Automated noninvasive epithelial cell counting in phase contrast microscopy images with automated parameter selection.

Cell counting is commonly used to determine proliferation rates in cell cultures and for adherent cells it is often a 'destructive' process requiring disruption of the cell monolayer resulting in the inability to follow cell growth longitudinally. This process is time consuming and utilises significant resource. In this study a relatively inexpensive, rapid and widely applicable phase contrast microscopy-based technique has been developed that emulates the contrast changes taking place when bright field microscope images of epithelial cell cultures are defocused. Processing of the resulting images produces an image that can be segmented using a global threshold; the number of cells is then deduced from the number of segmented regions and these cell counts can be used to generate growth curves. The parameters of this method were tuned using the discrete mereotopological relations between ground truth and processed images. Cell count accuracy was improved using linear discriminant analysis to identify spurious noise regions for removal. The proposed cell counting technique was validated by comparing the results with a manual count of cells in images, and subsequently applied to generate growth curves for oral keratinocyte cultures supplemented with a range of concentrations of foetal calf serum. The approach developed has broad applicability and utility for researchers with standard laboratory imaging equipment.

Potential role of periodontal pathogens in compromising epithelial barrier function by inducing epithelial-mesenchymal transition.

BACKGROUND AND OBJECTIVE: Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells acquire a mesenchymal-like phenotype and this may be induced by exposure to gram-negative bacteria. It has been proposed that EMT is responsible for compromising epithelial barrier function in the pathogenesis of several diseases. However, the possible role of EMT in the pathogenesis of periodontitis has not previously been investigated. The aim of this study therefore was to investigate whether gram-negative, anaerobic periodontal pathogens could trigger EMT in primary oral keratinocytes in vitro. MATERIAL AND METHODS: Primary oral keratinocytes were harvested from labial mandibular mucosa of Wistar Han rats. Cells were exposed to heat-killed Fusobacterium nucleatum and Porphyromonas gingivalis (100 bacteria/epithelial cell) and to 20 µg/mL of Escherichia coli lipopolysaccharide over an 8-day period. Exposure to bacteria did not significantly change epithelial cell number or vitality in comparison with unstimulated controls at the majority of time-points examined. Expression of EMT marker genes was determined by semiquantitative RT-PCR at 1, 5, and 8 days following stimulation. The expression of EMT markers was also assessed by immunofluorescence (E-cadherin and vimentin) and using immunocytochemistry to determine Snail activation. The loss of epithelial monolayer coherence, in response to bacterial challenge, was determined by measuring trans-epithelial electrical resistance. The induction of a migratory phenotype was investigated using scratch-wound and transwell migration assays. RESULTS: Exposure of primary epithelial cell cultures to periodontal pathogens was associated with a significant decrease in transcription (~3-fold) of E-cadherin and the upregulation of N-cadherin, vimentin, Snail, matrix metalloproteinase-2 (~3-5 fold) and toll-like receptor 4. Bacterial stimulation (for 8 days) also resulted in an increased percentage of vimentin-positive cells (an increase of 20% after stimulation with P. gingivalis and an increase of 30% after stimulation with F. nucleatum, compared with controls). Furthermore, periodontal pathogens significantly increased the activation of Snail (60%) and cultures exhibited a decrease in electrical impedance (P < .001) in comparison with unexposed controls. The migratory ability of the cells increased significantly in response to bacterial stimulation, as shown by both the number of migrated cells and scratch-wound closure rates. CONCLUSION: Prolonged exposure of primary rat oral keratinocyte cultures to periodontal pathogens generated EMT-like features, which introduces the possibility that this process may be involved in loss of epithelial integrity during periodontitis.

Automatic thresholding from the gradients of region boundaries.

