Limulus Amebocyte Lysate Research Articles

Antimicrobial and anti-endotoxin activity of N-acetylcysteine, calcium hydroxide and their combination against Enterococcus faecalis, Escherichia coli and lipopolysaccharides.

The management of endodontic infections is a complex challenge, mainly due to the involvement of diverse microorganisms and their by-products. This study aimed to evaluate the efficacy of N-acetylcysteine (NAC), calcium hydroxide (Ca(OH)2), and their combined application as intracanal medications in combating Enterococcus faecalis, Escherichia coli, and lipopolysaccharides (LPS) from E. coli. A total of 60 single-rooted human teeth were carefully selected and divided into six groups. These tooth canals were deliberately exposed to E. faecalis (ATCC 29212) and E. coli (ATCC 25922) to induce biofilm formation. Subsequently, the specimens were treated with NAC, Ca(OH)2, or a combination of both substances. Three samples of the root canals were collected at three moments: the first sample (S1) was to confirm the initial contamination, the second sample (S2) was immediately post-instrumentation, and the third sample (S3) was collected after the use of the intracanal medication. The antimicrobial efficacy of these intracanal medications was assessed by enumerating colony-forming units per milliliter (CFU/mL). In addition to this, the kinetic chromogenic Limulus Amebocyte Lysate (LAL) assay by Lonza was used to quantify LPS from E. coli. Data tested for normality; then, Kruskal-Wallis and Friedman tests were used, and Dunn's for multiple comparisons. The findings of this study showed significant reductions in the microbial load of E. faecalis and E. coli by S3. Notably, there were no statistically significant differences among the treatment groups concerning these microorganisms. However, it was observed that only the combination of NAC and Ca(OH)2 led to a noteworthy decrease in the quantity of E. coli's LPS after 7-days, demonstrating a statistically significant difference from the other treatment groups. NAC + Ca(OH)2 combination, applied for a duration of 7-days, proved to be more suitable in reducing the presence of E. faecalis, E. coli, and LPS from E. coli within the context of endodontic infections.

COMPARATIVE CHARACTERISTICS OF THE INFORMATIVENESS OF THE LAL TEST AND PROCALCITONIN IN THE DIAGNOSIS OF INFECTIOUS COMPLICATIONS IN ACUTE ALCOHOLIC PANCREATITIS

Acute pancreatitis (AP) is an inflammatory condition of the pancreas, characterized by acute, intense abdominal pain. AP accounts for 20-30% of hospitalizations due to abdominal symptoms. The incidence of AP is increasing globally by 2-5% annually, ranging from 3.4 to 73.4 cases per 100,000 people. A critical concern in AAP is the potential development of infectious complications, which may require surgical intervention and increase mortality rates. Effective management and monitoring of these complications are essential. Research Justification. The diagnosis of AAP in clinical practice commonly relies on serum levels of α-amylase, lipase, C-reactive protein, and leukocyte counts. However, these markers have limitations and do not fully reflect the level of intoxication. The accuracy of C-reactive protein as a marker is not well established and is used mainly as a prognostic indicator. Serum α-amylase levels tend to decrease sharply after the fifth day of illness, regardless of the patient's condition. Lipase levels in diabetic patients can exceed reference values even without pathology, necessitating adjustments for AAP diagnosis. These criteria are most effective during the enzymatic phase of AAP (days 1-5). To predict the course of AAP and assess the adequacy of therapy, as well as to determine the need for surgical intervention, serum procalcitonin levels are commonly used. However, some studies question the diagnostic accuracy of procalcitonin, particularly regarding its correlation with organ failure, infectious complications, and patient mortality. Materials and Methods: This study involved 55 AAP patients, 51 men (92.7%) and 4 women (7.3%), with an average age of 49.49±9.87 years. Patients were categorized based on the severity of their condition: moderate (39 patients, 70.9%) and severe (16 patients, 29.1%). The control group consisted of 10 healthy individuals (9 men, 90.0%; 1 woman, 10.0%; average age 45.22±7.55 years). AAP was classified according to the Atlanta classification of acute pancreatitis. The study included patients with moderate to severe AAP who were hospitalized within 48 hours of symptom onset. All patients received treatment according to clinical guidelines, including intensive fluid therapy with Ringer's lactate. The Limulus Amebocyte Lysate (LAL) test and procalcitonin levels were measured on days 5, 8, 11, and 14 of hospitalization. For suspected infected necrosis, patients underwent contrast-enhanced computed tomography (CT) on day 14, with fine-needle aspiration biopsy or surgical intervention if necessary. Results. On day 5, LAL test values in the moderate and severe AAP groups were within reference ranges, and procalcitonin levels did not exceed 0.5 ng/ml. By day 14, a significant increase in LAL test values was noted in both groups, with a corresponding rise in procalcitonin levels. The ROC analysis for LAL test values on day 14 in the moderate AAP group showed an area under the curve (AUC) of 0.966, with 92.86% sensitivity and 96% specificity. For procalcitonin, the AUC was 0.723, with 71.43% sensitivity and 92% specificity. Conclusion. The LAL test is a sensitive and specific marker for detecting endotoxemia and monitoring the progression of AAP, particularly in predicting the development of infectious complications. The study demonstrates that LAL test values significantly correlate with the severity of AAP and may provide better diagnostic accuracy than procalcitonin, especially in the later stages of the disease.

