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.

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.

Is dialysis water a safe component for hemodialysis treatment in São Paulo State, Brazil?

Failure on the water treatment poses hemodialysis patients at risk of injury and death. Identifying if the patients are exposed to water quality related microbiological risks is an important objective to reduce the mortality for chronic renal patients and is the main issue of this study. We evaluated the microbiological water quality used by 205 dialysis services in São Paulo State, Brazil between 2010 to 2016. The study included heterotrophic bacteria count, total coliforms research, and bacterial endotoxin determination in 1366 dialysis water samples. The number of unsatisfactory clinics for at least one microbiological parameter decreased 16.0% between 2010 to 2015 but increased 57.2% in 2016. In 2010, the most frequent unsatisfactory parameter was related to heterotrophic bacteria count (54.8%) followed by endotoxin determination (45.2%). However, in 2013 an opposite situation was observed: endotoxin determination as the parameter of the higher incidence of nonconformities. Total coliform was verified at a lower frequency. We highlighted the importance of regular monitoring of dialysis water quality to prevent infections caused by dialytic procedures and to ensure that the water is a safe component of the treatment.

Effect of edta and qmix ultrasonic activation on the reduction of microorganisms and endotoxins in ex vivo human root canals

Abstract The present study aimed to compare the effectiveness of QMiX and 17% EDTA associated to passive ultrasonic irrigation (PUI) or manual agitation (MA) on the reduction of E. faecalis, E. coli and LPS from root canals. Forty single rooted human teeth were randomly divided into four groups (n=10), according to the final irrigation protocol: EDTA+MA, QMiX+MA, EDTA+PUI, QMiX+PUI. Sample collections were obtained from the root canal content immediately before preparation (baseline-S1), after instrumentation (S2), after final irrigation protocol (S3) and 7 days after instrumentation and final irrigation (S4). The antimicrobial effectivity and on endotoxin content were analyzed by culture procedure (CFU/mL) and LAL assay (EU/mL), respectively. The results were statistically analyzed by Kruskal-Wallis and Friedman test (α=5%). QMiX+MA and QMiX+PUI reduced 100% of E. coli and E. faecalis bacteria and also prevented E. faecalisregrowth at S4. EDTA significantly reduced E. coli, but it was not effective in reducing E. faecalis. All protocols reduced EU/mL when compared to S1, however at S4 there was a significant reduction of EU/mL only in the QMiX+MA and QMiX+PUI groups in relation to S3 and S2, respectively. Final irrigation with QMiX associated with MA or PUI had superior antibacterial efficacy compared to EDTA, eliminating 100% of E. coli and E. faecalis strains. In addition, QMiX+PUI reduced 97.61% of the initial content of LPS.

Resumo O presente estudo objetivou comparar a eficácia do QMiX e do EDTA 17% associado à irrigação ultrassônica passiva (PUI) ou agitação manual (MA) na redução de E. faecalis, E. coli e LPS de canais radiculares. Quarenta dentes humanos unirradiculares foram divididos aleatoriamente em quatro grupos (n = 10), de acordo com o protocolo final de irrigação: EDTA+MA, QMiX+MA, EDTA+PUI, QMiX+PUI. Coletas das amostras foram obtidas a partir do conteúdo do canal radicular imediatamente antes do preparo (inicial-S1), após a instrumentação (S2), após o protocolo final de irrigação (S3) e 7 dias após a instrumentação e irrigação final (S4). A eficácia antimicrobiana e o conteúdo de endotoxina foram analisados ​​por procedimento de cultura (UFC/mL) e ensaio LAL (EU/mL), respectivamente. Os resultados foram analisados estatisticamente pelo teste de Kruskal-Wallis e Friedman (α = 5%). QMiX+MA e QMiX+PUI reduziram 100% das bactérias E. coli e E. faecalis e também preveniram a recolonização de E. faecalis em S4. O EDTA reduziu significativamente E. coli, mas não foi eficaz na redução de E. faecalis. Todos os protocolos reduziram EU/mL quando comparados com S1, no entanto, no S4 houve uma redução significativa de EU/mL apenas nos grupos QMiX+MA e QMiX+PUI em relação a S3 e S2, respectivamente. A irrigação final com QMiX associada a MA ou PUI apresentou eficácia antibacteriana superior em relação ao EDTA, eliminando 100% das cepas de E. coli e E. faecalis. Além disso, QMiX+PUI reduziu 97,61% do conteúdo inicial de LPS.

A Protocol to Perform Systemic Lipopolysacharide (LPS) Challenge in Rats / Protocolo para realizar reto sistémico con lipopolisacárido (LPS) en ratas

Abstract 19. Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria. In animals, intraperitoneal administration of LPS, stimulates innate immunity and the production of proinflammatory cytokines. LPS provides an inflammatory stimulus that activates the neuroimmune and neuroendocrine systems resulting in a set of responses termed sickness behavior. The purpose of this protocol is to describe step-by-step the preparation and procedure of application of intraperitoneal injection of LPS in rats, as a guide for those researchers that want to use this assay to mount an inflammatory response. LPS intraperitoneal challenge in rats has been widely used to evaluate antiinflammatory reagents and to address basic scientific questions.

Resumen 23. El lipopolisacárido (LPS) es un componente de la membrana externa de las bacterias Gram negativas. En animales, la administración intraperitoneal de LPS estimula la inmunidad innata y la producción de citoquinas proinflamatorias. El LPS proporciona un estímulo inflamatorio que activa el sistema neuroinmunológico y el sistema neuroendocrino, lo que da como resultado un conjunto de respuestas denominadas conductas de enfermedad. El propósito de este protocolo es describir paso a paso la preparación y el procedimiento de aplicación de la inyección intraperitoneal de LPS en ratas, como una guía para aquellos investigadores que desean utilizar este método para estimular una respuesta inflamatoria en el animal. La estimulación con LPS en ratas, aplicada intraperitonealmente, se ha utilizado ampliamente para evaluar reactivos antiinflamatorios y para abordar preguntas básicas de investigación científica.

