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.

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.

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.

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.

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.

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.

Developmental and photosynthetic regulation of δ-endotoxin reveals that engineered sugarcane conferring resistance to 'dead heart' contains no toxins in cane juice.

The phosphoenolpyruvate (PEP) carboxylase is regulated at the levels of transcription and post-translation in C4 plants in light and abundantly accumulates in leaf mesophyll cells. We report here developmental and photosynthetic regulation of stably accumulated Bacillus thuringiensis δ-endotoxin under the control of PEP-C promoter in transgenic sugarcane. In young leaves of plants, the transprotein is accumulated to 39% of the levels in mature leaves (135 ng mg(-1)), and is induced with the cell development, from base to tip. Nevertheless, these levels are decreased up to 99.98% in non-photosynthetic cells as cane matures, from top to bottom, suggesting the photosynthesis regulation of δ-endotoxin in cane cells. Further, transgenic plants are highly resistant to 'dead heart'. In these studies, Scirpophaga nivela larvae causing 'dead heart' were killed within one hour of release to the transgenic plants. Therefore, this report may be regarded as the first report that provides a better strategy for developing transgenic sugarcane lines with absolute protection against invading larvae and no toxin residues in cane juice.

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.

Bt maize fed-prey mediated effect on fitness and digestive physiology of the ground predator Poecilus cupreus L. (Coleoptera: Carabidae).

We investigated the effects of a Bt maize hybrid on fitness and digestive physiology of the ground-dwelling predator Poecilus cupreus L., as compared with the near-isogenic hybrid. A tritrophic assay revealed that there was a great decline in the detection of Cry1Ab toxin through the trophic chain, the concentration of the toxin being 945, 349 and 37 ng g(-1) of fresh weight in Bt maize leaves, Spodoptera littoralis (Boisduval) larvae and P. cupreus larvae, respectively. Moreover, the toxin was only detected in 8% of the P. cupreus adults collected from fields growing Bt maize. Developmental time of both larvae and pupae of P. cupreus was not adversely affected by the Cry1Ab toxin via fed-prey. To elucidate potential detrimental effects due to a reduction in the quality of the prey, we assessed the digestive proteolytic activities of P. cupreus adults from a laboratory culture and insects collected in commercial Bt and non-Bt maize fields. Field-collected P. cupreus adults had higher proteolytic activities than those reared in the laboratory, whereas no significant differences were found between P. cupreus adults reared on Bt and non-Bt maize fed-S. littoralis or between P. cupreus adults collected in commercial Bt and non-Bt maize fields.

Analysis of Cry1Ab toxin bioaccumulation in a food chain of Bt rice, an herbivore and a predator.

Lessons from organophosphorus pesticides, which could be bioaccumulated in non-target organisms at different trophic levels and caused unexpected negative impacts, necessitate a study of the possibility of biotransfer and bioaccumulation of Bacillus thuringiensis (Bt) insecticidal toxin(s) expressed in Bt plants. Using ELISA, we evaluated the transfer of Cry1Ab toxin in a food chain of Bt rice (KMD1 and KMD2), the target insect, Cnaphalocrocis medinalis, and its predator, Pirata subpiraticus. Cry1Ab was detected in C. medinalis and P. subpiraticus. However, the concentration of Cry1Ab detected from C. medinalis and P. subpiraticus did not increase as feeding or preying time increased. A binding study of Cry1Ab to the brush border membrane vesicle of C. medinalis and P. subpiraticus indicated that P. subpiraticus does not have binding receptors in its midgut to Cry1Ab, while C. medinalis does. Survivorship and fecundity of P. subpiraticus preying on Bt rice-fed C. medinalis were not significantly different from those preying on non-Bt rice-fed C. medinalis. Developmental time of P. subpiraticus was significantly longer when it preyed on Bt rice-fed C. medinalis than on non-Bt rice-fed prey. However, a 3-year field trial indicated that Bt rice did not significantly affect the density of P. subpiraticus.

