Endotoxin Levels Research Articles

Interlaboratory comparison of endotoxin contamination assessment of nanomaterials.

Endotoxin contamination is a significant hurdle to the translation of nanomaterials for biomedical applications. Multiple reports now describe that more than one-third of nanomaterials fail early pre-clinical assessment due to levels of endotoxin above regulatory requirements. Additionally, most immunological studies or in vivo studies testing nanomaterials in the literature lack inclusion of this assessment, which may lead to false-positive or false-negative results if high levels of the contaminant are present. The currently approved methods for endotoxin contamination assessment rely on enzymatic activity and wavelength absorbance as their endpoint, and many nanomaterials can interfere with such assays. For this reason, we devised an interlaboratory comparison of endotoxin contamination assessment for a range of nanomaterials to challenge the current international organization for standardization and pharmacopeia standards. Herein, we show that detected endotoxin levels could vary considerably between groups, and, in some instances, nanomaterials could both pass and fail regulatory endotoxin limits for medical devices depending on the group undertaking the assessment, all while passing all quality criteria standards. This work emphasises the requirement for multiple assays to fully assess the endotoxin levels in a nanomaterial and highlights the need for additional assays to be developed in this space.

The Effect of a HealiAid ® Collagen Wound Dressing on Wound Healing in Patients: A Randomized Double-Blind Pilot Clinical Trial

Wound healing is a complex, multi-phase process. With rising healthcare costs, an aging population, and increasing chronic disease prevalence, the burden of wound care is escalating. Thus, finding safe and effective methods to accelerate wound healing is crucial. This study aims to explore the safety and efficacy of HealiAid® Collagen Wound Dressing in treating various types of wounds, including venous ulcers, diabetic foot ulcers, pressure ulcers, and burns. A clinical trial was conducted on 18 subjects with venous ulcers, diabetic foot wounds, pressure ulcers, and burn wounds. The study assessed the effectiveness of HealiAid in different wound types by analyzing its thermal stability, porous structure, biocompatibility, and clinical efficacy. Wound healing was strictly monitored, and the safety and tolerability of the dressing were evaluated. The results showed that HealiAid Collagen Wound Dressing has excellent thermal stability and structural integrity. Its porous microstructure enhances cell adhesion and tissue regeneration, demonstrating significant healing effects across various wound types, including venous ulcers, diabetic foot wounds, pressure ulcers, and burns. No serious adverse reactions were reported, and endotoxin levels were well below safety standards. This study confirms the safety and efficacy of HealiAid Collagen Wound Dressing in treating multiple wound types. Its outstanding thermal stability, porous structure, and biocompatibility make it a promising option for clinical wound care

Interaction Between Peroxisome Proliferator-Activated Receptors and Cannabidiol in the Gut of Chickens Applied to Different Challenge Conditions

Peroxisome proliferator-activated receptors (PPARs) are important targets for cannabidiol (CBD), which mediate many of its biological actions. The hypothesis of the present research assumed that PPARs affect the gut response to different challenge factors in chickens (C. perfringens vs. lipopolysaccharides (LPS) from E. coli), and that CBD can mediate the pathways of this response. The study proved that CBD and the challenge factors significantly affect the expression level of PPARα (p = 0.001) and selected genes determining gut barrier function. A positive correlation was demonstrated between PPARs and genes involved in the formation of tight junctions, immune, and oxidative stress responses in chickens. Dietary supplementation with CBD actively mediated the expression rate of PPARs, but the mechanism of interaction between CBD and PPARs was different depending on the stress factor used. The addition of CBD to the birds’ diets did not contribute to reducing intestinal permeability under induced stress conditions nor cause stress, as indicated by the absence of elevated blood cortisol and endotoxin levels. CBD also supported the mechanisms of protecting intestinal cells from the cytotoxic effects in a C. perfringens challenge through the levels of genes involved in oxidative stress. This study indicates the importance of research toward understanding the mechanisms of PPARs as a target for enhancing intestinal barrier function, provides new results on the biological action of CBD in chickens, and shows a constant PPAR association with the jejunum mucosa of birds.

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.