Correlation between volume of root canal, cultivable bacteria, bacterial complexes and endotoxins in primary infection

Abstract This clinical study was conducted to correlate the levels of endotoxins and culturable bacteria found in primary endodontic infection (PEI) with the volume of root canal determined by using Cone Beam Computed Tomography (CBCT); and to evaluate the bacterial diversity correlating with clinical features. Twenty patients with PEI were selected and clinical features were recorded. The volume (mm3) of root canal was determined by CBCT analysis. Root canal samples were analyzed by using kinetic LAL-assay test to determine the levels of endotoxins and anaerobic technique to determine the bacterial count (CFU/mL). DNA was extracted from all samples to determine bacterial diversity and quantified by using Checkerboard-DNA-DNA- Hybridization. Culturable bacteria and endotoxins were detected in 100% of the root canal samples. Linear regression analysis revealed a correlation between root canal volume and presence of anaerobic bacteria (p<0.05). Positive correlations were found between bacteria species and presence of different clinical features (p<0.05). After grouping the bacteria species into bacterial complexes, positive associations were found between green, orange and red complexes with presence of sinus tract (p<0.05). This clinical study revealed that larger root canals hold higher levels of culturable bacteria in PEI. Thus, the interaction of different virulent bacteria species in complexes seems to play an important role in the development of clinical features.

Resumo Este estudo clínico foi conduzido para correlacionar os níveis de endotoxinas e bactérias cultiváveis encontradas na infecção endodôntica primária (IEP) com o volume do canal radicular determinado pelo uso da Tomografia Computadorizada de Feixe Cônico (TCFC); e avaliar a diversidade bacteriana correlacionada com características clínicas. Vinte pacientes com IEP foram selecionados e as características clínicas foram registradas. O volume (mm3) do canal radicular foi determinado pela análise TCFC. As amostras do canal radicular foram analisadas usando o teste cinético de análise LAL para determinar os níveis de endotoxinas e técnicas anaeróbicas para determinar a contagem bacteriana (UFC/mL). O DNA foi extraído de todas as amostras para determinar a diversidade bacteriana e quantificado utilizando o teste Checkerboard-DNA-DNA-Hybridization. Bactérias cultiváveis e endotoxinas foram detectadas em 100% das amostras do canal radicular. A análise de regressão linear revelou uma correlação entre o volume do canal radicular e a presença de bactérias anaeróbicas (p<0,05). Foram encontradas correlações positivas entre espécies de bactérias e presença de diferentes características clínicas (p<0,05). Após agrupamento das espécies dos micro-organismos em complexos bacterianos, foram encontradas associações positivas entre os complexos verde, laranja e vermelho com presença de fístula (p<0,05). Este estudo clínico revelou que os canais radiculares mais amplos possuem níveis mais elevados de bactérias cultiváveis na IEP. Assim, a interação de diferentes espécies de bactérias virulentas em complexos parece desempenhar um papel importante no desenvolvimento de características clínicas.

Avaliação do Teste de Ativação de Monócitos (MAT) para a detecção de pirógenos em Vacina Zika Inativada experimental

Injectable products including vaccines must be safe and pyrogen free. Pyrogens are molecules that once recognized by immune system can result in inflammatory responses of IL-1β e IL-6 cytokines release, leading to fever. In order to reduce number of laboratory animals, this study aimed to evaluate Monocyte Activation Test (MAT) efficiency at pyrogen detection in Zika Vaccine. The test was performed with PyroDetect System Merck Millipore, according supplier instructions. Zika vaccine was tested pure and diluted at 1/10, 1/20 and 1/40. Pyrogen recovery was tested by using spiked and non-spiked samples. After incubation with human cryopreserved blood Cryoblood® 16-18h, 37°C and CO2 5%, the incubation mixture blood and samples was collected to quantify IL-1β levels by ELISA. The results indicate low cytokine levels at tested samples, including spiked samples. This suggests vaccine had some interferers that might hinder the monocytes response. Furthermore, standard curves resulted lower absorbance values than expected, which would lead to misestimated endotoxin concentration in the samples. To assess test accuracy the calculated endotoxin recovery must be between 80 and 120% of spiked endotoxin. The results demonstrated variable recovery to same endotoxin concentration. We concluded this test seemed instable and it did not comply with accuracy and reproducibility parameters, for Zika Vaccine in the tested condition.

As vacinas, bem como todas as formulações injetáveis para uso humano, devem ser seguras e livres de pirogênio. Os pirógenos podem ser descritos como substâncias que, quando reconhecidas pelo sistema imune inato, desencadeiam respostas inflamatórias que resultam na liberação de citocinas, como IL-1β e IL-6, e podem ocasionar diversos sintomas, entre eles a febre. Com o intuito de substituir o uso de coelhos, esse estudo buscou verificar a eficiência do Teste de Ativação de Monócitos (MAT) para sua validação analítica na detecção de pirógenos, lipopolissacarídeos (LPS) e ácido lipoteicóico (LTA), no concentrado da Vacina Zika Inativada através da quantificação do mediador inflamatório IL-1β. Para isso, foi utilizado o PyroDetect System Merck Millipore, seguindo as orientações do fabricante. O concentrado da vacina Zika foi testado puro e em diluições de 1/10, 1/20 e 1/40, com e sem a contaminação por LPS ou LTA. As amostras foram incubadas com sangue humano criopreservado Cryoblood® – Kit PyroDetect por 16-18h a 37oC e, em seguida, foi realizado um ensaio de ELISA para quantificar os níveis de IL-1β liberados no sobrenadante. Os resultados dos testes indicaram baixos níveis de detecção de IL-1β nas diluições testadas, inclusive nas amostras que foram contaminadas por endotoxina. Esses resultados sugerem que a vacina Zika possa ter algum componente que cause interferência no teste. Além disso, verificou-se menor liberação de IL-1β nos ensaios do que o esperado, o que poderia levar a resultados superestimados da concentração de endotoxina nas amostras. Para determinar a exatidão do teste, foram calculadas as taxas de recuperação de endotoxina para cada ensaio, e estas deveriam estar em uma faixa entre 80 e 120% para a validação. No entanto, os resultados demonstraram taxas de recuperação fora da faixa esperada. Sendo assim, concluímos que os parâmetros de exatidão, precisão e reprodutibilidade não foram atingidos para a Vacina Zika nas condições testadas.

