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

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

[Box: see text].

Applicability of mouse models for induction of severe acute lung injury.

Acute lung injury (ALI) is a significant clinical challenge associated with high morbidity and mortality. Worldwide, it affects approximately 200.000 individuals annually, with a staggering 40 % mortality rate in hospitalized cases and persistent complications in out-of-hospital cases. This review focuses on the key immunological pathways underlying bacterial ALI and the exploration of mouse models as tools for its induction. These models serve as indispensable platforms for unraveling the inflammatory cascades and biological responses inherent to ALI, while also facilitating the evaluation of novel therapeutic agents. However, their utility is not without challenges, mainly due to the stringent biosafety protocols required by the diverse bacterial virulence profiles. Simple and reproducible models of pulmonary bacterial infection are currently available, including intratracheal, intranasal, pleural and, intraperitoneal approaches. These models use endotoxins such as commercially available lipopolysaccharide (LPS) or live pathogens such as Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Streptococcus pneumoniae, all of which are implicated in the pathogenesis of ALI. Combining murine models of bacterial lung infection with in-depth studies of the underlying immunological mechanisms is a cornerstone in advancing the therapeutic landscape for acute bacterial lung injury.

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

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

Disinfectant effectiveness of chlorhexidine gel compared to sodium hypochlorite: a systematic review with meta-analysis.

This study aimed to compare the disinfectant ability of chlorhexidine (CHX) gel and sodium hypochlorite (NaOCl). Systematic searches were conducted from inception until December 8th, 2022 (MEDLINE/PubMed, Cochrane Library, Web of Science, Scopus, Embase, and Grey Literature databases). Only randomized clinical trials were included. The revised Cochrane risk of bias tools for randomized trials were used to assess the quality of studies. Meta-analyses were performed. The overall quality of evidence was assessed through the Grading of Recommendations Assessment, Development, and Evaluation tool. Six studies were included. Five had a low risk of bias and 1 had some concerns. Three studies assessed bacterial reduction. Two were included in the meta-analysis for bacterial reduction (mean difference, 75.03 [confidence interval, CI, -271.15, 421.22], p = 0.67; I2 = 74%); and 3 in the meta-analysis for cultivable bacteria after chemomechanical preparation (odds ratio, 1.03 [CI, 0.20, 5.31], P = 0.98; I2 = 49%). Five studies assessed endotoxin reduction. Three were included in a meta-analysis (mean difference, 20.59 [CI, -36.41, 77.59], p = 0.48; I2 = 74%). There seems to be no difference in the disinfectant ability of CHX gel and NaOCl, but further research is necessary.

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

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

Neonatal overnutritional programming impairs the hypophagia and neuron activation induced by acute lipopolysaccharide in adult male rats.

Nutritional status during critical windows in early development can challenge metabolic functions and physiological responses to immune stress in adulthood, such as the systemic inflammation induced by lipopolysaccharide (LPS). The aim of this study was to investigate the long-term effects of post-natal over- and undernutrition on the anorexigenic effect of LPS and its association with neuronal activation in the brainstem and hypothalamus of male rats. Animals were raised in litters of 3 (small - SL), 10 (normal - NL), or 16 (large - LL) pups per dam. On post-natal day 60, male rats were treated with LPS (500 µg/Kg) or vehicle for the evaluation of food intake and c-Fos expression in the area postrema (AP), nucleus of solitary tract (NTS), and paraventricular (PVN), arcuate (ARC), ventromedial (VMH), and dorsomedial (DMH) nuclei of the hypothalamus. SL, NL, and LL animals showed a decreased food consumption after LPS treatment. In under- and normonourished animals, peripheral LPS induced an increase in neuronal activation in the brainstem, PaV, PaMP, and ARC and a decrease in the number of c-Fos-ir neurons in the DMH. Overnourished rats showed a reduced hypophagic response, lower neuron activation in the NTS and PaMP, and no response in the DMH induced by LPS. These results indicate that early nutritional programming displays different responses to LPS, by means of neonatal overnutrition decreasing LPS-mediated anorexigenic effect and neuronal activation in the NTS and hypothalamic nuclei.

