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.

Monoclonal antibodies to human butyrylcholinesterase reactive with butyrylcholinesterase in animal plasma.

Five mouse anti-human butyrylcholinesterase (BChE) monoclonal antibodies bind tightly to native human BChE with nanomolar dissociation constants. Pairing analysis in the Octet system identified the monoclonal antibodies that bind to overlapping and independent epitopes on human BChE. The nucleotide and amino acid sequences of 4 monoclonal antibodies are deposited in GenBank. Our goal was to determine which of the 5 monoclonal antibodies recognize BChE in the plasma of animals. Binding of monoclonal antibodies 11D8, B2 18-5, B2 12-1, mAb2 and 3E8 to BChE in animal plasma was measured using antibody immobilized on Pansorbin cells and on Dynabeads Protein G. A third method visualized binding by the shift of BChE activity bands on nondenaturing gels stained for BChE activity. Gels were counterstained for carboxylesterase activity. The three methods agreed that B2 18-5 and mAb2 have broad species specificity, but the other monoclonal antibodies interacted only with human BChE, the exception being 3E8, which also bound chicken BChE. B2 18-5 and mAb2 recognized BChE in human, rhesus monkey, horse, cat, and tiger plasma. A weak response was found with rabbit BChE. Monoclonal mAb2, but not B2 18-5, bound pig and bovine BChE. Gels stained for carboxylesterase activity confirmed that plasma from humans, monkey, pig, chicken, and cow does not contain carboxylesterase, but plasma from horse, cat, tiger, rabbit, guinea pig, mouse, and rat has carboxylesterase. Rabbit plasma carboxylesterase hydrolyzes butyrylthiocholine. In conclusion monoclonal antibodies B2 18-5 and mAb2 can be used to immuno extract BChE from the plasma of humans, monkey and other animals.

Pharmacokinetics and immunogenicity of a recombinant human butyrylcholinesterase bioscavenger in macaques following intravenous and pulmonary delivery.

Recombinant (r) and native butyrylcholinesterse (BChE) are potent bioscavengers of organophosphates (OPs) such as nerve agents and pesticides and are undergoing development as antidotal treatments for OP-induced toxicity. Because of the lethal properties of such agents, regulatory approval will require extensive testing under the Animal Rule. However, human (Hu) glycoprotein biologicals, such as BChE, present a challenge for assessing immunogenicity and efficacy in heterologous animal models since any immune responses to the small species differences in amino acids or glycans between the host and biologic may alter pharmacodynamics and preclude accurate efficacy testing; possibly underestimating their potential protective value in humans. To establish accurate pharmacokinetic and efficacy data, an homologous animal model has been developed in which native and PEGylated forms of CHO-derived rMaBChE were multiply injected into homologous macaques with no induction of antibody. These now serve as controls for assessing the pharmacokinetics and immunogenicity in macaques of multiple administrations of PEGylated and unmodified human rBChE (rHuBChE) by both intravenous (IV) and pulmonary routes. The results indicate that, except for maximal concentration (Cmax), the pharmacokinetic parameters following IV injection with heterologous PEG-rHuBChE were greatly reduced even after the first injection compared with homologous PEG-rMaBChE. Anti-HuBChE antibody responses were induced in all monkeys after the second and third administrations regardless of the route of delivery; impacting rates of clearance and usually resulting in reduced endogenous MaBChE activity. These data highlight the difficulties inherent in assessing pharmacokinetics and immunogenicity in animal models, but bode well for the efficacy and safety of rHuBChE pretreatments in homologous humans.

Detection of cresyl phosphate-modified butyrylcholinesterase in human plasma for chemical exposure associated with aerotoxic syndrome.