Aplicabilidade de métodos alternativos no monitoramento da qualidade microbiológica da água tratada para diálise / Applicability of alternative methods for the monitoring microbiological quality of treated water for dialysis

A fim de garantir a qualidade final de produtos os laboratórios de análise microbiológica fornecem dados sobre a qualidade dos mesmos em todas as suas etapas de produção. A crescente preocupação com a saúde dos pacientes conduz à busca de métodos que forneçam resultados precisos e rápidos, pois possibilitam que ações corretivas sejam tomadas em tempo real. O presente trabalho teve por objetivo avaliar o potencial de tecnologia alternativa no monitoramento de endotoxina bacteriana na água tratada para diálise e dialisato e avaliar o potencial da citometria de fluxo na análise de água. Para isso utilizou-se Portable Test System (PTS®) como método alternativo para detecção de endotoxina bacteriana no monitoramento da água tratada para diálise e dialisato, o qual foi validado frente ao método convencional farmacopeico. Paralelamente realizou-se revisão narrativa da literatura a fim de avaliar a aplicabilidade da citometria de fluxo em análises de água. A análise dos diferentes parâmetros de validação para endotoxina bacteriana no método alternativo mostrou que, exceto para a menor diluição analisada, houve linearidade e precisão nos resultados. Por outro lado a concentração de 0,25 UE/mL foi a menor que apresentou exatidão e especificidade. Observou-se ainda, que o limite de detecção foi de 0,125UE/mL e o de quantificação de 0,25 UE/mL, portanto o intervalo foi de 0,25-1,0 UE/mL. Adicionalmente pela análise de resistência pode-se perceber que ao variar analistas não houve diferença significativa. Em relação ao tempo de análise em uma condição de rotina laboratorial com muitas amostras, o PTS® mostrou-se demorado. Ressalta ainda, que seria importante que a legislação vigente determinasse a análise mensal de endotoxinas no dialisato. A revisão da literatura evidencia o potencial da tecnologia de citometria de fluxo, pois a mesma mostrou-se satisfatória quando comparada a metodologias convencionais para análise de água. O trabalho desenvolvido permitiu concluir que o PTS®) mostrou-se adequado para analisar amostras in loco, permitindo análises em tempo real, que para as quais haja a expectativa de ausência de endotoxinas ou de concentração respeitando o intervalo de 0,25 UE/mL a 1,0 UE/mL. Quanto a citometria de fluxo, esta mostrou-se uma tecnologia promissora em analisar amostras de água, sendo portanto recomendável proceder a estudos de validação e aplicabilidade

In order to guarantee the final quality of products, the microbiological analysis laboratories provide data about their quality at all production stages. The growing concern for patients' health leads to the search for methods that provide accurate and fast results, as they enable corrective actions to be taken in real time. The present work aimed to evaluate the alternative technology potential in the monitoring of bacterial endotoxin in treated water for dialysis and dialysate and to evaluate the potential of flow cytometry in water analysis. The different validation parameters analysis for bacterial endotoxin in alternative method showed that, except for the lowest dilution analyzed, there was linearity and precision in the results. On the other hand, the concentration of 0.25 EU / mL was the lowest that presented accuracy and specificity. It was further observed that the detection limit was 0.125UE / mL and the quantification limit was 0.25 EU / mL, so the range was 0.25-1.0 EU / mL. Additionally by the ruggedness analysis it was possible to perceived that when varying analysts there was no significant difference. Regarding the analysis time in a laboratory routine condition with many samples, the PTS® was was time consuming. It was also observed that it would be important to determine monthly analysis of endotoxins in dialysate. The literature review evidence the flow cytometry technology potential of the because it was satisfactory when compared to conventional methodologies for water analysis. The research showed that the PTS® was suitable for analyzing samples in loco, allowing real-time analyzes, for which there is expectation of endotoxins absence or concentration respecting the range of 0.25 EU / mL to 1.0 EU / mL. For the flow cytometry, it was shown to be a promising technology for analyzing water samples, and it is therefore advisable to carry out validation and applicability studies

Clinical and hematological changes in sheep induced by Escherichia coli lipopolysaccharide / alterações clínicas e hematológicas induzidas pela administração de lipopolisscarídeo de Escherichia coli em ovelhas / Alteraciones clínicas y hematológicas inducidas por la administración de lipopoliscárido de Escherichia coli en ovejas

