Current Considerations and Practical Solutions for Overcoming Nanoparticle Interference with LAL Assays and Minimizing Endotoxin Contamination.

Monitoring endotoxin contamination in drugs and medical devices is required to avoid pyrogenic responses and septic shock in patients receiving these products. Endotoxin contamination of engineered nanomaterials and nanotechnology-based medical products represents a significant translational hurdle. Nanoparticles often interfere with an in vitro limulus amebocyte lysate (LAL) assay commonly used in the pharmaceutical industry for the detection and quantification of endotoxin. Such interference challenges the preclinical development of nanotechnology-formulated drugs and medical devices containing engineered nanomaterials. Protocols for the analysis of nanoparticles using LAL assays have been reported before. Here, we discuss considerations for selecting an LAL format and describe a few experimental approaches for overcoming nanoparticle interference with the LAL assays to obtain more accurate estimations of endotoxin contamination in nanotechnology-based products. The discussed approaches do not solve all types of nanoparticle interference with the LAL assays but could be used as a starting point to address the problem. This chapter also describes approaches to prevent endotoxin contamination in nanotechnology-formulated products.

A cross-sectional study on occupational exposure to microorganisms, endotoxin, hydrogen sulfide, and dust during work at drilling waste treatment plants.

This cross-sectional study aims to obtain knowledge about workers' exposure to airborne dust, bacterial and fungal species, endotoxin, biofilm formation, and hydrogen sulfide (H2S) in drilling waste treatment plants. In total, 408 full-shift personal samples, 66 work areas, 40 drilling waste, and reference (outdoor air and seawater) samples were analyzed. Some workers were exposed to high levels of endotoxin (207 EU/m3), bacteria (3.8 × 104 colony forming units (CFU)/m3 and 9.8 × 104 DNA copies/m3), or fungi (1.4 × 107 CFU/m3 and 3,600 copies/m3). The exposure levels to endotoxin, bacteria, and peaks of H2S were dependent on the treatment technique. All types of drilling waste contained large concentrations of bacteria compared to the seawater references. Elevated concentrations of airborne bacteria were found close to drilling waste basins. In total, 116, 146, and 112 different bacterial species were found in workers' exposure, work areas, and the drilling waste, respectively. An overlap in bacterial species found in the drilling waste and air (personal and work area) samples was found. Of the bacterial species found, 49 are classified as human pathogens such as Escherichia coli, Enterobacter cloacae, and Klebsiella oxytoca. In total, 44 fungal species were found in the working environment, and 6 of these are classified as human pathogens such as Aspergillus fumigatus. In conclusion, across the drilling waste treatment plants, human pathogens were present in the drilling waste, and workers' exposure was affected by the drilling waste treated at the plants with elevated exposure to endotoxin and bacteria. Elevated exposure was related to working as apprentices or chemical engineers, and working with cleaning, or slop water, and working in the daytime.

Production of pro-inflammatory mediators stimulated by exposure of poultry house workers to airborne dust particulates.

INTRODUCTION AND OBJECTIVE: Poultry house employees spend a significant part of their work shift being exposed to airborne particulate pollutants. The aim of this study was to assess their exposure at different stages of chicken production cycle, based on quantification of pro-inflammatory mediators (IL-1ß, IL-6, IL-8, and TNFα) in nasal lavage (NAL) samples. MATERIAL AND METHODS: The concentrations of airborne dust at 3 different stages of the production cycle (i.e. empty poultry house, with 7- and 42-day-old chickens) were stationary measured using Grimm spectrometer, as well as CIS and Button samplers. The dust collected by the latter 2 samplers was analyzed for endotoxin and (1→3)-ß-D-glucan content. NAL samples were collected from employees after their work shift to determine the pro-inflammatory mediator levels. RESULTS: The maximum particulate aerosol, endotoxin, and (1→3)-ß-D-glucan concentrations at workplaces reached the levels of 4.12 mg/m3, 45.21 ng/m3, and 56.54 ng/m3, respectively. The IL-1ß, IL-6, and IL-8 concentrations in NAL samples ranged between 0.62-18.12 pg/mL, <0.70-25.37 pg/mL, and <3.50-259.5 pg/mL, respectively. All TNFα levels were below 4 pg/mL. There were no significant differences between these cytokine concentrations in NAL samples collected at different stages of chicken breeding in either 'winter' or 'summer' seasons. CONCLUSIONS: Inhalation stimulation with poultry dust containing endotoxins and (1→3)-ß-D-glucans resulted in the production of pro-inflammatory mediators, which proves the course of immunological processes in the exposed employees that may lead to adverse effects. The use of nasal lavage fluid in the control of such exposure confirms that NAL analysis is a reliable laboratory tool for assessing the impact of poultry dust on exposed farm workers.

