Effects of ciliary muscle plasmid electrotransfer of TNF-alpha soluble receptor variants in experimental uveitis.

Intraocular inflammation has been recognized as a major factor leading to blindness. Because tumor necrosis factor-alpha (TNF-alpha) enhances intraocular cytotoxic events, systemic anti-TNF therapies have been introduced in the treatment of severe intraocular inflammation, but frequent re-injections are needed and are associated with severe side effects. We have devised a local intraocular nonviral gene therapy to deliver effective and sustained anti-TNF therapy in inflamed eyes. In this study, we show that transfection of the ciliary muscle by plasmids encoding for three different variants of the p55 TNF-alpha soluble receptor, using electrotransfer, resulted in sustained intraocular secretion of the encoded proteins, without any detection in the serum. In the eye, even the shorter monomeric variant resulted in efficient neutralization of TNF-alpha in a rat experimental model of endotoxin-induced uveitis, as long as 3 months after transfection. A subsequent downregulation of interleukin (IL)-6 and iNOS and upregulation of IL-10 expression was observed together with a decreased rolling of inflammatory cells in anterior segment vessels and reduced infiltration within the ocular tissues. Our results indicate that using a nonviral gene therapy strategy, the local self-production of monomeric TNF-alpha soluble receptors induces a local immunomodulation enabling the control of intraocular inflammation.

Biophysical characterization of the interaction of endotoxins with hemoglobins.

Bacterial endotoxin (lipopolysaccharide, LPS) is the major component of the outer leaflet of the outer membrane in gram-negative bacteria. During severe infections, bacteria may reach the blood circuit of humans, and endotoxins may be released from the bacteria due to cell division or cell death. In particular enterobacterial forms of LPS represent extremely strong activator molecules of the human immune system causing a rapid induction of cytokine production in monocytes and macrophages. Various mammalian blood proteins have been documented to display LPS binding activities mediating normally decreasing effects in the biological activity of LPS. In more recent studies, the essential systemic oxygen transportation protein hemoglobin (Hb) has been shown to amplify LPS-induced cytokine production on immune cells. The mechanism responsible for this effect is poorly understood. Here, we characterize the interaction of hemoglobin with LPS by using biophysical methods. The data presented, revealing the changes of the type and size of supramolecular aggregates of LPS in the presence of Hb, allow a better understanding of the hemoglobin-induced increase in bioactivity of LPS.

Histopathological and ultrastructural effects of delta-endotoxins of Bacillus thuringiensis serovar israelensis in the midgut of Simulium pertinax larvae (Diptera, Simuliidae)

The bacterium Bacillus thuringiensis (Bt) produces parasporal crystals containing delta-endotoxins responsible for selective insecticidal activity on larvae. Upon ingestion, these crystals are solubilized in the midgut lumen and converted into active toxins that bind to receptors present on the microvilli causing serious damage to the epithelial columnar cells. We investigated the effect of these endotoxins on larvae of the Simulium pertinax, a common black fly in Brazil, using several concentrations during 4 h of the serovar israelensis strain IPS-82 (LFB-FIOCRUZ 584), serotype H-14 type strain of the Institute Pasteur, Paris. Light and electron microscope observations revealed, by time and endotoxin concentration, increasing damages of the larvae midgut epithelium. The most characteristic effects were midgut columnar cell vacuolization, microvilli damages, epithelium cell contents passing into the midgut lumen and finally the cell death. This article is the first report of the histopathological effects of the Bti endotoxins in the midgut of S. pertinax larvae and the data obtained may contribute to a better understanding of the mode of action of this bacterial strain used as bioinsecticide against black fly larvae.

