Bacterial contamination of coffee and personal exposure to inhalable dust and endotoxin in primary coffee processing factories in Ethiopia.

INTRODUCTION AND OBJECTIVE: Endotoxins from gram-negative bacteria might be released when the coffee cherries are processed and may cause respiratory health problems among workers in the coffee industry. The relationship between bacterial contamination and occupational exposure to endotoxin levels has not been thoroughly explored previously in primary coffee processing factories in Ethiopia, or elsewhere. The aim of this study was to characterize the level of personal endotoxin exposure and its relations with bacterial contamination of coffee cherries in such factories in Ethiopia. MATERIAL AND METHODS: A cross-sectional study was conducted from March 2020 - February 2021 in 9 primary coffee processing factories in 3 regions in Ethiopia. A total of 180 personal air samples were collected to analyze workers' exposure to inhalable dust and endotoxin. Correlation tests were performed to assess the relationship between total bacteria and endotoxin levels and between inhalable dust and endotoxin levels. RESULTS: The geometric mean (GM) of personal inhalable dust exposure among machine room workers and hand pickers were 9.58 mg/m3 and 2.56 mg/m3, respectively. The overall GM of endotoxin exposure among machine room workers and hand pickers were 10,198 EU/m3 and 780 EU/m3, respectively. Gram-negative bacteria were found in all 54 coffee samples. The correlation between inhalable dust and endotoxin exposure was significant (r=0.80; P <0.01). CONCLUSIONS: About 92% of the samples from hand pickers and all samples from machine room workers exceeded the occupational exposure limit of 90 EU/m3 recommended by the Dutch Expert Committee on Occupational Standards. Prevention and control of bacterial contamination of the coffee in primary coffee processing are suggested to reduce endotoxin exposure that might cause respiratory health problems among coffee workers.

Endotoxin Contamination and Reaction Interfering Substances in the Plant Extract Library.

Endotoxin is an unintentional contaminant that has numerous activities and can affect various biological experiments using cells. In this study, we measured the endotoxin activity of samples from a plant extract library (PEL) and determined their degrees of contamination. Endotoxin was detected in approx. 48% (n = 139) and approx. 4% (n = 5) of field-collected and crude drug samples, respectively, and in concentrations >5.0 EU/mL in some samples. The concentrations of endotoxin that affect cells in vitro vary depending on the target cell type. Although the degree of contamination varied in the present study, it was considered to have little effect on the cell experiments. More than 150 PEL samples had problems with reaction courses or recovery rates of Limulus amoebocyte lysate (LAL) tests. In the LAL tests, using three plant extracts [Sanguisorba officinalis L. (Rosaceae), Oenothera biennis L. (Onagraceae), and Lythrum salicaria L. (Lythraceae)], the polyphenolic compounds in the plant extracts affected LAL test and their effects differed depending on the plant species. When the 16 single polyphenol compounds were added to the LAL tests, the compounds with caffeoyl and pyrogallol moieties were found to affect the LAL reaction course and recovery rate. Furthermore, none of the compounds had any effects at concentrations of 1 µM. Because the plant extracts contained analogs of various polyphenolic compounds, they were presumed to actually act synergistically. Our findings demonstrated that attention must be paid to the recovery rate and reaction process of LAL tests with samples containing polyphenolic compounds.

Occupational exposure during treatment of offshore drilling waste and characterization of microbiological diversity.

The exposure for workers handling and recycling offshore drilling waste are previously not described, and given the potential for exposure to hazardous components, there is a need for characterizing this occupational exposure. In this study five plants recycling offshore drilling waste with different techniques were included. Measurements were conducted in both winter and summer to include seasonal exposure variations. Altogether >200 personal air-exposure measurements for oil mist, oil vapor, volatile organic compounds (VOC), hydrogen sulfide (H2S) and solvents were carried out respectively. Microorganisms related to drilling waste were identified in bulk samples and in stationary air measurements from two of the plants. The exposure to oil mist and oil vapor were below 10% of the current Norwegian occupational exposure limits (OEL) for all measured components. The plants using the Resoil or TCC method had a statistically significant higher exposure to oil vapor than the plant using complete combustion (p-value <0.05). No statistically significant difference was found between the different treatment methods for oil mist. The exposure to solvents was generally low (additive factor < 0.03). Endotoxin measurements done during winter showed a median concentration of 5.4 endotoxin units (EU)/m3. Levels of H2S above the odor threshold of 0.1 ppm were measured at four plants. Both drill mud and slop water contained a high number and diversity of bacteria (2-4 × 104 colony forming unit (CFU)/mL), where a large fraction was Gram-negative species. Some of the identified microorganisms are classified as potentially infectious pathogens for humans and thus might be a hazard to workers.

