Entacapone enhances the response to levodopa in parkinsonian patients with motor fluctuations. Nomecomt Study Group.

OBJECTIVE: To study the effect and safety of entacapone as an adjunct to levodopa treatment in patients with PD with wearing-off motor fluctuations. BACKGROUND: Entacapone is a catechol-O-methyltransferase (COMT) inhibitor that has been shown to increase the area under the concentration-time curve of plasma levodopa by decreasing its systemic elimination, thereby promoting and improving therapeutic response to it. METHODS: A total of 171 parkinsonian patients with wearing-off-type motor fluctuations participated in a 6-month randomized, placebo-controlled, double-blind, parallel-group study. The extent of therapeutic response was elicited in the first hand with home diary recordings of "on" and "off" times by the patient and with Unified Parkinson's Disease Rating Scale scoring by the examiner. The patients took either 200 mg entacapone or identical placebos concomitantly with each daily levodopa dose (four to 10 times a day). RESULTS: Patients' home diaries indicated that entacapone increased the mean (+/- SD) "on" time significantly (9.3 +/- 2.2 to 10.7 +/- 2.2 hours; p < 0.01) and correspondingly decreased the "off" time significantly (5.3 +/- 2.2 to 4.2 +/- 2.2 hours; p < 0.001). The average benefit derived from a daily levodopa dose as related by the patients was increased significantly (p < 0.01). The daily levodopa dose was reduced significantly in the entacapone group, the difference between the groups being 102 mg (p < 0.01). The entacapone-derived increase in the benefit from levodopa was lost almost completely following its withdrawal. Entacapone was well tolerated. Dopaminergic adverse events, which increased, were ameliorated by reducing the levodopa dose. Diarrhea was the most common nondopaminergic adverse event. CONCLUSIONS: Long-term entacapone treatment effectively prolonged the beneficial response to levodopa in parkinsonian patients with the wearing-off phenomenon. The improvement occurred irrespective of the reduction of the levodopa dose.

Dyslipoproteinemia in patients with ischemic cerebro-vascular disease: a study of stroke before the age of 55.

Serum lipoproteins were determined 8-12 weeks after the onset of ischemic cerebro-vascular disease (ICD) in 61 patients, 38 males and 23 females, before the age of 55. The results were compared with those of a matched control material. The diagnosis was based on clinical findings, CSF spectrophotometry, computer tomography, and angiography. Hyperlipoproteinemia was no common finding in these young and middle-aged patients with ICD. The normal mean total serum cholesterol concentration was the result of a slight increase in VLDL cholesterol and a concomitant HDL cholesterol reduction. In men, the HDL cholesterol concentration was lower than expected for any VLDL-TG concentration. The mean value of the HDL cholesterol concentration in the patients was 18% lower than in the control group. On agarose electrophoresis the lipoprotein variants "late prebeta", "sinking prebeta" and "rapid beta" lipoproteins could be demonstrated in the same frequency as in controls. There was no significant correlation between the degree of atherosclerosis, estimated by angiography, and any serum lipoprotein fraction. Several recent studies have stressed the importance of a low HDL concentration as an independent risk factor for atherosclerosis. The decreased HDL cholesterol levels found in the present material require further attention to the possible beneficial role of HDL in ICD.

Increased cerebrospinal fluid concentration of nitrite in Parkinson's disease.

The concentration of nitrite, a metabolite of nitric oxide (NO), was increased in the cerebrospinal fluid (CSF) of untreated patients with Parkinson's disease and in patients treated with L-DOPA in comparison with a group of patients without dopaminergic dysfunction. There was no difference in the concentration of L-arginine (ARG), a precursor of NO, between the groups. There was a highly significant, linear relationship between the concentration of nitrite and ARG in the CSF suggesting that the production of NO is dependent on the availability of ARG. The results support the possibility that production of NO is increased in the brain in Parkinson's disease.

Inhibition of DNA synthesis in human gliomas by roscovitine.

