The purpose of this study was evaluate the interobserver reliability of endodontists in the diagnosis the presence or absence of pulpal and/or periradicular disease. The study used 47 patients presenting to the VCU School of Dentistry for screening appointments as a test population under the rules and regulations of the VCU IRB. The patients were examined separately by two endodontists, using a thorough patient history, clinical exam, and radiographs. The answer to the question was then answered, does the patient have pulpal and/or periradicular disease, and compared. The data was analyzed using Kappa and the standard error was determined to test for statistical significance. Observers agreed 88% of the time with a Kappa of 0.74. This was determined to represent a bona fide reliability with p<.0001. The results indicate that agreement among endodontists is very good when patients are evaluated for pulpal and/or periradicular disease.

Epilepsy is one of the most common neurological disorders, affecting more than 2% of children and 1% of adults in the U.S. Emerging research has demonstrated that calcium, as a major second messenger system, underlies many of these injury-induced plasticity changes associated with the development of epilepsy. Recent evidence has suggested that long term elevations in neuronal resting calcium levels play a role in initiating and maintaining epileptogenesis (the development of epilepsy). Collaborations between our lab and others have produced microarray data that suggests that a major calcium-binding protein, calbindin D-28k, mRNA levels are decreased in epileptic rats even up to one year following pilocarpine treatment. The goal of this research effort was to determine if epileptogenesis alters basal calcium levels by producing a long-term change in the expression of the major calcium binding protein in neurons, calbindin D-28k. Immunohistochemistry (MC) and western blot experiments have been conducted to test the hypothesis that epileptogenesis produces a long lasting decrease in the expression of calbindin in the hippocampus in the rat pilocarpine model of acquired epilepsy. IHC experiments indicated that changes in calbindin expression occur gradually over a 2-4 week interval after the initial injury. Significant decreases in calbindin immunoreactivity are seen in the hippocampus of epileptic animals, at one month, four months, and six months post-pilocarpine treatment. However, these changes were not seen as early as 4 days post-status epilepticus. Western blots quantitated differences between epileptic animals and naive controls. Long lasting decreases in calbindin may play an important role in the altered calcium homeostatic mechanisms observed in epileptic neurons. These findings will help to elucidate one of many changes that occurs in epilepsy.

In the past decade, Staphylococcus aureus has developed two distinct vancomycin resistance mechanisms. First, the bacterium is capable of generating a thickened, poorly crosslinked cell wall that creates false targets. These targets cause vancomycin to bind at the periphery of the thickened peptidoglycan, allowing normal cell wall formation to continue at the cell membrane. Second, S. aureus has acquired genes from Enterococcus that encode an alternative stem peptide. The genes, known as van genes, alter the target of vancomycin, rendering vancomycin treatment ineffectual.In this work, we attempted to further characterize both mechanisms of vancomycin resistance. First, a potential link between up-regulated purine biosynthesis and increased vancomycin resistance due to a thickened cell wall was examined. Despite exploration of multiple mechanisms to increase purine levels within the cell, increased purine synthesis did not provide S. aureus with any advantage in the presence of vancomycin. However, during the investigation, purine biosynthesis in S. aureus was further characterized by confirming purr as the repressor of the purine pathway and demonstrating its sensitivity to mutation.Next, the relationship between homotypic oxacillin resistance and increased vancomycin resistance in the absence of the van genes was investigated. Vancomycin passage of two heterotypic methicillin resistant S. aureus (MRSA) caused these strains to convert to homotypic oxacillin resistance in the absence of oxacillin exposure. Additionally, conversion of heterotypic oxacillin resistant strains to homotypy by oxacillin passage increased strain survival in vancomycin. The SOS response was examined as the possible link between conversion to homotypic oxacillin resistance and increasing vancomycin resistance due to a thickened cell wall. The current study, however, detected no induction of the SOS response during vancomycin exposure.Lastly, the relationship between oxacillin resistance and vancomycin resistance due to the acquisition of the van genes was examined. In vitro and in vivo methods were utilized to determine the effectiveness of a combination of β-lactam antibiotics and vancomycin to treat vancomycin resistant S. aureus (VRSA) infections. Combination therapy provided a significant advantage over untreated control or either antibiotic alone in the rabbit model of endocarditis.