Identification of Sources of Endotoxin Exposure as Input for Effective Exposure Control Strategies.

Objective: Aim of the present study is to investigate the levels of endotoxins on product samples from potatoes, onions, and seeds, representing a relevant part of the agro-food industry in the Netherlands, to gather valuable insights in possibilities for exposure control measures early in the process of industrial processing of these products. Methods: Endotoxin levels on 330 products samples from companies representing the potato, onion, and seed (processing) industry (four potato-packaging companies, five potato-processing companies, five onion-packaging companies, and four seed-processing companies) were assessed using the Limulus Amboecyte Lysate (LAL) assay. As variation in growth conditions (type of soil, growth type) and product characteristics (surface roughness, dustiness, size, species) are assumed to influence the level of endotoxin on products, different types, and growth conditions were considered when collecting the samples. Additionally, waste material, rotten products, felt material (used for drying), and process water were collected. Results: A large variation in the endotoxin levels was found on samples of potatoes, onions, and seeds (overall geometric standard deviation 17), in the range between 0.7 EU g-1 to 16400000 EU g-1. The highest geometric mean endotoxin levels were found in plant material (319600 EU g-1), followed by soil material (49100 EU g-1) and the outer side of products (9300 EU g-1), indicating that removal of plant and soil material early in the process would be an effective exposure control strategy. The high levels of endotoxins found in the limited number of samples from rotten onions indicate that these rotten onions should also be removed early in the process. Mean endotoxin levels found in waste material (only available for seed processing) is similar to the level found in soil material, although the range is much larger. On uncleaned seeds, higher endotoxin levels were found than on cleaned seeds, indicating that cleaning processes are important control measures and also that the waste material should be handled with care. Conclusions: Although endotoxin levels in batches of to-be-processed potatoes, onions, and seeds vary quite dramatically, it could be concluded that rotten products, plant material, and waste material contain particularly high endotoxin levels. This information was used to propose control measures to reduce exposure to endotoxins of workers during the production process.

Bitrophic and Tritrophic Effects of Transgenic cry1Ab/cry2Aj Maize on the Beneficial, Nontarget Harmonia axyridis (Coleoptera: Coccinellidae).

Harmonia axyridis (Pallas) is a common and abundant predator in China and may be exposed to Cry toxins that are produced in Bt crops either by feeding on plant parts or by feeding on target or nontarget herbivorous insects. A new Bt maize line, expressing the Cry1Ab/Cry2Aj fused protein, has been developed and should be rigorously assessed for the ecological risks on the natural enemy. Laboratory experiments were carried out to study the effects of this Bt maize on nontarget predator H. axyridis via bitrophic interaction of adult H. axyridis feeding on Bt maize pollen and tritrophic interaction of H. axyridis consuming the lepidopteran prey. Spodoptera exigua (Hübner) neonate larvae were used to transfer Bt protein because they could survive after ingesting transgenic cry1Ab/cry2Aj maize kernels in the previous study. ELISA bioassays confirmed that the Bt protein could be transferred, but diluted through Bt maize-prey-predator. Life history parameters such as survival, development, weight, fecundity, and egg hatching rate were not significantly different when H. axyridis consumed prey that had been reared on Bt maize compared with prey reared on a nontransformed parental control. Furthermore, feeding directly on Bt maize pollen also had no detrimental effects on fitness, survival, and weight of female and male adults. In conclusion, our results indicate that transgenic cry1Ab/cry2Aj maize poses no ecological risks on the nontarget predator H. axyridis.

