A prospective study of the financial costs of multiple sclerosis at different stages of the disease.

The aim of our study was to estimate the costs of multiple sclerosis (MS) in Poland according to severity of disease. Total, direct and indirect costs were compared in 148 patients divided into three groups categorized by disease severity: stage I Expanded Disability Status Scale (EDSS <3.5), stage II (EDSS 4.0-6.0) and stage III (EDSS >6.5). Cost evaluation was performed from the societal perspective and covered the 5-month period. Simple sensitivity analysis was performed by varying the tariffs and valuing caregiving at 40% of the average wage. The mean total cost/patient for 5 months was estimated at 10,955, 15, 603 and 18, 464 PLN for stage I, II and III, respectively [exchange rate: 4 PLN=1 EUR; purchasing power pariety: 1 EUR=2.05 PLN] (P <0.0001). Regardless of EDSS stage indirect costs exceeded direct costs. Both direct and indirect costs increased with MS progression. For indirect cost the main item was productivity loss. This study confirms that MS represents a high economic burden, with indirect costs greatly exceeding direct costs. As costs increase with disease progression, treatment efforts should focus on patients in the early stages of MS.

Dolichol phosphate mannose synthase from the filamentous fungus Trichoderma reesei belongs to the human and Schizosaccharomyces pombe class of the enzyme.

Dolichol phosphate mannose (DPM) synthase activity, which is required in N:-glycosylation, O-mannosylation, and glycosylphosphatidylinositol membrane anchoring of protein, has been postulated to regulate the Trichoderma reesei secretory pathway. We have cloned a T.reesei cDNA that encodes a 243 amino acid protein whose amino acid sequence shows 67% and 65% identity, respectively, to the Schizosaccharomyces pombe and human DPM synthases, and which lacks the COOH-terminal hydrophobic domain characteristic of the Saccharomyces cerevisiae class of synthase. The Trichoderma dpm1 (Trdpm1) gene complements a lethal null mutation in the S.pombe dpm1(+) gene, but neither restores viability of a S.cerevisiae dpm1-disruptant nor complements the temperature-sensitivity of the S. cerevisiae dpm1-6 mutant. The T.reesei DPM synthase is therefore a member of the "human" class of enzyme. Overexpression of Trdpm1 in a dpm1(+)::his7/dpm1(+) S.pombe diploid resulted in a 4-fold increase in specific DPM synthase activity. However, neither the wild type T. reesei DPM synthase, nor a chimera consisting of this protein and the hydrophobic COOH terminus of the S.cerevisiae DPM synthase, complemented an S.cerevisiae dpm1 null mutant or gave active enzyme when expressed in E.coli. The level of the Trdpm1 mRNA in T.reesei QM9414 strain was dependent on the composition of the culture medium. Expression levels of Trdpm1 were directly correlated with the protein secretory capacity of the fungus.

Glycosylation defects corrected by the changes in GDPmannose level.

GDPMan is a key substrate in glycoprotein formation. This is especially true for lower eukaryotes where, in addition to the involvement in N-glycan biosynthesis and GPI-anchor formation, GDPMan takes part in the process which is unique for yeast and fungi i.e. O-mannosylation. Several lines of evidence have been presented that the level of GDPMan affects the process occurring in the Golgi compartment i.e. the elongation of outer mannose chain of glycoproteins in Saccharomyces cerevisiae. Results from our laboratory indicate that the availability of GDPMan affects also the early steps of glycoprotein formation ascribed to the endoplasmic reticulum, i.e. assembly of the dolichol-linked oligosaccharide as well as mannosyl-phosphodolichol (MPD) formation. The biochemical basis of carbohydrate deficient glycoprotein syndrome, a severe neurological disorder related to the GDPMan deficiency, is also discussed.

Heterologous expression of genes in filamentous fungi.

Isolation of some biologically important proteins from natural sources was found to be too expensive or scarcely possible (human proteins). The problem could be solved by expression of heterologous genes. Many biologically active proteins have been successfully expressed in filamentous fungi, some of them, however, at a low level. Thus, improvement of this technique appears to be a very important task. The process comprises several steps. Some of them, such as efficient transformation, vector construction, processing of signal sequences, post-translational modifications and secretion of the expressed proteins, have been intensively investigated. This review presents obstacles and problems encountered in expression of heterologous genes and discusses strategies of development in this area.