Purpose: This study seeks to determine 1) whether the type of advertising exposure is associated with adolescent health perceptions of tobacco use, and 2) whether the type of media exposure is associated with initiation plans (non-smokers) or quitting plans (smokers). Methods: This was a cross-sectional survey of middle school students (n=l1,128).Psychosocial variables were knowledge of the risks of tobacco use (range: 3-15) and benefits of being tobacco free (range: 7-35) with higher scores indicating greater understanding. Intentions to quit or initiate were construed as binary variables. Exposure variables were tobacco advertisements or anti-tobacco media messages. Covariates were gender, race, grade level, and parental closeness. The data were analyzed using SAS, version 9.1 and hierarchical regression was used to account for random effects of students nested within organizations.Results: Exposure to tobacco advertising was associated with higher knowledge (12.6 v. 12.4, pth graders, exposure to tobacco messages was associated with higher percentages planning to initiate (19.7 v. 16.2, p=0.008) whereas anti-tobacco exposure was associated with lower percentages (16.3 v. 20.3, p=0.024). Exposure to more than one anti-tobacco message was associated with higher knowledge and benefit.Conclusions: Counter-advertising can make adolescents more aware of health benefits of remaining tobacco free. Placing tighter restrictions on tobacco advertising directed at adolescents is warranted. The "dose-response" suggests disseminating anti-tobacco messages in many venues.

Neisseria gonorrhoeae is an obligate human pathogen that requires iron for its survival within the host. N. gonorrhoeae expresses high-affinity iron acquisition systems to acquire iron from host iron binding proteins. The gonococcal transferrin-iron uptake system is composed of two transferrin binding proteins, TbpA and TbpB. TbpA is a TonB-dependent, outer membrane transporter, while TbpB is a surface-exposed lipoprotein. Unlike TbpA, TbpB is not required for transferrin utilization, but makes the process more efficient. The precise mechanism by which TbpA and TbpB function to mediate transferrin-iron uptake has not been fully characterized. However, the mechanism of iron acquisition from transferrin is distinct from characterized TonB-dependent ferric-siderophore uptake systems. The transferrin-iron uptake system is unique in two ways: the involvement of the TbpB lipoprotein component and the process of iron acquisition and internalization. Unlike siderophore transporters, the transferrin-iron uptake system requires the removal of iron from transferrin for its subsequent internalization. Based on analogy with characterized TonB-dependent transporters, TbpA is proposed to consist of two distinct domains: a b-barrel and plug domain. Previous studies suggest that the plug domain has a specific role in iron internalization and this study addresses the role of the plug domain in transferrin-iron acquisition. It is thought that the TbpA plug domain facilitates iron removal from transferrin and subsequent iron binding and transport. To analyze this, iron binding by the TbpA plug domain was performed and site-directed substitution mutagenesis of putative iron-coordinating residues was carried out. From these analyses, it can be concluded that the plug domain binds iron and likely plays an active role in the process of iron internalization. Mutagenesis revealed specific residues of the plug domain critical for transferrin-iron uptake, but defects imparted by these mutations were compensated for by TbpB. Thus, this study also attempts to characterize the compensatory function provided by TbpB. Through mutagenesis, critical domains involved in the efficiency of transferrin-iron acquisition were identified. One additional study describes and characterizes a novel mechanism of TonB-independent transferrin-iron acquisition. Overall, these studies further elucidate mechanisms utilized by Neisseria gonorrhoeae in the process of iron acquisition from human transferrin.

Iron, an essential nutrient for most microorganisms, is sequestered in the host by iron-binding proteins, such as lactoferrin and transferrin. Neisseria gonorrhoeae utilizes transferrin as an iron source and its iron acquisition system is composed of two transferrin binding proteins: TbpA and TbpB. TbpA is a TonB-dependent, outer membrane transporter and TbpB is a bilobed, surface exposed lipoprotein. TbpB can distinguish between apo- and holo-transferrin which is involved in increasing the efficiency of iron uptake through the Tbps. It is anchored in the outer leaflet of the outer membrane by its lipid moiety. We aimed to identify the mechanism of TbpB export to the cell surface. No conclusions could be made from our results but we identified a protein that could potentially be involved in lipoprotein transport. TbpB is a bilobed protein with controversy over which lobe is involved in transferrin binding. In this study, we constructed a C-lobe deletion of TbpB to determine the role of the C-lobe in TbpB function. Results presented here showed deletion of the C-lobe caused degradation of TbpB and the minimal protein expressed was unable to bind transferrin both in vitro and in vivo. We were also able to demonstrate the TbpB C-lobe deletion is able to support limited transferrin-mediated growth, indicating some function of TbpB is retained. These results confirmed that both lobes are necessary for wild-type function of TbpB.

