Role of α7-Nicotinic Acetylcholine Receptors of B Cells in the Immunotoxic Effect of Organophosphorus Compounds.

Experiments on white non-inbred rats demonstrated that treatment with organophosphorus compound dimethyl dichlorovinyl phosphate (DDVP) decreased T cell-independent antibody production by B cells and blood levels of IL-10 and IL-12; a similar effect was produced by GTS-21, a selective agonist of α7-nicotinic acetylcholine receptor. N-nicotinic receptor antagonist chlorisondamine in combination with DDVP partially prevented suppression of antibody production in comparison with the effect observed during intoxication with DDVP.

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.

The role of cholinergic balance perturbation in neurological diseases.

The maintenance of a balanced cholinergic homeostasis is crucial for the function of the central nervous system, peripheral nervous system and the neuromuscular junction. However, it appears that the cholinergic system is not restricted to neurons and synapses but may also involve immune reactions. In the present review we reassess the role of the cholinergic balance in myasthenia gravis and Alzheimer's disease which for a long time are known to be associated with cholinergic transmission perturbation. We have included neuroinflammation, particularly multiple sclerosis in this group of neurological disorders in light of the relatively new studies involving the immune cholinergic system. In all the aforementioned disorders, treatment with acetylcholinesterase inhibitors can attenuate inflammation. This is performed by increasing the acetylcholine (ACh) concentration near immune cells and making it available for interaction with alpha(7) nicotinic ACh receptor, expressed on these cells. This outcome is additional to the effect of acetylcholinesterase inhibitors on neurons and synapses.

Enzyme-linked immunosorbent assays for the insecticide fenitrothion. Influence of hapten conformation and sample matrix on assay performance.

This study aimed at developing competitive enzyme-linked immunosorbent assays (ELISAs) for the organophosphorus (OP) insecticide fenitrothion using a monoclonal antibody. The hapten used to obtain the antibody had an ideal structural feature that allowed minimal functional group sacrifice. By using the antibody and a coating antigen, a competitive indirect ELISA was developed, which showed an IC50 of 14 ng mL(-1) with a detection limit of 3.0 ng mL(-1). A competitive direct ELISA using an enzyme tracer was also developed, which showed an IC50 of 17 ng mL(-1) with a detection limit of 1.6 ng mL(-1). The antibodies in both assays showed negligible cross-reactivity with the metabolites of fenitrothion and other OP pesticides except with the insecticides parathion-methyl and parathion-ethyl. Recoveries of fenitrothion from fortified rice and lettuce samples were determined and the bias in the recovery values was rationalized by using the standard curves obtained in the matrix extract.

Anticholinesterase mechanism as a factor of immunotoxicity of various chemical compounds.

Experiments on Wistar rats showed that acute poisoning with chemicals in a dose of 0.75 LD(50) (dimethyl dichlorovinyl phosphate, sarin, VX substance, sulfur yperite, lewisite, tetraethyl lead, dichloroethane) inhibiting platelet acetylcholine esterase, alpha-naphthyl-AS-acetate esterase, and alpha-naphthyl-butyrate esterase suppressed T cell-mediated immune reactions.

B-cell autoepitopes on the acetylcholinesterase-homologous region of human thyroglobulin: association with Graves' disease and thyroid eye disease.

OBJECTIVE: Thyroglobulin (Tg) is a large autoantigen involved in autoimmune thyroid diseases. Tg epitopes have, so far, been identified within large peptides. In the present study, we used small synthetic peptides to finely map serological epitopes on the highly immunogenic C-terminal region of Tg. Homology of this region to acetylcholinesterase (AChE) has been implicated in the pathogenesis of thyroid eye disease (TED) through cross-reactive antibodies. METHODS: We tested total IgG purified from four pilot Graves' disease (GD) sera reactive with both Tg and AChE and from three healthy controls, for reactivity against overlapping 20mer peptides (pin synthesis) covering the sequence 2171-2748 of human Tg. Antibody-reactive peptides were subsequently synthesized by a solid-phase technique for confirmation with a large number of sera: 99 GD, 32 Hashimoto's thyroiditis (HT) and 45 healthy controls. RESULTS: Peptides TgP15, TgP26 and TgP41 (amino acids 2339-2358, 2471-2490 and 2651-2670 respectively) were found to be targets of autoantibodies on intact Tg, recognized by a statistically significant proportion of GD sera (22.2%, 35.4% and 30.3% respectively), compared with HT (0%, 15.6% and 6.3% respectively) and healthy controls (0%, 4.4% and 4.4% respectively). The majority of GD sera (56.6%) were positive for at least one of the three peptides. In GD, TgP26 reactivity was found to be associated with TED (48.6% with TED versus 25.5% without TED, P<0.05). CONCLUSION: Some epitopes on the C-terminal region of Tg are associated with GD. A subset of Tg-reactive autoantibodies, directed to this region, is associated with TED and may be involved in the development of the disease.

