Functional characterization of the human Huntington's disease gene promoter.

The genetic basis of neurodegeneration in Huntington's disease (HD) has been identified as a (CAG)(>37) repeat expansion in a gene of unknown function. Interestingly, patients with the same expanded (CAG)(n) repeat length may have markedly different ages at onset. Based on experiences in animal models the level of expression might be one of the modifying factors. To gain insight into the regulation of the human HD gene we functionally analyzed 2266 bp of the HD gene promoter region. This region lacks a TATA and a CAAT box, is GCrich, and it has several consensus sequences for SP1, AP-2 and AP-4 binding sites. The stretch between nucleotides -49 and -198 relative to the first ATG is highly conserved between human and rodents and it harbors several potential binding sites for transcription factors. We analyzed deletion mutants fused with the chloramphenicol acetyltransferase (CAT) reporter gene in transfected, huntingtin expressing neuronal (NS20Y) and non-neuronal (CHO) cell lines. Partial deletion of the evolutionarily conserved part of the promoter significantly reduces the activity in both neuronal and non-neuronal cells indicating that the core promoter activity is located between nucleotides -221 and 4, relative to the +1 translation start site. Binding affinities of DNA-protein interactions were defined by electrophoretic mobility shift assays and the protected nucleotide positions were determined by DNase I footprinting.

Photostability of dithranol.

The influence of light on the antipsoriatic drug dithranol was investigated. A Suntest CPS with xenon lamp and liquid cooling was used as light source for the test. Solutions of dithranol in different organic solvents and in therapeutical concentrations in excipients for the preparation of topical formulations were tested under defined conditions. The extent and rate of photodegradation was determined and compared with the degradation of light-protected solutions. The drug content in the solutions was measured by HPLC. Degradation products were characterised and identified by diode array technique and HPLC-mass spectrometry coupling. The results showed a strong dependency of the photodegradation on the excipient or solvent used.

Two-point vibrotactile discrimination related to parameters of pulse burst stimulus.

Tactile spatial resolution is an important factor in the design of vibrotactile arrays. The two-point discrimination distance is used as a measure of tactile spatial resolution. An experimental study is presented showing the effect of pulse burst stimulus parameters, pulse repetition period and duty cycle on two-point vibrotactile spatial discrimination. An array of piezoceramic vibrators is used to measure two-point spatial discrimination on the index finger. In a group of 14 subjects, the average two-point discrimination distance for a pulse repetition period of 1/25s is 2.1 mm (SD = 1.0), whereas for 1/500 s it is 5.1 mm (SD = 0.9). Differences in discrimination distances are statistically significant according to the ANOVA analysis (p < 0.001). Results show that the two-point discrimination distance is better for longer pulse repetition periods. Therefore the pulse repetition period in an excitatory waveform composed of bursts of pulses is important for tactile resolution. No statistically significant differences in discrimination distances are found between bursts of pulses of 50% duty cycle and those of lower duty cycle. The latter result indicates that, by choosing low-duty cycle waveforms for vibrotactile stimulation, the power can be reduced with no loss in two-point discrimination capacity.

Power requirements for vibrotactile piezo-electric and electromechanical transducers.

Human-machine information transfer through tactile excitation has addressed new applications in virtual reality, robotics, telesurgery, sensory substitution and rehabilitation for the handicapped in the past few years. Power consumption is an important factor in the design of vibrotactile displays, because it affects energy needs and the size, weight, heat dissipation and cost of the associated electronics. An experimental study is presented on the power required to reach tactile thresholds in electromechanical and piezo-electric transducers. Three different waveforms are considered, with an excitatory period formed by a burst of rectangular 50% duty cycle pulses (R50), rectangular low duty cycle pulses (RLO) and sinusoidal pulses (SIN). Ten different pulse repetition periods (RPs) were considered in the range 1/550-1/25 s. The voltage and current waveforms applied to the transducers at sensation thresholds in a group of 12 healthy subjects were sampled and stored in a digital oscilloscope. The average power was determined for each subject, and differences of two orders of magnitude were measured between the electromechanical and the piezo-electric transducer power consumption. Results show that, for the electromechanical transducer, a smaller power consumption of 25 microW was determined for RP = 1/25 s and the RLO waveform. In the case of the piezo-electric transducer, power of 0.21 microW was determined for SIN excitation and RP = 1/250 s. These results show the advantages of reducing power requirements for vibrotactile displays, which can be optimised by the choice of appropriate types of transducer, excitatory waveforms and pulse repetition periods.

Expert-system classification of sleep/waking states in infants.