Indoor air quality in remote first nations communities in Ontario, Canada.

A recent study of the health of Indigenous children in four First Nations Communities in remote northwestern Ontario found that 21% of children had been admitted to hospital for respiratory infections before age 2 years. Here we report a detailed analysis of the housing conditions in these communities. We employed a variety of statistical methods, including linear regression, mixed models, and logistic regression, to assess the correlations between housing conditions and loadings of biocontaminants (dust mite allergens, fungal glucan, and endotoxin) and indoor concentrations of PM2.5, CO2, benzene, and formaldehyde. The houses (n = 101) were crowded with an average of approximately 7 people. Approximately 27% of the homes had sustained CO2 concentrations above 1500 ppm. Most homes had more than one smoker. Commercial tobacco smoking and the use of non-electric heating (e.g., wood, oil) were associated with increased fine particle concentrations. Over 90% of the homes lacked working Heat Recovery Ventilators (HRVs), which was associated with increased fine particle concentrations and higher CO2. Of the 101 homes, 12 had mold damage sufficient to increase the relative risk of respiratory disease. This resulted from roof leaks, through walls or around the windows due to construction defects or lack of maintenance. A similar percentage had mold resulting from condensation on windows. Endotoxin loadings were much higher than any previous study in Canada. This work provides evidence for the need for more effort to repair existing houses and to ensure the HRVs are properly installed and maintained.

Work in nursing homes and occupational exposure to endotoxin and bacterial and fungal species.

Indoor microbial exposure may cause negative health effects. Only little is known about the occupational microbial exposure in nursing homes and the factors that influence the exposure. The exposure in nursing homes may be increased due to close contact with elderly persons who may carry infectious or antimicrobial-resistant microorganisms and due to handling of laundry, such as used clothing and bed linen. We investigated the microbial exposure in 5 nursing homes in Denmark, by use of personal bioaerosol samples from different groups of staff members taken during a typical working day, stationary bioaerosol measurements taken during various work tasks, sedimented dust samples, environmental surface swabs, and swabs from staff members' hands. From the samples, we explored bacterial and fungal concentrations and species composition, endotoxin levels, and antimicrobial resistance in Aspergillus fumigatus isolates. Microbial concentrations from personal exposure samples differed among professions, and geometric means (GM) were 2,159 cfu/m3 (84 to 1.5 × 105) for bacteria incubated on nutrient agar, 1,745 cfu/m3 (82 to 2.0 × 104) for bacteria cultivated on a Staphylococcus selective agar, and 16 cfu/m3 air for potential pathogenic fungi incubated at 37 °C (below detection limit to 257). Bacterial exposures were elevated during bed making. On surfaces, the highest bacterial concentrations were found on bed railings. The majority of bacterial species found were related to the human skin microflora, such as different Staphylococcus and Corynebacterium species. Endotoxin levels ranged from 0.02 to 59.0 EU/m3, with a GM of 1.5 EU/m3. Of 40 tested A. fumigatus isolates, we found one multiresistant isolate, which was resistant towards both itraconazole and voriconazole, and one isolate resistant towards amphotericin B. In conclusion, we give an overview of the general microbial exposure in nursing homes and show that microbial exposures are higher for staff with more care and nursing tasks compared with administrative staff.