Preventing Gut Leakiness and Endotoxemia Contributes to the Protective Effect of Zinc on Alcohol-Induced Steatohepatitis in Rats.

BACKGROUND: Zinc deficiency has been well documented in alcoholic liver disease. OBJECTIVE: This study was undertaken to determine whether dietary zinc supplementation provides beneficial effects in treating alcohol-induced gut leakiness and endotoxemia. METHODS: Male Sprague Dawley rats were divided into 3 groups and pair-fed (PF) Lieber-DeCarli liquid diet for 8 wk: 1) control (PF); 2) alcohol-fed (AF; 5.00-5.42% wt:vol ethanol); and 3) AF with zinc supplementation (AF/Zn) at 220 ppm zinc sulfate heptahydrate. The PF and AF/Zn groups were pair-fed with the AF group. Hepatic inflammation and endotoxin signaling were determined by immunofluorescence and quantitative polymerase chain reaction (qPCR). Alterations in intestinal tight junctions and aldehyde dehydrogenases were assessed by qPCR and Western blot analysis. RESULTS: The AF rats had greater macrophage activation and cytokine production (P < 0.05) in the liver compared with the PF rats, whereas the AF/Zn rats showed no significant differences (P > 0.05). Plasma endotoxin concentrations of the AF rats were 136% greater than those of the PF rats, whereas the AF/Zn rats did not differ from the PF rats. Ileal permeability was 255% greater in the AF rats and 19% greater in the AF/Zn rats than in the PF rats. The AF group had reduced intestinal claudin-1, occludin, and zona occludens-1 (ZO-1) expression, and the AF/Zn group had upregulated claudin-1 and ZO-1 expression (P < 0.05) compared with the PF group. The intestinal epithelial expression and activity of aldehyde dehydrogenases were elevated (P < 0.05) in the AF/Zn rats compared with those of the AF rats. Furthermore, the ileal expression and function of hepatocyte nuclear factor 4α, which was impaired in the AF group, was significantly elevated in the AF/Zn group compared with the PF group. CONCLUSIONS: The results demonstrate that attenuating hepatic endotoxin signaling by preserving the intestinal barrier contributes to the protective effect of zinc on alcohol-induced steatohepatitis in rats.

Accuracy of Turbidimetric Limulus Amebocyte Lysate Assay for the Recovery of Endotoxin Interacted with Commonly Used Antimicrobial Agents of Endodontic Therapy.

INTRODUCTION: This study was conducted to investigate whether the interaction between the turbidimetric limulus amebocyte lysate (LAL) substrate for endotoxin measurement and the substances/antimicrobial agents used in endodontic therapy can lead to the inhibition/enhancement of endotoxin recovery. METHODS: Ten microliters of a suspension of Escherichia coli endotoxin (O55:B55) was inoculated and kept in contact for 1 hour with different substances categorized as follows: group 1: auxiliary chemical substances: 5.25% and 2.5% sodium hypochlorite solutions, 2% chlorhexidine (CHX) (gel and solution), 1% Natrosol gel (Drogal Chemicals and Pharmaceuticals Ltd, Piracicaba, SP, Brazil), 17% EDTA, 10% citric acid, 3% hydrogen peroxide, 5% sodium thiosulfate, and 0.5% Tween 80 associated with 0.07% soy lecithin (Drogal Chemicals and Pharmaceuticals Ltd) and group 2: intracanal medications: neomycin/polymyxin B/hydrocortisone (Otosporin; Glaxo Wellcome, Rio de Janeiro, RJ, Brazil); calcium hydroxide (Ca[OH]2); Ca(OH)2 + 2% CHX gel; Ca(OH)2 + 2% CHX gel + zinc oxide eugenol; Ca(OH)2 + camphorated paramonochlorophenol (Calen; S.S. White, Rio de Janeiro, RJ, Brazil); triple antibiotic paste; mineral trioxide aggregate (MTA); and iodoform. Positive and negative controls consisted of root canal hemorrhagic exudate and pyrogen-free sterile water, respectively. All samples were diluted up to a 10:4 dilution. Each dilution was individually examined by the turbidimetric LAL assay. Collected data were analyzed through performance characteristics of the LAL assay such as linearity, coefficient of variation percentage, and product positive control (PPC) values. RESULTS: Correlation coefficient (≥0.980) and coefficient of variation percentage (<10%) of the standard curve in triplicate showed the tests' linearity. Spike recovery of auxiliary chemical substances achieved PPC values ranging from 50%-197%, showing no interferences with LAL substrate. Conversely, 3% hydrogen peroxide achieved product inhibition in which endotoxin values were underestimated even after the 10:4 dilutions. Regarding intracanal medicaments, neomycin/polymyxin B/hydrocortisone also inhibited endotoxin detection in all dilutions investigated (PPC values <50%). In contrast, Ca(OH)2 + 2% CHX gel + ZOE as well as triple antibiotic paste led to the enhancement of endotoxin detection in which endotoxin values could not be validated by the turbidimetric kinetic LAL assay (PPC value >200%). CONCLUSIONS: The performance characteristics of the kinetic turbidimetric assay for endotoxin measurement, such as precision and reproducibility, are modulated by the interaction of the LAL substrate with the substances/antimicrobial agents used in endodontic therapy.

