Endotoxins Induced ECM-Receptor Interaction Pathway Signal Effect on the Function of MUC2 in Caco2/HT29 Co-Culture Cells.

Endotoxins are toxic substances that widely exist in the environment and can enter the intestine with food and other substances. Intestinal epithelial cells are protected by a mucus layer that contains MUC2 as its main structural component. However, a detailed understanding of the mechanisms involved in the function of the mucus barrier in endotoxin penetration is lacking. Here, we established the most suitable proportion of Caco-2/HT-29 co-culture cells as a powerful tool to evaluate the intestinal mucus layer. Our findings significantly advance current knowledge as focal adhesion and ECM-receptor interaction were identified as the two most significantly implicated pathways in MUC2 small interfering RNA (siRNA)-transfected Caco-2/HT-29 co-culture cells after 24 h of LPS stimulation. When the mucus layer was not intact, LPS was found to damage the tight junctions of Caco-2/HT29 co-cultured cells. Furthermore, LPS was demonstrated to inhibit the integrin-mediated focal adhesion structure and damage the matrix network structure of the extracellular and actin microfilament skeletons. Ultimately, LPS inhibited the interactive communication between the extracellular matrix and the cytoskeleton for 24 h in the siMUC2 group compared with the LPS(+) and LPS(-) groups. Overall, we recognized the potential of MUC2 as a tool for barrier function in several intestinal bacterial diseases.

Microbial Products and Metabolites Contributing to Alcohol-Related Liver Disease.

As a serious public health concern, alcohol-related liver disease is associated with dysregulations in the intestinal barrier function and the gut microbiota. The liver and gut communicate via the gut-liver axis, through which microbial products and metabolites translocate to the liver. Here, the current knowledge of various microbial products and metabolites which contribute to the alcohol-related liver diseases, including bile acids, indole-3-acetic acid, butyrate, long-chain fatty acids, endotoxin, cytolysin, ß-glucan, and candidalysin is reviewed. Some of these might serve as therapeutic targets for alcohol-related liver disease.

Gut derived-endotoxin contributes to inflammation in severe ischemic acute kidney injury.

BACKGROUND: Recent researches indicate that the intestinal consequences of renal ischemia reperfusion (IR) would predispose to the translocation of gut-derived endotoxin. Here, we designed experiments to test the hypothesis that the gut-derived endotoxin has a potential role in mediating local inflammatory processes in the acutely injured kidney. METHODS: Rats were performed sham or renal IR surgery (60 min of bilateral renal ischemia, then 24 h of reperfusion) (n = 5). The intestinal structural and mucosa permeability were evaluated. Serum endotoxin and bacterial load in liver and mesenteric lymph nodes (MLN) were measured. Separate groups were pretreated with oral norfloxacin 20 mg/kg/day or saline for 4 weeks and divided into sham plus saline, sham plus norfloxacin, renal IR plus saline and renal IR plus norfloxacin group. Serum biochemistry and endotoxin were determined. Kidney pathological changes were scored. Protein or mRNA expression of toll-like receptor 4 (TLR4) and proinflammatory mediators were measured in kidney homogenate. RESULTS: Renal IR led to marked intestinal integrity disruption and increase in intestinal permeability. These are accompanied by low grade of endotoxemia as well as increased bacterial load in liver and MLN. The group pretreated with norfloxacin showed significant attenuation of the increase in serum urea, ALAT, ASAT and endotoxin. The increased renal protein or mRNA of TLR4 and proinflammatory mediators (IL-6 and MCP-1) in the unpretreated animals was significantly attenuated in the norfloxacin-pretreated animals. However, norfloxacin pretreatment did not produce any protective effects on renal tubular integrity. CONCLUSIONS: Our results show for the first time that gut-derived endotoxin, resulting from an increased intestinal permeability after severe renal IR, subsequently amplifies intrarenal inflammatory response by activation renal TLR4 signaling. Our study results do not establish that antibiotic administration was effective in improving the overall renal outcome. However, our findings may be the first step to understanding how to tailor therapies to mitigate intrarenal inflammation in select groups of patients.

