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Nerve agents with low volatility such as VX are primarily absorbed through the skin when released during combat or a terrorist attack. The barrier function of the stratum corneum may be compromised during certain stages of development, allowing VX to more easily penetrate through the skin. However, age-related differences in the lethal potency of VX have yet to be evaluated using the percutaneous (pc) route of exposure. Thus, we estimated the 24 and 48 h median lethal dose for pc exposure to VX in male and female rats during puberty and early adulthood. Pubescent, female rats were less susceptible than both their male and adult counterparts to the lethal effects associated with pc exposure to VX possibly because of hormonal changes during that stage of development. This study emphasizes the need to control for both age and sex when evaluating the toxicological effects associated with nerve agent exposure in the rat model.

Soman (GD) and VX are chemical warfare agents that can be absorbed through the skin. We determined the median lethal dose (MLD) for the cutaneous application of GD and VX in anesthetized haired guinea pigs and then tested the ability of a currently fielded decontamination kit, the M291 Skin Decontamination Kit (SDK), and decontaminating foam made by SANDIA Labs to decontaminate areas that have been exposed to cutaneous applications of GD and VX. The fur of guinea pigs was clipped on the left flank 24h prior to exposure. Animals were anesthetized and 5 min later neat GD or neat VX was applied. The MLD for percutaneous exposure to GD was 11.6 mg/kg, and to VX it was 0.10mg/kg. To test the ability of the M291 SDK, either GD or VX was applied and removed 1 min later with the pads of the M291 SDK clasped in a pair of forceps and wiped across the flank of the animal. The MLDs for GD and VX removed with the M291 SDK pads were 76.9 mg/kg and 0.87 mg/kg, respectively. When neat GD or neat VX was applied and removed 1 min later in the same manner with gauze soaked in SANDIA foam (MDF-100), the MLDs were 412 mg/kg and 10.4 mg/kg respectively. These data demonstrate that GD and VX are significantly less potent when applied cutaneously than previously reported for subcutaneous injections and indicate that improvement is needed on the limited protective ratio provided by the M291 SDK., (Published by Elsevier Ireland Ltd.)

Objective: This report, first in a series of five, directly compares the efficacy of 4 decontamination products and Skin Exposure Reduction Paste Against Chemical Warfare Agents (SERPACWA) in the haired guinea pig model following exposure to VX., Methods: In all experiments, guinea pigs were close-clipped and given anesthesia. In the decontamination experiments, the animals were challenged with VX and decontaminated after a 2-minute delay for the standard procedure or at longer times for the delayed-decontamination experiments. Skin Exposure Reduction Paste Against Chemical Warfare Agents was applied as a thin coating (0.1 mm thick), allowed to dry for 15 minutes, and challenged with VX. After a 2-hour challenge, any remaining VX was blotted off the animal, but no additional decontamination was done. Positive control animals were challenged with VX in the same manner as the treated animals, except that they received no treatment. In addition, the positive control animals were always challenged with 5% VX in isopropyl alcohol (IPA) solution, whereas the treatment animals received either neat (undiluted) VX or 5% VX in IPA solution. All animals were observed during the first 4 hours and again at 24 hours after exposure for signs of toxicity and death. The protective ratio (PR, defined as the median lethal dose [LD(50)] of the treatment group divided by the LD(50) of the untreated positive control animals) was calculated from the probit dose-response curves established for each treatment group and nontreated control animals. Significance in this report was defined as p < .05., Results: In the standard 2-minute neat VX decontamination experiments, the calculated PRs for Reactive Skin Decontamination Lotion (RSDL), 0.5% bleach, 1% soapy water, and the M291 Skin Decontamination Kit (SDK) were 66, 17, 16, and 1.1, respectively. Reactive Skin Decontamination Lotion was by far the most effective decontamination product tested and was significantly better than any of the other products. Bleach and soapy water provided equivalent and good (PR > 5) protection. They were both significantly better than the M291 SDK. The M291 SDK did not provide significant protection compared with positive controls. In the neat VX delayed-decontamination experiments, the calculated LT(50) (the delayed-decontamination time at which 50% of the animals died in the test population following a 5-LD(50) challenge) values for RSDL, 0.5% bleach, and 1% soapy water were 31, 48, and 26 minutes, respectively. The results showed that SERPACWA provided significant, but modest (PR < 5), protection against neat VX, with a PR of 2.1., Conclusions: Several conclusions can be drawn from this study: 1) RSDL provided superior protection against VX compared with the other products tested; 2) 0.5% bleach and 1% soapy water were less effective than RSDL, but still provided good protection against VX; 3) the M291 SDK was the least effective decontamination product and did not provide significant protection against VX; 4) the agent was observed to streak when using the M291 SDK, and efficacy may improve if the agent is first blotted, followed by wiping with a new or clean part of the M291 SDK pad; 5) RSDL, 0.5% bleach, and 1% soapy water provided significant protection against a 5-LD(50) challenge of VX, even when decontamination was delayed for up to about 30 minutes; and 6) SERPACWA provided significant, but modest, protection against VX.