Schistosoma mansoni antigen-driven interleukin-10 production in infected asthmatic individuals

Asthmatics infected with Schistosoma mansoni have a less severe course of asthma and an inhibition of the Th2 inflammatory response that seems to be mediated by interleukin (IL-10). The objective of this study was to evaluate the capacity of some S. mansoni antigens to stimulate IL-10 production in vitro by cells of asthmatic infected individuals. Peripheral bloods mononuclear cells were stimulated with the S. mansoni recombinant antigens Sm22.6, Sm14, P24, and PIII antigen. IL-10 was measured in the supernatants of cultures. As the recombinant antigens were cloned in Escherichia coli, we blocked contaminant endotoxin with polymyxin B added to the cultures. We demonstrated that all antigens used drove high production of IL-10 in S. mansoni infected individuals (n = 13, 408 ± 514 and 401 ± 383 pg/ml, 484 ± 245 pg/ml, 579 ± 468 pg/ml, respectively). In asthmatics infected with S. mansoni (n = 21) rP24 induced higher levels of IL-10 (565 ± 377 pg/ml) when compared to PIII, rSm14 and rSm22.6 (184 ± 209 pg/ml; 292 ± 243 pg/ml; 156 ± 247 pg/ml, respectively). Conclusion: the S. mansoni antigens evaluated in this study stimulated IL-10 production by cells from infected individuals and therefore they have the potential to be used as a modulator of the inflammatory response in asthma.

Anti-vascular endothelial growth factor acts on retinal microglia/macrophage activation in a rat model of ocular inflammation.

PURPOSE: To evaluate whether anti-vascular endothelial growth factor (VEGF) neutralizing antibodies injected in the vitreous of rat eyes influence retinal microglia and macrophage activation. To dissociate the effect of anti-VEGF on microglia and macrophages subsequent to its antiangiogenic effect, we chose a model of acute intraocular inflammation. METHODS: Lewis rats were challenged with systemic lipopolysaccharide (LPS) injection and concomitantly received 5 µl of rat anti-VEGF-neutralizing antibody (1.5 mg/ml) in the vitreous. Rat immunoglobulin G (IgG) isotype was used as the control. The effect of anti-VEGF was evaluated at 24 and 48 h clinically (uveitis scores), biologically (cytokine multiplex analysis in ocular media), and histologically (inflammatory cell counts on eye sections). Microglia and macrophages were immunodetected with ionized calcium-binding adaptor molecule 1 (IBA1) staining and counted based on their differential shapes (round amoeboid or ramified dendritiform) on sections and flatmounted retinas using confocal imaging and automatic quantification. Activation of microglia was also evaluated with inducible nitric oxide synthase (iNOS) and IBA1 coimmunostaining. Coimmunolocalization of VEGF receptor 1 and 2 (VEGF-R1 and R2) with IBA1 was performed on eye sections with or without anti-VEGF treatment. RESULTS: Neutralizing rat anti-VEGF antibodies significantly decreased ocular VEGF levels but did not decrease the endotoxin-induced uveitis (EIU) clinical score or the number of infiltrating cells and cytokines in ocular media (interleukin [IL]-1β, IL-6, tumor necrosis factor [TNF]-α, and monocyte chemoattractant protein [MCP]-1). Eyes treated with anti-VEGF showed a significantly decreased number of activated microglia and macrophages in the retina and the choroid and decreased iNOS-positive microglia. IBA1-positive cells expressed VEGF-R1 and R2 in the inflamed retina. CONCLUSIONS: Microglia and macrophages expressed VEGF receptors, and intravitreous anti-VEGF influenced the microglia and macrophage activation state. Taking into account that anti-VEGF drugs are repeatedly injected in the vitreous of patients with retinal diseases, part of their effects could result from unsuspected modulation of the microglia activation state. This should be further studied in other ocular pathogenic conditions and human pathology.