Organic response to infection is characterized by a systemic reaction known as acute phase response (APR). In order to know the effect of the administration of Escherichia coli lipopolysaccharide (LPS) on physiological, hematological and biochemical variables, 10 sheep weighing 45 ± 5 kg were divided in two groups: Experimental group treated with 3 doses of 1 µg.kg-1 LPS and control group treated with saline solution (SS) at the same frequency as experimental group. Body temperature (BT°), heart rate (HR) and respiratory rate (RR) were monitored. Blood samples for hemogram and enzyme activity for aspartate amino transferase (AST) and gamma glutamyl transferase (GGT) were collected between 1 and 24 h post-LPS. LPS-treated sheep presented mean values of BT (41.2 ± 0.4°C), HR (132 ± 12.3 beats/min) and RR (107.2 ± 25 cycles/min) higher than those observed in control sheep (39.8 ± 0.2°C, 88.8 ± 8.7 beats/min and 53.6 ± 17.1 cycles/min respectively). Between 4 and 8 hours post-injection (hpi) of LPS the leukocyte count was associated with lymphopenia, followed by leukocytosis at 24 hours. No changes were observed in the activity of AST and GGT enzymes. The results characterize APR induced by LPS in sheep, representing a useful model to study cardiovascular, hematological and biochemical responses to infection. (AU)

A resposta orgânica à infecção é caracterizada por uma reação sistêmica conhecida como resposta de fase aguda (RFA). Para conhecer o efeito da administração de lipopolissacárideo (LPS) de Escherichia coli sobre as variáveis fisiológicas, hematológicas e bioquímicas, 10 carneiros pesando 45 ± 5 kg foram divididos em dois grupos: o grupo experimental tratado com três doses de 1 µg.kg -1 de LPS e grupo controle tratado com solução salina (SS) na mesma frequência que o grupo experimental. Temperatura corporal (T°C), frequência cardíaca (FC) e frequência respiratória (FR) foram monitoradas. As amostras de sangue foram tomadas para hemograma e atividade da enzima aspartato aminotransferase (AST) e gama- -glutamiltransferase (GGT) entre uma e 24 h pós-LPS. Ovelhas tratadas com LPS apresentaram valores médios de T°C (41,2 ± 0,4°C), FC (132 ± 12,3 batimentos/min) e FR (107,2 ± 25 ciclos/min) acima dos observados em ovelhas do tratamento controle (39,8 ± 0,2°C, 88,8 ± 8,7 batimentos/min e 53,6 ± 17,1 ciclos/min, respectivamente). Entre 4 e 8 horas após a injeção de LPS, a contagem de leucocitos foi asociado com linfopenia, seguida de leucocitose as 24 horas. Nenhuma mudança na atividade das enzimas AST e GGT foi observada. Os resultados caracterizam uma resposta de fase aguda induzida por LPS em ovelhas, o que representa um modelo útil para estudar os sistemas cardiovascular, hematológico e bioquímico em resposta à infecção.(AU)

La respuesta orgánica a la infección se caracteriza por una reacción sistémica conocida como respuesta de fase aguda (RFA). Para conocer el efecto de la administración de lipopolisacárido (LPS) de Escherichia coli sobre las variables fisiológicas, hematológicas y bioquímicas, 10 ovejas con un peso de 45 ± 5 kg se dividieron en dos grupos: grupo experimental tratado con 3 dosis de 1 µg.kg-1de LPS y grupo control tratado con solución salina (SS) en la misma frecuencia que el grupo experimental. Temperatura corporal (T°C), frecuencia cardíaca (FC) y frecuencia respiratoria (FR) fueron monitoreadas. Se tomaron muestras de sangre para hemograma y actividad enzimática para el aspartato amino transferasa (AST) y gamma glutamil transferasa (GGT) entre 1 y 24 h post-LPS. Las ovejas tratadas con LPS presentaron valores medios de T°C (41.2 ± 0.4°C), FC (132 ± 12.3 latidos / min) y FR (107.2 ± 25 ciclos/min) por encima de los observados en ovinos controles (39.8 ± 0.2°C, 88.8 ± 8.7 latidos/min y 53.6 ± 17.1 ciclos/min respectivamente). Entre las 4 y 8 horas después de la inyección de LPS, el recuento de leucocitos se asoció a linfopenia, seguida de leucocitosis a las 24 horas. No se observaron cambios en la actividad de las enzimas AST y GGT. Los resultados caracterizan una respuesta de fase aguda inducida por LPS en ovinos, representando un modelo útil para estudiar los sistemas cardiovascular, hematológico y bioquímico en respuesta a la infección.(AU)

Validação de metodologia analítica para determinação de endotoxina bacteriana em água para diálise por meio de método cromogênico cinético / Analytical methodology validation for determining the bacterialendotoxin in the dialysis water by means of kinetic chromogenic technique

O nível de endotoxina presente na água tratada para hemodiálise é um importante indicador dequalidade, uma vez que altas concentrações de endotoxina atuam como a principal fonte de inflamaçãocrônica em pacientes submetidos à diálise. Este estudo visa validar o método analítico para determinarquantitativamente a endotoxina bacteriana em amostras de água de hemodiálise pelo métodocromogênico cinético e de comparar com o método de coagulação em gel. Os ensaios pelo métodode coagulação em gel foram realizados de acordo com a Farmacopeia Brasileira em três amostrasde água de hemodiálise. A validação do método cromogênico cinético foi realizada utilizando-se asmesmas amostras por meio de sistema de teste portátil. As médias geométricas das concentrações dospontos finais obtidos nos testes de confirmação de sensibilidade LAL e de interferência por métodode coagulação em gel apresentaram resultado de 0,125 UE/mL. Os resultados obtidos pelo métodocromogênico para a recuperação do controle positivo do produto variaram de 89 a 186% e o coeficientede variação de 2,5 a 18,2%, demonstrando que as amostras não apresentaram interferência. Foramobtidos resultados equivalentes em ambos os métodos, o que permite a implementação do método emlaboratórios de saúde pública.