The early effect of 1-100 microM roscovitine, a purine analogue and cyclin-dependent kinase inhibitor, was studied on tissue specimens from eight human malignant gliomas. The tissue was incubated immediately after resection with DMEM containing [3H]methylthymidine plus vehicle alone or the proper concentration of roscovitine for 30-90 min. The DNA synthesis rate was assessed by measurement of [3H]methylthymidine incorporation into trichloroacetic acid insoluble material/mg protein/min. In all gliomas, 100 microM roscovitine inhibited DNA synthesis by 71-97% (average 89 +/- 8%, p<0.0001). This inhibitory effect of roscovitine appeared within 30 min of incubation and was concentration dependent.

Isoelectric focusing and crossed immunoelectrofocusing of CSF immunoglobulins in MS.

The 3 main Ig classes and the presence of free light chains were studied by isoelectric focusing and crossed immunoelectrofocusing in 100 CSF samples from patients with clinically definitie or probable MS. Minute quantities of IgM and free light-chain (mostly lambda) components were found in 2 out of 11 and 5 out of 14 samples respectively. IgG and IgA were detected in all samples examined for these proteins and were found in the pI-ranges of 5.3-9.8 and 4.6-6.4 pH-units respectively. The gammaglobulin abnormalities found on isoelectric focusing were identified as microheterogeneous, oligoclonal IgG with predominantly kappa light-chain determinants. The IgG immunoprecipitates differed from those of normal subjects and the major abnormal components most frequently exhibited pI-values greater than 8 pH-units. The IgA immunoprecipitates had 2-4 main components with some tendency to discontinuous subfractionation. This Ig class, however, did not exhibit the marked tendency to oligoclonal distribution found for IgG.

Abnormal CSF immunoglobulin components detected by isoelectric focusing.

The results from thin-layer isoelectric focusing of 4000 paired CSF and serum samples were examined for the occurrence of abnormal CSF immunoglobulin components. Such changes, not referable to serum protein abnormalities, were detected in 630 samples from 465 patients. All but 3 of these subjects had symptoms and signs of an affection of the nervous system, predominantly MS and encephalitic or myelitic/radiculitic disorders of known or presumed infectious etiology. The abnormal, microheterogeneous immunoglobulin components were mostly focused in the pI range approximately 7.5--9 with generally a shift towards pI-values > 8 pH units; a distribution differing from that of M components in plasma cell dyscrasias.

Crossed immunoelectrofocusing of cerebrospinal fluid immunoglobulins.

The combination of the high separation capacity of isoelectric focusing and the specificity of immunoelectrophoresis gives a valuable tool for protein studies. This technic can be applied to immunoglobulin examinations by using KOCN-treated antibodies isoelectric at pH 5 and performing the immunoelectrophoretic procedure at this pH. CSF and serum samples of patients exhibiting CSF immunoglobulin abnormalities and blood-CSF barrier damages were examined by this method.

Examinations of cerebrospinal fluid immunoglobulin G by chromatofocusing.

IgG components in the CSF and paired sera were examined by chromatofocusing. The separations were performed using the FPLC system with a mono P column and were based on a pH gradient between 9.5 and 6.0. A total amount of 0.1 mg IgG was applied to the column. All samples with oligoclonal IgG were easily identified and the findings differed clearly from those of normal IgG. The sensitivity, resolution capacity and relatively fast as well as easy performance make chromatofocusing a promising tool for investigations of IgG in the CSF.

Isoelectric focusing of CSF proteins and the future evolution of multiple sclerosis: a clinical follow-up.

A clinical follow-up covering a period of 5-10 years after onset was performed in 150 patients with optic neuritis or other potential onset symptoms of MS. Thin-layer isoelectric focusing had been used for the initial CSF-protein analysis. No evidence for a more probable alternative diagnosis appeared in 147 patients while a non-MS diagnosis was established in 3 patients. Among these 147 subjects the planned follow-up was accomplished in 131 patients, but not in 16. An evolution into clinically definite MS occurred in 59 subjects, in whom oligoclonal CSF immunoglobulin was found in 92%. Further clinical activity without spatial dissemination--i.e. lesser degrees of diagnostic probability for MS--were found in 35 patients in whom oligoclonal CSF immunoglobulin components were detected in 86%. Among the 131 patients with a complete follow-up, 45 remained free from further clinical activity; oligoclonal CSF immunoglobulin components occurred in 40% of these patients. The frequency of further clinical activity with or without spatial dissemination was significantly higher in subjects exhibiting oligoclonal CSF immunoglobulin components than in those without such changes.