We present an approach for automatic threshold segmentation of greyscale images. The procedure is inspired by a reinterpretation of the strategy observed in human operators when adjusting thresholds manually and interactively by means of 'slider' controls. The approach translates into two methods. The first one is suitable for single or multiple global thresholds to be applied globally to images and consists of searching for a threshold value that generates a phase whose boundary coincides with the largest gradients in the original image. The second method is a variation, implemented to operate on the discrete connected components of the thresholded phase (i.e. the binary regions) independently. Consequently, this becomes an adaptive local threshold procedure, which operates relative to regions, rather than to local image subsets as is the case in most local thresholding methods previously published. Adding constraints for specifying certain classes of expected objects in the images can improve the output of the method over the traditional 'segmenting first, then classify' approach.

Fractals in microscopy.

Fractal geometry, developed by B. Mandelbrot, has provided new key concepts necessary to the understanding and quantification of some aspects of pattern and shape randomness, irregularity, complexity and self-similarity. In the field of microscopy, fractals have profound implications in relation to the effects of magnification and scaling on morphology and to the methodological approaches necessary to measure self-similar structures. In this article are reviewed the fundamental concepts on which fractal geometry is based, their relevance to the microscopy field as well as a number of technical details that can help improving the robustness of morphological analyses when applied to microscopy problems.

Digital enhancement of haematoxylin- and eosin-stained histological images for red-green colour-blind observers.

Individuals with red-green colour-blindness (CB) commonly experience great difficulty differentiating between certain histological stain pairs, notably haematoxylin-eosin (H&E). The prevalence of red-green CB is high (6-10% of males), including among medical and laboratory personnel, and raises two major concerns: first, accessibility and equity issues during the education and training of individuals with this disability, and second, the likelihood of errors in critical tasks such as interpreting histological images. Here we show two methods to enhance images of H&E-stained samples so the differently stained tissues can be well discriminated by red-green CBs while remaining usable by people with normal vision. Method 1 involves rotating and stretching the range of H&E hues in the image to span the perceptual range of the CB observers. Method 2 digitally unmixes the original dyes using colour deconvolution into two separate images and repositions the information into hues that are more distinctly perceived. The benefits of these methods were tested in 36 volunteers with normal vision and 11 with red-green CB using a variety of H&E stained tissue sections paired with their enhanced versions. CB subjects reported they could better perceive the different stains using the enhanced images for 85% of preparations (method 1: 90%, method 2: 73%), compared to the H&E-stained original images. Many subjects with normal vision also preferred the enhanced images to the original H&E. The results suggest that these colour manipulations confer considerable advantage for those with red-green colour vision deficiency while not disadvantaging people with normal colour vision.

Periodontal pathogens promote epithelial-mesenchymal transition in oral squamous carcinoma cells in vitro.

Epithelial-mesenchymal transition (EMT) is potentially involved in increasing metastasis of oral squamous cell carcinoma (OSCC). Periodontal pathogens are well-known for their ability to induce intense immune responses and here we investigated whether they are involved in inducing EMT. Cultures of OSCC cell line (H400) were treated separately with heat-killed periodontal pathogens F. nucleatum, or P. gingivalis or E. coli LPS for 8 d. EMT-associated features were assayed using sq-PCR and PCR-arrays, for EMT-related markers, and ELISAs for TGF-ß1, TNF-α, and EGF. The migratory ability of cells was investigated using scratch and transwell migration assays. E-cadherin and vimentin expression was assessed using immunofluorescence while Snail activation was detected with immunocytochemistry. In addition, the integrity of the cultured epithelial layer was investigated using transepithelial electrical resistance (TEER). PCR data showed significant upregulation after 1, 5, and 8 d in transcription of mesenchymal markers and downregulation of epithelial ones compared with unstimulated controls, which were confirmed by immunofluorescence. Periodontal pathogens also caused a significant increase in level of all cytokines investigated which could be involved in EMT-induction and Snail activation. Exposure of cells to the bacteria increased migration and the rate of wound closure. Downregulation of epithelial markers also resulted in a significant decrease in impedance resistance of cell monolayers to passage of electrical current. These results suggested that EMT was likely induced in OSCC cells in response to stimulation by periodontal pathogens.