Peritoneal resorption capacity for the inflammatory mediators in acute experimental Staphylococcus aureus peritonitis.

BACKGROUND: Elevations in both endotoxin and interleukin-6 (IL-6) concentrations in peritoneal exudates are a thousand times higher than their respective concentrations in the peripheral blood in patients with gram-positive or gram-negative peritonitis. We aimed in this study to evaluate the resorption capacity of the peritoneum for endotoxin and IL-6 in a model of bacterial (gram-positive) peritonitis. METHODS: Intraperitoneal (i.p.) injection of mucin-pretreated staphylococci in phosphate buffered saline (PBS) or of PBS alone was performed in 93 male Wistar rats. Studies of resorption were undertaken at time points of 4 hours (h), 8h, 12h and 24h. Endotoxin was intraperitoneally injected in 44 rats and IL-6 in 49 rats. After 0, 5, 10, 15, 30 and 60 minutes (min), blood was sampled. Endotoxin and IL-6 were measured using the limulus-amoebocyte-lysate (LAL) test and ELISA technique, respectively. RESULTS: No endotoxin or IL-6 was measured in the blood of controls. Plasma endotoxin and IL-6 levels were significantly high in the peritonitis groups. There was no further increase in endotoxin plasma levels after i.p. injection of endotoxin. Following i.p. injection of IL-6, there was an increase in IL-6 level over the time of sampling in the peripheral blood at 4h of peritonitis. CONCLUSION: There was a clear reduction in peritoneal resorption of endotoxin and IL-6 in this acute model of gram-positive peritonitis.

Bacillus thuringiensis Cry1Ac toxin-binding and pore-forming activity in brush border membrane vesicles prepared from anterior and posterior midgut regions of lepidopteran larvae.

It is generally accepted that Bacillus thuringiensis Cry toxins insert into the apical membrane of the larval midgut after binding to specific receptors, and there is evidence that the distribution of binding molecules along the midgut is not uniform. By use of the voltage-sensitive dye DiSC(3)(5) and (125)I-labeled Cry1Ac, we have measured the effect of Cry1Ac in terms of permeabilization capacity and of binding parameters on brush border membrane vesicles (BBMV) prepared from the anterior and the posterior regions of the larval midgut from two insect species, Manduca sexta and Helicoverpa armigera. The permeabilizing activity was significantly higher with BBMV from the posterior region than with the one observed in the anterior region in both insect species. Instead, (125)I-Cry1Ac bound specifically to BBMV from the two midgut regions, with no significant differences in the binding parameters between the anterior and posterior regions within an insect species. N-acetylgalactosamine inhibition patterns on pore formation and binding differed between anterior and posterior midgut regions and between species, providing evidence of a multifaceted involvement of the sugar in the Cry1Ac mode of action. The analysis of binding and pore formation in different midgut regions could be an effective method to study differences in the mode of action of Cry1Ac toxin in different species.

The in vivo digestive fate of the Cry3Bb1 protein in laying hens fed diets containing MON 863 corn.

Two trials were conducted to assess the fate of the Cry3Bb1 protein from YieldGard rootworm corn (MON 863) when fed to laying hens. In the first trial, 2 diets, 1 formulated with MON 863 and 1 with conventional corn, were fed to laying hens (12 replicate cages with 4 hens/cage per treatment) for 8 wk. Daily feed intake (FI), egg production (EP), and BW were measured. Prestudy fecal samples, wk 4 and 8 egg and fecal samples, and hepatic and pectoralis tissue samples were collected from 12 killed hens and were tested for the Cry3Bb1 protein. Corn source had no significant effects on FI, EP, or BW. Feces from hens fed diets containing MON 863 were positive for the Cry3Bb1 protein or proteolytic fragments (1.5 to 4.0 ppm fecal dry matter). The Cry3Bb1 protein could not be determined in eggs due to the presence of an interfering substance in all test and control eggs. No Cry3Bb1 protein was detected in hepatic and pectoralis tissue. In the second trial, the same test and control diets were fed to 12 hens each. Six hens/treatment were sampled after 7 and 28 d. Samples included blood, feces, and digesta (crop, small and large intestine, and ceca). The Cry3Bb1 protein could not be determined in blood due to the presence of an interfering substance in all test and control blood samples. The Cry3Bb1 protein or partially digested fragments, or both, were found in the digesta sampled from all sections of the digestive tract. About 98 to >99% of the dietary Cry3Bb1 protein was digested. Overall, MON 863, when fed to laying hens, had no significant effects on FI, EP, or BW. The Cry3Bb1 protein was extensively digested, similar to that of other dietary proteins, and was not detected in hepatic or muscle tissue.

