Endotoxin Neutralization Research Articles

Study of endotoxin (LPS) neutralizing capacity in serum of patients with cirrhosis

Study of endotoxin (LPS) neutralizing capacity in serum of patients with cirrhosis

Autologous cryo-shocked neutrophils enable targeted therapy of sepsis via broad-spectrum neutralization of pro-inflammatory cytokines and endotoxins.

Background: Sepsis is a life-threatening disease characterized by multiple organ failure due to excessive activation of the inflammatory response and cytokine storm. Despite recent advances in the clinical use of anti-cytokine biologics, sepsis treatment efficacy and improvements in mortality remain unsatisfactory, largely due to the mechanistic complexity of immune regulation and cytokine interactions. Methods: In this study, a broad-spectrum anti-inflammatory and endotoxin neutralization strategy was developed based on autologous "cryo-shocked" neutrophils (CS-Neus) for the management of sepsis. Neutrophils were frozen to death using a novel liquid nitrogen "cryo-shock" strategy. The CS-Neus retained the source cell membrane structure and functions related to inflammatory site targeting, broad-spectrum inflammatory cytokines, and endotoxin (LPS) neutralizing properties. This strategy aimed to disable harmful pro-inflammatory functions of neutrophils, such as cytokine secretion. Autologous cell-based therapy strategies were employed to avoid immune rejection and enhance treatment safety. Results: In both LPS-induced sepsis mouse models and clinical patient-derived blood samples, CS-Neus treatment significantly ameliorated cytokine storms by removing inflammatory cytokines and endotoxin. The therapy showed notable anti-inflammatory therapeutic effects and improved the survival rate of mice. Discussion: The results of this study demonstrate the potential of autologous "cryo-shocked" neutrophils as a promising therapeutic approach for managing sepsis. By targeting inflammatory organs and exhibiting anti-inflammatory activity, CS-Neus offer a novel strategy to combat the complexities of sepsis treatment. Further research and clinical trials are needed to validate the efficacy and safety of this approach in broader populations and settings.

Heparin enables the reliable detection of endotoxin in human serum samples using the Limulus amebocyte lysate assay

The determination of lipopolysaccharide (endotoxin) in serum or plasma samples using Limulus amebocyte lysate (LAL)-based assays is currently not sufficiently reliable in clinical diagnostics due to numerous interfering factors that strongly reduce the recovery of LPS in clinical samples. The specific plasma components responsible for the endotoxin neutralizing capacity of human blood remain to be identified. There are indications that certain endotoxin-neutralizing proteins or peptides, which are part of the host defense peptides/proteins of the innate immune system may be responsible for this effect. Based on our finding that several antimicrobial peptides can be neutralized by the polyanion heparin, we developed a heparin-containing diluent for serum and plasma samples, which enables reliable quantification of LPS measurement in clinical samples using the LAL assay. In a preclinical study involving 40 donors, this improved protocol yielded an over eightfold increase in LPS recovery in serum samples, as compared to the standard protocol. This modified protocol of sample pretreatment could make LPS measurement a valuable tool in medical diagnostics.

Novel Antibacterial Agents SAAP-148 and Halicin Combat Gram-Negative Bacteria Colonizing Catheters.

The antibiotic management of catheter-related infections (CRIs) often fails owing to the emergence of antimicrobial-resistant strains and/or biofilm/persister apparitions. Thus, we investigated the efficacy of two novel antimicrobial agents, i.e., the synthetic peptide SAAP-148 and the novel antibiotic halicin, against Gram-negative bacteria (GNB) colonizing catheters. The antibacterial, anti-biofilm, and anti-persister activities of both agents were evaluated against Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae strains. The enrolled strains were isolated from catheters and selected based on their resistance to at least three antibiotic classes and biofilm formation potential. Furthermore, the hemolysis and endotoxin neutralization abilities of these agents were explored. The bactericidal activity of both agents was reduced in urine and plasma as compared to buffered saline. In a dose-dependent manner, SAAP-148 and halicin reduced bacterial counts in 24 h preformed biofilms on silicone elastomer discs and eliminated persisters originating from antibiotic-exposed mature 7-day biofilms, with halicin being less effective than SAAP-148. Importantly, SAAP-148 and halicin acted synergistically on E. coli and K. pneumoniae biofilms but not on A. baumannii biofilms. The peptide, but not halicin, decreased the production of IL-12p40 upon exposure to UV-killed bacteria. This preliminary study showed that SAAP-148 and halicin alone/in combination are promising candidates to fight GNB colonizing catheters.

