Low temperature-synthesized MgAl2 O4 :Eu3+ nanophosphors and their structural validations using density functional theory: photoluminescence, photocatalytic, and electrochemical properties for multifunctional applications.

A low temperature-assisted and oxalyl dihydrazide fuel-induced combustion synthesized series of uncalcined MgAl2 O4 :Eu3+ nanophosphors showed an average crystallite size of ~20 nm, and bandgap energy (Eg ) of 4.50-5.15 eV, and were validated using density functional theory and found to match closely with the experimental values. The photoluminescence characteristic emission peaks of Eu3+ ions were recorded between 480 and 680 nm. The nanophosphors excited at 392 nm showed f-f transitions assigned as 5 D0 →7 FJ (J = 0, 1, 2, and 3). The optimized MgAl2 O4 phosphors had Commission Internationale de l'Eclairage coordinates in the red region, a correlated colour temperature of 2060 K, and a colour purity of 98.83%. The estimated luminescence quantum efficiency ( η ) was observed to be ~63% using Judd-Ofelt analysis. Electrochemical and photocatalytic performance were explored and indicated its multifunctional applications. Therefore, MgAl2 O4 :Eu3+ nanophosphors could be used for the fabrication of light-emitting diodes, industrial dye degradation, and as electrodes for supercapacitor applications.

Regulatory Considerations on the Development of mRNA Vaccines.

Developing traditional viral vaccines for infectious diseases usually takes years, as these are usually produced either by chemical inactivation of the virus or attenuation of the pathogen, processes that can take considerable time to validate and also require the live pathogen. With the advent of nucleic-acid vaccines (DNA and mRNA), the time to vaccine design and production is considerably shortened, since once the platform has been established, all that is required is the sequence of the antigen gene, its synthesis and insertion into an appropriate expression vector; importantly, no infectious virus is required. mRNA vaccines have some advantages over DNA vaccines, such as expression in non-dividing cells and the absence of the perceived risk of integration into host genome. Also, generally lower doses are required to induce the immune response. Based on experience in recent clinical trials, mRNA-based vaccines are a promising novel platform that might be useful for the development of vaccines against emerging pandemic infectious diseases. This chapter discusses some of the specific issues that mRNA vaccines raise with respect to production, quality, safety and efficacy, and how they have been addressed so as to allow their evaluation in clinical trials.

Monoclonal antibodies to fetal bovine serum acetylcholinesterase distinguish between acetylcholinesterases from ruminant and non-ruminant species.

Two types of cholinesterases (ChEs) are present in mammalian blood and tissues: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). While AChE regulates neurotransmission by hydrolyzing acetylcholine at the postsynaptic membranes and neuromuscular junctions, BChE in plasma has been suggested to be involved in detoxifying toxic compounds. This study was undertaken to establish the identity of circulating ChE activity in plasmas from domestic animals (bovine, ovine, caprine, porcine and equine) by assessing sensitivity to AChE-specific inhibitors (BW284c51 and edrophonium) and BChE-specific inhibitors (dibucaine, ethopropazine and Iso-OMPA) as well as binding to anti-FBS AChE monoclonal antibodies (MAbs). Based on the inhibition of ChE activity by ChE-specific inhibitors, it was determined that bovine, ovine and caprine plasma predominantly contain AChE, while porcine and equine plasma contain BChE. Three of the anti-FBS AChE MAbs, 4E5, 5E8 and 6H9, inhibited 85-98% of enzyme activity in bovine, ovine and caprine plasma, confirming that the esterase in these plasmas was AChE. These MAbs did not bind to purified recombinant human or mouse AChE, demonstrating that these MAbs were specific for AChEs from ruminant species. These MAbs did not inhibit the activity of purified human BChE, or ChE activity in porcine and equine plasma, confirming that the ChE in these plasmas was BChE. Taken together, these results demonstrate that anti-FBS AChE MAbs can serve as useful tools for distinguishing between AChEs from ruminant and non-ruminant species and BChEs.

Predictive modeling of hypoglycemia for clinical decision support in evaluating outpatients with diabetes mellitus.