Limitations of Limulus amebocyte lysate test for endotoxin control in raw materials for liposomal nanoformulations.

Aim: To evaluate the applicability of Limulus amebocyte lysate (LAL) assay for endotoxin determination in lipid compounding liposomal nanoformulations.Materials & methods: Spiked cholesterol, hydrogenated soy phosphatidylcholine and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG 2000) samples with endotoxins, simulating contaminated samples or in-process contamination were analyzed by chromogenic LAL assay.Results: Recovery of spiked endotoxins was achieved from DSPE-PEG 2000 suspended in water, whereas recovery was not achieved from spiked cholesterol and hydrogenated soy phosphatidylcholine suspended in methanol, and from multilamellar vesicles. Conclusion: Endotoxins, when in contact with organic solvents, no longer react in the LAL assay as they do in aqueous media. This indicates limitations of the LAL assay for endotoxin control in raw materials for liposomal nanoformulations.

Endotoxin Detection in Magnetic Resonance Imaging Contrast Agent Using Optimising Chromogenic Limulus Amebocyte Lysate Assay.

Endotoxin contamination in magnetic resonance imaging (MRI) contrast agents can pose a risk to patient safety causing immune reactions. Strict endotoxin limits are enforced for implants and catheters inserted into the body, but there are not clear rules for MRI contrast agents. Here, we investigated the efficacy of chromogenic LAL assay test for screening endotoxin activity in MRI contrast media manufactured in Malaysia. The powdered agent was dissolved in water for injection and endotoxin levels were measured. The coefficient of efficiency value for the standard curve, exhibiting r 2 ≥ 0.98, along with the absence of interfering substances and endotoxin activity below the regulatory threshold of 0.5 EU/mL, support the conclusion that the agent is unlikely to be pyrogenic or induce pyrogenic effect.

A-294 Exocytosis Inhibitor Increases yield of Limulus Amebocyte Lysate (LAL), Used for adjunct diagnosis of Invasive Fungal infection

Abstract Background The limulus amebocyte lysate (LAL) was widely used in endotoxin detection in vitro and the clinical diagnosis of invasive fungal infection. The previous data showed that a caffeine collection solution could prevent degranulation during the blood collection procedure, and caffeine-derived LAL could work in both the chromogenic test and turbidimetric method assays. However, the cell pellets collected by caffeine collection solution aggregates after re-suspension in 5 mM CaCl2. No similar phenomenon was observed with PBS collection buffer. The purpose of this study is to set up a PBS-caffeine bleed solution to improve the yield of LAL Methods During the 2023 bleed season, experiments were carried out to test the results of PBS-caffeine solution on the degranulation during bleed process. 6 crabs were bled once a week for a total of 60 crabs by the PBS-caffeine collection solution. The blood of each crab was collected by PBS caffeine collection solution. The cell pellets were finally re-suspended into 5 mM CaCl2 to promote degranulation. The LAL activity was tested by both chromogenic and turbidimetric assay methods. Results The data demonstrates that PBS-caffeine collection solution could prevent degranulation for over 4 hours, and the activity yield of PBS-caffeine-derived LAL was higher. There was no discernable impact on the activity yield of the crab’s size during the bleed season. PBS-caffeine-collection solution could reduce amebocyte aggregation/clot formation during processing. Studies described the biochemical characteristics of PBS-caffeine-derived LAL. Moreover, PBS-caffeine-derived LAL could work in both the chromogenic and turbidimetric assays. Conclusions First, PBS-caffeine collection solutions prevent amebocyte degranulation during blood collection and processing. Next, a PBS-caffeine collection solution could reduce cell aggregation/clotting during processing, and the activity of PBS-caffeine LAL is higher. Finally, PBS LAL is functional in both the chromogenic test and turbidimetric assays

