Release Of Lipopolysaccharide Research Articles

Beneficial effect of garlic on d-galactosamine and lipopolysaccharide-induced acute hepatic failure in male albino rats

Background and aimsActivation of the pro-inflammatory and anti-inflammatory cytokine cascade, including tumour necrosis factor (TNF)-alpha and interleukin (IL)-4, is considered to play an important role in severe liver injury. Kupffer cells, resident macrophages of the liver, activated with lipopolysaccharide (LPS) release pro-inflammatory cytokine. d-Galactosamine (d-GalN), a hepatocyte-specific inhibitor of RNA synthesis, is known to sensitise animals to the lethal effects of LPS. In the present study we seek to reverse some altered parameters, immunological and histopathological, to normal values of rats pre-treated with garlic. MethodsAcute hepatic failure was induced in male albino rats by the intraperitoneal injection of 500mg d-GalN and 50μg LPS/kg body weight. Expression levels of TNF-α and IL-4 were detected by ELISA. Leukocytes proliferation was carried out by differential count. For histopathology, liver sections were stained with haematoxylin and eosin. Data were analysed by SPSS program version 13.0. ResultsThe data showed significant increase in the numbers of granulocytes, but with significant decreases in lymphocyte and monocytes proliferation and the TNF-alpha and IL-4 levels in d-GalN/LPS-induced group. Garlic pre-treatment of liver-injured rats induced significant amelioration in the numbers of monocytes and lymphocytes, with significant increase in granulocytes numbers, TNF-α level and IL-4 level. ConclusionsResults of this study revealed that garlic could afford a significant protection in the alleviation of d-GalN/LPS-induced hepatocellular injury.

Bench-to-bedside review: Clinical experience with the endotoxin activity assay

Endotoxin detection in human patients has been a difficult challenge, in part due to the fact that the conserved active portion of the molecule (lipid A) is a relatively small epitope only amenable to binding by a single ligand at any one instance and low levels (pg/ml) are capable of stimulating the immune system. The endotoxin activity assay, a bioassay based on neutrophil activation by complement opsonized immune complexes of lipopolysaccharide (LPS), has allowed the specific detection of the lipid A epitope of LPS in a rapid whole blood assay format. This review summarizes diagnostic studies utilizing the endotoxin activity assay in a variety of hospital patient populations in whom endotoxin is postulated to play a significant role in disease etiology. These include ICU patients at risk of developing 'sepsis syndrome', abdominal and cardiovascular surgery patients and patients with serious traumatic injury. Significant features of these studies include the high negative predictive value of the assay (98.6%) for rule out of Gram-negative infection, ability to risk stratify patients progressing to severe sepsis (odds ratio 3.0) and evidence of LPS release in patients with gut hypoperfusion. Preliminary studies have successfully combined the assay with anti-LPS removal strategies to prospectively identify patients who might benefit from this therapy with early evidence of clinical benefit.

Change in Temperature Profile May Precede Fever and be an Early Indicator of Sepsis

Change in Temperature Profile May Precede Fever and be an Early Indicator of Sepsis

In vitro antioxidant, antiplatelet and anti-inflammatory activity of Carpobrotus rossii (pigface) extract

