Higher incidence of the SNP Met 788 Ile in the coding region of A20 in diffuse large B cell lymphomas.

Genetic alterations causing constitutive activation of the nuclear factor kappa B (NF-κB) signaling pathway has been associated with the development of lymphomas. A20 (TNFAIP3) is a key regulator of NF-κB signaling. Its suppressor functions are often inactivated by deletions and/or mutations in various hematologic malignancies. Since we recently found the rs143002189 polymorphism in the A20 loci in our multiple myeloma samples, we further investigated this polymorphism in different lymphoid neoplasias. For this purpose, we tested 479 cases of the most common B cell malignancies for the presence of the rs143002189 polymorphism. We found a significant higher occurrence of the rs143002189 polymorphism in diffuse large B cell lymphoma (DLBCL) compared to non-neoplastic controls and other types of B cell malignancies. Furthermore, structure analyses of the mutated A20 protein led to the assumption that the new steric interaction within the protein is responsible for a reduced or inactivated A20 protein. Our data indicates that in a significant fraction of patients, rs143002189 might contribute to the development of DLBCL.

Genetic variants of the NOTCH3 gene in the elderly and magnetic resonance imaging correlates of age-related cerebral small vessel disease.

Cerebral small vessel disease-related brain lesions such as white matter lesions and lacunes are common findings of magnetic resonance imaging in the elderly. These lesions are thought to be major contributors to disability in old age, and risk factors that include age and hypertension have been established. The radiological, histopathologic and clinical phenotypes of age-related cerebral small vessel disease remarkably resemble autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy, which is caused by mutations in NOTCH3. We hypothesized that genetic variations in NOTCH3 also play a role in age-related cerebral small vessel disease. We directly sequenced all 33 exons, the promoter and 3'-untranslated region of NOTCH3 in 195 participants with either coalescent white matter lesions or lacunes and compared the results to 82 randomly selected participants with no focal changes on magnetic resonance images in the Austrian Stroke Prevention Study. We detected nine common and 33 rare single nucleotide polymorphisms, of which 20 were novel. All common single nucleotide polymorphisms were genotyped in the entire cohort (n = 888), and four of them, rs1043994, rs10404382, rs10423702 and rs1043997, were associated significantly with both the presence and progression of white matter lesions. The association was confined to hypertensives, a result which we replicated in the Cohorts for Heart and Ageing Research in Genomic Epidemiology Consortium on an independent sample of 4773 stroke-free hypertensive elderly individuals of European descent (P = 0.04). The 33 rare single nucleotide polymorphisms were scattered over the NOTCH3 gene with three being located in the promoter region, 24 in exons (18 non-synonymous), three in introns and three in the 3'-untranslated region. None of the single nucleotide polymorphisms affected a cysteine residue. Sorting Intolerant From Tolerant, PolyPhen2 analyses and protein structure simulation consistently predicted six of the non-synonymous single nucleotide polymorphisms (H170R, P496L, V1183M, L1518M, D1823N and V1952M) to be functional, with four being exclusively or mainly detected in subjects with severe white matter lesions. In four individuals with rare non-synonymous single nucleotide polymorphisms, we noted anterior temporal lobe hyperintensity, hyperintensity in the external capsule, lacunar infarcts or subcortical lacunar lesions. None of the observed abnormalities were specific to cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy. This is the first comprehensive study investigating (i) the frequency of NOTCH3 variations in community-dwelling elderly and (ii) their effect on cerebral small vessel disease related magnetic resonance imaging phenotypes. We show that the NOTCH3 gene is highly variable with both common and rare single nucleotide polymorphisms spreading across the gene, and that common variants at the NOTCH3 gene increase the risk of age-related white matter lesions in hypertensives. Additional investigations are required to explore the biological mechanisms underlying the observed association.

Correlation of image analysis features and visual morphology in melanocytic skin tumours using in vivo confocal laser scanning microscopy.

