Cytokine Adsorption Effects of a Novel Hemofiltration Column for the Treatment of Experimental Endotoxemia.

INTRODUCTION: The TKM-101 is a new hemofiltration column packed with a polymer alloy membrane consisting of polyethersulfone, polyvinylpyrrolidone, and sulfonated poly (arylene ether) copolymers. We examined the ability of the TKM-101 column to remove cytokines and humoral mediators from blood in vitro and the effects of extracorporeal treatment with the TKM-101 column on the mortality rate and inflammatory responses to endotoxic shock in vivo. METHODS: In vitro and in vivo laboratory investigations were conducted. In the in vitro experiment, the adsorption abilities of TKM-101, AN69-ST, and control columns for cytokine-related sepsis in blood were compared using human serum samples. In the in vivo experiment, male Sprague-Dawley rats were anesthetized and injected with Escherichia coli endotoxin (15 mg/kg, intravenously). Afterward, the rats were assigned (in a double-blind manner) to one of three groups (n = 17 per group): apheresis with a control column (control group), apheresis with an AN69-ST column (AN69-ST group), or apheresis with a TKM-101 column (TKM-101 group). Outcomes were compared among the groups. RESULTS: In vitro, the concentrations of all evaluated cytokines significantly decreased with the TKM-101 column compared to those with the control column; however, there were no significant differences between the TKM-101 and AN69-ST columns. In vivo, the mortality rates 8 h after endotoxin injection were 65%, 29%, and 29% for the control, AN69-ST, and TKM-101 groups, respectively. Hypotension and elevated plasma cytokine concentrations were less prominent in the TKM-101 and AN69-ST groups compared to those in the control group. CONCLUSIONS: TKM-101 effectively removed proteins of varying sizes, from small-sized proteins such as interleukin (IL)-8 to mid-sized protein such as IL-10 in vitro. Moreover, TKM-101 treatment reduced mortality and had inhibitory effects on inflammatory responses in endotoxemic rats. These findings suggest that TKM-101 treatment may be available for use in patients with sepsis and/or endotoxemia.

Endotoxemia Correlates with Kidney Function and Length of Stay in Critically Ill Patients.

INTRODUCTION: Endotoxin is a key driver of sepsis, which frequently causes acute kidney injury (AKI). However, endotoxins may also be found in non-bacteremic critically ill patients, likely from intestinal translocation. Preclinical models show that endotoxins can directly injure the kidneys, and in COVID-19 patients, endotoxemia correlated with AKI. We sought to determine correlations between endotoxemia and kidney and hospital outcomes in a broad group of critically ill patients. METHODS: In this single-center, serial prospective study, 124 predominantly Caucasian adult patients were recruited within 48 h of admission to Stony Brook University Hospital Intensive Care Unit (ICU). Demographics, vital signs, laboratory data, and outcomes were collected. Circulating endotoxin was measured on days 1, 4, and 8 using the endotoxin activity assay (EAA). The association of EAA with outcomes was examined with EAA: (1) categorized as <0.6, ≥0.6, and nonresponders (NRs); and (2) used as a continuous variable. RESULTS: Patients with EAA ≥0.6 had a higher prevalence of proteinuria, and lower arterial oxygen saturation (SaO2) to fraction of inspired oxygen (FiO2) (SaO2/FiO2) ratio versus patients with EAA <0.6. EAA levels positively correlated with serum creatinine (sCr) levels on day 1. Patients whose EAA level stayed ≥0.6 had a slower decline in sCr compared to those whose EAA started at ≥0.6 and subsequently declined. Patients with AKI stage 1 and EAA ≥0.6 on day 1 showed slower decline in sCr compared to patients with stage 1 AKI and EAA <0.6. EAA ≥0.6 and NR patients had longer hospital stay and delayed ICU discharge versus EAA <0.6. CONCLUSIONS: High EAA levels correlated with worse kidney function and outcomes. Patients whose EAA levels fell, and those with AKI stage I and day 1 EAA <0.6 recovered more quickly compared to those with EAA ≥0.6, suggesting that removal of circulating endotoxins may be beneficial in critically ill patients.

Lack of change in the respiratory quotient during oxygen supply dependence in endotoxemic shock: a subanalysis of an experimental controlled study.