Panorama da qualidade biológica da água destinada ao tratamento dialítico em pacientes sob cuidados críticos em Unidades de Terapia Intensiva / Overview of the biological quality of water intended for dialysis treatment in patients under critical care in Intensive Care Units

A incidência da lesão renal aguda caracteriza-se como evento frequente em pacientes críticos internados em Unidades de Terapia Intensiva e está associada ao aumento de mortalidade, causando grande impacto à Saúde Pública. As intercorrências clínicas são minimizadas com intervenções dialíticas, acarretando a exposição do paciente a volumes expressivos de água tratada durante a terapia renal em leito. As análises microbiológicas e de determinação de endotoxinas bacterianas em amostras de água tratada e em soluções de dialisato foram executadas em dois hospitais públicos do município de São Paulo, seguindo metodologias analíticas preconizadas em compêndios oficiais. A avaliação demonstrou que a porcentagem de resultados satisfatórios no período de 2010 a 2022 variou entre 35,2 a 100% e de 40 a 100% para as unidades hospitalares I e II para a água tratada, respectivamente; e, 100% para as soluções de dialisato para a unidade hospitalar I. A eficácia de ações delineadas pelas equipes técnicas das unidades hospitalares, na adequação da água destinada à terapia dialítica, aponta para a importância em estimular outras instituições hospitalares na padronização e implantação de melhoria contínua de seus sistemas de tratamento de água para uso em procedimento dialítico, prevenindo riscos adicionais aos pacientes expostos à terapia renal.

The incidence of acute kidney is high among critically ill patients admitted to Intensive Care Units and is associated with increased mortality, having a major impact on public health. Clinical complications are minimized with dialysis interventions, which expose patients to significant volumes of treated water during in-bed renal therapy. Microbiological analyzes and determination of bacterial endotoxins were performed on treated water samples and dialysate solutions in two public hospitals in São Paulo city, using analytical methodologies recommended in official compendia. The evaluation showed that the percentage of satisfactory results for treated water ranged from 35.2% to 100% in Hospital Unit I and from 40% to 100% in Hospital Unit II between 2010 and 2022. For dialysate solutions in Hospital Unit I, the percentage of satisfactory results was 100% during the same period. The effectiveness of actions implemented by the technical hospital teams, in adapting water for dialysis therapy, points to the importance of encouraging other hospital institutions to standardize and implement a program of continuous improvement for their water treatment systems used in dialysis procedures. This will help to prevent additional risks to patients exposed to renal therapy.

An algorithm for quantitatively estimating occupational endotoxin exposure in the biomarkers of exposure and effect in agriculture study: II. Application to the study population.

BACKGROUND: We developed an algorithm to quantitatively estimate endotoxin exposure for farmers in the Biomarkers of Exposure and Effect in Agriculture (BEEA) Study. METHODS: The algorithm combined task intensity estimates derived from published data with questionnaire responses on activity duration to estimate task-specific cumulative endotoxin exposures for 13 tasks during four time windows, ranging from "past 12 months" to "yesterday/today." We applied the algorithm to 1681 participants in Iowa and North Carolina. We examined correlations in endotoxin metrics within- and between-task. We also compared these metrics to prior day full-shift inhalable endotoxin concentrations from 32 farmers. RESULTS: The highest median task-specific cumulative exposures were observed for swine confinement, poultry confinement, and grind feed. Inter-quartile ranges showed substantial between-subject variability for most tasks. Time window-specific metrics of the same task were moderately-highly correlated. Between-task correlation was variable, with moderately-high correlations observed for similar tasks (e.g., between animal-related tasks). Prior day endotoxin concentration increased with the total metric and with task metrics for swine confinement, clean other animal facilities, and clean grain bins. SIGNIFICANCE: This study provides insight into the variability and sources of endotoxin exposure among farmers in the BEEA study and summarizes exposure estimates for future investigations in this population.

An algorithm for quantitatively estimating occupational endotoxin exposure in the Biomarkers of Exposure and Effect in Agriculture (BEEA) study: I. Development of task-specific exposure levels from published data.