Fish antimicrobial peptides: at a glance

Generally, antimicrobial peptides (AMPs) are considered as an important part of innate immunity, due to which they provide the first line of defence against various pathogens. Additionally, they also kill pathogens that show resistance towards many antibiotics. Fishes are regularly challenged by various pathogens which not only affect their health but the risk of becoming resistant to conventional antibiotics is also increasing. As fishes shows more dependence on innate immunity, AMPs can aid as important defensive weapon in fishes. In general, AMPs exhibit various multidimensional characteristics such as neutralization of pathogens (viral, fungal & bacterial), rapidly diffuse to the infection site, and other immune cells recruitment to the infected tissues. AMPs also show various biological effects such as immunomodulation, neutralization of endotoxin and angiogenesis induction. There are numerous AMPs that have been isolated from fishes but not fully characterized at molecular level. In this review we basically focus on approaches used to design new AMP, machine learning approach, current objectives of AMPs and future prospects.

ApoM binds endotoxin contributing to neutralization and clearance by High Density Lipoprotein

BackgroundHDL possesses anti-inflammatory properties, however, the exact mechanism is not fully understood. Endotoxin is a potent inducers of TLR4 signaling, leading to inflammatory mediators’ release. It has been estimated that TLR4 recognizes about 5% of circulating lipopolysaccharide whereas 95% is cleared by plasma lipoproteins, mainly HDL. ApoM is required for HDL biogenesis and 95% of plasma ApoM is found associated with HDL, both are significantly reduced during sepsis. AimThe aim of this study is to investigate whether ApoM binds endotoxin and contributes to anti-inflammatory activity of HDL. MethodsIsothermal Titration Calorimetry (ITC) was used to determine the binding of ultrapure E. coli LPS to the recombinant ApoM protein. Purified human HDL and recombinant ApoM was used to investigate LPS neutralization using human and murine macrophages and computational simulation was performed. ResultApoM shows high affinity for E. coli LPS, forming 1:1 complexes with Kd values below 1 μΜ, as revealed by ITC. The binding process is strongly exothermic and enthalpy-driven (ΔrH = −36.5 kJ/mol), implying the formation of an extensive network of interactions between ApoM and LPS in the bound state. Computational simulation also predicted high-affinity binding between ApoM and E. coli LPS and the best scoring models showed E. coli LPS docking near the calyx of ApoM without blocking the pocket. The biological significance of this interaction was further demonstrated in macrophages where purified HDL neutralized an E. coli LPS effect and significantly reduced TNFα release from human THP-1 cells. ConclusionApoM binds LPS to facilitate endotoxin neutralization and clearance by HDL.

Neutrophil-inspired photothermo-responsive drug delivery system for targeted treatment of bacterial infection and endotoxins neutralization

BackgroundP. aeruginosa, a highly virulent Gram-negative bacterium, can cause severe nosocomial infections, and it has developed resistance against most antibiotics. New therapeutic strategies are urgently needed to treat such bacterial infection and reduce its toxicity caused by endotoxin (lipopolysaccharide, LPS). Neutrophils have been proven to be able to target inflammation site and neutrophil membrane receptors such as Toll-like receptor-4 (TLR4) and CD14, and exhibit specific affinity to LPS. However, antibacterial delivery system based on the unique properties of neutrophils has not been reported.MethodsA neutrophil-inspired antibacterial delivery system for targeted photothermal treatment, stimuli-responsive antibiotic release and endotoxin neutralization is reported in this study. Specifically, the photothermal reagent indocyanine green (ICG) and antibiotic rifampicin (RIF) are co-loaded into poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP-ICG/RIF), followed by coating with neutrophil membrane to obtain antibacterial delivery system (NM-NP-ICG/RIF). The inflammation targeting properties, synergistic antibacterial activity of photothermal therapy and antibiotic treatment, and endotoxin neutralization have been studied in vitro. A P. aeruginosa-induced murine skin abscess infection model has been used to evaluate the therapeutic efficacy of the NM-NP-ICG/RIF.ResultsOnce irradiated by near-infrared lasers, the heat generated by NP-ICG/RIF triggers the release of RIF and ICG, resulting in a synergistic chemo-photothermal antibacterial effect against P. aeruginosa (~ 99.99% killing efficiency in 5 min). After coating with neutrophil-like cell membrane vesicles (NMVs), the nanoparticles (NM-NP-ICG/RIF) specifically bind to inflammatory vascular endothelial cells in infectious site, endowing the nanoparticles with an infection microenvironment targeting function to enhance retention time. Importantly, it is discovered for the first time that NMVs-coated nanoparticles are able to neutralize endotoxins. The P. aeruginosa murine skin abscess infection model further demonstrates the in vivo therapeutic efficacy of NM-NP-ICG/RIF.ConclusionThe neutrophil-inspired antibacterial delivery system (NM-NP-ICG/RIF) is capable of targeting infection microenvironment, neutralizing endotoxin, and eradicating bacteria through a synergistic effect of photothermal therapy and antibiotic treatment. This drug delivery system made from FDA-approved compounds provides a promising approach to fighting against hard-to-treat bacterial infections.