Factors Affecting Reduction of Reference Endotoxin Standard Activity Caused by Chelating Agent/Detergent Matrices: Kinetic Analysis of Low Endotoxin Recovery.

Reduction of reference standard endotoxin activity was kinetically analyzed under low endotoxin recovery conditions and was considered as an apparent first-order reaction. Temperature, pH, and salt concentrations affected the rates of reduction of reference standard endotoxin activity. Temperature appeared to be the most important factor affecting low endotoxin recovery. Components of low endotoxin recovery matrices, such as citrate and polysorbate 20, showed similar low endotoxin recovery effect at concentrations commonly used. Phosphate concentrations showed negative correlation against the half-life of reference standard endotoxin activity in solutions containing phosphate buffer and polysorbate 20. Activation energy for low endotoxin recovery with naturally occurring endotoxin was higher than that with reference standard endotoxin, and this explained one of the reasons for naturally occurring endotoxin resistance to low endotoxin recovery. Lower temperature, lower pH, and a higher salt concentration are preferable to avoid low endotoxin recovery in a hold-time study. This study provides useful data for anticipation of the severity of the low endotoxin recovery effect and future hold-time studies in the biopharmaceutical field.LAY ABSTRACT: Endotoxin derived from Gram-negative bacteria is potentially harmful when it is parenterally administrated. Therefore, injectables and medical devices are tested by the bacterial endotoxins test to detect contamination by endotoxin of those products. Low endotoxin recovery is a phenomenon of reduction of detectable standard endotoxin activity by certain matrices of biopharmaceutical products containing a chelating agent and a detergent, and it is a controversial topic because its mechanism and clinical risks are unknown. The author analyzed the kinetics of low endotoxin recovery to elucidate the mechanism of low endotoxin recovery and to propose conditions to avoid low endotoxin recovery.

Determination of a No-Observable Effect Level for Endotoxin Following a Single Intravitreal Administration to Dutch Belted Rabbits.

Purpose: The purpose of this study was to characterize the inflammatory response and determine a no-observable effect level (NOEL) in rabbit eyes after endotoxin intravitreal (ITV) injection. Methods: Fifty-three naïve male Dutch Belted rabbits were treated with a single 50-µL ITV injection ranging from 0.01 to 0.75 endotoxin units/eye (EU/eye) and monitored for up to 42 days post treatment. Ophthalmic examination included slit-lamp biomicroscopy and indirect ophthalmoscopy. Laser flare photometry was performed in a subset of animals. On days 2, 8, 16, and 43, a subset of animals was necropsied and eyes processed for histopathological evaluation. Results: Intravitreal injection of endotoxin at ≥0.05 EU/eye resulted in a dose-related anterior segment inflammation response. No aqueous flare or cell response was noted in the 0.01 EU/eye dose group. A more delayed posterior segment response characterized by vitreal cell response was observed beginning on day 5, peaking on day 9, and decreasing starting on day 16 that persisted at trace to a level of 1+ on day 43. Microscopy findings of infiltrates of minimal mixed inflammatory cells in the vitreous and subconjunctiva and proteinaceous fluid in the anterior chamber and/or vitreous were observed in eyes given ≥0.1 EU/eye. Conclusions: We defined the NOEL for ITV endotoxin to be 0.01 EU/eye, suggesting that the vitreal cavity is more sensitive to the effects of endotoxin than the anterior segment and aqueous chamber. These data highlight the importance of assessing endotoxin level in intravitreal formulations, as levels as low as 0.05 EU/eye may confound the safety evaluations of intravitreal therapeutics in rabbits.

Gut microbiota in chronic kidney disease. / Microbiota intestinal en la enfermedad renal crónica.