Overexpression of the Saccharomyces cerevisiae mannosylphosphodolichol synthase-encoding gene in Trichoderma reesei results in an increased level of protein secretion and abnormal cell ultrastructure.

Production of extracellular proteins plays an important role in the physiology of Trichoderma reesei and has potential industrial application. To improve the efficiency of protein secretion, we overexpressed in T. reesei the DPM1 gene of Saccharomyces cerevisiae, encoding mannosylphosphodolichol (MPD) synthase, under homologous, constitutively acting expression signals. Four stable transformants, each with different copy numbers of tandemly integrated DPM1, exhibited roughly double the activity of MPD synthase in the respective endoplasmic reticulum membrane fraction. On a dry-weight basis, they secreted up to sevenfold-higher concentrations of extracellular proteins during growth on lactose, a carbon source promoting formation of cellulases. Northern blot analysis showed that the relative level of the transcript of cbh1, which encodes the major cellulase (cellobiohydrolase I [CBH I]), did not increase in the transformants. On the other hand, the amount of secreted CBH I and, in all but one of the transformants, intracellular CBH I was elevated. Our results suggest that posttranscriptional processes are responsible for the increase in CBH I production. The carbohydrate contents of the extracellular proteins were comparable in the wild type and in the transformants, and no hyperglycosylation was detected. Electron microscopy of the DPM1-amplified strains revealed amorphous structure of the cell wall and over three times as many mitochondria as in the control. Our data indicate that molecular manipulation of glycan biosynthesis in Trichoderma can result in improved protein secretion.

Isolation of a Trichoderma reesei cDNA encoding GTP: a-D-mannose-1-phosphate guanyltransferase involved in early steps of protein glycosylation.

A cDNA coding for GTP: alpha-d-mannose-1-phosphate guanyltransferase (MPG1 transferase) (EC 2.7.7.13) was isolated from a cDNA library of the Trichoderma reesei RutC-30 strain by suppression of the yeast Saccharomyces cerevisiae mutation in the DPM1gene encoding mannosylphosphodolichol (MPD) synthase. The nucleotide sequence of the 1.6 kb-long cDNA revealed an ORF which encodes a protein of 364 amino acids. Sequence comparisons demonstrate 70% identity with the S. cerevisiae guanyl transferase gene (MPG1) and 75% identity with the Schizosaccharomyces pombe homologue. No similarity was found with the MPD synthase encoded by the S. cerevisiae DPM1 gene. The possibility that cloned cDNA encodes a product with a MPD synthase activity was also excluded by transforming a heterozygous S. cerevisiae dpm1::LEU2/DPM1 diploid, which did not lead to the restoration of viability of the dpm1 spores. Simultaneously, a significant increase in MPG transferase activity, as compared with the wild-type yeast, was observed in cellular extracts when the mpg1 cDNA from Trichoderma was expressed in the S. cerevisiae dpm1-6 mutant.

Solubilization and one-step purification of mannosylphosphodolichol synthase from Trichoderma reesei.

Mannosylphosphodolichol synthase (MPD-synthase) (EC 2.4.1.830) catalyzing formation of MPD from GDPMan and dolichylphosphate (PD) has been purified from T. reesei cellular membranes almost to homogeneity. Selective solubilization of the enzyme was followed by one step purification on Phenyl-Sepharose column. SDS/ PAGE of the purified enzyme fraction revealed the presence of a protein band of 31 kDa corresponding to the apparent molecular mass of the MPD-synthase purified from S. cerevisiae [Babczinski, P. et al. (1980) Eur. J. Biochem. 105, 509-515; Haselbeck A. (1989) Eur. J. Biochem. 181, 663-668]. During solubilization, the enzyme was stabilized by the presence of a lipophilic substrate dolichylphosphate and phospholipids as well as by protease inhibitors. The Phenyl-Sepharose purified enzyme had an absolute requirement for dolichylphosphate and was activated by cAMP dependent protein kinase.

[Diagnostic value of oligoclonal band detection in cerebrospinal fluid using PhastSystem device]. / Wartosc diagnostyczna wykrywania pasm oligoklonalnych w plynie mózgowo-rdzeniowym z zastosowaniem aparatu PhastSystem.