BACKGROUND/PURPOSE: In vivo confocal laser scanning microscopy (CLSM) represents a novel imaging tool that allows the non-invasive examination of skin cancer morphology at a quasi histological resolution without biopsy. Previous studies dealt with the search for diagnostic, but subjective visual criteria. In this study we examined the correlation between objectively reproducible image-analysis features und visual morphology in melanocytic skin tumours using CLSM. METHODS: Eight hundred and fifty-seven CLSM tumour images including 408 benign nevi and 449 melanoma images were evaluated. Image analysis was based on features of the wavelet transform and classification tree analysis (CART) was used for classification purposes. In a second step, morphologic details of CLSM images, which have turned out to be of diagnostic significance by the classification algorithm were evaluated. RESULTS: CART analysis of the whole set of CLSM images correctly classified 97.55% of all melanoma images and 96.32% of all nevi images. Seven classification tree nodes seemed to indicate benign nevi, whereas six nodes were suggestive for melanoma morphology. The visual examination of selected nodes demonstrated that monomorphic melanocytic cells and melanocytic cell nests are characteristic for benign nevi whereas polymorphic melanocytic cells, disarray of melanocytic architecture and poorly defined or absent keratinocyte cell borders are characteristic for melanoma. CONCLUSION: Well-known, but subjective CLSM criteria could be objectively reproduced by image analysis features and classification tree analysis. Moreover, features not accessible to the human eye seem to contribute to classification success.

Diagnostic image analysis of malignant melanoma in in vivo confocal laser-scanning microscopy: a preliminary study.

BACKGROUND/PURPOSE: In this study we assessed the applicability of image analysis and a machine learning algorithm on diagnostic discrimination of benign and malignant melanocytic skin tumours in in vivo confocal laser-scanning microscopy (CLSM). METHODS: A total of 857 CLSM tumour images including 408 benign nevi and 449 melanoma images was evaluated. Image analysis was based on features of the wavelet transform. For classification purposes we used a classification tree software (CART). Moreover, automated image analysis results were compared with the prediction success of an independent human observer. RESULTS: CART analysis of the whole set of CLSM tumour images correctly classified 97.55% and 96.32% of melanoma and nevi images. In contrast, sensitivity and specificity of 85.52% and 80.15% could be reached by the human observer. When the image set was randomly divided into a learning (67% of the images) and a test set (33% of the images), overall 97.31% and 81.03% of the tumour images in the learning and test set could be classified correctly by the CART procedure. CONCLUSION: Provided automated decisions can be used as a second opinion. This can be valuable in assisting diagnostic decisions in this new and exciting imaging technique.

Frequent down regulation of the tumor suppressor gene a20 in multiple myeloma.

Multiple myeloma (MM) is a malignant clonal expansion of plasma cells in the bone marrow and belongs to the mature B-cell neoplams. The pathogenesis of MM is associated with constitutive NF-κB activation. However, genetic alterations causing constitutive NF-κB activation are still incompletely understood. Since A20 (TNFAIP3) is a suppressor of the NF-κB pathway and is frequently inactivated in various lymphoid malignancies, we investigated the genetic and epigenetic properties of A20 in MM. In total, of 46 patient specimens analyzed, 3 single base pair exchanges, 2 synonymous mutations and one missense mutation were detected by direct sequencing. Gene copy number analysis revealed a reduced A20 gene copy number in 8 of 45 (17.7%) patients. Furthermore, immunohistochemical staining confirmed that A20 expression correlates with the reduction of A20 gene copy number. These data suggest that A20 contributes to tumor formation in a significant fraction of myeloma patients.

Virtopsy: forensic traumatology of the subcutaneous fatty tissue; multislice computed tomography (MSCT) and magnetic resonance imaging (MRI) as diagnostic tools.

Traumatic lesions of the subcutaneous fatty tissue provide important clues for forensic reconstruction. The interpretation of these patterns requires a precise description and recording of the position and extent of each lesion. During conventional autopsy, this evaluation is performed by dissecting the skin and subcutaneous tissues in successive layers. In this way, depending on the force and type of impact (right angle or tangent), several morphologically distinct stages of fatty tissue damage can be differentiated: perilobular hemorrhage (I), contusion (II), or disintegration (III) of the fat lobuli, and disintegration with development of a subcutaneous cavity (IV). In examples of virtopsy cases showing blunt trauma to the skin and fatty tissue, we analyzed whether these lesions can also be recorded and classified using multislice computed tomography (MSCT) and magnetic resonance imaging (MRI). MSCT has proven to be a valuable screening method to detect the lesions, but MRI is necessary in order to properly differentiate and classify the grade of damage. These noninvasive radiological diagnostic tools can be further developed to play an important role in forensic examinations, in particular when it comes to evaluating living trauma victims.

