[Compliance with treatment in Wilson's disease: On the interest of a multidisciplinary closer follow-up]. / L'observance dans la maladie de Wilson : intérêt d'un suivi rapproché au long cours.

BACKGROUND: Compliance with treatment is very important for patients who suffer from Wilson's disease, a rare genetic disorder. They can benefit a long-life and effective treatment. The purpose of our study is to identify the level of compliance in Wilson's disease patients and features associated with compliance as well. METHODS: This is a prospective study carried out in the National Reference Center for Wilson Disease (based in Paris and Lyon) over a 8 months period. Patients were evaluated on the first (M0) and last month (M8) with a questionnaire evaluating the number of missed treatment doses, a self-questionnaire collecting the reasons for non-compliance, and analogic scales analyzing the doctor-patient relationship and their behavior towards the treatment. The severity of depression symptoms was investigated by the Beck Depression Inventory (BDI). A detailed phone call interview was conducted by a psychologist every two months to evaluate their compliance and feeling. RESULTS: Thirty-nine patients were included. The mean age of patients was 34 years (±9.9). At M0, 84.6% had a poor compliance with treatment. They were diagnosed more recently (P=0.049) with a higher proportion involving neurological disorders (P=0.007). Age, the type of treatment and the quality of the doctor-patient relationship were not associated with the outcome; 38.5% suffered from depressive symptoms. At M8, 56.8% of patients were poor compliants and 21.6% presented depressive symptoms. CONCLUSION: Wilsons's disease patients have important problems with compliance, without necessary being depressed. A close follow-up may help them become compliant, particularly those with neurological symptoms.

Cognitive profile in Wilson's disease: a case series of 31 patients.

BACKGROUND: Wilson's disease (WD) is a rare autosomal recessive disorder of copper metabolism. If untreated, WD, which is initially a liver disease, can turn into a multi-systemic disease with neurological involvement. Very few studies have described cognitive impairment in WD. The aim of this study is to report the cognitive profile of 31 treated WD patients. METHODS: Patients were classed into two groups using the Unified Wilson Disease Rating Scale (UWDRS): WD patients without neurological signs (WD-N(-)) (n=13), and WD patients with neurological signs (WD-N(+)) (n=18). The patients participated in a neuropsychological assessment evaluating memory, executive function and visuo-spatial abilities. RESULTS: Both groups performed well for verbal intelligence and episodic memory skills. However, the majority of these patients exhibited altered performance for at least one cognitive test, particularly in the executive domain. The WD-N(+) group performed less well than the WD-N(-) group on cognitive tests involving rapid motor function, abstract thinking, working memory and top-down inhibitory control. CONCLUSIONS: Cognitive impairment in treated WD patients essentially affects executive function involving fronto-striatal circuits. Verbal intelligence and episodic memory abilities seem to be remarkably preserved. Neuropsychological assessment is a valuable tool to evaluate the presence and the consequences of these cognitive impairments in WD patients with or without neurological signs in the course of this chronic disease.

Relevance of 10-min delayed recall in dementia screening.

Within the context of early diagnosis of Alzheimer's disease (AD), there is a growing interest in neuropsychological screening tests. Amongst these tests, we focused on the largely used Memory Impairment Screen (MIS). The objective of the present work was to show that adding a 10-min delayed recall to the MIS, improves the test psychometric characteristics in order to detect dementia in the earliest stages. A prospective study was carried out on a cohort of 270 consecutive elderly ambulatory subjects attending the Broca Hospital Memory Clinic: normal controls (n = 67), mild cognitive impairment subjects (n = 98) and mildly demented patients [n = 105, Mini Mental State Examination (MMSE) = 23 +/- 4]. This study consisted in testing the advantage of the 10-min delayed recall entitled MIS-D compared with the MIS. At a cut-off score of 6, the MIS-D revealed satisfying psychometric characteristics with a sensitivity of 81% and a specificity of 91%, whilst the MIS alone indicated a sensitivity of 60% and a specificity of 88% in detecting dementia. In demented patients with MMSE score > or =26, MIS-D properties still remained satisfying (sensitivity: 75%, specificity: 92%). MIS-D is a more relevant screening test than MIS alone at very early stages of dementia.

Accidental MCI in healthy subjects: a prospective longitudinal study.

