Liposome-based dry powder vaccine immunization targeting the lungs induces broad protection against pneumococcus.

Streptococcus pneumoniae is an important human pathogen. Currently used conjugate vaccines are effective against invasive disease, but protection is restricted to serotypes included in the formulation, leading to serotype replacement. Furthermore, protection against non-invasive disease is reported to be considerably lower. The development of a serotype-independent vaccine is thus important and Pneumococcal surface protein A (PspA) is a promising vaccine candidate. PspA shows some diversity and can be classified in 6 clades and 3 families, with families 1 and 2 being the most frequent in clinical isolates. The ideal vaccine should thus induce protection against the two most common families of PspA. The aim of this work was to develop a liposome-based vaccine containing PspAs from family 1 and 2 and to characterize its immune response. Liposomes (LP) composed of dipalmitoylphosphatidylcholine (DPPC) and 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) with or without α-galactosylceramide (α-GalCer) were produced by microfluidics, encapsulating PspA from clade 1 (PspA1, family 1) and/or clade 4 (PspA4Pro, family 2) followed by spray-drying with trehalose to form nanocomposite microparticles carriers (NCMP). LP/NCMPs showed good stability and preservation of protein activity. LP/NCMPs containing PspA1 and/or PspA4Pro were used for immunization of mice targeting the lungs. High serum IgG antibody titers against both PspA1 and PspA4Pro were detected in animals immunized with LP/NCMPs containing α-GalCer, with a balance of IgG1 and IgG2a titers. IgG in sera from immunized mice bound to pneumococcal strains from different serotypes and expressing different PspA clades, indicating broad recognition. Mucosal IgG and IgA were also detected. Importantly, immunization with LP/NCMPs induced full protection against strains expressing PspAs from family 1 and 2. Furthermore, CD4+ resident memory T cells were detected in the lungs of the immunized animals that survived the challenge.

Liposome-based dry powder vaccine immunization targeting the lungs induces broad protection against pneumococcus

Streptococcus pneumoniae is an important human pathogen. Currently used conjugate vaccines are effective against invasive disease, but protection is restricted to serotypes included in the formulation, leading to serotype replacement. Furthermore, protection against non-invasive disease is reported to be considerably lower. The development of a serotype-independent vaccine is thus important and Pneumococcal surface protein A (PspA) is a promising vaccine candidate. PspA shows some diversity and can be classified in 6 clades and 3 families, with families 1 and 2 being the most frequent in clinical isolates. The ideal vaccine should thus induce protection against the two most common families of PspA. The aim of this work was to develop a liposome-based vaccine containing PspAs from family 1 and 2 and to characterize its immune response. Liposomes (LP) composed of dipalmitoylphosphatidylcholine (DPPC) and 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) with or without α-galactosylceramide (α-GalCer) were produced by microfluidics, encapsulating PspA from clade 1 (PspA1, family 1) and/or clade 4 (PspA4Pro, family 2) followed by spray-drying with trehalose to form nanocomposite microparticles carriers (NCMP). LP/NCMPs showed good stability and preservation of protein activity. LP/NCMPs containing PspA1 and/or PspA4Pro were used for immunization of mice targeting the lungs. High serum IgG antibody titers against both PspA1 and PspA4Pro were detected in animals immunized with LP/NCMPs containing α-GalCer, with a balance of IgG1 and IgG2a titers. IgG in sera from immunized mice bound to pneumococcal strains from different serotypes and expressing different PspA clades, indicating broad recognition. Mucosal IgG and IgA were also detected. Importantly, immunization with LP/NCMPs induced full protection against strains expressing PspAs from family 1 and 2. Furthermore, CD4+ resident memory T cells were detected in the lungs of the immunized animals that survived the challenge.

Pharmacological blockage of the AHR-CYP1A1 axis: a call for in vivo evidence.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that can be activated by structurally diverse compounds arising from the environment and the microbiota and host metabolism. Expanding evidence has been shown that the modulation of the canonical pathway of AHR occurs during several chronic diseases and that its abrogation might be of clinical interest for metabolic and inflammatory pathological processes. However, most of the evidence on the pharmacological abrogation of the AHR-CYP1A1 axis has been reported in vitro, and therefore, guidance for in vivo studies is needed. In this review, we cover the state-of-the-art of the pharmacodynamic and pharmacokinetic properties of AHR antagonists and CYP1A1 inhibitors in different in vivo rodent (mouse or rat) models of disease. This review will serve as a road map for those researchers embracing this emerging therapeutic area targeting the AHR. Moreover, it is a timely opportunity as the first AHR antagonists have recently entered the clinical stage of drug development.