A-274 The Yield of the Limulus Amebocyte Lysate (LAL), Which is Used for Endotoxin Detection and Fungal Diagnosis is Increased by Caffeine Collection Buffer

Abstract Background Limulus Amebocyte Lysate (LAL), which is a major component of Fungal infection diagnosis kit, is used for the adjunct diagnosis of invasive fungal infection. This study used different blood collection buffers to find the optimal blood collection buffer to maximize the yield of LAL. The PBS collection buffer was found to prevent degranulation during blood collection procedure and the PBS-derived Limulus Amebocyte Lysate (LAL) was observed to work in both the chromogenic test and the turbidimetric method assays. A similar phenomenon was observed with the caffeine collection buffer. Methods To further confirm this observation, we used a caffeine collection buffer to collect the blood of horseshoe crabs in the 2023 bleeding season (from July to October). Once a week, 3 crabs were bled for a total of 30 crabs. The blood of each crab was collected using the caffeine collection buffer and the 3% NaCl collection buffer, respectively. The cell pellets were resuspended into LAL reagent water (LRW) or 5 mM CaCl2. The final LAL activity was tested by both chromogenic and turbidimetric assay methods. Results The results showed that a caffeine collection buffer could prevent degranulation for over 1 hr and the activity yield of caffeine-derived LAL is much higher than that of the 3% NaCl solution. There was no discernible impact upon activity yield of crab size, period of the bleed season, or the vendors. Interestingly, caffeine-derived LAL was observed to work in both the chromogenic and turbidimetric assays. The enzyme characteristic of the LAL was also described. Conclusions Caffeine collection buffer prevents amebocyte degranulation during blood collection and processing. The activity of caffeine LAL is much higher than that of 3% NaCl. Caffeine LAL works in both chromogenic test and turbidimetric assay.

In Vitro and In Vivo Biocompatibility of Bacterial Cellulose.

Bacterial cellulose is a unique biomaterial produced by various species of bacteria that offers a range of potential applications in the biomedical field. To provide a cost-effective alternative to soft-tissue implants used in cavity infills, remodeling, and subdermal wound healing, in vitro cytotoxicity and in vivo biocompatibility of native bacterial cellulose were investigated. Cytotoxicity was assessed using a metabolic assay on Swiss 3T3 fibroblasts and INS-1832/13 rat insulinoma. Results showed no cytotoxicity, whether the cells were seeded over or under the bacterial cellulose scaffolds. Biocompatibility was performed on Sprague-Dawley rats (males and females, 8 weeks old) by implanting bacterial cellulose membranes subcutaneously for 1 or 12 weeks. The explanted scaffolds were then sliced and stained with hematoxylin and eosin for histological characterization. The first series of results revealed acute and chronic inflammation persisting over 12 weeks. Examination of the explants indicated a high number of granulocytes within the periphery of the bacterial cellulose, suggesting the presence of endotoxins within the membrane, confirmed by a Limulus amebocyte lysate test. This discovery motivated the development of non-pyrogenic bacterial cellulose scaffolds. Following this, a second series of animal experiments was done, in which materials were implanted for 1 or 2 weeks. The results revealed mild inflammation 1 week after implantation, which then diminished to minimal inflammation after 2 weeks. Altogether, this study highlights that unmodified, purified native bacterial cellulose membranes may be used as a cost-effective biomedical device provided that proper endotoxin clearance is achieved.