A study was realized on 130 healthy and autonomous volunteers (60-80 years old) who met specific medical and functional inclusion criteria. A comprehensive battery of neuropsychological tests was performed at baseline (M0), 6 and 12 months (M6, M12). At M0 the results indicated that 65% were cognitively normal on each of all the neuropsychological tests, whereas 35% presented a cognitive deficit on one or more tests. At M12, 52% of the subjects who had a cognitive deficit at M0 remained impaired, whereas 48% normalized their scores: they performed as well as the subjects classified normal at M0. The results also indicated that the subjects who remained impaired at M12, had at M0 low scores on three tests or more, whereas the ones who normalized their scores had one or two failed tests. This study focuses on the risk of false positive cases and shows that low scores can be accidental. The authors propose decision rules allowing to reduce the risk of false positive cases. The observation of accidental impairment invites to be cautious and makes this 1-year follow-up study particularly relevant, since a 1-year follow-up is generally needed to diagnose very mild dementia.

[A global intervention program for institutionalized demented patients]. / Méthode de prise en charge globale non médicamenteuse des patients déments institutionnalisés.

INTRODUCTION: Over the last decades many approaches have been developed to manage cognitive and behavioral disturbances in dementia. The present work describes a global intervention program carried out with moderately to severely demented institutionalized patients. The aims of the intervention program are to stimulate and maintain the preserved abilities of demented patients in a supportive context, to decrease the behavioral disturbance and to avoid burnout of care-unit staff. METHODS: This intervention combines different means: psychosocial care (validation therapy, social interaction), cognitive stimulation (memory and verbal training), and motor and sensitive stimulation. The global intervention program requires a special trained team composed of a supervisor, six aid-nurses, an occupational therapist, a speech therapist, a psychomotor therapist and a psychologist. The team cared for the patients five days per week over a three-month period. Assessments were conducted before and after the intervention program to measure the benefit. RESULTS: Positive effects were shown for cognitive abilities, nutritional problems and staff burnout. However, due to the small sample size for this study, more research is needed to verify the effectiveness of this global intervention program, particularly the implications for nutrition. CONCLUSION: This global intervention combined with pharmacological treatment seems to be useful for managing psychological and behavioral disorders of institutionalized demented patients.

Implicit memory for words presented in short texts is preserved in Alzheimer's disease.

BACKGROUND: The level of efficiency of implicit memory in Alzheimer's disease remains unclear as previous studies using stem completion tasks have led to contradictory results. METHOD: The present study used target words embedded in significant short texts that subjects were required to read aloud (i.e. to enhance semantic processing). Texts were presented in two perceptual situations: 'simple' (blank spaces delimitating words) and complex' (spaces were filled by '8's). In the completion phase, patients had to write the first word that came to mind in order to complete a three-letter stem. The recognition phase explored explicit memory performance. The performance of 24 Alzheimer patients was compared to a matched sample of healthy controls. RESULTS: Reading times differed between groups and were shorter for healthy controls. Recognition was dramatically lower in patients, thus confirming the alteration of explicit memory in this pathology. However, a significant priming effect (e.g. the tendency to complete the stem with the aid of a previously explored word) was present in both groups and did not differ between patients and healthy controls. CONCLUSIONS: The absence of a correlation between priming and recognition scores suggests that this result cannot be explained by an explicit memory bias. Moreover, as the priming level was identical whatever the perceptual aspect of the text, we suggest that the priming effect is not only mediated by perceptual processes but also by lexical and conceptual processes, which to some extent are preserved during the light and moderate stages of this disease.

Protein purification in multicompartment electrolyzers with isoelectric membranes.

Preparative purification of proteins under isoelectric conditions is reviewed, with particular regard to novel equipment, a multicompartment electrolyzer with isoelectric membranes, which can capture any desired protein into an isoelectric trap as the sole, ultra-pure component. This novel machine is based on the Immobiline chemistry, i.e. the novel generation of non-amphoteric buffers, based on the chemistry of acrylamides, which can be insolubilized onto polyacrylamide supports. After a description of the instrument and of its performance, a number of protein purification protocols are described, leading to truly homogeneous (by the most stringent criterion of surface charge) protein fractions. Such a high charge purity has been found to be often a fundamental prerequisite for the growth of protein crystals. Interfacing the electrolyzer with mass spectrometry has permitted the decoding of the structure of minor components generated from a parental molecule, especially ones having a higher pI. It was found that these species were often generated either by proteolytic cleavage or by the formation of a trisulphide bridge between two Cys residues. A unique application of the electrolyzer is finally described: its use as an immobilized enzyme reactor under an electric field. The performance of this reaction is outstanding, in that the kinetic parameters of the immobilized enzyme are identical to those of a free enzyme form.

Investigation on minor degraded derivatives of the recombinant hirudin variant HM2 from Hirudinaria manillensis isolated by isoelectric focusing in multicompartment electrolyzers.