Enhancing infection control in dialysis at a resource limited public healthcare institute: A cross-sectional study on microbiological quality assessment of dialysis water and dialysate

PurposeTo evaluate the microbiological quality of dialysis water and dialysate samples from hemodialysis units at a tertiary care government hospital and to assess the use of culture, endotoxin and periodicity of testing these assays to guide the actions of monitoring the quality of hemodialysis fluids, implement preventive and corrective actions, and improve the safety and outcomes of the dialysis process. MethodsA cross-sectional study was conducted at a 250-bedded super-specialty government hospital with a 24/7 hemodialysis unit equipped with 40 dialysis machines. Dialysis water and dialysate samples were collected monthly and analysed for microbial contamination and endotoxin levels as per AAMI guidelines. Bacterial cultures were done using Reasoner's Agar plates, and endotoxin analysis was performed using gel clot assay. Interpretation of results was based on predefined thresholds. ResultsAmong the 740 samples processed for microbial culture 19.6 % and 80.4% were unacceptable and acceptable respectively.Among the acceptable samples 10.5 % were at action level. At the end of 2 days of incubation, 15.2%dialysis water samples and 5.1%dialysate samples had unacceptable levels of bacterial colonies and at 5days additional 10.6 % dialysis water samples and 7.7%dialysate samples were unacceptable. 21.5 % of the samples tested for endotoxin had unacceptable levels. ConclusionsRegular monitoring of water quality in government healthcare hemodialysis units is crucial for quality of care, timely preventive and corrective actions for mitigating adverse outcomes. Processing cultures for 5–7 days is essential for detecting all contaminated specimens. While there might be a link between endotoxin levels and bacterial contamination, both endotoxin testing and bacterial culture independently are vital for evaluating water quality in dialysis settings. A comprehensive approach integrating various testing methods is necessary to uphold patient safety and enable necessary improvements.

A weekly 4-methylpyrazole treatment attenuates the development of non-obese metabolic dysfunction-associated steatotic liver disease (MASLD) in male mice: Role of JNK.

4-methylpyrazole (4MP, fomepizole) is a competitive inhibitor of alcohol dehydrogenase (ADH) preventing the metabolism of ethylene glycol and methanol, respectively, into their toxic metabolites. 4MP seems also to possess a potential in the treatment of intoxication from other substance, for example, acetaminophen, and to modulate JNK-dependent signalling. Here, we determined if a treatment with 4MP once weekly affects the development of diet-induced non-obese metabolic dysfunction-associated steatotic liver disease (MASLD) in C57BL/6 mice. Male C57BL/6 mice (6-8 weeks old, n = 7-8/group) were pair-fed either a liquid control diet (C) or a liquid sucrose-, fat- and cholesterol-rich diet (SFC) for 8 weeks while being concomitantly treated with 4MP (50 mg/kg bw i.p.) or vehicle once a week. Liver damage, inflammatory markers and glucose tolerance were assessed. Moreover, in endotoxin-challenged J774A.1 cells pretreated with 4MP, pro-inflammatory markers were assessed. The concomitant treatment of SFC-fed mice with 4MP attenuated the increase in JNK phosphorylation and pro-inflammatory markers like IFNγ, IL-6 and 3-nitrotyrosine protein adducts in liver tissue found in vehicle-treated SFC-fed mice, while not affecting impairments of glucose tolerance or the increase in portal endotoxin levels. Moreover, a pretreatment of endotoxin-stimulated J774A.1 cells with 4MP significantly attenuated the increases in JNK phosphorylation and pro-inflammatory mediators like IL-6 and Mcp1. Taken together, our results suggest that a treatment with 4MP once weekly attenuates the activation of JNK and dampens the development of non-obese MASLD in mice.