Isoelectric focusing of CSF and serum proteins in neurological disorders combined with benign and malignant proliferations of reticulocytes, lymphocytes and plasmocytes.

The clinical data and the CSF and serum findings on isoelectric focusing (IEF) and electrophoresis were studied in nine patients with neurological disorders combined with proliferations of reticulocytes, lymphocytes or plasmocytes. Paraneoplastic neurological manifestations were considered in some of the subjects. CSF findings compatible with intrathecal synthesis of oligoclonal immunoglobulins were observed in two patients with lymphoreticular neoplasms. One of these had an IEF band spectrum differing from those found with multiple sclerosis (MS), while the other had changes indistingusihable from those with MS. On IEF the CSF and serum M components of the patients with plasma cell dyscrasias, as well as the serum M components of nine other patients with myelomatosis, exhibited considerable microheterogeneity with 3-18 abnormal bands. The isoelectric point (pI) of the individual bands was 5.8-9.2, mostly greater than 6.4; the band spectra exhibited pI ranges of 0.4-2.5 pH units, most frequently 0.4-1.2. The CSF findings, possible modified by the blood-CSF barrier, reflected the serum changes of plasma cell dyscrasias. The immunoglobulins produced in such disorders gave IEF band spectra differing from those of intrathecally produced immunoglobulins in MS.

Isoelectric focusing of cerebrospinal fluid proteins in ischemic cerebrovascular disease.

The cerebrospinal fluid (CSF) protein patterns, in ischemic cerebrovascular disease (ICD), of varying extension were studied by isoelectric focusing (IEF) in 100 patients at different intervals after the onset of symptoms. The diagnoses were based on conventional clinical examinations and CSF spectrophotometry in all cases. Computed tomography was performed on 52 cases. One or more CSF protein aberrations were noted in 94 patients. Some of these findings were most common with small lesions including TIA. Other aberrations were most frequent with the more extensive infarctions. A regional increase in the gammaglobulin range was found in six cases. The findings were most frequent in the first days after the stroke except for barrier damage which reached a maximum during the second week. The IEF findings of CSF seem to be of diagnostic value. Taken together with the clinical signs and CT findings, they could conceivably give prognostic information.

Peripheral neuropathy in patients with benign monoclonal gammopathy--a pilot study.

It is well known that peripheral neuropathy occurs in patients with myeloma or macroglobulinaemia, but its pathogenesis is still obscure. In recent years, neuropathy has also been reported in association with benign monoclonal or oligoclonal gammopathy. Modern histo-immunological methods have revealed evidence of antibody production to peripheral nerve tissue, probably the myelin sheath. The present study included 21 unselected, consecutive patients with benign monoclonal gammopathy observed in the Division of Haematology. Clinical and laboratory investigations included electrophysiological examination and analyses of the M components. Of the 21 patients 11 had noticed slight neuropathic symptoms in their extremities; in 5 both clinical and electrophysiological findings were compatible with neuropathy; 6 showed positive clinical signs of neuropathy; 4 had either positive electromyographic or electroneurographic findings. In summary, 15 of 21 patients had some signs of peripheral neuropathy. In spite of the screening design of the study, this strikingly high frequency is comparable with other recent reports. Haematological studies did not reveal any significant differences between the patient groups with positive or negative neurological findings. The findings indicate that even benign gammopathies may be associated with peripheral neuropathy.

Plasma levels of 24S-hydroxycholesterol in patients with neurological diseases.