On isoelectric focusing in immobilized pH gradients (IPG) a preparation of recombinant hirudin from Hirudinaria manillensis, purified to homogeneity, was found to still contain a total of 5% minor components: three with higher pI values (pIs 4.10, 4.25 and 4.31), one with a lower pI value (pI 3.98) as compared with the main form (pI 4.03). Multicompartment electrolyzers with isoelectric membranes and micropreparative IPG gel slabs allowed the recovery of pure fractions of such minor components, which were further characterized by electrospray mass spectra, limited proteolysis, and sequence analysis. All four minor isoforms were found to be cleavage products of the parent, full-length hirudin molecule (molecular mass 6797 Da), as follows: the pI 4.31 (5032 Da) had lost sixteen amino acids from the N-terminus, the pI 4.25 (6212 Da) lacked five amino acids from the C-terminus, the pI 4.10 (2980 Da) was a cleavage product at residue Cys37, and the pI 3.98 (6610 Da) lacked the dipeptide Val-Ser at the N-terminus. Combining the extreme resolving power of IPGs with the high accuracy of mass spectra was found to be an attractive strategy in decoding post-synthetic modifications often encountered in r-DNA proteins.

Detection of traces of a trisulphide derivative in the preparation of a recombinant truncated interleukin-6 mutein.

A new mutein of interleukin-6, called delta 22-IL-6 Cys 3,4, characterized by the deletion of the first 22 amino acids at the N-terminal end and by the substitution of the first two cysteines (Cys23 and Cys29) with serine residues, was produced in Escherichia coli and was found to maintain the structural and functional properties of the human native form. A partially purified preparation still showed in isoelectric focusing a minor acidic component (pI 6.10) and a more basic component (pI 6.70), the native form having a pI of 6.56. This preparation was further fractionated in a multi-compartment electrolyser with isoelectric membranes, which allowed the collection of the more alkaline species for characterization. Mass spectra of the pI 6.70 form gave an additional mass of 32 atomic mass units (amu), suggesting the addition of two oxygen atoms (a potential oxidation of two methionine residues to sulphoxide). However, the five methionine residues in this higher pI form were identified after enzymatic hydrolysis and peptide mapping and were found to be in a reduced state. In addition, the pI 6.70 form was quickly converted into the native form by mild reductive treatment. On digestion and fingerprinting, the peptide from residues 50 to 65 of the pI 6.70 species (containing the only two cysteine residues of the molecule) exhibited a more hydrophobic behaviour in reversed-phase high-performance liquid chromatography and retained a mass increase of 32 amu. These experimental findings more likely suggest the addition of an extra sulphur atom to the only disulphide bridge to give an unusual protein trisulphide molecule.

Protein microheterogeneity and crystal habits: the case of epidermal growth factor receptor isoforms as isolated in a multicompartment electrolyzer with isoelectric membranes.

A purified, soluble form of the epidermal growth factor receptor (sEGFR) was found, by isoelectric focusing in immobilized pH gradients, to consist of three major isoforms (with pI values 6.45, 6.71 and 6.96, respectively) and ca. a dozen minor components. This wild-type sEGFR, while producing crystals, has so far defied any attempt at decoding the structure, due to the very poor diffraction pattern. When the wild-type sEGFR was purified in a multicompartment electrolyzer with isoelectric Immobiline membranes, it yielded the three major isoforms as single-pI components, collected in three separate chambers of the recycling electrolyzer. The pI 6.71 and the pI 6.96 isoforms produced large crystals of apparent good quality. However, while the former produced a high-quality diffraction pattern, which may lead to decoding of three-dimensional structure, the pI 6.96 produced crystals which did not diffract at all. It is concluded that, in the case of "tough" proteins (large size, heterogeneous glycosylation, high water content of crystals), purification to single-charge components might be an essential step for growing proper crystals. The unique advantage of purification via isoelectric membranes is that the protein is collected both isoelectric and isoionic, i.e. uncontaminated by soluble buffers (such as the carrier ampholytes used in conventional focusing).

Purification of human recombinant superoxide dismutase by isoelectric focusing in a multicompartment electrolyzer with zwitterionic membranes.