Objective: This report, the second in a series of five, directly compares the efficacy of Reactive Skin Decontamination Lotion (RSDL), the M291 Skin Decontamination Kit (SDK), 0.5% bleach (sodium or calcium hypochlorite solution), 1% soapy water, and Skin Exposure Reduction Paste Against Chemical Warfare Agents (SERPACWA) in the haired guinea pig model following exposure to soman (GD)., Methods: In all experiments, guinea pigs were close-clipped and given anesthesia. In the decontamination experiments, the animals were challenged with GD and decontaminated after a 2-minute delay for the standard procedure or at longer times for the delayed-decontamination experiments. Positive control animals were challenged with GD in the same manner as the treated animals, except that they received no treatment. All animals were observed during the first 4 hours and again at 24 hours after exposure for signs of toxicity and death. The protective ratio (PR, defined as the median lethal dose [LD(50)] of the treatment group divided by the LD(50) of the untreated positive control animals) was calculated from the derived probit dose-response curves established for each treatment group and nontreated control animals. SERPACWA was applied as a thin coating (0.1 mm thick), allowed to dry for 15 minutes, and challenged with GD. After a 2-hour challenge, any remaining GD was blotted off the animal, but no additional decontamination was done. Significance in this report is defined as p <.05. Neat (undiluted) GD was used to challenge all animals in these studies., Results: In the standard 2-minute GD decontamination experiments, the calculated PRs for RSDL, 0.5% bleach, 1% soapy water, and M291 SDK were 14, 2.7, 2.2, and 2.6, respectively. RSDL was by far the most effective decontamination product tested and significantly better than any of the other products. Bleach, soapy water, and the M291 SDK provided equivalent and modest protection. Since only RSDL provided at least good protection (PR > 5), it was the only decontamination product evaluated for delayed decontamination. In the GD delayed-decontamination experiments, the calculated LT(50) (the delayed-decontamination time at which 50% of the animals die in the test population following a 5-LD(50) challenge) value for RSDL was only 4.0 minutes., Conclusions: Several conclusions can be drawn from this study: 1) Reactive Skin Decontamination Lotion provided superior protection against GD compared with the other products tested; 2) The 0.5% bleach solution, the 1% soapy water solution, and the M291 SDK were less effective than RSDL, but still provided modest (2 < PR < 5) protection against GD; 3) Reactive Skin Decontamination Lotion, the best product tested, did not provide significant protection against GD when decontamination was delayed for more than 3 minutes; 4) Skin Exposure Reduction Paste Against Chemical Warfare Agents provided significant, but modest, protection against GD; 5) There was good correlation between using the rabbit model and the guinea pig model for decontamination efficacy evaluations; and 6) Soman (GD) is an agent of real concern because it is very difficult to decontaminate and the effects of exposure are difficult to treat.

The administration of purified human plasma-derived butyrylcholinesterase (HuBuChE) as a pretreatment has been demonstrated to enhance survival and protect against decreased cognitive function after exposure to organophosphorus poisons (OPs). Based on efficacy data obtained with guinea pigs and non-human primates and the lack of behavioral side effects, plasma-derived HuBuChE has been granted investigational new drug status by the US Food and Drug Administration. The recent availability of a recombinant form of HuBuChE (rHuBuChE) from the milk of transgenic goats has now allowed us to determine the pharmacokinetics of that material in guinea pigs and use it as a therapy following exposure to the VX. The rHuBuChE was expressed as a dimer and following intramuscular (i.m.) administration had more a rapid adsorption and clearance profile in guinea pigs than the plasma-derived material. Based on those data, we administered rHuBuChE i.m. 1h after a percutaneous exposure of guinea pigs to either 2xLD(50) or 5xLD(50) of VX. Post-exposure therapy with rHuBuChE provided improved survival at both challenge levels, 90% and 33% respectively versus 20% or 0% respectively for animals that did not receive therapy. These studies showed that BuChE can be efficacious as a therapy against percutaneous exposure to VX., (Published by Elsevier Ireland Ltd.)