Objective: Hypoglycemia occurs in 20-60% of patients with diabetes mellitus. Identifying at-risk patients can facilitate interventions to lower risk. We sought to develop a hypoglycemia prediction model. Methods: In this retrospective cohort study, urban adults prescribed a diabetes drug between 2004 and 2013 were identified. Demographic and clinical data were extracted from an electronic medical record (EMR). Laboratory tests, diagnostic codes and natural language processing (NLP) identified hypoglycemia. We compared multiple logistic regression, classification and regression trees (CART), and random forest. Models were evaluated on an independent test set or through cross-validation. Results: The 38,780 patients had mean age 57 years; 56% were female, 40% African-American and 39% uninsured. Hypoglycemia occurred in 8128 (539 identified only by NLP). In logistic regression, factors positively associated with hypoglycemia included infection, non-long-acting insulin, dementia and recent hypoglycemia. Negatively associated factors included long-acting insulin plus sulfonylurea, and age 75 or older. The models' area under curve was similar (logistic regression, 89%; CART, 88%; random forest, 90%, with ten-fold cross-validation). Conclusions: NLP improved identification of hypoglycemia. Non-long-acting insulin was an important risk factor. Decreased risk with age may reflect treatment or diminished awareness of hypoglycemia. More complex models did not improve prediction.

Implementation of Basal-Bolus Therapy in Type 2 Diabetes: A Randomized Controlled Trial Comparing Bolus Insulin Delivery Using an Insulin Patch with an Insulin Pen.

Background: Barriers to mealtime insulin include complexity, fear of injections, and lifestyle interference. This multicenter, randomized controlled trial evaluated efficacy, safety, and self-reported outcomes in adults with type 2 diabetes, inadequately controlled on basal insulin, initiating and managing mealtime insulin with a wearable patch versus an insulin pen. Methods: Adults with type 2 diabetes (n = 278, age: 59.2 ± 8.9 years), were randomized to patch (n = 139) versus pen (n = 139) for 48 weeks, with crossover at week 44. Baseline insulin was divided 1:1 basal: bolus. Using a pattern-control logbook, subjects adjusted basal and bolus insulin weekly using fasting and premeal glucose targets. Results: Glycated hemoglobin (HbA1c) change (least squares mean ± standard error) from baseline to week 24 (primary endpoint) improved (P < 0.0001) in both arms, -1.7% ± 0.1% and -1.6% ± 0.1% for patch and pen (-18.6 ± 1.1 and -17.5 ± 1.1 mmol/mol), and was maintained at 44 weeks. The coefficient of variation of 7-point self-monitoring blood glucose decreased more (P = 0.02) from baseline to week 44 for patch versus pen. There were no differences in adverse events, including hypoglycemia (three severe episodes per arm), and changes in weight and insulin doses. Subject-reported treatment satisfaction, quality of life, experience ratings at week 24, and device preferences at week 48 significantly favored the patch. Most health care providers preferred patch for mealtime insulin. Conclusions: Bolus insulin delivered by patch and pen using an algorithm-based weekly insulin dose titration significantly improved HbA1c in adults with type 2 diabetes, with improved subject and health care provider experience and preference for the patch.

New design of highly sensitive and selective MoO3:Eu3+ micro-rods: Probing of latent fingerprints visualization and anti-counterfeiting applications.