Lymphocyte activation and trafficking are indispensable to the immune system. CD44, an adhesion molecule, plays important roles in T cell activation, lymphocyte homing/trafficking, and tumor metastasis. Although the functions of CD44 have been shown in T cells and other leukocytes, little is known about its role in B cells. The effects of CD44 cross-linking on murine B cell activation via CD40L/IL-4 was explored using the anti-CD44 mAbs RK3G9 and IM7. Immobilized RK3G9 and IM7 could strongly inhibit B cell proliferation and Ig production, with IgE inhibition being prominent. Soluble anti-CD44 had no effect. The inhibitory effect of RK3G9 was not influenced by addition of anti-FCγRII, indicating no role for the inhibitory receptor. The effects of delayed addition of immobilized anti-CD44 mAbs were studied, and the results indicated no inhibition after 96 hrs of culture. B cells were also activated by either LPS or anti-IgM F(ab')2. While LPS-induced B cell activation was inhibited by immobilized anti-CD44 mAbs, anti-IgM activation was refractory. Interestingly, addition of both anti-IgM and CD40L or LPS resulted in some modulation of the inhibitory activity. Additionally, FACS and Elispot revealed that RK3G9-treated cells had reduced numbers of plasma cells. Taken together, these results suggest that CD44 cross-linking could control polyclonal B cell activation by CD40L, but allow sIgM/CD40L activation to continue.

Traumatic brain injury (TBI) produces axonal damage and deafferentation, triggering injury-induced synaptogenesis, a process influenced by matrix metalloproteinases (MMP) and their substrates. Here we report results of studies examining the expression and potential role of two membrane-bound MMPs, membrane-type 5-MMP (MT5-MMP) and a disintegrin and metalloproteinase-10 (ADAM-10), along with their common synaptic substrate N-cadherin, during the period of reactive synaptogenesis. Protein and mRNA expression of MT5-MMP, ADAM-10 and N-cadherin were compared in two TBI models, one exhibiting adaptive plasticity (unilateral entorhinal cortex lesion; UEC) and the other maladaptive plasticity (fluid percussion injury + bilateral EC lesions; TBI+BEC), targeting 2, 7, and 15d postinjury intervals. In adaptive UEC plasticity, membrane-bound MMP expression was elevated during synaptic degeneration (2d) and regeneration (7d), and normalized at 15d. By contrast, N-cadherin expression was significantly decreased at 2 and 7d after UEC, but increased during 15d synaptic stabilization. In maladaptive plasticity, 2d membrane-bound MMP expression was dampened compared to UEC, with persistent ADAM-10 elevation and reduced N-cadherin protein level at 15d. These results were supported by 7d microarray and qRT-PCR analyses, which showed transcript shifts in both hippocampus and dentate molecular layer (ML) for each model. Parallel immunohistochemistry revealed significant MT5-MMP, ADAM-10 and N-cadherin localization within ML reactive astrocytes, suggesting a glial synthetic or phagocytotic role for their processing during recovery. We also investigated the effect of MMP inhibition on molecular, electrophysiological, behavioral and structural outcome at 15d following TBI+BEC. MMP inhibitor GM6001 was administered at 6 and 7d postinjury, during elevated MT5-MMP/ADAM-10 expression and synapse regeneration. MMP inhibition showed: 1) reduced ADAM-10 and elevated N-cadherin protein expression, generating profiles similar to 15d post-UEC, 2) attenuation of deficits in the initiation phase of long-term potentiation, and 3) improved hippocampal dendritic and synaptic ultrastructure. Collectively, our results provide evidence that membrane-bound MMPs and N-cadherin influence both adaptive and maladaptive plasticity in a time and injury-dependent manner. Inhibition of membrane-bound MMPs during maladaptive plasticity produces more adaptive conditions, improving synaptic efficacy and structure. Thus, targeting MMP function and expression have potential to translate maladaptive plasticity into an adaptive process, facilitating improved recovery.