OBJECTIVE: To evaluate if the reductions in systemic and renal oxygen consumption are associated with the development of evidence of anaerobic metabolism. METHODS: This is a subanalysis of a previously published study. In anesthetized and mechanically ventilated sheep, we measured the respiratory quotient by indirect calorimetry and its systemic, renal, and intestinal surrogates (the ratios of the venous-arterial carbon dioxide pressure and content difference to the arterial-venous oxygen content difference. The Endotoxemic Shock Group (n = 12) was measured at baseline, after 60 minutes of endotoxemic shock, and after 60 and 120 minutes of fluid and norepinephrine resuscitation, and the values were compared with those of a Control Group (n = 12) without interventions. RESULTS: Endotoxemic shock decreased systemic and renal oxygen consumption (6.3 [5.6 - 6.6] versus 7.4 [6.3 - 8.5] mL/minute/kg and 3.7 [3.3 - 4.5] versus 5.4 [4.6 - 9.4] mL/minute/100g; p < 0.05 for both). After 120 minutes of resuscitation, systemic oxygen consumption was normalized, but renal oxygen consumption remained decreased (6.3 [5.9 - 8.2] versus 7.1 [6.1 - 8.6] mL/minute/100g; p = not significance and 3.8 [1.9 - 4.8] versus 5.7 [4.5 - 7.1]; p < 0.05). The respiratory quotient and the systemic, renal and intestinal ratios of the venous-arterial carbon dioxide pressure and content difference to the arterial-venous oxygen content difference did not change throughout the experiments. CONCLUSION: In this experimental model of septic shock, oxygen supply dependence was not associated with increases in the respiratory quotient or its surrogates. Putative explanations for these findings are the absence of anaerobic metabolism or the poor sensitivity of these variables in detecting this condition.

Protective Effect of Electroacupuncture on the Barrier Function of Intestinal Injury in Endotoxemia through HO-1/PINK1 Pathway-Mediated Mitochondrial Dynamics Regulation.

Background and Aims: Endotoxemia (ET) is a common critical illness in patients receiving intensive care and is associated with high mortality and prolonged hospital stay. The intestinal epithelial cell dysfunction is regarded as the "engine" of deteriorated ET. Although electroacupuncture (EA) can mitigate endotoxin-induced intestinal epithelial cell dysfunction in ET, the mechanism through which EA improves endotoxin-induced intestinal injury for preventing ET deterioration needs further investigation. Methods: An in vivo ET model was developed by injecting lipopolysaccharide (LPS) in wild-type and PINK1-knockout mice. An in vitro model was also established by incubating epithelial cells in the serum samples obtained from both groups of mice. Hemin and zinc protoporphyrin IX (ZnPP) were applied to activate or inhibit heme oxygenase 1 (HO-1) production. EA treatment was performed for 30 min consecutively for 5 days before LPS injection, and on the day of the experiment, EA was performed throughout the process. Samples were harvested at 6 h after LPS induction for analyzing tissue injury, oxidative stress, ATP production, activity of diamine oxidase (DAO), and changes in the levels of HO-1, PTEN-induced putative kinase 1 (PINK1), mitochondrial fusion and fission marker gene, caspase-1, and interleukin 1 beta (IL-1ß). Results: In the wild-type models (both in vivo and vitro), EA alleviated LPS-induced intestinal injury and mitochondrial dysfunction, as indicated by decreased reactive oxygen species (ROS) production and oxygen consumption rate (OCR) and reduced levels of mitochondrial fission proteins. EA treatment also boosted histopathological morphology, ATP levels, DAO activity, and levels of mitochondrial fusion proteins in vivo and vitro. The effect of EA was enhanced by hemin but suppressed by Znpp. However, EA + AP, Znpp, or hemin had no effects on the LPS-induced, PINK1-knocked out mouse models. Conclusion: EA may improve the HO-1/PINK1 pathway-mediated mitochondrial dynamic balance to protect the intestinal barrier in patients with ET.

Application of Bone Marrow Mesenchymal Stem Cells Effectively Eliminates Endotoxemia to Protect Rat from Acute Liver Failure Induced by Thioacetamide.

BACKGROUND: Endotoxemia is related to worse clinical outcomes in acute liver failure (ALF), but its management remains unsatisfactory. In this study, we aimed to assess whether the application of bone marrow mesenchymal stem cells (BMSCs) could eliminate endotoxemia and protect rats against ALF induced by thioacetamide (TAA). METHODS: BMSCs were isolated from rats and identified by the specific morphology, differentiation potential, and surface markers. The optimal dose of TAA for this study was explored and TAA-induced ALF rats were randomized to three groups: the normal control group (Saline), ALF group (TAA + Saline), and BMSCs-treated group (TAA + BMSCs). The intestinal migration and differentiation of BMSCs was tracked in vivo, and intestinal permeability, endotoxin and inflammatory cytokines, histology, and mortality were analyzed. Moreover, we added the inhibitor of the PI3K/AKT/mTOR signaling pathway into the co-culture system of BMSCs with enterocytes and then performed CK and Villin expression experiments to assess the role of PI3K/AKT/mTOR signal pathway in the intestinal differentiation of BMSCs. RESULTS: BMSCs migrated to the intestinal injury sites and differentiated into enterocytes, intestinal permeability was decreased compared with the ALF group. The higher expression of endotoxin and inflammatory cytokines were reversed after BMSCs transplantation in rats with ALF. Mortality and intestinal lesion were significantly decreased. Blocking the PI3K/AKT/mTOR signal pathway inhibited BMSCs' intestinal differentiation in vitro. CONCLUSION: BMSCs can eliminate endotoxemia and reduce mortality in rats with ALF, and the PI3K/AKT/mTOR signal pathway is involved in intestinal differentiation. BMSCs transplantation could be a potential candidate for the treatment of endotoxemia in ALF.

