Beata Sokołowska

Matrix metalloproteinases and their tissue inhibitors in serum and cerebrospinal fluid of patients with amyotrophic lateral sclerosis

Background and purpose:  Matrix metalloproteinases (MMPs) are implicated in the pathogenesis of motor neuron degeneration in amyotrophic lateral sclerosis (ALS). We investigated the expression of MMPs and tissue inhibitors of matrix metalloproteinases (TIMPs) in serum and cerebrospinal fluid (CSF) correlating the results with age, disease duration and the clinical course.

Chloroacetaldehyde-induced mutagenesis in Escherichia coli: The role of AlkB protein in repair of 3,N4-ethenocytosine and 3,N4-α-hydroxyethanocytosine

Etheno (epsilon) adducts are formed in reaction of DNA bases with various environmental carcinogens and endogenously created products of lipid peroxidation. Chloroacetaldehyde (CAA), a metabolite of carcinogen vinyl chloride, is routinely used to generate epsilon-adducts. We studied the role of AlkB, along with AlkA and Mug proteins, all engaged in repair of epsilon-adducts, in CAA-induced mutagenesis. The test system used involved pIF102 and pIF104 plasmids bearing the lactose operon of CC102 or CC104 origin (Cupples and Miller (1989) [17]) which allowed to monitor Lac(+) revertants, the latter arose by GC-->AT or GC-->TA substitutions, respectively, as a result of modification of guanine and cytosine. The plasmids were CAA-damaged in vitro and replicated in Escherichia coli of various genetic backgrounds. To modify the levels of AlkA and AlkB proteins, mutagenesis was studied in E. coli cells induced or not in adaptive response. Formation of varepsilonC proceeds via a relatively stable intermediate, 3,N(4)-alpha-hydroxyethanocytosine (HEC), which allowed to compare repair of both adducts. The results indicate that all three genes, alkA, alkB and microg, are engaged in alleviation of CAA-induced mutagenesis. The frequency of mutation was higher in AlkA-, AlkB- and Mug-deficient strains in comparison to alkA(+), alkB(+), and microg(+) controls. Considering the levels of CAA-induced Lac(+) revertants in strains harboring the pIF plasmids and induced or not in adaptive response, we conclude that AlkB protein is engaged in the repair of epsilonC and HEC in vivo. Using the modified TTCTT 5-mers as substrates, we confirmed in vitro that AlkB protein repairs epsilonC and HEC although far less efficiently than the reference adduct 3-methylcytosine. The pH optimum for repair of HEC and epsilonC is significantly different from that for 3-methylcytosine. We propose that the protonated form of adduct interact in active site of AlkB protein.

The role of AlkB protein in repair of 1,N6-ethenoadenine in Escherichia coli cells

Etheno (ε) DNA adducts, including 1,N(6)-ethenoadenine (εA), are formed by various bifunctional agents of exogenous and endogenous origin. The AT→TA transversion, the most frequent mutation provoked by the presence of εA in DNA, is very common in critical codons of the TP53 and RAS genes in tumours induced by exposure to carcinogenic vinyl compounds. Here, using a method that allows examination of the mutagenic potency of a metabolite of vinyl chloride, chloroacetaldehyde (CAA), but eliminates its cytotoxicity, we studied the participation of alkA, alkB and mug gene products in the repair of εA in Escherichia coli cells. The test system used comprised the pIF105 plasmid bearing the lactose operon of CC105 origin, which allowed monitoring of Lac(+) revertants that arose by AT→TA substitutions due to the modification of adenine by CAA. The plasmid was CAA-modified in vitro and replicated in E.coli of various genetic backgrounds (wt, alkA, alkB, mug, alkAalkB, alkAmug and alkBmug). To modify the levels of the AlkA and AlkB proteins, mutagenesis was studied in E.coli cells induced or not in adaptive response to alkylating agents. Considering the levels of CAA-induced Lac(+) revertants in strains harbouring the CAA-modified pIF105 plasmid and induced or not in adaptive response, we conclude that the AlkB dioxygenase plays a major role in decreasing the level of AT→TA mutations, thus in the repair of εA in E.coli cells. The observed differences of mutation frequencies in the various mutant strains assayed indicate that Mug glycosylase is also engaged in the repair of εA, whereas the role the AlkA glycosylase in this repair is negligible.

