Clostridium difficile is the most common infectious cause of nosocomial diarrhea, affecting thousands of patients annually and exacting enormous costs on the U.S. health care system. Early diagnosis is critical to prevent transmission and reduce morbidity and mortality, yet sensitive and specific diagnostic tests with a quick turnaround time are lacking. The objective of this study was to determine if a new commercially available real time polymerase chain reaction (PCR) test would prove more rapid, sensitive and specific than standard methods for the diagnosis of C. difficile infection (CDI). BD GeneOhm Cdiff assay, a real-time PCR assay for detection of C. difficile toxin B (tcdB) gene, was compared with Tox A/B II ELISA and a two-step algorithm which includes C. Diff Chek-60 Glutamate Dehydrogenase (GDH)-antigen assay followed by cytotoxin neutralization. Four-hundred liquid or semisolid stools submitted for diagnostic C. difficile testing were selected: 200 GDH antigen-positive and 200 GDH antigen-negative. All samples were tested by the C. Diff Chek-60 GDH antigen, cytotoxin neutralization, Toxin A/B II ELISA, and BD GeneOhm Cdiff assay. Discrepant specimens were tested by toxigenic culture as an independent gold standard. Chart review was performed on patients with discrepant specimens. As BD GeneOhm Cdiff assay was not FDA-cleared at the time of study, PCR results were not clinically reported. Of 200 GDH-positive samples, 71 were positive by Tox A/B II, 88 were positive by the two-step method, 93 were positive by PCR, and 96 were positive by GDH-antigen only. Of 200 GDH-negative samples, 3 were positive by PCR only. Toxigenic culture was performed on 41 samples with discrepant results and 39 were culture-positive. After culture resolution of discrepants, Tox A/B II detected 70 (66.7%), the two-step method detected 87 (82.9%), and PCR detected 96 (91.4%) of 105 true positives. The BD Gene-Ohm Cdiff assay was more sensitive in detecting toxigenic C. difficile than Tox A/B II (p <0.0001); however, the difference between PCR and the two-step method was not significant (p=0.1237). The BD GeneOhm Cdiff assay took a similar amount of time to perform as the Tox A/B II and was more rapid than the two-step method. Chart review revealed that 18 patients with cytotoxin-negative, PCR-positive discrepant samples were given 1-2 days of therapy (n=8), or no treatment at all (n=10). Yet symptoms resolved and no further C. difficile testing was requested for 13 of 18 patients for 6-8 months after hospital discharge. Only one patient had a subsequent cytotoxin positive stool submitted 22 days after the study sample was tested. Enhanced sensitivity and rapid turnaround time make the BD GeneOhm Cdiff assay an important advance in the diagnosis of toxigenic C. difficile infection. The BD GeneOhm Cdiff assay is significantly more sensitive than a commonly-used ELISA toxin assay and has a sensitivity and specificity comparable to the two-step method. Its turnaround time is similar to ELISA toxin assays and more rapid than the two-step method. Disadvantages to implementation of BD GeneOhm Cdiff assay include increased cost and potential treatment of asymptomatic carriers and mild, self-resolving disease.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 72-81).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 98-106).

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 121-148). Also issued on the Internet.

No description available.

L-glutamate (Glu) is the predominant excitatory neurotransmitter inthe mammalian central nervous system (CNS) and is associated with a widevariety of functions including motor behavior and sensory perception. Whilemicrodialysis methods have been used to record tonic levels of Glu, little isknown about the more rapid changes in Glu signals that may occur in awakeanimals. We have previously reported acute recording methods using anenzyme-based microelectrode array (MEA) with fast temporal resolution (800msec), that is minimally invasive and is capable of detecting low levels of Glu (andlt;0.2 ??M) in anesthetized animals with little interference from other analytes. Wehave made a series of modifications to the MEA design to allow for reliablemeasures in the brain of awake behaving rats. In these studies, wecharacterized the effects of chronic implantation of the MEA into the striatum andprefrontal cortex (PFC) of Fischer 344 and Long Evans rats. We measuredresting levels of Glu and local application of Glu for 7 days without a significantloss of sensitivity and determined that Glu measures due to exogenous Gluvaried between rat strain and brain region. In addition, we determined theviability of the recordings in the brains of awake animals. We performed studiesof tail-pinch induced stress which caused an increase in Glu in the striatum andPFC of Long Evans and Fischer 344 rats. Histological data show that chronicimplantation of our MEAs caused minimal injury to the CNS. Taken together, ourdata support that chronic recordings of tonic and phasic Glu can be carried out inawake rats reliably for 7 days in vivo allowing for longer term studies of Gluregulation in behaving rats.