The occurrence of endotoxin in the treated water for hemodialysis is an important indicator of quality,since high concentrations of endotoxin constitute the main source for causing chronic inflammationin patients undergoing dialysis. This study aims at validating the analytical method for determiningquantitatively the bacterial endotoxin in hemodialysis water samples. The data from the kineticchromogenic method were compared with the results obtained from the gel coagulation technique.The gel coagulation assays were performed in three samples of hemodialysis water, according to theBrazilian Pharmacopoeia. The validation of the kinetic chromogenic method was performed usingthe same samples through the portable test system. The geometric means of the concentrations ofthe endpoints obtained from the tests for confirming the LAL sensitivity and the interference by gelcoagulation method showed a result of 0.125 EU/mL. The results obtained by the chromogenic methodfor recovering the product positive control varied from 89 to 186% and the coefficient of variationfrom 2.5 to 18.2%, demonstrating that the samples did not show interference. Equivalent results wereobtained in both methods, therefore being viable the implementation of this methodology in the publichealth laboratories.

Validação de metodologia analítica para determinação de endotoxina bacteriana em água para diálise por meio de método cromogênico cinético / Analytical methodology validation for determining the bacterial endotoxin in the dialysis water by means of kinetic chromogenic technique

O nível de endotoxina presente na água tratada para hemodiálise é um importante indicador de qualidade, uma vez que altas concentrações de endotoxina atuam como a principal fonte de inflamação crônica em pacientes submetidos à diálise. Este estudo visa validar o método analítico para determinar quantitativamente a endotoxina bacteriana em amostras de água de hemodiálise pelo método cromogênico cinético e de comparar com o método de coagulação em gel. Os ensaios pelo método de coagulação em gel foram realizados de acordo com a Farmacopeia Brasileira em três amostras de água de hemodiálise. A validação do método cromogênico cinético foi realizada utilizando-se as mesmas amostras por meio de sistema de teste portátil. As médias geométricas das concentrações dos pontos finais obtidos nos testes de confirmação de sensibilidade LAL e de interferência por método de coagulação em gel apresentaram resultado de 0,125 UE/mL. Os resultados obtidos pelo método cromogênico para a recuperação do controle positivo do produto variaram de 89 a 186% e o coeficiente de variação de 2,5 a 18,2%, demonstrando que as amostras não apresentaram interferência. Foram obtidos resultados equivalentes em ambos os métodos, o que permite a implementação do método em laboratórios de saúde pública.

The occurrence of endotoxin in the treated water for hemodialysis is an important indicator of quality, since high concentrations of endotoxin constitute the main source for causing chronic inflammation in patients undergoing dialysis. This study aims at validating the analytical method for determining quantitatively the bacterial endotoxin in hemodialysis water samples. The data from the kinetic chromogenic method were compared with the results obtained from the gel coagulation technique. The gel coagulation assays were performed in three samples of hemodialysis water, according to the Brazilian Pharmacopoeia. The validation of the kinetic chromogenic method was performed using the same samples through the portable test system. The geometric means of the concentrations of the endpoints obtained from the tests for confirming the LAL sensitivity and the interference by gel coagulation method showed a result of 0.125 EU/mL. The results obtained by the chromogenic method for recovering the product positive control varied from 89 to 186% and the coefficient of variation from 2.5 to 18.2%, demonstrating that the samples did not show interference. Equivalent results were obtained in both methods, therefore being viable the implementation of this methodology in the public health laboratories.

Continuous blood purification treatment for endotoxin-induced acute respiratory distress syndrome

This study aimed to explore the effects of continuous blood purification (CBP) treatment in pigs affected with acute respiratory distress syndrome (ARDS). A total of 12 healthy male pigs, weighing 12±1.8 kg, were randomly and equally assigned to the control and experimental groups. The ARDS pig model was prepared by intravenous injections of endotoxin (20 µg/kg). The control group was given conventional supportive therapy, while the experimental group was given continuous veno-venous hemofiltration therapy. During the treatment process, the variations in dynamic lung compliance, oxygenation index, hemodynamics, and urine volume per hour at different times (Baseline, 0, 2, 4, and 6 h) were recorded. The levels of tumor necrosis factor (TNF-α), interleukin 6 (IL-6), and IL-10 in serum and bronchoalveolar lavage fluid (BALF) were measured using the enzyme-linked immunosorbent assay. The histomorphological changes of the lung, heart, and kidney were visualized using a light microscope. The nuclear factor κB p65 protein content of the heart, lung, and kidney tissues was also detected using western blot. The experimental group outperformed the control group in both respiratory and hemodynamic events. CBP treatment cleared TNF-α, IL-6, and IL-10 partially from serum and BALF. The pathological examination of the heart, lung, and kidney tissues revealed that the injury was less severe in the experimental group. CBP treatment can improve the organ functions of pigs affected with endotoxin-induced ARDS and protect these organs to some extent.

Investigation of Bacterial Contents From Persistent Endodontic Infection and Evaluation of Their Inflammatory Potential

Abstract This clinical study investigated and quantified cultivable bacteria and their levels of endotoxins in persistent endodontic infection, determining their antigenicity against macrophages and fibroblast cells by IL-1β and TNF-α secretion and evaluating their relationship with clinical and radiographic features. Samples from the root canals were obtained after root filling removal. Culture techniques were used to determine the bacterial count and the endotoxins were determined by LAL-assay. PCR analysis (16S rDNA) was used for bacterial detection. Raw 264.5 macrophages and V79 fibroblast were stimulated with endodontic contents. ELISA assay measured the amounts of IL-1ß/TNF-?#61537; secretion. Bacteria and endotoxin medians were 1.24x105 CFU/mL and 9.62 EU/mL, respectively. Porphyromonas endodontalis was the most frequently detected species. Higher levels of endotoxins were found in teeth with pain on palpation (23.56 EU/mL) rather than in its absence (8.21 EU/mL). Larger areas of bone destruction were related to higher levels of endotoxins and IL-1β and TNF-α secretion. The study findings revealed the presence of Gram-negative bacteria species in persistent endodontic infection, with their endotoxins related to both severity of bone destruction and development of symptomatology. Moreover, larger areas of bone destruction were related to higher levels of IL-1β and TNF-α secreted by macrophages and fibroblast cells.