Influence of different cleaning methods on the concentration of airborne endotoxins and microbial aerosols in the oral clinical environment

AimThis study aims to evaluate the effectiveness of various cleaning methods in reducing airborne endotoxin and microbial aerosols during oral cleaning procedures.MethodForty patients undergoing oral cleaning procedures were randomly assigned to one of four groups (n = 10 per group). Group A received strong suction alone; Group B received strong suction combined with an air disinfection machine; Group C received strong suction combined with a dental electric suction machine; Group D received strong suction in conjunction with both an air disinfection machine and a dental electric suction machine. Airborne aerosol concentrations were assessed at four-time points: before treatment, 30 min into treatment, immediately after treatment, and 60 min after treatment ended. Samples were collected at distances of 20 cm, 60 cm, and 1 m from the patient’s oral cavity using the natural sedimentation method. T-test was used to evaluate the difference among tested groups.ResultsAirborne endotoxins and microbial aerosols levels increased significantly during treatment, with the highest levels observed at 20 cm from the patient’s mouth. During treatment, groups with additional cleaning methods (Groups B, C, and D) exhibited higher levels of airborne endotoxins and microbial aerosols compared to Group A (strong suction alone). However, post-treatment analysis revealed that Group D demonstrated the lowest level of airborne endotoxins and microbial aerosols, while Group A exhibited the highest.ConclusionsImplementing effective aerosol management strategies can significantly reduce aerosol dispersion in the oral clinical environment. Continuous monitoring aerosol concentrations and the application of appropriate control measures are essential for minimizing infection risks for both patients and healthcare providers during oral cleaning procedures.

The Hidden Relationship between Intestinal Microbiota and Immunological Modifications in Preeclampsia Pathogenesis.

Preeclampsia is a multifactorial gestational syndrome characterized by increased blood pressure during pregnancy associated with multiorgan involvement. The impact of this disease on maternal and neonatal health is significant, as it can lead to various fetal comorbidities and contribute to the development of maternal comorbidities later in life. Consistent evidence has shown that the microbiota acts as a regulator of the immune system, and it may, therefore, influence the development of preeclampsia by modulating immune factors. This narrative review aims to investigate the role of the immune system in the pathogenesis of preeclampsia and to summarize the most recent literature on the possible link between preeclampsia and alterations in the intestinal microbiota. To this end, we conducted a literature search, aiming to perform a narrative review, on PubMed and Embase from January 1990 to March 2024, focusing on the latest studies that highlight the main differences in microbial composition between patients with and without preeclampsia, as well as the effects of microbial metabolites on the immune system. From the review of 28 studies assessing the intestinal microbiota in preeclamptic women, preeclampsia could be associated with a state of dysbiosis. Moreover, these patients showed higher plasmatic levels of endotoxin, pro-inflammatory cytokines, and T helper 17 cells; however, the findings on specific microbes and metabolites that could cause immune imbalances in preeclampsia are still preliminary.

The Mechanism of Bacterial Endotoxin Invasion Pathways in Porcine Reproductive and Respiratory Syndrome Virus-Positive Porcine Endometrial Epithelial Cells

Porcine reproductive and respiratory syndrome virus (PRRSV) causes abortions, stillbirths, and dummy pregnancies. Previous studies found that PRRSV can promote secondary bacterial infections and elevate bacterial endotoxin levels, further increasing the abortion rate in sows. However, the pathways by which bacterial endotoxins invade the bodies of PRRSV(+) sows and aggravate their clinical symptoms are unknown. In this study, we established a model of PRRSV and lipopolysaccharide (LPS) working together on porcine endometrial epithelial cells (PEECs). We speculate that PRRSV and LPS affect PEECs through viral protein interaction with cytokines and cytokine receptors, natural killer cell-mediated cytotoxicity, and regulation of actin cytoskeleton signaling pathways by analyzing seq-RNA. The PRRSV proteins act on inflammatory factors and their receptors to activate chemokines-5 (CCL5), chemokines-4 (CCL4), and chemokines-8 (CCL8) mRNA expression, causing severe inflammatory reactions. In addition, the elevation of MEK1/2 factors and the integrins acting on NK cells promote the upregulation of VAV1/Tiam1, RAC, and IRSp53, leading to increased expression of Arp2/3 and F-actin in PEECs in the PRRSV + LPS(+) groups. However, the highly expressed cell microfilaments and cytoskeleton disrupt the original network structure, causing changes in the original physiological function of the PEECs. In summary, the PRRSV protein interacts with cytokines and cytokine receptors of PEECs, thereby enhancing virus-mediated chemokine factors and their receptor activity, accelerating bacterial endotoxin entry into the body and the invasion of cells. They destroy the cytoskeletal structure of the cells and increase damage to uterine tissue.