Reduction in Ventilation-Induced Diaphragmatic Mitochondrial Injury through Hypoxia-Inducible Factor 1α in a Murine Endotoxemia Model.

Mechanical ventilation (MV) is essential for patients with sepsis-related respiratory failure but can cause ventilator-induced diaphragm dysfunction (VIDD), which involves diaphragmatic myofiber atrophy and contractile inactivity. Mitochondrial DNA, oxidative stress, mitochondrial dynamics, and biogenesis are associated with VIDD. Hypoxia-inducible factor 1α (HIF-1α) is crucial in the modulation of diaphragm immune responses. The mechanism through which HIF-1α and mitochondria affect sepsis-related diaphragm injury is unknown. We hypothesized that MV with or without endotoxin administration would aggravate diaphragmatic and mitochondrial injuries through HIF-1α. C57BL/6 mice, either wild-type or HIF-1α-deficient, were exposed to MV with or without endotoxemia for 8 h. MV with endotoxemia augmented VIDD and mitochondrial damage, which presented as increased oxidative loads, dynamin-related protein 1 level, mitochondrial DNA level, and the expressions of HIF-1α and light chain 3-II. Furthermore, disarrayed myofibrils; disorganized mitochondria; increased autophagosome numbers; and substantially decreased diaphragm contractility, electron transport chain activities, mitofusin 2, mitochondrial transcription factor A, peroxisome proliferator activated receptor-γ coactivator-1α, and prolyl hydroxylase domain 2 were observed (p < 0.05). Endotoxin-stimulated VIDD and mitochondrial injuries were alleviated in HIF-1α-deficient mice (p < 0.05). Our data revealed that endotoxin aggravated MV-induced diaphragmatic dysfunction and mitochondrial damages, partially through the HIF-1α signaling pathway.

The protective effect of Escherichia coli Nissle 1917 on the intestinal barrier is mediated by inhibition of RhoA/ROCK2/MLC signaling via TLR-4.

AIMS: This study investigated the protective effect of Escherichia coli Nissle 1917 (EcN) on intestinal barrier and the mechanism in the context of acute severe inflammation. MATERIALS AND METHODS: In this study, mice received lipopolysaccharide (LPS) intraperitoneal injection with or without EcN administration to construct a mouse model of endotoxemia. Clinical scores, intestinal permeability, inflammatory cytokines and histopathological analysis of four main organs from different groups were assessed. The expression of tight junction proteins and activation of RhoA/ROCK2/MLC signaling were examined using western blotting. The localization of tight junction proteins was examined by immunofluorescence. Caco-2 monolayers with or without TLR-4 knockdown were incubated with EcN or TNF-α/IFN-γ and the monolayer barrier function was assessed by transepithelial electrical resistance (TER) and FITC-dextran 4000 Da (FD-4) flux. The expression of tight junction proteins and activation of RhoA/ROCK2/MLC signaling were examined by western blotting. The localization of tight junction proteins was examined by immunofluorescence. KEY FINDINGS: We found that EcN downregulated the RhoA/ROCK2/MLC signaling pathway to preserve barrier function and alleviated systemic inflammation in mouse model. And EcN also protected barrier function of Caco-2 monolayers by inhibiting the activation of RhoA/ROCK2/MLC signaling via TLR-4. SIGNIFICANCE: The results indicated that EcN protected the intestinal barrier function in endotoxemia through inhibiting the activation of RhoA/ROCK2/MLC signaling via TLR-4.

Lactobacillus acidophilus ameliorates obesity in mice through modulation of gut microbiota dysbiosis and intestinal permeability.