The chronicle of the ups and downs that made Bacillus thuringiensis a natural insecticide

Bacillus thuringiensis is a worldwide known bacterium for its capacity to control insect pests thanks to the action of its parasporal crystal. The objective of this paper deals with the history, in some cases unknown, of the study of Bacillus thuringiensis that led it to be a crucial biological alternative in controlling pest insects. How the mode of action for killing insects was understood, as well as the field tests that were carried out to evaluate its effectiveness and to develop the first commercial products, are reflected in this review that presents and discusses the scientific successes and failures that marked the course of B. thuringiensis.

Bacillus thuringiensis es una bacteria conocida mundialmente por su capacidad para controlar insectos plaga, gracias a la acción de su cristal parasporal. El objetivo de esta revisión trata de la historia, en algunos casos desconocida, del estudio de Bacillus thuringiensis que la llevó a ser una importante alternativa biológica en el control de insectos plaga. Cómo se llegó a comprender el modo de acción para matar insectos, así como las pruebas de campo que se realizaron para evaluar su efectividad y lograr desarrollar los primeros productos comerciales están plasmados en esta revisión que presenta y discute los aciertos y desaciertos científicos que marcaron el rumbo de B. thuringiensis.

In vitro study of structural and mechanical properties of latex and non-latex intermaxillary orthodontic elastics.

PURPOSE: We evaluated bacterial endotoxin adhesion, superficial micromorphology and mechanical properties of latex and non-latex intermaxillary orthodontic elastics. METHODS: To quantify the adhered bacterial endotoxin, elastics were divided into 5 groups: experimental (n = 12) latex and non-latex elastics, previously contaminated by an endotoxin solution, negative control (n = 6) latex and non-latex elastics without contamination, and positive control (n = 6) stainless steel specimens (metallic replicas), contaminated by an endotoxin solution. In parallel, the structural micromorphology (n = 6) and surface roughness of latex and non-latex intermaxillary orthodontic elastics were assessed using confocal laser microscopy. Force degradation (g) and deformation of the internal diameter change (mm) were also evaluated. Structural micromorphology, surface roughness (µm), force degradation (g) and internal diameter (mm) change were evaluated at time 0 and after 24 and 72 h in a deformation test. Data were analyzed by the Shapiro-Wilk, Kruskal-Wallis, Dunn, ANOVA and Bonferroni tests (α = 5%). RESULTS: Endotoxin adhered similarly to both types of elastics with scores of 3 (> 1.0 EU/mL). The surface microstructure of both types of elastics showed irregularities and porosities at all times. Initially, the latex elastics had a higher surface roughness (p < 0.001) than the non-latex ones. After 24 h loading, surface roughness of the latex elastics was significantly reduced (p < 0.001), while after 72 h, the values were similar for both types (p > 0.05). The non-latex elastics had significantly higher force generation values (p < 0.05) at 0, 24 and 72 h compared with the latex elastics, although there was a significant reduction (p < 0.001) in force over time for both elastics. Despite similar initial values, non-latex elastics had a significantly larger internal diameter (p < 0.001) after the loading periods of 24 and 72 h compared with the latex elastics. CONCLUSION: Both elastics showed high affinity with endotoxin and microstructural irregularities of their surface. The non-latex elastics generated higher force values but demonstrated greater deformation of the internal diameter after loading.

Lipopolysaccharide effects on activation and apoptosis of primordial ovarian follicles in heifers / Efeitos de lipopolissacarídeos na ativação e apoptose de folículos ovarianos primordiais em novilhas

The objective of the present study was to evaluate the effect of lipopolysaccharide (LPS) administration on activation and apoptosis of primordial follicles. There was no difference in the total number of follicles as well as in the different types of follicles. Furthermore, the LPS challenge didn't modulate the expression of genes related with ovarian reserve (HAM), oocyte survival (Survivin), activation rate (Pten, KIT, KITL1, KITL2, AKT1, SIRT1), and follicular abnormalities. Therefore, the LPS exposure with 24h interval had no effect on activation rate and primordial follicles abnormalities, and also had no effect on expression of anti-apoptotic genes and genes related with ovarian reserve, oocyte survival, activation rate, and primordial follicles abnormalities.