Red colour light emitting Eu3+ ions activated MoO3 nanophosphors (NPs) were prepared through an ultrasound assisted sonochemical method using Aloe Vera (A.V.) gel as a bio-surfactant. Properties like crystal structure, morphology, optical band gap, luminescent properties, radiative parameters of prepared samples and their use in latent fingerprint (LFPs) visualization were reported. Powder X-ray diffraction results revealed the single orthorhombic crystal structure of the prepared samples, which specifies effective substitution of dopant ions. Morphology of NPs exhibits the hexagonal rod-like structures with size of ∼10 nm. Photoluminescence (PL) emission spectra exhibited sharp, intense peaks at ∼539 nm, 593 nm, 615 nm, 651 nm and 702 nm attributed to 5D0 → 7F0, 5D0 → 7F1, 5D0 → 7F2, 5D0 → 7F3 and 5D0 → 7F4 transitions of Eu3+ ions respectively. Judd-Ofelt (J-O) theory was used to estimate the PL intensity parameters and Eu-O ligand behavior. The International Commission on Illumination coordinates of the prepared samples located in the pure red region. The optimized sample can be explored as a novel sensing material for the visualization of LFPs on various surfaces under 254 nm UV light. Clear level-3 patterns (sweat pores) were observed in the LFPs and their decay was very slow compared to the LFPs obtained from commercial powders. The photometric characterization of the prepared samples reveals the suitability of the MoO3:Eu3+ NPs for pure red emission in light-emitting diode, better visualization of LFPs and anti-counterfeiting applications.

A single phase, red emissive Mg2SiO4:Sm3+ nanophosphor prepared via rapid propellant combustion route.

Mg2SiO4:Sm3+ (1-11 mol%) nanoparticles were prepared by a rapid low temperature solution combustion route. The powder X-ray diffraction (PXRD) patterns exhibit orthorhombic structure with α-phase. The average crystallite size estimated using Scherer's method, W-H plot and strain-size plots were found to be in the range 25-50 nm and the same was confirmed by Transmission Electron Microscopy (TEM). Scanning electron microscopy (SEM) pictures show porous structure and crystallites were agglomerated. The effect of Sm3+ cations on luminescence of Mg2SiO4 was well studied. Interestingly the samples could be effectively excited with 315 nm and emitted light in the red region, which was suitable for the demands of high efficiency WLEDs. The emission spectra consists of four main peaks which can be assigned to the intra 4-f orbital transitions of Sm3+ ions 4G5/2→6H5/2 (576 nm), 4G5/2→6H7/2 (611 nm), 4G5/2→6H9/2 (656 nm) and 4G5/2→6H11/2 (713 nm). The optimal luminescence intensity was obtained for 5 mol% Sm3+ ions. The CIE (Commission International de I'Eclairage) chromaticity co-ordinates were calculated from emission spectra, the values (0.588, 0.386) were close to the NTSC (National Television Standard Committee) standard value of red emission. Coordinated color temperature (CCT) was found to be 1756 K. Therefore optimized Mg2SiO4:Sm3+ (5 mol%) phosphor was quite useful for solid state lighting.

Zn2TiO4:Eu(3+) nanophosphor: self explosive route and its near UV excited photoluminescence properties for WLEDs.

A simple and low-cost solution combustion method was used to prepare Eu(3+) (1-11mol%) doped Zn2TiO4 nanophosphors at 500°C using zinc nitrates as precursors and oxalyl di-hydrazide (ODH) as fuel. The final product was calcined at 1100°C for 3h and then characterized by powder X-ray diffraction (PXRD), fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and UV-visible absorption (UV-Vis). The PXRD patterns of the sample calcined at 1100°C show pure cubic phase. The crystallite size was estimated using Scherrer's method and found to be in the range 20-25nm and the same was confirmed by TEM studies. Effects of Eu(3+) (1-11mol%) cations on the luminescence properties of Zn2TiO4 nanoparticles were studied. The samples exhibit intense red emission upon 395nm near ultra violet (NUV) excitation. The characteristic emission peaks recorded at ∼578, 592, 613 and 654nm may be attributed to the 4f-4f intra shell transitions ((5)D0→(7)Fj=0,1,2,3) of Eu(3+) cations. The CIE chromaticity co-ordinates and CCT were calculated from emission spectra and the values (x, y) were very close to NTSC standard values for red emission and CCT was close to Plankian locus. Therefore, the present phosphor may be highly useful for display applications.

Development and validation of a simple assay for the determination of cholinesterase activity in whole blood of laboratory animals.