Caspase-11 signaling promotes damage to hippocampal CA3 to enhance cognitive dysfunction in infection

BackgroundCognitive dysfunction caused by infection frequently emerges as a complication in sepsis survivor patients. However, a comprehensive understanding of its pathogenesis remains elusive.MethodsIn our in vivo experiments, an animal model of endotoxemia was employed, utilizing the Novel Object Recognition Test and Morris Water Maze Test to assess cognitive function. Various techniques, including immunofluorescent staining, Western blotting, blood‒brain barrier permeability assessment, Limulus Amebocyte Lysate (LAL) assay, and Proximity-ligation assay, were employed to identify brain pathological injury and neuroinflammation. To discern the role of Caspase-11 (Casp11) in hematopoietic or non-hematopoietic cells in endotoxemia-induced cognitive decline, bone marrow chimeras were generated through bone marrow transplantation (BMT) using wild-type (WT) and Casp11-deficient mice. In vitro studies involved treating BV2 cells with E. coli-derived outer membrane vesicles to mimic in vivo conditions.ResultsOur findings indicate that the deficiency of Casp11-GSDMD signaling pathways reverses infection-induced cognitive dysfunction. Moreover, cognitive dysfunction can be ameliorated by blocking the IL-1 effect. Mechanistically, the absence of Casp11 signaling significantly mitigated blood‒brain barrier leakage, microglial activation, and synaptic damage in the hippocampal CA3 region, ultimately leading to improved cognitive function.ConclusionThis study unveils the crucial contribution of Casp11 and GSDMD to cognitive impairments and spatial memory loss in a murine sepsis model. Targeting Casp11 signaling emerges as a promising strategy for preventing or treating cognitive dysfunction in patients with severe infections.

The component composition of the total flavonoid fractions from some <i>Ericaceae</i> family and their effect on the NO-stimulating activity of peritoneal macrophages

Introduction. Representatives of the Ericaceae family are quite common in Russia and are promising for the creation of new medicines of plant origin. At the same time, only 4 of them are official. The study of biologically active substances and pharmacological activity of Andromeda polifolia L., Chamaеdaphne calyculata (L.) Moench, Ledum palustre L., Empetrum nigrum L. with rich resource reserves is promising.Aim. Comparative study of the composition of total flavonoid fractions from A. polifolia, C. calyculata, L. palustre, E. nigrum and study of their effect on the NO-stimulating activity of peritoneal macrophagesMaterials and methods. The crushed aboveground part (leafy shoots) was previously depigmented with chloroform, treated with 70 % aqueous acetone, then acetone was removed. Flavonoids were extracted with ethyl acetate from the aqueous phase. The identification of flavonoids was carried out by HPLC (UltiMate 3000 chromatograph) according to the coincidence of retention times and spectral characteristics, the calculation of the content was carried out by simple normalization. The effect of the samples on nitric oxide production was studied on macrophages of C57BL/6 mice. Endotoxin control in the samples was carried out using a LAL test and cell incubation in the presence of polymyxin B.Results and discussion. The composition of flavonoid fractions from A. polifolia, C. calyculata, L. palustre and E. nigrum has been studied. 8 phenolic compounds were found in C. calyculata shoots, including isoquercitrin, herbacetin, naringenin and naringin – for the first time for this species. 5 compounds were detected in A. polifolia shoots, including isoquercitrin and herbacetin, for the first time for this species. 5 and 4 compounds were identified in L. palustre and E. nigrum shoots, respectively, while quercetin glycosides are predominant in all samples: isoquercitrin, hyperoside and rutin. The fraction of C. calyculata flavonoids at doses of 1, 5, 10 mcg/ml inhibits the production of nitric oxide by macrophages by 30 %, and E. nigrum flavonoids at doses of 100 and 200 mcg/ml, on the contrary, enhance the production of nitrites by macrophages by 33 and 37 %, respectively.Conclusion. A comparative study of the composition of total flavonoid fractions from A. polifolia, C. calyculata, L. palustre, and E. nigrum, which are capable of activating both M1 and M2 polarization of peritoneal macrophages in mice, has been conducted, which requires further in-depth study. The flavonoids C. calyculata and E. nigrum are promising for further study.

Evaluation of dust and endotoxin exposure among rice mill workers in northeast India

Exposure to particulate matter and endotoxin can lead to acute and chronic respiratory problems in workers. A study was conducted to quantify particulate matter with an aerodynamic diameter of ≤10 μm (PM10) and endotoxin levels in rice mills in northeast India. Sixty-four PM10 dust samples were collected from eight rice mills at two locations: the feeding and polishing sections for two varieties of paddy: Ranjit and Hali. Endotoxin exposures were analyzed using the Limulus Amoebocyte Lysate (LAL) gel clot test. The results showed that the geometric mean of the 8-h time-weighted average (TWA) PM10 dust concentration and endotoxin level in the rice mills were 10.69 mg/m³ and 2.2 EU/m³, respectively. Dust and endotoxin exposure were higher in the feeding section than in the polishing section. Endotoxin exposure in the feeding section during the milling of the Hali variety of paddy was 56.0 % higher than the milling of the Ranjit variety. Additionally, endotoxin exposure during the milling of the Hali variety was 24.2 % higher than that of the milling of the Ranjit variety in the polishing section. The dust exposure in the rice mills was 194.5 % higher than the standards set up by the United States Environmental Protection Agency. However, the exposure to endotoxin in rice mills was approximately 40.10 times below the recommended limit of 90 EU/m3.