O objetivo do presente estudo foi avaliar o efeito da administração de lipopolissacarídeo (LPS) na ativação e a apoptose de folículos primordiais. Dez novilhas saudáveis (Bos taurus taurus), com idade média de 14 meses, alojadas em sistema de confinamento e alimentadas com TMR, foram utilizadas neste experimento. Os animais foram distribuídos aleatoriamente em dois grupos: grupo LPS (LPS; n = 5), que recebeu duas injeções intravenosas de 0,5µg/kg de peso corporal de lipopolissacarídeo (Sigma Aldrich®) diluído em 2mL de solução salina (0,9% de NaCl), com intervalo de 24h; e grupo controle (CTR; n = 5), que recebeu duas injeções intravenosas de 2mL de solução salina (0,9% de NaCl), com intervalo de 24h. A primeira injeção de LPS foi realizada no d 1, e no d 5 os animais foram abatidos, os ovários foram pesados e as amostras dos ovários foram coletadas para avaliação histológica e molecular. Não houve diferença no número total de folículos, bem como nos diferentes tipos de folículos. Além disso, o desafio com LPS não modulou a expressão de genes relacionados à reserva ovariana (HAM), à sobrevivência oocitária (Survivin), à taxa de ativação (Pten, KIT, KITL1, KITL2, AKT1, SIRT1) e às anormalidades foliculares. Portanto, a exposição ao LPS com intervalo de 24h não teve efeito sobre a taxa de ativação e as anormalidades dos folículos primordiais, bem como não teve efeito sobre a expressão de genes antiapoptóticos e de genes relacionados com a reserva ovariana, a sobrevivência oocitária, a taxa de ativação e as anormalidades dos folículos primordiais.

Glucose metabolism is upregulated in the mononuclear cell proteome during sepsis and supports endotoxin-tolerant cell function.

Metabolic adaptations shape immune cell function. In the acute response, a metabolic switch towards glycolysis is necessary for mounting a proinflammatory response. During the clinical course of sepsis, both suppression and activation of immune responses take place simultaneously. Leukocytes from septic patients present inhibition of cytokine production while other functions such as phagocytosis and production of reactive oxygen species (ROS) are preserved, similarly to the in vitro endotoxin tolerance model, where a first stimulation with lipopolysaccharide (LPS) affects the response to a second stimulus. Here, we sought to investigate how cellular metabolism is related to the modulation of immune responses in sepsis and endotoxin tolerance. Proteomic analysis in peripheral blood mononuclear cells (PBMCs) from septic patients obtained at intensive care unit admission showed an upregulation of proteins related to glycolysis, the pentose phosphate pathway (PPP), production of ROS and nitric oxide, and downregulation of proteins in the tricarboxylic acid cycle and oxidative phosphorylation compared to healthy volunteers. Using the endotoxin-tolerance model in PBMCs from healthy subjects, we observed increased lactate production in control cells upon LPS stimulation, while endotoxin-tolerant cells presented inhibited tumor necrosis factor-α and lactate production along with preserved phagocytic capacity. Inhibition of glycolysis and PPP led to impairment of phagocytosis and cytokine production both in control and in endotoxin-tolerant cells. These data indicate that glucose metabolism supports leukocyte functions even in a condition of endotoxin tolerance.

Participation of ecto-5'-nucleotidase in the inflammatory response in an adult zebrafish (Danio rerio) model.