The intestinal microflora maintains a symbiotic relationship with the host under normal conditions, but its imbalance has recently been associated with several diseases. In chronic kidney disease (CKD), dysbiotic intestinal microflora has been reported with an increase in pathogenic flora compared to symbiotic flora. An enhanced permeability of the intestinal barrier, allowing the passage of endotoxins and other bacterial products to the blood, has also been shown in CKD. By fermenting undigested products that reach the colon, the intestinal microflora produce indoles, phenols and amines, among others, that are absorbed by the host, accumulate in CKD and have harmful effects on the body. These gut-derived uraemic toxins and the increased permeability of the intestinal barrier in CKD have been associated with increased inflammation and oxidative stress and have been involved in various CKD-related complications, including cardiovascular disease, anaemia, mineral metabolism disorders or the progression of CKD. The use of prebiotics, probiotics or synbiotics, among other approaches, could improve the dysbiosis and/or the increased permeability of the intestinal barrier in CKD. This article describes the situation of the intestinal microflora in CKD, the alteration of the intestinal barrier and its clinical consequences, the harmful effects of intestinal flora-derived uraemic toxins, and possible therapeutic options to improve this dysbiosis and reduce CKD-related complications.

Membrane adsorber for endotoxin removal

ABSTRACT The surface of flat-sheet nylon membranes was modified using bisoxirane as the spacer and polyvinyl alcohol as the coating polymer. The amino acid histidine was explored as a ligand for endotoxins, aiming at its application for endotoxin removal from aqueous solutions. Characterization of the membrane adsorber, analysis of the depyrogenation procedures and the evaluation of endotoxin removal efficiency in static mode are discussed. Ligand density of the membranes was around 7 mg/g dry membrane, allowing removal of up to 65% of the endotoxins. The performance of the membrane adsorber prepared using nylon coated with polyvinyl alcohol and containing histidine as the ligand proved superior to other membrane adsorbers reported in the literature. The lack of endotoxin adsorption on nylon membranes without histidine confirmed that endotoxin removal was due to the presence of the ligand at the membrane surface. Modified membranes were highly stable, exhibiting a lifespan of approximately thirty months.

RESUMO A superfície de membranas planas de nylon foi modificada utilizando-se bisoxirano como espaçador e poli(álcool vinílico) para recobrimento das membranas. O aminoácido histidina foi utilizado como ligante para endotoxinas, visando à sua aplicação na remoção de endotoxinas a partir de soluções aquosas. São discutidas as etapas de caracterização do adsorvedor com membranas, análise do procedimento de despirogenização e avaliação da eficiência de remoção em modo estático. A densidade de ligantes nas membranas foi em torno de 7 mg/g membrana (massa seca), permitindo uma remoção de endotoxinas de até 65%. O desempenho das membranas preparadas com nylon e recobertas com poli(álcool vinílico) contendo histidina como ligante foi superior ao de outros adsorvedores com membranas descritos na literatura. A ausência de adsorção de endotoxinas em membranas sem histidina confirma que a remoção das endotoxinas deve-se exclusivamente à presença do ligante na superfície da membrana. As membranas modificadas mostraram-se bastante estáveis, exibindo um tempo de vida superior a 30 dias.

Use of sevelamer in chronic kidney disease: beyond phosphorus control.

Sevelamer is a non-calcium phosphate binder used in advanced chronic kidney disease (CKD) and in dialysis for hyperphosphataemia control. Several experimental, observational studies and clinical trials have shown that sevelamer has pleiotropic effects, beyond hyperphosphataemia control, including actions on inflammation, oxidative stress, lipid profile and atherogenesis, vascular calcification, endothelial dysfunction and the reduction of several uremic toxins. This is the biological basis for its global effect on cardiovascular morbidity and mortality in patients with chronic kidney disease. This review focuses on these pleiotropic actions of sevelamer and their impact on cardiovascular health, with the experience published after more than ten years of clinical expertise.

IL-1 receptor blockade alleviates endotoxin-mediated impairment of renal drug excretory functions in rats.

The aim of our study was to investigate whether two potent anti-inflammatory agents, dexamethasone and anakinra, an IL-1 receptor antagonist, may influence acute kidney injury (AKI) and associated drug excretory functions during endotoxemia (LPS) in rats. Ten hours after LPS administration, untreated endotoxemic rats developed typical symptoms of AKI, with reduced GFR, impaired tubular excretion of urea and sodium, and decreased urinary excretion of azithromycin, an anionic substrate for multidrug resistance-transporting proteins. Administration of both immunosuppressants attenuated the inflammatory response, liver damage, AKI, and increased renal clearance of azithromycin mainly by restoration of GFR, without significant influence on its tubular secretion. The lack of such an effect was related to the differential effect of both agents on the renal expression of individual drug transporters. Only dexamethasone increased the urinary clearance of bile acids, in accordance with the reduction of the apical transporter (Asbt) for their tubular reabsorption. In summary, our data demonstrated the potency of both agents used for the prevention of AKI, imposed by endotoxins, and for the restoration of renal drug elimination, mainly by the improvement of GFR. The influence of both drugs on altered tubular functions and the expression of drug transporters was differential, emphasizing the necessity of knowledge of transporting pathways for individual drugs applied during sepsis. The effect of anakinra suggests a significant contribution of IL-1 signaling to the pathogenesis of LPS-induced AKI.