Current methods for measuring acetylcholinesterase (AChE) activities in whole blood use butyrylcholinesterase (BChE)-selective inhibitors. However, the poor selectivity of these inhibitors results in the inhibition of AChE activity to some degree, leading to errors in reported values. The goal of this study was to develop and validate a simple assay for measuring AChE and BChE activities in whole blood from humans as well as experimental animals. Blood was fractionated into plasma and erythrocytes, and cholinesterase activities were titrated against ethopropazine and (-)-huperzine A to determine the lowest concentration of ethopropazine that inhibited BChE completely without affecting AChE activity and the lowest concentration of (-)-huperzine A that inhibited AChE completely without interfering with BChE activity. Results indicate that 20 µm ethopropazine can be successfully used for the accurate measurement of AChE activity in blood from humans as well as animals. Use of (-)-huperzine A is not required for measuring BChE activity in normal or 'exposed' blood samples. The method was validated for blood from several animal species, including mice, rats, guinea pigs, dogs, minipigs, and African green, cynomolgus and rhesus monkeys. This method is superior to all reported methods, does not require the separation of erythrocyte and plasma fractions, and is suitable for measuring cholinesterase activities in fresh or frozen blood from animals that were exposed to nerve agents or those that were administered high doses of BChE. The method is simple, direct, reproducible, and reliable and can easily be adapted for high-throughput screening of blood samples. Published 2012. This article is a US Government work and is in the public domain in the USA.

Amino acid residues at the N- and C-termini are essential for the folding of active human butyrylcholinesterase polypeptide.

Human serum butyrylcholinesterase (HuBChE) is currently the most suitable bioscavenger for the prophylaxis of highly toxic organophosphate (OP) nerve agents. A dose of 200mg of HuBChE is envisioned as a prophylactic treatment that can protect humans from an exposure of up to 2 × LD50 of soman. The limited availability and administration of multiple doses of this stoichiometric bioscavenger make this pretreatment difficult. Thus, the goal of this study was to produce a smaller enzymatically active HuBChE polypeptide (HBP) that could bind to nerve agents with high affinity thereby reducing the dose of enzyme. Studies have indicated that the three-dimensional structure and the domains of HuBChE (acyl pocket, lip of the active center gorge, and the anionic substrate-binding domain) that are critical for the binding of substrate are also essential for the selectivity and binding of inhibitors including OPs. Therefore, we designed three HBPs by deleting some N- and C-terminal residues of HuBChE by maintaining the folds of the active site core that includes the three active site residues (S198, E325, and H438). HBP-4 that lacks 45 residues from C-terminus but known to have BChE activity was used as a control. The cDNAs for the HBPs containing signal sequences were synthesized, cloned into different mammalian expression vectors, and recombinant polypeptides were transiently expressed in different cell lines. No BChE activity was detected in the culture media of cells transfected with any of the newly designed HBPs, and the inactive polypeptides remained inside the cells. Only enzymatically active HBP-4 was secreted into the culture medium. These results suggest that residues at the N- and C-termini are required for the folding and/or maintenance of HBP into an active stable, conformation.

Characterization of human serum butyrylcholinesterase in rhesus monkeys: behavioral and physiological effects.

The effects of a large dose of human serum butyrylcholinesterase (HuBChE) were evaluated in rhesus monkeys using a serial-probe recognition (SPR) task designed to assess attention and short-term memory. Each monkey received an intravenous injection of 150 mg (105,000 U or 30 mg/kg) of HuBChE 60 min prior to testing on the SPR task. Concurrent with the cognitive-behavioral assessment, blood was collected at various time points throughout the study and was analyzed for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities, anti-BChE antibody production and gross clinical pathology (i.e., complete blood count and clinical chemistry panel). HuBChE revealed a peak blood activity of 227 U/ml at 5 min after intravenous injection and a mean residence time of approximately 72 h. No cognitive-behavioral decrements of any kind in SPR performance and no toxic signs in clinical pathology were detected in any of the blood assays during the 5 weeks of observation. Anti-HuBChE antibodies peaked at about 14 days after injection, with no concomitant behavioral changes. These results demonstrate the behavioral and physiological safety of HuBChE in rhesus monkeys and support its development as a bioscavenger for the prophylaxis of chemical warfare agent toxicity in humans.

