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Introduction. Inflammation is characterized by complex interactions between pro- and anti- inflammatory cytokines. Recent research has probed the role of the nervous system in inflammation, part of which includes the cholinergic anti-inflammatory pathway that regulates immunologically-mediated inflammation. In this pathway, norepinephrine release from the splenic nerves binds to beta-2-adrenergic receptors on T cells, causing release of acetylcholine (ACh). ACh subsequently suppresses macrophage production and release of pro-inflammatory cytokines. In this project, we aim to develop a method to quantify the ACh release in splenocytes when challenged with different mediators in this pathway. Our method utilizes liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for quantification of ACh and choline (Ch) in cell culture media. Methods. Literature review revealed that the hydrophilic interaction liquid chromatography (HILIC) is the most appropriate separation mechanism for ACh and Ch. The developed LC-MS/MS method utilizes an isocratic separation (14% 10mM ammonium formate, pH 3, and 86% acetonitrile) on an Atlantis HILIC column (2.1 x 100 mm, 3 micron). The MS operates in positive electrospray (+ESI) mode, monitoring ions specific for ACh, Ch, and their corresponding deuterium labeled internal standards. The calibration range for ACh was 0.5 - 5 micrograms/ml (3.4 - 34 mM) and 10 - 50 micrograms/ml (96 - 480 mM) for Ch. Cell culture media contained neostigmine to inhibit cholinesterase. Cell culture media samples are prepared by freeze drying, reconstituting in acetonitrile, and filtering (0.2micron). Potential loss of ACh through degradation during cell culture was evaluated by monitoring d4-labeled ACh with and without the presence of splenocytes for 4 and 24 hours. Results. Correlation coefficients (R2) indicate linearity for ACh and Ch in acetonitrile and culture media in the aforementioned calibration range. This linearity applies to external calibration as well as calibration utilizing the deuterium labeled internal standard. Calibration curve slopes differed between samples prepared in culture media and those prepared in acetonitrile. As such, experimental samples will be matrix matched by preparing calibrants in media. During the six-min separation, ACh elutes at 3.8 min and Ch at 5.1 min. Deuterium-labeled ACh, when incubated for 4 and 24 hours, showed statistically significant loss, compared to control, of ACh after 24 hours in media and media + splenocytes. Despite this, the average loss of ACh by hydrolysis averaged less than 10%. Media and media + splenocyte samples incubated for 4 hr showed no statistically significant difference from control. Conclusions. The developed LC-MS/MS assay for quantification of ACh and Ch in cell culture media can be applied to the investigation of the cholinergic anti-inflammatory pathway. The method provides a rapid separation of ACh and Ch, with the successful incorporation of stable-isotope labeled internal standards.

In this study, the single-cell relative quantitative mRNA expression of three Calcium-binding proteins (CaBPs; calbindin, calretinin, parvalbumin) and eight nicotinic acetylcholine receptor (nAChR) subunits (alpha2-alpha5, alpha7, beta2-beta4) from interneurons from the stratum radiatum or stratum oriens within the CA1 region of rat hippocampi was analyzed using quantitative real time RT-PCR. Eighty-seven percent of the interneurons examined expressed CaBP mRNA. Parvalbumin mRNA was detected in 64%, while calbindin and calretinin expression was detected in 26% and 40% of interneurons, respectively. CaBP expression was not exclusive; the average number of CaBP mRNA detected per interneuron of the 47 interneurons examined for CaBP was 1.3. There was no significant difference between the proportion of CaBPs expressed in the stratum radiatum compared to the stratum oriens. However, interneurons from the stratum radiatum expressed significantly higher relative levels of mRNA for calbindin. Eighty-four percent of the 31 interneurons examined for both CaBP and nAChR subunits expressed nAChR subunit mRNA; the average number of nAChR subunits detected per interneuron was 2.9. Furthermore, of the 24, 140, and 168 possible combinations of 2-, 3-, and 4-way co-expression between CaBP+nAChR mRNA, respectively, only two significant 3-way combinations were detected: parvalbumin+a3+a5 and parvalbumin+alpha5+beta4. This study reports that subpopulations of nAChR-containing interneurons defined by quantitative CaBP mRNA expression or CaBP+nAChR co-expression do exist within the CA1 region of the hippocampus.

Whole hippocampus preparations, isolated bilaterally, from untreated Wistar rats at various ages (10-90 days old) were analyzed for the mRNA expression of the alpha 2, alpha 3, alpha 4, alpha 5, alpha 7, beta 2, beta 3, and beta 4 neuronal nicotinic acetylcholine receptor subunits. To do so, RNA was isolated from acutely isolated hippocampal samples, converted to cDNA by means of a reverse transcription reaction, then analyzed with quantitative real-time PCR to determine the relative levels of the mRNAs the cells were expressing at the age when the samples were obtained. The relative expression of the levels of RNA were then compared across age groups by subunits and across subunits by ages. The results suggest that all eight subunits are expressed throughout the life of the rat and that the subunit expression for the Hippocampus varies only slightly as a rat develops.

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Malaria continues to cause significant mortality in sub-Saharan Africa and elsewhere, and existing vector control measures are being threatened by growing resistance to pyrethroid insecticides.  With the goal of developing new human-safe, resistance-breaking insecticides we have explored several classes of acetylcholinesterase inhibitors.  In vitro assay studies have shown that trifluoromethyl ketones (TFK's) are potent inhibitors of An. gambiae AChE (AgAChE), that inhibit the enzyme by making a covalent adduct with the catalytic serine of the enzyme.  However research in the Carlier group has shown that trifluoromethyl ketones bearing benzene and pyrazole cores have shown very little toxicity to An. gambiae, perhaps due to hydration and rapid clearance. Focus was directed towards synthesis of oximes, oxime ethers, and hydrazones as potential prodrugs to prevent immediate hydration and reach the central nervous system.  The synthesis of various oximes, oxime ethers, and hydrazones has been shown to give cimpounds toxic to Anopheles gambiae within 3- to 4-fold of the toxicity of propoxur. However, thus far we have not been able to link the toxicity of these compounds to a cholinergic mechanism.  Pre-incubation studies suggest that significant hydrolysis of these compounds to TFKs does not occur or 22 h at pH 7.7 or 5.5.   Future work will be directed towards TFKs that have better pharmacokinetic properties.  Work will also be directed at synthesis of oxime and hydrazone TFK isosteres to determine the mechanism of action of these compounds. / Master of Science