Limulus Amoebocyte Lysate Assay Research Articles

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.

Detection of Beta-Glucan Contamination in Nanoparticle Formulations.

Beta-glucans with diverse chemical structures are produced by a variety of microorganisms and are commonly found in microbial cell walls. β-(1,3)-D-glucans are present in yeast and fungi, and, for this reason, their traces are commonly used as a sign of yeast or fungal infection or contamination. Despite being less immunologically active than endotoxins, beta-glucans are pro-inflammatory and can activate cytokines and other immunological responses via their cognate pattern recognition receptors. Unlike endotoxins, there is no established threshold pyrogen dose for beta-glucans; as such, their quantity in pharmaceutical products is not regulated. Nevertheless, regulatory agencies recognize the potential contribution of beta-glucans to the immunogenicity of protein-containing drug products and recommend assessing beta-glucans to aid the interpretation of immunotoxicity studies and assess the risk of immunogenicity. The protocol for the detection and quantification of β-(1,3)-D-glucans in nanoparticle formulations is based on a modified limulus amoebocyte lysate assay. The results of this test are used to inform immunotoxicity studies of nanotechnology-based drug products.

Clinical efficacy of endodontic protocols on reducing cultivable bacteria and endotoxin in infected root canal in patients submitted to head and neck radiotherapy: a randomised clinical trial.

Assess the efficacy of biomechanical preparation using a reciprocating system followed by final irrigation protocols, then intracanal medication, on reducing endotoxins and cultivable bacteria of infected teeth in irradiated patients. Twenty-two infected single-rooted canals in patients submitted to head and neck radiotherapy were prepared by reciprocating motion and 2.5% NaOCl. Patients were randomly divided into two groups of 11 patients before the final irrigation protocol: apical positive pressure (APP) or passive ultrasonic activation (PUA). Both groups were treated in two sessions, using Ca(OH)2 as intracanal medication for 14 days. Root canal content sampling was performed after canal access (S1), after biomechanical preparation plus the irrigation protocol (S2), and after intracanal medication (S3). Chromogenic limulus amoebocyte lysate assay measured endotoxin levels (EU/mL), and bacterial load was determined by culture techniques (CFU/mL). Treatment protocols reduced bacterial counts after S2 in both groups (p = 0.01). S3 differed from S1 (p = 0.01), but not from S2 (p = 0.4). Endotoxin levels were reduced in both groups after S2 (P = 0.03) and were lower in S3 than in S2, with significant differences in the APP group (p = 0.03). Biomechanical preparation using a reciprocating system and 2.5% NaOCl in irradiated teeth, followed by the irrigation protocol (APP or PUA), demonstrated efficacy in reducing endodontic contaminants. Ca(OH)2 as intracanal medication should be performed in irradiated patients with infected root canals. This clinical study demonstrated that endodontic treatment in irradiated patients is efficacious at reducing bacterial load and endotoxin levels.

Neisseria meningitidis accumulate in large organs during meningococcal sepsis.