Vitamin D deficiency and coronary artery calcification in subjects with type 1 diabetes.

OBJECTIVE: The objective of this study is to examine the relationship among serum levels of 25-hydroxyvitamin D (25[OH]D), polymorphisms in vitamin D-associated genes, and the presence and progression of coronary artery calcification (CAC) in adults with type 1 diabetes. RESEARCH DESIGN AND METHODS: This prospective study included 374 non-Hispanic white individuals with type 1 diabetes (mean age 40 ± 9 years; 46% were male). CAC was measured at the baseline and 3- and 6-year follow-up visits were determined by electron beam computed tomography. Serum 25[OH]D levels were measured by liquid chromatography tandem mass spectrometry at the 3-year visit. RESULTS: Normal (>30 ng/mL), insufficient (20-30 ng/mL), and deficient (<20 ng/mL) 25-[OH]D levels were present in 65%, 25%, and 10% of the individuals with type 1 diabetes, respectively. 25[OH]D deficiency was associated with the presence of CAC at the 3-year visit, odds ratio (OR) = 3.3 (95% CI 1.6-7.0), adjusting for age, sex, and hours of daylight. In subjects free of CAC at the 3-year visit, 25[OH]D deficiency predicted the development of CAC over the next 3 years in those with the vitamin D receptor M1T CC genotype (OR = 6.5 [1.1-40.2], P = 0.04) than in those with the CT or TT genotype (OR = 1.6 [0.3-8.6], P = 0.57). CONCLUSIONS: Vitamin D deficiency independently predicts prevalence and development of CAC, a marker of coronary artery plaque burden, in individuals with type 1 diabetes.

A potent mitogenic lectin from the mycelia of a phytopathogenic fungus, Rhizoctonia bataticola, with complex sugar specificity and cytotoxic effect on human ovarian cancer cells.

A lectin with strong mitogenic activity towards human peripheral blood mononuclear cells (PBMCs) and cytotoxic effect on human ovarian cancer cells has been purified from the mycelium of a phytopathogenic fungus, Rhizoctonia bataticola, using ion exchange chromatography and affinity chromatography on asialofetuin-Sepharose. The lectin, termed RBL, is a tetramer of 11-kDa subunits and has unique amino acid sequence at its blocked N-terminus. The purified RBL was blood group nonspecific and its hemagglutination activity was inhibited by mucin (porcine stomach), fetuin (fetal calf serum) and asialofetuin. Glycan array analysis revealed high affinity binding of RBL towards N-glycans and also the glycoproteins containing complex N-glycan chains. Interestingly, the lectin showed high affinity for glycans which are part of ovarian cancer marker CA125, a high molecular weight mucin containing high mannose and complex bisecting type N-linked glycans as well core 1 and 2 type O-glycans. RBL bound to human PBMCs eliciting strong mitogenic response, which could be blocked by mucin, fetuin and asialofetuin demonstrating the carbohydrate-mediated interaction with the cells. Analysis of the kinetics of binding of RBL to PBMCs revealed a delayed mitogenic response indicating a different signaling pathway compared to phytohemagglutinin-L. RBL had a significant cytotoxic effect on human ovarian cancer cell line, PA-1.

Latent autoimmune diabetes in adults.

CONTEXT: Autoantibodies that are reactive to islet antigens are present at the time of diagnosis in most patients with type 1 diabetes. Additionally, approximately 10% of phenotypic type 2 diabetic patients are positive for at least one of the islet autoantibodies, and this group is often referred to as "latent autoimmune diabetes in adults (LADA)." These patients share many genetic and immunological similarities with type 1 diabetes, suggesting that LADA, like type 1 diabetes, is an autoimmune disease. However, there are differences in autoantibody clustering, T cell reactivity, and genetic susceptibility and protection between type 1 diabetes and LADA, implying important differences in the underlying disease processes. EVIDENCE ACQUISITION AND SYNTHESIS: In this clinical review, we will summarize the current understanding of LADA based on the MEDLINE search of all peer-reviewed publications (original articles and reviews) on this topic between 1974 and 2009. CONCLUSIONS: In LADA, diabetes occurs earlier in the beta-cell-destructive process because of the greater insulin resistance. Complexities arise also because of variable definitions of LADA and type 1 diabetes in adults. As immunomodulatory therapies that slow or halt the type 1 diabetes disease process are discovered, testing these therapies in LADA will be essential.