Protective effects of Hydrocotyle umbellata var. bonariensis Lam. (Araliaceae) on memory in sleep-impaired female mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Hydrocotyle umbellata var.bonariensis Lam. (Hb), popularly known in Brazil as acariçoba and outside Brazil Hb by a number of names including marsh-pennywort, and many-flower, has traditionally been used in Ayurvedic medicine in the retardation of aging (Rasayana effect). AIM OF THE STUDY: The present study evaluated the effect of Hb treatment before and during paradoxical sleep deprivation (PSD) and sleep restriction (SR) on learning, memory, and acetylcholinesterase (AChE) brain activity. MATERIAL AND METHODS: Adult Swiss nulliparous female mice were randomly distributed among the experimental groups. The treated groups received the aqueous solution of Hb leaves orally at concentrations of 500 and 1.000 mg/kg. PSD and SR were induced by the multiple platform method, in which the animals remained for 3-days in PSD or 15-days in SR for 22 h per day. The collection of the vaginal epithelium occurred daily to determine the estrous cycle. Body mass gain was determined. The animals were submitted to the passive avoidance test and were then euthanized for the collection of brain tissue and the determination of cerebral cholinesterase activity. RESULTS: The aqueous solution of Hb was associated with a significant reduction in cholinesterase activity at both doses in the SR model, and at the dose of 1.000 mg/kg in the PSD model. Regarding the learning and memory test, the PSD group treated with 1.000 mg/kg presented significant improvement, whereas in the SR experiment none of the treated-groups showed any improvement in learning and memory. In the analysis of SR/PSD interference and/or Hb treatment on the estrous cycle, it was possible to observe that the treatment acted as a protector in the SR group, maintaining a normal cycle. CONCLUSIONS: The analyses showed that Hb was safe to use during periods of SR or PSD, acting as an adaptogen for these situations, in addition to being able to reduce cholinesterase activity, which suggests its neuroprotective action. In relation to the estrous cycle, Hb can act as a protector in SR situations.

Regenerative potential, metabolic profile, and genetic stability of Brachypodium distachyon embryogenic calli as affected by successive subcultures.

Brachypodium distachyon, a model species for forage grasses and cereal crops, has been used in studies seeking improved biomass production and increased crop yield for biofuel production purposes. Somatic embryogenesis (SE) is the morphogenetic pathway that supports in vitro regeneration of such species. However, there are gaps in terms of studies on the metabolic profile and genetic stability along successive subcultures. The physiological variables and the metabolic profile of embryogenic callus (EC) and embryogenic structures (ES) from successive subcultures (30, 60, 90, 120, 150, 180, 210, 240, and 360-day-old subcultures) were analyzed. Canonical discriminant analysis separated EC into three groups: 60, 90, and 120 to 240 days. EC with 60 and 90 days showed the highest regenerative potential. EC grown for 90 days and submitted to SE induction in 2 mg L-1 of kinetin-supplemented medium was the highest ES producer. The metabolite profiles of non-embryogenic callus (NEC), EC, and ES submitted to principal component analysis (PCA) separated into two groups: 30 to 240- and 360-day-old calli. The most abundant metabolites for these groups were malonic acid, tryptophan, asparagine, and erythrose. PCA of ES also separated ages into groups and ranked 60- and 90-day-old calli as the best for use due to their high levels of various metabolites. The key metabolites that distinguished the ES groups were galactinol, oxaloacetate, tryptophan, and valine. In addition, significant secondary metabolites (e.g., caffeoylquinic, cinnamic, and ferulic acids) were important in the EC phase. Ferulic, cinnamic, and phenylacetic acids marked the decreases in the regenerative capacity of ES in B. distachyon. Decreased accumulations of the amino acids aspartic acid, asparagine, tryptophan, and glycine characterized NEC, suggesting that these metabolites are indispensable for the embryogenic competence in B. distachyon. The genetic stability of the regenerated plants was evaluated by flow cytometry, showing that ploidy instability in regenerated plants from B. distachyon calli is not correlated with callus age. Taken together, our data indicated that the loss of regenerative capacity in B. distachyon EC occurs after 120 days of subcultures, demonstrating that the use of EC can be extended to 90 days.