Flight crews complain of illness following a fume event in aircraft. A chemical in jet engine oil, the neurotoxicant tri-o-cresyl phosphate, after metabolic activation to cresyl saligenin phosphate makes a covalent adduct on butyrylcholinesterase (BChE). We developed a mass spectrometry method for detection of the cresyl phosphate adduct on human BChE as an indicator of exposure. Monoclonal mAb2, whose amino acid sequence is provided, was crosslinked to cyanogen bromide-activated Sepharose 4B and used to immunopurify plasma BChE treated with cresyl saligenin phosphate. BChE was released with acetic acid, digested with pepsin, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MSMS) on the Triple TOF 5600 mass spectrometer. Peptide FGES198AGAAS with an added mass of 170 Da from cresyl phosphate on serine 198 (Ser198) was detected as parent ion 966.4 Da. When characteristic daughter ions were monitored in the MSMS spectrum, the limit of detection was 0.1% cresyl saligenin phosphate inhibited plasma BChE. This corresponds to 2×10(-9) g in 0.5 ml or 23×10(-15) moles of inhibited BChE in 0.5 ml of plasma. In conclusion, a sensitive assay for exposure to tri-o-cresyl phosphate was developed. Laboratories that plan to use this method are cautioned that a positive result gives no proof that tri-o-cresyl phosphate is toxic at low levels.

Inhibitors of acetylcholinesterase and butyrylcholinesterase meet immunity.

Acetylcholinesterase (AChE) inhibitors are widely used for the symptomatic treatment of Alzheimer's disease and other dementias. More recent use is for myasthenia gravis. Many of these inhibitors interact with the second known cholinesterase, butyrylcholinesterase (BChE). Further, evidence shows that acetylcholine plays a role in suppression of cytokine release through a "cholinergic anti-inflammatory pathway" which raises questions about the role of these inhibitors in the immune system. This review covers research and discussion of the role of the inhibitors in modulating the immune response using as examples the commonly available drugs, donepezil, galantamine, huperzine, neostigmine and pyridostigmine. Major attention is given to the cholinergic anti-inflammatory pathway, a well-described link between the central nervous system and terminal effector cells in the immune system.

Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli.

Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1-14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1-14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1-14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1-14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis.

Monoclonal antibodies to mouse butyrylcholinesterase.

Our immunization strategy introduced recombinant mouse butyrylcholinesterase (BChE) to naïve BChE knockout mice. An extraordinarily strong immune reaction gave rise to a whole spectrum of antibodies with different properties. Two selective and highly efficient monoclonal anti-mouse BChE antibodies 4H1 (IgG1) and 4 C9 (IgG2a), with Kd values in the nanomolar range were generated. ELISA detected BChE in as little as 20-50 nl of mouse plasma using 2 µg (4H1) or 4 µg (4C9). Both antibodies cross-reacted with BChE in dog plasma but only 4 H1 reacted with rat BChE, suggesting that the antibodies are targeted towards different epitopes. Surprisingly, neither recognized human BChE. The anti-mouse BChE antibodies were used in immunohistochemistry analysis of mouse muscle where they specifically stained the neuromuscular junction. The antibodies enable visualization of the BChE protein in the mouse tissue, thus complementing activity assays. They can be used to study a long-lasting question about the existence of mixed acetylcholinesterase/BChE oligomers in mouse tissues. Moreover, monoclonal anti-mouse BChE antibodies can provide a simple, fast and efficient way to purify mouse BChE from small amounts of starting material by using a single-step immunomagnetic bead-based protocol.

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.

Enzyme-linked immunosorbent assay for detection of organophosphorylated butyrylcholinesterase: a biomarker of exposure to organophosphate agents.

A sandwich enzyme-linked immunosorbent assay (sELISA) has been developed for detection of organophosphorylated butyrylcholinesterase (OP-BChE), a potential biomarker for human exposure to organophosphate insecticides and nerve agents. A pair of antibodies specific to OP-BChE adduct were identified through systematic screening of several anti BChE antibodies (anti-BChE) and anti-phosphoserine antibodies (anti-P(ser)) from different sources. The selected anti-BChE (set as capture antibody) antibodies recognize both phosphorylated and nonphosphorylated BChE. These antibodies can therefore be used to capture both BChE and OP-BChE from the sample matrices. The anti-P(ser) (set as detecting antibody) was used to recognize the OP moiety of OP-BChE adducts. With the combination of the selected antibody pair, several key parameters (such as the concentration of anti-BChE and anti-P(ser), and the blocking agent) were optimized to enhance the sensitivity and selectivity of the sELISA. Under the optimal conditions, the sELISA has shown a wide linear range from 0.03 nM to 30 nM, with a detection limit of 0.03 nM. Furthermore, the sELISA was successfully applied to detect OP-BChE using in vitro biological samples such as rat plasma spiked with OP-BChE with excellent adduct recovery (z>99%). These results demonstrate that this novel approach holds great promise to develop an ELISA kit and offers a simple and cost-effective tool for screening/evaluating exposure to organophosphate insecticides and nerve agents.