Yeast Expressed Hybrid Peptide CLP Abridged Pro-Inflammatory Cytokine Levels by Endotoxin Neutralization.

The aim of this study was to apply a strategy to express a recombinant CLP peptide and explore its application as a product derived from natural compounds. The amphiphilic CLP peptide was hybridized from three parent peptides (CM4, LL37, and TP5) and was considered to have potent endotoxin-neutralizing activity with minimal cytotoxic and hemolytic activity. To achieve high secretion expression, an expression vector of pPICZαA-HSA-CLP was constructed by the golden gate cloning strategy before being transformed into Pichia pastoris and integrated into the genome. The recombinant CLP was purified through the Ni-NTA affinity chromatography and analyzed by SDS-PAGE and mass spectrometry. The Limulus amebocyte lysate (LAL) test exhibited that the hybrid peptide CLP inhibited lipopolysaccharides (LPS) in a dose-dependent manner and was significantly (p < 0.05) more efficient compared to the parent peptides. In addition, it essentially diminished (p < 0.05) the levels of nitric oxide and pro-inflammatory cytokines (including TNF-α, IL6, and IL-1β) in LPS-induced mouse RAW264.7 macrophages. As an attendant to the control and the parental peptide LL37, the number of LPS-induced apoptotic cells was diminished compared to the control parental peptide LL37 (p < 0.05) with the treatment of CLP. Consequently, we concluded that the hybrid peptide CLP might be used as a therapeutic agent.

Activation of Canine, Mouse and Human TLR2 and TLR4 by Inactivated Leptospira Vaccine Strains.

Canine Leptospira vaccines contain inactivated strains of pathogenic Leptospira, the causative agents of leptospirosis. For an effective response to vaccination, activation of the innate immune system via pattern recognition receptors such as TLRs is crucial. However, it is not known which TLRs are activated by Leptospira in dogs. To investigate the involvement of canine TLR2, TLR4, and TLR5 in the recognition of Leptospira, we stimulated canine moDC and reporter cells expressing canine TLR2 with either whole-inactivated bacteria or purified LPS of Leptospira strains, representing the serogroups generally used in canine leptospirosis vaccines. Using the endotoxin neutralizing reagent polymyxin B and TLR4 antagonist RS-LPS, we demonstrate that Leptospira LPS and canine TLR4 are involved in IL-1β production as well as in the uptake of inactivated Leptospira in canine moDC. Furthermore, polymyxin B only partially inhibited IL-1β production induced by inactivated Leptospira, suggesting that next to TLR4, also other TLRs may be involved. The observed activation of canine TLR2-expressing reporter cells by inactivated Leptospira strains indicates that TLR2 could be one of these TLRs. Next, we analyzed TLR2 and TLR4 activating capabilities by the same Leptospira strains using human and mouse TLR-expressing reporter cells. Inactivated Leptospira and leptospiral LPS activated not only mouse, but also human TLR4 and this activation was shown to be LPS dependent in both cases. Additionally, inactivated Leptospira activated mouse and human TLR2-expressing reporter cell lines. In our study, we could not identify significant species differences in the recognition of Leptospira by TLR2 and TLR4 between dog, human and mouse. Lastly, we show that these inactivated Leptospira strains are recognized by both mouse and human TLR5 reporter cells only after exposure to additional heat-treatment. Unfortunately, we were not able to confirm this in the canine system. Our data show that TLR2 and TLR4 are involved in the recognition of Leptospira strains used in the production of canine Leptospira vaccines. This study contributes to the understanding of Leptospira-induced innate immune responses in dogs, humans, and mice. Future studies are needed to further explore the role of canine TLR2, TLR4 and TLR5 in the induction of vaccine-mediated immunity against Leptospira.