Obesity associated with low-grade chronic inflammation and intestinal dysbiosis is considered as a worldwide public health crisis. In the meanwhile, different probiotics have demonstrated beneficial effects on this condition, thus increasing the interest in the development of probiotic treatments. In this context, the aim of this study is to investigate the anti-obesity effects of potential probiotic Lactobacillus acidophilus isolated from the porcine gut. Then, it is found that L. acidophilus reduces body weight, fat mass, inflammation and insulin resistance in mice fed with a high-fat diet (HFD), accompanied by activation in brown adipose tissue (BAT) as well as improvements of energy, glucose and lipid metabolism. Besides, our data indicate that L. acidophilus not only reverses HFD-induced gut dysbiosis, as indicated by the decreased Firmicutes-to-Bacteroidetes ratios and endotoxin bearing Gram-negative bacteria levels, but also maintains intestinal barrier integrity, reduces metabolic endotoxemia, and inhibits the TLR4 / NF- κB signaling pathway. In addition, the results of microbiome phenotype prediction by BugBase and bacterial functional potential prediction using PICRUSt show that L. acidophilus treatment improves the gut microbiota functions involving metabolism, immune response, and pathopoiesia. Furthermore, the anti-obesity effect is transmissible via horizontal faeces transfer from L. acidophilus-treated mice to HFD-fed mice. According to our data, it is seen that L. acidophilus could be a good candidate for probiotic of ameliorating obesity and associated diseases such as hyperlipidemia, nonalcoholic fatty liver diseases, and insulin resistance through its anti-inflammatory properties and alleviation of endothelial dysfunction and gut dysbiosis.

Endotoxin Adsorbent Therapy in Severe COVID-19 Pneumonia.

INTRODUCTION: Uncontrolled systemic inflammation may occur in severe coronavirus disease 19 (COVID-19). We have previously shown that endotoxemia, presumably from the gut, may complicate COVID-19. However, the role of endotoxin adsorbent (EA) therapy to mitigate organ dysfunction in COVID-19 has not been explored. METHODS: We conducted a retrospective observational study in COVID-19 patients who received EA therapy at the King Chulalongkorn Memorial Hospital, Bangkok, Thailand, between March 13 and April 17, 2020. Relevant clinical and laboratory data were collected by inpatient chart review. RESULTS: Among 147 hospitalized COVID-19 patients, 6 patients received EA therapy. All of the 6 patients had severe COVID-19 infection with acute respiratory distress syndrome (ARDS). Among these, 5 of them were mechanically ventilated and 4 had complications of secondary bacterial infection. The endotoxin activity assay (EAA) results of pre-EA therapy ranged from 0.47 to 2.79. The choices of EA therapy were at the discretion of attending physicians. One patient was treated with oXiris® along with continuous renal replacement therapy, and the others received polymyxin B hemoperfusion sessions. All patients have survived and were finally free from the mechanical ventilation as well as had improvement in PaO2/FiO2 ratio and decreased EAA level after EA therapy. CONCLUSIONS: We demonstrated the clinical improvement of severe COVID-19 patients with elevated EAA level upon receiving EA therapy. However, the benefit of EA therapy in COVID-19 ARDS is still unclear and needs to be elucidated with randomized controlled study.

[Parasympathetic innervation contributes to the increase of survival rate and anti-inflammatory effect of electroacupuncture at "Ciliao"(BL32) in rats with lethal endotoxemia].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Ciliao" (BL32) on the survival rate and serum inflammatory cytokine levels in rats with lethal endotoxemia, and to explore its parasympathetic mechanism in suppressing severe systemic inflammation. METHODS: A total of 82 male SD rats were used in the present study. In the first part of this study, 40 rats were randomized into model and EA-BL32 groups (n=20/group). The endotoxemia model was established by intraperitoneal injection of lethal amount of lipopolysaccharide (LPS, 10 mg/kg). EA (30 Hz, 6 mA) was applied to bilateral BL32 for 30 min before and after LPS injection. The survival rate in 7 days was then recorded. In the second part of this study, 42 rats were randomized into normal control, model, EA-BL32, EA-BL32+cervical vagotomy, EA-BL32+truncal (subdiagrammatical) vagotomy and EA-BL32+pelvic neurectomy groups (n=7/group). The endotoxemia model was established by intraperitoneal injection of LPS (6 mg/kg) 30 min after the neurectomy. Rats of the control group received intraperitoneal injection of 6 mg/kg saline. EA with the same parameters mentioned above was applied to bilateral BL32 for 30 min before and after LPS injection. Blood sample was collected from the abdominal aorta 3 h after LPS injection for detecting the levels of TNF-α, IL-1ß and IL-6 by ELISA. RESULTS: ① The EA survival rate was 25% in the model group and 60% in the EA -BL32group, being significantly improved after EA (P<0.05). ② The contents of serum TNF-α, IL-1ß and IL-6 were significantly higher in the model group than those in the control group (P<0.000 1). After EA intervention, and compared with the model group, the levels of TNF-α, IL-1ß and IL-6 were significantly decreased in the EA-BL32, EA-BL32+cervical vagotomy, EA-BL32+truncal vagotomy and EA-BL32+pelvic neurectomy groups (P<0.000 1，P<0.01). After neurectomy and compared to the EA-BL32 group, the contents of TNF-α and IL-6 in the EA+cervical vagotomy and EA+pelvic neurectomy groups, IL-1ß in the EA+pelvic neurotomy group were significantly higher (P<0.0000 1, P<0.05), suggesting an elimination of EA effects after neurectomy. No significant differences were found among the 3 neurectomy groups in the levels of TNF-α, IL-1ß and IL-6 (P>0.05). CONCLUSION: EA of BL32 can improve the survival rate and attenuate the level of inflammatory cytokines in rats with lethal endotoxemia, which is closely related to the intact of parasympathetic pathway including the vagus nerve and pelvic nerve.