Neisseria meningitidis (Nm) is the cause of epidemic meningitis and fulminant meningococcal septicemia. The clinical presentations and outcome of meningococcal septic shock is closely related to the circulating levels of lipopolysaccharides (LPS) and of Neisseria meningitidis DNA (Nm DNA). We have previously explored the distribution of Nm DNA in tissues from large organs of patients dying of meningococcal septic shock and in a porcine meningococcal septic shock model. 1) To explore the feasibility of measuring LPS levels in tissues from the large organs in patients with meningococcal septic shock and in a porcine meningococcal septic shock model. 2) To evaluate the extent of contamination of non-specific LPS during the preparation of tissue samples. Plasma, serum, and fresh frozen (FF) tissue samples from the large organs of three patients with lethal meningococcal septic shock and two patients with lethal pneumococcal disease. Samples from a porcine meningococcal septic shock model were included. Frozen tissue samples were thawed, homogenized, and prepared for quantification of LPS by Pyrochrome® Limulus Amoebocyte Lysate (LAL) assay. N. meningitidis DNA and LPS was detected in FF tissue samples from large organs in all patients with meningococcal septic shock. The lungs are the organs with the highest LPS and Nm DNA concentration followed by the heart in two of the three meningococcal shock patients. Nm DNA was not detected in any plasma or tissue sample from patients with lethal pneumococcal infection. LPS was detected at a low level in all FF tissues from the two patients with lethal pneumococcal disease. The experimental porcine meningococcal septic shock model indicates that also in porcinis the highest LPS and Nm DNA concentration are detected in lungs tissue samples. The quantification analysis showed that the highest concentration of both Nm DNA and LPS are in the organs and not in the circulation of patients with lethal meningococcal septic shock. This was also shown in the experimental porcine meningococcal septic shock model. Our results suggest that LPS can be quantified in mammalian tissues by using the LAL assay.

Indoor Airborne Microbiome and Endotoxin: Meteorological Events and Occupant Characteristics Are Important Determinants.

Airborne bacteria and endotoxin may affect asthma and allergies. However, there is limited understanding of the environmental determinants that influence them. This study investigated the airborne microbiomes in the homes of 1038 participants from five cities in Northern Europe: Aarhus, Bergen, Reykjavik, Tartu, and Uppsala. Airborne dust particles were sampled with electrostatic dust fall collectors (EDCs) from the participants' bedrooms. The dust washed from the EDCs' clothes was used to extract DNA and endotoxin. The DNA extracts were used for quantitative polymerase chain (qPCR) measurement and 16S rRNA gene sequencing, while endotoxin was measured using the kinetic chromogenic limulus amoebocyte lysate (LAL) assay. The results showed that households in Tartu and Aarhus had a higher bacterial load and diversity than those in Bergen and Reykjavik, possibly due to elevated concentrations of outdoor bacterial taxa associated with low precipitation and high wind speeds. Bergen-Tartu had the highest difference (ANOSIM R = 0.203) in β diversity. Multivariate regression models showed that α diversity indices and bacterial and endotoxin loads were positively associated with the occupants' age, number of occupants, cleaning frequency, presence of dogs, and age of the house. Further studies are needed to understand how meteorological factors influence the indoor bacterial community in light of climate change.

Application of a novel chemical assay for the quantification of endotoxins in bacterial bioreactor samples

A novel chemical assay, the so-called Kdo-DMB-liquid chromatography (LC) assay, was used for the accurate and cost-effective determination of the endotoxin content in supernatants of Gram-negative bacteria bioreactor samples. During mild acid hydrolysis, the endotoxin-specific sugar acid 3-deoxy-D-manno-oct-2-ulsonic acid (Kdo) is quantitatively released. Kdo is reacted with 1,2-diamino-4,5-methylenedioxybenzene (DMB) to obtain the highly fluorescent derivate Kdo-DMB. It is separated from the reaction mixture by reversed phase-(U)HPLC and detected by fluorescence. From the Kdo content the endotoxin content of the sample is calculated. For three batch cultivations of Escherichia coli K12 and a fed-batch cultivation of Pseudomonas putida KT2440, the evolution of the endotoxin content in dependence on the cultivation time was monitored. Under optimal, constant cultivation conditions a linear correlation between the endotoxin content and the easy-to-access bioreactor parameters optical density at 600 nm and dry cell weight was found for both endotoxin kinds. Under stress cultivation conditions the E. coli K12 cultivation showed a stronger increase of the endotoxin content at harvest in comparison to optimal conditions. Optical density and dry cell weight may be used for production reactors as an economic real-time estimation tool to determine the endotoxin content at different cultivation time points and conditions. The optical density can further be used to establish straightforward sample dilution schemes for endotoxin quantification in samples of unknown endotoxin content. The endotoxin content [ng mL−1] measured by the Kdo-DMB-LC assay and the endotoxin activity [EU mL−1] obtained by the compendial Limulus Amoebocyte Lysate assay show a high correlation for the bacterial bioreactor samples tested.