This work is part of a project to develop an expert system for automated classification of the sleep/waking states in human infants; i.e. active or rapid-eye-movement sleep (REM), quiet or non-REM sleep (NREM), including its four stages, indeterminate sleep (IS) and wakefulness (WA). A model to identify these states, introducing an objective formalisation in terms of the state variables characterising the recorded patterns, is presented. The following digitally recorded physiological events are taken into account to classify the sleep/waking states: predominant background activity and the existence of sleep spindles in the electro-encephalogram; existence of rapid eye movements in the electro-oculogram; and chin muscle tone in the electromyogram. Methods to detect several of these parameters are described. An expert system based on artificial ganglionar lattices is used to classify the sleep/waking states, on an off-line minute-by-minute basis. Algorithms to detect patterns automatically and an expert system to recognise sleep/waking states are introduced, and several adjustments and tests using various real patients are carried out. Results show an overall performance of 96.4% agreement with the expert on validation data without artefacts, and 84.9% agreement on validation data with artefacts. Moreover, results show a significant improvement in the classification agreement due to the application of the expert system, and a discussion is carried out to justify the difficulties of matching the expert's criteria for the interpretation of characterising patterns.

Polysomnographic pattern recognition for automated classification of sleep-waking states in infants.

A robust, automated pattern recognition system for polysomnography data targeted to the sleep-waking state and stage identification is presented. Five patterns were searched for: slow-delta and theta wave predominance in the background electro-encephalogram (EEG) activity; presence of sleep spindles in the EEG; presence of rapid eye movements in an electro-oculogram; and presence of muscle tone in an electromyogram. The performance of the automated system was measured indirectly by evaluating sleep staging, based on the experts' accepted methodology, to relate the detected patterns in infants over four months of post-term age. The set of sleep-waking classes included wakefulness, REM sleep and non-REM sleep stages I, II, and III-IV. Several noise and artifact rejection methods were implemented, including filters, fuzzy quality indices, windows of variable sizes and detectors of limb movements and wakefulness. Eleven polysomnographic recordings of healthy infants were studied. The ages of the subjects ranged from 6 to 13 months old. Six recordings counting 2665 epochs were included in the training set. Results on a test set (2,369 epochs from five recordings) show an overall agreement of 87.7% (kappa 0.840) between the automated system and the human expert. These results show significant improvements compared with previous work.

[A study of the epidemiological behaviour of bovine paratuberculosis using time series in Tandil in the province of Buenos Aires, Argentina]. / Estudio del comportamiento epidemiológico de la paratuberculosis bovina mediante series cronológicas en Tandil, provincia de Buenos Aires, Argentina.

The authors present the results of a study conducted using time series data from the 1993--2001 period in order to characterise the clinical behaviour of bovine paratuberculosis. The case data, confirmed by macroscopic examination, enzyme-linked immunosorbent assay (ELISA) and comparative tuberculin test, came from the herd health register, located in Tandil in the province of Buenos Aires, Argentina. The upper limit of customary variation was observed in April, with an incidence of 2.5%. In 1997, 1999, 2000 and 2001 the disease was epidemic and a peak of 5.6% occurred in March 1999. Over the long term a rise in the total annual incidence was observed, from 0.7% in 1993 to 10.2% in 2001. Knowledge about the epidemiology of paratuberculosis will help to control the disease and minimise its impact on the national economy, and will also provide new information for use in public health.

Stabilization of dithranol in topical formulations.

The influence of different factors such as drug concentration, temperature, pH value, humidity, solubility and stabilizers on the antipsoriatic drug dithranol in topical formulations has been investigated. Dithranol was examined in lipophilic, hydrophilic and cream bases under defined conditions. Additionally, stabilizers have been investigated to protect dithranol against degradation. The drug stability in the formulations was measured by a selective HPLC system. The results showed a strong dependency of the degradation on the drug concentration. The influence of temperature is not of the same importance. Besides the pH value also the type of puffer system must be considerated. The most important effect on dithranol stability which has been directed is the solubility of the drug substance in the ointment base. With higher solubility a significantly accelerated degradation occurs. Degradations of dithranol in hydrophilic and lipophilic bases can be reduced by different stabilizers. Among others succinic acid and tartaric acid being most effective.

Combined therapy with cyclodextrin/allopregnanolone and miglustat improves motor but not cognitive functions in Niemann-Pick Type C1 mice.