Carpobrotus rossii (CR) has a history of use as a food and therapeutic agent by Australian indigenous peoples and early European settlers and is believed to contain a number of pharmacologically active polyphenolic compounds. Oxidation of low density lipoprotein (LDL), platelet aggregation, and inflammation contribute to the development and progression of atherosclerosis. The aim of the present study was to investigate the antioxidant, antiplatelet and anti-inflammatory activity of CR extract using human blood components. An assay employing in vitro copper-induced oxidation of serum lipids was used to assess antioxidant activity of CR extract (and tannin, flavonoid and pre- and post-flavonoid fractions). The effects of CR extract on ADP- and collagen-induced platelet aggregation, and on basal (unstimulated) and lipopolysaccharide (LPS)- and phytohaemagglutinin A (PHA)-stimulated cytokine release from peripheral blood mononuclear cells (PBMC) were also investigated. CR extract increased the lag time of serum oxidation (maximum of ∼4-fold at 20μg/ml) in a concentration-dependent manner. The antioxidant activity resided only in the tannin and post-flavonoid fractions. CR had no effect on ADP-induced platelet aggregation, but significantly decreased collagen-induced platelet aggregation. LPS, but not PHA, significantly increased the release of IL-1β and TNF-α from PBMC. CR extract alone inhibited monocyte chemoattractant protein (MCP)-1 release and in the presence of LPS, inhibited IL-10, TNF-α and MCP-1 release compared to LPS alone. CR has significant in vitro antioxidant, antiplatelet and, potentially, anti-inflammatory activity.

Evidence for the complementary and synergistic effects of the three-alkaloid combination regimen containing berberine, hypaconitine and skimmianine on the ulcerative colitis rats induced by trinitrobenzene-sulfonic acid

Ulcerative colitis involves complicated etiology and presents diverse symptoms including intestine inflammation, bowel pain and diarrhea. Anti-inflammatory drugs are the mainstay in patient care, accompanied with antidiarrhea and analgesic agents used as symptomatic treatment. A classic traditional Chinese medicine formula, Fructus Mume pill (FMP), showed remarkable therapeutic efficacy in treating ulcerative colitis. However, since it contains many herbs and countless chemicals, the underlying mechanism is not clear. In this study, we selected three alkaloids from FMP, namely, berberine, hypaconitine and skimmianine to study the individual drug effect and compare these results with the BHS combination on: 1) The recovery of ulcerative colitis rats induced by trinitrobenzene-sulfonic acid. 2) Mice with xylene-induced acute exudative edema and acetic acid-induced writhing. 3) Gastrointestinal transit inhibition, and 4) the response of HT29 cells after treatment with lipopolysaccharide. We found that the compound hypaconitine showed a potent analgesic effect, while skimmianine acted as an antidiarrhea agent and the component berberine was the key agent exerting anti-inflammatory effect. However, since berberine killed the commensal bacteria and induced lipopolysaccharide release, it could at the same time aggravate colon inflammation. The three-alkaloid combination BHS produced complementary and synergistic effects in colon inflammation recovery, relieving acetic acid-induced bowel pain and xylene-induced acute exudative edema. BHS also decreased lipopolysaccharide production and enhanced the therapeutic efficacy. It is hoped that this study will lay the foundation to further dissect and understand the FMP formula to improve the treatment with simplified and well defined drug combinations for this dreadful disease.

Antibiotic-induced Porphyromonas gingivalis LPS release and inhibition of LPS-stimulated cytokines by antimicrobial peptides

Bacterial lipopolysaccharide (LPS) release during periodontal infection is a significant component of periodontal disease. We hypothesized that some bacterial LPS release results from bacterial exposure to antibiotics. Therefore, we examined the ability of various classes of antibiotics to induce LPS release from Porphyromonas gingivalis as well as the ability of antimicrobial peptides (AMPs) to inhibit purified LPS. All antibiotics tested against P. gingivalis were able to liberate 1.9–12.9 times more LPS as compared to untreated bacteria. Among the three AMPs tested, LL-37 was found to be the most potent inhibitor of cytokine (tumor necrosis factor-α, interleukin-1β, interleukin-6) production and completely neutralized purified P. ginigivalis LPS activity in the chromogenic limulus amebocyte lysate (LAL) and whole blood cytokine stimulation assays. These observations suggest that therapeutic approaches utilizing AMPs as adjuncts to neutralize released LPS should be considered.

Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane

Lipopolysaccharide (LPS) is the major glycolipid that is present in the outer membranes (OMs) of most Gram-negative bacteria. LPS molecules are assembled with divalent metal cations in the outer leaflet of the OM to form an impervious layer that prevents toxic compounds from entering the cell. For most Gram-negative bacteria, LPS is essential for growth. In Escherichia coli, eight essential proteins have been identified to function in the proper assembly of LPS following its biosynthesis. This assembly process involves release of LPS from the inner membrane (IM), transport across the periplasm, and insertion into the outer leaflet of the OM. Here, we describe the biochemical characterization of the two-protein complex consisting of LptD and LptE that is responsible for the assembly of LPS at the cell surface. We can overexpress and purify LptD and LptE as a stable complex in a 1:1 stoichiometry. LptD contains a soluble N-terminal domain and a C-terminal transmembrane domain. LptE stabilizes LptD by interacting strongly with the C-terminal domain of LptD. We also demonstrate that LptE binds LPS specifically and may serve as a substrate recognition site at the OM.

GroEL and Lipopolysaccharide fromFrancisella tularensisLive Vaccine Strain Synergistically Activate Human Macrophages

Francisella tularensis, the causative agent of tularemia, interacts with host cells of innate immunity in an atypical manner. For most Gram-negative bacteria, the release of lipopolysaccharide (LPS) from their outer membranes stimulates an inflammatory response. When LPS from the attenuated live vaccine strain (LVS) or the highly virulent Schu S4 strain of F. tularensis was incubated with human umbilical vein endothelial cells, neither species of LPS induced expression of the adhesion molecule E-selectin or secretion of the chemokine CCL2. Moreover, a high concentration (10 microg/ml) of LVS or Schu S4 LPS was required to stimulate production of CCL2 by human monocyte-derived macrophages (huMDM). A screen for alternative proinflammatory factors of F. tularensis LVS identified the heat shock protein GroEL as a potential candidate. Recombinant LVS GroEL at a concentration of 10 microg/ml elicited secretion of CXCL8 and CCL2 by huMDM through a TLR4-dependent mechanism. When 1 microg of LVS GroEL/ml was added to an equivalent amount of LVS LPS, the two components synergistically activated the huMDM to produce CXCL8. Schu S4 GroEL was less stimulatory than LVS GroEL and showed a lesser degree of synergy when combined with Schu S4 LPS. These findings suggest that the intrinsically low proinflammatory activity of F. tularensis LPS may be increased in the infected human host through interactions with other components of the bacterium.

FK506 Protects Against Various Immune Responses and Secondary Degeneration Following Cerebral Ischemia

The immunosuppressant FK506 (1 mg/kg, i.p.) reduces the infarct size following 90 min occlusion of the middle cerebral artery (MCAo) in adult rat brain. Here we have investigated the effect of FK506 on cerebral immune cells that are considered to contribute to neurodegeneration. FK506 substantially attenuated the response of resident and peripheral immune cells following transient ischemia. Between 24 hr and 5 days after MCAo, FK506 reduced the T-cell infiltration in the infarct area as well as the presence of activated and/or phagocytic OX-18, OX-42, GSA-IB4, Iba1, and ED1 positive microglia/macrophages. FK506 also lowered the protein levels of TNFalpha and IL-2 in ischemic brain areas. Repetitive application of FK506 over 20 days attenuated the activation of microglia in the substantia nigra (SN), an area of secondary degeneration. Importantly, FK506 conferred also lasting protection of the neurons of SN; these neurons degenerate by withdrawal of neurotrophic factors from the striatum that undergoes necrotic death as part of the ischemic core. To understand the molecular basis of FK506 effects in cerebral immune cells, we determined in primary postnatal day 0/1 (P0/P1) microglia (i) the expression of the FK506 binding proteins FKBP12, FKBP52, and FKPB65 and (ii) that FK506 (1-100 ng/mL) lowered the number of resting or lipopolysaccharide stimulated microglia as well as we induced the lipopolysaccharide release of TNFalpha in a dose-dependent manner. In summary, FK506 confers rescue of brain tissue following cerebral ischemia not only by neuronal protection, but also by suppression of microglial activation and peripheral immune responses.