Exposure levels of animal allergens, endotoxin, and β-(1,3)-glucan on a university campus of veterinary medicine.

The study aimed to determine the allergen, endotoxin and β-(1,3)-glucan concentrations at various areas on a university campus of veterinary medicine. Dust samples were collected four times a year for three years using electrostatic dust collectors (EDC) at 25 different locations on a campus of veterinary medicine and in laboratories of inorganic chemistry as a control area representing animal-free environment. Major animal allergens from dog, cat, horse, cattle and mouse, domestic mite (DM) allergens, and β-(1,3)-glucan were measured using enzyme immunoassays and endotoxin using the limulus amoebocyte lysate (LAL) assay. Seasonal, annual and local influences on exposure levels were analyzed using Bayesian mixed models. With the exception of mouse allergens, all other determinants were found in almost all locations on the campus and in the control area, but in up to 10.000-fold variable concentrations. By far the highest levels of feline, canine, equine and bovine allergens were detected in buildings where the respective species were examined. The highest levels of mouse and DM allergens, β-(1,3)-glucan and endotoxin occurred together and were associated with locations where large animals were present. In buildings without animals, allergen levels were considerably lower but still elevated at several locations compared to the control area, especially for dog and horse allergens, and β-(1,3)-glucan. Significant seasonal effects were observed for dog, cat, horse and DM allergens, and β-(1,3)-glucan. Variations between years were less apparent than between seasons (except for β-(1,3)-glucan). The strongest influencing factor on the concentration of mammalian allergens was the presence of the corresponding animal at the collection site. Seasonal influence on allergen concentrations was observed, while the overall exposure remained constant over the years. At locations with horses, elevated levels of mite allergens, endotoxin, and β-(1,3)-glucan can be expected, probably due to passive transfer from stable environment.

Occupational Bioaerosol Exposures Associated with Poultry Farming

ABSTRACT This study aimed to investigate occupational exposure to particulate dust, endotoxin, and (1–3)-β-D-glucan among workers involved in various poultry farming activities. A total of 298 personal samples were collected from randomly selected individuals from exposure groups based on distinct poultry farming activities comprising broiler farms, rearing, laying, hatchery, and catching activities. Aside from the inhalable particulate dust concentration that was determined, filter extracts were also analyzed for (1–3)-β-D-glucan and endotoxin using the endpoint Glucatell® and Limulus amoebocyte lysate (LAL) assays, respectively. Data were analyzed using STATA 12 and linear regression models developed. The mean (GM) dust particulate concentration was 11.04 mg/m3 (GSD = 3.87); 2298 endotoxin units (EU/m3) (GSD = 10.56) and 149 ng/m3 for (1–3)-β-D-glucan (GSD = 4.62). A modest positive correlation was observed between log-transformed endotoxin and (1–3)-β-D-glucan concentrations (Pearson r = 0.44, p < .001), whilst a moderate negative correlation was observed for inhalable dust particulate and (1–3)-β-D-glucan (Pearson r = −0.33, p < .001). However, there was a very poor correlation between inhalable dust and endotoxin (Pearson r = −0.02, p < .001). In the regression models, exposure group based on the nature of farming activity explained 50% of the variability in dust particulate and glucan levels. For dust particulate, rearing activities were significant predictors of higher dust levels compared to hatchery work, while rearing, laying, broiler and catching activities were significant predictors of higher endotoxin or glucan levels. Furthermore, working in a small broiler was a significant determinant of elevated glucan exposures. Farms using automated laying activities had significantly higher particulate levels compared to those using manual laying activities. This study revealed that workers engaged in poultry farming activities were exposed to significantly high levels of inhalable particulate dust, endotoxin, and (1–3)-β-D-glucan concentrations, posing an increased risk for adverse respiratory health effects in these farm workers.