Efficacy, safety and feasibility of intravenous iloprost in the domiciliary treatment of patients with ischemic disease of the lower limbs.

OBJECTIVE: Intravenous iloprost is an important option in the treatment of ischemic disease of the lower limbs; however, the administration of therapy is frequently compromised because of the need for long cycles of infusion in a hospital setting. The aim of the study is to evaluate the efficacy, safety, feasibility, and the economic impact of infusion therapy in the outpatient setting. PATIENTS AND METHODS: Twenty-four consecutive patients were treated with iloprost at their homes where they were administered a slow rate of infusion for 24 hours a day, during 9.9 ± 2.3 days, with a portable syringe pump (Infonde®). RESULTS: The clinical condition of patients evaluated with the modified SVS/ISCVS scale significantly improved after treatment (+1.29 ± 1.04 points vs. baseline, p<0.001). The drug was well tolerated; neither significant adverse events associated with medication nor problems related to venous access were recorded at home. Ninety-six percent of patients successfully completed the entire treatment cycle, and the evaluation questionnaire showed a high acceptance of the therapy. From the perspective of the hospital authority, lower direct medical costs were estimated for the domiciliary infusion process compared with the inpatient infusion setting. CONCLUSIONS: Treatment with iloprost in the outpatient setting is effective, safe, feasible, and more acceptable to patients than infusion at the hospital. In addition, it has a favorable economic and organizational impact on the medical ward.

X-ray Study of Human Dental Tissues Affected by Erythroblastosis Fetalis.

Numerous diseases are known to cause microstructural alteration of dental tissues structure. One type in particular is associated with neonatal jaundice and circulation of bilirubin in blood at high concentration due to increased hemolysis in conditions such as erythroblastosis fetalis, septicemia, biliary atresia, and other causes of hyperbilirubinemia. In those conditions, the products of the catabolism of hemoglobin end up deposited in various tissues, including teeth, where they can present clinically as visibly stained brown/green teeth. There is almost no information on the nature or extent of the structural changes taking place in these conditions. Here, advanced nondestructive wide-angle synchrotron X-ray scattering techniques combined with scanning microscopy methods were used to investigate for the first time the ultrastructure of the dental hard tissues in an archival case of intrinsically pigmented green teeth. Despite no obvious elemental variation across the pigmented tissue region, the high-resolution crystallographic properties probed by wide-angle synchrotron X-ray scattering revealed an ultrastructural variation (orientation, particle size, and lattice parameter of hydroxyapatite crystallites) associated with a pigmentation line in dentine and with a distinct neonatal line in enamel.

Characteristics, outcome, and care of stroke associated with atrial fibrillation in Europe: data from a multicenter multinational hospital-based registry (The European Community Stroke Project).

BACKGROUND AND PURPOSE: The role of atrial fibrillation (AF) as a determinant of stroke outcome is not well established. Studies focusing on this topic relied on relatively small samples of patients, scarcely representative of the older age groups. We aimed at evaluating clinical characteristics, care, and outcome of stroke associated with AF in a large European sample. METHODS: In a European Concerted Action involving 7 countries, 4462 patients hospitalized for first-in-a-lifetime stroke were evaluated for demographics, risk factors, clinical presentation, resource use, and 3-month survival, disability (Barthel Index), and handicap (Rankin scale). RESULTS: AF was present in 803 patients (18.0%). AF patients, compared with those without AF, were older, were more frequently female, and more often had experienced a previous myocardial infarction; they were less often diabetics, alcohol consumers, and smokers (all P:<0.001). At 3 months, 32.8% of the AF patients were dead compared with 19.9% of the non-AF patients (P:<0.001). With control for baseline variables, AF increased by almost 50% the probability of remaining disabled (multivariate odds ratio 1.43, 95% CI 1.13 to 1.80) or handicapped (multivariate odds ratio 1.51, 95% CI 1.13 to 2.02). Before stroke, only 8.4% of AF patients were on anticoagulants. The chance of being anticoagulated was reduced by 4% per year of increasing age. AF patients underwent CT scan and other diagnostic procedures less frequently and received less physiotherapy or occupational therapy. CONCLUSIONS: Stroke associated with AF has a poor prognosis in terms of death and function. Prevention and care of stroke with AF is a major challenge for European health systems.