Resumo Este estudo clínico investigou e quantificou bactérias cultiváveis e seus níveis de endotoxinas na infecção endodôntica persistente, determinando a sua antigenicidade contra macrófagos e células de fibroblastos através de IL-1β e TNF-α; e avaliando sua relação com características clínicas e radiográficas. As amostras dos canais radiculares foram obtidas após a desobturação. Técnicas de cultura foram utilizadas para determinar a contagem de bactérias e a quantificação de endotoxinas foram determinadas por ensaio de LAL. Análise por PCR (16S rDNA) foi utilizada para a detecção bacteriana. Células 264,5 macrófagos e fibroblastos V79 foram estimuladas com conteúdo endodôntico. IL-1β e TNF-α produzidas pelas células avaliadas foram medidas por ensaio de ELISA. As medianas de bactérias e endotoxinas foram 1,24x105 UFC/mL e 9,62 EU/mL, respectivamente. Porphyromonas endodontalis foi a espécie mais frequentemente detectada. Níveis mais elevados de endotoxinas foram encontrados em dentes com dor à palpação (23,56 EU/mL) quando comparado a sua ausência (8,21 EU/mL). Maiores áreas de destruição óssea foram relacionados com níveis mais elevados de endotoxinas e IL-1β e TNF-α. O estudo revelou presença de espécies de bactérias Gram-negativas em infecção endodôntica persistente, com níveis elevados de endotoxinas relacionados a maior destruição óssea periapical e presença de sintomatologia. Além disso, grandes áreas de destruição óssea foram relacionados com níveis mais elevados de IL-1β e TNF-α secretadas por macrófagos e fibroblastos.

Comparison of Fusobacterium nucleatum and Porphyromonas gingivalis Lipopolysaccharides Clinically Isolated from Root Canal Infection in the Induction of Pro-Inflammatory Cytokines Secretion

Abstract The aim of this study was to compare the biological activity of lipopolysaccharides (LPS) purified from Fusobacterium nucleatum and Porphyromonas gingivalis strains, both isolated from primary endodontic infection (PEI) in the levels of IL-1β and TNF-α released by macrophage cells. Moreover, LPS was purified from F. nucleatum and P. gingivalis American Type Collection (ATCC) and its biological activity was compared to respectively clinical isolates strains. F. nucleatum and P. gingivalis strains clinically isolated from PEI had their identification confirmed by sequencing the 16S rRNA gene. LPS from F. nucleatum and P. gingivalis and their respective ATCC strains were extracted by using Tri-reagent method. Macrophages (Raw 264.7) were stimulated with LPS at 100 ng/mL for 4, 8 and 12 h. Secretion of IL-1 β and TNF-α was also determined. Paired t-test, repeated measures ANOVA and one-way ANOVA were employed. All LPS induced significant production of IL-1β and TNF-α, with the former being secreted at higher levels than the latter in all time-points. F. nucleatum induced a higher expression of both cytokines compared to P. gingivalis (p<0.05). No differences were observed between clinical and ATCC strains, as both presented the same potential to induce pro-inflammatory response. It was concluded that F. nucleatum and P. gingivalis LPS presented different patterns of activation against macrophages as seen by the IL-1β and TNF-α production, which may contribute to the immunopathogenesis of apical periodontitis. Moreover, clinical and ATCC strains grown under the same in vitro environment conditions presented similar biological activity.

Resumo O objetivo deste estudo foi comparar a atividade biológica de lipopolissacarídeos (LPS) purificados a partir de linhagens de Fusobacterium nucleatum e Porphyromonas gingivalis, ambas isoladas de infecções endodônticas primárias (IEP) nos níveis de IL-1β e TNF-α produzidos por macrófagos. Adicionalmente, LPS foi purificado de F. nucleatum e P. gingivalis "American Type Collection" (ATCC) e sua atividade comparada às respectivas linhagens clinicamente isoladas. Linhagens de F. nucleatum e P. gingivalis isoladas clinicamente de IEP tiveram sua identificação confirmada por sequenciamento do gene 16S rRNA. LPS de F. nucleatum e P. gingivalis e das respectivas linhagens foram extraídos com o uso do método "Tri-reagent". Macrófagos (Raw 264.7) foram estimulados com LPS a 100 ng/mL por 4, 8 e 12 h. A secreção de IL-1β e de TNF-α foi determinada. Foram usados os testes t-pareado, ANOVA de medidas repetidas e ANOVA de um fator. Todos os LPS induziram a produção significante de IL-1β e TNF-α, sendo o primeiro secretado em mais altas concentrações que o último em todos os tempos avaliados. F. nucleatum induziu uma maior expressão de ambas as citocinas comparativamente ao P. gingivalis (p<0,05). Não foram observadas diferenças entre as linhagens clínica e ATCC, uma vez que ambas apresentaram o mesmo potencial de indução da resposta pró-inflamatória. Conclui-se que F. nucleatum e P. gingivalis possuem diferentes padrões de ativação dos macrófagos, como visto pela produção de IL-1β e TNF-α, o que pode contribuir para a imunopatogênese da periodontite apical. Ainda, linhagens clínica e ATCC mantidas no mesmo ambiente in vitro apresentaram ativação biológica semelhante.

