Actinobacillus actinomycetemcomitans leukotoxin requires lipid microdomains for target cell cytotoxicity.

Actinobacillus actinomycetemcomitans produces a leukotoxin (Ltx) that kills leukocyte function-associated antigen-1 (LFA-1)-bearing cells from man, the Great Apes and Old World monkeys. The unique specificity of Ltx for the beta2 integrin, LFA-1, suggests it is capable of providing insight into the pathogenic mechanisms of Ltx and other RTX toxins. Using the Jurkat T cell line and an LFA-1-deficient Jurkat mutant (Jbeta2.7) as models, we found the initial effect of Ltx is to elevate cytosolic Ca2+ [Ca2+]c, an event that is independent of the Ltx/LFA-1 interaction. [Ca2+]c increases initiate a series of events that involve the activation of calpain, talin cleavage, mobilization to, and subsequent clustering of, LFA-1 in cholesterol and sphingolipid-rich regions of the plasma membrane known as lipid rafts. The association of Ltx and LFA-1 within lipid rafts is essential for cell lysis. Jbeta2.7 cells fail to accumulate Ltx in their raft fractions and are not killed, while cholesterol depletion experiments demonstrate the necessity of raft integrity for Ltx function. We propose that toxin-induced Ca2+ fluxes mobilize LFA-1 to lipid rafts where it associates with Ltx. These findings suggest that Ltx utilizes the raft to stimulate an integrin signalling pathway that leads to apoptosis of target cells.

E2F4 expression patterns in SIV encephalitis.

The E2F1 transcriptional regulator has been shown to exhibit altered expression and localization in HIVE and SIVE. However, other E2F family members are expressed in mature neurons and participate in neuronal differentiation. In an in vitro model of neuronal differentiation, E2F4 protein levels have been shown to increase. Further reduction in E2F4 leads to loss of neurites in this model. Neuritic damage and loss are also seen in progression of HIVE and SIVE. To determine if changes in E2F4 may contribute to altered neuronal morphology and survival, we assessed E2F4 immunostaining in caudate and mid-frontal cortex from SIVE macaques and non-encephalitic controls. We found that E2F4 was expressed in neurons and localized to nuclei in both SIVE and non-encephalitic controls. Quantification of E2F4 fluorescence intensity indicated that there was an overall decrease in E2F4 in caudate of SIVE macaques as compared to non-encephalitic controls, which correlated with a decrease in the neuronal phenotypic marker, MAP2. In contrast, we observed a slight increase in E2F4 in mid-frontal cortex of SIVE despite a significant decrease in MAP2. When E2F4 is normalized to MAP2, we found an increase in E2F4 fluorescence intensity per MAP2 in SIVE mid-frontal cortex. These findings suggest changes in E2F4 may be contributing to altered neuronal morphology or survival in SIVE.

Assessing the accuracy of caries diagnosis via radiograph. Film versus print.

BACKGROUND: The authors conducted this study to determine if proximal caries diagnoses made using bitewing radiographic images printed on photographic paper were comparable with diagnoses made using traditional radiographic film images. METHODS: The authors digitized 15 posterior bitewing radiographs that contained 74 carious and 127 sound unrestored proximal surfaces and printed them on photographic paper. Fourteen dentists evaluated the radiographs and two printed image formats (4 x 3 centimeters and 8 x 6 cm) for evidence of caries. The diagnostic accuracy and interobserver agreement for caries diagnoses obtained in the two printed image formats were compared with those for radiographic film images. RESULTS: Overall, the diagnostic accuracy of printed images did not differ significantly from radiographic film images for dentinal caries. However, for caries limited to the enamel surface, a decrease in sensitivity was noted in six of the 14 observers for the smaller print images, while no significant differences in the diagnoses of enamel caries were observed among any of the observers in the enlarged print format. CONCLUSION: This study provides evidence that printed images can be used to diagnose dental caries reliably. CLINICAL IMPLICATIONS: The results of this study indicate that the diagnostic information obtained by viewing printed images is equivalent to that obtained by viewing standard radiographs. Size of the printed image also may be important in caries diagnosis and care must be taken to print bitewing radiographic images at a size that optimizes interpretation. Other factors that must be considered are the type of printer, printer resolution, paper quality and type of ink used. With careful consideration of printing parameters, clinicians can be assured of diagnostic quality in printed images.