Human recombinant superoxide dismutase (SOD), purified to homogeneity, is resolved by both conventional isoelectric focusing and immobilized pH gradients into three bands, with isoelectric points (pIs) in the pH range 4.8 to 5.1, the pI 4.80 form representing the minor component. Due to the fact that this enzyme is expressed in E. coli, N-terminal acetylation or glycosylation should be ruled out. When purified by small-scale preparative isoelectric focusing in immobilized pH gradient gels, it was found that, upon subsequent analysis, the pI 5.07 form would band in the same position, but the intermediate pI 4.92 band would split into the upper (pI 5.07) and the lower (pI 4.80) species, in nearly the same amounts, whereas the lowest pI component would always generate both the intermediate and upper forms. Enzymatic essays pointed out that these three isoforms had nearly the same specific activity, slightly higher than that of the starting material. Metal analysis indicated that all three forms contained the same metal/protein ratio, approaching the value Cu2Zn2-SOD, as reported in the literature. Circular dichroism spectra of the pI 4.80 and 5.07 forms showed the same profile in the 190-240 nm range, but marked differences in the 250-350 nm region. Treatment with EDTA produces 1-2 additional, slightly higher pI isoforms, whereas treatment with KCN generates a number of higher pI components, reaching pI values as high as pH 7, with nearly complete disappearance of the three major SOD isoforms. It is concluded that these three isoforms could represent interconvertible species, the highest pI component representing the most stable conformer.

Isoelectric focusing in a multicompartment electrolyzer with zwitterionic membranes, exemplified by purification of glucoamylase.

A highly purified preparation of glucoamylase G1 from Aspergillus niger was found, by isoelectric focusing in immobilized pH gradients, to contain a major form with a pI of 3.50 and a number of minor, more acidic and more basic contaminants. The major isoform was purified to homogeneity by recycling isoelectric focusing in a multicompartment electrolyzer, by confining this form in between two zwitterionic membranes, with pI 3.49 at the anodic side and pI 3.52 at the cathodic side. Recoveries were high (90%) and, notwithstanding the rather low operational pH, the electrosmotic flow was minimal and no protein precipitation occurred up to concentrations of 2.5 mg/ml (at the isoelectric point). The forms resolved in an analytical focusing gel were subjected to two types of in situ enzyme detections, by the glucose oxidase peroxidase (GOP) test and by the starch-iodine test. By both criteria all resolved zones exhibited enzyme activity, the GOP assay, however, following more closely the Coomassie blue stained protein profile. By computer modelling, it is shown that it is impossible to obtain linear pH gradients at such low pH values (pH 2.5-4.5 intervals) when the mixture has a low buffering power (beta = 2.0 mequiv.l-1 pH-1). When the beta power was gradually raised (beta = 4, beta = 6, beta = 8) the pH gradient became progressively linear until, in a recipe with beta = 10 mequiv.l-1 pH-1 full linearity of the pH gradient could be obtained. This is shown to be due to the substantial buffering power of bulk water in the pH 2.5-3.5 region.

Conventional isoelectric focusing and immobilized pH gradients in 'macroporous' polyacrylamide gels.

Lateral aggregation in presence of a hydrophilic polymer (e.g. 10 kDa polyethylene glycol) in the gelling solution (Righetti et al., Electrophoresis 1992, 13, 587-595) is not inhibited by high ionic strength nor in the pH 4-10 interval. However, the bundles are disaggregated by glycerol (Tm at 20%) and by ethylene glycol (Tm at 24.5%) as well as by pH extremes (pH 3 and pH 11). Supercoiling is also strongly inhibited in a copolymer, formed by acrylamide an N,N-dimethylacrylamide or N-methylacrylamide. A level of 50% uncoiling is obtained well before a 1:1 ratio, already at a level of 18% N,N-dimethylacrylamide. All the above data strongly suggest that the nascent chains are held together in bundles by hydrogen bonds prior to the cross-linking event, instead of having a random orientation and distribution in the solvent. However, it is not possible to distinguish between H-bonds oriented perpendicular to the chain axis vs. H-bonds occurring within a single polymer filament, and the two types of H-bonds probably coexist. Macroporous gels perform well in steady-state electrophoretic techniques, such as conventional isoelectric focusing and immobilized pH gradients, where a large-pore structure is necessary for fast protein migration and for attainment of equilibrium conditions.

Purification to single isoforms of a secreted epidermal growth factor receptor in a multicompartment electrolyzer with isoelectric membranes.

A purified, size-homogeneous (100 kDa), desialylated form of a truncated, soluble form of the epidermal growth factor receptor secreted by A431 human tumor cells has been found, by isoelectric focusing in immobilized pH gradients, to consist of two major isoforms (with pIs of 6.96 and 6.71), one intermediate form (pI 6.45) and a number (> 10) of minor components. The two major components have been purified to charge homogeneity by isoelectric focusing in a multicompartment electrolyzer with buffering isoelectric membranes having the following pI values: 5.90, 6.63, 6.76, 6.92, 7.05 and 7.35. Such single pI species are presently used for attempts at crystal growing.