Purificación y caracterización de lipopolisacáridos de Eikenella corrodens 23834 y Porphyromonas gingivalis W83 / Purification and characterization of lipopolysaccharide from Eikenella corrodens 23834 and Porphyromonas gingivalis W83

La purificación de lipopolisacáridos (LPS) o endotoxinas y su caracterización es un aspecto esencial para estudios que buscan aclarar el papel de estas biomoléculas de bacterias Gram negativas presentes en la cavidad oral y su relación con enfermedades locales periodontales y sistémicas. Este estudio implementa una metodología para la extracción, purificación y caracterización de LPS a partir de bacteria completa de Eikenella corrodens 23834 y Porphyromonas gingivalis W83, utilizando técnicas previamente descritas. La extracción cruda de LPS se realizó con fenol-agua caliente; la purificación se realizó con tratamiento enzimático con nucleasas y proteasa, seguido de cromatografía de exclusión por tamaño (Sephacryl S-200 HR) con deoxicolato de sodio como fase móvil. La caracterización de los extractos purificados se realizó por barrido espectrofotométrico, pruebas bioquímicas de electroforesis SDS-PAGE, ensayo Purpald y la prueba cromogénica de LAL. Como control para la identificación y caracterización de los extractos purificados se utilizaron LPS comerciales de Escherichia coli, Salmonella typhimurium, Rodobacter sphaeroides y Porphyromonas gingivalis. La metodología implementada permitió la obtención de LPS de elevada pureza con la identificación de KDO o heptosas, un quimiotipo de LPS-S (liso) para E. corrodens y LPS-SR (semi-rugoso) para P. gingivalis W83. Ambos LPS purificados mostraron capacidad endotóxica a bajas concentraciones. La metodología implementada en este estudio para la purificación y caracterización de LPS a partir de bacteria completa fue eficiente al compararla con los LPS comerciales.

Purification of lipopolysaccharide (LPS) or endotoxins and its characterization is an important aspect for studies aimed at clarify the role of these biomolecules from Gram negative bacteria present in the oral cavity and its relationship with periodontal and systemic diseases. This study describes an extraction, purification and characterization method of LPS from Eikenella corrodens 23834 and Porphyromonas gingivalis W83. LPS extraction was performed by using hot phenol-water; the purification was done with nuclease and protease enzymatic treatment, followed by size-exclusion chromatography (Sephacryl S-200 HR) with sodium deoxycholate as mobile phase. The characterization of the purified extracts was performed by spectrophotometric scanning, SDS-PAGE biochemical tests, Purpald assay and chromogenic LAL test. As control, commercial LPS from Escherichia coli, Salmonella typhimurium, P. gingivalis, and Rodobacter sphaeroides were used. The methodology mentioned above had allowed obtaining high purity LPS by identifying KDO or heptoses, a chemotype S-LPS (smooth) to E. corrodens; SR-LPS (semi-rough) for P. gingivalis W83. Both purified LPS showed endotoxic capacity at low concentrations. The methodology used in this study for purification and characterization of LPS from the whole bacteria was efficient when it was compared with commercial LPS.

Neonatal lipopolysaccharide exposure induces sexually dimorphic sickness behavior in adult rats

The aim of the present study was to evaluate whether neonatal exposure to lipopolysaccharide (LPS; 50 µg/kg, i.p., on postnatal day 2) induces depressive- and/or anxiety-like effects and sexually dimorphic responses in rats challenged with LPS (100 µg/kg, i.p.) in adulthood. The results revealed that males presented a less depressive state in the forced swim test and exhibited no changes in general motor activity in the open field test. Females exhibited an increase in sickness behavior, revealing different behavioral strategies in response to a bacterial disease. The male rats also exhibited higher cell proliferation, reflected by bone marrow and peripheral blood counts, and female rats exhibited a decrease in corticosterone levels. No changes were observed in the elevated plus maze or peripheral cytokine levels (interleukin-1β and tumor necrosis factor-α). Neonatal exposure to LPS induced sexually dimorphic behavioral, neuroendocrine, and immune effects after an LPS challenge in adulthood, differentially affecting male and female susceptibility to disease later in life...

Mesenteric lymph reperfusion exacerbates spleen injury caused by superior mesenteric artery occlusion shock

The intestinal lymph pathway plays an important role in the pathogenesis of organ injury following superior mesenteric artery occlusion (SMAO) shock. We hypothesized that mesenteric lymph reperfusion (MLR) is a major cause of spleen injury after SMAO shock. To test this hypothesis, SMAO shock was induced in Wistar rats by clamping the superior mesenteric artery (SMA) for 1 h, followed by reperfusion for 2 h. Similarly, MLR was performed by clamping the mesenteric lymph duct (MLD) for 1 h, followed by reperfusion for 2 h. In the MLR+SMAO group rats, both the SMA and MLD were clamped and then released for reperfusion for 2 h. SMAO shock alone elicited: 1) splenic structure injury, 2) increased levels of malondialdehyde, nitric oxide (NO), intercellular adhesion molecule-1, endotoxin, lipopolysaccharide receptor (CD14), lipopolysaccharide-binding protein, and tumor necrosis factor-α, 3) enhanced activities of NO synthase and myeloperoxidase, and 4) decreased activities of superoxide dismutase and ATPase. MLR following SMAO shock further aggravated these deleterious effects. We conclude that MLR exacerbates spleen injury caused by SMAO shock, which itself is associated with oxidative stress, excessive release of NO, recruitment of polymorphonuclear neutrophils, endotoxin translocation, and enhanced inflammatory responses.