Physiological-based pharmacokinetic modeling of endotoxin in the rat.

We have previously measured the distribution and pharmacokinetics of biosynthetically radiolabeled endotoxin of Salmonella typhimurium following intraperitoneal (IP) dosing (200 µg/kg) in Sprague-Dawley rats. In our experiments, the fatty acid residues were labeled with (3)H and the glucosamine residues were labeled with (14)C. To predict the dynamics of endotoxin exposure, we developed a physiological-based pharmacokinetic model using our measured distribution results. The model was validated with published low-dose (30 µg/kg) IP exposure results in rats. Endotoxin pharmacokinetics depended on dose and route. At high IP doses, absorption was followed by biphasic decay over 48 h in plasma. There were tissue accumulations of the fatty acid and glucosamine residues in various target organs, including the brain. We also found that the glucosamine and fatty acid components separated in vivo about 3 h after IP injection. At the lower IP dose, a smaller fraction of the dose was distributed to the tissues, with most of the dose remaining in the blood. Each component had its own dynamic behavior and target tissue distribution in the rat. The fatty acid components tended to remain in the brain stem, caudate nucleus, cerebellum, frontal cortex, hippocampus, and hypothalamus. Other organs (spleen, kidney, meninges, and choroid plexus) had similar biphasic distribution. The liver had the unique accumulation of both glucosamine and fatty acid residues.

Distribution and pharmacokinetics of double-radiolabeled endotoxin in the rat brain and peripheral organs.

The endotoxin, lipopolysaccharide (LPS), of Salmonella typhimurium was biosynthetically labeled with (3)H and (14)C incorporated into the fatty acyl chains and glucosamine residues, respectively. The radio-labeled LPS was isolated from the bacteria and then injected into Sprague-Dawley rats. The distribution of (14)C and (3)H-LPS in plasma and other organs was determined following intraperitoneal (IP) doses of (14)C and (3)H-LPS (200 µg/kg). Plasma concentrations of both fatty acyl chains and glucosamine residues were biphasic, with a relatively rapid decay followed by a slow decline for 48 h. Similar biphasic results were found in the peripheral organs (kidney and heart) and brain barrier tissues (meninges and choroid plexus). In other brain tissues (brain stem, caudate nucleus, hypothalamus, frontal cortex, cerebellum and hippocampus), the glucosamine residue was biphasic, whereas the fatty acyl chains showed accumulation. Highest concentrations of LPS were found in the plasma, spleen and the liver. In addition, in the liver, sustained elevations of (14)C-glucosamine and (3)H-fatty acyl chains were observed. This indicates LPS accumulation in the liver. By contrast, the spleen showed biphasic decay of glucosamine residues and accumulation of fatty acyl chains. In the brain barrier tissues, peak LPS concentrations were significantly reduced (about 70%) and were further reduced (about 95%) in other brain tissues. The high elevation of LPS in the spleen is considered indicative of an immune response. Our findings highlight the potential significant role of lipid A as shown with the sustained elevation of (3)H-fatty acyl chains in the brain.

Unlinked genetic loci control the reduced transcription of aminopeptidase N 1 and 3 in the European corn borer and determine tolerance to Bacillus thuringiensis Cry1Ab toxin.