Novel therapeutics to overcome the toxic effects of organophosphorus (OP) chemical agents are needed due to the documented use of OPs in warfare (e.g. 1980-1988 Iran/Iraq war) and terrorism (e.g. 1995 Tokyo subway attacks). Standard OP exposure therapy in the United States consists of atropine sulfate (to block muscarinic receptors), the acetylcholinesterase (AChE) reactivator (oxime) pralidoxime chloride (2-PAM), and a benzodiazepine anticonvulsant to ameliorate seizures. A major disadvantage is that quaternary nitrogen charged oximes, including 2-PAM, do not cross the blood brain barrier (BBB) to treat brain AChE. Therefore, we have synthesized and evaluated pro-2-PAM (a lipid permeable 2-PAM derivative) that can enter the brain and reactivate CNS AChE, preventing seizures in guinea pigs after exposure to OPs. The protective effects of the pro-2-PAM after OP exposure were shown using (a) surgically implanted radiotelemetry probes for electroencephalogram (EEG), (b) neurohistopathology of brain, (c) cholinesterase activities in the PNS and CNS, and (d) survivability. The PNS oxime 2-PAM was ineffective at reducing seizures/status epilepticus (SE) in diisopropylfluorophosphate (DFP)-exposed animals. In contrast, pro-2-PAM significantly suppressed and then eliminated seizure activity. In OP-exposed guinea pigs, there was a significant reduction in neurological damage with pro-2-PAM but not 2-PAM. Distinct regional areas of the brains showed significantly higher AChE activity 1.5h after OP exposure in pro-2-PAM treated animals compared to the 2-PAM treated ones. However, blood and diaphragm showed similar AChE activities in animals treated with either oxime, as both 2-PAM and pro-2-PAM are PNS active oximes. In conclusion, pro-2-PAM can cross the BBB, is rapidly metabolized inside the brain to 2-PAM, and protects against OP-induced SE through restoration of brain AChE activity. Pro-2-PAM represents the first non-invasive means of administering a CNS therapeutic for the deleterious effects of OP poisoning by reactivating CNS AChE., (Published by Elsevier Ireland Ltd.)

The purpose of this study was to evaluate the efficacy of a novel barrier cream formulation at reducing the percutaneous toxicity of a 2xLD(50) liquid challenge of nerve agent (VX). The study was conducted in vitro and in vivo using the domestic pig. Pretreatment of the (inner ear skin) exposure site with barrier cream eliminated mortality, reduced cholinesterase inhibition and prevented any physiological or biochemical signs of intoxication. In contrast, untreated animals exposed to VX exhibited severe signs of intoxication, near total AChE inhibition and generally died within the (3 hr) exposure period (5/6 animals). Application of the barrier cream caused a significant decrease in the area of skin contaminated by VX. It was tentatively concluded that spreading was predominantly a surface phenomena (possibly mediated by capillary movement of the agent through the microrelief or between hair follicles) with little or no contribution from lateral diffusion within the stratum corneum. There was a disparity between the in vitro and in vivo skin absorption measurements that was ascribed to the absence of systemic clearance in vitro. However, both models indicated a substantial reservoir of VX within the skin, providing a potential strategy for future investigations into "catch-up therapies". In summary, the novel barrier cream formulation was effective against a 2xLD(50) (liquid, percutaneous) dose of VX applied for 3 hr. Further work should be conducted to investigate more pragmatic issues such as optimal reapplication frequency and environmental effects such as temperature and humidity.

The purpose of this study was to characterize the skin absorption and distribution of VX (O-ethyl-S-[2 (diisopropylamino)ethyl] methylphosphonothioate) in the domestic pig in order to evaluate the animal as a potential model for assessing pretreatments against toxic anti-cholinesterase compounds. A liquid droplet (equivalent to a 2 x LD50 dose) of radiolabelled VX was applied to the inner ear-skin of each anaesthetized animal. Blood and tissue samples (liver, lung, kidney, heart and skin exposure sites) were obtained post-mortem. The amount of radioactivity in each sample was measured by liquid scintillation counting, from which the skin absorption rate and dose distribution of VX were calculated. A substantial proportion (22 +/- 3%) of the applied dose remained within the skin at the site of application. It is conceivable that strategies to minimize or remove this reservoir may be of benefit in the early treatment of VX-exposed casualties. Image analysis of autoradiographs of exposed skin sites indicated that each milligram of radioactive VX covered an area of 1.2 +/- 0.5 cm2. The average skin absorption rate of 14C-VX was 661 +/- 126 microg/cm2 per hour. Comparison of these data with previous studies suggests that human skin is less permeable to VX than pig skin, but VX spreads over a greater surface area when applied to human skin. Thus, paradoxically, while pig-ear skin is more permeable than human skin, the difference in skin surface spreading may lead to the absorption of an equivalent systemic dose.

British anti-Lewisite (BAL) (2,3-dimercapto-1-propanol) is a potential therapeutic compound when used against the effects of cutaneous sulfur mustard, and a method for its determination in plasma has been developed. BAL and the internal standard (IS) ethane dithiol were isolated from plasma samples through solid-phase extraction and then reacted with 1-pentafluoropropionylimidazole, forming stable pentafluoropropionyl derivates that are sensitive to gas chromatographic-mass spectrometric analysis. Examination of concentration versus peak-area ratios of the BAL and IS derivatives demonstrated the method to be linear over a concentration range of 0.48 to 124 ng/mL in plasma when fit to a weighted (1/y2) least-squares regression. Correlation coefficients were 0.9943 to 0.9995 for six runs, and coefficients of variation (CV) were 2.5 to 8.7% over the eight concentrations tested. The intra- and interday accuracy and precision of this method was measured by examining six groups of eight unknown test samples (n = 6). Intraday accuracy, as expressed by percent error, was found to range from -15.4 to 0.21%, whereas the precision, expressed as %CV, was less than 9.8% over all sample concentrations. Interday test unknown sample results were similar in that the accuracy was shown to be -7.1 to 0.4%, and precision was 4.7 to 9.5%. BAL levels in frozen plasma (-70 degrees C) remained constant for more than 14 days with a CV of less than 10% for the eight concentrations tested. The data indicate that the method will provide accurate and precise determination of BAL at concentrations down to approximately 1 ng/mL in plasma. This procedure has been applied to determine preliminary time-concentration profile studies of BAL in the hairless guinea pig.