Edaravone protects endotoxin-induced liver injury by inhibiting apoptosis and reducing proinflammatory cytokines

Studies have shown that edaravone may prevent liver injury. This study aimed to investigate the effects of edaravone on the liver injury induced by D-galactosamine (GalN) and lipopolysaccharide (LPS) in female BALB/c mice. Edaravone was injected into mice 30 min before and 4 h after GalN/LPS injection. The survival rate was determined within the first 24 h. Animals were killed 8 h after GalN/LPS injection, and liver injury was biochemically and histologically assessed. Hepatocyte apoptosis was measured by TUNEL staining; proinflammatory cytokines [tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)] in the liver were assayed by ELISA; expression of caspase-8 and caspase-3 proteins was detected by Western blot assay; and caspase-3 activity was also determined. Results showed that GalN/LPS induced marked elevations in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Edaravone significantly inhibited elevation of serum AST and ALT, accompanied by an improvement in histological findings. Edaravone lowered the levels of TNF-α and IL-6 and reduced the number of TUNEL-positive cells. In addition, 24 h after edaravone treatment, caspase-3 activity and mortality were reduced. Edaravone may effectively ameliorate GalN/LPS-induced liver injury in mice by reducing proinflammatory cytokines and inhibiting apoptosis.

Preferential protection of domains II and III of bacillus thuringiensis cry1Aa toxin by brush border membrane vesicles / Protección preferencial de los dominios II y III de la toxina cry1Aa de bacillus thuringiensis en vesículas de membrana de borde de cepillo

The surface exposed Leucine 371 on loop 2 of domain II, in Cry1Aa toxin, was mutated to Lysine to generate the trypsin-sensitive mutant, L371K. Upon trypsin digestion L371K is cleaved into approximately 37 and 26 kDa fragments. These are separable on SDS-PAGE, but remain as a single molecule of 65 kDa upon purification by liquid chromatography. The larger fragment is domain I and a portion of domain II (amino acid residues 1 to 371). The smaller 26-kDa polypeptide is the remainder of domain II and domain III (amino acids 372 to 609). When the mutant toxin was treated with high dose of M. sexta gut juice both fragments were degraded. However, when incubated with M. sexta BBMV, the 26 kDa fragment (domains II and III) was preferentially protected from gut juice proteases. As previously reported, wild type Cry1Aa toxin was also protected against degradation by gut juice proteases when incubated with M. sexta BBMV. On the contrary, when mouse BBMV was added to the reaction mixture neither Cry1Aa nor L371K toxins showed resistance to M. sexta gut juice proteases and were degraded. Since the whole Cry1Aa toxin and most of the domain II and domain III of L371K are protected from proteases in the presence of BBMV of the target insect, we suggest that the insertion of the toxin into the membrane is complex and involves all three domains.

La superficie de la toxina Cry1Aa, en el asa 2 del dominio II contiene expuesta la leucina 371, la cual fue modificada a lisina produciendo una mutante sensible a la tripsina, L371K. Esta mutante produce dos fragmentos de 37 y 26 kDa por acción de la tripsina que son separables por SDS-PAGE, pero que a la purificación por cromatografía líquida se mantienen como una sola molécula de 65 kDa. El fragmento grande contiene al dominio I y una parte del dominio II (aminoácidos 1 al 371). El polipéptido de 26 kDa contiene la parte restante del dominio II y dominio III (aminoácidos 372 al 609). Cuando la toxina mutante fue tratada con dosis altas de jugo intestinal de Manduca sexta, ambos fragmentos fueron degradados. Sin embargo, cuando fueron incubados en VMBC de M. sexta, el fragmento de 26 kDa fue protegido preferencialmente de las proteasas intestinales. Como se ha reportado, la toxina silvestre Cry1Aa también es protegida de la degradación de las proteasas cuando es incubada en VMBC de M. sexta. Sin embargo, cuando se adicionó VMBC de ratón a la mezcla de reacción, ni la toxina Cry1Aa ni la mutante L371K mostraron resistencia a las proteasas y fueron degradadas. Dado que la toxina completa de Cry1Aa y casi todo de los dominios II y III de L371K están protegidos de proteasas en presencia de VMBC del insecto, este estudio sugiere que la inserción de la toxina en la membrana involucra los tres dominios.

Influence of radiation on endotoxin test using the PTS TM for 18-FDG radiopharmaceutical / Influência da radiação sobre o teste de endotoxina usando o PTS TM para 18 radiofármaco FDG

F-18 FDG (2-[18-F] fluoro-2-deoxy-D-glucose) is the most frequently used radiopharmaceutical for PET and PET CT imaging exams. The FDA recently approved the use of the PTS TM (Portable Test System) as an alternative to the standard test proposed by the United States Pharmacopeia using the LAL (Limulus Amebocyte Lysates), that takes longer to perform (about 1h) than the PTS TM (15 min). Recent studies have demonstrated that radiation could interfere with the PTS TM test. In order to study the effects of radiation on the PTS TM test and/or equipment, 27 batches of F-18 FDG produced in the Nuclear Engineering Institute were analyzed. The results showed that no direct correlation with radiation was found in any of the cases.

O FDG-18 é o radiofármaco mais utilizado nos exames de PET e PET CT. O FDA recentemente aprovou o uso do PTS TM (Portable Test System) como método alternativo ao teste padrão de endotoxina, proposto pela Farmacopéia Americana, considerando que no primeiro há um tempo de espera de 1 hora frente a somente 15 minutos do segundo. Estudo recentes demonstram que a radiação poderia interferir no teste do PTS TM. De modo a avaliar os efeitos da radiação no teste PTS TM foram analisados 27 lotes de F-18 FDG produzidos no Instituto de Engenharia Nuclear. Os resultados demonstraram que em todos os casos nenhuma correlação direta com a radiação foi observada.