Fetal Alz-50 clone 1 interacts with the human orthologue of the Kelch-like Ech-associated protein.

The fetal Alz-50 reactive clone 1 (FAC1) protein exhibits altered expression and subcellular localization during neuronal development and neurodegenerative diseases such as Alzheimer's disease. Using the yeast two-hybrid screen, the human orthologue of Keap1 (hKeap1) was identified as a FAC1 interacting protein. Keap1 is an important regulator of the oxidative stress response pathway through its interaction with the Nrf family of transcription factors. An interaction between full-length FAC1 and hKeap1 proteins has been demonstrated, and the FAC1 binding domain of hKeap1 has been identified as the Kelch repeats. In addition, FAC1 colocalizes with endogenous Keap1 within the cytoplasm of PT67 cells. Exogenously introduced eGFP:hKeap1 fusion protein redistributed FAC1 to colocalize with eGFP:hKeap1 in perinuclear, spherical structures. The interaction between FAC1 and hKeap1 is reduced by competition with the Nrf2 protein. However, competition by Nrf2 for hKeap1 is reduced by diethylmaleate (DEM), a known disrupter of the Nrf2:Keap1 interaction. DEM does not affect the ability of FAC1 to bind hKeap1 in our assay. These results suggest that hKeap1 regulates FAC1 in addition to its known role in control of Nrf2. Furthermore, the observed competition between FAC1 and Nrf2 for binding hKeap1 indicates that the interplay between these three proteins has important implications for neuronal response to oxidative stress.

Bone structure effect on root resorption.

OBJECTIVES: To explore if alveolar bone shape and density might promote external apical root resorption. SETTING AND SAMPLE POPULATION: Panoramic radiographs of 700 patients who had orthodontic treatment at Temple University were reviewed and 22 patients with radiographic evidence of root resorption on the lower incisors were selected for the study. Exclusion criteria included a history of systemic diseases, craniofacial abnormalities, tooth injury, endodontically treated teeth, and impacted teeth. METHODS: Pre-treatment (T1) and post-treatment (T2) cephalometric radiographs were converted into digital format and enhanced to reduce contrast variability and improve edge definition. Tooth length, root length, root area, alveolar area around the root including cortical area, area of medullary bone, and area of the symphysis were measured using an interactive software algorithm. A region of interest within the symphysis was also defined and trabecular space area and fractal dimension calculated as an estimate of bone density. RESULTS: Root area and tooth length were correlated negatively with changes in root area, tooth area, and root length. Larger teeth demonstrated a greater amount of root resorption. Dentoalveolar complex dimensions remained relatively unchanged during tooth movement. The amount of alveolar bone around the root, thickness of cortical bone, density of the trabecular network, and fractal dimension showed no significant correlation with the extent of the external apical root resorption. CONCLUSIONS: The results of this study suggest that the density and morphology of the dentoalveolar complex are not significant factors in the etiology of external apical root resorption.

Quantifying labial blood flow using optical Doppler tomography.