Fast, Easy, and Reproducible Fingerprint Methods for Endotoxin Characterization in Nanocellulose and Alginate-Based Hydrogel Scaffolds.

Nanocellulose- and alginate-based hydrogels have been suggested as potential wound-healing materials, but their utilization is limited by the Food and Drug Administration (FDA) requirements regarding endotoxin levels. Cytotoxicity and the presence of endotoxin were assessed after gel sterilization using an autoclave and UV treatment. A new fingerprinting method was developed to characterize the compounds detected in cellulose nanocrystal (CNC)- and cellulose-nanofiber (CNF)-based hydrogels using both positive- and negative-ion mode electrospray ionization Fourier transform ion cyclotron resonance mass spectroscopy (ESI FT-ICR MS). These biobased hydrogels were used as scaffolds for the cultivation and growth of human dermal fibroblasts to test their biocompatibility. A resazurin-based assay was preferred over all other biocompatibility methodologies since it allowed for the evaluation of viability over time in the same sample without causing cell lysis. The CNF dispersion of 6 EU mL-1 was slightly above the limits, and it did not affect the cell viability, whereas CNC hydrogels induced a reduction of metabolic activity by the fibroblasts. The chemical structure of the detected endotoxins did not contain phosphates, but it encompassed hydrophobic sulfonate groups, requiring the development of new high-pressure sterilization methods for the use of cellulose hydrogels in medicine.

Associations between bioaerosols, lung function work-shift changes and inflammatory markers: A study of recycling workers.

We investigated associations between bioaerosol exposures and work-shift changes in lung function and inflammatory markers among recycling workers. Inhalable dust was measured with personal samplers and analyzed for endotoxin, bacteria, and fungi (incubated at 25 °C and 37 °C) levels. Lung function (FEV1, FVC) was measured before and after work-shifts and serum concentrations of inflammatory markers (CRP, SAA, CC16, IL1B, IL2, IL4, IL5, IL6, IL8, IL10, IL13, and TNF) after the shift. Associations were explored by linear mixed-effects models. We included 170 measurements from 88 production workers exposed to inhalable dust, endotoxin, bacteria, and fungi (25 °C and 37 °C) at geometric mean levels of 0.6 mg/m3, 10.7 EU/m3, 1.6×104 CFU/m3, 4.4×104 CFU/m3, and 103 CFU/m3, respectively, and 14 administrative workers exposed at 7-fold lower levels. No associations were observed between bioaerosol exposures and work-shift change in lung function. IL2, IL6, IL10, and TNF concentrations were positively associated with inhalable dust levels, SAA and IL6 with bacteria, CRP, SAA, IL8, and TNF with fungi (25 °C or 37 °C), with the latter being the only statistically significant finding (exp(β) 1.40, 95% confidence interval 1.01-1.96). This study of recycling workers exposed to bioaerosol levels generally below those of farmers and compost workers and above background levels did not indicate any acute effect on lung function. Several inflammatory markers tended to increase with exposure, suggesting a systemic effect. Future research should combine data from bioaerosol-exposed workers to uncover health risks that may form the basis for health-based occupational exposure limits.

Efficacy of prophylactic antibiotics in the adjuvant treatment of alcohol-related liver disease (ALD): A systematic review and meta-analysisProphylactic antibiotics in ALD