The ecto-5'-nucleotidase is an important source of adenosine in the extracellular medium. Adenosine modulation appears early in evolution and performs several biological functions, including a role as an anti-inflammatory molecule. Here, we evaluate the activity and mRNA expression of ecto-5'-nucleotidase in response to lipopolysaccharide (LPS) using zebrafish as a model. Adult zebrafish were injected with LPS (10 µg/g). White blood cell differential counts, inflammatory markers, and ecto-5'-nucleotidase activity and expression in the encephalon, kidney, heart, and intestine were evaluated at 2, 12, and 24 h post-injection (hpi). At 2 hpi of LPS, an increase in neutrophils and monocytes in peripheral blood was observed, which was accompanied by increased tnf-α expression in the heart, kidney, and encephalon, and increased cox-2 expression in the intestine and kidney. At 12 hpi, monocytes remained elevated in the peripheral blood, while tnf-α expression was also increased in the intestine. At 24 hpi, the white blood cell differential count no longer differed from that of the control, whereas tnf-α expression remained elevated in the encephalon but reduced in the kidney compared with the controls. AMP hydrolysis in LPS-treated animals was increased in the heart at 24 hpi [72 %; p = 0.029] without affecting ecto-5'-nucleotidase gene expression. These data indicate that, in most tissues studied, inflammation does not affect ecto-5'-nucleotidase activity, whereas in the heart, a delayed increase in ecto-5'-nucleotidase activity could be related to tissue repair.

Metabolic trade-offs favor regulated hypothermia and inhibit fever in immune-challenged chicks.

The febrile response to resist a pathogen is energetically expensive, while regulated hypothermia seems to preserve energy for vital functions. We hypothesized here that immune-challenged birds facing metabolic trade-offs (reduced energy supply/increased energy demand) favor a regulated hypothermic response at the expense of fever. To test this hypothesis, we compared 5 day old broiler chicks exposed to fasting, cold (25°C), and fasting combined with cold with a control group fed under thermoneutral conditions (30°C). The chicks were injected with saline or with a high dose of endotoxin known to induce a biphasic thermal response composed of a drop in body temperature (Tb) followed by fever. Then Tb, oxygen consumption (metabolic rate), peripheral vasomotion (cutaneous heat exchange), breathing frequency (respiratory heat exchange) and huddling behavior (heat conservation indicator) were analyzed. Irrespective of metabolic trade-offs, chicks presented a transient regulated hypothermia in the first hour, which relied on a suppressed metabolic rate for all groups, increased breathing frequency for chicks fed/fasted at 30°C, and peripheral vasodilation in chicks fed/fasted at 25°C. Fever was observed only in chicks kept at thermoneutrality and was supported by peripheral vasoconstriction and huddling behavior. Fed and fasted chicks at 25°C completely eliminated fever despite the ability to increase metabolic rate for thermogenesis in the phase correspondent to fever when it was pharmacologically induced by 2,4-dinitrophenol. Our data suggest that increased competing demands affect chicks' response to an immune challenge, favoring regulated hypothermia to preserve energy while the high costs of fever to resist a pathogen are avoided.

ClearColi as a platform for untagged pneumococcal surface protein A production: cultivation strategy, bioreactor culture, and purification.

Several studies have searched for new antigens to produce pneumococcal vaccines that are more effective and could provide broader coverage, given the great number of serotypes causing pneumococcal diseases. One of the promising subunit vaccine candidates is untagged recombinant pneumococcal surface protein A (PspA4Pro), obtainable in high quantities using recombinant Escherichia coli as a microbial factory. However, lipopolysaccharides (LPS) present in E. coli cell extracts must be removed, in order to obtain the target protein at the required purity, which makes the downstream process more complex and expensive. Endotoxin-free E. coli strains, which synthesize a nontoxic mutant LPS, may offer a cost-effective alternative way to produce recombinant proteins for application as therapeutics. This paper presents an investigation of PspA4Pro production employing the endotoxin-free recombinant strain ClearColi® BL21(DE3) with different media (defined, auto-induction, and other complex media), temperatures (27, 32, and 37 °C), and inducers. In comparison to conventional E. coli cells in a defined medium, ClearColi presented similar PspA4Pro yields, with lower productivities. Complex medium formulations supplemented with salts favored PspA4Pro yields, titers, and ClearColi growth rates. Induction with isopropyl-ß-D-thiogalactopyranoside (0.5 mM) and lactose (2.5 g/L) together in a defined medium at 32 °C, which appeared to be a promising cultivation strategy, was reproduced in 5 L bioreactor culture, leading to a yield of 146.0 mg PspA4Pro/g dry cell weight. After purification, the cell extract generated from ClearColi led to 98% purity PspA4Pro, which maintained secondary structure and biological function. ClearColi is a potential host for industrial recombinant protein production. KEY POINTS: • ClearColi can produce as much PspA4Pro as conventional E. coli BL21(DE3) cells. • 10.5 g PspA4Pro produced in ClearColi bioreactor culture using a defined medium. • Functional PspA4Pro (98% of purity) was obtained in ClearColi bioreactor culture. Graphical abstract.