Poly-quercetin-based nanoVelcro as a multifunctional wound dressing for effective treatment of chronic wound infections

Comprehensive wound dressings for bacterial wound treatment requires a combination of antibacterial, antioxidative, and anti-inflammatory effects and endotoxin neutralization. Here, we report a novel nanoVelcro dressing made from quercetin (QNV) through alkaline-mediated catalytic polymerization and crosslinking. These dressings can effectively inhibit the growth of Gram-negative, Gram-positive and multidrug-resistant bacteria in complicated medium (e.g., human plasma) through ultrastrong bacterial wrapping. QNV shows a minimum inhibitory concentration (MIC) of 10 μg mL−1 against the tested bacteria, which is 5-fold lower than that of monomeric quercetin. In addition to its superior antibacterial activity, QNV exhibits the inherent antioxidative and inflammatory activities of quercetin. More importantly, QNV wrapping further prevents bacteria from releasing toxins during infection, inhibiting the subsequent inflammatory response. QNV, in contrast to silver nanoparticles and 3 M wound care dressings, effectively inhibits bacterial growth and wound inflammation in methicillin-resistant Staphylococcus aureus (MRSA) − infected wounds in diabetic mice by accelerating the wound closure rate. Our results reveal that the use of QNV in the design of an ultrastrong bacterial binder is promising for the development of multifunctional wound dressings for the treatment of chronic infectious wounds.

An in vitro study on factors affecting endotoxin neutralization in human plasma using the Limulus amebocyte lysate test

Endotoxin neutralization, caused by plasma components, makes it difficult to detect endotoxins in human blood. In this study, we investigated which factors influence the recovery of endotoxins using limulus ameobocyte lysate (LAL)-based assays. The individual factors that were examined in more detail were lipoprotein content, type of blood anticoagulation, kinetics and serum levels of divalent cations. Furthermore, it was investigated whether there is a direct correlation between LAL activity and monocyte activation. We could show that polyanionic heparin increases endotoxin recovery in blood, while citrate anticoagulation promotes endotoxin neutralization. Furthermore, we could show that the endotoxin activity in human plasma and serum decreases strongly over time. Time-dependent endotoxin neutralization reaches its maximum after 4–6 h incubation. By means of filtration tests we could determine that endotoxins in the plasma bind to lipoproteins but do not influence their activity. Comparative measurements have shown that high LAL activity of endotoxins in plasma simultaneously possesses high monocyte activating properties in whole blood. For the maximum recovery of endotoxins in human blood the physiological calcium and magnesium concentrations are sufficient. In this study, it was shown that the endotoxin neutralizing plasma components have a molecular weight similar to β2-microglobulin (11.7 kDa). For the exact identification of the endotoxin neutralizing plasma components, which caused a modulation of the immunostimulating endotoxin activity, further investigations have to be carried out in the future.

Abstract PO001: Results from a preclinical study to evaluate the efficacy of polyvalent immunoglobulins on the imbalance of (anti-)inflammatory immune cell responses in clinical colorectal cancer