Assessment of Pyrogenic Response of Medical Devices and Biomaterials by the Monocyte Activation Test (MAT): A Systematic Review.

Pyrogens are fever-inducing substances routinely investigated in health products through tests such as the Rabbit Pyrogen Test (RPT), the Limulus Amebocyte Lysate (LAL), and the Monocyte Activation Test (MAT). However, the applications of the MAT for medical devices and biomaterials remain limited. This work aimed to overview the studies evaluating the pyrogenicity of medical devices and biomaterials using the MAT, highlighting its successes and potential challenges. An electronic search was performed by December 2023 in PubMed, Scopus, and Web of Science, identifying 321 records which resulted in ten selected studies. Data were extracted detailing the tested materials, MAT variants, interferences, and comparisons between methods. Methodological quality was assessed using the ToxRTool, and the results were synthesized descriptively. The selected studies investigated various materials, including polymers, metals, and natural compounds, employing the different biological matrices of the MAT. Results showed the MAT's versatility, with successful detection of pyrogens in most materials tested, though variability in sensitivity was noted based on the material and testing conditions. Challenges remain in optimizing protocols for different material properties, such as determining the best methods for direct contact versus eluate testing and addressing the incubation conditions. In conclusion, the MAT demonstrates significant potential as a pyrogen detection method for medical devices and biomaterials. However, continued research is essential to address existing gaps, optimize protocols, and validate the test across a broader range of materials.

It is time to reconsider the use of naturally-occurring endotoxins in endotoxin recovery studies: Part 2 of a BioPhorum harmonized endotoxin recovery study

The evaluation of Naturally Occurring Endotoxins (NOEs) for Low Endotoxin Recovery (LER) studies has been a topic in the industry and regulatory agencies have been hesitant to endorse NOE use in LER studies over purified Lipopolysaccharide (LPS) standards such as Control Standard Endotoxin (CSE) or Reference Standard Endotoxin (RSE). In a recent study involving 11 BioPhorum member companies across 13 sites, NOEs prepared in high and low nutrient conditions were evaluated in two common monoclonal antibody buffer formulations: 10 mM Sodium Citrate, 0.05 % Polysorbate 80, pH 6.0 and 20 mM Histidine, 0.05 % Polysorbate 80, pH 6.0. 12 g-negative bacterial isolates were used to prepare NOE analytes, which were spiked into the formulation buffers. Additionally, the NOEs were spiked into Limulus Amebocyte Lysate (LAL) reagent water as controls and purified LPS into the citrate/polysorbate buffer as the LER control. Results showed the average of three runs per organism was >50 % recovery, at the conclusion of the 7-day period, regardless of nutrient culture preparation conditions. Furthermore, purified LPS controls became undetectable (<50 % recovery) in the citrate/polysorbate buffer, highlighting the presence of LER. These findings highlight the potential value of using NOEs from relevant manufacturing facilities to assess overall risk when purified LPS recovery is insufficient.

Gut Permeability and Immune-Mediated Inflammation in Heart Failure.

Heart failure (HF) is characterized by low-grade immune-mediated inflammation due to increased Toll-like receptor (TLR) expression as response to endotoxin increase and dysregulated gut barrier permeability. We investigated TLR expression and possible gut dysbiosis in HF patients compared to a control group. We enrolled 80 Caucasian HF patients and 20 controls. Low-grade immune-mediated inflammation was evaluated by TLR expression, while gut dysbiosis by the detection of zonulin and bacterial endotoxin activity in a semi-quantitative (endotoxin activity assay [EAA]) and quantitative (limulus amebocyte lysate [LAL] test) way. Compared to controls, patients with HF showed significantly higher age and blood pressure values, worse metabolic profile and kidney function, higher inflammatory biomarkers levels, and lower levels of zonulin and endotoxin activity. When dividing failing patients in those with reduced ejection fraction (HF-rEF) and those with preserved ejection fraction (HF-pEF), HF-rEF patients showed significantly higher values of inflammatory biomarkers and TLR expression than HF-pEF patients. Gut permeability biomarkers inversely correlated with the severity of HF and positively with renal function. eGFR was retained as an independent predictor of zonulin variation in all the three groups of failing patients. Present data work to extend current knowledge about the role of gut microbiota in immune-mediated inflammation in HF.