The study confirming the presence of oligoclonal IgG in cerebrospinal fluid by isoelectric focusing (IEF) with PhastSystem equipment was carried out in 68 patients with clinically definite MS, in 23 with clinically probable MS and in 23 with other neurological diseases. Other indicators intrathecal synthesis of IgG were olso marked. According to the results it was confirmed that the most sensitive method of detection of intrathecal synthesis of immunoglobulins is finding of the oligoclonal IgG. Using the PhastSystem evidently shorties the diagnostic process. The sensivity of the method equals that of MRI and both have similar clinical value.

[Ganglioside GM1 in early strokes]. / Zastosowanie gangliozydu GM1 we wczesnej fazie udaru mózgu.

The study was taken up to compare the effect of treatment with ganglioside GM1 (Sygen, FIDIA-Italy) and typical treatment in the course of the disease. The study included 98 patients aged 40-82 in good or moderately good physical condition, with the ischaemic stroke confirmed by CT scan, in early stage of stroke (within 48 hours after onset). Patients with severe physical diseases were excluded. The patients were divided at random into two groups. Group I (50 patients) was treated typically and group II (48 patients) was given both typical treatment and ganglioside GM1 administered in 100 mg daily doses over 30 days, i.v. during first 5 days, then i.m. The neurological state was assessed according to the Canadian Neurological Scale (CNS), general fitness according to the Ranking Classification of Neurologic Disability Status (RS) at the admission and after 30 days of the treatment. After 30 days of the treatment no difference between the two groups was found in: 1) mortality, 2) mean survival time, 3) neurological state, 4) patient general fitness. According to the above results the beneficial influence of Sygen treatment of ischaemic stroke was not confirmed.

Modulation of mannosylphosphodolichol synthase and dolichol kinase activity in Trichoderma, related to protein secretion.

It has been postulated that exoprotein secretion in Trichoderma is related to their O-glycosylation. In the present paper the involvement of phosphodolichol in this process is described and the key role of mannosylphosphodolichol (MPD) synthase in protein O-mannosylation is discussed. The effect of water soluble phospholipid precursors such as choline and Tween 80, known also to increase secretion of cellulases when added to the medium, on MPD-synthase activity is presented. This effect is positive in the Trichoderma reesei QM 9414 (a low producing strain) but has no influence on the enzyme activity from the RUT C-30 strain selected to overproduce secretion of exoproteins and known to contain an increased cellular amount of endoplasmic reticulum. The positive effect of addition of choline and Tween to the medium on the level of dolichol kinase activity is also demonstrated. The influence of cultivation temperature on the activity of the various enzymes involved in dolichol-dependent protein glycosylation i.e. MPD-synthase, dolichyl kinase and MPD/Protein mannosyl transferase was tested. For all enzymes cultivation at 35 degrees C led to the elevated activity, which was most striking for dolichol kinase, whereas for MPD-synthase and MPD/Protein mannosyl transferase the difference was only apparent in the assay when endogenous phosphodolichol was used as a substrate. Furthermore, lipid extract from the membranes cultivated at elevated temperature, when added to the enzyme obtained from Trichoderma grown at 25 degrees C, enhanced the dolichol kinase activity measured in the absence of exogenous dolichol.(ABSTRACT TRUNCATED AT 250 WORDS)

[Secretion of tumor necrosis factor alpha (TNF-alpha) by peripheral blood monocytes in patients with multiple sclerosis and subacute sclerosis panencephalitis]. / Wydzielanie czynnika nekrotycznego guza alpha (tumor necrosis factor alpha TNF-alpha) przez monocyty krwi obwodowej pacjentów ze stwardnieniem rozsianym i podostrym stwardniajacym zapaleniem mózgu.

TNF alpha production by peripheral blood monocytes was studied in seventeen patients with a recent exacerbation of MS, thirteen with remission of MS and fourteen patients with SSPE. Monocytes from both MS groups spontaneously secreted high amounts of TNF alpha in vitro. Addition of lipopolisaccharide could not stimulate further synthesis of TNF alpha. In SSPE spontaneous and stimulated TNF alpha release did not differ from that in the control group. The observed changes in TNF alpha release in MS patients could play a role in the pathogenesis of the disease.

[Giant arteriovenous angiomas of the brain with scant clinical manifestations]. / Olbrzymie naczyniaki tetniczo-zylne mózgu skapoobjawowe klinicznie.