Analysis of postural sway in patients with normal pressure hydrocephalus: effects of shunt implantation

Poor postural balance is one of the major risk factors for falling in normal pressure hydrocephalus (NPH). Postural instability in the clinic is commonly assessed based upon force platform posturography. In this study we focused on the identification of changes in sway characteristics while standing quiet in patients with NPH before and after shunt implantation. Postural sway area and sway radius were analyzed in a group of 9 patients and 46 controls of both genders. Subjects spontaneous sway was recorded while standing quiet on a force platform for 30-60 s, with eyes open and then closed. Both analyzed sway descriptors identified between-group differences and also an effect of shunt implantation in the NPH group. Sway radius and sway area in patients exhibited very high values compared with those in the control group. Importantly, the effect of eyesight in patients was not observed before shunt implantation and reappeared after the surgical treatment. The study documents that static force platform posturography may be a reliable measure of postural control improvement due to shunt surgery.

The LITAF/SIMPLE I92V sequence variant results in an earlier age of onset of CMT1A/HNPP diseases

Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) represent the most common heritable neuromuscular disorders. Molecular diagnostics of CMT1A/HNPP diseases confirm clinical diagnosis, but their value is limited to the clinical course and prognosis. However, no biomarkers of CMT1A/HNPP have been identified. We decided to explore if the LITAF/SIMPLE gene shared a functional link to the PMP22 gene, whose duplication or deletion results in CMT1A and HNPP, respectively. By studying a large cohort of CMT1A/HNPP-affected patients, we found that the LITAF I92V sequence variant predisposes patients to an earlier age of onset of both the CMT1A and HNPP diseases. Using cell transfection experiments, we showed that the LITAF I92V sequence variant partially mislocalizes to the mitochondria in contrast to wild-type LITAF which localizes to the late endosome/lysosomes and is associated with a tendency for PMP22 to accumulate in the cells. Overall, this study shows that the I92V LITAF sequence variant would be a good candidate for a biomarker in the case of the CMT1A/HNPP disorders.

Posturography in Differential Diagnosis of Normal Pressure Hydrocephalus and Brain Atrophy

Differentiation between normal pressure hydrocephalus (NPH) and brain atrophy is difficult in clinical practice. The purpose of this paper was to apply two advanced statistical, pattern recognition methods: discriminant analysis (DA) and k-nearest neighbour (K-NN) for the classification of NPH and atrophy patients to approach computer aided differential diagnosis. The classification is based on a few measures of the center of foot pressure (COP) movements (radius, area, and length). The posturography method gives a measure of current postural stability by a quantitative evaluation of postural sways. Measurements have been performed in the standing upright position in two conditions: with eyes open (EO) and closed (EC). The study comprises 18 patients (mean age 64 ±13 years) diagnosed as normal pressure hydrocephalus and qualifying for shunt implantation. The patients were evaluated by static posturography twice: before and after surgery. The NPH patients were compared with 36 atrophy patients (mean age 64±13 years) and 47 healthy persons (mean age 60 ±7 years). There were two basic dissimilarities in the NPH patients before surgery in comparison with the other groups: very large sways and their independence from vision. Over 90% of the NPH cases both before and after surgery were correctly classified. There also were over 90% of correctly classified patients if we compared the before surgery NPH and atrophy patients. Further posturographic measurements and data collection are needed to verify these results.

Osteopontin – a fibrosis-related marker – in dilated cardiomyopathy in patients with Emery-Dreifuss muscular dystrophy

Abstract Background: As osteopontin (OPN) may be assumed to have diagnostic/prognostic value in heart diseases, it is worth assessing whether it is also involved in the pathogenesis and can be applied in the diagnosis of the dilated cardiomyopathy (DCM) in Emery-Dreifuss muscular dystrophy (EDMD). Methods: Serum levels of osteopontin were quantified by means of sandwich immunoassay in 25 EDMD patients (10 laminopathies AD-EDMD and 15 emerinopathies – X-EDMD), eight carriers of X-EDMD, nine disease controls (patients with dystrophinopathy) and 20 age-matched healthy controls. Results: The levels of circulating OPN were elevated in all AD-EDMD and X-linked EDMD patients, as well as in X-EDMD carriers and patients suffering progressive muscular dystrophy. There was no correlation between the osteopontin level and different cardiac parameters, including left-ventricular end-diastolic diameter, left atrial diameter, the left ventricular ejection fraction and the CK-MB level. There was a slight negative correlation with the ages of the patients. Conclusions: The presented results indicate that assessments of circulating OPN levels may help to identify EDMD patients at risk of dilated cardiomyopathy and might be therefore included among the set of biomarkers referred to with a view to appropriate early cardiologic diagnosis and therapy being commenced with in time.