Niemann-Pick Type C1 (NPC1) is an autosomal recessive disorder characterized by the accumulation of cholesterol and glycosphingolipids. Combination-treatment utilizing cyclodextrin, allopregnanolone and miglustat (CYCLO/ALLO/miglustat) can ameliorate NPC1 disease in a mutant mouse model. The present study was designed to add behavioral analysis in NPC1 mutant mice upon CYCLO/ALLO/miglustat therapy. NPC1 mutant (BALB/cJ NPC1NIH) and control mice were used. For the combination treatment mice were injected with CYCLO/ALLO weekly, starting at P7. The miglustat injection was performed daily from P10 till P23. Starting at P23, miglustat was added to the powdered chow. For the sham treatment of control and mutant mice the same schedule was used with 0.9% NaCl injection. Locomotor activity was assessed in open field, elevated plus maze and accelerod tests. For assessment of spatial learning and memory the Morris water maze test was conducted. Electron microscopy has been performed to support the behavioral data. The sham-treated mutant mice exhibited motor impairments in all performed tests. In the water maze the sham-treated mutants exhibited impairment in remembering the location of the hidden platform. CYCLO/ALLO/miglustat treatment positively influenced motor dysfunction: total distance and number of visits significantly increased, and accelerod performance improved. The spatial learning, however, did not benefit from therapy. At the morphological level, an excessive accumulation of electron-dense material was seen in the cerebellar Purkinje cells of mutant mice. A regression of these autophagosomal inclusions was seen upon therapy. CYCLO/ALLO/miglustat therapy ameliorates motor but not cognitive deficits in NPC1 mutant mice, suggesting unequal vulnerability of different brain areas to the treatment.

Medical expert system on fuzzy analog ganglionar lattices: explication and prospection based on sensitivity.

A reasoning method to explicate the conclusions-a posteriori- and to prospect for the next test-a priori-applied to medical expert systems based on fuzzy analog ganglionar lattices is presented. This method is founded on the sensitivity criterion of the consequent respect to the antecedents. It proves to be suited for problems of any complexity, specially when they are formulated on well-established intermediate concepts, such as syndromes, clinical finding, laboratory procedures, etc., as is usually the case in medical procedures. This type of expert system uses the structural properties of the ganglionar lattice to produce explications for its conclusions at different levels of abstraction, and to an arbitrary, but fixed, explicative degree. A measure to evaluate the consequent's achieved preciseness (certainty) is also supplied. A full example of application in the diagnosis of cardiac insufficiency with hypertensive myocardiopathy is shown, and a computational implementation of this analog procedure is described. Finally, the reasoning methods for explicating and prospecting are discussed.

A fuzzy model for medical diagnosis.

A medical diagnosis support system based on a fuzzy model for illnesses is proposed. The model for each disease is a lattice structure composed by unidirectional fuzzy relations among intermediate diagnostic units (IDU). Its evaluation is sequential: it starts at the bottom units, the observable medical evidence (symptoms), and progresses through the IDUs to the cusp unit, representing the disease. Three types of IDUs are defined: for associated, non-associated and excluding evidence. These units represent medical concepts gathering knowledge under a common characteristic or criterion of diagnostic interest. Fuzzy unidirectional relations are used to quantify the medically known dependence between IDUs. The model is specified for six cardiopathies, identifying and calibrating its parameters using data from patient's records. The model performance is evaluated comparing results with the diagnosis provided by three medical observers on new patient's records. Concordance histograms are shown as an objective measure for the model performance. A discussion about application of the diagnosis sensitivity respect to symptoms and respect to IDUs relations is performed.

Methodology for a medical expert system on fuzzy analog ganglionar lattices. Non-approximate reasoning with multiple antecendents of different relative importance and limited uncertainty.

This work presents an Expert System based on fuzzy analog ganglionar lattices. Its reasoning scheme is designed analogously to the expert's mental organization and it is realized on an (analog) operator called the ganglionar lattice. It is a connectionist system that uses the medical knowledge to define its architecture. The operator evokes some similarities to higher order neural networks and performs as the knowledge base and inference engine of the expert system, in a unified manner. A main feature of this operator is that it exhibits the variables corresponding to all intermediate concepts identified by the expert; this characteristic is shown to be most valuable for assessing, explicating and prospecting in medical applications. Further, it is capable of (i) evaluating a consequent for a variety of non-approximate reasonings with multiple antecendents of different relative importance under limited uncertainty; (ii) explicating the conclusions at different levels of abstraction to suit the user; and (iii) prospecting for the best 'a priori' sequence of unevaluated antecedents, from which to choose following tests. These procedures are based on the objective criterion of the consequent's uncertainty decrease (entropy). All results are produced in numerical form and may be translated into restricted natural language. A simple example of this technology is fully developed. Finally the method's potentials are discussed for future applications.

Interpretation module for screening normal ECG.