Hydrolysis of organophosphorus nerve agent soman by the monoclonal antibodies elicited against an oxyphosphorane hapten.

The antibody-mediated hydrolysis of the nerve agent O-1,2,2-trimethylpropyl methylphosphonofluoridate (soman) 1 has now been established with two monoclonal antibodies raised against the cyclic pentacovalent methyloxyphosphorane hapten 10 that mimics the pentacoordinated trigonal bipyramidal transition-state of the reaction. The hydrolysis reaction was studied using molecular orbital methods at the MP2/6-31 + G*/(/)HF/6-31 + G* level of accuracy. According to the ab initio calculations, the reaction seems to proceed via three separate transition-states. The calculations are in good agreement with the experimental results. The 1,3-dioxabenzophosphole hapten 10 was synthesized, coupled to the carrier protein and the antibodies were obtained by the hybridoma technique. Two antibodies, DB-108P and DB-108Q were found to enhance the rate of soman hydrolysis and they were kinetically characterised.

Allosteric control of acetylcholinesterase activity by monoclonal antibodies.

Previous studies showed that monoclonal antibodies raised against phosphorylated fetal bovine serum acetylcholinesterase appeared to modulate the catalytic activity of the enzyme by binding to a conformational epitope located at or near the region of the peripheral anionic site. The mechanism of inhibition of acetylcholinesterase by these monoclonal antibodies was further investigated by determining their effect on (i) substrate inhibition due to the binding of excess substrate to the peripheral anionic site and (ii) binding of peripheral anionic site ligands, such as propidium and fasciculin. Results of these experiments demonstrate that the accessibility of substrate to the peripheral anionic site in these complexes was restricted but not completely blocked, as none of the monoclonal antibodies eliminated the phenomenon of excess substrate inhibition. The results also show that propidium clearly slowed the inhibition of fetal bovine serum acetylcholinesterase by all six inhibitory monoclonal antibodies but to different levels. Complexation of fetal bovine serum acetylcholinesterase with monoclonal antibodies 25B1, 4E5, 6H9, and 5E8 interfered with the binding of fasciculin to the complexed enzyme, suggesting that part of their epitope overlapped with the fasciculin binding site. These monoclonal antibodies bind, in part, at the peripheral anionic site, since polyclonal anti-idiotypic antibodies generated against two monoclonal antibodies, 25B1 and 6H9, bound stoichiometric amounts of propidium. Like fasciculin, binding of these monoclonal antibodies in the vicinity of the peripheral anionic site at the rim of the active site gorge allosterically affects the orientation of W86 located at the base of the gorge, resulting in inhibition of enzyme activity.

An explanation on the limited efficacy of detoxication against VX toxicity by purified specific antibodies.

Studies on the mechanism of detoxication against VX toxicity by purified rabbit anti-VX antibodies were carried out in mice. VX (25 micrograms/kg) was injected subcutaneously immediately following intravenous injection of purified anti-VX antibodies. Blood concentration of free VX declined linearly as the antibody dose increased. Free VX concentration in brain was far lower than that in blood. When the dose of purified anti-VX antibodies was 8 and 6 mg/kg, respectively, free VX concentration in brain and blood approached 0. At the same time, the cholinesterase (ChE) activity in brain increased along with increasing antibody dose, with 70% of normal control value detected when 8 mg/kg antibodies were administered. On the other hand, anti-VX antibodies were not able to protect blood ChE unless the VX dose was lower than 10 micrograms/kg. The studies suggest that the protective action of purified rabbit anti-VX antibodies is from VX antibodies combining with the free VX in blood and extravascular tissue to reduce the amount of VX entering into the brain.