Extraction of biomedical traits for patients with Amyotrophic Lateral Sclerosis using parallel and hierarchical classifiers

This paper concerns the analysis and interpretation of results obtained from biomedical experiments. The biomedical approach allows differentiating patients by testing their EPO concentration in serum and cerebrospinal fluids. The diagnostic tests can be applied in patient monitoring, or future therapeutic research. The paper presents the possibility of recognition of the clinical status of patients with Amyotrophic Lateral Sclerosis (ALS) by the use of parallel and hierarchical classifiers. The proposed method can be used in biometric identification of ALS patients. The results demonstrate that the pattern recognition methods may be helpful in evaluation of the clinical progress of ALS.

Posturography in Patients with Rheumatoid Arthritis and Osteoarthritis

The purpose of the paper was to investigate the usefulness of posturographic analysis in patients with rheumatoid arthritis (RA) and osteoarthritis (OA). RA is a chronic inflammatory disorder responsible for destruction of active and passive components of joints. It is the most common autoimmune disease, and the second most common form of arthritis after OA. OA is a chronic disorder characterized by irreversible changes in the joint structure developing with advancing age. Both diseases lead to the destruction of many parts of the motor system, cause pain, weakness, and damage of ligaments, muscles, bones, and articular cartilage. The etiology of the diseases remains unknown. In the present study, evaluation of body balance in the standing position was performed by means of Pro-Med force plate system. Three posturographic tests were applied: with eyes open, closed, and with the biofeedback--under conscious visual control of body movements. The following posturographic parameters were measured: the radius of sways, the developed area, and the total length of posturograms, and also two directional components of sways: the length of left-right (in frontal plane) and forward-backward (in sagittal plane) motions. The results demonstrate that the biofeedback test is most useful in the evaluation of instability in rheumatic patients; it is more powerful than the other posturographic tests evaluated.

Natriuretic Peptides Assessment in Dilated Cardiomyopathy in Patients with Emery-Dreifuss Muscular Dystrophy

Introduction: Levels of natriuretic peptides in blood are often tested for as screening for heart disease and their progress assessed. Dilated cardiomyopathy (DCM) with conduction disturbances is one of the leading serious manifestations in genetically transmitted Emery-Dreifuss muscular dystrophy (EDMD). However, the potential significance of and variability to brain-natriuretic peptides (BNP) and atrial-natriuretic peptides (ANP) in this disease has not been tested hitherto. It seemed worth considering whether estimation of natriuretic peptides might help in defining cardiac dysfunction in the early stages of the disease, prior to the appearance of echocardiographic changes. This is perceived especially important in cardiologically asymptomatic patients, who are still at risk of cardiac sudden death. Patients and Methods: Serum levels of BNP, NT-proBNP, ANP and NT-proANP were quantified by ELISA sandwich immunoassay in 25 EDMD patients (10 autosomal-dominant AD-EDMD, 15 X-linked EDMD), 8 X-EDMD carriers, 9 patients with dystrophinopathy as disease controls, and 20 age-matched healthy controls. Results: Serum levels of BNP, NT-pro-BNP, ANP, and NT-proANP were elevated in the blood of about 50% of patients with both the AD-EDMD and the X-EDMD form. Values were distributed from normal through to highly elevated. In the X-EDMD group there was a marked increase in the ANP and NT-proANP values. The X-EDMD group also manifested a correlation between level of atrial natriuretic peptides, echocardiographic parameters and severity of cardiac symptoms. Conclusions: The presented results indicate that assessment of circulating natriuretic peptides is of limited value in identifying cardiac involvement in EDMD. However, when included to the panel of other cardiologic biomarkers the estimation of natriuretic peptides may offer additional information in respect of proper diagnosis, prognosis, monitoring of appropriate treatment, prediction of outcome. and help to prevent cardiac decompensation and sudden death.