Molecular diagnosis and prognosis with DNA microarrays.

Microarray analysis makes it possible to determine thousands of gene expression values simultaneously. Changes in gene expression, as a response to diseases, can be detected allowing a better understanding and differentiation of diseases at a molecular level. By comparing different kinds of tissue, for example healthy tissue and cancer tissue, the microarray analysis indicates induced gene activity, repressed gene activity or when there is no change in the gene activity level. Fundamental patterns in gene expression are extracted by several clustering and machine learning algorithms. Certain kinds of cancer can be divided into subtypes, with different clinical outcomes, by their specific gene expression patterns. This enables a better diagnosis and tailoring of individual patient treatments.

Computer-aided diagnosis of melanocytic skin tumors by use of confocal laser scanning microscopy images.

OBJECTIVE: To check the applicability of machine learning algorithms for the computer-aided diagnosis of confocal laser scanning microscopy (CLSM) views of skin lesions. STUDY DESIGN: Features, based on spectral properties of the wavelet transform, are very suitable for the automatic analysis because architectural structures at different scales play an important role in diagnosis of CLSM views. The images are discriminated by several machine learning algorithms, based on Bayes-, tree-, rule-, function (numeric)-, and lazy-classifiers. RESULTS: The function and lazy classifiers delivered best classification results. However, these algorithms deliver no information about the inference mechanism leading to the classification. The tree classifiers provided better results than the rule classifiers. To obtain more insight into the inference process, and to compare it with the diagnostic guidelines of the dermopathologists, we combined the advantages of tree, numerical, and rule classifiers and choose the classification and regression trees (CART) algorithm, which automatically generates accurate inferring rules. The classification results were relocated to the images by use of the inferring rules as diagnostic aid. CONCLUSION: The discriminated elements of the skin lesions images show tissue with features in good accordance with typical diagnostic CLSM features.

Homology modelling: a review about the method on hand of the diabetic antigen GAD 65 structure prediction.

This introductory paper describes the basic principles and clinical applications of the protein 3D structure prediction by homology modelling. The paper mainly addresses physicians and medical chemists. Because many proteins are of immediate clinical importance, the determination of their structures is crucial for molecular medicine. In homology modelling, a protein sequence with unknown structure is aligned with sequences of known protein structures. By exploiting structural information from the known configurations, the new structure can be predicted. The necessary condition for successful homology modelling is a sufficient similarity between the protein sequences. Because in the near future for every protein family at least one member with a known structure will be available, the importance and applicability of homology modelling is steadily increasing. We demonstrate the principles of homology modelling on hand of the Glutamic Acid Decarboxylase (GAD 65) structure prediction, which is a typical autoantigen involved in Diabetes Mellitus Type 1.

The tumour necrosis factor as a mediator of vessel inflammation: importance of exposed receptor residues for its neutralization.

BACKGROUND: The analysis of the macromolecular tumour necrosis factor (TNF)-receptor interface helps to understand the antigenicity of this inflammatory protein. METHOD: The calculations are based on structural data from the protein database. The residues of the macromolecular interface are identified in the interface contact matrix, a plot of pair-wise interactions between adjacent residues in the TNF-receptor complex. Starting from the matrix elements, the most exposed residues of the receptor, together with their relative contribution to the interface, are determined. This is done by Voronoi tessellation, a unique and well defined partition of the protein into polyhedral cells defining the proprietary space of the associated amino acid and its contact faces with neighboured residue cells. RESULTS: Several interfacial receptor residues, contributing with a total amount of 63% to the macromolecular interface, could be identified. CONCLUSION: Based on the assumption that residues with higher interfacial exposure values are playing the most important role in TNF-receptor complex, they will be the original material for further developments in engineering more efficient TNF blockers.