Transgenic expression of Bacillus thuringiensis (Bt) crystalline (Cry) toxins by crop plants result in reduced insect feeding damage, but sustainability is threatened by the development of resistance traits in target insect populations. We investigated Bt toxin resistance trait in a laboratory colony of the European corn borer, Ostrinia nubilalis, selected for increased survival when exposed to Cry1Ab and correlated survival on Cry1Ab toxin with a constitutive ∼146.2 ± 17.3-fold reduction in midgut aminopeptidase N1 (apn1) transcript levels. A 7.1 ± 1.9-fold reduction apn3 transcript level was also correlated with Cry1Ab resistance. Quantitative trait locus (QTL) mapping identified a single major genome region controlling Cry1Ab resistance on linkage group 24 (LG24), and a minor QTL on LG27. Both QTL were independent of apn1 and apn3 loci on LG02. Positional mapping identified genetic markers that may assist in the identification of causal gene(s) within QTL intervals. This study indicates that genetic factor(s) may act in trans to reduce both apn1 and apn3 expression in Cry1Ab resistant O. nubilalis larvae, and suggest that gene regulatory pathways can influence Bt resistance traits. These findings show that gene interactions (epistasis) may influence Bt resistance in target insect populations.

Utilidad de la hemoperfusión en el tratamiento del paciente séptico grave. Actualización / Usefulness of haemoperfusion in the treatment of the severe septic patient: an update

La hemoperfusión es un procedimiento extracorpóreo que consiste en la retirada de endotoxina y/o mediadores inflamatorios por un mecanismo de adsorción durante el paso de la sangre por un filtro específico. La mayor parte de los estudios publicados han empleado la polimixina B como adsorbente. Este tratamiento se basa en la premisa de que la eliminación de endotoxina y mediadores de la circulación atenúa la respuesta inflamatoria en la sepsis. Se revisan las bases teóricas y los resultados clínicos publicados con el uso de la hemoperfusión. Si bien la mayoría de los estudios que emplean esta técnica presentan resultados positivos, existen dudas acerca de la idoneidad de los métodos empleados (grupos pequeños, baja calidad en el diseño de los estudios, mortalidad excesiva en los grupos control). También existen inconsistencias en la base teórica de su uso (ausencia de beneficios tras la eliminación de endotoxina por otros mecanismos, discrepancias en el momento de iniciar la terapia, aparente utilidad en enfermedades sin elevación de endotoxina). Los autores opinan que la hemoperfusión es prometedora en el tratamiento de la sepsis, pero requiere su confirmación en estudios bien diseñados antes de ser incluida en los protocolos habituales de tratamiento(AU)

Haemoperfusion is an extracorporeal technique that removes endotoxin and/or inflammatory mediators by means of an adsorptive mechanism during the passage of the blood through a porous filter. Most of the studies in the literature use polymyxin B as the adsorptive agent. This treatment is based on the assumption that the removal of endotoxin and inflammatory mediators from the circulation attenuates the inflammatory response in sepsis. This review summarizes the theoretical basis, and the experimental and clinical results published to date with the use of haemoperfusion. Although most of the studies show positive results, some doubts have arisen about the suitability of the methods described (small number of cases, low quality of the experimental design, and excessive mortality in the control groups). There are also some inconsistencies regarding the theoretical basis of its use (lack of positive effects after the removal of endotoxin from the circulation using alternative mechanisms, discrepancies regarding the best moment to initiate the therapy, unexplained beneficial effects in the absence of increased endotoxin levels). It is the opinion of the authors that haemoperfusion represents a promising therapy for the treatment of sepsis, but consider that its usefulness requires confirmation in well designed studies before being included in protocols(AU)

The ability of endotoxin adsorption during a longer duration of direct hemoperfusion with a polymyxin B-immobilized fiber column in patients with septic shock.

The patients' hemodynamic conditions of septic shock due to intra-abdominal infection were improved by the longer duration of direct hemoperfusion with a polymyxin B-immobilized fiber column (PMX), reducing plasma endotoxins measured by the novel endotoxin detection method, named endotoxin scattering photometry (ESP) method; however, turbidimetric method could not detect endotoxins. We also observed the reduction in the endotoxin after passing through column by ESP method even after the longer duration of PMX. ESP method may more sensitively detect endotoxins than the ordinary turbidimetric method. Moreover, we demonstrated the ability of endotoxin adsorption in spite of the longer duration of PMX.

Immunology in alcoholic liver disease.