Evaluation of imipenem for prophylaxis and therapy of Yersinia pestis delivered by aerosol in a mouse model of pneumonic plague.

It has been previously shown that mice subjected to an aerosol exposure to Yersinia pestis and treated with ß-lactam antibiotics after a delay of 42 h died at an accelerated rate compared to controls. It was hypothesized that endotoxin release in antibiotic-treated mice accounted for the accelerated death rate in the mice exposed to aerosol Y. pestis. Imipenem, a ß-lactam antibiotic, binds to penicillin binding protein 2 with the highest affinity and produces rounded cells. The binding of imipenem causes cells to lyse quickly and thereby to release less free endotoxin. Two imipenem regimens producing fractions of time that the concentration of free, unbound drug was above the MIC (fT>MIC) of approximately 25% (6/24 h) and 40% (9.5/24 h) were evaluated. In the postexposure prophylaxis study, the 40% and 25% regimens produced 90% and 40% survivorship, respectively. In the 42-h treatment study, both regimens demonstrated a 40 to 50% survivorship at therapy cessation and some deaths thereafter, resulting in a 30% survivorship. As this was an improvement over the results with other ß-lactams, a comparison of both endotoxin and cytokine levels in mice treated with imipenem and ceftazidime (a ß-lactam previously demonstrated to accelerate death in mice during treatment) was performed and supported the original hypotheses; however, the levels observed in animals treated with ciprofloxacin (included as an unrelated antibiotic that is also bactericidal but should cause little lysis due to a different mode of action) were elevated and significantly (7-fold) higher than those with ceftazidime.

Daily changes of peak expiratory flow and respiratory symptom occurrence around a soy processing factory.

OBJECTIVES: To evaluate sensitization and acute respiratory health effects in inhabitants living in the vicinity of a factory producing soy oil. METHODS: Two panels of potential responders were created on the basis of a response to a short screening questionnaire sent to random samples of 1,000 exposed and 1,000 non-exposed individuals living around the factory and a control area. Individuals responding to the questionnaire were invited for a medical evaluation, including a respiratory symptom questionnaire and skin prick testing, for a panel of common allergens and a soy allergen extract. This resulted in 53 atopic and/or asthmatic inhabitants from the area surrounding the factory and 30 comparable control subjects. In these subjects, morning and evening Peak Expiratory Flow (PEF), respiratory symptoms and medication use were recorded daily during a 10-week period in the autumn. At the same time, soy allergen and endotoxin concentrations were determined in airborne dust in the exposed and the control area. The wind direction relative to the location of a subjects' house and the factory was used to determine whether an individual was exposed on a particular day. RESULTS: Only few of the atopic subjects were sensitized to soy. PEF showed a decrease, respiratory symptoms and bronchodilator use, an increase among soy sensitized subjects after having been downwind from the factory. Airborne soy allergen was found more frequently in the area surrounding the factory and levels were higher than in the control area. Highest levels were found on the factory premises. Only a weak association was found with wind direction. Airborne endotoxin concentrations did not show a consistent pattern with distance, but levels were clearly higher on the factory premises. CONCLUSION: Sensitization to soy allergen was not increased among the population sample living in the vicinity of the factory. Soy sensitized individuals living in the surroundings of the factory reported more respiratory symptoms, used bronchodilators more often and had a lower PEF after having been downwind of the factory.