Tumour necrosis factor and its receptor: a basic structural analysis of two counterparts.

Inflammation of vessels is partially caused by tumour necrosis factor (TNF). Although the pharmacological understanding of the main inflammatory protein data is well characterised, basic structural information is rare. For this reason, we developed a method for the representation and analysis of the macromolecular interface between TNF and its receptor, enabling a better understanding of their interaction. In this paper we use structural information on the TNF-receptor complex in the protein (PDB) database as input to calculate an interface contact matrix, based on the distance between individual residues of each counterpart. The two-dimensional matrix is a plot of pairwise interactions between adjacent residues of the two chains in the protein complex. The residue names within each chain are plotted on the respective axis and an entry is made wherever two residues come into close contact. The matrix elements are annotated with physicochemical properties. The interface contact matrix is linked to a 3D visualisation of the macromolecular structure in such a way that mouse clicking on the appropriate part of the interface contact matrix highlights the corresponding residues in the 3D structure. Additionally the residues in the matrix are used to define the molecular surface at the interface. The interface contact matrix enables an overview representation of the residue distribution at the macromolecular interface and an evaluation of interfacial 'hot spots'. The selection of the residues in the interface contact matrix and the highlighting in the 3D structure allow an easy retrieval of the desired information out of the wealth of structural information. The representation with molecular surfaces shows complementary shapes. Many forms of treatment have been developed to reduce excessive TNF activity and their success is based on knowledge of the active sites of TNF. Our macromolecular interface analysis system will help us to define better receptor and acceptor molecules for the neutralisation and excretion of TNF.

DNA microarray analysis: principles and clinical impact.

In recent years, a new technology, allowing the measurements of the expression of thousands of genes simultaneously, has emerged in medicine. This method, called DNA microarray analysis, is today one of the most promising method in functional genomics. Fundamental patterns in gene expression are extracted by several clustering methods like: hierarchical clustering, self organizing maps and support vector machines. Changes in gene expression, as a response to changing environment conditions, diseases, drug treatment or chemotherapy medications, can be detected allowing insights into the dynamic of the genome. Microarrays seem to be an important tool for diagnosis of diseases at a molecular level. Applications are for example the improvement of diagnosis and treatment of cancer and the improvement of the effectiveness of drug treatment. In this introductory paper, we present the principles of DNA microarray experiments, selected clustering methods for gene expression analysis and the impact to clinical research.

Evaluation of texture features in spatial and frequency domain for automatic discrimination of histologic tissue.

OBJECTIVE: To investigate the applicability of different texture features in automatic discrimination of microscopic views from benign common nevi and malignant melanoma lesions. STUDY DESIGN: In tissue counter analysis (TCA) the images are dissected into square elements used for feature calculation. The first class of features is based on the histogram, the co-occurrence matrix and the texture moments. The second class is derived from spectral properties of the wavelet Daubechie 4 and the Fourier transform. Square elements from images of a training set are classified by Classification and Regression Trees analysis. RESULTS: Features from the histogram and the co-occurrence matrix enable correct classification of 94.7% of nevi elements and 92.6% of melanoma elements in the training set. Classification results are applied to individual test set cases. Discriminant analysis based on the percentage of "malignant elements" showed correct classification of all nevi cases and 95% of melanoma cases. Features derived from the wavelet and Fourier spectrum showed correct results for 88.8% and 79.3% of nevi and 85.6% and 81.5% of melanoma elements, respectively. CONCLUSION: TCA is a potential diagnostic tool in automatic analysis of melanocytic skin tumors. Histogram and co-occurrence matrix features are superior to the wavelet and the Fourier features.

Insights into molecular medicine: development of new diagnostic and prognostic parameters.