HDL and LDL have distinct, opposing effects on LPS-induced brain inflammation

Endotoxemia and sepsis induce neuroinflammation and increase the risk of neurodegenerative disorders although the mechanism by which peripheral infection leads to brain inflammation is not well understood. While circulating serum lipoproteins are known immunometabolites with the potential to modulate the acute phase response and cross the blood brain barrier, their contribution to neuroinflammation during systemic infection is unknown. The objective of this study was to elucidate the mechanisms by which lipoprotein subclasses modulate lipopolysaccharide (LPS)-induced neuroinflammation. Adult C57BL/6 mice were divided into 6 treatment groups, including a sterile saline vehicle control group (n = 9), an LPS group (n = 11), a premixed LPS + HDL group (n = 6), a premixed LPS + LDL group (n = 5), a HDL only group (n = 6) and an LDL only group (n = 3). In all cases injections were administered intraperitoneally. LPS was administered at 0.5 mg/kg, and lipoproteins were administered at 20 mg/kg. Behavioural testing and tissue collection was performed 6 h post-injection. The magnitude of peripheral and central inflammation was determined by qPCR of pro-inflammatory genes in fresh liver and brain. Metabolite profiles of liver, plasma and brain were determined by 1H NMR. Endotoxin concentration in the brain was measured by the Limulus Amoebocyte Lysate (LAL) assay. Co-administration of LPS + HDL exacerbated both peripheral and central inflammation, whilst LPS + LDL attenuated this inflammation. Metabolomic analysis identified several metabolites significantly associated with LPS-induced inflammation, which were partially rescued by LDL, but not HDL. Endotoxin was detected at significantly greater concentrations in the brains of animals that received LPS + HDL compared to LPS + saline, but not those that received LPS + LDL. These results suggest that HDL may promote neuroinflammation through direct shuttling of endotoxin to the brain. In contrast, LDL was shown to have anti-neuroinflammatory properties in this study. Our results indicate that lipoproteins may be useful targets in neuroinflammation and neurodegeneration associated with endotoxemia and sepsis.

Effect of antimicrobial photodynamic therapy on the reduction of bacteria and virulence factors in teeth with primary endodontic infection.

To evaluate the effectiveness of supplemental photodynamic therapy for improving the bacterial removal and the levels of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) by conducting a clinical trial. Twenty-four root canals with pulp necrosis and periapical lesion were selected and randomly divided into conventional group using endodontic treatment with chemo-mechanical preparation (CMP) alone (n=12) and a group using antimicrobial photodynamic therapy (aPDT) after CMP (n=12). The samples were collected before and after CMP (conventional group) and after photodynamic therapy (aPDT group). A photosensitizer (0.005% methylene blue) was applied to the root canal for 3 minutes after CMP, whereas aPDT was performed by using a red laser with a power of 30Mw and energy density of 9J/cm2 for 90 s per root canal. Culture technique was performed to determine the bacterial colony forming units. LPS and LTA levels were quantified by using limulus amoebocyte lysate (LAL) assay and enzyme-linked immunosorbent assay (ELISA), respectively. All samples showed growth of viable bacteria on Fastidious Anaerobe Agar (FAA), with an average of 5.19×105 CFU/ mL. CMP was effective in decreasing viable bacteria (p<0.05), whereas there was a significant decrease (p<0.05) in the samples treated with aPDT compared to those submitted to CMP. LPS and LTA were detected in all initial samples, with mean values of 20.561 EU/mL and 430.91 pg/mL, respectively. Both CMP and aPDT groups significantly decreased the levels of LPS and LTA (p<0.05), with a statistical difference between the groups regarding aPDT (p<0.05). Photodynamic therapy as an adjunct to CMP proved to be effective in improving root canal disinfection and reducing the LPS and LTA levels in teeth with primary endodontic infection.