Abstract Introduction: The purpose of this study was to determine the efficacy of an immunoglobulin concentrate on dysregulated inflammatory responses in patients with colorectal cancer (CRC). Recent findings have shown that CRC is associated with dysbiosis of the gut microbiome and frequently a compromised gut barrier, resulting in prolonged and smoldering endotoxin leakage into the circulation. Subclinical local and systemic microinflammation is suggested to facilitate metastasis formation. Previous studies have pointed to the capacity of polyvalent immunoglobulins to neutralize endotoxins and pathogens on gut epithelial surfaces to abrogate inflammatory responses and thereby tumor recurrence. The effects of KMP01D, a colostrum preparation with a multitude of immunoglobulins against gram-negative/positive bacteria and viruses, on pro-/anti-inflammatory cytokine responses and apoptosis were determined ex vivo. Methods: Peripheral blood and tumor-derived mononuclear cells (MNC) from CRC patients at UICC stages I–IV (n=48) operated at our Center for Operative Medicine/Comprehensive Cancer Center and treated under current guidelines for (neo-)adjuvant therapy were further investigated. Expression of CD14, CD68, Toll-like receptor(TLR) 4, and insulin-like growth factor (IGF)-1 with/without co-incubation of KMP01D as well as immune cell apoptosis was analyzed by flow cytometry, RT PCR, immunohistochemistry, and Elisa in each patient. Results: KMP01D increased interleukin (IL)-10 and IL-13 anti-inflammatory cytokine expression in peripheral blood MNCs (UICC I-III). More interestingly, it likewise decreased secretion of IL-1β, IL-6, IFN-γ, TNF-α, and IL-12 driven inflammation as well as IGF-1. Moreover, CD14/CD68 and TLR4 expression critically involved in endotoxin signaling was downregulated in PBMCs and tumor-infiltrating MNCs. CD14+Fas+/TUNEL+ PBMCs and tumor-derived MNCs indicating apoptosis were likewise enhanced under KMP01D co-incubation. Addition of vitamin D3 known as a regulatory factor in immune responses demonstrated additive anti-inflammatory effects in vitro. Conclusions: Inflammatory immune responses are significantly and stage-dependently upregulated which negatively impacts anti-tumor-specific immune reactivity, immune cell apoptosis and prognosis in CRC patients despite modern adjuvant treatment protocols. KMP01D produced from natural bovine sources and composed of a multitude of immunoglobulin activity (IgG1/2 and Lactoferrin) demonstrates highly ex vivo efficacy on downregulation of inflammatory cytokine responses in PBMCs and normalized immune cell apoptosis in CRC patients. Together with previous clinical data our results strongly suggest that KMP01-mediated endotoxin and pathogen neutralization and thereby mitigated chronic inflammation can be a highly valuable add-on instrument in the treatment of CRC patients to downregulate stage-dependently their local and systemic inflammation and risk for metastasis formation. Citation Format: Martin Gasser, Reinhard Lissner, Li-Li Hsiao, Ana Maria Waaga-Gasser. Results from a preclinical study to evaluate the efficacy of polyvalent immunoglobulins on the imbalance of (anti-)inflammatory immune cell responses in clinical colorectal cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO001.

Bioinformatic Analysis of 1000 Amphibian Antimicrobial Peptides Uncovers Multiple Length-Dependent Correlations for Peptide Design and Prediction

Amphibians are widely distributed on different continents, except for the polar regions. They are important sources for the isolation, purification and characterization of natural compounds, including peptides with various functions. Innate immune antimicrobial peptides (AMPs) play a critical role in warding off invading pathogens, such as bacteria, fungi, parasites, and viruses. They may also have other biological functions such as endotoxin neutralization, chemotaxis, anti-inflammation, and wound healing. This article documents a bioinformatic analysis of over 1000 amphibian antimicrobial peptides registered in the Antimicrobial Peptide Database (APD) in the past 18 years. These anuran peptides were discovered in Africa, Asia, Australia, Europe, and America from 1985 to 2019. Genomic and peptidomic studies accelerated the discovery pace and underscored the necessity in establishing criteria for peptide entry into the APD. A total of 99.9% of the anuran antimicrobial peptides are less than 50 amino acids with an average length of 24 and a net charge of +2.5. Interestingly, the various amphibian peptide families (e.g., temporins, brevinins, esculentins) can be connected through multiple length-dependent relationships. With an increase in length, peptide net charge increases, while the hydrophobic content decreases. In addition, glycine, leucine, lysine, and proline all show linear correlations with peptide length. These correlations improve our understanding of amphibian peptides and may be useful for prediction and design of new linear peptides with potential applications in treating infectious diseases, cancer and diabetes.