Autonomic Disbalance During Systemic Inflammation is Associated with Oxidative Stress Changes in Sepsis Survivor Rats.

Sepsis affects 31.5 million people worldwide. It is characterized by an intense drop in blood pressure driving to cardiovascular morbidity and mortality. Modern supportive care has increased survival in patients; however, after experiencing sepsis, several complications are observed, which may be potentiated by new inflammatory events. Nevertheless, the interplay between sepsis survivors and a new immune challenge in cardiovascular regulation has not been previously defined. We hypothesized that cecal ligation and puncture (CLP) cause persistent cardiovascular dysfunctions in rats as well as changes in autonomic-induced cardiovascular responses to lipopolysaccharide (LPS). Male Wistar rats had mean arterial pressure (MAP) and heart rate (HR) recorded before and after LPS or saline administration to control or CLP survivor rats. CLP survivor rats had similar baseline MAP and HR when compared to control. LPS caused a drop in MAP accompanied by tachycardia in control, while CLP survivor rats had a noteworthy enhanced MAP and a blunted tachycardia. LPS-induced hemodynamic changes were related to an autonomic disbalance to the heart and resistance vessels that were expressed as an increased low- and high-frequency power of pulse interval in CLP survivors after saline and enhancement in the low-frequency power of systolic arterial pressure in control rats after LPS. LPS-induced plasma interferon γ, but not interleukin-10 surges, was blunted in CLP survivor rats. To further access whether or not LPS-induced autonomic disbalance in CLP survivor rats was associated with oxidative stress dysregulation, superoxide dismutase (SOD) activity and thiobarbituric acid reactive substances (TBARS) plasma levels changes were measured. LPS-induced oxidative stress was higher in CLP survivor rats. These findings indicate that key changes in hemodynamic regulation of CLP survivors rats take place in response to LPS that are associated with oxidative stress changes, i.e., reduced SOD activity and increased TBARS levels.

ClearColi as a platform for untagged pneumococcal surface protein a production: cultivation strategy, bioreactor culture, and purification

Several studies have searched for new antigens to produce pneumococcal vaccines that are more effective and could provide broader coverage, given the great number of serotypes causing pneumococcal diseases. One of the promising subunit vaccine candidates is untagged recombinant pneumococcal surface protein A (PspA4Pro), obtainable in high quantities using recombinant Escherichia coli as a microbial factory. However, lipopolysaccharides (LPS) present in E. coli cell extracts must be removed, in order to obtain the target protein at the required purity, which makes the downstream process more complex and expensive. Endotoxin-free E. coli strains, which synthesize a nontoxic mutant LPS, may offer a cost-effective alternative way to produce recombinant proteins for application as therapeutics. This paper presents an investigation of PspA4Pro production employing the endotoxin-free recombinant strain ClearColi® BL21(DE3) with different media (defined, auto-induction, and other complex media), temperatures (27, 32, and 37 °C), and inducers. In comparison to conventional E. coli cells in a defined medium, ClearColi presented similar PspA4Pro yields, with lower productivities. Complex medium formulations supplemented with salts favored PspA4Pro yields, titers, and ClearColi growth rates. Induction with isopropyl-β-D-thiogalactopyranoside (0.5 mM) and lactose (2.5 g/L) together in a defined medium at 32 °C, which appeared to be a promising cultivation strategy, was reproduced in 5 L bioreactor culture, leading to a yield of 146.0 mg PspA4Pro/g dry cell weight. After purification, the cell extract generated from ClearColi led to 98% purity PspA4Pro, which maintained secondary structure and biological function. ClearColi is a potential host for industrial recombinant protein production.

Standardized pyrogen testing of medical products with the bacterial endotoxin test (BET) as a substitute for rabbit Pyrogen testing (RPT): A scoping review.