The effects of co-administration of probiotics with herbal medicine on obesity, metabolic endotoxemia and dysbiosis: a randomized double-blind controlled clinical trial.

BACKGROUND & AIMS: Probiotics help maintain balance in composition of the gut microbiota, and have been considered as a potential treatment for obesity. This study was conducted in order to assess the effects of probiotics when combined with herbal medicine in treatment of obesity. Probiotics were tested for the ability to modulate gut microbiota, gut permeability, and endotoxin level, which may have correlation with factors involved in obesity. METHODS: A randomized, double-blind, placebo controlled study was conducted, in which patients with higher BMI (>25 kg/m(2)) and waist circumference (>85 cm) were enrolled and randomly assigned to receive Bofutsushosan with either probiotics or placebo capsules for a period of eight weeks. Assessment of body composition parameters, metabolic biomarkers, endotoxin level, gut permeability, and fecal bacteria in stool was performed at baseline and at week 8. The study was registered at the Clinical Research Information Service, approved by the Korea National Institute of Health (KCT0000386). RESULTS: Although both groups showed a significant reduction in weight and waist circumference (p = 0.000), no significant differences in body composition and metabolic markers were observed. In correlation analysis, change in body composition showed positive correlation with endotoxin level (r = 0.441, p < 0.05 for BW; and r = 0.350, p < 0.05 for fat mass) and the population of gut Lactobacillus plantarum (r = 0.425, p < 0.05 for BW; and r = 0.407, p < 0.05 for BMI). The Gram negative bacterial population in gut also exhibited positive correlation with changes in body composition (WC) and total cholesterol level (r = 0.359, and 0.393, for the former and later parameters, respectively, p < 0.05 for both). While, the profile of gut Bifidobacterium breve population showed negative correlation with endotoxin level (r = -0.350, p < 0.05). CONCLUSIONS: Correlations between gut microbiota and change in body composition indicate that probiotics may influence energy metabolism in obesity. Correlation between endotoxin level and weight reduction indicates that probiotics may play an important role in prevention of endotoxin production, which can lead to gut microbiota dysbiosis associated with obesity.

Distribution and pharmacokinetics of double-radiolabeled endotoxin in the rat brain and peripheral organs.

The endotoxin, lipopolysaccharide (LPS), of Salmonella typhimurium was biosynthetically labeled with (3)H and (14)C incorporated into the fatty acyl chains and glucosamine residues, respectively. The radio-labeled LPS was isolated from the bacteria and then injected into Sprague-Dawley rats. The distribution of (14)C and (3)H-LPS in plasma and other organs was determined following intraperitoneal (IP) doses of (14)C and (3)H-LPS (200 µg/kg). Plasma concentrations of both fatty acyl chains and glucosamine residues were biphasic, with a relatively rapid decay followed by a slow decline for 48 h. Similar biphasic results were found in the peripheral organs (kidney and heart) and brain barrier tissues (meninges and choroid plexus). In other brain tissues (brain stem, caudate nucleus, hypothalamus, frontal cortex, cerebellum and hippocampus), the glucosamine residue was biphasic, whereas the fatty acyl chains showed accumulation. Highest concentrations of LPS were found in the plasma, spleen and the liver. In addition, in the liver, sustained elevations of (14)C-glucosamine and (3)H-fatty acyl chains were observed. This indicates LPS accumulation in the liver. By contrast, the spleen showed biphasic decay of glucosamine residues and accumulation of fatty acyl chains. In the brain barrier tissues, peak LPS concentrations were significantly reduced (about 70%) and were further reduced (about 95%) in other brain tissues. The high elevation of LPS in the spleen is considered indicative of an immune response. Our findings highlight the potential significant role of lipid A as shown with the sustained elevation of (3)H-fatty acyl chains in the brain.

Application of capillary electrophoretic chips in protein profiling of plant extracts for identification of genetic modifications of maize.