The brain is the exclusive or almost exclusive site of formation of 24S-hydroxycholesterol and we have shown that the circulating level of 24S-hydroxycholesterol is dependent upon the relation between cerebral production and hepatic clearance. In the present work we determined plasma levels of 24S-hydroxycholesterol in patients with various neurological diseases. Eleven subjects with brain death occurring 6-10 h before collection of the plasma samples had markedly reduced circulating levels of 24S-hydroxycholesterol (-43%, P<0.001). Patients with advanced Alzheimer's disease and cerebral inflammatory diseases had slightly lower levels of 24S-hydroxycholesterol in plasma when compared to matched controls. Patients with acute ischemic stroke, multiple sclerosis and primary brain tumors had levels not significantly different from those of controls. The conditions leading to reduced plasma levels of 24S-hydroxycholesterol had no significant effect on plasma levels of another side-chain oxidized oxysterol, 27-hydroxycholesterol. Except for conditions characterized by very marked destruction of the central nervous system, different severe neurological diseases seem to have relatively small effects on the flux of 24S-hydroxycholesterol from the brain.

Evidence for an anti-aggregatory effect of adenosine at physiological concentrations and for its role in the action of dipyridamole.

The effects of physiological adenosine concentrations on platelet aggregation in vitro were studied. Furthermore, we evaluated the effect of elevated adenosine levels in vivo, produced by the administration of dipyridamole, on platelet aggregation in whole blood. Platelet aggregation in plasma was significantly inhibited in vitro by adenosine at all concentrations tested in the physiological range (0.1-1.0 microM, 14-63% inhibition). Dipyridamole by itself had no effect at a therapeutic plasma concentration in vitro. Ten patients with ischaemic cerebrovascular disease were given 100 mg dipyridamole orally, and the level of adenosine increased from 0.22 to 0.29 microM (p less than 0.05). This was accompanied by a decrease in ADP-induced platelet aggregation in whole blood (17 to 15 ohms, p less than 0.05). When dipyridamole was infused in 11 healthy subjects, the adenosine level was not significantly elevated but the platelet aggregation was inhibited (from 13 to 11 ohms, p less than 0.05). It is concluded that adenosine may be of importance in the physiological regulation of platelet aggregation. Furthermore, dipyridamole treatment is associated with an anti-aggregatory effect that is probably mediated by its effect on endogenous adenosine levels.

Isoelectric focusing of CSF proteins in known or probable infectious neurological diseases and the Guillain-Barré syndrome.

The CSF and serum proteins of 120 patients with known or probable infectious neurological diseases or the Guillain-Barré syndrome were examined with thin-layer IEF. All but two of these patients exhibited one or combinations of different CSF-protein aberrations in the acidic and alkaline range. Aberrant non-Ig fractions (including transferrin, the tau-fraction and gamma-trace protein) were found in frequencies varying between 4 and 48%. CSF Ig components of restricted heterogeneity, i.e. oligoclonal bands and/or regional increases of gamma-globulins, were more frequent in patients with (meningo-)encephalitic or (meningo-)-myelitis/radiculitic disorders (respectively 69 and 48%) than in subjects with meningitis or Guillain-Barré syndrome (17%). The occurrence of such Ig abnormalities was higher in subacute or chronic than in acute disease and in subjects examined greater than 4 weeks after the onset rather than earlier. Ig-band spectra with marked anodal extension were found predominantly in (meningo-)encephalitic disorders with infratentorial symptoms. Age and sex were not found to influence the occurrence of abnormal Ig fractions. Such components could be detected in spite of pronounced blood-CSF barrier defects.

CSF protein examinations with thin-layer isoelectric focusing in multiple sclerosis.

Thin-layer IEF, due to its extremely high resolving capacity, has been found to be quite valuable for CSF protein examinations, one important advantage of the technique being its excellent capacity for separation of immunoglobulins. The CSF and serum proteins of 230 patients with clinically verified or probable MS and 20 subjects with optic neuritis were examined with thin-layer IEF and the findings were compared with clinical data and results of other CSF examinations. All but 3 of the MS patients and about two thirds of the subjects with optic neuritis inhibited one or combinations of different CSF protein aberrations in the acidic and alkaline range. Oligoclonal bands and/or regional increases of Ig fractions, changes compatible with intrathecal Ig synthesis, were detected in respectively 95 and 80% of patients with clinically verified and probable MS and 30% of subjects with optic neuritis. Other aberrant CSF protein fractions (including transferrin, the taufraction and gamma-trace protein) were found in about half of the cases; some of these fractions had the highest occurrence in patients with the most extensive Ig abnormalities. The diverse CSF protein aberrations seemed to be influenced by the duration and course of the disorder as well as the probable sites of lesions; further factors might be the release of decomposition products from destroyed tissues, the genetically determined reactivity of the individual and the presence of possible agents.