Isoflurane preconditioning reduces mouse microglial activation and injury induced by lipopolysaccharide and interferon-γ

Activation and injury of microglial cells are involved in a broad range of brain diseases including stroke, brain infection and neurodegenerative diseases. However, there is very little information regarding how to reduce microglial reaction and preserve these cells to provide neuroprotection. Here, we showed that the incubation of C8-B4 mouse microglial cells with lipopolysaccharide (LPS) plus interferon-γ (IFNγ) for 24 h decreased the viability of these cells. Pretreatment of these cells with 1%, 2% or 3% isoflurane, a commonly used volatile anesthetic, for 1 h at 30 min before the exposure to LPS plus IFNγ attenuated the reduction of cell viability (preconditioning effect). LPS plus IFNγ also activated these microglial cells to express inducible nitric oxide synthase (iNOS) and to induce accumulation of nitrite, a stable oxidation product of nitric oxide, in the incubation medium. Isoflurane preconditioning attenuated these LPS plus IFNγ effects on the iNOS expression and nitrite accumulation. Aminoguanidine, an iNOS inhibitor, attenuated the LPS plus IFNγ-induced glutamate release and decrease of microglial viability. Isoflurane preconditioning also reduced LPS plus IFNγ-induced glutamate release. Exogenous glutamate decreased microglial viability. Finally, the isoflurane preconditioning-induced protection was abolished by chelerythrine, a protein kinase C inhibitor. These results suggest that LPS plus IFNγ activates the iNOS–nitric oxide–glutamate pathway to induce microglial injury and that this activation is attenuated by isoflurane preconditioning. Protein kinase C may be involved in the isoflurane preconditioning effects.

Inhibition of Lipopolysaccharide Activation of Kupffer Cells by Transition Metals

Bacterial endotoxins are the principal agents causing sepsis and septic shock. Cytokine cascades produced by cellular interactions to endotoxins can cause cardiovascular failure followed by multi-organ failure and death. Endotoxin intravenously administered to mice can have fatal consequences. Previous studies have shown that the transition metals Mn2+ and Cr3+ can be protective. The effects of Mn2+, Cr3+, Zn2+, and Cu2+ on lipopolysaccharide (LPS) binding to rat Kupffer cell extracts were analyzed using dot-blots, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western transfer. Kupffer cells were isolated from rat livers by collagenase perfusion, differential centrifugation, and adhesion to plastic. Five millimolar of Mn2+, Zn2+, Cr3+, and Cu2+ completely inhibited LPS binding. Isolated Kupffer cells were also exposed to Mn2+ and to LPS and tumor necrosis factor-alpha release measured. The presence of Mn2+ significantly (P < 0.05) reduced tumor necrosis factor-alpha production by Kupffer cells in response to LPS. Experiments to determine if these effects were mediated by binding to LPS-binding proteins showed this was not the case. More likely a complex occurs between the metal and LPS. We also showed significantly enhanced uptake of LPS into Kupffer cells in the presence of Mn2+. The data are consistent with the metals binding to LPS via its two phosphate groups and neutralizing their charge. These data also support the hypothesis that there is enhanced cellular uptake by non-receptor-mediated methods such as absorptive pinocytocis. At the same time receptor binding and activation of the cells is inhibited. This can explain the effects of transition metals on LPS toxicity.