OBJECTIVES: Changes in the oral microvasculature occur in a variety of diseases. Optical Doppler tomography (ODT) combines laser Doppler flowmetry with optical coherence tomography (OCT) to produce high-resolution tomographic images of biological tissues that also detect the velocity and direction of blood flow. The objective of this study was to determine the feasibility of ODT to image labial blood flow. A prototype ODT imaging system was constructed that characterized and measured labial blood flow in healthy subjects. MATERIALS AND METHODS: A prototype ODT instrument was constructed using a diode light source with a central wavelength of 1300 nanometers, a 40-nanometer spectral width and 2.4 microwatts output power. To verify the accuracy of the system, the flow rates of a phantom material (Intralipid) pumped through a capillary tube at various speeds was measured. To evaluate the clinical feasibility of the ODT prototye, the mucosal aspect of the upper and lower lips at the midline was imaged in 9 healthy volunteers. The sample arm of the instrument consisted of a fiberoptic probe with a 2-mm in diameter polished glass lens attached to the end. The probe was placed approximately 3 mm from the mucosal surface of the lip and oriented perpendicular to the surface. A motorized translation stage moved the fiber in a superior to inferior direction while the subject's head was stabilized by placing the chin into a chin rest. Imaging time for a 12-mm x 2.5-mm scan was approximately 64 seconds. RESULTS: The phantom experiments revealed that accuracy of this novel ODT prototype to measure flow was within 5%. In vivo labial blood flow velocity ranged from 11.8 to 43.1 mm/second in the upper lip and 8.2 to 53.2 mm/second in the lower lip. There were no statistically significant differences between flow rates in the upper and lower lips. OCT images and Doppler velocity signals were successfully integrated producing in vivo images of labial blood in all of the subjects (15 images). The resulting cross-sectional images revealed microscopic details of labial structures and, to the best of our knowledge, are the first ODT images of the labial microvasculature. CONCLUSIONS: The results of this in vivo study prove the feasibility of ODT to quantify labial blood flow and produce high spatial resolution images specifically localizing vessels anatomically. ODT provides both flow speed and flow direction information. ODT is noninvasive and offers the advantages of high volumetric flow sensitivity.

Doppler angle and flow velocity mapping by combined Doppler shift and Doppler bandwidth measurements in optical Doppler tomography.

Accurate estimation of flow velocity requires measurement of Doppler angle, which is not available in general clinical applications. We describe a novel method of direct Doppler angle and flow velocity mapping that uses a conventional single-beam optical Doppler tomography system. The Doppler angle is estimated by combination of Doppler shift and Doppler bandwidth measurements, and flow velocity is calculated from the Doppler shift and the estimated Doppler angle. In vivo study of lip microvascularization demonstrates that this method is capable of providing both flow speed and flow direction information.

Identification of occlusal sealants using optical coherence tomography.

OBJECTIVE: Optical coherence tomography (OCT) is a new imaging technique that uses light to image dental structures interferometrically. OCT creates cross-sectional images that have potential diagnostic value for dental applications. When used in epidemiological studies, OCT offers a safe, non-invasive technique to discriminate occlusal sealants and composite restorations. This paper summarizes a study in which dentists were asked to interpret and discriminate between OCT images. METHODOLOGY: Twenty-one dentists were asked to interpret OCT images of nine extracted premolars that were either not restored, contained an occlusal sealant or were restored with a composite restoration. RESULTS: Although the dentists were previously unfamiliar with OCT images, they adapted well and felt confident in their diagnoses using this new technology. The sensitivity of OCT to discriminate composite and sealants was > 0.92, while the specificity of discrimination was > 0.94. The capacity of OCT to discriminate sealants from non-restored occlusal surfaces was slightly less (sensitivity 0.88; specificity 0.86), but still within a clinically acceptable level. Inter- and intra-rater reliability, as measured by the kappa statistic, also revealed excellent performance by dentists using this new imaging technology. Intra-rater reliability was very good, ranging from 0.82 to 1.0. Inter-rater reliability was also high, predominately in the "Good" to "Very Good" agreement range. CONCLUSION: This preliminary study indicates OCT imaging may be an important new technology for discriminating occlusal sealants and composite restorations.

Quantitative assessment of flow velocity-estimation algorithms for optical Doppler tomography imaging.

We present a quantitative comparison of three categories of velocity estimation algorithms, including centroid techniques (the adaptive centroid technique and the weighted centroid technique), the sliding-window filtering technique, and correlation techniques (autocorrelation and cross correlation). We introduce, among these five algorithms, two new algorithms: weighted centroid and sliding-window filtering. Simulations and in vivo blood flow data are used to assess the velocity estimation accuracies of these algorithms. These comparisons demonstrate that the sliding-window filtering technique is superior to the other techniques in terms of velocity estimation accuracy and robustness to noise.