Genetic parameters and path analysis in cowpea genotypes grown in the Cerrado/Pantanal ecotone.

Estimating genetic parameters in plant breeding allows us to know the population potential for selecting and designing strategies that can maximize the achievement of superior genotypes. The objective of this study was to evaluate the genetic potential of a population of 20 cowpea genotypes by estimating genetic parameters and path analysis among the traits to guide the selection strategies. The trial was conducted in randomized block design with four replications. Its morphophysiological components, components of green grain production and dry grain yield were estimated from genetic use and correlations between the traits. Phenotypic correlations were deployed through path analysis into direct and indirect effects of morphophysiological traits and yield components on dry grain yield. There were significant differences (P < 0.01) between the genotypes for most the traits, indicating the presence of genetic variability in the population and the possibility of practicing selection. The population presents the potential for future genetic breeding studies and is highly promising for the selection of traits dry grain yield, the number of grains per pod, and hundred grains mass. A number of grains per green pod is the main determinant trait of dry grain yield that is also influenced by the cultivar cycle and that the selection for the dry grain yield can be made indirectly by selecting the green pod mass and green pod length.

Microscopic agglutination test on captive rattlesnakes : Data on serovars and titers.

The microscopic agglutination test (MAT) is considered the "golden standard" leptospirosis serodiagnostic test, but there is little information about it as it pertains to snakes. To fill this information gap, we provide data on serovars and titers of fifty-six Crotalus durissus collilineatus sera samples that tested positive by MAT (10.1016/j.actatropica.2016.02.006 (Rodrigues et al., 2016) [5]). These data are presented in a table, along with a description of the methodology used for sample collection and serologic testing.

Anti-Leptospira spp. antibodies in Crotalus durissus collilineatus kept in captivity and its zoonotic relevance.

Leptospirosis is a worldwide spread zoonosis that can affect all groups of vertebrates, including reptiles. Because it has been little studied in snakes, this study focused on determining the occurrence of anti-Leptospira spp. antibodies in 64 Crotalus durissus collilineatus kept in captivity and on identifying the most common serovars in these animals, using the microscopic agglutination test. Of these, almost 90% were positive and there were reactions to the 22 serovars used in the study. The most common serovar in these snakes was Javanica, Andamana and Patoc. Most frequent titers were 25 and 50, although high titers (such as 1600) were also recorded, despite the absence of clinical symptoms. The possibility should be considered of captive snakes serving as a serious source of leptospiral infection in humans, which is why it is essential to study, prevent and control the disease in breeding centers and serpentariums.

Hypertension is the metabolic syndrome component most strongly associated with microvascular complications and coronary artery calcification in Type 1 diabetes.

AIM: To evaluate the association of metabolic syndrome (MetS) and its individual components with microvascular complications and coronary artery calcification (CAC) in patients with Type 1 diabetes. MATERIAL/SUBJECTS AND METHODS: Cross-sectional study included 261 patients with Type 1 diabetes. Patients were assessed regarding the presence of MetS according to National Cholesterol Education Program (NCEP) criteria. CAC score was measured in a subset of 100 patients without known cardiovascular disease. RESULTS: The prevalence of MetS was 13.4% according to the NCEP criteria. Microvascular complications and CAC were more frequent in patients with MetS. In a multiple logistic regression analysis, MetS remained associated with nephropathy [OR: 6.33 (95% CI 2.54-15.77), p<0.001], but not with retinopathy and CAC. Among the MetS components, hypertension was associated with presence of retinopathy [OR: 4.04 (95% CI 1.65- 9.90), p=0.002], nephropathy [OR: 5.92 (95% CI 2.42-14.4), p<0.001] and CAC [OR: 2.97 (95% CI 1.06-8.30), p=0.03]. CONCLUSIONS: Hypertension was the only MetS component associated with retinopathy, nephropathy and the presence of CAC. Hypertension was better associated with CAC than MetS itself.

Coronary artery calcification is associated with insulin resistance index in patients with type 1 diabetes.