Simulation of corneal neovascularization by inverted diffusion limited aggregation.

PURPOSE: The authors modeled the normally avascular mammalian cornea response to injury by neovascularization. The nature of this process remains obscure, although diffusion mechanisms are thought to be involved. METHODS: Corneal neovascularization was simulated using fractal stochastic computer models for nonequilibrium diffusion (inverted diffusion limited aggregation) and a stochastic ballistic aggregation. RESULTS: The inverted diffusion limited aggregation model was found to generate patterns strikingly similar to those observed in pathologic neovascularization of the cornea in vivo. CONCLUSIONS: This result supports the role of random diffusion mechanisms modified by environmental factors, such as the release of angiogenic factors in corneal angiogenesis.

Local connected fractal dimensions and lacunarity analyses of 60 degrees fluorescein angiograms.

PURPOSE: The retinal vascular tree exhibits fractal characteristics. These findings relate to the mechanisms involved in the vascularization process and to the objective morphologic characterization of retinal vessels using fractal analysis. Although normal retinas show uniform patterns of blood vessels, in pathologic retinas with central vein or artery occlusions, the patterns are irregular. Because the generalized box fractal dimension fails to differentiate successfully between normal and abnormal retinal vessels in 60 degrees fluorescein angiograms, the authors have further investigated this problem using the local connected fractal dimension (alpha). METHODS: The authors studied 24 digitized 60 degrees fluorescein angiograms of patients with normal retinas and 5 angiograms of patients with central retinal vein or artery occlusion. The pointwise method estimated the local complexity of the angiogram within a finite window centered on those pixels that belong to the retinal vessels. Color-coded dimensional images of the angiograms were constructed by plotting the pixels forming the object with a color that corresponded to specific values of alpha +/- delta alpha. RESULTS: The color-coded representation allowed recognition of areas with increased or decreased local angiogram complexity. The alpha distributions showed differences between normal and pathologic retinas, which overcomes problems encountered when using the methods of calculating the generalized fractal dimensions. A multivariate linear discriminant function using parameters from the alpha distribution and a further fractal parameter--lacunarity--reclassified 23 of the 24 normal and 4 of the 5 pathologic angiograms in their original groups (total: 92.1% correct). CONCLUSIONS: This methodology may be used for automatic detection and objective characterization of local retinal vessel abnormalities.

Investigation of primary cell-biomaterial interactions using silver nitrate staining of nucleolar organising regions.

The quantification of silver nitrate staining of nucleolar organising regions (AgNORs) within the nucleus of the cell has been shown to give a relative measure of the metabolic activity of the cell. In the present study, silver nitrate staining was utilised to identify metabolic variations in cells cultured on different surfaces and compared with proliferative activity assessed using bromodeoxyuridine (BrdU) uptake. Primary osteoblast and periosteal cells, isolated from the calvaria of neonate rats, were cultured on tissue culture-grade (TCPS) and bacteriological-grade (BACPS) polystyrene petri dishes for 3, 5, 7 and 9 days (silver nitrate) or 14 days (BrdU). The phenotype of the cells was examined using RT-PCR of the mRNA for osteocalcin, collagen 1a, alkaline phosphatase and osteopontin. The number and area of AgNORs and the proportion of BrdU positive cells were statistically different in cells cultured on TCPS compared with BACPS at each culture period tested. The results suggest that the metabolic activity and proliferation of cells were affected by the substrate which they colonise.