Two patients are described with large intracranial arteriovenous angioma with minimal neurological symptoms. The first patient suffered from repeated right fronto-temporal headache. Migraine was diagnosed and he was treated with analgesic drugs. We carried out scintigraphic investigation and computerized tomography of the brain. The results of these examinations suggested the presence of large brain lesion (tumour or vascular malformation). Bilateral carotid angiography and aortic arch angiography were done in this patient. A large arteriovenous angioma was discovered in the right occipital area. The second patient complained of throbbing headaches. A bruit could be heard over the left eye which was exophthalmic. Angiograms showed a carotid-cavernous fistula on the left side and a large angioma in the fronto-parietal area. The angioma was clinically "silent". It diminished the symptoms of the carotid-cavernous fistula through the uptake of blood. Both patients were regarded as unsuitable for surgery. The reasons were the great dimension of these angiomas and a large number of supplying arteries.

[Two cases of ophthalmic zoster follow by hemiplegia]. / Dwa przypadki pólpasca ocznego z nastepowym niedowladem polowiczym.

Two cases of ophthalmic zoster are reported in which several weeks after the appearance of skin changes hemiplegia developed. In one case the clinical course was unfavourable, and on autopsy extensive vasculitis was found in the brain with ischaemic foci situated mainly on the side of zoster. In the second case with favourable outcome CT demonstrated ischaemic foci probably of vascular origin, again on the side of zoster.

Interleukin 1 and 2 production by peripheral blood mononuclear cells in subacute sclerosing panencephalitis and exacerbation of multiple sclerosis.

Il-1 and Il-2 production by peripheral blood mononuclear cells was studied in 10 patients with SSPE and 15 patients in the acute stage of MS. Both SSPE and MS cells in vitro released spontaneously high amounts of Il-1, and could not be further stimulated to synthesis of Il-1 by latex. The Il-2 production was decreased in both processes but in MS after the recovery from the acute stage the production of Il-2 increased. The role of the observed changes in both Il-1 and Il-2 secretion in the pathogenesis of SSPE and MS is unknown.

Influence of increased environmental temperature on oxidation processes in rat liver mitochondria.

Exposure of rats to elevated temperature of 28 degrees C or 35 degrees C for 3 days six hours daily resulted in a decreased rate of oxidation with succinate or glutamate + malate as substrates, by the mitochondria of liver. The higher decrease was observed in environment temperature of 35 degrees C. There was no change in ADP/O ratio. The activities of NADH: cytochrome c reductase and cytochrome oxidase were stimulated but activities of succinate dehydrogenase and succinate cytochrome reductase were decreased.

[The clinical picture of stenosis and occlusion of the extracranial cerebral arteries]. / Obraz kliniczny zwezen i niedroznosci tetnic mózgowych pozaczaszkowych.

The authors report a clinical analysis of 200 cases of various forms of stenosis and occlusion of the extracranial arteries supplying the brain. The cases were collected in a period of 15 years. Follow-up investigations were conducted in 100 conservatively treated patients and 100 surgically treated ones. Syndromes of cerebral, ophthalmic, trophic and vascular signs are described, their incidence is discussed and the most salient features are emphasized.

[Visualization of the venous vessels in cerebral arteriograms in various types of strokes]. / Uwidocznienie naczyn zylnych w arteriografii mózgowej w róznych postaciach udarów mózgowych.

The authors analysed 1468 internal carotid angiograms including 945 performed in patients with strokes and 523 with brain tumours. Three phases were evaluated: arterial, middle and venous, directing attention to brain venous system filling in the arterial phase. Carotid arteriography carried out within 14 days after stroke onset visualizes early filling of the veins and this sign may be helpful in localizing the site of brain damage.

[Evaluation of sulocton action in patients with atherosclerotic brain ischemia]. / Ocena dzialania suloctonu u chorych z niedokrwieniem mózgu w przebiegu miazdzycy.

Sulocton effect was evaluated in 33 patients with symptoms of brain ischaemia during atherosclerosis. Thirty patients received the drug orally for 2 months in doses of 100 mg thrice daily. Three patients discontinued the treatment earlier (two of them discontinued it because of side effects). After two months of treatment a significant improvement was observed in such disturbances important for wellbeing and social contacts as: anxiety and fear, mood depression, disequilibrium of emotion, motivation and initiative. In many patients headaches, dizziness and gait disturbances disappeared or diminished. Sulocton was useful in the treatment of patients with cerebral atherosclerosis.