Characterization of monoclonal antibodies that strongly inhibit Electrophorus electricus acetylcholinesterase.

In this study, we describe three different monoclonal antibodies (mAbs Elec-403, Elec-408, and Elec-410) directed against Electrophorus electricus acetylcholinesterase (AChE) which were selected as inhibitors for this enzyme. Two of these antibodies (Elec-403 and Elec-410), recognized overlapping but different epitopes, competed with snake venom toxin fasciculin for binding to the enzyme, and thus apparently recognized the peripheral site of AChE. In addition, the binding of Elec-403 was antagonized by 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284C51) and propidium, indicating that the corresponding epitope encompassed the anionic site involved in the binding of these low-molecular-mass inhibitors. The third mAb (Elec-408), was clearly bound to another site on the AChE molecule, and its inhibitory effect was cumulative with those of Elec-403, Elec-410, and fasciculin. All mAbs bound AChE with high affinity and were as strong inhibitors with an apparent Ki values less than 0.1 nM. Elec-403 was particularly efficient with an inhibitory activity similar to that of fasciculin. Inhibition was observed with both charged (acetylthiocholine) and neutral substrates (o-nitrophenyl acetate) and had the characteristics of a non-competitive process. Elec-403 and Elec-410 probably exert their effect by triggering allosteric transitions from the peripheral site to the active site. The epitope recognized by mAb Elec-408 has not been localized, but it may correspond to a new regulatory site on AChE.

Production, characterization and application of monoclonal antibodies against the organophosphorus nerve agent Vx.

Two monoclonal antibodies (Vx-BB8 and Vx-EA11) to the chemical warfare agent Vx were produced and characterized. A competitive inhibition enzyme immunoassay was developed to detect Vx concentrations as low as 3.7 x 10(-7) - 3.7 x 10(-6) mol/l in biological samples. Vx-BB8 400 micrograms given intravenously immediately before 1 x LD95 Vx or 400 micrograms Vx-BB8 intraperitoneally 1.5 h-3 days before 1 x LD95 Vx could protect all the tested mice from death.

Immunochemical characterization of anti-acetylcholinesterase inhibitory monoclonal antibodies.

Monoclonal antibodies (mAbs) were prepared against native or DFP-inhibited Torpedo californica acetylcholinesterase and native or DFP-, MEPQ-, and soman-inhibited fetal bovine serum acetylcholinesterase. The cross reactivity of these antibodies with acetylcholinesterases from various species and their ability to inhibit catalytic activity were determined. Eight antibodies were found to inhibit catalytic activity of either Torpedo or fetal bovine serum enzyme. In all cases the antibodies bound to the native form of the enzymes and in some cases even to the denatured form. None of the antibodies recognized human or horse serum butyrylcholinesterase. Sucrose density gradient centrifugation of enzyme-antibody complexes provided two types of profiles, one with multiple peaks, indicating numerous complexes between tetrameric forms of the enzyme, and the other with single peaks, demonstrating complex formation within the tetrameric form. Different antibodies appeared to interact with slightly different regions, but in all cases the binding encompassed the peripheral anionic site. Decrease in catalytic activity of the enzyme was most likely caused by conformational changes in the enzyme molecule resulting from interaction with these mAbs.

Production and characterization of antibodies directed against organophosphorus nerve agent VX.