Introduction and ObjectivesThis research aims to evaluate the efficacy and safety of prophylactic antibiotics in patients with alcohol-related liver disease (ALD). Materials and MethodsWe systematically searched databases including PubMed, Embase, Cochrane, and Web of Science up to October 2023. Our scope encompassed the influence of prophylactic antibiotics on all-cause mortality, infection, variceal bleeding, hepatic encephalopathy (HE), hepatorenal syndrome (HRS), adverse events (AE), fungal infection, clostridioides difficile infection (CDI), and multidrug-resistant (MDR) bacterial infection. Additionally, total bilirubin, creatinine, platelet counts, and plasma endotoxin levels were also analyzed. ResultsAfter comprehensive selection, 10 studies with 974 participants were included for further analysis. The study demonstrated that prophylactic antibiotic therapy was associated with reductions in infection rates, HE incidence, variceal bleeding, and all-cause mortality. The treatment did not increase the incidence of AE, fungal infection, and CDI, but it did raise the MDR bacteria infection rate. The analysis revealed no significant protective effect of antibiotic prophylaxis on total bilirubin and creatinine levels. Furthermore, the administration of antibiotics led to marginal increases in platelet counts, a minor reduction in endotoxin concentrations, and a subtle enhancement in HRS; however, these changes did not reach statistical significance. ConclusionsProphylactic antibiotic therapy was an effective and safe treatment for advanced ALD. To mitigate the risk of MDR bacterial infections, a strategy of selective intestinal decontamination could be advisable. Future investigations should prioritize varied ALD patient populations with extended follow-up periods and assorted antibiotic regimens to solidify the efficacy and safety of ALD treatments.

Switching from acetate to citrate dialysate in a central concentrate delivery system for high-volume online hemodiafiltration: a retrospective cohort study

Interest in citrate-based dialysate (Cit-D) is growing due to its benefits, including anticoagulation and dialysis efficacy. However, research on safety and efficiency of Cit-D in high-volume hemodiafiltration (HDF) via central concentrate delivery system (CCDS) is scarce. This study aimed to investigate the safety and efficacy of Cit-D when switching from acetate-based dialysate (Acet-D) in high-volume HDF via CCDS. This is a retrospective analysis of 28 patients who underwent post-dilution online HDF via CCDS, who switched from Acet-D to Cit-D. The study period was divided into 3 periods for analysis: 12 weeks using Acet-D (AD period), the first 12 weeks using Cit-D (CD-1 period), and the second 12 weeks using Cit-D (CD-2 period). We collected the laboratory, dialysis, and safety parameters in each period from electrical medical records. After switching from Acet-D to Cit-D, heparin dosage decreased by 17%, whereas the incidence of complications did not increase. Kt/VBUN and urea reduction ratio increased by 4.6% and 2.1%, respectively. Pre-dialysis beta2-microglobulin concentration decreased after using Cit-D. The corrected calcium levels decreased in the CD-1 period compared to the AD period, but in CD-2, they subsequently increased to levels similar to those observed during the AD period. Symptomatic hypocalcemia did not occur, and there was no significant difference in the incidence of hyperparathyroidism. Endotoxin levels and the bacterial culture of ultrapure dialysate were unremarkable throughout all periods. These results might suggest that Cit-D could potentially offer advantages over Acet-D, such as reducing the heparin dose and increasing dialysis efficiency, in patients undergoing high-volume HDF using CCDS.

Hesperidin Helps Improve the Intestinal Structure, Maintain Barrier Function, and Reduce Inflammation in Yellow-Feathered Broilers Exposed to High Temperatures.

To investigate the possible protective effect of hesperidin on intestinal damage caused by high-temperature heat stress in yellow-feathered broilers, 960 broilers aged 21 days were randomly divided into four groups: HT, HT300, HT450, and HT600, with each group receiving different amounts of hesperidin supplementation (0, 300, 450, and 600 mg/kg). The dietary supplementation of hesperidin could mitigate the elevation of corticosterone (CORT) and adrenocorticotropic hormone (ATCH) levels in serum from yellow-feathered broilers induced by heat stress. The supplementation of 300 mg/kg and 450 mg/kg of hesperidin reduced crypt depth and increased the V/C ratio in the small intestine compared to the HT group. The dietary supplementation of hesperidin decreased endotoxin and D-lactic acid levels in the blood, and dietary supplementation of 300 mg/kg of hesperidin increased the expression of claudin-1 and ZO-1 mRNA in the jejunum compared with the HT group. Furthermore, the dietary supplementation of 300 mg/kg of hesperidin decreased serum IL-1β and IL-6 levels. In comparison, supplementation with 300 mg/kg and 450 mg/kg of hesperidin decreased serum TNF-α levels in yellow-feathered broilers compared to the HT group. Moreover, the dietary supplementation of hesperidin decreased NF-κB mRNA levels. Overall, these data suggest that dietary supplementation with hesperidin potentially improves intestinal injury caused by heat stress in yellow-feathered broilers.