The limited availability of human embryonic tissue for dopamine cell transplants in Parkinson's patients has led to an increased interest in using xenogeneic donor tissue. Unfortunately, without aggressive immunosuppression, such brain xenografts are rejected by the host immune system. Chronic brain xenograft rejection is largely mediated by helper T cells, which require presentation of xenoantigens by major histocompatability complex (MHC) class II for their activation. We examined survival and function of xenografts of E13 mouse mesencephalon deficient in either MHC class I, class II, or both after transplantation into adult hemiparkinsonian rats without immunosuppression. Recipients received grafts from C57BL/6 mice that were either: 1) wild-type (wt), 2) MHC class I knockout (KO), 3) MHC class II KO, 4) MHC class I and II double KO, or 5) saline sham transplants. At 6 weeks after transplantation, recipients of MHC class I KO, class II KO, and double KO xenografts significantly reduced methamphetamine-induced circling rate while rats with wt xenografts and sham-operated rats showed no improvement. MHC class II KO grafts had the greatest number of surviving dopamine neurons. All transplants, including saline sham controls, contained infiltrating host MHC class II-positive cells. Saline sham grafts and MHC class II KO xenografts contained significantly fewer infiltrating host MHC class II-positive cells than did wt grafts. Our results show that MHC class II-deficient xenografts survive transplantation for at least 6 weeks in the absence of immunosuppression, reduce rotational asymmetry, and provoke lesser immune reaction than wt grafts.

To promote dopamine cell survival in human fetal tissue strands transplanted into immunosuppressed 6-OHDA-lesioned rats, we have preincubated tissue in insulin-like growth factor-I (IGF-I, 150 ng/ml) and basic fibroblast growth factor (bFGF, 15 ng/ml) in vitro for 2 weeks. Growth factor treatment did not affect the rate of homovanillic acid production in vitro but increased overall dopamine neuron survival in animals after transplant from 1240 +/- 250 to 2380 +/- 440 neurons (P < 0.05). Animals in the growth factor-treated group had a significantly greater reduction in methamphetamine-induced rotation (66%) compared to control transplants (30%, P < 0.05). We conclude that in vitro preincubation of human fetal tissue strands with IGF-I and bFGF improves dopamine cell survival and the behavioral outcome of transplants., (Copyright 2001 Academic Press.)

Fetal cell transplantation therapies are being developed for the treatment of a number of neurodegenerative disorders including Parkinson's disease [10-12,21,22,24,36,43]. Massive apoptotic cell death is a major limiting factor for the success of neurotransplantation. We have explored a novel protein kinase pathway for its role in apoptosis of dopamine neurons. We have discovered that inhibitors of p38 MAP kinase (the pyridinyl imidazole compounds: PD169316, SB203580, and SB202190) improve survival of rat dopamine neurons in vitro and after transplantation into hemiparkinsonian rats. In embryonic rat ventral mesencephalic cultures, serum withdrawal led to 80% loss of dopamine neurons due to increased apoptosis. Incubation of the cultures with p38 MAP kinase inhibitors at the time of serum withdrawal prevented dopaminergic cell death by inhibiting apoptosis. In the hemiparkinsonian rat, preincubation of ventral mesencephalic tissue with PD169316 prior to transplantation accelerated behavioral recovery and doubled the survival of transplanted dopamine neurons. We conclude that inhibitors of stress-activated protein kinases improve the outcome of cell transplantation by preventing apoptosis of neurons after grafting.

Over the past 13 years approximately 300 patients with Parkinson's disease have received transplants of human fetal dopamine cells in an attempt to reduce or control disease symptoms. Many of these patients have had improvements in their motor skills and a reduction in their daily levodopa administration. However, improvements are far from guaranteed and questions need to be answered before this technique can be widely applied. To help address some of these issues, a search of all the published results of patients with Parkinson's disease transplanted with human fetal tissue was conducted. This generated a database of 70 transplant recipients who had their levodopa administration and clinical benefit reported both prior to transplant and at least 6 months post-transplant. Furthermore, the number of years of disease onset prior to transplant was available for all recipients. This database was examined for motor improvement and reduction in levodopa dosage for up to 2 years post-transplant to determine the effects of time on transplant outcome. The database showed that most recipients had significant improvements in motor skills and levodopa administration, and that most benefits were observed in the first 6 months post-transplant. In addition, the database demonstrated that the number of years of disease onset prior to transplantation was not a predictor of patient outcome 1-year post-transplant. Current and future directions in fetal tissue transplantation research and replacements for fetal tissue are discussed.