Clara cell protein and surfactant protein B in garbage collectors and in wastewater workers exposed to bioaerosols

OBJECTIVES: Inhalation of bioaerosols has been hypothesised to cause "toxic pneumonitis" that should increase lung epithelial permeability at the bronchioloalveolar level. Serum Clara cell protein (CC16) and serum surfactant protein B (SPB) have been proposed as sensitive markers of lung epithelial injury. This study was aimed at looking for increased lung epithelial permeability by determining CC16 and SPB in workers exposed to bioaerosols from wastewater or garbage. METHODS: Subjects (778 wastewater, garbage and control workers; participation 61%) underwent a medical examination, lung function tests [American Thoracic Society (ATS) criteria], and determination of CC16 and SPB. Symptoms of endotoxin exposure and several potential confounders (age, gender, smoking, kidney function, obesity) were looked for. Results were examined with multiple linear or logistic regression. RESULTS: Exposure to bioaerosols increased CC16 concentration in the wastewater workers. No effect of exposure on SPB was found. No clue to work-related respiratory diseases was found. CONCLUSIONS: The increase in CC16 in serum supports the hypothesis that bioaerosols cause subclinical "toxic pneumonitis", even at low exposure. [Authors]

Biliverdin inhibits Toll-like receptor-4 (TLR4) expression through nitric oxide-dependent nuclear translocation of biliverdin reductase.

The cellular response to an inflammatory stressor requires a proinflammatory cellular activation followed by a controlled resolution of the response to restore homeostasis. We hypothesized that biliverdin reductase (BVR) by binding biliverdin (BV) quells the cellular response to endotoxin-induced inflammation through phosphorylation of endothelial nitric oxide synthase (eNOS). The generated NO, in turn, nitrosylates BVR, leading to nuclear translocation where BVR binds to the Toll-like receptor-4 (TLR4) promoter at the Ap-1 sites to block transcription. We show in macrophages that BV-induced eNOS phosphorylation (Ser-1177) and NO production are mediated in part by Ca(2+)/calmodulin-dependent kinase kinase. Furthermore, we show that BVR is S-nitrosylated on one of three cysteines and that this posttranslational modification is required for BVR-mediated signaling. BV-induced nuclear translocation of BVR and inhibition of TLR4 expression is lost in macrophages derived from Enos(-/-) mice. In vivo in mice, BV provides protection from acute liver damage and is dependent on the availability of NO. Collectively, we elucidate a mechanism for BVR in regulating the inflammatory response to endotoxin that requires eNOS-derived NO and TLR4 signaling in macrophages.

L-canavanine, an inhibitor of inducible nitric oxide synthase, improves venous return in endotoxemic rats.

OBJECTIVE: To investigate the hemodynamic effects of L-canavanine (an inhibitor of inducible, but not of constitutive, nitric oxide synthase) in endotoxic shock. DESIGN: Controlled, randomized, experimental study. SETTING: Animal laboratory. SUBJECTS: Wistar rats. INTERVENTIONS: Rats were anesthetized with pentobarbital, and hemodynamically monitored. One hour after an intravenous challenge with 5 mg/kg of Escherichia coli endotoxin, the rats were randomized to receive a continuous infusion of either L-canavanine (20 mg/kg/hr; n = 8) or vehicle only (isotonic saline, n = 11). In all animals, the infusion was given over 5 hrs at a rate of 2 mL/kg/hr. These experiments were repeated in additional rats challenged with isotonic saline instead of endotoxin (sham experiments). MEASUREMENTS AND MAIN RESULTS: Arterial blood pressure, heart rate, thermodilution cardiac output, central venous pressure, mean systemic filling pressure, urine output, arterial blood gases, blood lactate concentration, and hematocrit were measured. In sham experiments, hemodynamic stability was maintained throughout and L-canavanine had no detectable effect. Animals challenged with endotoxin and not treated with L-canavanine developed progressive hypotension and low cardiac output. After 6 hrs of endotoxemia, both central venous pressure and mean systemic filling pressure were significantly below their baseline values, indicating relative hypovolemia as the main determinant of reduced cardiac output. In endotoxemic animals treated with L-canavanine, hypotension was less marked, while cardiac output, central venous pressure, and mean systemic filling pressure were maintained throughout the experiment. L-canavanine had no effect on the time-course of hematocrit. L-canavanine significantly increased urine output and reduced the severity of lactic acidosis. CONCLUSIONS: Six hours after an endotoxin challenge in rats, low cardiac output develops, which appears to be primarily related to relative hypovolemia. L-canavanine, a selective inhibitor of the inducible nitric oxide synthase, increases the mean systemic filling pressure, thereby improving venous return, under these conditions.