In this study, the chip gel electrophoresis with LIF detection was applied in protein profiling of fractionated and total extracts of maize standards. The sensitivity of such determinations can be enhanced by lyophilization of extracts or employing filtering and preconcentration with cutoff filters. Combinatorial peptide ligand library applied for sample processing prior to the electrophoretic analysis was, especially, an effective pretreatment step in the determination of low-abundance proteins. Several repeatable differences were observed for protein profiles between maize standards not containing the genetically modified organisms (GMOs) and those containing GMO, which can be potentially employed for identification of GMO in maize samples and foods of maize origin.

Potato flour mediated solid-state fermentation for the enhanced production of Bacillus thuringiensis-toxin.

In this study, we explored the efficacy of raw potato flour (PF) as supplement to the conventional LB medium (LB control, designated as M1) for enhancing the concomitant production of endospores and δ-endotoxin from Bacillus thuringiensis subsp. kurstaki by solid-state fermentation (SSF). Of different concentrations and combinations of media tested, 10% (w/v) PF supplemented LB medium (M2) was found as the best source for the maximum yield of toxin. After 12 h submerged fermentation (SmF) at 37°C and 125 rpm, M2 was made into a wet-solid matter for SSF by removing the supernatant (1000 ×g, 10 min); the resultant pellet subsequently incubated statically (37°C) for the production of B. thuringiensis subsp. kurstaki toxin (Btk-toxin). In comparison to M1, yield of δ-endotoxin purified by sucrose density gradient centrifugation method from M2 was about 6-fold higher (53% recovery). This maximum yield from M2 was obtained at 48 h (as against 72 h from M1), thus the gestation period of M2 was reduced by 24 h with higher yield. In addition to the quantitative data, qualitative photomicrographs taken by image analyzer, scanning electron and fluorescent microscopes and digital camera showed physical evidences for the upper hand of SSF over conventional SmF for the enhanced production of Btk-toxin. SDS-PAGE image of the purified δ-endotoxin showed three major fractions with apparent MWs 66, 45 and 30 kDa. Briefly, if low-cost agricultural products like PF is used as supplement to LB, by SSF strategy, production of Btk-toxin could be enhanced to 6-fold in short gestation time without losing its entomotoxicity efficiency.

Respiratory symptoms, exhaled nitric oxide, and lung function among workers in Tanzanian coffee factories.

OBJECTIVE: To compare chronic respiratory symptoms, fractional exhaled nitric oxide (FENO), and lung function between Robusta and Arabica coffee workers and a control group. METHODS: Chronic respiratory symptoms were assessed by a questionnaire (n = 138 coffee workers and n = 120 controls). The FENO was measured by NIOX MINO device (Aerocrine AB, Solna, Sweden). Lung function was examined by a portable spirometer. RESULTS: Coffee workers had higher prevalence of chronic respiratory and asthma symptoms than controls. Robusta coffee workers were exposed to higher levels of endotoxin and had more asthma symptoms than Arabica coffee workers (38% vs. 18%). Coffee workers had reduced lung function associated with cumulative exposure to total dust and endotoxin. CONCLUSION: Work in coffee factories is associated with small but significant lung function impairment. These changes were not associated with the level of FENO.

Personal exposure to dust and endotoxin in Robusta and Arabica coffee processing factories in Tanzania.

INTRODUCTION: Endotoxin exposure associated with organic dust exposure has been studied in several industries. Coffee cherries that are dried directly after harvest may differ in dust and endotoxin emissions to those that are peeled and washed before drying. The aim of this study was to measure personal total dust and endotoxin levels and to evaluate their determinants of exposure in coffee processing factories. METHODS: Using Sidekick Casella pumps at a flow rate of 2l/min, total dust levels were measured in the workers' breathing zone throughout the shift. Endotoxin was analyzed using the kinetic chromogenic Limulus amebocyte lysate assay. Separate linear mixed-effects models were used to evaluate exposure determinants for dust and endotoxin. RESULTS: Total dust and endotoxin exposure were significantly higher in Robusta than in Arabica coffee factories (geometric mean 3.41 mg/m(3) and 10 800 EU/m(3) versus 2.10 mg/m(3) and 1400 EU/m(3), respectively). Dry pre-processed coffee and differences in work tasks explained 30% of the total variance for total dust and 71% of the variance for endotoxin exposure. High exposure in Robusta processing is associated with the dry pre-processing method used after harvest. CONCLUSIONS: Dust and endotoxin exposure is high, in particular when processing dry pre-processed coffee. Minimization of dust emissions and use of efficient dust exhaust systems are important to prevent the development of respiratory system impairment in workers.