BACKGROUND/OBJECTIVE: Farmers conduct numerous tasks with potential for endotoxin exposure. As a first step to characterize endotoxin exposure for farmers in the Biomarkers of Exposure and Effect in Agriculture (BEEA) Study, we used published data to estimate task-specific endotoxin concentrations. METHODS: We extracted published data on task-specific, personal, inhalable endotoxin concentrations for agricultural tasks queried in the study questionnaire. The data, usually abstracted as summary measures, were evaluated using meta-regression models that weighted each geometric mean (GM, natural-log transformed) by the inverse of its within-study variance to obtain task-specific predicted GMs. RESULTS: We extracted 90 endotoxin summary statistics from 26 studies for 9 animal-related tasks, 30 summary statistics from 6 studies for 3 crop-related tasks, and 10 summary statistics from 5 studies for 4 stored grain-related tasks. Work in poultry and swine confinement facilities, grinding feed, veterinarian services, and cleaning grain bins had predicted GMs > 1000 EU/m3 . In contrast, harvesting or hauling grain and other crop-related tasks had predicted GMs below 100 EU/m3 . SIGNIFICANCE: These task-specific endotoxin GMs demonstrated exposure variability across common agricultural tasks. These estimates will be used in conjunction with questionnaire responses on task duration to quantitatively estimate endotoxin exposure for study participants, described in a companion paper.

Ophthalmic viscoelastics commonly used in cataract surgery: A microbiota investigation.

PURPOSE: To survey commonly used, sterile ophthalmic viscoelastic materials used during routine cataract surgery for the presence of bacterial DNA and/or viable bacteria and endotoxin quantification. METHODS: Samples from three different ophthalmic viscoelastic manufacturers and three different production lots per manufacturer were collected for 16 S ribosomal ribonucleic acid (rRNA) sequencing and conventional aerobic and capnophilic bacterial culture. Other samples of viscoelastic material from the same three manufacturers were collected for endotoxin quantification using a commercially available Limulus amebocyte lysate (LAL) assay. Statistical analysis was performed using Sigma Plot 14.0, and R v4.0.2.0. Differences (p ≤ .05) between sample collection sites in total DNA concentration, microbial richness, mean intra-group distances, and endotoxin quantification alongside reagent controls were evaluated. RESULTS: Culture yielded two isolates, identified as Staphylococcus epidermidis and Bacillus megaterium. 16 S rRNA sequencing revealed no differences between brands in richness or overall composition. The most common bacterial DNA detected across all brands was Staphylococcus sp., Cutibacterium sp., Flavobacterium sp., and Lactobacillus sp. A significant difference was found between the median endotoxin concentration between Anvision and Hyvisc® viscoelastic (Anvision: 0.171 EU/mL, Hyvisc®: 0.03 EU/mL; p < .001). CONCLUSIONS: No brand-specific differences in bacterial DNA were detected in the viscoelastic materials. Staphylococcus, Cutibacterium, Flavobacterium, and Lactobacillus were the dominant contributors to the bacterial DNA detected. Although Anvision viscoelastic samples contained significantly more endotoxin than Hyvisc® viscoelastic samples, endotoxin concentrations were below the FDA limit of 0.2 EU/mL for both manufacturers. These data further the understanding of inflammatory outcomes following cataract surgery.

Protocol to measure endotoxin from opaque tissues in mice using an optimized kinetic limulus amebocyte lysate assay.

Endotoxin accumulation has been widely noted in several pathologies ranging from metabolic dysregulation to bacterial infection. Using limulus amebocyte lysate (LAL) assays to detect endotoxin load has been the only reliable way to assess endotoxin accumulation, but assays optimized for detection in opaque tissues are still lacking. We optimized a sensitive Kinetic LAL assay for endotoxin detection from murine tissues. In this protocol, we describe tissue collection and homogenization, followed by the procedure to run the assay and data analysis. For complete details on the use and execution of this protocol, please refer to Ceasrine et al. (2022).

A simple scheme for large scale purification of urine - Derived Bence Jones Kappa protein.