A new insight into the oscillation characteristics of endosonic files used in dentistry.

The aim of this study was to assess the oscillation characteristics of unconstrained endosonic files using a scanning laser vibrometer (SLV). Factors investigated included file vibration frequency and node/antinode location as well as the variation in file displacement amplitude due to increasing generator power setting. A 30 kHz Mini Piezon generator (Electro-Medical Systems, Switzerland) was used in conjunction with a #15 and #35 K-file. Each file was fixed in position with the long axis of the file perpendicular to the SLV camera head. The laser from the SLV was scanned over the length of the oscillating file for generator power settings 1 to 5 (minimum to half power). Measurements were repeated ten times. The fundamental vibration frequency for both files was 27.50 kHz. Scans of each file showed the positions of nodes/anti-nodes along the file length. The #15 file demonstrated no significant variation in its mean maximum displacement amplitude with increasing generator power, except at power setting 5, where a decrease in displacement amplitude was observed. The #35 file showed a general increase in mean maximum displacement amplitude with increasing power setting, except at power setting 4 where a 65% decrease in displacement amplitude occurred. In conclusion, scanning laser vibrometry is an effective method for assessing endosonic file vibration characteristics. The SLV was able to demonstrate that (unloaded) file vibration displacement amplitude does not increase linearly with increasing generator power. Further work is being performed on a greater variety of files and generators. Vibration characteristics of files under various loads and varying degrees of constraint should also be investigated.

Fractal properties of herpes simplex dendritic keratitis.

Fractal geometry can be used to analyze the complexity and self-similarity of many natural forms and structures. By using three techniques for fractal dimension estimation, a group of 11 herpes simplex virus dendritic corneal ulcers was analyzed and found to possess outlines with fractal properties. The mean surface fractal dimension approached 1.4 over 1.5 decades of scale. The demonstration and quantification of the fractal dimension of dendritic lesions is important in the understanding of their behaviour and, at a more fundamental level, the planar spread of viral infection.

Videodensitometrical study of the alveolar bone crest in periodontal disease.

The relative mineral content per unit volume of bone in the alveolar bone crest was determined in 81 bone crests from 25 human autopsy cases by means of contact microradiography and videodensitometry of 100 microns thick sections. Each bone crest was divided in 4 areas: crestal, mesial, distal, and trabecular; and the relative mineral content was determined for each area. The data were analyzed statistically and it was found that the crestal area had a significantly lower mineral content than the other 3 areas. Histologically, the crestal area had a reduced number of Sharpey's fibers and it was speculated that these features lower the probability of restoring the natural crestal shape and the fiber reattachment, increasing the risk of bone loss. No correlation was found between relative mineral content and age or extent of inflammatory infiltration of the periodontal tissues.

Odontogenic epithelial hamartoma of the gingiva: a case report.

A rare case of odontogenic epithelial hamartoma of the gingiva in a 63-year-old Japanese female is reported. This tumor-like lesion probably originated from the reduced tooth-forming tissues such as rests of dental lamina lying dormant in the gingiva after odontogenesis. The differential diagnosis of this lesion from many other odontogenic and glandular tumors is important; however, through careful clinico-pathological examinations, it would not be a very difficult task.

Increased fractal complexity of the epithelial-connective tissue interface in the tongue of 4NQO-treated rats.