Visual Detection of LPS at the Femtomolar Level Based on Click Chemistry-Induced Gold Nanoparticles Electrokinetic Accumulation.

Lipopolysaccharide (LPS) presents a significant threat to human health. Herein, a novel method for detecting LPS was developed by coupling hybridization chain reaction (HCR), gold nanoparticles (AuNPs) agglutination (AA) triggered by a Cu(I)-catalyzed azide-alkyne cycloaddition click chemistry (CuAAC), and electrokinetic accumulation (EA) in a microfluidic chip, termed the HCR-AA-EA method. Thereinto, the LPS-binding aptamer (LBA) was coupled with the AuNP-coated Fe3O4 nanoparticle, which was connected with the polymer of H1 capped on CuO (H1-CuO) and H2-CuO. Upon LPS recognition by LBA, the polymers of H1- and H2-CuO were released into the solution, creating a "one LPS-multiple CuO" effect. Under ascorbic acid reduction, CuAAC was initiated between the alkyne and azide groups on the AuNPs' surface; then, the product was observed visually in the microchannel by EA. Finally, LPS was quantified by the integrated density of AuNP aggregates. The limit of detections were 29.9 and 127.2 fM for water samples and serum samples, respectively. The levels of LPS in the injections and serum samples by our method had a good correlation with those from the limulus amebocyte lysate test (r = 0.99), indicating high accuracy. Remarkably, to popularize our method, a low-cost, wall-power-free portable device was developed, enabling point-of-care testing.

Membrane filtration enhanced by magnetic field for reducing endotoxin from dialysis water

AimThis study investigates the impact of magnetic field-assisted filtration on endotoxin reduction in dialysis water, focusing on the efficacy of ultrafiltration and nanofiltration membranes. MethodsThe starting material was synthetic water with a known amount of endotoxin (0.48 EU/ml). The experiment was conducted in a small-lab-scale unit, likely to test and optimize the purification method before large-scale application. Limulus amebocyte lysate (LAL) assay is a standard test used to detect and quantify endotoxin levels in liquids. ResultsThe study found that all treatment strategies effectively reduced endotoxin levels, with the best treatment being a third hybrid treatment involving magnetic field, UF, and NF exposure. This kind of treatment reduced the endotoxin from 0.169 ± 0.09 EU/ml to 0.06 ± 0.06 EU/ml at an exposure time of 20 min for a magnetic field. ConclusionThe integration of magnetically treated water in dialysis treatment plants holds promise for enhancing the quality of dialysis fluid and aligning with international standards. By employing physical methods like magnetic treatment, facilities can address concerns surrounding water purity in dialysis, which is crucial for patient safety and treatment effectiveness.

Soy Protein Concentrate Diets Inversely Affect LPS-Binding Protein Expression in Colon and Liver, Reduce Liver Inflammation, and Increase Fecal LPS Excretion in Obese Zucker Rats.