Antimicrobial and anti-inflammatory activities of short dodecapeptides derived from duck cathelicidin: Plausible mechanism of bactericidal action and endotoxin neutralization

Antimicrobial peptides (AMPs) have gained increasing attention to combat antibiotic-resistant pathogens. dCATH (duck cathelicidin) is a 20-residue avian cathelicidin with potent bactericidal activity. However, its therapeutic application is limited due to high mammalian cell cytotoxicity. To develop therapeutically useful AMPs with enhanced antimicrobial and cell-selective property, we designed a series of 12-meric (dodeca) short amphiphilic peptides based on dCATH. Among these, Trp and Lys-rich dCATH 12–4 and dCATH 12–5 exhibited higher selectivity towards bacterial cells than erythrocytes and macrophages. Additionally, these AMPs significantly reduced NO and TNF-α secretion in LPS-stimulated macrophage cells, suggesting their anti-inflammatory properties. Various fluorophore-based studies and confocal microscopic observations demonstrated that dCATH 12–4 and dCATH 12–5 could penetrate the bacterial cell membrane and accumulate in the cytoplasm, without disrupting membrane integrity. Results from the microscopic examination and gel-retardation DNA binding assay suggested that both the designed AMPs could bind with bacterial DNA, subsequently leading to cell death via arrest of DNA synthesis. Fluorescence spectroscopy and flow cytometry analysis revealed that the designed AMPs induced strong binding to LPS oligomers which resulted in dissociation of LPS aggregates, thereby preventing LPS from binding to the carrier protein lipopolysaccharide-binding protein (LBP) or alternatively to CD14 receptors of macrophage cells. Additionally, both dCATH 12–4 and dCATH 12-5 demonstrated synergistic actions with various conventional antibiotics against antibiotic resistant pathogens, thus indicating their ability as promising adjuncts to combination therapy. In summary, these findings contribute to the design of short AMPs with bactericidal and immunomodulatory properties for combating bacterial infection and sepsis.

Is There an Obesity Paradox in Critical Illness? Epidemiologic and Metabolic Considerations.

Obesity represents a global epidemic with serious implications in public health due to its increasing prevalence and its known association with a high morbidity and mortality burden. However, a growing number of data support a survival benefit of obesity in critical illness. This review summarizes current evidence regarding the obesity paradox in critical illness, discusses methodological issues and metabolic implications, and presents potential pathophysiologic mechanisms. Data from meta-analyses and recent studies corroborate the obesity-related survival benefit in critically ill patients as well as in selected populations such as patients with sepsis and acute respiratory distress syndrome, but not trauma. However, this finding warrants a cautious interpretation due to certain methodological limitations of these studies, such as the retrospective design, possible selection bias, the use of BMI as an obesity index, and inadequate adjustment for confounding variables. Main pathophysiologic mechanisms related to obesity that could explain this phenomenon include higher energy reserves, inflammatory preconditioning, anti-inflammatory immune profile, endotoxin neutralization, adrenal steroid synthesis, renin-angiotensin system activation, cardioprotective metabolic effects, and prevention of muscle wasting. The survival benefit of obesity in critical illness is supported from large meta-analyses and recent studies. Due to important methodological limitations, more prospective studies are needed to further elucidate this finding, while future research should focus on the pathophysiologic role of adipose tissue in critical illness.

Structural and Morphological Changes in the Liver Due to Intestinal Endotoxins.

Under normal physiological conditions, endotoxin (ET) released during self-renewal of the colibacillus pool is an obligate stimulus for the formation of the immune system and homeostasis of the body. Violation of the barrier function of the intestinal wall and the mechanisms of neutralization of endotoxin lead to systemic endotoxemia of intestinal origin. Its development is facilitated by stress, intoxication, a decrease in nonspecific resistance of the body, as well as damage to the intestinal mucosa and dysbiosis, where the mucous membrane is more vulnerable and permeable to endotoxin. The aim of this study is to compare and assess the severity and nature of hepatocyte damage from endotoxin exposure and the degree of manifestation of stress due to oxidation, to determine the characteristics of structural changes in hepatocytes and to assess the oxidation stress during endotoxin intoxication in the experiment with biochemical markers. The experiments were conducted on 40 non-linear rats, divided into two groups of 20 animals. Group 1 animals received intraperitoneal injections of ET of Escherichia coli drug (Sigma USA K-235) for seven days at a rate of 0.1 mg/kg of the body weight. Animals of the second group served as the control group. Character and stage of liver damage were studied using morphological methods, including electron and light microscopy. In studying oxidizing stress, biochemical methods were used to define the changes, such as conjugated dienes and dienketones, spontaneous oxidizing modification of proteins. 1. The severity and depth of morphological changes in the liver during endotoxin intoxication were correlated with the dynamics of the content of lipid oxidation products (CD and DK, MDA) and proteins. There was a tendency for a more significant increase in the oxidative modification of proteins in serum. This confirms the data on the primary damage of proteins by free radicals. 2. When exposed to intestinal microflora endotoxin, pronounced dyscirculatory changes, fatty and hydropic degeneration of hepatocytes with signs of toxic damage to their nuclei were determined, but at the same time, the increased hyperplastic activity of sinusoidal cells remained associated with the effects of endotoxin. These changes are associated with both the direct toxic effect of endotoxin, and the effects of oxidative stress, in which endotoxin is a potent inducer.