Aerobic exercise improves LPS-induced sepsis via regulating the Warburg effect in mice.

We investigated the impact of aerobic exercise (AE) on multiple organ dysfunction syndrome (MODS), aortic injury, pathoglycemia, and death during sepsis. ICR mice were randomized into four groups: Control (Con), Lipopolysaccharide (LPS), Exercise (Ex), and Exercise + LPS (Ex + LPS) groups. Mice were trained with low-intensity for 4 weeks. LPS and Ex + LPS mice received 5 mg/kg LPS intraperitoneally for induction of sepsis. Histopathological micrographs showed the organ morphology and damage. This study examined the effects of AE on LPS-induced changes in systemic inflammation, pulmonary inflammation, lung permeability, and bronchoalveolar lavage fluid (BALF) cell count, oxidative stress-related indicators in the lung, blood glucose levels, plasma lactate levels, serum insulin levels, plasma high-mobility group box 1 (HMGB1) levels, glucose transporter 1 (Glut1) and HMGB1, silent information regulator 1 (Sirt-1), and nuclear factor erythroid 2-related factor 2 (Nrf-2) mRNA expression levels in lung tissue. AE improved sepsis-associated multiple organ dysfunction syndrome (MODS), aortic injury, hypoglycemia, and death. AE prominently decreased pulmonary inflammation, pulmonary edema, and modulated redox balance during sepsis. AE prominently decreased neutrophil content in organ. AE prominently downregulated CXCL-1, CXCL-8, IL-6, TNF-α, Glu1, and HMGB1 mRNA expression but activated IL-1RN, IL-10, Sirt-1, and Nrf-2 mRNA expression in the lung during sepsis. AE decreased the serum levels of lactate and HMGB1 but increased blood glucose levels and serum insulin levels during sepsis. A 4-week AE improves sepsis-associated MODS, aortic injury, pathoglycemia, and death. AE impairs LPS-induced lactate and HMGB1 release partly because AE increases serum insulin levels and decreases the levels of Glut1. AE is a novel therapeutic strategy for sepsis targeting aerobic glycolysis.

Effect of probiotic supplementation along with calorie restriction on metabolic endotoxemia, and inflammation markers in coronary artery disease patients: a double blind placebo controlled randomized clinical trial.

PURPOSE: Alterations in the gut microbiome (dysbiosis) has been associated with increased microbial translocation, leading to chronic inflammation in coronary artery disease (CAD). It has been proposed that modulation of gut microbiota by probiotic might modify metabolic endotoxemia. Therefore, the purpose of this study was to examine the effects of Lactobacillus rhamnosus GG (LGG) on endotoxin level, and biomarkers of inflammation in CAD participants. METHODS: This study was a 12-weeks randomized, double-blind, and intervention on 44 patients with CAD. Patients were randomly allocated to receive either one LGG capsule 1.6 × 109 colony-forming unit (CFU) or the placebo capsules for 12 weeks. In addition, all the participants were also prescribed a calorie-restricted diet. Serum levels of interleukin-1ß (IL-1ß), Toll-like receptor 4 (TLR4), interleukin-10 (IL-10), and lipopolysaccharide (LPS), were assessed before and after the intervention. RESULTS: A significant decrease in IL1-Beta concentration (- 1.88 ± 2.25, vs. 0.50 ± 1.58 mmol/L, P = 0.027), and LPS levels (- 5.88 ± 2.70 vs. 2.96+ 5.27 mg/L, P = 0.016), was observed after the probiotic supplementation compared with the placebo. Participants who had ≥2.5 kg weight loss showed significantly improved cardiovascular-related factors, compared to patients with < 2.5 kg weight reduction, regardless of the supplement they took. CONCLUSION: These data provide preliminary evidence that probiotic supplementation has beneficial effects on metabolic endotoxemia, and mega inflammation in participants with CAD.

Induced normothermia ameliorates the procoagulant host response in human endotoxaemia.