Hepatic fibrosis is a known consequence of long-term use of alcohol and is regarded as a turning point in alcohol-induced liver disease because it can lead to cirrhosis. The mechanisms of injury are not well understood, but recent studies have helped advance the understanding of the earliest events in the process that eventually leads to hepatic injury and, in some cases, fibrosis. It is hoped that increasing understanding of the role played by the immune system in the process will lead to the development of new therapies for these patients.

The dose-dependent effects of endotoxin on protein metabolism in two types of rat skeletal muscle

Endotoxin administration is frequently used as a model of systemic inflammatory response which is considered the important pathogenetic factor in muscle wasting development in severe illness, such as sepsis, cancer, injury, AIDS and others. The main purpose of this study was determining the effect of various doses of endotoxin on protein and amino acid metabolism in two types of rat skeletal muscle. Sepsis was induced by intraperitoneal administration of endotoxin in a dose of 1, 3 and 5 mg/kg body weight (bw); control animals received a corresponding volume of the saline solution. After 24 h, extensor digitorum longus (EDL) and soleus (SOL) muscles were isolated and used for determination of total and myofibrillar proteolysis, protein synthesis, activity of cathepsins B and L, chymotrypsin-like activity of proteasome and amino acid release. The endotoxemia induced the body weight loss, the rise of total cholesterol and triglyceride plasma concentration and the protein catabolic state in skeletal muscle, which was caused by a higher increase in protein breakdown (due to activation of the proteasome system) than protein synthesis. The more significant effect of endotoxin was seen in EDL than SOL. The dose of 5 mg of endotoxin/kg bw induced the most significant changes in parameters of the protein and amino acid metabolism measured and could be therefore considered appropriate for studies of protein catabolism in young rat skeletal muscle at 24 h after endotoxin treatment (AU)

Metformin protects against the development of fructose-induced steatosis in mice: role of the intestinal barrier function.

To test the hypothesis that metformin protects against fructose-induced steatosis, and if so, to elucidate underlying mechanisms, C57BL/6J mice were either fed 30% fructose solution or plain water for 8 weeks. Some of the animals were concomitantly treated with metformin (300 mg/kg body weight/day) in the drinking solution. While chronic consumption of 30% fructose solution caused a significant increase in hepatic triglyceride accumulation and plasma alanine-aminotransferase levels, this effect of fructose was markedly attenuated in fructose-fed mice concomitantly treatment with metformin. The protective effects of the metformin treatment on the onset of fructose-induced non-alcoholic fatty liver disease (NAFLD) were associated with a protection against the loss of the tight junction proteins occludin and zonula occludens 1 in the duodenum of fructose-fed mice and the increased translocation of bacterial endotoxin found in mice only fed with fructose. In line with these findings, in metformin-treated fructose-fed animals, hepatic expression of genes of the toll-like receptor-4-dependent signalling cascade as well as the plasminogen-activator inhibitor/cMet-regulated lipid export were almost at the level of controls. Taken together, these data suggest that metformin not only protects the liver from the onset of fructose-induced NAFLD through mechanisms involving its direct effects on hepatic insulin signalling but rather through altering intestinal permeability and subsequently the endotoxin-dependent activation of hepatic Kupffer cells.

Effects of chitosan-nanoparticle-mediated tight junction opening on the oral absorption of endotoxins.

Recently, we reported a pH-responsive nanoparticle (NP) system shelled with chitosan (CS), which could effectively increase the oral absorption of insulin and produce a hypoglycemic effect, presumably due to the CS-mediated tight junction (TJ) opening. It has been often questioned whether CS can also enhance the absorption of endotoxins present in the small intestine. To address this concern, we studied the effect of CS NPs on the absorption of lipopolysaccharide (LPS), the most commonly found toxin in the gastrointestinal tract. To follow their biodistribution by the single-photon emission computed tomography/computed tomography, LPS and insulin were labeled with (99m)Tc-pertechnetate ((99m)Tc-LPS) and (123)iodine ((123)I-insulin), respectively. The (99m)Tc-LPS was ingested 1 h prior to the administration of the (123)I-insulin-loaded NPs to mimic the physiological conditions. The confocal and TEM micrographs show that the orally administered CS NPs were able to adhere and infiltrate through the mucus layer, approach the epithelial cells and mediate to open their TJs. The radioactivity associated with LPS was mainly restricted to the gastrointestinal tract, whereas (123)I-insulin started to appear in the urinary bladder at 3 h post administration. This observation indicates that the insulin-loaded in CS NPs can traverse across the intestinal epithelium and enter the systemic circulation, whereas LPS was unable to do so, probably because of the charge repulsion between the anionic LPS in the form of micelles and the negatively charged mucus layer. Our in vivo toxicity study further confirms that the enhancement of paracellular permeation by CS NPs did not promote the absorption of LPS. These results suggest that CS NPs can be used as a safe carrier for oral delivery of protein drugs.