Preliminary study of antimicrobial peptide cathelicidin-PY therapy in mice with acute liver failure

Objective: To investigate the feasibility of cationic antimicrobial peptide cathelicidin-PY(PY) therapy through a mouse model of acute liver failure. Methods: The ability of different concentrations of antimicrobial peptide PY to neutralize endotoxin / lipopolysaccharide (LPS) in vitro was detected by Limulus Amebocyte Lysate (LAL) assay. Cell counting kit-8 (CCK-8) was used to detect the toxic effect of different concentrations of antimicrobial peptide PY on mouse monocyte macrophages (RAW264.7). An in vitro hemolysis experiment was used to evaluate the activity of antimicrobial peptide PY on healthy human erythrocytes. D-galactosamine combined with LPS- induced mouse model of acute liver failure was constructed. The antimicrobial peptide PY effect on survival rate of mouse model was observed. HE staining was used to observe the pathological changes of liver tissue. Immunohistochemistry and Western blotting were used to detect the expression of apoptosis-associated protein caspase-3. Intra-group comparisons were performed using t-test and analysis of variance. χ (2) test was used for the comparison of rates. Results: An in vitro experiment showed that the endotoxin neutralization rate was higher at very low dose (0.01 μmol/L), and exceeded 70% at medium-dose (10-40 μmol/L), and the difference between groups with different concentration was statistically significant (F = 569.22, P < 0.05). Medium-dose antimicrobial peptide PY had strong endotoxin neutralizing effect, low cytotoxicity and hemolytic activity. Moreover, in vivo experiments showed that the degree of liver injury and survival rate of mouse model was significantly improved with the medium-dose of antimicrobial peptide PY. Immunohistochemistry results showed that the expression of caspase-3 in the liver tissue was significantly depleted in the medium-dose group than that of the liver failure group, and the results were consistent with protein immunoblotting testing. Conclusion: Antimicrobial peptide PY possesses a strong ability to neutralize endotoxin and few toxic side effects. A specific dose of antimicrobial peptide PY can attenuate hepatocyte apoptosis and significantly improve the survival rate of animal model, and thus provides a new idea for the liver failure treatment.

Escherichia coli Lipopolysaccharide Modulates Biological Activities of Human-β-Defensin Analogues but Not Non-Ribosomally Synthesized Peptides.

Human-β-defensins (HBD1-3) are antibacterial peptides containing three disulphide bonds. In the present study, the effect of Escherichia coli lipopolysaccharide (LPS) on the antibacterial activities of HBD2-3, C-terminal analogues having a single disulphide bond, Phd1-3, and their corresponding myristoylated analogues MPhd1-3 were investigated. The effect of LPS on the activities of linear amphipathic peptides melittin, LL37 and non-ribosomally synthesized peptides, polymyxin B, alamethicin, gramicidin A, and gramicidin S was also examined. The antibacterial activity of HBD 2-3, Phd1-3, and MPhd1-3 in the presence of LPS against E. coli and Staphylococcus aureus was inhibited. While LPS inhibited the antibacterial activity of LL37, the inhibition of melittin activity was partial. The hemolytic activity exhibited by MPhd1, MPhd3, melittin, and LL37 was inhibited in the presence of LPS. HBD2-3, Phd1-3, and MPhd1-3 also showed endotoxin neutralizing activity. The antibacterial and hemolytic activities of polymyxin B, alamethicin, gramicidin A, and gramicidin S were not inhibited in the presence of LPS. Fluorescence assays employing dansyl cadaverine showed that HBD2-3 and defensin analogues bind to LPS more strongly as compared to alamethicin, gramicidin A, and gramicidin S. Electron microscopy images indicated that peptides disintegrate the structure of LPS. The inhibition of the antibacterial activity of native defensins and analogues in the presence of LPS indicates that the initial interaction with the bacterial surface is similar. The native defensin sequence or structure is also not essential, although cationic charges are necessary for binding to LPS. Hydrophobic interaction is the main driving force for association of non-ribosomally synthesized polymyxin B, alamethicin, gramicidin A, and gramicidin S with LPS. It is likely that these peptides rapidly insert into membranes and do not interact with the bacterial cell surface, whereas cationic peptides such as β-defensin and their analogues, melittin and LL37, first interact with the bacterial cell surface and then the membrane. Our results suggest that evaluating interaction of antibacterial and hemolytic peptides with LPS is a compelling way of elucidating the mechanism of bacterial killing or hemolysis.