Two-dimensional electrophoresis as a tool for control of quality and consistency in production systems using animal cells. Two-dimensional electrophoresis in animal cell culture technology.

A nonrecombinant human melanoma cell line and recombinant chinese hamster ovary (CHO) cells were used as examples for long-term in vitro cultivation in protein-free media. The method used to monitor the consistency of protein release by these mammalian cells was two-dimensional electrophoresis with immobilized pH gradient. Secreted proteins from a melanoma cell line cultivated in a continuous fermentation system over a period of 22 months were monitored. Two-dimensional patterns of secreted proteins were compared and the stability of their composition was determined over a period of nearly 14 months, with significant pattern variation being observed after 14 months. The protein pattern from this extended in vitro culture was compared to those of the very same melanoma cell line recultivated after being frozen in liquid nitrogen for more than 2 years. Due to the high resolution of complex polypeptide mixtures and the possibility to detect even minor differences in the composition of protein patterns, we propose the two-dimensional electrophoresis as a tool for quality assessment in animal cell culture technology.

Crystallization of the Fab from a human monoclonal antibody against gp 41 of human immunodeficiency virus type I.

A monoclonal IgG antibody directed against gp 41 from the human immunodeficiency virus (HIV-1) has been crystallized in both intact and Fab forms. Crystals of the intact antibody grow as tetragonal-like prisms too small for conventional X-ray analysis. However, the Fab portion of the antibody produces suitable plate-like crystals which belong to the space group P2(1)2(1)2(1) with unit cell constants of a = 66.5 A, b = 74.3 A and c = 105.3 A. There is one molecule of Fab in the asymmetric unit. The Fab crystals show diffraction to d-spacings less than 3.0 A.

Shifts of isoelectric points between cellular and secreted antibodies as revealed by isoelectric focusing and immobilized pH gradients.

Charge microheterogeneity of monoclonal antibodies, as revealed by isoelectric focusing in carrier ampholytes, has been known for a long time. Here we demonstrate, in the case of monoclonals against the gp-41 of the HIV-1 virus, that this heterogeneity is already present within the cell sap of hybridoma cells during antibody synthesis. When the monoclonals are secreted extracellularly, the same isoelectric point (pI) spectrum is maintained, but there is marked redistribution of the relative isoform abundance towards the lower pI components. This suggests in vivo processing of such forms, possibly via glycosylation or deamidation. The secreted antibodies are also analyzed by immobilized pH gradients (IPG), where they demonstrate an even more extensive heterogeneity, due to the marked increment in resolving power. Single bands are purified by preparative IPGs in a multicompartment electrolyzer and are shown to be stable with time. Thus, artefactual heterogeneity produced by the focusing technique is completely excluded and cellular processing is clearly established.

Isoelectric focusing in immobilized pH gradients of phosphoglucomutase and esterases from the spiny lobster.

A method is described for detecting polymorphisms of cephalothorax and tail homogenates of 25 puerulus staged Panulirus argus in phosphoglucomutase (PGM) and esterases. Isoelectric focusing in immobilized pH gradients was used. In the pH 6.0-8.0 interval for phosphoglucomutase and in the pH 3.5-5.0 and 4.2-4.9 ranges for esterases, both enzymes appeared as polymorphic band patterns. These could be explained by one locus with 2 alleles for phosphoglucomutase and 3 loci with 2, 3 and 4 alleles for esterases. Esterases exhibit a more extensive polymorphism in immobilized pH gradients than in polyacrylamide gel electrophoresis.

Capillary zone electrophoresis for monitoring r-DNA protein purification in multi-compartment electrolysers with immobiline membranes.

Isoforms of human monoclonal antibodies against the gp-41 of AIDS virus and of human recombinant superoxide dismutase have been purified to homogeneity by isoelectric focusing (IEF) in a multi-compartment electrolyser with isoelectric, immobiline membranes. This system allows the processing of large sample volumes and gram-scale protein loads and can resolve isoforms as close as 0.001 in pI difference. The purification progress was usually monitored by analytical IEF in immobilized pH gradients (IPG). Capillary zone electrophoresis (CZE) was applied to the monitoring of the content of each chamber of the electrolyser. CZE was found to be superior in terms of speed of analysis and quantification (but only by UV reading at 200-210 nm, i.e., in the region of the peptide bond) but, notwithstanding the millions of theoretical plates reported, was no match for the resolving power of IPGs, at least for protein analysis. When compared also with chromatofocusing, the resolving power decreases in the order IPG greater than CZE much greater than chromatofocusing.