This work concerns the development of a module for automatic recognition of normal ECGs, i.e. those whose waves morphology do not suggest physiopathological or structural heart alterations. Such a unit constitutes the central part of an administrative system under development which will aid in the management of ECG testing in the primary care delivery level in Chile. This system greatly contributes to the optimal allocation of resources in order to increase the test delivery coverage and to reduce the social and private costs involved. A main feature of the system is that it operates through the screening of normal ECG. This process is performed in a computerized unit whose core is the interpretation module. The design of such a module uses concepts and methods previously developed for the general problem of medical diagnosis, based on fuzzy set theory. The interpretation module parameters were adjusted considering hypothetical and real data covering a wide variety of normal and pathological cases. Then its performance was tested using more than one hundred patients' records chosen at random. Results of this test are given and a discussion, including a comparison with similar commercial equipment, is provided.

Polymorphisms of the alpha-synuclein promoter: expression analyses and association studies in Parkinson's disease.

Mutations of the alpha-synuclein gene have shown to be relevant in some rare families with autosomal dominant Parkinson's disease (PD). Furthermore, alpha-synuclein protein is a major component of the Lewy bodies also in sporadic PD patients. Increased levels of wildtype alpha-synuclein in the cell leads to increased intracellular hydrogen peroxide levels and causes death of dopaminergic neurons in rat primary culture. Subsequently, oxidative stress has been directly linked with alpha-synuclein aggregation in vitro. This raises the question whether increased alpha-synuclein expression might be linked to higher susceptibility to PD and whether alpha-synuclein promoter polymorphisms are associated with PD. Here, two polymorphisms (-116C>G and -668T>C) of the alpha-synuclein promoter defining four haplotypes have been characterized in 315 German PD patients. The influence of the four haplotypes on gene expression was studied by CAT reporter gene assays in neuronal SK-N-AS cells. The -668C/-116G haplotype revealed significant higher CAT expression than the -668T/-116G or the -668T/-116C haplotype, respectively. Although the -668C/-116G haplotype was more common in PD patients, this difference was not significant.

Isolation and characterization of the rat huntingtin promoter.

Huntington's disease (HD) is a neurodegenerative disorder caused by a (CAG)>37 repeat expansion in a novel gene of unknown function. Although the huntingtin gene is expressed in neuronal and non-neuronal tissues, the disease affects nerve cells of selected regional areas of the central nervous system. To gain insight into the regulation of the HD gene we analysed 1348 bp of the rat huntingtin promoter region. This region lacks a TATA and a CAAT box, is rich in GC content and has several consensus sequences for binding sites for SP1, PEA3, Sif and H2A. The stretch between nucleotides -56 and -206 relative to the first ATG is highly conserved between human and rodents and it harbours several potential binding sites for transcription factors. We analysed deletion mutants fused with the chloramphenicol acetyltransferase reporter gene in transfected, HD-expressing neuronal (NS20Y, NG108-15) and non-neuronal Chinese hamster ovary cell lines. Hence these cells should contain the required trans-acting factors necessary for HD gene expression. Partial deletion of the evolutionarily conserved part of the promoter significantly decreases the activity in both neuronal and non-neuronal cells, indicating that the core promoter activity is located between nucleotides -332 and -15. DNase I footprinting and electrophoretic mobility-shift assays were used to define the nucleotide positions and binding affinity of DNA-protein interactions.

Whole blood capcellia CD4/CD8 immunoassay for enumeration of CD4+ and CD8+ peripheral T lymphocytes.

We evaluated the Whole Blood Capcellia(R) CD4/CD8, an immunoenzymatic method that provides absolute counts of CD4+ and CD8+ T cells in peripheral blood. The assay is based on the separation of T cells by use of an anti-CD2 magnetic bead suspension, followed by reaction of the CD4 or CD8 molecules with the corresponding monoclonal antibody coupled to peroxidase. CD4-positive monocytes were excluded from the assay. Freeze-dried magnetic bead-T-cell complexes were used as calibrators. Capcellia counts from HIV-1-infected patients were compared with those obtained by flow cytometry as the comparison method. The results by Capcellia correlated well with those by flow cytometric analysis: r2 = 0.95; P <0.001; (y = 0.96x - 22.1); Sy|x = 64 for CD4; r2 = 0.81; P <0.001; (y = 1.26x - 76.4); Sy|x = 139 for CD8; n = 76. The correlation between CD4+ T-cell counts determined by two trained experimenters was significant (r2 = 0.96). Our results indicate that this new ELISA technique for lymphocyte immunophenotyping is an efficient alternative to flow cytometry.