Pharmacokinetics and immunologic consequences of repeated administrations of purified heterologous and homologous butyrylcholinesterase in mice.

AIM: To assess the consequences of repeated administrations of purified human serum butyrylcholinesterase (Hu BChE) and mouse serum (Mo) BChE into mice. MAIN METHODS: Purified Hu BChE and Mo BChE isolated from the sera of CD-1 mice were administered into Balb/c or CD-1 mice. The enzymes were delivered by i.m. injections of approximately 100U (0.15mg) on day 1 and on day 28, respectively. The effects of two injections were monitored by following blood BChE and anti-BChE IgG levels. KEY FINDINGS: Hu BChE displayed a mean residence time (MRT) of 50h, and an area under the curve (AUC) of 1220U/ml.h in Balb/c or CD-1 mice. Mo BChE exhibited an MRT of 78h and an AUC of 1815U/ml.h in Balb/c mice; the AUC increased to 2504U/ml.h in CD-1 mice. A second injection of Hu BChE in both strains exhibited a marked reduction in circulatory stability. The circulatory stability of the second injection of Mo BChE was reduced in Balb/c mice, but was almost identical to the first injection in CD-1 mice. Consistent with these observations, circulating anti-BChE IgGs were observed in mice injected with Hu BChE; low levels of anti-BChE IgGs were observed only in Balb/c mice injected with Mo BChE. No antibody response was detected in CD-1 mice following either injection of homologous Mo BChE. SIGNIFICANCE: The identical pharmacokinetic profiles and the absence of an immunologic response following a second administration of homologous BChE support the development of Hu BChE as a detoxifying drug in humans.

Use of continuous glucose monitoring in patients with type 1 diabetes.

The prevalence of type 1 diabetes continues to increase worldwide at a rate higher than previously projected, while the number of patients achieving American Diabetes Association (ADA) glycated hemoglobin (A1c) goals remains suboptimal. There are numerous barriers to patients achieving A1c targets including increased frequency of severe hypoglycemia associated with lowering plasma glucose as measured by lower A1c values. Continuous glucose monitoring (CGM) was first approved for retrospective analysis and now has advanced to the next step in diabetes management with the approval of real-time glucose sensing. Real-time CGM, in short term studies, has been shown to decrease A1c values, improve glucose variability (GV), and minimize the time and number of hypoglycemic events in patients with type 1 diabetes. These products are approved for adjunctive use to self-monitoring of blood glucose (SMBG), but future long-term studies are needed to document their safety, efficacy, ability to replace SMBG as a tool of monitoring, and ultimately utility into closed-loop insulin delivery systems. New algorithms will need to be developed that account for rapid changes in the glucose values, so that accuracy of the sensor data can be maintained. In addition, for better clinical care and usage, algorithms also need to be developed for both patients and the providers to guide them for their ongoing diabetes care.

Effect of polyethylene glycol modification on the circulatory stability and immunogenicity of recombinant human butyrylcholinesterase.