The Bacterial Endotoxin Test (BET) is a method for exclusion of endotoxin-related pyrogen contamination in pharmaceutical products, as an alternative to the Rabbit Pyrogen Test (RPT). However, BET does not detect a broad range of biologically relevant pyrogens, and interferences can limit its practical use for different medical products. This work aimed to scope the evidence in the scientific literature for case-by-case validity assessments of BET in different uses for medical products. A search strategy was conducted in PubMed, Scopus, and Web of Science in April 2020, according to the PRISMA-ScR statement. Twenty-two references were included, evaluating medical products for endotoxin contamination through both BET and RPT according to standardized protocols. A critical appraisal was performed through ToxRTool, followed by data extraction and qualitative synthesis of outcomes and methodological issues. Four classes of products assessed by BET were identified, including nanoparticles, drugs, blood and biological products. A considerable variation was observed on the BET methods used. Collectively, the evidence indicates different factors influencing the outcome of BET, including the chemical nature of samples that may cause interference depending on the selected method. While some applications to medical products appear adequate, others, such as nanoparticles, may require the use of different in vitro pyrogen testing methods, reinforcing the need for case-by-case validation for each BET method and type of medical product.

Bacterial levels and amount of endotoxins in carious dentin within reversible pulpitis scenarios.

OBJECTIVES: the objective of the present exploratory study was to determine bacterial diversity and endotoxin levels in deep carious lesions of teeth presenting symptoms of reversible pulpitis. MATERIALS AND METHODS: Twenty patients with deep carious lesions, reporting clinical symptomatology compatible with reversible pulpitis (n = 10) or not reporting clinical symptomatology (n = 10), were selected. Carious dentin samples were obtained with the aid of sterile and pyrogen-free spoon excavators and harvested in two steps: before and after infected dentin removal. Samples were collected for checkerboard and for kinetic chromogenic LAL assay for determination of microbial profile and quantitation of endotoxin, respectively. Data were analyzed by Mann Whitney for bacteria and two-way ANOVA for endotoxins (5%). RESULTS: No difference on the studied bacteria was detected between the superficial and deep dentin layers. Symptomatic teeth showed greater presence of Lactobacillus species, Capnocytophaga sputigena, and Leptotrichia buccalis. For the endotoxins, symptomatic teeth resulted in greater quantity of endotoxins (p = 0.047), being 4.13 log10 EU/mL/µg dentin and 3.45 log10 EU/mL/µg dentin, for symptomatic and asymptomatic teeth, respectively. Dentin collected in different areas presented similar number of endotoxins (p = 0.139). CONCLUSION: The amount of the studied bacteria does not seem to be related to reported symptomatology of deep carious lesions, while endotoxins quantity is greater in symptomatic scenarios, regardless of the harvesting area. CLINICAL RELEVANCE: The understanding of bacterial amount in reversible pulpitis is important to establish a clinical protocol of treatment.

Investigation of microbial profile, levels of endotoxin and lipoteichoic acid in teeth with symptomatic irreversible pulpitis: a clinical study.

AIM: To investigate the microbial profile, and levels of endotoxin (LPS) and lipoteichoic acid (LTA), in infected dentine (ID) and root canals (RC) at different phases of root canal treatment in teeth with symptomatic irreversible pulpitis. METHODOLOGY: Ten volunteers were included, and samples were collected from infected dentine (ID) and the root canal lumen (RC) using sterile excavators and paper points, respectively. RC samples were taken before (S1) and after (S2) chemo-mechanical canal preparation (CMP), and after intracanal medication (ICM; S3). Checkerboard DNA-DNA hybridization was used for microbial analysis. The levels of LPS and LTA were evaluated using the limulus amebocyte lysate assay and ELISA, respectively. Shapiro-Wilk's test was used to verify data normality. Friedman's test was used to evaluate statistical differences using checkerboard DNA-DNA hybridization in the ID and RC at the different phases of the RC treatment. Post hoc Dunn's multiple comparison test was used to verify significant differences recorded at the different time-points. The levels of LPS and LTA were analysed statistically by using repeated measures anova and Tukey's post hoc test to evaluate differences in both sites. The significance level was set at 5% (P < 0.05). RESULTS: A total of 40 DNA probes were used for microbial investigation of ID and RC samples using checkerboard DNA-DNA hybridization. The levels and complexity of bacteria were similar in the ID and initial RC samples. The levels of LPS and LTA in ID were significantly higher than the initial RC samples (S1; P < 0.05). Canal preparation was effective in significantly decreasing the levels of bacteria, LPS and LTA (P < 0.05). ICM did not provide additional reduction in the levels of bacteria and LPS (P > 0.05). However, a significant reduction in the levels of LTA was observed after ICM (P < 0.05). CONCLUSION: The microbial profile of infected dentine and root canals of teeth with irreversible pulpitis was complex, harbouring different species including Gram-positive and Gram-negative, cocci and bacilli, and facultative and strict anaerobes. Root canal preparation was effective in reducing the levels of bacteria, LPS and LTA from the root canals of teeth with pulpitis.