Simplified Head-to-Tail Cyclic Polypeptides as Biomaterial-Associated Antimicrobials with Endotoxin Neutralizing and Anti-Inflammatory Capabilities.

The therapeutic application of antimicrobial peptides (AMPs), a potential type of peptide-based biomaterial, is impeded by their poor antimicrobial activity and potential cytotoxicity as a lack of understanding of their structure–activity relationships. In order to comprehensively enhance the antibacterial and clinical application potency of AMPs, a rational approach was applied to design amphiphilic peptides, including head-to-tail cyclic, linear and D-proline antimicrobial peptides using the template (IR)nP(IR)nP (n = 1, 2 and 3). Results showed that these amphiphilic peptides demonstrated antimicrobial activity in a size-dependent manner and that cyclic peptide OIR3, which contained three repeating units (IR)3, had greater antimicrobial potency and cell selectivity than liner peptide IR3, DIR3 with D-Pro and gramicidin S (GS). Surface plasmon resonance and endotoxin neutralization assays indicated that OIR3 had significant endotoxin neutralization capabilities, which suggested that the effects of OIR3 were mediated by binding to lipopolysaccharides (LPS). Using fluorescence spectrometry and electron microscopy, we found that OIR3 strongly promoted membrane disruption and thereby induced cell lysis. In addition, an LPS-induced inflammation assay showed that OIR3 inhibited the pro-inflammatory factor TNF-α in RAW264.7 cells. OIR3 was able to reduce oxazolone-induced skin inflammation in allergic dermatitis mouse model via the inhibition of TNF-α, IL-1β and IL-6 mRNA expression. Collectively, the engineered head-to-tail cyclic peptide OIR3 was considerable potential candidate for use as a clinical therapeutic for the treatment of bacterial infections and skin inflammation.

Antimicrobial peptides conjugated with fatty acids on the side chain of D-amino acid promises antimicrobial potency against multidrug-resistant bacteria

With the alarming burden of antibiotic resistance, antimicrobial peptides (AMPs) seem to be novel antimicrobial alternatives for infection treatment due to their rapid broad-spectrum antimicrobial activity and low tendency for bacterial resistance. To obtain promising AMPs, a series of new peptides were designed and synthesized by conjugating various lengths of fatty acid chains onto the side chain of the position 4 or 7 D-amino acid of Ano-D4,7 (analogue of anoplin with D-amino acid substitutions at positions 4 and 7). The new peptides exhibited excellent antimicrobial activity against a range of bacteria, especially multidrug-resistant bacteria in contrast to conventional antibiotics. Moreover, the new peptides conjugated with fatty acid chains ranging from 8 to 12 carbons in length presented preferable antimicrobial selectivity and anti-biofilm activity. Additionally, the new peptides also exerted high stability to trypsin, serum, salts and different pH environments. Most notably, the new peptides showed a low tendency to develop bacterial resistance and they displayed optimal antimicrobial activity against the obtained resistant strains. Furthermore, the results from the outer/inner membrane permeabilization and cytoplasmic membrane depolarization assays and flow cytometry and scanning electron microscopy analyses demonstrated that the new peptides exert antimicrobial effects by typical non-receptor-mediated membrane mechanisms, as well as intracellular targets characterized by gel retardation and reactive oxygen species (ROS) generation assays. Furthermore, the new peptides presented remarkable in vivo antimicrobial potency, anti-inflammatory activity, and endotoxin neutralization. Collectively, the conjugation of fatty acids to the side chains of D-amino acids is a potential strategy for designing hopeful antimicrobial alternatives to tackle the risk of bacterial resistance.