Weakening of Salmonella with Selected Microbial Metabolites of Berry-Derived Phenolic Compounds and Organic Acids

Gram-negative bacteria are important food spoilage and pathogenic bacteria. Their unique outer membrane (OM) provides them with a hydrophilic surface structure, which makes them inherently resistant to many antimicrobial agents, thus hindering their control. However, with permeabilizers, compounds that disintegrate and weaken the OM, Gram-negative cells can be sensitized to several external agents. Although antimicrobial activity of plant-derived phenolic compounds has been widely reported, their mechanisms of action have not yet been well demonstrated. The aim of our study was to elucidate the role of selected colonic microbial metabolites of berry-derived phenolic compounds in the weakening of the Gram-negative OM. The effect of the agents on the OM permeability of Salmonella was studied utilizing a fluorescence probe uptake assay, sensitization to hydrophobic antibiotics, and lipopolysaccharide (LPS) release. Our results show that 3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 3-(3,4-dihydroxyphenyl)propionic acid (3,4-diHPP), 3-(4-hydroxyphenyl)propionic acid, 3-phenylpropionic acid, and 3-(3-hydroxyphenyl)propionic acid efficiently destabilized the OM of Salmonella enterica subsp. enterica serovar Typhimurium and S. enterica subsp. enterica serovar Infantis as indicated by an increase in the uptake of the fluorescent probe 1-N-phenylnaphthylamine (NPN). The OM-destabilizing activity of the compounds was partially abolished by MgCl2 addition, indicating that part of their activity is based on removal of OM-stabilizing divalent cations. Furthermore, 3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, and 3,4-diHPP increased the susceptibility of S. enterica subsp. enterica serovar Typhimurium strains for novobiocin. In addition, organic acids present in berries, such as malic acid, sorbic acid, and benzoic acid, were shown to be efficient permeabilizers of Salmonella as shown by an increase in the NPN uptake assay and by LPS release.

Un nouveau schéma pour mieux comprendre les maladies de la surface oculaire

The mechanistic view of dry eye disease aims at completing the classic etiological approach that classifies the disease as parallel ocular surface disorders leading to lacrimal film impairment and dry eye. This approach proposes two levels of ocular surface impairment (with standard etiologies, previously validated in the NEI/Industry workshop), which may not be independent diseases but rather risk factors and/or ways to enter a self-stimulated biological process involving the ocular surface. All external disorders proposed in this model, although unlikely to be fully exhaustive, are classical mechanisms considered to be causes of tear film impairment and ocular surface damage, by tear instability and evaporation, tear hyposecretion, or both. These mechanisms, sometimes alone--when severe or becoming chronic or repeatedly present on the ocular surface and when two or more are present--may cause the patient to enter the self-stimulated loop. Tear film instability/imbalance can be considered as the key point of dry eye disease. It will cause local or diffuse hyperosmolarity of the tear film and therefore of superficial epithelial cells of the cornea and/or conjunctiva, stimulating epithelial cells and resident inflammatory cells. Cell damage in the cornea and conjunctiva, by means of apoptosis and direct mechanical and/or osmotic stress, will stimulate the reflex neurosensory arc, in turn stimulating lacrimal gland and neurogenic inflammation, with inflammatory cytokine release, MMP activation, and inflammatory involvement of the conjunctival epithelium. Goblet cell loss is thus directly related to chronic inflammation and surface cell apoptosis subsequent to cell hyperosmolarity and chronic damage, resulting in further tear film instability/imbalance. On the other hand, bacterial changes and an imbalance resulting from specific diseases or from tear film abnormalities may trigger release of endotoxins, lipopolysaccharides, and/or lipase activation, causing eyelid inflammation, meibomian gland dysfunction, and lipidic changes, directly influencing tear film stability and favoring tear evaporation. The lipidic hypothesis therefore participates in the vicious circle as a parallel, independent, or complementary loop. This mechanistic approach proposes a synthetic combination of mechanisms previously validated independently, with two levels of ocular surface impairment, a first level including many possible acute or chronic causes that favor or trigger the imbalance and can be reversible if correctly and specifically managed when possible, and the further involvement of a series of biological cascades centered by tear film imbalance and inflammatory stimulation, finally acting as an independent vicious circle, however the patient entered the loop. Clinically, this approach may explain examples of dry eye syndrome occurring after ocular surgery, contact lens wear, chronic allergy or systemic or topical drugs, and the long-lasting effect even though all causal factors have been removed or have disappeared. This model should be considered as a basis for further reflection on biological mechanisms that could be even more complex but individually constitute potential leads for targeting therapeutic strategies to allow patients to leave the loop even though the triggering factors are still present or can only be attenuated, such as in Sjögren syndrome or ocular rosacea. It also should be considered a complement to more classic etiological and severity classifications aimed at understanding and classifying the large number of diseases that may cause dry eye disease and better assessing the major impairment it causes on the patient's quality of life.