Exposure to dust, endotoxins and microbes in Finnish vegetable greenhouses.

In Finland and other northern countries, vegetable greenhouse workers are exposed to airborne dust and microbes all year round. The aim of the study was to assess respiratory exposure to dust, endotoxins and microbes, and to identify the risk phases of respiratory and skin exposure to promote safe working methods. Work in greenhouses was observed and recorded in a structured form. 23 personal samples and eight stationary samples of dust, endotoxins and viable microbes were collected from eight tomato or cucumber greenhouses in Finland. Dust samples were analysed gravimetrically. Endotoxins were analysed on filters by chromogenic Limulus amoebocyte lysate assay. Microbes were collected on filters, incubated and counted in three different culture media. Eight additional stationary samples of microbes were collected on a six-phase impactor. The greenhouses were generally clean and well organised, but skin protection was deficient. The median of personal dust exposure was 0.24 mg/m3: 0.21 mg/m3 in the tomatohouses and 0.62 mg/m3 in the cucumber greenhouses. The mean level of bacteria in the tomato houses was 2.7 × 103 and in the cucumber houses 1.6×104. Those of fungi were 3.6 × 105 and 1.7 × 105 on Hagem and 8.1 × 105 and 1.9 × 105 on DG-18, respectively. The microbes made up about 1/10 of these values in the stationary samples. Very low concentrations of endotoxins were found in 5/23 air samples. The clean appearance of the greenhouses was reflected in the low to moderate levels of dust. Risk phases of high exposure, such as lifting leaf debris, were identified. The microbe species were typical of this climate, and their levels were comparable to other greenhouse studies, but lower than in farming. Prevention among the foreign workforce is challenging due to the lack of a common language.

Er:YAG laser irradiation enhances bacterial and lipopolysaccharide clearance and human gingival fibroblast adhesion on titanium discs

To investigate the effect of Er:YAG laser treatment on lipopolysaccharide (LPS) clearance and fibroblast adhesion on titanium disks. Grade IV titanium discs (n = 216) were used and allocated to 6 groups. Group 1 was the negative control without Porphyromonas gingivalis inoculation. Discs in Groups 2–6 were incubated with P. gingivalis to form a biofilm. Group 3 received 0.12% chlorhexidine irrigation and Group 4 received titanium curettage to remove the biofilm. Group 5 was treated with Er:YAG laser irradiation and Group 6 was treated with titanium curettage plus Er:YAG laser irradiation. The contact angle and surface roughness were measured after the various treatments. The surface microstructure and residual bacteria were examined using scanning electron microscopy and confocal laser scanning microscopy, respectively. Residual LPS was examined using a limulus amoebocyte lysate assay and human gingival fibroblast adhesion was quantified using fluorescent microscopy. Curettage plus Er:YAG laser irradiation was the most effective method for removing bacteria and LPS. No significant difference in the amount of fibroblast adhesion was found between the control and Group 6. Combined use of Er:YAG laser irradiation and curettage optimizes LPS clearance and fibroblast adhesion on titanium discs.

Comparing the concentration levels of allergens and endotoxins in employees\u2019 homes and offices

ObjectiveThe aim of the study was to find out whether allergen and endotoxin concentrations in offices differ from those measured at the homes of employees, and identify the parameters that influence exposure.MethodsElectrostatic dust collectors (EDCs) were placed in five office buildings (68 rooms, 436 EDCs), as well as the homes of the office workers (145 rooms, 405 EDCs) for 14 days, four times a year. In addition, surface samples were collected from the offices four times a year by vacuuming the carpeted floors. Domestic mite (DM), and the major cat and dog allergens (Fel d 1 and Can f 1) were quantified in all samples using fluorescence enzyme immunoassays. Endotoxin was measured in the EDC samples, using the Limulus amoebocyte lysate assay. The allergen and endotoxin concentrations were log transformed and analysed with multilevel models.ResultsEndotoxin concentrations were significantly higher in personal homes compared to levels measured in the offices, and depended on the number of persons living in each household, as well as the presence of a dog. DM allergens were significantly higher in households than in offices, and were significantly higher in bedrooms compared to living rooms. Offices occupied by cat owners had significantly higher Fel d 1 concentrations than offices or homes without. Additionally, Can f 1 concentrations were significantly higher in offices occupied by dog owners compared to those without.ConclusionsPet owners appear to transfer cat and dog allergens to their offices. Therefore, in case of allergy complaints at the office, employers and physicians might consider possible contamination by cat and dog allergens.