The therapeutic value of human serum butyrylcholinesterase (Hu BChE) as a bioscavenger of chemical warfare agents is due to its high reactivity with organophosphorus compounds and prolonged circulatory stability. Native Hu BChE is mostly tetrameric in form while the enzyme produced using molecular cloning technology is a mixture of tetramers, dimers, and monomers. Previous studies revealed that monomers and dimers of recombinant human (rHu) BChE cleared rapidly from the circulation of mice compared to tetrameric rHu BChE and native Hu BChE, which have mean residence times (MRTs) of 18h and 45h, respectively. It was also shown that polyethylene glycol-20K (PEG) modification of tetrameric rHu BChE prolonged its circulatory stability and bioavailability in vivo. The goal of this study was to determine if modification with PEG could prolong the circulatory stability and eliminate the immunogenicity of monomeric rHu BChE. Monomeric rHu BChE was expressed in human 293A cells using a cDNA lacking the 45 amino acid tetramerization domain from the carboxyl terminus and the adenovirus expression system. The catalytic and inhibitory properties of purified monomeric rHu BChE were similar to those for native Hu BChE and were not affected by PEG modification. As expected, monomeric rHu BChE rapidly cleared from the circulation of mice (MRT=3.2+/-0.3h) while monomeric PEG-rHu BChE demonstrated significant improvement in its bioavailability and circulatory stability in blood (MRT=31.4+/-5.4h). However, a second injection of monomeric PEG-rHu BChE, 28 days after the first, displayed a much shorter MRT=11.6+/-0.4h, and circulating anti-monomeric PEG-rHu BChE antibodies were detected in the blood of mice. These results suggest that PEG modification increased the circulatory stability of monomeric rHu BChE but failed to reduce or eliminate its immunogenicity.

A repeated injection of polyethyleneglycol-conjugated recombinant human butyrylcholinesterase elicits immune response in mice.

Human serum butyrylcholinesterase (Hu BChE) serves as an efficacious bioscavenger of highly toxic organophosphorus (OP) compounds. Since there is a concern that the supply of native Hu BChE may be limited, monomeric and tetrameric forms of recombinant Hu BChE (rHu BChE) were evaluated as replacements and found that they lacked sufficient stability in vivo. However, their in vivo stability could be significantly prolonged by conjugation with polyethyleneglycol-20K (PEG) suggesting that monomeric and tetrameric PEG-rHu BChE could function as bioscavengers. Here, the immunogenicity of PEG-rHu BChE was evaluated in mice following two injections given four weeks apart. In addition to pharmacokinetic parameters, such as mean residence time, maximal concentration, time to reach the maximal concentration, elimination half-life and area under the plasma concentration-time curve extrapolated to infinity, the presence of circulating anti-rHu BChE antibodies was also determined. Although the pharmacokinetic parameters were significantly improved for the first injection of monomeric and tetrameric PEG-rHu BChEs, they were much lower for the second injection. Anti-rHu BChE antibodies were detected in the blood of mice following the first and second enzyme injections and their levels were approximately higher by 5-fold and 2-fold in mice injected with monomeric and tetrameric PEG-rHu BChEs as compared to mice injected with unconjugated enzymes. The findings that the rapid clearance of a repeat injection of PEG-rHu BChEs in mice which coincides with the presence of circulating anti-rHu BChE antibodies suggest that PEG conjugation prolonged the circulatory stability of rHu BChE but failed to eliminate its immunogenicity in mice.

Comparison of methods used for the determination of cholinesterase activity in whole blood.

Cholinesterases (ChEs) are classified as either acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) based on their substrate and inhibitor specificity. Organophosphate and carbamate compounds commonly represented by herbicides, pesticides, and nerve gases irreversibly inhibit ChEs. Therefore, exposure to organophosphates and carbamates is normally assessed by measuring ChE activity in blood. There are two approaches for measuring AChE and BChE activity present in whole blood: (1) separating blood into erythrocytes, which contain only AChE, and plasma which contains only BChE, to measure their activity individually, or (2) use a BChE-specific inhibitor to measure the activity of AChE in whole blood. A number of studies have reported the use of different inhibitors for the simultaneous measurement of AChE and BChE activities. However, the inhibitors used for completely inhibiting BChE activity also inhibited AChE activity leading to errors in reported values. The goal of this study was to find the most accurate and simple method for the simultaneous determination of AChE and BChE activity in animal whole blood. Solutions containing human AChE and BChE in various proportions were prepared and AChE and BChE activities were measured using three reported methods. Results demonstrate that ethopropazine and (-) huperzine A appear to be the most specific ChE inhibitors. Preliminary results with human and animal whole blood suggest that 20 microM ethopropazine and 500 nM (-) huperzine A can be used for measuring AChE and BChE activities across species.