A strategy is described to raise high-affinity antibodies directed against the organophosphorus nerve agent VX [O-ethyl S-(2-diisopropylamino)ethyl)methyl phosponothionate]. Ten chemical derivatives of VX (haptens) have been synthesized. Their structures differ principally from VX structure by substitution of S-atom by an O-atom or CH2-group and by introduction of a reactive group (carboxylic acid, arylamine or primary amine) on the O-ethyl side chain. None of these haptens, except one, exhibit potential toxicity as tested by their inhability to inhibit acetylcholinesterase (E.C. 3.1.1.7.). After coupling with a protein carrier, they were injected intradermally to rabbits. Nine of these immunogenic conjugates led to the appearance of antibodies able to bind VX in a competitive solid phase immunoassay. The apparent titer and affinity of the antisera differed greatly depending on the hapten used. The highest affinity (9 nM) was observed with the VX derivative bearing O-S substitution and O-ethyl-carboxylic side chains. The antibodies appear specific for VX, since cross-reactivity with other nerve agents (Soman, Sarin or Tabun) was low. However, two haptens elicited antibodies with affinity to Soman or Sarin in the micromolar range. Antibodies were able to neutralize VX inhibition of acetylcholinesterase in vitro but not in vivo.

A homogeneous immunological detection system for soman using the in vitro protection of acetylcholinesterase by a monoclonal antibody.

In this study a monoclonal antibody (MAb) based soman detection system was investigated. Since the MAb F71D7 recognizes the pinacolyl group of soman, non-toxic soman analogues are also detected when using an indirect competitive ELISA. This can lead to falsely positive results. The toxic effect of soman is, however, independent of the pinacolyl group. In the described homogeneous enzyme immunoassay (EIA), the inhibitory effect of soman on acetylcholinesterase (AChE) was combined with its specific binding to the MAb F71D7 in order to minimize false positive results and enhance the specificity of the detection system. In this rapid EIA no incubation or washing steps are necessary, so only time for pipetting and reaction have to be considered. Soman could be detected in concentrations of 1.6-25 nM using the EIA. This corresponds to 8 pg soman per 25 microliters sample and means that compared to other ELISA systems, besides enhanced specificity, the limit of detection could be improved by 3 orders of magnitude.

Immunologic protection against VX intoxication in experimental animals.

Rabbits immunized with an artificial VX-antigen could survive 1 x LD95 of VX challenge on the 7th day and the 31st day after the last immunization. One hundred microliters of rabbit anti-VX antiserum given intravenously immediately before 1 x LD95 of VX or 200 microliters of antiserum intraperitoneally 1-10 days before 1 x LD95 VX protected all the tested mice from death. The antiserum could prevent the in vitro inhibition of Torpedo AChE activity by VX and reduced its effect on brain AChE activity in vivo. No protective effect of the antiserum was observed on the Torpedo AChE activity inhibited by sarin and soman.

Detection of the organophosphorus nerve agent soman by an ELISA using monoclonal antibodies.

The development of a specific and sensitive immunologic ELISA detection system for methylphosphonoflouridic acid. 1,2,2-trimethylpropylester (soman) by the use of monoclonal antibodies (MAbs) is described. The monoclonal antibodies F71D7, F71H10, F71B12 and F71H9 originally produced against the soman derivative methyl phosphonic acid, p-aminophenyl 1,2,2-trimethylpropyldiester (MATP) also reacted with soman in a previously developed, direct competitive ELISA. After optimizing the ELISA system by varying the reaction mixture and the solvents for the organophosphate, 5.0 x 10(-7) mol/l soman (80% purity), e.g. 2.5 ng or 2 ng pure soman per 25 microliters test buffer, could be detected after a total test duration of 40 min. A shortening of the incubation time to 10 min resulted in a drop of sensitivity to 1.8 x 10(-6) mol/l soman. Various alcohols which may be used as extraction media for soman from various materials (isopropanol, ethanol and methanol) were shown to inhibit peroxidase activity and thereby reduce the sensitivity of the test. However, the influence of alcohols decreased with the shortening of incubation time. All monoclonal antibodies showed little cross reactivity to sarin and no cross reactivity to tabun and VX. Judging on the reactivity of the MAbs with MATP and soman oxidazed by 1,2-dihydrobenzol, some reactivity with some other (non-toxic) soman analogues containing the same pinacolyl group can be expected. There was no evidence for stereoselectivity of the MAbs tested. Finally, soman could be detected in different biological samples like human serum, goat serum, rabbit serum, chicken serum, milk, and tap water in concentrations between 1.3 x 10(-6) and 2.0 x 10(-6) mol/l.