate the utility of our fluid membrane array technology to ligand/receptor studies, we investigated the multivalent binding of the cholera toxin B-subunit (CTB) to the membrane ganglioside GM1. We have also displayed a number of bona fide drug targets, including bacterial endotoxin (also referred to as lipopolysaccharide (LPS)) and membrane proteins important in T cell activation. CONCLUSION: We have demonstrated the applicability of our fluid cell membrane array technology to both academic research applications and industrial drug discovery. Our technology facilitates the study of ligand/receptor interactions and cell-cell signaling, providing rich qualitative and quantitative information.