Extracts from skeletal muscle cell cultures have been shown to increase levels of the enzyme tyrosine hydroxylase (TH) and promote survival of different types of developing neurons in vitro. To determine the effect of muscle cell co-grafts on the survival of dopamine neurons in a rat model of Parkinson's disease, we transplanted an embryonic day (ED)-15 rat mesencephalic cell suspension alone or with neonatal muscle cells into 6-hydroxydopamine (6-OHDA) denervated rat striatum. In parallel experiments conducted in vitro, we cultured ED-15 rat mesencephalon or rat striatum in conditioned medium from neonatal rat muscle cultures (MC-CM). Our results showed that: (A) in vitro, MC-CM increased the number of TH-immunoreactive (TH-IR) neurons in embryonic mesencephalic cultures but did not induce expression of TH in embryonic striatal cultures; (B) in vivo, animals with co-grafts of muscle cells and ED-15 mesencephalon had more TH-IR in the grafted striatum compared to animals that received mesencephalic cells grafts alone, although the graft-induced reversal of circling behavior in response to methamphetamine was the same in both transplanted groups; and (C) grafts of muscle cells alone did not induce TH-IR in the denervated striatum and did not reduce methamphetamine-induced circling. These findings suggest that in vivo, neonatal muscle cells secrete factors that promote survival and/or outgrowth of fetal midbrain dopamine cells and improve the levels of TH-IR in grafted striatum.

6-Hydroxydopamine (6-OHDA) causes selective degeneration of dopaminergic neurons in the rat brain and has been used to produce an animal model of Parkinsonism. Recently, a clonal line of immortalized dopamine (DA) neurons (1RB3AN27), which expresses varying levels of tyrosine hydroxylase, dopamine transporter, neuron specific enolase, and nestin, was established. These DA neurons reduce behavioral deficits in 6-OHDA-lesioned rats. The relative sensitivity of fetal and adult neurons to potential neurotoxins is not well defined. The availability of immortalized DA neurons provides a unique opportunity to compare the relative neurotoxicity of 6-OHDA in differentiated and undifferentiated DA neurons in vitro and identify neuroprotective agents. Our results showed that 6-OHDA treatment for 24 hr decreased the viability of undifferentiated and differentiated immortalized DA neurons in vitro, as determined by the MTT assay, and increased the rate of apoptosis in differentiated DA neurons. The differentiated DA neurons (IC50 = 33 microM) were about 2-fold more sensitive to 6-OHDA than undifferentiated DA neurons (IC50 = 75 microM) in cell culture. Similarly, the differentiated DA neurons were more sensitive to another neurotoxin, 1-methyl-4-phenylpyridinium (MPP+), which is commonly used to induce Parkinsonism in animal models, than were the undifferentiated DA neurons in culture. Among growth factors tested, only glial cell line-derived neurotrophic factor (GDNF) partially protected differentiated DA neurons against 6-OHDA-induced toxicity. These results suggest that undifferentiated and differentiated immortalized DA neurons can be a useful experimental model to study relative sensitivity to neurotoxins and neuroprotective agents that could have relevance to fetal and adult neurons.

Since 1988, patients with Parkinson's disease have participated in clinical trials evaluating the efficacy of transplantation of human fetal dopamine cells into the caudate and putamen. Transplantation of fetal tissue leads to clinical benefits in some patients which is associated with a reduction of the amount of LDOPA administered. Major issues in transplant research need to be addressed before this technique can be widely applied. In this review, a pool of 35 patients was generated from the published cases of human fetal tissue transplantation. This group of transplant recipients was examined for motor improvement and reduction in L-DOPA dosage at one year post-transplant. Issues addressed in this review include the benefits of unilateral vs bilateral transplantation, age of the transplant recipient, solid vs suspensions of fetal mesencephalon and the number of fetal donors per recipient.

The success of embryonic neural transplants as a treatment for patients with Parkinson's disease has been limited by poor survival of transplanted dopamine neurons. To see if a new partially intact tissue preparation method improves survival, we have developed a technique for extruding embryonic tissue into strands. We expected this method to reduce cell damage and improve transplant survival as well as provide improved tissue delivery. We have compared transplants of tissue strands with mechanically dispersed suspensions of embryonic day 15 rat ventral mesencephalon. Tissue from ventral mesencephalon was transplanted into a single site in dopamine denervated striatum of unilateral 6-hydroxydopamine (6-OHDA) lesioned rats. To evaluate the effects of striatal cografts and growth factors on dopamine cell survival, dispersed mesencephalic cells were cotransplanted with dispersed striatal cells. Another group had dispersed mesencephalic cells cotransplanted with striatal cells incubated in the cold for 2 h with glial cell line-derived neurotrophic factor (GDNF, 100 ng/ml), insulin-like growth factor-I (IGF-I, 1500 ng/ml), and basic fibroblast growth factor (bFGF, 150 ng/ml). Behavioral improvement was assessed monthly by changes in methamphetamine-induced rotational behavior. Animals were sacrificed after 3 months, and dopamine neurons were identified by tyrosine hydroxylase (TH) immunohistochemistry. Transplants of tissue strands produced better dopamine neuron survival and led to more robust behavioral restoration than did cell suspensions even when suspensions were supported with cografts of striatal cells or pretreatment with growth factors., (Copyright 1998 Elsevier Science B.V.)