Immunoglobulin light chain patterns in the cerebrospinal fluid. A study with special reference to the occurrence of free light chains in cerebrospinal fluid with and without oligoclonal immunoglobulin G.

The immunoglobulin light chain patterns were studied in paired cerebrospinal fluid (CSF) and serum samples from 30 controls, 70 multiple sclerosis (MS) patients, 18 subjects with other inflammatory neurological diseases and 17 patients with other non-inflammatory neurological disorders. In MS, all CSF samples examined by two-dimensional gel electrophoresis exhibited clonally restricted light chain components. Isoelectric focusing and immunoblotting detected free light chains in around 90% of CSF samples from patients with MS or other inflammatory diseases. These components were clonally restricted, appeared in both mono- and dimeric forms and occurred in CSF samples with as well as without oligoclonal immunoglobulin G bands. Generally, the positive CSF samples contained kappa as well as lambda free lights chains. Such components were not detected in the sera, nor in the CSF samples from controls or patients with non-inflammatory diseases.

A longitudinal study on IL-2, sIL-2R, IL-4 and IFN-gamma in multiple sclerosis CSF and serum.

The cerebrospinal fluid (CSF) and serum levels of interleukin-2 (IL-2), soluble IL-2 receptor (sIL-2R), interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) were longitudinally investigated in 20 multiple sclerosis (MS) patients. There were 80 paired CSF and serum samples (range 2-8 per patient) covering a 1-5 year (mean 2.5 year) period. Increased levels of IL-2 and sIL-2R were found in 56 and 71%, respectively, of MS sera. In all patients, one or several sera (totally 89%) exhibited values above the normal range for either one of the components or both. The occurrence of IL-2 or sIL-2R positive CSF specimens was much lower, 15 and 9%, respectively. Only 3 MS sera (from one patient) had clearly detectable IL-4 and no CSF samples were definitely positive. IFN-gamma was undetectable in all serum and CSF specimens. No correlations were found between the immunological parameters and the clinical disease activity. The cytokine patterns in MS give strong support for the presence of a systemic T-cell activation. Furthermore, the data argue for a predominant activation of an IL-2- and sIL-2R-producing but not IL-4-producing T-helper (Th) lymphocyte subpopulation, Th1/CD4 + CD45R + cells.

Evidence of a local immune activation in cystic brain tumors.

The fluid of cystic brain tumors was characterized with regard to the protein content. In most malignant tumors, the concentrations of immunoglobulins G and M (IgG and IgM) were higher relative to other proteins in the cyst fluid than in the serum of the same patient. A markedly elevated ratio of monomeric to pentameric IgM was detected in the cyst fluid of two patients with glioblastomas. The results indicate a local immunoglobulin synthesis in malignant cystic brain tumors. It is hypothesized that higher-than-expected concentrations of IgG and IgM in cyst fluid as compared to plasma are a sign of an ongoing immune response triggered by the tumor.

Chromatofocusing.

Investigations of different protein species in complex biological fluids, e.g., cerebrospinal fluid (CSF) and serum, are important tasks in clinical laboratory work as well as in biochemical research. One crucial part of such investigations is the application of a suitable method in order to separate the individual proteins and/or resolve a certain protein into subtypes or microheterogeneous components. There are two major categories of procedures that are used to achieve these separations. One is represented by electromigration techniques, such as electrophoresis or isoelectric focusing, which separate the sample molecules according to size or charge; an immunoelectrophoretic step may also be used, i.e., a separation according to antigenicity. The second major category is formed by the liquid chromatographies, which give a partition of the sample molecules on the basis of size, charge, hydrophobicity, or binding to a biospecific ligand.