Estradiol and progesterone strongly inhibit the innate immune response of mononuclear cells in newborns.

Newborns are particularly susceptible to bacterial infections due to qualitative and quantitative deficiencies of the neonatal innate immune system. However, the mechanisms underlying these deficiencies are poorly understood. Given that fetuses are exposed to high concentrations of estradiol and progesterone during gestation and at time of delivery, we analyzed the effects of these hormones on the response of neonatal innate immune cells to endotoxin, bacterial lipopeptide, and Escherichia coli and group B Streptococcus, the two most common causes of early-onset neonatal sepsis. Here we show that at concentrations present in umbilical cord blood, estradiol and progesterone are as powerful as hydrocortisone for inhibition of cytokine production by cord blood mononuclear cells (CBMCs) and newborn monocytes. Interestingly, CBMCs and newborn monocytes are more sensitive to the effects of estradiol and progesterone than adult peripheral blood mononuclear cells and monocytes. This increased sensitivity is associated with higher expression levels of estrogen and membrane progesterone receptors but is independent of a downregulation of Toll-like receptor 2 (TLR2), TLR4, and myeloid differentiation primary response gene 88 in newborn cells. Estradiol and progesterone mediate their anti-inflammatory activity through inhibition of the NF-κB pathway but not the mitogen-activated protein kinase pathway in CBMCs. Altogether, these results suggest that elevated umbilical cord blood concentrations of estradiol and progesterone acting on mononuclear cells expressing high levels of steroid receptors contribute to impair innate immune responses in newborns. Therefore, intrauterine exposure to estradiol and progesterone may participate in increasing susceptibility to infection during the neonatal period.

Hemoglobin enhances the biological activity of synthetic and natural bacterial (endotoxic) virulence factors: a general principle.

Although hemoglobin (Hb) is mainly present in the cytoplasm of erythrocytes (red blood cells), lower concentrations of pure, cell-free Hb are released permanently into the circulation due to an inherent intravascular hemolytic disruption of erythrocytes. Previously it was shown that the interaction of Hb with bacterial endotoxins (lipopolysaccharides, LPS) results in a significant increase of the biological activity of LPS. There is clear evidence that the enhancement of the biological activity of LPS by Hb is connected with a disaggregation of LPS. From these findings one questions whether the property to enhance the biological activity of endotoxin, in most cases proven by the ability to increase the cytokine (tumor-necrosis-factor-alpha, interleukins) production in human mononuclear cells, is restricted to bacterial endotoxin or is a more general principle in nature. To elucidate this question, we investigated the interaction of various synthetic and natural virulence (pathogenicity) factors with hemoglobin of human or sheep origin. In addition to enterobacterial R-type LPS a synthetic bacterial lipopeptide and synthetic phospholipid-like structures mimicking the lipid A portion of LPS were analysed. Furthermore, we also tested endotoxically inactive LPS and lipid A compounds such as those from Chlamydia trachomatis. We found that the observations made for endotoxically active form of LPS can be generalized for the other synthetic and natural virulence factors: In every case, the cytokine-production induced by them is increased by the addition of Hb. This biological property of Hb is connected with its physical property to convert the aggregate structures of the virulence factors into one with cubic symmetry, accompanied with a considerable reduction of the size and number of the original aggregates.