The objective of the present study was to evaluate the risk factors associated with the presence of coronary artery calcification (CAC) in patients with type 1 diabetes (T1D). A cross-sectional study was conducted on 100 consecutive T1D patients without coronary artery disease, with at least 5 years of diabetes and absence of end-stage renal disease. Mean age was 38 ± 10 years and 57% were males. CAC score was measured by multidetector computed tomography (Siemens Sensation 64 Cardiac). The insulin resistance index was measured using the estimated glucose disposal rate (eGDR). The eGDR was lower among CAC-positive patients than among CAC-negative patients, suggesting an increased insulin resistance. In a logistic regression model adjusted for age (at 10-year intervals), eGDR, diabetic nephropathy and gender, CAC was associated with age [OR = 2.73 (95%CI = 1.53-4.86), P = 0.001] and with eGDR [OR = 0.08 (95%CI = 0.02-0.21), P = 0.004]. In T1D subjects, insulin resistance is one of the most important risk factors for subclinical atherosclerosis.

Coronary artery calcification is associated with insulin resistance index in patients with type 1 diabetes

The objective of the present study was to evaluate the risk factors associated with the presence of coronary artery calcification (CAC) in patients with type 1 diabetes (T1D). A cross-sectional study was conducted on 100 consecutive T1D patients without coronary artery disease, with at least 5 years of diabetes and absence of end-stage renal disease. Mean age was 38 ± 10 years and 57 percent were males. CAC score was measured by multidetector computed tomography (Siemens Sensation 64 Cardiac). The insulin resistance index was measured using the estimated glucose disposal rate (eGDR). The eGDR was lower among CAC-positive patients than among CAC-negative patients, suggesting an increased insulin resistance. In a logistic regression model adjusted for age (at 10-year intervals), eGDR, diabetic nephropathy and gender, CAC was associated with age [OR = 2.73 (95 percentCI = 1.53-4.86), P = 0.001] and with eGDR [OR = 0.08 (95 percentCI = 0.02-0.21), P = 0.004]. In T1D subjects, insulin resistance is one of the most important risk factors for subclinical atherosclerosis.

Effect of metabolic syndrome and of its individual components on renal function of patients with type 2 diabetes mellitus

The objective of this study was to evaluate the effect of metabolic syndrome (MetS) and its individual components on the renal function of patients with type 2 diabetes mellitus (DM). A cross-sectional study was performed in 842 type 2 DM patients. A clinical and laboratory evaluation, including estimated glomerular filtration rate (eGFR) calculated by the modification of diet in renal disease formula, was performed. MetS was defined according to National Cholesterol Education Program - Adult Treatment Panel III criteria. Mean patient age was 57.9 ± 10.1 years and 313 (37.2 percent) patients were males. MetS was detected in 662 (78.6 percent) patients. A progressive reduction in eGFR was observed as the number of individual MetS components increased (one: 98.2 ± 30.8; two: 92.9 ± 28.1; three: 84.0 ± 25.1; four: 83.8 ± 28.5, and five: 79.0 ± 23.0; P < 0.001). MetS increased the risk for low eGFR (<60 mL·min-1·1.73 (m²)-1) 2.82-fold (95 percentCI = 1.55-5.12, P < 0.001). Hypertension (OR = 2.2, 95 percentCI = 1.39-3.49, P = 0.001) and hypertriglyceridemia (OR = 1.62, 95 percentCI = 1.19-2.20, P = 0.002) were the individual components with the strongest associations with low eGFR. In conclusion, there is an association between MetS and the reduction of eGFR in patients with type 2 DM, with hypertension and hypertriglyceridemia being the most important contributors in this sample. Interventional studies should be conducted to determine if treatment of MetS can prevent renal failure in type 2 DM patients.

Effect of metabolic syndrome and of its individual components on renal function of patients with type 2 diabetes mellitus.