BACKGROUND: Dysregulation of coagulation occurs commonly in sepsis, ranging from mild coagulopathy with decreased platelets to disseminated intravascular coagulation (DIC). We investigated the effect of induced normothermia on coagulation during lipopolysaccharide (LPS)-induced endotoxaemia in healthy volunteers. METHODS: Twelve volunteers received an infusion of bacterial lipopolysaccharide (Escherichia coli; 2 ng kg-1) and were assigned to either induced normothermia or control. Induced normothermia to maintain core temperature at 37°C consisted of external surface cooling, cold i.v. fluids, and medication to reduce shivering (buspirone, clonidine, and magnesium sulphate). The primary outcome was the DIC score (International Society on Thrombosis and Haemostasis guideline). Prothrombin time (PT), activated partial thromboplastin time (aPTT), D-dimer, plasma von Willebrand factor (vWf), and rotational thromboelastometry (ROTEM) were measured before and 1, 3, 6, and 8 h after LPS infusion. Differences between groups were tested with a mixed effects model. RESULTS: In control subjects, lipopolysaccharide caused a fever, transiently decreased platelet levels and lowered activated partial thromboplastin time, while prolonging prothrombin time and increasing D-Dimer and vWf levels. Normothermia prevented the DIC-score exceeding 4, which occurred in 50% of control subjects. Normothermia also reduced the fall in platelet count by 67x109 L-1([95%CI:27-107]; p=0.002), aPTT (mean difference:3s [95%CI:1-5]; p=0.005) and lowered vWf levels by 89% ([95%CI:6-172]; p=0.03), compared to the fever group. ROTEM measurements were unaffected by lipopolysaccharide. CONCLUSION: In human endotoxaemia, induced normothermia decreases markers of endothelial activation and DIC. Maintaining normothermia may reduce coagulopathy in hyperinflammatory states.

Splenic Nerve Neuromodulation Reduces Inflammation and Promotes Resolution in Chronically Implanted Pigs.

Neuromodulation of the immune system has been proposed as a novel therapeutic strategy for the treatment of inflammatory conditions. We recently demonstrated that stimulation of near-organ autonomic nerves to the spleen can be harnessed to modulate the inflammatory response in an anesthetized pig model. The development of neuromodulation therapy for the clinic requires chronic efficacy and safety testing in a large animal model. This manuscript describes the effects of longitudinal conscious splenic nerve neuromodulation in chronically-implanted pigs. Firstly, clinically-relevant stimulation parameters were refined to efficiently activate the splenic nerve while reducing changes in cardiovascular parameters. Subsequently, pigs were implanted with a circumferential cuff electrode around the splenic neurovascular bundle connected to an implantable pulse generator, using a minimally-invasive laparoscopic procedure. Tolerability of stimulation was demonstrated in freely-behaving pigs using the refined stimulation parameters. Longitudinal stimulation significantly reduced circulating tumor necrosis factor alpha levels induced by systemic endotoxemia. This effect was accompanied by reduced peripheral monocytopenia as well as a lower systemic accumulation of CD16+CD14high pro-inflammatory monocytes. Further, lipid mediator profiling analysis demonstrated an increased concentration of specialized pro-resolving mediators in peripheral plasma of stimulated animals, with a concomitant reduction of pro-inflammatory eicosanoids including prostaglandins. Terminal electrophysiological and physiological measurements and histopathological assessment demonstrated integrity of the splenic nerves up to 70 days post implantation. These chronic translational experiments demonstrate that daily splenic nerve neuromodulation, via implanted electronics and clinically-relevant stimulation parameters, is well tolerated and is able to prime the immune system toward a less inflammatory, pro-resolving phenotype.

Therapeutic Rationale for Endotoxin Removal with Polymyxin B Immobilized Fiber Column (PMX) for Septic Shock.

Endotoxin removal therapy with polymyxin B immobilized fiber column (PMX) has been clinically applied for sepsis and septic shock patients since 1994. The effectiveness and usefulness of this therapy have been demonstrated for more than a quarter of a century. However, a documented survival benefit has not yet been demonstrable in a large, multicenter, randomized and controlled trial. Following the findings derived from a large sepsis clinical trial with PMX in North America, a new trial is ongoing to determine if PMX has a long-term survival benefit when administered to septic patients. Another approach to support a survival benefit from intervention with PMX is to utilize a detailed analysis available from a large clinical data base. The endotoxin adsorption capacity of PMX columns in vitro and the effectiveness of PMX columns can be further demonstrable in animal models. The capability of PMX and details of its mechanism of action to intervene in the sepsis cascade and impede organ dysfunction in septic patients is not fully understood. The surface antigen expression in monocytes and neutrophils are improved after PMX therapy. Immunomodulatory effects as a result of endotoxin removal and/or other mechanisms of action have been suggested. These effects and other potential immune effects may explain some of the improved effects upon organ dysfunction of sepsis and septic shock patients. Endotoxemia may be involved in the pathophysiology of other diseases than sepsis. A rapid diagnostic method to detect and target endotoxemia could allow us to practice precision medicine and expand the clinical indications of endotoxin removal therapy.