Alterations in the gut microbiota of Eimeria infected broiler chickens fed diets supplemented with varying levels of dietary calcium and phosphorus, along with 25-hydroxycholecalciferol

The purpose of the study was to investigate the effects of the reduced dietary calcium (Ca) and phosphorus (P) level and supplementation of 25-hydroxycholecalciferol (25-OHD3) on the expression of vitamin D receptor (VDR) and antimicrobial peptides and gut microbiota of broiler chickens with/without Eimeria challenge. A total of 576 fourteen-day-old broiler chicks were randomly allocated according to a 2 × 2 × 2 factorial design with main effects including Eimeria challenging (125,000 Eimeria acervulina, 25,000 Eimeria maxima, and 25,000 Eimeria tenella), dietary Ca and P levels (0.84% Ca and 0.42% available P or 0.64% Ca and 0.22% available P), and supplementation of 25-OHD3 (3,000 IU/kg) of 6 replicates. Three-way ANOVA was performed, and the effects of 3 main factors and their interactions were investigated. The reduced dietary Ca and P level downregulated cathelicidins 3 (CATH3) in the upper jejunum in the Eimeria challenging condition (interaction; P < 0.05). The reduced dietary Ca and P level decreased the relative mRNA expression of jejunal avian beta defensin 5 (AvBD5) in the Eimeria challenging condition (interaction; P < 0.05). The reduced dietary Ca and P level tended to decrease the relative mRNA expression of jejunal AvBD9 in the Eimeria challenging condition (interaction; P = 0.051). The reduced dietary Ca and P level decreased observed features (alpha diversity parameter for richness) in the upper jejunal microbiota in the Eimeria challenging condition (interaction; P < 0.05). The supplementation of 25-OHD3 decreased the relative abundance of the phylum Bacteroidetes (P < 0.05) and increased the relative abundance of the family Ruminococcaceae (P < 0.05) in the cecal digesta. The supplementation of 25-OHD3 decreased the serum endotoxin level in the Eimeria challenging condition (interaction; P < 0.05). Therefore, the reduced dietary Ca and P level modulated the upper jejunal microbiota via modulating the expression of antimicrobial peptides, and the supplementation of 25-OHD3 favorably modulated the cecal microbiota in broiler chickens with/without Eimeria challenge.

Low temperature exposure influences nitrogen metabolism resulting in decreased Cry1Ac insecticidal endotoxin content in cotton seeds

BackgroundSudden temperature drops, resulting from extreme weather events, often occur during the boll-setting period of cotton in Xinjiang, China, causing decreased expression of Bacillus thuringiensis (Bt) insecticidal proteins in cotton bolls. The precise threshold temperatures and durations that lead to significant changes in Cry1Ac endotoxin levels under low temperatures remain unclear. To address this, we investigated the effects of different temperatures and stress durations on Cry1Ac endotoxin levels in cotton bolls. In 2020–2021, two Bt transgenic cotton varieties, conventional Sikang1 and hybrid Sikang3, were selected as experimental materials. Various low temperatures (ranging from 16 to 20 °C) with different durations (12 h, 24 h and 48 h) were applied during the peak boll-setting period.ResultsAs the temperature decreased, the Cry1Ac endotoxin content in the boll shell, fiber, and seed exhibited a declining trend. Moreover, the threshold temperature which caused a significant reduction in Cry1Ac endotoxin content increased with the prolonged duration of low-temperature stress. Among the components of cotton bolls, seeds were most affected by low-temperature stress, with the threshold temperature for a significant reduction in Cry1Ac endotoxin content ranging from 17 °C to 19 °C. Correlation analysis indicated that low temperatures led to a decrease in protein synthesis capacity and an increase in degradation ability, resulting in reduced Cry1Ac endotoxin content. Pathway analysis revealed that both free amino acid and peptidase had significant negative effects on Cry1Ac endotoxin content.ConclusionIn summary, when the daily average temperature was ≤ 19 °C, implementing cultural practices to reduce free amino acid content and peptidase activity could serve as effective cold defense strategies for Bt cotton production.