The effect of the carcinogen 4-nitroquinoline 1-oxide (0.001% in drinking water) on the irregularity of the epithelial connective tissue interface (ECTI) of the ventral surface of the tongue was quantified in Dark Agouti and Wistar Furth rats. Histological tongue sections stained with the Azan-Mallory method were digitised (111 images, resolution 1 pixel = 3.1 microns), and the limit between epithelium and stroma of the ventral surface was extracted and analysed using a fractal geometry technique (local connected dimension). The results showed that although none of the images included carcinomas on the ventral surface of the tongue (all cases had other oral carcinomas), the epithelial profiles of the treated cases showed a statistically significant increase in irregularity when compared to controls. Canonical discriminant analysis of the parameters describing the irregularity of the ECTI classified 81.1% of the images in the original groups (treated or control). Fractal analysis is capable of detecting subtle architectural changes in the oral epithelium of the rat occurring after exposure to the carcinogen, even when full malignant transformation has not yet taken place. Fractal analysis, which may prove useful for monitoring the progression of carcinogenesis in this animal model, is a morphometrical parameter in the diagnosis of oral epithelial dysplasia.

Size dependent variation in the fractal dimensions of herpes simplex epithelial keratitis.

Dendritic ulcers, the commonest manifestation of herpes simplex epithelial keratitis (HSEK) are fractals. It is likely that their fractal properties alter if they progress to a geographic appearance. This study investigates the relationship of maximum ulcer diameter (Feret's diameter) to area (Dm) and perimeter fractal dimensions (Ds), parameters of the complexity of their areas and outlines respectively. For dendritic ulcers in the size range of 1.6-3.2 mm, Dm = 1.41 +/- 0.06 and Ds = 1.40 +/- 0.04 (mean +/- SD). With increasing ulcer size, a progressive divergence of the values of Dm and Ds occurred, such that values of 1.75 and 1.22 respectively were found at a maximum diameter of 8.4 mm. These results imply that as ulcers enlarge, their outlines become less irregular and they fill more of a 2-dimensional plane. Dm and Ds are useful parameters in quantifying the progression of HSEK from dendritic to amoeboid morphology and could have a role in the assessment of ulcer response to pharmacological intervention. A knowledge of the fractal properties of HSEK may increase understanding of the mechanisms of ulcer formation and viral spread.

Fractal analysis of the normal human retinal fluorescein angiogram.

The fractal dimension of the retinal vasculature and isolated venous and arterial trees down to a caliber of 40 microns was estimated in 23 routine fluorescein angiograms of normal retinas. Fractal dimension was determined with a method based on the box counting theorem. This method is less susceptible to the radial architecture of the retinal vascular tree than those previously reported (mass-radius relation and density-density correlation function). Two scale ranges with different fractal dimension were consistently present. The estimated fractal dimensions showed no significant difference between isolated arterial and venous trees which is not supported by previous reports. This method was designed for simple application in a clinical setting.

The influence of mixing ratio on the toughening mechanisms of a hand-mixed zinc phosphate dental cement.

OBJECTIVE: A range of mixing ratios occur for zinc phosphate cements in clinical usage and trials. The compressive strength of these cements is dependent on mixing ratio and at a critical ratio, a sudden rise in compressive strength is known to occur. This study investigated the mechanisms behind the cement curve behavior in an attempt to explain the strength increase with mixing ratio. METHODS: Crack propagation from indentations produced in cements were examined to identify if any variation in toughening mechanism existed over the range of mixing ratios investigated (1.7-3.2g/ml). Pore distribution within the cylindrical specimens was determined using an image analysis technique. RESULTS: Increasing the powder content from 2.3 to 2.4g/ml increased the number of powder agglomerates formed in the cement mix over individual particles. The likelihood of introducing pores in excess of 42 microm diameter was enhanced when the ratio exceeded 2.6g/ml. SIGNIFICANCE: It is proposed that crack deflection by agglomerates in the cement reduces the energy of the crack fronts emanating from indentations more than would occur with individual powder particles. This decreases the cracks ability to progress and is likely to result in the marked strength increase from 2.3 to 2.4g/ml identified previously. It is suggested that cement pores in excess of 42 microm diameter were probably generated on filling the sample molds with more viscous cements. These results emphasise that the properties of cements manipulated under optimum conditions provide little information on the cement characteristics present in clinical practice.