A novel system for the efficient generation of antibodies following immunization of unique knockout mouse strains.

BACKGROUND: We wished to develop alternate production strategies to generate antibodies against traditionally problematic antigens. As a model we chose butyrylcholinesterase (BChE), involved in termination of cholinergic signaling, and widely considered as a poor immunogen. METHODOLOGY/PRINCIPAL FINDINGS: Jettisoning traditional laborious in silico searching methods to define putative epitopes, we simply immunized available BChE knock-out mice with full-length recombinant BChE protein (having been produced for crystallographic analysis). Immunization with BChE, in practically any form (recombinant human or mouse BChE, BChE purified from human serum, native or denatured), resulted in strong immune responses. Native BChE produced antibodies that favored ELISA and immunostaining detection. Denatured and reduced BChE were more selective for antibodies specific in Western blots. Two especially sensitive monoclonal antibodies were found capable of detecting 0.25 ng of BChE within one min by ELISA. One is specific for human BChE; the other cross-reacts with mouse and rat BChE. Immunization of wild-type mice served as negative controls. CONCLUSIONS/SIGNIFICANCE: We examined a simple, fast, and highly efficient strategy to produce antibodies by mining two expanding databases: namely those of knock-out mice and 3D crystallographic protein-structure analysis. We conclude that the immunization of knock-out mice should be a strategy of choice for antibody production.

Demonstration of in vivo stability and lack of immunogenicity of a polyethyleneglycol-conjugated recombinant CHO-derived butyrylcholinesterase bioscavenger using a homologous macaque model.

Human serum and recombinant butyrylcholinesterase (rHuBChE) are the most advanced prophylactics against organophosphate (OP) toxicity due to nerve agent or insecticide exposure. For ethical reasons, such potential multi-use treatments cannot be tested in humans and will require extensive testing in animal models and the "Animal Rule" 21 (21 CFR 601.90) for regulatory approval. This will involve multiple injections of rHuBChE into heterologous animals, e.g. macaques, rodents with inevitable immunogenicity and subsequent elimination of the enzyme on repeat injections. In order to accurately assess pharmacokinetics, efficacy and safety of a candidate rBChE in an "antibody free" system, a homologous macaque (Ma) model has been developed. In these studies, macaques received single or multiple intravenous injections of native MaBChE as well as unmodified or PEG-conjugated forms of rMaBChE produced in CHO cells. Compared to the poor plasma retention of unmodified rBChE (MRT: <10h), three injections of 1.5-2.3mg/kg of PEG-conjugated tetrameric rBChE resulted in high circulatory stability (MRT: >134h) and lack of immunogenicity similar to native MaBChE. PEG-conjugation of the monomeric rMaBChE form also exhibited pharmacokinetic profiles comparable to the tetrameric form (MRT: >113h). However, despite the increased bioavailability of PEG-rBChE, antigenicity studies using sandwich ELISA showed that while macaque BChE was not immunogenic in macaques, PEGylation of rMaBChE did not prevent binding to anti-BChE antibodies, suggesting PEGylation may not be sufficient to mask non-human epitopes on rBChE. This homologous model can provide necessary preclinical protection data for the use of PEG-rHuBChE in humans and bodes well for a safe and efficacious CHO-derived rHuBChE therapeutic.

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.

Pharmacokinetics and immunologic consequences of exposing macaques to purified homologous butyrylcholinesterase.