Topomimetics of amphipathic beta-sheet and helix-forming bactericidal peptides neutralize lipopolysaccharide endotoxins.

Release of lipopolysaccharide (LPS) endotoxin from Gram negative bacterial membranes triggers macrophages to produce large quantities of cytokines that can lead to septic shock and eventual death. Agents that bind to and neutralize LPS may provide a means to clinically prevent septic shock upon bacterial infection. Previously, we reported the design of antibacterial helix peptide SC4 and beta-sheet-forming betapep peptides that neutralize LPS in vitro. We hypothesized that the ability of these and other such peptides to neutralize LPS rested in the common denominator of positively charged amphipathic structure. Here, we describe the design and synthesis of nonpeptide, calixarene-based helix/sheet topomimetics that mimic the folded conformations of these peptides in their molecular dimensions, amphipathic surface topology, and compositional properties. From a small library of topomimetics, we identified several compounds that neutralize LPS in the 10-8 M range, making them as effective as bactericidal/permeability increasing protein and polymyxin B. In an endotoxemia mouse model, three of the most in vitro effective topomimetics are shown to be at least partially protective against challenges of LPS from different bacterial species. NMR studies provide mechanistic insight by suggesting the site of molecular interaction between topomimetics and the lipid A component of LPS, with binding being mediated by electrostatic and hydrophobic interactions. This research contributes to the development of pharmaceutical agents against endotoxemia and septic shock.

Controlled release of lipopolysaccharide in the subarachnoid space of rabbits induces chronic vasospasm in the absence of blood

Objective Leukocyte-endothelial cell interactions appear to play a role in the development of vasospasm after SAH. Using a purely inflammatory protein, LPS, we evaluated the effect of inflammation on the development of chronic vasospasm in the absence of blood and compared it to SAH-induced vasospasm in rabbits. Methods Lipopolysaccharide was incorporated into EVAc polymers to produce 20% LPS/EVAc polymers (wt/wt). Rabbits (n = 23) were randomized to 4 experimental groups: (1) empty polymer (n = 6), (2) SAH (n = 5), (3) 0.7 mg/kg polymeric LPS dose (n = 6), and (4) 1.4 mg/kg polymeric LPS dose (n = 6). Blood and polymers were inserted into the cisterna magna. The rabbits were killed 3 days postoperatively, and the basilar arteries were harvested for morphometric analysis. Clinical response and lumen patencies were analyzed using ANOVA and a post hoc Newman-Keuls Multiple Comparisons test. Results Significant narrowing of the basilar artery was observed by insertion of 20% LPS/EVAc polymers into the subarachnoid space at a polymeric dose of 1.4 mg/kg (actual dose, 66 μg kg −1 d −1) (75.4% ± 4.2%; P < .01) and by SAH (80.3% ± 8.1%; P < .01) as compared with the empty polymer group. A trend toward narrowing was observed in the 0.7 mg/kg polymeric LPS dose group (actual dose, 33 μg kg −1 d −1) (85.2% ± 2.6%; P > .05). Symptoms associated with SAH were noted in 50% of the rabbits in the 0.7 mg/kg LPS group and in 100% of rabbits in the 1.4 mg/kg LPS group. Conclusion Controlled release of LPS into the subarachnoid space of rabbits produced chronic vasospasm in a dose-dependent manner. At a polymeric dose of 1.4 mg/kg, LPS-induced vasospasm was equivalent to that induced by SAH. This suggests that LPS and SAH may induce vasospasm through similar mechanisms and provides further evidence that inflammation plays a central role in the etiology of chronic vasospasm.