The objective of this study was to evaluate the effect of metabolic syndrome (MetS) and its individual components on the renal function of patients with type 2 diabetes mellitus (DM). A cross-sectional study was performed in 842 type 2 DM patients. A clinical and laboratory evaluation, including estimated glomerular filtration rate (eGFR) calculated by the modification of diet in renal disease formula, was performed. MetS was defined according to National Cholesterol Education Program - Adult Treatment Panel III criteria. Mean patient age was 57.9 +/- 10.1 years and 313 (37.2%) patients were males. MetS was detected in 662 (78.6%) patients. A progressive reduction in eGFR was observed as the number of individual MetS components increased (one: 98.2 +/- 30.8; two: 92.9 +/- 28.1; three: 84.0 +/- 25.1; four: 83.8 +/- 28.5, and five: 79.0 +/- 23.0; P < 0.001). MetS increased the risk for low eGFR (<60 mL x min(-1) x 1.73 (m2)(-1)) 2.82-fold (95%CI = 1.55-5.12, P < 0.001). Hypertension (OR = 2.2, 95%CI = 1.39-3.49, P = 0.001) and hypertriglyceridemia (OR = 1.62, 95%CI = 1.19-2.20, P = 0.002) were the individual components with the strongest associations with low eGFR. In conclusion, there is an association between MetS and the reduction of eGFR in patients with type 2 DM, with hypertension and hypertriglyceridemia being the most important contributors in this sample. Interventional studies should be conducted to determine if treatment of MetS can prevent renal failure in type 2 DM patients.

Higher fibrinogen levels predict progression of coronary artery calcification in adults with type 1 diabetes.

AIM: To determine whether fibrinogen levels predict independently progression of coronary artery calcification (CAC) in adults with type 1 diabetes. METHODS: Data from a prospective cohort--the Coronary Artery Calcification in Type 1 Diabetes Study--were evaluated. Fibrinogen levels at baseline were separated into quartiles. CAC was measured twice and averaged at baseline and at follow-up 2.4+/-0.4 years later. CAC progressors were defined as participants whose square-root transformed CAC volume increased by >or=2.5 mm3 or development of clinical coronary artery disease during the follow-up period. RESULTS: Fibrinogen levels were higher in progressors than in non-progressors (276+/-61 mg/dl versus 259+/-61 mg/dl, p=0.0003). CAC progression, adjusted for known cardiovascular risk factors, increased in the highest quartile. CONCLUSIONS: Higher fibrinogen levels predict CAC progression in type 1 diabetes subjects, independent of standard cardiovascular risk factors.

Degree of catecholamine hypersecretion is the most important determinant of intra-operative hemodynamic outcomes in pheochromocytoma.

Pheochromocytoma resection is often complicated by intra-operative hypertension and post-resection hypotension. Factors associated with these hemodynamic alterations are not well defined. The aim of this study was to analyse the clinical-laboratory features associated with hemodynamic parameters during pheochromocytoma resection. Twenty-seven patients submitted to tumor resection - either open (no.=18) or video laparoscopic - between 1978-2007 were included. Nineteen received pre-operative alpha-blockers. Intra-operative hemodynamic data analysed were: maximum and minimum mean arterial blood pressure (MABP), no. of severe hypertensive (systolic BP >200 mmHg) and hypotensive episodes (MABP <60 mmHg), maximum and minimum heart rate (HR), no. of episodes of tachycardia and bradycardia, need to receive iv intra-operative treatment for hypertension and hypotension and the volume of fluids administered during surgery. Patients were 39.4+/-14.4-yr-old, 66% women. Intra-operative hemodynamic parameters were not different in patients submitted to open or video laparoscopic resection. Maximum intraoperative HR and the percentage of patients with HR>100 beats/min were higher in patients without pre-operative alpha- blocker treatment (no.=8). Pre-operative urinary vanylmandelic acid was positively associated with intra-operative maximum MABP (r=0.535, p=0.047) and with maximum transoperative systolic BP (r=0.805, p=0.016). Pre-operative urinary catecholamine (Pearson correlation r=0.575, p=0.03) and vanylmandelic acid (Pearson correlation r=0.605, p=0.04) levels were associated with maximum intra- operative MABP, adjusted for the presence of pheochromocytoma symptoms, surgical approach and pre-operative alpha-blockers. In conclusion, the degree of pre-operative catecholamine secretion was the most important aspect of transoperative BP control.