Resolution of Nasal Sinus Tract after Endodontic Therapy: A Case Report with Microbial Analysis.

This case report describes the resolution of a 20-year misdiagnosed nasal sinus tract after root canal therapy with multiple sessions of calcium hydroxide (Ca[OH]2) intracanal medication. Clinical evaluation, including diagnostic testing and sinus tract tracing, was performed followed by a cone-beam computed tomographic scan and 3-dimensional reconstruction of the apical lesion. Bacteria and endotoxin analyses were performed from the nasal sinus tract and paired root canal infection before (s1) and after instrumentation (s2) and after 7 (s3), 14 (s4), and 21 (s5) days of Ca(OH)2 medication. The bacteria analysis was performed using the checkerboard DNA-DNA hybridization method and endotoxin quantified by the limulus amebocyte lysate method. A similar microbiota profile was found in the sinus tract and paired root canal infection. No target bacterial species were detected in the root canal at s2, s3, and s5. In contrast, Actinomyces israellii and Eubacterium nodatum were detected at s4. Differences in bacterial detection were found between s1 × s2, s3 × s4, and s4 × s5 (all P < .05). Endotoxin was detected in the root canal at all sampling times. Differences in the levels of endotoxin were found between s1 × s2, s2 × s3, and s3 × s4 (all P < .05).The bacterial analysis revealed similar microbiota profiles present in the nasal sinus tract and paired root canal infection with the participation of a wide variety of gram-positive and -negative species. Additionally, root canal therapy with multiple sessions of Ca(OH)2 intracanal medication for 21 days was effective in disinfecting the root canal system and resolving the nasal sinus tract.

Photosensitizers attenuate LPS-induced inflammation: implications in dentistry and general health.

Antimicrobial photodynamic therapy (aPDT) is a complementary therapeutic modality for periodontal and endodontic diseases, in which Gram-negative bacteria are directly involved. Currently, there are few evidences regarding the effects of aPDT on bacterial components such as lipopolysaccharide (LPS) and it would represent a major step forward in the clinical use of this therapy. In this context, this study aimed to evaluate the efficacy of different photosensitizers (PSs) used in aPDT in LPS inhibition. Four PSs were used in this study: methylene blue (MB), toluidine blue (TBO), new methylene blue (NMB), and curcumin (CUR). Different approaches to evaluate LPS interaction with PSs were used, such as spectrophotometry, Limulus amebocyte lysate (LAL) test, functional assays using mouse macrophages, and an in vivo model of LPS injection. Spectrophotometry showed that LPS decreased the absorbance of all PSs used, indicating interactions between the two species. LAL assay revealed significant differences in LPS concentrations upon pre-incubation with the different PSs. Interestingly, the inflammatory potential of LPS decreased after previous treatment with the four PSs, resulting in decreased secretion of inflammatory cytokines by macrophages. In vivo, pre-incubating curcumin with LPS prevented animals from undergoing septic shock within the established time. Using relevant models to study the inflammatory activity of LPS, we found that all PSs used in this work decreased LPS-induced inflammation, with a more striking effect observed for NMB and curcumin. These data advance the understanding of the mechanisms of LPS inhibition by PSs.