Effects of EDTA and Tween60 on biodegradation of n-hexadecane with two strains of Pseudomonas aeruginosa

Two Pseudomonas aeruginosa strains with different hydrocarbon uptake modes were observed in the previous research. One strain produces biosurfactant during the process of hydrocarbon uptake, and was named as P. aeruginosa S. The other one utilizes hydrocarbon directly and was named as P. aeruginosa Y. For comparison of the characteristics of the two strains, the effects of EDTA and Tween60 on alkane biodegradation and cell surface characteristics were investigated in this paper. The results showed that the bacteria with different hydrocarbon uptake mode also have different response to the addition of EDTA and Tween60. As a result, in the case of P. aeruginosa S, the addition of EDTA inhibited the cell growth and hexadecane biodegradation, but enhanced the lipopolysaccharide (LPS) release and increased the cell surface potential, while the influence of the addition of Tween60 was smaller than that of EDTA. On the other hand, in the case of P. aeruginosa Y, the addition of EDTA or Tween60 not only inhibited the cell growth and hexadecane biodegradation, but also decreased the cell surface hydrophobicity (CSH) dramatically.

Weakening Effect of Cell Permeabilizers on Gram-Negative Bacteria Causing Biodeterioration

Gram-negative bacteria play an important role in the formation and stabilization of biofilm structures on stone surfaces. Therefore, the control of growth of gram-negative bacteria offers a way to diminish biodeterioration of stone materials. The effect of potential permeabilizers on the outer membrane (OM) properties of gram-negative bacteria was investigated and further characterized. In addition, efficacy of the agents in enhancing the activity of a biocide (benzalkonium chloride) was assessed. EDTA, polyethylenimine (PEI), and succimer (meso-2,3-dimercaptosuccinic) were shown to be efficient permeabilizers of the members of Pseudomonas and Stenotrophomonas genera, as indicated by an increase in the uptake of a hydrophobic probe (1-N-phenylnaphthylamine) and sensitization to hydrophobic antibiotics. Visualization of Pseudomonas cells treated with EDTA or PEI by atomic force microscopy revealed damage in the outer membrane structure. PEI especially increased the surface area and bulges of the cells. Topographic images of EDTA-treated cells were compatible with events assigned for the effect of EDTA on outer membranes, i.e., release of lipopolysaccharide and disintegration of OM structure. In addition, the effect of EDTA treatment was visualized in phase-contrast images as large areas with varying hydrophilicity on cell surfaces. In liquid culture tests, EDTA and PEI supplementation enhanced the activity of benzalkonium chloride toward the target strains. Use of permeabilizers in biocide formulations would enable the use of decreased concentrations of the active biocide ingredient, thereby providing environmentally friendlier products.

Rhamnolipids Are Virulence Factors That Promote Early Infiltration of Primary Human Airway Epithelia byPseudomonas aeruginosa

The opportunistic bacterium Pseudomonas aeruginosa causes chronic respiratory infections in cystic fibrosis and immunocompromised individuals. Bacterial adherence to the basolateral domain of the host cells and internalization are thought to participate in P. aeruginosa pathogenicity. However, the mechanism by which the pathogen initially modulates the paracellular permeability of polarized respiratory epithelia remains to be understood. To investigate this mechanism, we have searched for virulence factors secreted by P. aeruginosa that affect the structure of human airway epithelium in the early stages of infection. We have found that only bacterial strains secreting rhamnolipids were efficient in modulating the barrier function of an in vitro-reconstituted human respiratory epithelium, irrespective of their release of elastase and lipopolysaccharide. In contrast to previous reports, we document that P. aeruginosa was not internalized by epithelial cells. We further report that purified rhamnolipids, applied on the surfaces of the epithelia, were sufficient to functionally disrupt the epithelia and to promote the paracellular invasion of rhamnolipid-deficient P. aeruginosa. The mechanism involves the incorporation of rhamnolipids within the host cell membrane, leading to tight-junction alterations. The study provides direct evidence for a hitherto unknown mechanism whereby the junction-dependent barrier of the respiratory epithelium is selectively altered by rhamnolipids.