Dopamine (DA) deficiency is one of the primary lesions in the pathogenesis of Parkinson disease (PD). Because of long-term toxicity of L-DOPA therapy, the grafting of fetal mesencephalic tissue containing dopamine neurons or homogeneous populations of DA neurons into striatum appears to be rational. Fetal tissue transplants have many problems which include legal (in some countries), ethical, paucity of tissue availability, heterogenicity of cell populations, and the presence of antigen-presenting cells that are responsible for rejection of allogeneic grafts. In order to resolve the above problems, we have established immortalized DA neurons from fetal rat mesencephalon by inserting the large T-antigen (LTa) gene of the SV40 virus into the cells. A clone of DA neurons (1RB3AN27) was isolated, characterized, and tested in 6-hydroxydopamine (6-OHDA)-lesioned rats (a model of PD). These cells divided with a doubling time of about 26 h, expressed the LTa gene, and contained the tyrosine hydroxylase and dopamine transporter proteins and their respective mRNAs, which became elevated upon differentiation. These cells were nontumorigenic and nonimmunogenic and improved the symptoms of neurological deficits (methamphetamine-induced rotation) in 6-OHDA-lesioned rats. The differentiated DA neurons were more effective than undifferentiated ones. These studies suggest that immortalized DA neurons generated in vitro by LTa gene insertion may be used in transplant therapy without fear of tumor formation or rejection., (Copyright 1998 Academic Press.)

Parkinson's disease symptoms can be improved by transplanting fetal dopamine cells into the putamen of parkinsonian patients. Because the supply of human donor tissue is limited and variable, an alternative and genetically modifiable non-human source of tissue would be valuable. We have generated cloned transgenic bovine embryos, 42% of which developed beyond 40 days. Dopamine cells collected from the ventral mesencephalon of the cloned fetuses 42 to 50 days post-conception survived transplantation into immunosuppressed parkinsonian rats and cells from cloned and wild-type embryos improved motor performance. Somatic cell cloning can efficiently produce transgenic animal tissue for treating parkinsonism.

Embryonic dopamine neurons survive poorly after transplant into models of Parkinson's disease, possibly due to programmed cell death (apoptosis). Apoptosis in cultured dopamine neurons can be reduced by growth factors such as glial cell line-derived neurotrophic factor (GDNF) or a combination of insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF). To improve the survival of dopamine neurons in grafts, strands of E15 rat ventral mesencephalon were pretreated with a combination of GDNF, IGF-I, and bFGF and then transplanted into 6-hydroxydopamine-lesioned rats. In control animals, only 32% of dopamine neuron profiles survived the first 24 h after transplant. Growth factor pretreatment increased survival to 49% on day 1. Growth factors reduced the apoptotic rate of transplanted cells, just as they had in the previous in vitro experiments. Apoptotic nuclear morphology was observed in the transplanted dopamine neurons. We conclude that the majority of transplanted dopamine neurons die in grafts within the first 24 h after transplant, most likely by an apoptotic mechanism. Prevention of apoptosis with anti-apoptotic agents may improve the viability of dopamine neurons grafted for Parkinson's disease., (Copyright 1998 Elsevier Science B.V.)

The replacement of dopamine (DA) by DA neuron transplants in the treatment of advanced Parkinson disease (PD) is a rational approach. Because of limitations associated with fetal tissue transplants, a clone (1RB3AN27) of simian virus 40 large tumor antigen (LTa) gene-induced immortalized DA neurons were used in this study. These allogeneic immortalized dopamine neurons, when grafted into striata of normal rats, did not divide, did not form tumors, did not produce LTa, did not extend neurites to host neurons, and were not rejected, for as long as 13 months after transplantation. Grafted cells when recultured in vitro resumed cell proliferation and LTa production, suggesting the presence of a LTa gene-inhibiting factor in the brain. The grafting of undifferentiated and differentiated 1RB3AN27 cells or differentiated murine neuroblastoma (NBP2) cells into striata of 6-hydroxydopamine-lesioned rats (an animal model of PD) caused a time-dependent improvement in neurological deficits (reduction in the methamphetamine-induced turning rate). At 3 months after transplantation, 100% of the animals receiving differentiated 1RB3AN27 cells, 63% of the animals receiving undifferentiated 1RB3AN27 cells, 56% of the animals receiving differentiated NBP2 cells, and 0% of the sham-transplanted animals showed improvements in neurological deficits. At 6 months after transplantation, there was a progressive increase in spontaneous recovery in sham-transplanted animals. These results suggest that immortalized DA neurons should be further studied for their potential use in transplant therapy in advanced PD patients.