Dietary soy protein and soy isoflavones have anti-inflammatory properties. Previously, we reported that feeding soy protein concentrate diet (SPC) with low or high isoflavone (LIF or HIF) to young (seven-week-old) obese (fa/fa) Zucker rats inhibits lipopolysaccharide (LPS) translocation and decreases liver inflammation compared to a casein control (CAS) diet. The current study investigated whether SPC-LIF and SPC-HIF diets would reduce liver inflammation in adult obese Zucker rats fed a CAS diet. A total of 21 six-week-old male obese (fa/fa) Zucker rats were given CAS diet for 8 weeks to develop obesity then randomly assigned to CAS, SPC-LIF, or SPC-HIF (seven rats/group) diet for an additional 10 weeks. The expression of LPS-translocation, inflammation, and intestinal permeability markers were quantified by qPCR in liver, visceral adipose tissue (VAT), and colon. LPS concentration was determined in both the colon content and fecal samples by a Limulus amebocyte lysate (LAL) test. SPC-LIF and SPC-HIF diets significantly decreased liver LPS-binding protein (LBP) expression compared to CAS diet (p < 0.01 and p < 0.05, respectively). SPC-HIF diet also significantly decreased liver MCP-1 and TNF-α expression (p < 0.05) and had a trend to decrease liver iNOS expression (p = 0.06). In the colon, SPC-HIF diet significantly increased LBP expression compared to CAS diet (p < 0.05). When samples from all three groups were combined, there was a negative correlation between colon LBP expression and liver LBP expression (p = 0.046). SPC diets did not alter the expression of intestinal permeability markers (i.e., occludin, claudin 3, and zonula occludens-1) in the colon or inflammation markers (i.e., TNF-α and iNOS) in VAT or the colon. LPS levels in the colon content did not differ between any groups. Fecal LPS levels were significantly higher in the SPC-LIF and SPC-HIF groups compared to the CAS group (p < 0.01). In conclusion, SPC, particularly SPC with HIF, reduces liver LBP expression and inflammation makers (i.e., TNF-α and MCP-1 expression) in adult obese Zucker rats, likely by reducing LPS translocation.

Protective Effect of Silibinin on Lipopolysaccharide-Induced Endotoxemia by Inhibiting Caspase-11-Dependent Cell Pyroptosis.

To explore the protective effect and the underlying mechanism of silibinin (SIB), one of the active compounds from Silybum marianum (L.) Gaertn in endotoxemia. Mouse peritoneal macrophage were isolated via intraperitoneally injection of BALB/c mice with thioglycolate medium. Cell viability was assessed using the cell counting kit-8, while cytotoxicity was determined through lactate dehydrogenase cytotoxicity assay. The protein expressions of interleukin (IL)-1 α, IL-1 β, and IL-18 were determined by enzyme-linked immunosorbent assay. Intracellular lipopolysaccharide (LPS) levels were measured by employing both the limulus amoebocyte lysate assay and flow cytometry. Additionally, proximity ligation assay was employed for the LPS and caspase-11 interaction. Mice were divided into 4 groups: the control, LPS, high-dose-SIB (100 mg/kg), and low-dose-SIB (100 mg/kg) groups (n=8). Zebrafish were divided into 4 groups: the control, LPS, high-dose-SIB (200 εmol/L), and low-dose-SIB (100 εmol/L) groups (n=30 for survival experiment and n=10 for gene expression analysis). The expression of caspase-11, gasdermin D (GSDMD), and N-GSDMD was determined by Western blot and the expressions of caspy2, gsdmeb, and IL-1 β were detected using quantitative real-time PCR. Histopathological observation was performed through hematoxylineosin staining, and protein levels in bronchoalveolar lavage fluid were quantified using the bicinchoninicacid protein assay. SIB noticeably decreased caspase-11 and GSDMD-mediated pyroptosis and suppressed the secretion of IL-1 α, IL-1 β, and IL-18 induced by LPS (P<0.05). Moreover, SIB inhibited the translocation of LPS into the cytoplasm and the binding of caspase-11 and intracellular LPS (P<0.05). SIB also attenuated the expression of caspase-11 and N-terminal fragments of GSDMD, inhibited the relative cytokines, prolonged the survival time, and up-regulated the survival rate in the endotoxemia models (P<0.05). SIB can inhibit pyroptosis in the LPS-mediated endotoxemia model, at least in part, by inhibiting the caspase-11-mediated cleavage of GSDMD. Additionally, SIB inhibits the interaction of LPS and caspase-11 and inhibits the LPS-mediated up-regulation of caspase-11 expression, which relieves caspase-11-dependent cell pyroptosis and consequently attenuates LPS-mediated lethality.

Purification technology design, biochemical and immunological characteristics of the recombinant chimeric antigen for evaluation of T cell immunity against coronavirus infection