Shiga Toxin 2 and Lipopolysaccharide Induce Human Microvascular Endothelial Cells To Release Chemokines and Factors That Stimulate Platelet Function

Shiga toxins (Stxs) produced by Shigella dysenteriae type 1 and enterohemorrhagic Escherichia coli are the most common cause of hemolytic-uremic syndrome (HUS). It is well established that vascular endothelial cells, mainly those located in the renal microvasculature, are targets for Stxs. The aim of the present research was to evaluate whether E. coli-derived Shiga toxin 2 (Stx2) incubated with human microvascular endothelial cells (HMEC-1) induces release of chemokines and other factors that might stimulate platelet function. HMEC-1 were exposed for 24 h in vitro to Stx2, lipopolysaccharide (LPS), or the Stx2-LPS combination, and chemokine production was assessed by immunoassay. More interleukin-8 was released than stromal cell-derived factor 1alpha (SDF-1alpha) or SDF-1beta and RANTES. The Stx2-LPS combination potentiated chemokine release, but Stx2 alone caused more release of SDF-1alpha at 24 h than LPS or Stx2-LPS did. In the presence of low ADP levels, HMEC-1 supernatants activated platelet function assessed by classical aggregometry, single-particle counting, granule secretion, P-selectin exposure, and the formation of platelet-monocyte aggregates. Supernatants from HMEC-1 exposed only to Stx2 exhibited enhanced exposure of platelet P-selectin and platelet-THP-1 cell interactions. Blockade of platelet cyclooxygenase by indomethacin prevented functional activation. The chemokine RANTES enhanced platelet aggregation induced by SDF-1alpha, macrophage-derived chemokine, or thymus and activation-regulated chemokine in the presence of very low ADP levels. These data support the hypothesis that microvascular endothelial cells exposed to E. coli O157:H7-derived Stx2 and LPS release chemokines and other factors, which when combined with low levels of primary agonists, such as ADP, cause platelet activation and promote the renal thrombosis associated with HUS.

Attenuation of Fever At Near Term: Is Interleukin‐6–STAT3 Signalling Altered?

Pregnant rats in late gestation show a reduced fever response after stimulation with lipopolysaccharide (LPS). This can result from either an increased action of endogenous antipyretics or a reduction in the production or action of endogenous pyrogens. Nonpregnant rats given LPS release interleukin (IL)-6, which causes nuclear translocation of the signal transducer and activator of transcription 3 (STAT3) in the vascular organ of the lamina terminalis (OVLT), followed by a significant increase in core body temperature. The present study investigated whether the reduced fever response in near-term pregnant rats is associated with a reduced nuclear STAT3 response. Rats at gestation day 15 (G15), gestation day 21 (G21, near term) and at lactation day 5 (L5) were injected with LPS (50 microg/kg, i.p.) or vehicle. Only near-term pregnant rats responded with an attenuated body temperature during the fever response. Immunohistological analysis indicated no significant difference in nuclear STAT3 in the OVLT of the different animal groups 2 h after LPS. Measurement of total and phosphorylated STAT3 protein in the OVLT with semiquantitative western blot revealed no significant differences of this protein among these immune challenged animal groups. IL-6 concentrations were also similar at G15, G21 and L5 2 h after injection of LPS. These results lead to the conclusion that the attenuation of the fever response at near-term pregnancy is not associated with a reduced amount of nuclear STAT3 in the OVLT, indicating a maintained IL-6-STAT3 signalling pathway in the OVLT.