Increased levels of circulating LPS during Tuberculosis prevails in patients with advanced pulmonary involvement.

Our earlier studies in tuberculosis (TB) patients indicate that in those where the process evolves to a larger pulmonary involvement, the immune endocrine response may promote an unfavorable environment. Chronic infectious diseases, and their persistent proinflammatory response, may affect mucosal barriers integrity favoring the translocation of gastrointestinal bacteria, leading to an increase of circulating lipopolysaccharides (LPS). Consequently, we quantified LPS levels in TB patients, with different degrees of pulmonary involvement, and controls (Co) and analyzed the possible relationship between LPS and inflammatory mediators i.e., C reactive protein (CRP), interleukin 6 (IL-6) and Interferon-gamma (IFN-γ), Erythrocyte Sedimentation Rate (ESR), steroid hormones (Cortisol and Dehydroepiandrosterone, DHEA), and inflammatory transcripts from peripheral blood mononuclear cells (IL-1β, IL-6, IFN-γ). LPS was assessed by the Limulus amoebocyte lysate assay and the ELISA technique was used to quantify hormones and cytokines in the plasma samples. Cytokine transcripts from PBMC were evaluated by qRT-PCR. Non-parametric tests were used. LPS levels were increased in TB patients, as did levels of CRP, IL-6, IFN-γ, cortisol and ESR. Severe patients had the highest amounts of circulating LPS; with moderate and severe cases showing much higher levels of CRP, ESR, IL-6, IFN-γ and cortisol/DHEA ratio, as an endocrine imbalance. Only in PBMC from severe cases was mRNA for IL-1β increased. Correlation analysis showed that levels of LPS from severe patients were positively associated with IL-6 and IFN-γ plasma concentrations and with IL-1β transcripts, while IL-6 had a positive correlation with the cortisol/DHEA ratio. The higher levels of circulating LPS during progressive TB may emerge as a contributing factor for the persistence of the greater immune endocrine imbalance distinctive of advanced disease, which might suggest a vicious cycle among LPS, inflammation and endocrine imbalance.

Animal Allergens, Endotoxin, and β-(1,3)-Glucan in Small Animal Practices: Exposure Levels at Work and in Homes of Veterinary Staff

ObjectivesIn veterinary settings, high exposures to animal allergens and microbial agents can be expected. However, occupational exposure levels are largely unknown. The objective of this study was to estimate the allergen, endotoxin, and β-(1,3)-glucan concentrations in small animal practices and in the homes of practice employees.MethodsDust samples were collected using electrostatic dust fall collectors in diverse rooms of 36 small animal practices, as well as in employees’ homes. Major animal allergens (Fel d 1, Can f 1, Ory c 3, Cav p 1, Equ c 1, Bos d 2), domestic mite (DM) allergens, and β-(1,3)-glucan levels were measured using enzyme immunoassays. Endotoxin was determined using the Limulus amoebocyte lysate assay. Influences on exposure levels were analyzed using multilevel models.ResultsThe levels of Can f 1, Fel d 1, Ory c 3, and Cav p 1 were up to 30 times higher in practices compared with homes without animals, but significantly lower compared with the homes with the respective pet. Although horses were not treated in the practices, Equ c 1 was found in 87.5% of samples, with the highest concentrations measured in changing rooms. DM levels were significantly lower in practices than in all private homes, and endotoxin levels were similar to those in homes with pets. In the practice itself, exposure levels were significantly influenced by animal presence, type of the room, and area per employee; whereas, room volume and diverse cleaning measures had mostly no effect.ConclusionsExposure to animal allergens is high in veterinary practices, but it does not reach levels of households with pets. Domestic mite allergen and endotoxin exposure seem to be low for workers in veterinary practices. The high Equ c 1 detection rate strongly indicates dispersal of allergens, most likely through clothing and hair.