Impulsive choice refers to the inability to delay gratification and is associated with increased drug abuse vulnerability. Understanding the underlying neural mechanisms linking impulsive choice and drug abuse can contribute to improved treatment options for individuals with substance use disorders. Evidence suggests a major role for nucleus accumbens core (NAcc) in impulsive choice and the reinforcing effects of drugs of abuse. The neurotransmitters glutamate (Glu) and dopamine (DA) are implicated in the neural adaptations observed in drug addiction; however, the role of intra-NAcc Glu and DA in impulsive choice is unclear. Rats were trained in a delay discounting task, in which animals chose between a small, immediate reinforcer and large, delayed reinforcer. Consistently choosing the small, immediate reinforcer was considered to reflect increased impulsivity. Following delay discounting, in vitro receptor autoradiography was performed to quantify the number of N-methyl-D-aspartate (NMDA) receptors and dopamine transporters (DAT) in NAcc and nucleus accumbens shell (NAcSh). In a separate experiment, rats were trained in delay discounting and were implanted with guide cannulae into NAcc. Following surgery, rats received microinfusions of either a) the Glu-selective ligands MK-801 (noncompetitive NMDA receptor channel blocker; 0, 0.3, and 1.0 μg), AP-5 (competitive NMDA receptor antagonist; 0, 0.3, and 1.0 μg), ifenprodil (NMDA NR2B subunit antagonist; 0, 0.3, and 1.0 μg), and CNQX (AMPA receptor antagonist; 0, 0.2, and 0.5 μg) or b) the DA-selective ligands SKF 38393 (D1-like receptor agonist; 0, 0.03, and 0.1 μg), SCH 23390 (D1-like receptor antagonist; 0, 0.3, and 1.0 μg), quinpirole (D2-like receptor agonist; 0, 0.3, and 1.0 μg), and eticlopride (D2-like receptor antagonist; 0, 0.3, and 1.0 μg). In NAcc and NAcSh, NMDA receptor and DAT expression did not differ between high and low impulsive rats. Furthermore, intra-NAcc administration of NDMA and DA receptor ligands did not significantly alter impulsive choice. These results suggest that Glu and DA systems within NAcc do not directly mediate impulsive decision making. Future work is needed to determine the precise role of NAcc in mediating impulsive choice.

Ketamine is a non-competitive NMDA antagonist that has been shown to have antidepressant properties both clinically as well as in preclinical studies when administered at a subanesthetic dose. In vivo electrophysiological recordings were carried in male Sprague Dawley rats 30 minutes following ketamine administration (10 mg/kg) to first assess its effects on monoaminergic firing. Whilst no change in the firing activity of serotonin (5-HT) neurons was observed in the dorsal raphe nucleus (DRN), an increase in the firing activity was observed for dopamine (DA) and noradrenergic (NE) neurons in the ventral tegmental area (VTA) and locus coeruleus (LC), respectively. The effect of ketamine on these electrophysiological parameters was prevented by pre-administration of the AMPA receptor antagonist NBQX 10 minutes prior to ketamine administration. In a second series of experiments, an increase in AMPA-evoked response was observed within 30 minutes in the CA3 layer of the hippocampus (HPC) following acute ketamine administration. These findings suggest that acute ketamine administration produces a prompt enhancement of AMPA transmission in the forebrain and also results in increased catecholaminergic activity. These effects may play a crucial role in the rapid antidepressant effects of ketamine observed shortly following its infusion in the clinic.