Factors promoting the exposure to bioaerosols among crop workers

Introduction. Agricultural workers are among the professional groups most at risk of developing acute or chronic respiratory problems. Despite this fact, the etiology of these occupational diseases is poorly known, even in important sectors of agriculture such as the crops sector. Cereals can be colonized by a large number of fungal species throughout the plants' growth, but also during grain storage. Some of these fungi deliver toxins that can have a serious impact on human health when they are ingested via wheat products. Although International and European legislation on contaminants in food, including mycotoxins, include measures to ensure protection of public health by setting down the maximum levels for certain contaminants, the risks associated with the inhalation of such molecules during grain handling remains poorly documented. Goal of study. This project's objective was to characterize worker exposure to pathogenic, irritative or allergenic microorganisms and to identify the abiotic or biotic factors that reduce the growth of these microorganisms in crops. Indeed, the proliferation of microorganisms on wheat is dependent on temperature, rainfall and human disturbance (e.g. usage of tillage, addition of fungicides). A change in the concentration of these microorganisms in the substrate will directly result in a change in the concentration of aerosolized particles of the same microorganisms. Therefore, the exposure of worker to bioaérosols will also change. The Vaud region of Switzerland is a perfect region for conduct such a project as weather conditions vary and agricultural land management programs are divers at a small geographic scale. Methods. Bioaerosols and wheat dust have been sampled during wheat harvesting of summer 2010 at 100 sites uniformly distributed in the Vaud region that are representative of the different agriculture practices. Personal exposure has been evaluated for different wheat related activities: harvesting, grain unload, baling straw, the cleaning of harvesters and silos. Aerosols have been sampled at a rate of 2L/min between 15 min to 4 hours (t) on a 5m PVC filter for estimating the total dust inhaled, on gelatine filter for the identification and quantification of molds, and on a 0.45um polycarbonate filter for endotoxin quantification. Altitude, temperature and annual average rainfall were considered for each site. The physical and chemical characteristics of soils were determined using the methods in effect at Sol Council (Nyon). Total dust has been quantified following NIOSH 0500 method. Reactive endotoxine activity has been determined with Limulus Amebocyte Lysate Assay. All molds have been identified by the pyrosequencing of ITS2 amplicons generated from bioaerosol or wheat dust genomic DNA. Results & Conclusions. Our results confirm the previous quantitative data on the worker exposure to wheat dust. In addition, they show that crop workers are systematically exposed to complex mixtures of allergens, irritants or cytotoxic components. The novelty of our study is the systematic detection of molds such as Fusarium - that is a mycotoxins producer - in the bioaerosols. The results are interpreted by taking in account the agriculture practice, the Phosphorus : Carbon : Nitrogen ratio of the soil, the altitude and the average of rainy days per year.

A Dose-Response Strategy Reveals Differences between Normal-Weight and Obese Men in Their Metabolic and Inflammatory Responses to a High-Fat Meal.

A dose-response strategy may not only allow investigation of the impact of foods and nutrients on human health but may also reveal differences in the response of individuals to food ingestion based on their metabolic health status. In a randomized crossover study, we challenged 19 normal-weight (BMI: 20-25 kg/m(2)) and 18 obese (BMI: >30 kg/m(2)) men with 500, 1000, and 1500 kcal of a high-fat (HF) meal (60.5% energy from fat). Blood was taken at baseline and up to 6 h postprandially and analyzed for a range of metabolic, inflammatory, and hormonal variables, including plasma glucose, lipids, and C-reactive protein and serum insulin, glucagon-like peptide-1, interleukin-6 (IL-6), and endotoxin. Insulin was the only variable that could differentiate the postprandial response of normal-weight and obese participants at each of the 3 caloric doses. A significant response of the inflammatory marker IL-6 was only observed in the obese group after ingestion of the HF meal containing 1500 kcal [net incremental AUC (iAUC) = 22.9 ± 6.8 pg/mL × 6 h, P = 0.002]. Furthermore, the net iAUC for triglycerides significantly increased from the 1000 to the 1500 kcal meal in the obese group (5.0 ± 0.5 mmol/L × 6 h vs. 6.0 ± 0.5 mmol/L × 6 h; P = 0.015) but not in the normal-weight group (4.3 ± 0.5 mmol/L × 6 h vs. 4.8 ± 0.5 mmol/L × 6 h; P = 0.31). We propose that caloric dose-response studies may contribute to a better understanding of the metabolic impact of food on the human organism. This study was registered at clinicaltrials.gov as NCT01446068.