Resuscitation with PEGylated carboxyhemoglobin preserves renal cortical oxygenation and improves skeletal muscle microcirculatory flow during endotoxemia.

PEGylated carboxyhemoglobin (PEGHbCO), which has carbon monoxide-releasing properties and plasma expansion and oxygen-carrying properties, may improve both skeletal microcirculatory flow and renal cortical microcirculatory Po2 (CµPo2) and, subsequently, limit endotoxemia-induced acute kidney injury. Anesthetized, ventilated Wistar albino rats (n = 44) underwent endotoxemic shock. CµPo2 was measured in exposed kidneys using a phosphorescence-quenching method. Rats were randomly assigned to the following five groups: 1) unresuscitated lipopolysaccharide (LPS), 2) LPS + Ringer's acetate (RA), 3) LPS + RA + 0.5 µg·kg·-1min-1 norepinephrine (NE), 4) LPS + RA + 320 mg/kg PEGHbCO, and 5) LPS + RA + PEGHbCO + NE. The total volume was 30 mL/kg in each group. A time control animal group was used. Skeletal muscle microcirculation was assessed by handheld intravital microscopy. Kidney immunohistochemistry and myeloperoxidase-stained leukocytes in glomerular and peritubular areas were analyzed. Endotoxemia-induced histological damage was assessed. Plasma levels of IL-6, heme oxygenase-1, malondialdehyde, and syndecan-1 were assessed by ELISA. CµPo2 was higher in the LPS + RA + PEGHbCO-resuscitated group, at 35 ± 6mmHg compared with 21 ± 12 mmHg for the LPS+RA group [mean difference: -13.53, 95% confidence interval: (-26.35; -0.7156), P = 0.035]. The number of nonflowing, intermittent, or sluggish capillaries was smaller in groups infused with PEGHbCO compared with RA alone (P < 0.05), while the number of normally perfused vessels was greater (P < 0.05). The addition of NE did not further improve CµPo2 or microcirculatory parameters. Endotoxemia-induced kidney immunohistochemistry and histological alterations were not mitigated by PEGHbCO 1 h after resuscitation. Renal leukocyte infiltration and plasma levels of biomarkers were similar across groups. PEGHbCO enhanced CµPo2 while restoring skeletal muscle microcirculatory flow in previously nonflowing capillaries. PEGHbCO should be further evaluated as a resuscitation fluid in mid- to long-term models of sepsis-induced acute kidney injury.

Effect of supportive therapy on the pharmacokinetics of intravenous marbofloxacin in endotoxemic sheep.

The purpose of this study was to determine the influences of supportive therapy (ST) on the pharmacokinetics (PK) of marbofloxacin in lipopolysaccharide (LPS)-induced endotoxemic sheep. Furthermore, minimum inhibitory concentration (MIC) of marbofloxacin against Escherichia coli, Mannheimia haemolytica, Pasteurella multocida, Klebsiella pneumoniae, Salmonella spp., and Staphylococcus aureus was determined. The study was performed using a three-period cross PK design following a 15-day washout period. In the first period, marbofloxacin (10 mg/kg) was administered by an intravenous (IV) injection. In the second and third periods, marbofloxacin was co-administered with ST (lactated ringer + 5% dextrose + 0.45% sodium chloride, IV, 20 ml/kg, dexamethasone 0.5 mg/kg, SC) and ST + LPS (E. coli O55:B5, 10 µg/kg), respectively. Plasma marbofloxacin concentration was measured using HPLC-UV. Following IV administration of marbofloxacin alone, the t 1 / 2 λ z , AUC0-∞ , ClT , and Vdss were 2.87 hr, 34.73 hr × µg/ml, 0.29 L hr-1  kg-1 , and 0.87 L/kg, respectively. While no change was found in the MBX + ST group in terms of the PK parameters of marbofloxacin, it was determined that the ClT of marbofloxacin decreased, AUC0-∞ increased, and t 1 / 2 λ z and MRT prolonged in the MBX + ST + LPS group. MIC values of marbofloxacin were 0.031 to >16 µg/ml for E. coli, 0.016 to >16 µg/ml for M. haemolytica, 0.016-1 µg/ml for P. multocida, 0.016-0.25 µg/ml for K. pneumoniae, 0.031-0.063 µg/ml for Salmonella spp., and 0.031-1 µg/ml for S. aureus. The study results show the necessity to make a dose adjustment of marbofloxacin following concomitant administration of ST in endotoxemic sheep. Also, the PK and pharmacodynamic effect of marbofloxacin needs to be determined in naturally infected septicemic sheep following concomitant administration of single and ST.

Metabolic endotoxemia and cardiovascular disease: A systematic review about potential roles of prebiotics and probiotics.