Exposure to organophosphorus compounds (OPs), in the form of nerve agents and pesticides poses an ever increasing military and civilian threat. In recent years, attention has focused on the use of exogenously administered cholinesterases as an effective prophylactic treatment for protection against OPs. Clearly, a critical prerequisite for any potential bioscavenger is a prolonged circulatory residence time, which is influenced by the size of protein, the microheterogeneity of carbohydrate structures, and the induction (if any) of anti-enzyme antibodies following repeated injections of the enzyme. Previously, it was demonstrated that multiple injections of equine butyrylcholinesterase (BChE) into rabbits, rats, or rhesus monkeys, resulted in a mean residence time spanning several days, and variable immune responses. The present study sought to assess the pharmacokinetics and immunological consequences of administration of purified macaque BChE into macaques of the same species at a dose similar to that required for preventing OP toxicity. An i.v. injection of 7,000 U of homologous enzyme in monkeys demonstrated much longer mean residence times in plasma (MRT = 225 +/- 19 h) compared to those reported for heterologous Hu BChE (33.7 +/- 2.9 h). A smaller second injection of 3,000 U given four weeks later, attained predicted peak plasma levels of enzyme activity, but surprisingly, the MRT in the four macaques showed wide variation and ranged from 54 to 357 h. No antibody response was detected in macaques following either injection of enzyme. These results bode well for the potential use of human BChE as a detoxifying drug in humans.

Comparison of cholinesterase activities in the excretion-secretion products of Trichinella pseudospiralis and Trichinella spiralis muscle larvae.

The presence of cholinesterases (ChE) is reported in T. pseudospiralis excretion-secretion products (ESP) by spectrophotometric method, using acetylthiocholine (ATCI) and butyrilthiocholine (BTCI) as substrates. By inhibition assays, we found that T. pseudospiralis release both acetyl- and butiryl-cholinesterases (AchE and BchE, respectively). The sedimentation coefficientes of these enzymes were determined by sucrose density gradient. We studied the in vivo ChE secretion by immunoblot assays using AchE from Electrophorus (electric eel) and sera from normal or infected mice with T. pseudospiralis or T. spiralis. The presence of anti-AchE antibodies was only demonstrated in the sera from T. pseudospiralis infected mice. Moreover the in vivo secretion was corroborated by the high difference determinate between the ChE activity of the immuno complexes from T. pseudospiralis infected sera and the immunocomplexes from T. spiralis infected sera as well as normal sera. Finally, we analyzed the effect of the organophosphate Neguvón (metrifonate) on the ChE activity from the T. pseudospiralis ESP. The drug inhibits in part this activity. Moreover Neguvón (metrifonate) showed a high activity against the T. pseudospiralis viability.

Examination of cross-antigenicity of acetylcholinesterase and butyrylcholinesterase using anti-acetylcholinesterase antibodies.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) share a high degree of homology and overlap in several biochemical properties. This study aimed to compare and contrast the antigenic reactivity of AChE and BuChE with several polyclonal antibodies. We have performed a detailed analysis of AChE and BuChE enzymatic activities with different substrates and different inhibitors. Immunoassays conducted with polyclonal amino-terminus-specific anti-AChE antibodies were selective for mouse and electric eel AChE (EEAChE). Polyclonal carboxy-terminus-specific anti-AChE antibodies reacted with EEAChE and human BuChE, indicating an unexpected cross-reactivity. Polyclonal antisera raised against the whole AChE protein cross-reacted with horse BuChE, but not human BuChE. These data demonstrate that AChE and BuChE are immunologically similar.

Identification of hybrid cholinesterase forms consisting of acetyl- and butyrylcholinesterase subunits in human glioma.