Chylomicrons alter the fate of endotoxin, decreasing tumor necrosis factor release and preventing death.

The hypertriglyceridemia of infection was traditionally thought to represent the mobilization of substrate to fuel the body's response to the infectious challenge. However, we have previously shown that triglyceride-rich lipoproteins can protect against endotoxin-induced lethality. The current studies examine the mechanism by which this protection occurs. Rats infused with a lethal dose of endotoxin preincubated with chylomicrons had a reduced mortality compared with rats infused with endotoxin alone (15 vs. 76%, P < 0.001). Preincubation with chylomicrons increased the rate of clearance of endotoxin from plasma and doubled the amount of endotoxin cleared by the liver (30 +/- 1 vs. 14 +/- 2% of the total infused radiolabel, P < 0.001). In addition, autoradiographic studies showed that chylomicrons directed more of the endotoxin to hepatocytes and away from hepatic macrophages. Rats infused with endotoxin plus chylomicrons also showed reduced peak serum levels of tumor necrosis factor as compared with controls (14.2 +/- 3.3 vs. 44.9 +/- 9.5 ng/ml, mean +/- SEM, P = 0.014). In separate experiments, chylomicrons (1,000 mg triglyceride/kg) or saline were infused 10 min before the infusion of endotoxin. Chylomicron pretreatment resulted in a reduced mortality compared with rats infused with endotoxin alone (22 vs. 78%, P < 0.005). Therefore, chylomicrons can protect against endotoxin-induced lethality with and without preincubation with endotoxin. The mechanism by which chylomicrons protect against endotoxin appears to involve the shunting of endotoxin to hepatocytes and away from macrophages, thereby decreasing macrophage activation and the secretion of cytokines.

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.

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.

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.

Effects of initial passage of endotoxin through the liver on the extent of acute lung injury in a rat model.

We hypothesized that the extent of acute lung injury (ALI) caused by lipopolysaccharide (LPS) is modified with its initial passage through the liver. We tested this hypothesis by administering LPS, 5 mg/kg, or saline to 120 male Wistar rats via the portal vein (PV) or the inferior vena cava (IVC) over 1 h. Four experimental groups of rats were administered saline into the PV, saline into the IVC, LPS into the PV (LPS-PV group), and LPS into the IVC (LPS-IVC group), respectively. At 15 and 30 min after onset of 51Chromium-LPS infusion, the gamma counts in the liver were higher in the LPS-PV group than that in the LPS-IVC group. The ratio of 125Iodine-albumin counts in lung tissue to that in plasma per unit of weight (as an assessment of pulmonary microvascular permeability) at 240 min after onset of LPS stimulation, the accumulation of polymorphonuclear cell (assessed by myeloperoxidase activity) and the concentration of tumor necrosis factor alpha in the lung at 60 and 240 min after onset of LPS infusion, were higher in the LPS-IVC group than in the LPS-PV group. Significant differences in several factors indicative of inflammation and in the extent of LPS-induced ALI were observed after the onset of LPS infusion, depending on whether it was delivered via the PV or the IVC. These observations suggest that the entrapping of LPS during its initial passage through the hepatic circulation may attenuate LPS-induced ALI within 4 h of initiation of LPS stimulation.