A sensitive method is described to detect isolysergic acid diethylamide (iso-LSD) in urine. The compound was extracted from urine and converted to a C-8 carbanion by sodium ethoxide in ethanol. Protonation of the carbanion by water selectively produced LSD. The conversion of iso-LSD to LSD was almost quantitative (98%). The product was purified by solid-phase fractionation and acid-base separation techniques. The trimethylsilyl derivative of LSD was detected by a gas chromatography-mass spectrometry method. The overall recovery of the procedure was approximately 69%. Quantification of iso-LSD was linear over the concentration range 50-2000 ng/L. In specimen analysis, iso-LSD was detected when the LSD concentration was below the limit of the detection (50 ng/L) of the procedure. Because iso-LSD is a byproduct of illicit preparation of LSD, presence of iso-LSD in urine is an indication of LSD use.

SV40 large T-antigen (LTa) gene-induced immortalized rat dopamine-producing nerve cells (IRB3AN27), which produce LTa protein and divide in vitro, do not divide and do not produce LTa protein when transplanted into striatum of adult rats. This suggests the presence of LTa gene-inhibiting factors in brain. Here we report that rat brain soluble fraction (SF) contains factors which specifically inhibit LTa gene activity in vitro. The brain SF inhibited LTa protein levels and the growth of IRB3AN27 cells and 2RSG cells (LTa gene-induced immortalized rat parotid acinar cells) in vitro, but it stimulated the growth of spontaneously immortalized human parotid acinar cells (2HPC8) and had no effect on the proliferation of murine neuroblastoma cells (NBP2) and rat glioma cells (C-6) in culture. In contrast, the liver SF inhibited the growth of all cell lines tested at varying degrees and thus lacked specificity with respect to LTa gene activity. The presence of specific LTa gene-inhibiting factors in the brain and general tumor growth-inhibiting factors in the liver may provide some of the mechanisms of protection against in vivo carcinogenesis.

Dopamine cell death is the primary problem limiting the value of neurotransplantation in human patients with Parkinson's disease. To address this problem, we added glial cell line-derived neurotrophic factor (GDNF) to cultures of embryonic dopaminergic neurons obtained from human and from Bonnet monkey (Macaca radiata) in an effort to reduce apoptotic cell death and improve overall cell survival. Tissue from three human embryos, 7-8 weeks post-conception, and one 9-week post-conception monkey embryo were dissociated and cultured in F-12 media with 5% human placental serum. GDNF (10 ng/ml) in human cultures nearly doubled dopamine neuron survival and reduced the rate of apoptosis from 6% to 3%. In monkey cultures, GDNF also enhanced dopamine neuron survival and reduced the apoptotic rate. We conclude that GDNF improves the survival of primate embryonic dopamine neurons in culture by reducing apoptosis.

To study the possibility that increasing striatal activity of aromatic L-amino acid decarboxylase (AADC; EC 4.1.1.28) can increase dopamine production in dopamine denervated striatum in response to L-3,4-dihydroxy-phenylalanine (L-DOPA) administration, we grafted Cos cells stably expressing the human AADC gene (Cos-haadc cells) into 6-hydroxydopamine denervated rat striatum. Before grafting, the catalytic activity of the enzyme was assessed in vitro via the generation of 14CO2 from L-[14C]DOPA. The Km value for L-DOPA in intact and disrupted cells was 0.60 and 0.56 mM, respectively. The cofactor, pyridoxal 5-phosphate, enhanced enzymatic activity with maximal effect at 0.1 mM. The pH optimum for enzyme activity was 6.8. Grafting Cos-haadc cells into denervated rat striatum enhanced striatal dopamine levels measured after systemic administration of L-DOPA. When measured 2 h after L-DOPA administration, the mean dopamine level in the striata of Cos-haadc-grafted animals was 2 micrograms/g of tissue, representing 31% of normal striatal dopamine concentration. The mean dopamine concentration in the striata grafted with untransfected Cos cells (Cos-ut cells) was 1 microgram/g. At 6-8 h after L-DOPA administration, striatal dopamine content in the Cos-haadc-grafted animals was 0.67 microgram/g of tissue weight, representing 9% of intact striatum dopamine content. By contrast, the average dopamine content in the Cos-ut-grafted animals was undetectable. These findings demonstrate that enhancing striatal AADC activity can improve dopamine bioformation in response to systemically administered L-DOPA.