Markedly reduced blood pressure responsiveness in endotoxemic rats; reversal by neuropeptide Y.

This study in conscious normotensive rats was performed to assess the effect of the vasoconstrictor peptide, neuropeptide Y (NPY), on blood pressure responsiveness to exogenous norepinephrine in endotoxaemia. NPY and endotoxin were infused at doses which had no effect on blood pressure, whether given alone or in combination. Endotoxin markedly reduced the pressor responses to bolus injections of norepinephrine. However, blood pressure responsiveness could be enhanced by infusing NPY simultaneously with the endotoxin. It is suggested that low dose NPY infusions may be clinically useful in reversing the reduced vascular responsiveness to pressor amines in shock.

New formulation of vasoactive intestinal peptide using liposomes in hyaluronic acid gel for uveitis.

We evaluated the benefits of a novel formulation of vasoactive intestinal peptide (VIP) based on the incorporation of VIP-loaded rhodamine-conjugated liposomes (VIP-Rh-Lip) within hyaluronic acid (HA) gel (Gel-VIP-Rh-Lip) for the treatment of endotoxin-induced uveitis (EIU) in comparison with VIP-Rh-Lip alone. In vitro release study and rheological analysis showed that interactions between HA chains and liposomes resulted in increased viscosity and reinforced elasticity of the gel. In vivo a single intravitreal injection of Gel-VIP-Rh-Lip was performed in rats 7 days prior to uveitis induction by subcutaneous lipopolysaccharide injection. The maximal ocular inflammation occurs within 16-24 h in controls (VIP-Rh-Lip, unloaded-Rh-Lip). Whereas intraocular injection of VIP-Rh-Lip had no effect on EIU severity compared with controls, Gel-VIP-Rh-Lip reduced significantly the clinical score and number of inflammatory cells infiltrating the eye. The fate of liposomes, VIP and HA in the eyes, regional and inguinal lymph nodes and spleen was analyzed by immunostaining and fluorescence microscopy. Retention of liposomes by HA gel was observed in vitro and in vivo. Inflammation severity seemed to impact on system stability resulting in the delayed release of VIP. Thus, HA gel containing VIP-Rh-Lip is an efficient strategy to obtain a sustained delivery of VIP in ocular and lymph node tissues.

Assessment of bioaerosols and inhalable dust exposure in Swiss sawmills

An assessment of wood workers' exposure to airborne cultivable bacteria, fungi, inhalable endotoxins and inhalable organic dust was performed at 12 sawmills that process mainly coniferous wood species. In each plant, samples were collected at four or five different work sites (debarking, sawing, sorting, planing and sawing cockpit) and the efficiency of sampling devices (impinger or filter) for determining endotoxins levels was evaluated. Results show that fungi are present in very high concentrations (up to 35 000 CFU m(-3)) in all sawmills. We also find that there are more bioaerosols at the sorting work site (mean +/- SD: 7723 +/- 9919 CFU m(-3) for total bacteria, 614 +/- 902 CFU m(-3) for Gram-negative, 19 438 +/- 14 246 CFU m(-3) for fungi, 7.0 +/- 9.0 EU m(-3) for endotoxin and 2.9 +/- 4.8 g m(-3) for dust) than at the sawing station (mean +/- SD: 1938 +/- 2478 CFU m(-3) for total bacteria, 141 +/- 206 CFU m(-3) for Gram-negative, 12 207 +/- 10 008 CFU m(-3) for fungi, 2.1 +/- 1.9 EU m(-3) for endotoxin and 0.75 +/- 0.49 mg m(-3) for dust). At the same time, the species composition and concentration of airborne Gram-negative bacteria were studied. Penicillinium sp. were the predominant fungi, while Bacillus sp. and the Pseudomonadacea family were the predominant Gram-positive and Gram-negative bacteria encountered, respectively. [Authors]