Molecular medicine leads us towards an understanding of some diseases at the molecular level. Examples are the analysis of immune complexes and receptor-antireceptor compounds used in clinical medicine. Structural changes of some serum proteins occur in inflammation, neoplasia and autoimmunity. The detection and analysis of such structural modifications may offer a new field for the diagnosis, prognosis and therapy of some diseases. Modern medicine requires new technologies with high sensitivity, specificity and applicability. For the first time in Austria we have combined fluorescence correlation spectroscope (FCS), surface enhanced laser desorption ionisation--time of flight (SELDI-TOF) and the molecular modelling and visualization system according to the computer enhanced programs. Experimental and computational methods are combined in such a way that clinical data can be interpreted by theoretical methods at a molecular level or vice versa, the computational output delivers input for new investigations. One method brings us single results. In view of the spectrum of parameters relevant to clinical entities, multiplexing is a new way of development. Since the technologies are new, the scientifically interested reader should be informed about the matters arising.

Density histogram analysis of unenhanced hepatic computed tomography in patients with diffuse liver diseases.

OBJECTIVE: The aim of the study was to assess the potential of density histogram analysis of unenhanced hepatic computed tomography (CT) in the diagnosis and differentiation of diffuse liver diseases. METHODS: Twenty-six patients with normal liver parenchyma, 35 patients with diffuse steatosis, 14 patients with acute steatohepatitis, 15 patients with active alcoholic cirrhosis, 23 patients with inactive alcoholic cirrhosis, 15 patients with virus-induced cirrhosis, and 8 patients with hemochromatosis underwent unenhanced hepatic CT. All diffuse liver diseases and the absence of diffuse liver disease were histologically proven. Quantitative analysis of unenhanced liver parenchyma was performed in each patient. RESULTS: The hepatic density histogram showed no significant differences in kurtosis and skewness between the groups (P > 0.05). Except for steatosis, active alcoholic cirrhosis, and hemochromatosis, diffuse liver diseases led to similar densities of liver parenchyma in unenhanced hepatic CT. CONCLUSION: A reliable diagnosis and differentiation of diffuse liver diseases on the basis of density histogram analysis is not possible.

Discrimination of benign common nevi from malignant melanoma lesions by use of features based on spectral properties of the wavelet transform.

OBJECTIVE: To evaluate the possibilities of describing and discriminating common nevi and malignant melanoma tissue with features based on spectral properties of the Daubechies 4 wavelet transform. STUDY DESIGN: Images of common nevi and malignant melanoma were dissected in square elements. The wavelet coefficients were calculated inside the square elements. The diagonal coefficients and related power spectra were used for further analysis. The analysis results served as guide for the selection of features, including standard deviations of wavelet coefficients inside the frequency bands and the energy of the frequency bands. These features describe properties of the frequency bands, representing information on different scales. To test the usefulness of the features for discrimination, a study set of 80 cases was classified by classification and regression trees analysis. The set was divided into a training set and a test set. RESULTS: In the case of benign common nevi, the energies of the lower frequency bands and higher, whereas malignant melanoma tissue shows more variability of the coefficients in higher-frequency bands. The influence on the detail properties of the images was studied by suppression of coefficients with low values, which are concentrated mainly in higher-frequency bands. In the case of benign common nevi the main information is contained in 15% of the coefficients and in the case of malignant melanoma, in 39%. The results of classification show a clear-cut difference between the cases. The classification correctly classified 95.78% of nevi elements and 94.22% of melanoma elements in the training set and 100% of cases of benign nevi and 80% of cases of malignant melanoma in the test set. CONCLUSION: Features based on the wavelet power spectrum contain sufficient information for differentiation between common nevi and malignant melanomas.

Retrograde arterialized venous flap: an experimental study.

An experimental model was established to study circulation in retrograde arterialized venous flaps (RAVF). Venous flaps measuring 7 x 4 cm with a matching venous system were harvested from both forearms of 10 fresh human cadavers. In each trial, both flaps were simultaneously perfused with heparinized human blood driven by a pulsatile circulation model. In each trial there was one flap with retrograde perfusion, and one flap with antegrade perfusion. Clinical assessment, measurement of outflow, and angiographic examination with digitally assisted assessment after 3 h of perfusion showed better results for retrograde perfusion in 8 of the 10 trials. This study indicates that blood circulation in the periphery of arterialized venous flaps can be enhanced by retrograde arterialization.