Effect of Zingiber officinale and propolis on microorganisms and endotoxins in root canals

The purpose of this study was to evaluate the effectiveness of glycolic propolis (PRO) and ginger (GIN) extracts, calcium hydroxide (CH), chlorhexidine (CLX) gel and their combinations as ICMs (ICMs) against Candida albicans,Enterococcus faecalis, Escherichia coli and endotoxins in root canals. Material and Methods: After 28 days of contamination with microorganisms, the canals were instrumented and then divided according to the ICM: CH+saline; CLX, CH+CLX, PRO, PRO+CH; GIN; GIN+CH; saline. The antimicrobial activity and quantification of endotoxins by the chromogenic test of Limulus amebocyte lysate were evaluated after contamination and instrumentation at 14 days of ICM application and 7 days after ICM removal. Results and Conclusion: After analysis of results and application of the Kruskal-Wallis and Dunn statistical tests at 5% significance level, it was concluded that all ICMs were able to eliminate the microorganisms in the root canals and reduce their amount of endotoxins; however, CH was more effective in neutralizing endotoxins and less effective against C. albicans and E. faecalis, requiring the use of medication combinations to obtain higher success.

Efficacy of endodontic treatment for endotoxin reduction in primarily infected root canals and evaluation of cytotoxic effects.

INTRODUCTION: Endotoxins are one of the etiologic agents involved in the pathogenesis of apical periodontitis. The objectives of this clinical study were to investigate the effects of endodontic treatment by using different irrigants on endotoxins in root canals with pulp necrosis and apical periodontitis and to evaluate the cytotoxic effects. METHODS: Thirty-six root canals were selected. Samples were collected before (S1) and after instrumentation (S2). The root canals were divided into 3 groups (n = 12) according to the irrigant combination used: CLX + LW, 2% chlorhexidine gel + calcium hydroxide (0.14%, limewater); CLX + PmB, chlorhexidine + polymyxin B; CLX (control), chlorhexidine + saline. The third sampling (S3) was performed after ethylenediaminetetraacetic acid and S4 after intracanal medication (CLX + calcium hydroxide for 14 days). Endotoxins were quantified by the chromogenic Limulus amebocyte lysate assay, and cytotoxic effects were evaluated by the production of cytokines (interleukin-1ß, tumor necrosis factor α) in macrophages (RAW 264.7) stimulated with the root canal content. RESULTS: Endotoxins were detected in all root canals before instrumentation (S1). Group CLX + LW presented the greatest endotoxin reduction after instrumentation (99.18%), which was similar to group CLX + PmB (96.42%, P > .05) and different from group CLX (90.78%, P < .05). The intracanal medication promoted important endotoxin neutralization, with a reduction of 99.2% to 100%. The root canal content induced a higher production of tumor necrosis factor α and interleukin-1ß in S1 samples compared with samples obtained after treatment. CONCLUSIONS: The combination of CLX and limewater as irrigant was the most effective in reducing endotoxins in root canals, and intracanal medication was important to neutralize the cytotoxic effects.

Detecting endotoxin contamination of ophthalmic viscosurgical devices: intracameral versus intravitreal assays in rabbits.