Engineering Janus micromotors with WS2 and affinity peptides for turn-on fluorescent sensing of bacterial lipopolysaccharides

Herein we describe an “OFF-ON” Janus micromotor approach for the fast (5 min) and sensitive determination (limit of detection, 120 pM) of Escherichia coli O111:B4 lipopolysaccharide (LPS) associated with sepsis shock in microliter samples. The OFF-ON strategy relies on the loading of a specifically designed rhodamine-labeled affinity peptide into WS2–Pt–Fe2O3 polycaprolactone Janus micromotors. Specific attachment of the peptide with the WS2 via electrostatic and hydrophobic interactions results in fluorescent quenching, which is subsequently recovered by the detachment of the probe in the presence of the target LPS. Peptide loading into the micromotor structure increases the overall stability for over 2 months without any change in its properties and excellent analytical performance. No fluorescence recovery is observed in the presence of LPS with a similar structure, illustrating the high selectivity of the protocol, along with quantitative recoveries in human serum and bacteria cultures. The method was validated against the gold standard Limulus Amoebocyte lysate assay in real bacteria culture containing naturally occurring LPS, with similar recoveries in both cases. The micromotors hold great potential to carry out analytical measurements in real-time with small amounts of sample and reagents, allowing for fast detection of deadly toxins with high clinical relevance.

Comparison of LAL and rFC Assays-Participation in a Proficiency Test Program between 2014 and 2019.

Endotoxin (lipopolysaccharide) testing of drugs is routinely required in pharmaceutical industries. Suitable compendial assays are defined by national pharmacopoeias. At this time, Limulus Amoebocyte Lysate (LAL) assays are the gold standard. LAL is used in vitro for specific detection of endotoxin based on endotoxin-activated Factor C-mediated clotting cascade. However, alternative mediated pathways (e.g., Factor G), impurities, and further factors may influence test results. Some of these influencing factors are eliminated by recombinant Factor C (rFC) test, which represents a promising alternative. rFC not only enables highly specific endotoxin testing, as interfering Horseshoe Crab blood components are eliminated, but also offers ethical and ecological advantages compared to classical LAL assays. However, the question remains whether rFC-based tests are robust test systems, equivalent or superior to LAL and suitable for routine bacterial endotoxin testing. Pharmaceutical test users have validated the test successfully for their specific products, but no long-term studies have been published that combine testing of unknown samples, inter-laboratory, -operator, and -lot changes. Thus, it was of great interest to investigate rFC test performance in a routine setting within a proficiency test program set-up. Over a period of six years comparative endotoxin testing was conducted with one kinetic chromogenic LAL assay and two rFC-based assays. Results of this study demonstrate that both rFC-based assays were comparable to LAL. All results met acceptance criteria defined by compendial bacterial endotoxin testing. RFC-based methods generated results with even better endotoxin recovery rates compared to LAL. Therefore, rFC-based tests were found to represent reliable methods, as equivalent or even superior to LAL assays and suitable for routine bacterial endotoxin testing.