To explore the mechanism by which glial cell line-derived neurotrophic factor (GDNF) improves cell survival, we measured the apoptotic rate of dopamine neurons incubated with GDNF. Cultures were prepared from embryonic day 15 rat mesencephalon in medium with 5% human placental serum. GDNF reduced the rate of apoptosis in dopamine neurons from 5% to 2%. By contrast, GDNF had no effect on apoptoisis in astrocytes from embryonic mesencephalon or from neonatal cortex. Co-cultures with embryonic striatum as well as with combinations of growth factors were also tested for effects on dopamine neuron survival. Neuronal survival was maximal during co-culture with striatal cells with or without added growth factors. We conclude that GDNF inhibits apoptotic cell death in dopamine neurons.

The acetylcholine transporter exhibits such low affinity and specificity for acetylcholine that it appeared possible it could fail to select against other neurotransmitters. Potential interactions of classical noncholinergic neurotransmitters with cholinergic synaptic vesicles purified from electric organ were studied. No active transport of [3H]serotonin, [3H]noradrenaline, or [3H]glutamate occurred. Serotonin, noradrenaline, and N-acetyl-aspartyl glutamate inhibited active transport of [3H]acetylcholine by the vesicles. Dopamine previously had been shown to inhibit transport. Glutamate and gamma-aminobutyric acid were shown here not to inhibit active transport of [3H]-acetylcholine. Noradrenaline was competitive with respect to [3H]acetylcholine in this effect. Serotonin, noradrenaline, and dopamine inhibited binding of [3H]vesamicol to the vesicles, and dopamine was a competitive inhibitor of the binding of this allosteric ligand of the acetylcholine transporter. The results indicate that the acetylcholine transporter does not transport any other classical neurotransmitter, but serotonin, noradrenaline, and dopamine bind to the acetylcholine site.

A previous structure-activity investigation of acetylcholine (ACh) revealed a positive correlation between additional hydrophobic bulk and increased potency for inhibition of active transport of [3H]ACh by synaptic vesicles isolated from the electric organ of Torpedo. In the current study, several ACh analogues that are significantly larger than previously studied "false transmitters" were synthesized in the tritiated form by chemical means and tested for active transport. These are analogue 14 [(+/-)-(cis,trans)-1-benzyl-1-methyl-3-acetoxypyrrolidinium iodide], analogue 15 [(+/-)-1,1-dimethyl-3-benzoyloxypyrrolidinium iodide], and analogue 16/17 [(+/-)-(cis,trans)-1-benzyl-1-methyl-3-benzoyloxypyrrolidinium iodide]. These analogues place significant additional hydrophobic bulk on one or the other (analogues 14 and 15) or both (analogue 16/17) of the two pharmacophores of a small, conformationally constrained analogue of ACh. [3H]Analogue 14 and [3H]analogue 15 are actively transported, with Vmax values the same as or less than that of ACh, depending on the vesicle preparation. The observation that Vmax is the same for an analogue and ACh in some vesicle preparations suggests that the rate-limiting step does not involve ACh bound to the transporter. [3H]Analogue 16/17 is actively transported very poorly. Km values for ACh and for transported ACh analogues vary by up to two- to threefold in different vesicle preparations. The ACh transporter is much less selective for transported substrates than anticipated.

The ligand binding relationship between the acetylcholine transporter (AcChT) and the vesamicol receptor (VR) and the kinetics of active transport were studied in synaptic vesicles purified from the Torpedo electric organ using analogues of AcCh and vesamicol. Methoxyvesamicol, which should exhibit better equilibration properties for kinetics measurements than the more potent parent, inhibits active transport in a nonlinear noncompetitive manner. AcCh analogues competitively inhibit binding of [3H]vesamicol with higher affinity in hyposmotically lysed vesicle ghosts than in intact vesicles, apparently due to removal of a competing internal, osmotically active factor. AcCh and actively transported analogues of AcCh that are up to 57% larger in van der Waals volume exhibit up to a 200-fold ratio for the dissociation constant measured by inhibition of vesamicol binding to ghosts (KIAg) compared to the Michaelis constant for transport (KM) or the IC50 value for inhibition of [3H]AcCh active transport. In contrast, two AcCh analogues that are about 120% larger and that almost surely are not transported exhibit a KIAg/IC50 ratio of about 1. The data demonstrate that the vesamicol family of compounds binds to an allosteric site in the AcChT. Initiation of active transport has no apparent effect on the affinities of vesamicol and AcCh analogues, which suggests that most of the AcChT-VR in purified vesicles is transport incompetent. Vesicle ghosts actively transport [3H]AcCh nearly as well as intact vesicles, which suggests that internal factor does not affect transport-competent AcChT-VR. A kinetics model is proposed that predicts that AcCh analogues exhibiting a KIAg/IC50 ratio significantly greater than 1 are actively transported. Some of the microscopic constants in the model are estimated. The AcChT binds AcCh very weakly with a dissociation constant of about 20-50 mM, but it transports substrates rapidly in a process exhibiting remarkably little selectivity for the detailed shape and volume of the transported ion.