Translocation of microbiome-derived lipopolysaccharide (LPS) to the bloodstream (metabolic endotoxaemia) is associated with a significantly increased risk of cardiovascular diseases (CVD); however, the direction of this association is not fully understood. It has been revealed by some studies that alterations in the intestinal microbiota (dysbiosis) lead to increased intestinal permeability and translocation of LPS to the blood circulation. LPS may trigger toll-like receptor 4- (TLR-4) mediated inflammatory responses; this could lead to a chronic low-grade pro-inflammatory condition named metabolic endotoxaemia (ME), which is typically observed in CVD patients. ME is promoted by increased intestinal permeability. Moreover, dysbiosis leads to production of trimethylamine-N-oxide (TMAO), a gut bacterial metabolite suggested as a new risk factor in CVD development. Probiotics, extensively reviewed for decades, are live microorganisms which, when taken in adequate amounts, have beneficial effects on the host metabolism. Prebiotics are a type of dietary fibre that act as nourishment for the good bacteria in the gut and decrease the population of pathogen bacteria that produce greater amounts of endotoxins. Although an association has been postulated between ME and CVD, the results of studies investigating the role of antibiotic therapy in preventing the disease have been inconsistent. In this review, we discuss how prebiotics and probiotics modulate gut microbiota and consequently might help with prevention and/or treatment of CVD associated with ME.

Association between endotoxemia and enterocyte injury and clinical course in patients with gram-positive septic shock: A posthoc analysis of a prospective observational study.

Endotoxemia often occurs in patients with gram-positive infections. The possible mechanism is thought to be bacterial translocation after enterocyte hypoperfusion injury. However, the association between endotoxemia and enterocyte injury among patients with gram-positive septic shock has never been assessed. The aim of this study was to evaluate the association between endotoxemia and enterocyte injury in gram-positive septic shock patients and to evaluate the association among endotoxemia, subsequent clinical course, and other related factors.This was a posthoc analysis of a prospective observational study that evaluated the capability of intestinal fatty acid-binding protein (I-FABP), an indicator of enterocyte injury, to predict mortality. Among 57 patients in septic shock, those whose causative microorganisms were gram positive were included. The correlation between endotoxin activity (EA), which indicates endotoxemia, and I-FABP levels upon admission to the intensive care unit (ICU), the clinical course, and other related factors were evaluated.A total of 21 patients were examined. One-third of the patients presented with high EA levels at the time of ICU admission. However, there was no significant correlation between EA and I-FABP levels (Spearman ρ = 0.002, P = .993). Additionally, high EA levels were not associated with abdominal complications after ICU admission or mortality. Similarly, high EA levels were not associated with severity scores, inotropic scores, or lactate levels upon ICU admission, which were previously reported to be factors related to high EA levels.In this posthoc analysis, no correlation was observed between endotoxemia and enterocyte injury among patients in gram-positive septic shock. Additionally, high EA levels were not associated with the clinical course and reported factors related to endotoxemia. Although our results need to be validated in a large prospective cohort study, hypoperfusion enterocyte injury might not be a cause of endotoxemia in these patients. Thus, if there is no correlation between EA and I-FABP levels, other mechanisms that induce high EA levels among patients with gram-positive septic shock should be elucidated.

Clearance and phenotype of extracellular vesicles after red blood cell transfusion in a human endotoxemia model.

BACKGROUND: In the critically ill, extracellular vesicles (EV) from red blood cells (RBC) have been related to adverse effects of blood transfusion. Stored RBC units contain high concentrations of RBC- EVs, thereby increasing the concentration of EVs in the circulation after transfusion. The mechanisms underlying the clearance of donor RBC-EVs after transfusion are unknown. This study investigates whether membrane markers that are associated with clearance of RBCs are also implicated in clearance of RBC-EVs in human endotoxemic recipients of a transfusion. METHODS: Six volunteers were injected with Escherichia coli lipopolysaccharide, and after two hours transfused with an autologous RBC unit donated 35 days earlier. Samples were collected from the RBC unit and the volunteers before and after transfusion. RBC-EVs were labeled with (anti) glycophorin A, combined with (anti) CD44, CD47, CD55, CD59, CD147, or lactadherin to detect phosphatidylserine (PS) and analyzed on a A50 Micro flow cytometer. RESULTS: In the RBC unit, RBC-EVs solely exposed PS (7.8%). Before transfusion, circulating RBC-EVs mainly exposed PS (22%) and CD59 (9.1%), the expression of the other membrane markers was much lower. After transfusion, the concentration of RBC- EVs increased 2.4-fold in two hours. Thereafter, the EV concentration decreased towards baseline levels. The fraction of EVs positive for all tested membrane markers decreased after transfusion. CONCLUSION: Besides a minor fraction of PS-exposing EVs, RBC-EVs produced during storage do not expose detectable levels of RBC membrane markers that are associated with clearance, which is in contrast to the EVs produced by the circulating RBCs.