Nicotine is one of the most commonly used drugs among adolescent populations. Given the fact that adolescence is a unique developmental stage, during which nicotine has long-term effects on future drug-taking behavior, it is essential to understand how early exposure to nicotine during adolescence may affect the abuse liability of other drugs. We hypothesize that repeated exposure to low doses of nicotine in adolescence induce age-specific enhancement of the rewarding effects of several drugs of abuse in the conditioned place preference (CPP) test. Furthermore, we predict that these changes in behavioral responses are mediated by nicotine-induced brain region-specific increases in the expression of ΔFosB, a member of the Fos family of transcription factors, through activation of neuronal nicotinic receptors. We used mice as a model system to investigate the effects of adolescent nicotine exposure on responses to cocaine, amphetamine, and morphine in adulthood. We found that exposure to nicotine during the early phase of adolescence (postnatal day 28) enhanced cocaine CPP, acute locomotor activity, and locomotor sensitization in adulthood. Our data demonstrate that nicotine priming effects on cocaine are affected by the dose, duration, method of administration, age of exposure, and mouse strain. These data strongly suggest that nicotine intake during adolescence may cross-sensitize the brain to the rewarding effects of cocaine. A follow-up study was undertaken to determine if this enhancement applies to other drugs of abuse. The repeated exposure to 0.5 mg/kg nicotine (subcutaneous) during early adolescence resulted in significant enhancement of amphetamine and morphine preference in a CPP test, but had no effect on the somatic signs of morphine withdrawal. In addition, we investigated the possible neuronal mechanisms underlining enhancements to behavioral responses using both in vivo and in vitro techniques. Our results showed that nicotinic antagonists, with varying subtype selectivity, administered during adolescence prior to nicotine exposure diminished cocaine enhancement in CPP. This suggests that the enhancement of cocaine behavioral responses is mediated by neuronal nicotine receptors (mainly β2* and α7). Finally, studies of ∆FosB revealed significant effects of age and nicotine pre-treatment in nucleus accumbens (NAc), but not in the prefrontal cortex (PFC). Indeed, nicotine pre-treatment was able to significantly increase ∆FosB levels in NAc of early adolescent mice compared to adult mice. This accumulation of ∆FosB persisted for several weeks. Further studies are needed to fully examine the mechanisms of action underlying the observed changes in cocaine rewards.

Alzheimer's disease (AD) is characterized by the accumulation of amyloid beta plaques, neurofibrillary tangles (NFT) and loss of cortical neurons that control memory and cognition. The cause of NFTs and Aβ plaques is not clear, though it is known that they are formed by enzymes which are preferentially sequestered to membrane domains called lipid rafts. Sulfatide (ST) is a glycosphingolipid that is essential for the proper structure and function of lipid rafts. In mice that lack ST, membrane domains that are normally maintained by adhesive contacts and functional lipid rafts are improperly formed and are unstable. In these ST null mice, voltage gated sodium channels, neuronal proteins that normally cluster at the nodes of Ranvier, initially accumulate in the node but are not retained with age. Taken together the findings from the ST null mice indicate that membrane organization is compromised. Recently, a published report demonstrated that ST is significantly reduced in AD. Based on this observation combined with the findings from the ST null mice, I propose that membrane architecture is also altered in AD and this alteration may facilitate AD pathogenesis. To test this hypothesis, I have used an immunohistochemical approach to assess neuronal membrane organization in AD and non-AD brain samples. Analysis of the sodium channel clusters was chosen since these nodal domains provide an easy assessment tool for membrane organization. In the current study, sodium channel domains were not altered and no change in isoform expression was observed. Based on these findings, membrane organization does not appear to be altered in AD. It is important to note, however, that sodium channel clusters are restricted to a specific region of the axon and thus membrane organization within other regions of the axon and in other regions of the neuron may be altered. Additionally, assessment of the brain samples, using thin layer chromatography, did not show a reduction in ST levels between the AD and non-AD brains. Therefore, my study strongly suggests that further analysis of ST levels in AD brains should be conducted to resolve the contrasting results between the current study and the previously published work.