Diagnosis of specific T cell immunity to the antigenic determinants of SARS-CoV-2 in patients seems to be an increasingly important task due to accumulation of data about the role of T cell immune response in course of coronavirus infection and clearance of SARS-CoV-2 in case of secondary infection. Previously, we designed the recombinant CorD_PS antigen for evaluation of T cell antiviral immunity, containing conservative and immunogenic sequences of structural proteins of the SARS-CoV-2 coronavirus. E. coli CorD_PS producing strain with stable expression of the recombinant CorD_PS antigen was obtained. Aim of the present work is to design a laboratory technology for the production of recombinant antigen CorD_PS, to control the quality of the obtained chimeric protein and to study its immunological activity. Development of the cultivation conditions for E. coli CorD_PS cells was carried out in conical flasks in LB-M_Km medium at 37 °C, then scaled in a fermenter with a volume of 30 liters. Expression was induced by the addition of IPTG. Expression was controlled in culture lysates in 12% SDS-PAGE. The resulting biomass was lysed using an ultrasonic disintegrator with followed by centrifugation. The compositions of lysing and solubilizing buffers were selected, as well as conditions for refolding of the recombinant protein. Cation exchange (SP-sepharose), hydrophobic (Butylsepharose) and exclusive (Sephacryl S-200 HR) chromatography were used sequentially to purify the dissolved protein. Protein impurities in the preparation were determined by reverse-phase HPLC and electrophoresis in 12% SDS-PAGE, residual lipopolysaccharides were determined using a gel-thrombin variant of the LAL test, residual proteins of the producer strain were determined by enzyme immunoassay, residual DNA of the producer strain was determined by binding with PicoGreen dye. Specificity was controlled by indirect enzyme immunoassay. The evaluation of cytokine production by CD4+T lymphocytes in response to their stimulation by recombinant antigen ex vivo was performed on a flow cytofluorimeter. The biomass yield during cultivation of E. coli CorD_PS in a 30L fermenter was up to 20 g/L for 4 hours of 0.1 mM IPTG induction. Sequential washing of inclusion bodies from bacterial cellular components and their subsequent solubilization in a buffer containing 8 M urea allowed to obtain a solution of denatured antigen with a concentration of 10 mg/mL. The efficiency of refolding by dilution was 75%. After three stages of chromatographic purification, protein samples with a concentration of 1.2-1.4 mg/mL, HPLC purity of 98.43%, corresponding to key quality parameters according to the OFS.1.7.1.0007.15, were obtained. The recombinant antigen showed specific binding to a sample of the First WHO International Standard and sample Company Reference Standard No. 3 of anti- SARS-CoV-2 human immunoglobulins in a specified concentration range. CD4+T lymphocytes effectively responded to recombinant antigen treatment by increasing IFNγ production. The optimal concentration of recombinant coronavirus antigen was 5 μg/mL. The developed technological process makes it possible to obtain 5-7 grams of coronavirus recombinant CorD_PS antigen in one cultivation cycle in 30 liters of fermentation medium with key quality parameters according to the OFS.1.7.1.0007.15. As a result of specific immunological activity studies of the recombinant coronavirus antigen CorD_PS, the concept of its possible use as a diagnostic tool for determining the formation of a T cell immune response was confirmed.

A Study on the Application of Recombinant Factor C (rFC) Assay Using Biopharmaceuticals.

Gram-negative bacterial endotoxins can cause pathophysiological effects such as high fever when introduced into the bloodstream. Therefore, endotoxin testing is necessary when producing injectable pharmaceuticals. The pharmaceutical industry has widely used Limulus amebocyte lysate (LAL) to certify product quality. However, ethical concerns have been raised and the increasing scarcity of Limulus polyphemus necessitates the development of novel testing techniques. Recombinant factor C (rFC) was developed using genetic engineering techniques. The aim of this study was to investigate the validity of rFC testing and compare it with the LAL method. The specificity, linearity, accuracy, precision, and robustness of the rFC assay were evaluated. After validation, the rFC assay was found to be suitable for endotoxin detection. We compared the accuracy of the rFC and LAL assays using reference standard endotoxin. The rFC assay was as accurate as the LAL assay. We also compared the two assays using biopharmaceuticals. Greater interference occurred in some samples when the rFC assay was used than when the LAL assay was used. However, the rFC assay overcame the interference when the samples were diluted. Overall, we suggest that rFC can be applied to test biopharmaceuticals.

Analysis of commercially available snake antivenoms reveals high contents of endotoxins in some products

As injectable therapeutics, snake antivenoms must meet specifications for endotoxin content. The Limulus amebocyte lysate (LAL) test was used to evaluate the endotoxin content in several commercially available antivenoms released for clinical use. It was found that some products have endotoxin concentrations higher than the accepted limit for these contaminants. These results emphasize the need to include endotoxin determination as part of the routine evaluation of antivenoms by manufacturers and regulatory agencies.