Fate of Cry1Ab protein in agricultural systems under slurry management of cows fed genetically modified maize (Zea mays L.) MON810: a quantitative assessment.

The objective of the study was to track the fate of recombinant Cry1Ab protein in a liquid manure field trial when feeding GM maize MON810 to dairy cows. A validated ELISA was applied for quantification of Cry1Ab in the agricultural chain from GM maize plants, feed, liquid manure and soil to crops grown on manured fields. Starting with 23.7 µg of Cry1Ab g(-1) dry weight GM maize material, a rapid decline of Cry1Ab levels was observed as 2.6% and 0.9% of Cry1Ab from the GM plant were detected in feed and liquid manure, respectively. Half of this residual Cry1Ab persisted during slurry storage for 25 weeks. After application to experimental fields, final degradation of Cry1Ab to below detectable levels in soil was reported. Cry1Ab exhibited a higher rate of degradation compared to total protein in the agricultural processes. Immunoblotting revealed a degradation of the 65 kDa Cry1Ab into immunoreactive fragments of lower size in all analyzed materials.

Differential ex vivo and in vivo endotoxin tolerance kinetics following human endotoxemia.

OBJECTIVES: Endotoxin (lipopolysaccharide) tolerance is characterized by a transient refractory state to a subsequent lipopolysaccharide challenge. Following human endotoxemia, ex vivo tolerance of circulating leukocytes to lipopolysaccharide resolves within 24 hrs. However, the duration of in vivo tolerance, assumed to be primarily mediated by tissue-resident macrophages, is unknown. DESIGN, SETTING, SUBJECTS, AND INTERVENTIONS: Clinical experimental study in 16 healthy male volunteers at an intensive care research unit. To compare ex vivo and in vivo tolerance kinetics, whole blood from healthy volunteers was stimulated with lipopolysaccharide before, 4 hrs after, and 1, 2, 3, and 4 wks following in vivo endotoxin (2 ng/kg; lipopolysaccharide derived from Escherichia coli O:113) administration. Furthermore, we compared the inflammatory response during two subsequent endotoxemia experiments in healthy volunteers with an interval of 2 wks. The cytokines tumor necrosis factor-α, interleukin-6, interleukin-10, interleukin-1 receptor antagonist, and transforming growth factor-ß were measured. MEASUREMENTS AND MAIN RESULTS: Four hours after in vivo lipopolysaccharide administration, production of tumor necrosis factor-α, interleukin-6, and interleukin-10, but not interleukin-1 receptor antagonist in ex vivo lipopolysaccharide-stimulated whole blood was diminished. Ex vivo lipopolysaccharide tolerance completely resolved within 1 week. In contrast, in vivo lipopolysaccharide tolerance was still apparent after 2 wks. Compared to the first lipopolysaccharide administration, plasma peak levels of tumor necrosis factor-α, interleukin-6, interleukin-10, interleukin-1 receptor antagonist, and transforming growth factor-ß were attenuated by 46%, 36%, 45%, 10%, and 14%, respectively (all p < .05). CONCLUSIONS: While ex vivo lipopolysaccharide tolerance quickly resolves, in vivo lipopolysaccharide tolerance persists for at least 2 wks. These findings strengthen the notion that the in vivo response to lipopolysaccharide is mediated by tissue-resident macrophages and that ex vivo stimulation does not accurately reflect the in vivo innate immune response. Intervention studies utilizing the human endotoxemia model should be performed using parallel groups rather than a crossover design.