We have investigated and optimized purification process, suitable for industrial scale, to obtain high purity grade Bence Jones Kappa Protein ('BJK-Protein'), while preserving its physiological properties and functions. BJK-Protein was obtained from a biological waste product i.e. human urine of renal failure patients. Isolated 'BJK-Protein' was analyzed by electrophoresis, western blotting, double immunodiffusion, SEC-HPLC assay and Mass Spectrometry (MS). The relative molecular mass of 'BJK-Protein' is 23054.2 Da. Moreover, dimer forms of 'BJK-Protein' were also detected in SDS-PAGE and mass spectrum corresponding to 46054.4 Da. The results of western blotting, immunoelectrophoresis, SEC-HPLC assay, and mass spectrometry analysis indicate a high purity (>99 %) of 'BJK-Protein'. Peptide mass fingerprint analysis of 'BJK-Protein' yielded peptides that partially matches the known database sequences of kappa variable region (KV139_HUMAN) of immunoglobulin. This protein was found to be stable up to 20 months at 2-8 °C temperature and also found negative for major undesirable viral markers as well as bacterial endotoxin. With this purification approach, the cost of purified 'BJK-Protein' is significantly reduced as compared to the current market price of Kappa light chain available in international market.

Gene Expression Changes Induced by Exposure of RAW 264.7 Macrophages to Particulate Matter of Air Pollution: The Role of Endotoxins.

Despite the variable chemical and physical characteristics of particulate air pollutants, inflammation and oxidative stress have been identified as common mechanisms for cell damage and negative health influences. These effects are produced by organic components, especially by endotoxins. This study analyzed the gene expression profile after exposure of RAW 264.7 cells to the standard particulate matter (PM) material, NIST1648a, and PM with a reduced organic matter content, LAp120, in comparison to the effects of lipopolysaccharide (LPS). The selected parameters of cell viability, cell cycle progression, and metabolic and inflammatory activity were also investigated. Both forms of PM negatively influenced the parameters of cell activity. These results were generally reflected in the gene expression profile. Only NIST1648a, excluding LAp120, contained endotoxins and showed small but statistically significant pro-inflammatory activity. However, the gene expression profiling revealed strong pro-inflammatory cell activation induced by NIST1648a that was close to the effects of LPS. Changes in gene expression triggered by LAp120 were relatively small. The observed differences in the effects of NIST1648a and LAp120 were related to the content of organic matter in which bacterial endotoxins play an important role. However, other organic compounds and their interactions with other PM components also appear to be of significant importance.

Occupational Exposure to ß-d-Glucans, Mould Allergens, Endotoxins and Cultivable Fungi in Pig Farms.

Airborne concentrations of organic dust on animal farms are known to be very high. This dust is partly composed of microorganisms such as bacteria, fungi and their components [endotoxins, (1→3)-ß-d-glucans, mould allergens, mycotoxins], recognised as being responsible for numerous health effects. Several cross-sectional studies have measured levels of airborne bacteria, fungi and endotoxins on pig farms. However, the temporal dynamics of organic dust's components throughout the year have rarely been assessed, and airborne concentrations of (1→3)-ß-d-glucans and mould allergens remain poorly understood in these work environments. This longitudinal, four-season study measured cultivable fungi, endotoxins, (1→3)-ß-d-glucans, Aspergillus versicolor (AveX), Aspergillus fumigatus (Asp f1) and Alternaria sp (Alt a1) allergens on 31 pig farms in Switzerland. Results showed that exposure to AveX occurred in all four seasons. Total mean airborne concentration of endotoxins were between 3 and 4 times higher than the Swiss recommended limit value of 1000 EU m-3 and mean airborne concentrations of fungi were between 30 and 50 times higher than the Swiss recommended limit value of 1000 cfu m-3. Finally, accumulations of faecal matter on floors, humidity and dusty pathways were associated with increased concentrations of (1→3)-ß-d-glucans. In conclusion, pig farmers require better information about biological occupational risks, and measures to improve air quality should be implemented, especially in winter.

Microbial contamination and metabolite exposure assessment during waste and recyclable material collection.

Waste workers are exposed to bioaerosols when handling, lifting and dumping garbage. Bioaerosol exposure has been linked to health problems such as asthma, airway irritant symptoms, infectious, gastrointestinal and skin diseases, and cancer. Our objective was to characterize the exposure of urban collectors and drivers to inhalable bioaerosols and to measured the cytotoxic effect of air samples in order to evaluate their health risk. Personal and ambient air sampling were conducted during the summer of 2019. Workers from 12 waste trucks collecting recyclables, organic waste or compost were evaluated. Bacteria and fungi were cultured, molecular biology methods were used to detect microbial indicators, cytotoxic assays were performed and endotoxins and mycotoxins were quantified. Domestic waste collectors were exposed to concentrations of bacteria and endotoxins above the recommended limits, and Aspergillus section Fumigati was detected at critical concentrations in their breathing zones. Cytotoxic effects were observed in many samples, demonstrating the potential health risk for these workers. This study establishes evidence that waste workers are exposed to microbial health risks during collection. It also demonstrates the relevance of cytotoxic assays in documenting the general toxic risk found in air samples. Our results also suggest that exposures differ depending on the type of waste, job title and discharge/unloading locations.