OBJECTIVE: To compare the sensitivities of intracameral and intravitreal assays in the rabbit model to determine the relative adequacy of these methods in detecting bacterial endotoxin contamination of ophthalmic viscosurgical devices (OVDs). DESIGN: Experimental, randomized animal study. PARTICIPANTS: Twenty New Zealand white rabbits. METHODS: Rabbits were randomized into 4 groups to receive a cohesive or a dispersive OVD via intracameral or intravitreal injection. All 40 treated eyes (10 eyes of 5 animals in each group) received bilateral injection of OVD spiked with bacterial endotoxin at 7.0 endotoxin units/ml. All eyes were evaluated by slit-lamp biomicroscopy for inflammatory response at 3, 6, 9, 24, 48, and 72 hours after exposure. Eyes that received intravitreal injection were also dilated at 24, 48, and 72 hours and were re-examined by slit-lamp biomicroscopy and by indirect ophthalmoscopy. MAIN OUTCOME MEASURES: Conjunctival inflammation, anterior chamber (AC) flare, cells and fibrin, vitreous haze and cells, iridal hyperemia, corneal clouding, lens opacities, and onset times. RESULTS: Intracamerally injected eyes frequently showed conjunctival congestion, AC cells and flare, iridal hyperemia, and fibrin within 6 hours. Up to 80% showed AC cells and flare at 9 hours, and up to 70% showed fibrin at 24 hours. These signs diminished within 48 hours. Fibrin and cells also were seen on the lens surface of most of the eyes. Intravitreally injected eyes showed no signs of inflammation within 24 hours, other than some conjunctival inflammation. After the 24-hour time point, in addition to some conjunctival inflammation, some other signs of inflammation were observed infrequently in the intravitreally injected eyes, including minor vitreous cell reaction in 2 eyes. Although there was 1 dispersive OVD-treated eye with cells and fibrin on the lens capsule at 48 hours, no aqueous cells or flare were seen in the AC of any intravitreally injected eyes at any time during the course of the study. CONCLUSIONS: The rabbit intravitreal assay, when limited to 72 hours, does not seem to have adequate sensitivity to detect endotoxin reliably in OVDs.

Biochemical, microbiological and phytochemical studies on aqueous-fermented extracts from Atropa belladonna L. Part 1--biochemistry and microbiology.

Extraction methods of fresh plants into aqueous-fermented extracts according to German Homoeopathic Pharmacopoeia (HAB), regulation nos. 33 and 34 were evaluated. In the course of production, the extraction is accompanied by fermentation and the resulting preparation is stored for at least 6 months until further processing. The present work aimed at revealing the underlying biochemical reactions during manufacture and storage. In addition, the responsible microorganisms were isolated and identified. To study the robustness of the preparation method, formulation components as well as production conditions were varied. Additionally, questions were addressed at the reproducibility of the method and a comparison with an ethanolic extract was also performed. From 2006 to 2009, 110 extracts from the fresh flowering herb of Atropa belladonna var. belladonna (L.) were produced and analyzed. The results show that lactic acid bacteria (LAB) are primarily involved in the fermentation process, mainly producing lactic acid besides acetic acid and ethanol. The homofermentative Lactobacillus plantarum and the heterofermentative Lactobacillus brevis were identified as predominant lactic acid bacteria. Finally, factors for a successful fermentation are proposed.

[Comparison of activated carbon and ultrafiltration technique in the production process of huoxue tongluo injection].

OBJECTIVE: To research the applicability of activated carbon and ultrafiltration technique in the production process of Huoxue Tongluo Injection. METHODS: The kinetic-turbidimetric method was used to determine the content of bacterial endotoxins in Huoxue Tongluo solution. Particle size change in Huoxue Tongluo solution was determined by nanometer particle size instrument before and after the use of different concentration activated carbon and different molecular weight ultrafiltration membrane. RESULTS: The removal efficacy of bacterial endotoxins was 65.2%, 77.5%, 80.4% by using three concentrations of active carbon at 0.05%, 0.10%, 0.30% in Huoxue Tongluo Injection, respectively. It was above 95% by using cutoff molecular weight both 5 kDa and 10 kDa ultrafiltration membrane. Measure results by nanometer particle size instrument showed that particle size of filter liquor by 10 kDa cutoff molecular weight ultrafiltration membrane was much smaller than that of by use of different concentration activated carbon. CONCLUSION: Ultrafiltration method is more suitable to the removal of bacterial endotoxins. The solution is more clear after using ultrafiltration method, and large particles of solution is removed. The ultrafiltration method provides the basis for injection production.