Comparison of Limulus Amoebocyte Lysate and Recombinant Factor C Assays for Endotoxin Detection in Four Human Vaccines with Complex Matrices

Endotoxins, heat-stable lipopolysaccharides from Gram-negative bacteria, are potential contaminants that can be introduced during manufacturing of pharmaceutical products, including vaccines. Parental pharmaceutical products undergo endotoxin testing because endotoxins are pyrogenic in humans and can induce severe physiological reactions. Currently, animal-derived Limulus amoebocyte lysate (LAL) assays are widely used. Assays using recombinant factor C (rFC), a nonanimal-derived reagent, have been proposed as alternatives. Some components in the matrices of pharmaceutical products can interfere with these assays. We compared two LAL- and two rFC-based assays for endotoxin detection in four complex human vaccine matrices. We showed that the results for the rFC-based assays were at least equivalent to those for the LAL-based assays, although the rFC-based assays were found to be adequate but slightly less suitable for one of the products that contained proteases as the methods used to inactivate the proteases reduced the assay performance. Likewise, LAL was adequate but less suitable for another product that contained glucans. The rFC assays offer a number of benefits, including compliance with the principles of the 3Rs, i.e., replacement, reduction, and refinement of animal testing by safeguarding animal welfare and promoting more ethical and sustainable use of animals for testing. After they are fully validated, as per the compendial requirements, they could be considered as suitable replacement assays for the detection of endotoxin in the manufacturing processes of pharmaceutical products. In summary, we demonstrated that both LAL and rFC assays are adequate for testing and releasing four vaccine products.

Highly Sensitive Endotoxin Assay Combining Peptide/Graphene Oxide and DNA-Modified Gold Nanoparticles.

Endotoxin is a highly toxic stimulator originated from the outer membrane of Gram-negative bacteria, which should be monitored sensitively and selectively for human health concerns. Traditional detection methods mainly rely on limulus amoebocyte lysate assay. However, it suffers drawbacks like the narrow detection range, and the results may be environment-dependent. In this work, we have developed a sensitive electrochemical biosensor for endotoxin assay. Peptide is first designed as specific recognition element toward endotoxin. Graphene oxide and DNA-modified gold nanoparticles are then used to enhance the electrochemical signal. The analytical performances are excellent with the limit of detection as low as 0.001 EU mL–1. This method has also been successfully applied in endotoxin assay in complex biological samples, which may have great potential use.

Occurrence of bacteria and endotoxins in fermented foods and beverages from Nigeria and South Africa

In Africa, fermented foods and beverages play significant roles in contributing to food security. Endotoxins are ubiquitous heat stable lipopolysaccharide (LPS) complexes situated in the outer cell membranes of Gram-negative bacteria. This study evaluated the microbiological quality of fermented foods (ogiri, ugba, iru, ogi and ogi baba) and beverages (mahewu and umqombothi) from selected Nigerian and South African markets. The bacterial diversity of the fermented foods was also investigated and the identity of the isolates confirmed by biochemical and molecular methods. Isolate grouping was established through hierarchal clustering and the samples were further investigated for endotoxin production with the chromogenic Limulus Amoebocyte Lysate assay. The total aerobic count of the samples ranged from 5.7 to 10.8 Log CFU/g. Fourteen bacteria genera were detected with most of the isolates being members of the Enterobacteriaceae family. Sphingomonas paucimobilis and Escherichia coli were the dominant Gram-negative bacterial species detected. There were considerable variations in the concentrations of endotoxins produced and the lowest endotoxin concentration was found in ogi (4.3 × 10 EU/g) and the highest in iru (5.5 × 104 EU/g) while, 44% of umqombothi samples had endotoxins. Ogi baba samples had better microbial quality than other samples due to its reduced bacterial load and endotoxin levels. There was a strong positive (r = 0.714, r = 0.996) and significant (p < 0.01) correlation between the endotoxin levels and bacterial loads of the samples. Some previously unreported species of bacteria found in the fermented foods included Aeromonas haemolyticus and Rhizobium radiobacter. This is the first comprehensive report on endotoxins in fermented foods and beverages in Africa. Furthermore, the occurrence of pathogenic bacteria and toxins in the foods and beverages is of serious concern that calls for immediate action.