A Guide to Chemical Considerations for the Pre-Clinical Development of Oligonucleotides.

Oligonucleotide therapeutics, a pioneering category of modern medicinal drugs, are at the forefront of utilizing innate mechanisms to modulate gene expression. With 18 oligonucleotide-based FDA-approved medicines currently available for treating various clinical conditions, this field showcases an innovative potential yet to be fully explored. Factors such as purity, formulation, and endotoxin levels profoundly influence the efficacy and safety of these therapeutics. Therefore, a thorough understanding of the chemical factors essential for producing high-quality oligonucleotides for preclinical studies is crucial in their development for further clinical application. This paper serves as a concise guide to these chemical considerations, aiming to inspire and equip researchers with the necessary knowledge to advance in this exciting and innovative field.

Standardized Production of Anti-Desmoglein 3 Antibody AK23 for Translational Pemphigus Vulgaris Research.

Antibody-mediated receptor activation is successfully used to develop medical treatments. If the activation induces a pathological response, such antibodies are also excellent tools for defining molecular mechanisms of target receptor malfunction and designing rescue therapies. Prominent examples are naturally occurring autoantibodies inducing the severe blistering disease pemphigus vulgaris (PV). In the great majority of patients, the antibodies bind to the adhesion receptor desmoglein 3 (Dsg3) and interfere with cell signaling to provoke severe blistering in the mucous membranes and/or skin. The identification of a comprehensive causative signaling network downstream of antibody-targeted Dsg3 receptors (e.g., shown by pharmacological activators or inhibitors) is currently being discussed as a basis to develop urgently needed first-line treatments for PV patients. Although polyclonal PV IgG antibodies have been used as proof of principle for pathological signal activation, monospecific anti-Dsg3 antibodies are necessary and have been developed to identify pathological Dsg3 receptor-mediated signal transduction. The experimental monospecific PV antibody AK23, produced from hybridoma cells, was extensively tested in our laboratory in both in vitro and in vivo models for PV and proved to recapitulate the clinicopathological features of PV when generated using the standardized production and purification protocols described herein. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Bovine IgG stripping from FBS and quality control Basic Protocol 2: AK23 hybridoma expansion and IgG production Basic Protocol 3: AK23 IgG purification Basic Protocol 4: AK23 IgG quality control Support Protocol 1: Detection of endotoxin levels Support Protocol 2: Detection and removal of mycoplasma.

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.

The Effect of Systemic Endotoxinemia on the Structure of the Brachiocephal Arteries Wall or a Potential Clinical Model for Assessing the Efficacy of Anti-Aging Measures

Integrated indicators of systemic endotoxemia (SEE): the level of endotoxin (ET) and the concentration of antibodies to the hydrophobic (AT-LPS-FOB) and hydrophilic (AT-LPS-PIL) forms of the lipopolysaccharide (LPS) molecule in the general circulation can be used to determine the risk category of atherosclerosis development and progression, which dynamics is an objective marker of aging rate. The comparison of images the structure of brachycephalic arteries wall in dynamics with changes in blood parameters is one of the most successful models for monitoring the atherosclerotic process in a clinical setting. 42 people were examined without the presence of recognized risk factors for the progression of atherosclerosis at baseline and after 1.5-2 years. It was found that lipid profile indicators, such as the concentration of total cholesterol, low and high density lipoproteins, triglycerides, atherogenic index had no prognostic value. Whereas in the group with the progression of damage to the arterial wall, an increase in the concentration of ET was noted with a decrease in the concentration of AT-LPS-FOB and AT-LPS-PIL. In the group with regression of atherosclerotic changes, on the contrary, against the background of a decrease in the concentration of ET, an increase in AT was noted. A prospective study revealed an extremely important fact of a decrease in the concentration of LPS in the blood of patients with positive dynamics of morphological manifestations of atherosclerosis, indicating both the reversibility of the atherosclerotic process and the participation of ET in atherogenesis, which is a real way for extending the life of an individual and indicates the direction to achieve this goal – “antiendotoxin therapy”.