Diabetic retinopathy is associated with early autonomic dysfunction assessed by exercise-related heart rate changes

Diabetic retinopathy has been associated with cardiac autonomic dysfunction in both type 1 and type 2 diabetes mellitus (DM) patients. Heart rate (HR) changes during exercise testing indicate early alterations in autonomous tonus. The aim of the present study was to investigate the association of diabetic retinopathy with exercise-related HR changes. A cross-sectional study was performed on 72 type 2 and 40 type 1 DM patients. Autonomic dysfunction was assessed by exercise-related HR changes (Bruce protocol). The maximum HR increase, defined as the difference between the peak exercise rate and the resting rate at baseline, and HR recovery, defined as the reduction in HR from the peak exercise to the HR at 1, 2, and 4 min after the cessation of the exercise, were determined. In type 2 DM patients, lower maximum HR increase (OR = 1.62, 95 percentCI = 1.03-2.54; P = 0.036), lower HR recovery at 2 (OR = 2.04, 95 percentCI = 1.16-3.57; P = 0.012) and 4 min (OR = 2.67, 95 percentCI = 1.37-5.20; P = 0.004) were associated with diabetic retinopathy, adjusted for confounding factors. In type 1 DM, the absence of an increase in HR at intervals of 10 bpm each during exercise added 100 percent to the odds for diabetic retinopathy (OR = 2.01, 95 percentCI = 1.1-3.69; P = 0.02) when adjusted for DM duration, A1c test and diastolic blood pressure. In conclusion, early autonomic dysfunction was associated with diabetic retinopathy. The recognition of HR changes during exercise can be used to identify a high-risk group for diabetic retinopathy.

Diabetic retinopathy is associated with early autonomic dysfunction assessed by exercise-related heart rate changes.

Diabetic retinopathy has been associated with cardiac autonomic dysfunction in both type 1 and type 2 diabetes mellitus (DM) patients. Heart rate (HR) changes during exercise testing indicate early alterations in autonomous tonus. The aim of the present study was to investigate the association of diabetic retinopathy with exercise-related HR changes. A cross-sectional study was performed on 72 type 2 and 40 type 1 DM patients. Autonomic dysfunction was assessed by exercise-related HR changes (Bruce protocol). The maximum HR increase, defined as the difference between the peak exercise rate and the resting rate at baseline, and HR recovery, defined as the reduction in HR from the peak exercise to the HR at 1, 2, and 4 min after the cessation of the exercise, were determined. In type 2 DM patients, lower maximum HR increase (OR = 1.62, 95%CI = 1.03-2.54; P = 0.036), lower HR recovery at 2 (OR = 2.04, 95%CI = 1.16-3.57; P = 0.012) and 4 min (OR = 2.67, 95%CI = 1.37-5.20; P = 0.004) were associated with diabetic retinopathy, adjusted for confounding factors. In type 1 DM, the absence of an increase in HR at intervals of 10 bpm each during exercise added 100% to the odds for diabetic retinopathy (OR = 2.01, 95%CI = 1.1-3.69; P = 0.02) when adjusted for DM duration, A1c test and diastolic blood pressure. In conclusion, early autonomic dysfunction was associated with diabetic retinopathy. The recognition of HR changes during exercise can be used to identify a high-risk group for diabetic retinopathy.

Flow-injection spectrophotometric determination of paraoxon by its inhibitory effect on the enzyme acetylcholinesterase.

A spectrophotometric enzymatic flow injection (FI) system for the determination of diethyl-p-nitrophenylphosphate (paraoxon) is proposed. The method was based on the determination of the acetic acid formed by the enzymatic reaction of the acetylcholinesterase, immobilized on glass beads, with the substrate acetylcholine. The acetic acid formed permeates through a PTFE membrane and is received by a solution (pH 7.0) containing the acid-base indicator Bromocresol Purple (B.C.P.), leading to a pH change and therefore to a color change. The variation of the absorbance of the solution is detected spectrophotometrically at 400 nm. The determination of paraoxon is related to its inhibitory action on the enzyme. Therefore the analytical signal is the difference between the signal that corresponds to the free and the one that corresponds to the inhibited enzyme, considering a fixed acetylcholine concentration. The correlation between the peak height and paraoxon concentration at a given acetylcholine concentration is linear in the range from 5.0 x 10(-7) mol L-1 to 5.0 x 10(-5) mol L-1 (r = 0.998) of paraoxon, with a relative estimated standard deviation (R.S.D.) of +/- 1.7% (n = 10) considering a solution containing 5.0 x 10(-6) mol L-1 of paraoxon and a solution containing 5.0 x 10(-3) mol L-1 of acetylcholine. Therefore, the quantitative limit detection is about 2.5 x 10(-7) of paraoxon (3 sigma). A 1,1'-trimethylene-bis(4-formylpyridinium bromide)dioxime (TMB-4) solution was used to reactivate the enzyme.