The neurobiology of anxiety-related disorders is associated with impaired neuroplasticity. The glutamate/NO/cGMP pathway has been proposed to play a key role in neuroplasticity and neurodevelopment. It was demonstrated in recent reports that chronic co-administration of the phosphodiesterase type 5 (PDE5) inhibitor sildenafil and the antimuscarinic agent atropine exerts antidepressive-like activity in rats, and that this effect is related to PDE5 inhibition, with consequent elevation of cGMP levels and enhanced protein kinase G stimulation. The current study investigated possible anxiolytic effects of the chronic co-administration of sildenafil and atropine in stress-sensitive Flinders Sensitive Line (FSL) rats. FSL rats received vehicle control, fluoxetine (15 mg/kg), atropine (1 mg/kg), sildenafil (10 mg/kg) or sildenafil plus atropine via intraperitoneal administration, either acutely 30 minutes prior to testing (acutely) or daily for 14 days (chronically). FRL control rats received only vehicle. Thereafter anxiety-like behaviour was evaluated in the social interaction test (SIT - acute) and elevated plus maze (EPM - acute and chronic). The current study also compared to different ways to score the EPM, namely the percentage time spend in the open arms of the EPM and both the number of full and half body open arm entries, and also implemented defecation on the EPM as a measure of anxiety. Vehicle-treated FSL rats exhibited more anxiety-like behaviour than FRL rats in both the SIT and EPM following acute treatment, and in the EPM following chronic treatment. Acute treatment with fluoxetine exerted anxiogenic activity in the SIT and EPM, but anxiolytic activity following chronic administration, as observed in the EPM. In acute treatments neither sildenafil nor sildenafil plus atropine yielded any significant effects on anxiety-like behaviour. However, following chronic treatment, sildenafil exerted anxiolytic activity in the EPM by increasing the time spend in the open arms (45.72% ± 9.94% vs. 20.80% ± 9.94%, P<0.001). Atropine exerted a small anxiolytic response (30.71% ± 8.40% vs. 20.80 ± 9.94%), whereas atropine co-administration was additive to sildenafil alone and yielded an enhanced anxiolytic effect in the elevated plus maze (59.56% ± 4.95% vs. 20.80% ± 9.94%, P<0.001), relative to vehicle control. The percentage time spend in the open arms was scored in the EPM, the results suggested that the chronic treatment with sildenafil plus atropine exert an anxiolytic-like effect in FSL rats and the number of fecal droppings did not increase which is also an indication of an anxiolytic-like effects of the treatment. The current study demonstrated that the chronic treatment with sildenafil, alone or in combination with atropine, exhibit an anxiolytic-like action in stress-sensitive rats. In addition, the data support the clinical potential of using PDE5 inhibitors as antidepressant and anxiolytic strategy and warrant further investigation. Furthermore the study supports the previously proposed key role of the glutamate/NO/cGMP pathway in the neurobiology of anxiety-like disorders, and as an important target for drug development. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2011

The synthesis of arborescent poly(gamma-benzyl L-glutamate) (PBG) molecules was achieved through successive grafting reactions of linear PBG chains. These linear PBG building blocks were obtained by the ring-opening polymerization of gamma-benzyl L-glutamic acid N-carboxyanhydride initiated with n-hexylamine. Cleavage of a fraction of the benzyl ester groups on a linear PBG substrate, followed by coupling with linear PBG side chains via standard peptide coupling techniques, yielded a comb-branched or generation zero (G0) arborescent PBG. Further cycles of partial deprotection and grafting reactions led to arborescent PBG molecules of the subsequent generations (G1-G3). Molecular weights reaching over 106 were obtained for G3 arborescent PBG, while maintaining narrow molecular weight distributions (Mw/Mn ≤ 1.06) for each generation. The arborescent PBG molecules displayed α-helix to randomly coiled chain conformation changes from N,N-dimethylformamide to dimethylsulfoxide. Amphiphilic copolymers were obtained by grafting the arborescent PBG substrates randomly with side chains of either poly(glycidol acetal), poly(ethylene oxide), or poly(γ-tert-butyl L-glutamate) via the same peptide coupling techniques used to generate arborescent PBG. Copolymers were also synthesized by a chain end grafting method, whereby the linear chain segments were coupled exclusively with the chain termini of the arborescent PBG substrates. Water-soluble species were obtained by removal of the acetal and tert-butyl protecting groups from the poly(glycidol acetal) and poly(γ-tert-butyl L-glutamate) side chains, respectively, while the copolymers with poly(ethylene oxide) side chains did not require further modifications. Dynamic light scattering (DLS) measurements on the arborescent copolymers in aqueous solutions revealed that unimolecular micelles were the dominant species for the chain end grafted arborescent copolymers, whereas the randomly grafted arborescent copolymers were either insoluble or displayed significant aggregation. The synthesis of arborescent copolymers with PBG cores was also achieved through “click” chemistry, using the copper-catalyzed azide-alkyne Huisgen cycloaddition (CuAAC) reaction. To that end, polyglycidol, poly(ethylene oxide), and poly(2-trimethylsilylethyl acrylate) chains terminally functionalized with azide groups were grafted onto either randomly or chain end alkyne-functionalized arborescent PBG substrates. DLS analysis revealed solubility trends similar to the arborescent copolymers obtained by the peptide coupling method. The CuAAC reaction enables the incorporation of a broader range of polymers into arborescent copolymer structures derived from PBG substrates.