Toxic effects of the combined cadmium and Cry1Ab protein exposure on the protective and transcriptomic responses of Pirata subpiraticus.

Cadmium (Cd) pollution poses a serious threat to agricultural production and paddy field fauna. Crystalline proteins (e.g., Cry1Ab and Cry1Ac) are secreted by Bacillus thuringiensis, which can manage pests via a complicated toxic mechanism and have been widely used for pest control due to the commercialization of transgenic crops (e.g., cotton and rice) that expresses Bt insecticidal proteins. Nonetheless, studies on the effects of combined stress of Cd and Cry1Ab protein on field indicator species are limited. In the present study, we showed that spiders, Pirata subpiraticus, fed with Cd-containing flies+Cry1Ab had dramatically higher Cd accumulation than that in the spiders fed with Cd-containing flies (p < 0.05). In addition, the enrichment of Cd led to the activation of the protective mechanism by elevating the concentrations of glutathione peroxidase, glutathione S-transferase, and metallothionein in the spiders (p < 0.05). An in-depth transcriptome analysis revealed that the activities of ion metal binding proteins, transporters, and channels might play essential roles in the Cd accumulation process. More importantly, the higher Cd concentration in the combined Cd+Cry1Ab exposure prolonged developmental duration of P. subpiraticus, due to the down-regulated cuticle proteins (CPs) encoding genes involved in the molting process, which was regulated by a series of putative transcriptional factors such as ZBTB and zf-C2H2. Collectively, this integrated analysis illustrates that the combined Cd+Cry1Ab exposure increases the adverse effects of Cd stress on the growth, antioxidase, and CPs encoding genes of P. subpiraticus, thus providing a research basis and prospect for the rationality of transgenic Cry1Ab crops in the cultivation of heavy metal contaminated soil.

An in vitro assessment of the toxicity of two-dimensional synthetic and natural layered silicates.

Natural Layered Silicates (NLS) and Synthetic Layered Silicates (SLS) are a diverse group of clay minerals that have attracted great interest in various branches of industry. However, despite growing demand for this class of material, their impact on human health has not been fully investigated. Therefore, the aim of this study was to evaluate and compare the potential toxic effects of a wide range of commercially available SLS and NLS of varying physicochemical properties (lithium (Li) or fluoride (F) content and size). Mouse BALB/c monocyte macrophage (J774A.1) and human monocyte-derived macrophages (MDMs) were chosen as in vitro models of alveolar macrophages. Montmorillonite, hectorite, Medium (med) F/High Li and Low F/Med Li particles, were cytotoxic to cells and induced potent pro-inflammatory responses. The remaining particles (No F/Very (V)Low Li, No F/Med Li, No F/Low Li, High F/Med Li and High F/Med Li washed) were non- to relatively low- cytotoxic and inflammogenic, in both type of cells. In an acellular condition none of the tested samples increased reactive oxygen species (ROS), while ROS generation was observed following exposure to sublethal concentrations of Med F/High Li, Low F/Med Li, montmorillonite and hectorite samples, in J774A.1 cells. Based on the results obtained in this study the toxic potency of tested samples was not associated with lithium or fluoride content, but appeared to be dependent on particle size, with the platelets of larger dimension and lower surface area being more potent than the smaller platelet particles with higher surface area. In addition, the increased bioactivity of Med F/High Li and Low F/Med Li was associated with endotoxin contamination. Obtained results demonstrated that layered silicate materials have different toxicological profiles and suggest that toxicological properties of a specific layered silicate should be investigated on an individual basis.