Brain and non-brain tumors contain acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) transcripts and enzyme activity. AChE and BuChE occur in tissues as a set of molecular components, whose distribution in a cyst fluid from a human astrocytoma we investigated. The fluid displayed high BuChE and low AChE activities. Three types of cholinesterase (ChE) tetramers were identified in the fluid by means of sedimentation analyses and assays with specific inhibitors, and their sedimentation coefficients were 11.7S (ChE-I), 11.1S (ChE-II), and 10.5S (ChE-III). ChE-I was unretained, ChE-II was weakly retained and ChE-III was adsorbed to edrophonium-agarose, confirming the AChE nature of the latter. ChE-I and ChE-II tetramers contained BuChE subunits as shown by their binding with an antiserum against BuChE. The ChE activity of the immunocomplexes made with ChE-II and anti-BuChE antibodies decreased with the AChE inhibitor BW284c51, revealing that ChE-II was made of AChE and BuChE subunits, in contrast to ChE-I, which only contained BuChE subunits. The binding of an anti-AChE antibody (AE1) to ChE-II and ChE-III, but not to ChE-I, demonstrated the hybrid composition of ChE-II. A substantial fraction of the AChE tetramers and dimers of astrocytomas and oligodendrogliomas bound both to anti-AChE and anti-BuChE antibodies, which revealed a mixed composition of AChE and BuChE subunits in them. The AChE components of brain, meningiomas and neurinomas were only recognized by AE1. In conclusion, our results demonstrate that aberrant ChE oligomers consisting of AChE and BuChE subunits are generated in astrocytomatous cyst and gliomas but not in brain, meningiomas or neurinomas.

High-level expression of human butyrylcholinesterase gene in Bombyx mori and biochemical-pharmacological characteristic study of its product.

The human butyrylcholinesterase (BChE, EC 3.1.1.8) gene was highly expressed in Bombyx mori using baculovirus vector, and the biochemical-pharmacological properties of its product were studied. BChE cDNA was cloned into transfer vector pBn96 and co-transfected with wild-type Bombyx mori nucleopolyhedrovirus (BmNPV) DNA into BmN cells. The recombinant virus with the highest enzyme activity was sorted out and purified. Once the BmN cells or silkworm larvae had been infected with the recombinant virus, recombinant human BChE (rhBChE) could be secreted into the culture medium or the hemolymph of the larvae at levels of 1.5 mg x L(-1) and 35 mg x L(-1), respectively. Western blot and enzymatic staining of the electrophoresis gel of non-denatured protein showed that rhBChE manifested similar antigenicity and enzyme activity to native human BChE (nhBChE). The production of rhBChE in the hemolymph was 23-fold higher than that in BmN cells and about 280-fold that in Chinese hamster overy cells (125 microg x L(-1)). This is the first report of human BChE expression in silkworm with the highest level of yield so far. rhBChE was highly similar to nhBChE in respect to substrate affinity, inhibitor sensitivity, and reactivity of the inhibited enzyme. It is suggested that rhBChE functions as well as nhBChE and has potential practical value.

Behavioral and immunological effects of exogenous butyrylcholinesterase in rhesus monkeys.

Although conventional therapies prevent organophosphate (OP) lethality, laboratory animals exposed to such treatments typically display behavioral incapacitation. Pretreatment with purified exogenous human or equine serum butyrylcholinesterase (Eq-BuChE), conversely, has effectively prevented OP lethality in rats and rhesus monkeys, without producing the adverse side effects associated with conventional treatments. In monkeys, however, using a commercial preparation of Eq-BuChE has been reported to incapacitate responding. In the present study, repeated administration of commercially prepared Eq-BuChE had no systematic effect on behavior in rhesus monkeys as measured by a six-item serial probe recognition task, despite 7- to 18-fold increases in baseline BuChE levels in blood. Antibody production induced by the enzyme was slight after the first injection and more pronounced following the second injection. The lack of behavioral effects, the relatively long in vivo half-life, and the previously demonstrated efficacy of BuChE as a biological scavenger for highly toxic OPs make BuChE potentially more effective than current treatment regimens for OP toxicity.

From pseudocholinesterase to human immunodeficiency virus.

Pseudocholinesterase is a protein for which no function exists in mammals including human beings. To date, no substrate has been identified for this 'enzyme'. Involvement of this protein in the aetiopathogenesis of many diseases, such as hyperlipoproteinaemia, is still actively debated. Here, we propose a theoretical method to immobilize pseudocholinesterase in hepatocytes using antibody bound to liposomes. Conceptually, this approach will have widespread application, especially in blocking human immunodeficiency virus replication in vivo.