Quantitation of endotoxin by gas chromatography-mass spectrometry in Neisseria meningitidis serogroups A, C, W, Y and X during polysaccharide purification used in conjugate vaccine.

Bacterial lipopolysaccharide (LPS) responsible for endotoxin effect induces inflammatory reactions. The endotoxins are difficult to separate from the gram-negative polysaccharide (PS) during polysaccharide purification. The most common method to quantify LPS is the limulus amebocyte lysate (LAL) test which interferes with the agents used during PS purification. The gas chromatography-mass spectrometry (GC-MS) provides a suitable alternative by estimating lipid-A chain anchored 3-hydroxy fatty acid methyl ester (FAME) to estimate LPS however, there are no reports of its application in natural polysaccharides used for vaccine preparation. The transesterification of LPS and meningococcal PS yielded primary target 3-O-acetylated myristic acid which was detected by GC-MS and provided quantitative estimation of endotoxin. The GC-MS method was found in agreement with the LAL values showing lower endotoxin content< 10Eu/µg in meningococcal C and Y serogroup polysaccharides in comparison to higher endotoxin 177-523 Eu/µg in meningococcal A, W and X serogroups. The high endotoxin content in purified polysaccharide was attributed to it being detected in its intermediate stage by GC-MS unlike the LAL test. Thus GC-MS serves as a valuable method for endotoxin monitoring and quantitation in gram-negative meningococcal intermediate and purified PS during vaccine preparation.

Effects of systemic inflammation due to hepatic ischemia-reperfusion injury upon lean or obese visceral adipose tissue

Purpose: To evaluate how the induction of liver damage by ischemia and reperfusion affects the adipose tissue of lean and obese mice. Methods: Lean and diet-induced obese mice were subjected to liver ischemia (30 min) followed by 6 h of reperfusion. The vascular stromal fraction of visceral adipose tissue was analyzed by cytometry, and gene expression was evaluated by an Array assay and by RT-qPCR. Intestinal permeability was assessed by oral administration of fluorescein isothiocyanate (FITC)-dextran and endotoxemia by serum endotoxin measurements using a limulus amebocyte lysate assay. Results: It was found that, after liver ischemia and reperfusion, there is an infiltration of neutrophils, monocytes, and lymphocytes, as well as an increase in the gene expression that encode cytokines, chemokines and their receptors in the visceral adipose tissue of lean mice. This inflammatory response was associated with the presence of endotoxemia in lean mice. However, these changes were not observed in the visceral adipose tissue of obese mice. Conclusions: Liver ischemia and reperfusion induce an acute inflammatory response in adipose tissue of lean mice characterized by an intense chemokine induction and leukocyte infiltration; however, inflammatory alterations are already present at baseline in the obese adipose tissue and liver ischemia and reperfusion do not injure further.

Parasporin A13-2 of Bacillus thuringiensis Isolates from the Papaloapan Region (Mexico) Induce a Cytotoxic Effect by Late Apoptosis against Breast Cancer Cells.

The protein A13-2 was obtained from Bacillus thuringiensis strains isolated from the Papaloapan watershed region (Oaxaca, Mexico). The cytotoxic activity of parasporal inclusions was studied against breast cancer cell line (MCF-7) and normal cell (human peripheral blood mononuclear cells). The MTT, the formation of reactive species, nitric oxide, free cell DNA, and the type of death cellular were assessed. The protein A13-2 shows the highest cytotoxic activity against MCF-7 (13% cell viability at 6 µg/mL), the extracellular DNA increases, and it shows no stress for reactive species or nitric oxide. Besides, the A13-2 parasporin shows no toxicity to peripheral blood mononuclear cells, and it does not generate changes in nitric oxide levels or free cell DNA. Due to that, the cytotoxic effect of A13-2 was specific for MCF-7, and it does not affect normal cells. According to microscopy and flow cytometry, A13-2 parasporin leads to the death of MCF-7 cells by late apoptosis together with necrosis and without allowing the triggering of the survival mechanisms. When analyzed together, our results show for the first time that the A13-2 protein isolated from Mexican strains of B. thuringiensis preferentially kills MCF- 7 (cancer cells) over HEK 293 and PBMC cell lines (normal cells), thus representing a promising alternative for the treatment of cancer breast.